Compositions and methods for treating citrus greening

Abstract

Certain embodiments of the invention provide a method of biological control of citrus disease using microbial strains or microbially derived products as described herein.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING CITRUS GREENING

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 570,612 that was filed on March 27, 2024 and United States Provisional Application Number 63 / 637,192 that was filed on April 22, 2024. The entire content of the applications referenced above is hereby incorporated by reference herein.

[0004] GOVERNMENT FUNDING

[0005] This invention was made with government support under 2017-70016-26053, and 2020- 70029-33202 awarded by the National Institute of Food and Agriculture, USDA and 16- SCBGP-CA-0035 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Citrus Huanglongbing (HLB), is a serious disease of citrus and is the major threat to citriculture worldwide. In the United States, HLB is associated with a gram-negative, phloemlimited, alphaproteobacteria: Candidatus Liberibacter asiaticus (CLas) and several different strains of CLas have been reported in association with citrus (Chen et al., 2010. Phytopathology 100:567-572; Kunta et al. 2017. Genome Announc 5(15):e00170-17; Zheng et al. 2017. Phytopathology 107:662-668). This bacterium is vectored by insect psyllid vectors and the primary psyllid in the United States of America (USA) is the Asian Citrus Psyllid (ACP), Diaphorina citri. Both the vector and the bacterium are invasive species to the USA. Symptoms of the disease include leaf chlorosis, limb dieback, root loss, and phloem plugging (Bove JM. 2006. J Plant Pathol 88:7-37; da Graga et al., 2016. J Integr Plant Biol 58:373-387). Diseased trees produce small, bitter, hard, green and misshapen fruit. These fruits are unmarketable for juicing because the disease results in acidic, salty and off-flavor juice. In addition to unpalatable flavor, fruit borne of trees with severe HLB symptoms, exhibit severe morphological distortions and seed discoloration rendering them unsuitable for fresh market sale (Bassanezi et al., 2009. European Journal of Plant Pathology, 125:565-572; Dagulo et al., 2010. Journal of Food Science, 75(2):C199-207). Infected trees decline rapidly and die within 3-5 years of becoming infected and HLB can spread throughout an orchard in under seven years (Narouei-Khandan et al., 2016. Eur J Plant Pathol 144:655-670). All cultivated citrus varieties are susceptible to HLB (Folimonova et al., 2009. Phytopathology 99: 1346-1354; Gottwald et al., 2012. Crop Prot 36:73-82). Current management of HLB is limited and relies heavily on vector control through the use of insecticides (Blaustein et al., 2018. Phytopathology 108:424-435; Editorial, 2019. Nature 567:283). Thus, there is a need for new therapies for the treatment of HLB in citrus.

[0008] SUMMARY OF THE INVENTION

[0009] Certain embodiments of the invention provide a compound, a microbial isolate (i.e., an isolated microbe), or a composition as described herein (e.g., for use in a method described herein).

[0010] For example, certain embodiments provide a microbial isolate(s) as described herein (e.g., as described in an Example included herein, such as Example 5 (see, e.g., Example 5, Table 1, Figure 37 or Supplementary Table 1)), or an extract thereof. In certain embodiments, the microbial isolate(s) is an isolated microbe comprising a sequence described herein (e.g., a genomic sequence described herein, such as in Example 5). Certain embodiments also provide a composition comprising one or more microbial isolates as described herein (e.g., a composition comprising a concentrated amount of a microbial isolate as described herein) or an extract(s) thereof.

[0011] Certain embodiments of the invention provide a method of treating HLB or inhibiting Candidatus Liberibacter asiaticus (CLas) growth in a plant, comprising introducing to the plant at least one compound, microbial isolate, or microbial isolate preparation (e.g., bacterial extract) as described herein.

[0012] For example, certain embodiments of the invention provide a method of inhibiting Candidatus Liberibacter asiaticus (CLas) growth, treating a CLas infection, and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof.

[0013] Certain embodiments of the invention provide a method of inhibiting Candidatus Liberibacter asiaticus (CLas) growth, treating a CLas infection and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound as described herein (e.g., amicoumacin compound(s), pumilacidin compound(s) or surfactin compound(s)), or a salt thereof. For example, certain embodiments also provide a method of treating a Candidates Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound selected from the group consisting of an amicoumacin compound, bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or a salt thereof.

[0014] Certain embodiments provide a composition as described herein. In certain embodiments, the composition further comprises at least one additional therapeutic agent.

[0015] Certain embodiments provide a composition comprising at least one amicoumacin compound, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof.

[0016] Certain embodiments provide at least one compound, or salt thereof, as described herein, at least one microbial isolate, or extract thereof, as described herein and / or composition as described herein for inhibiting Candidates Liberibacter asiaticus (CLas) growth, treating a CLas infection, and / or treating Huanglongbing (HLB) in a plant.

[0017] Certain embodiments provide the use of at least one compound, or salt thereof, as described herein, at least one microbial isolate, or extract thereof, as described herein and / or composition as described herein in the preparation of a medicament for inhibiting Candidates Liberibacter asiaticus (CLas) growth, treating a CLas infection, and / or treating Huanglongbing (HLB) in a plant.

[0018] The invention also provides processes disclosed herein that are useful for preparing an extract (e.g., an extract fraction, such as a Bacillus safensis extract fraction as described herein) or compound as described herein. Certain embodiments also provide a composition (e.g., an extract fraction) as described herein prepared by a method described herein.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1. The plant microbiome interactions and disease incidence.

[0021] Figure 2. HLB management strategies.

[0022] Figure 3. Disease-induced microbial shifts in citrus indicate microbiome-derived responses to Huanglongbing across the disease severity spectrum. Ginnan et al., 2020, Phytobiomes Journal, 4(4):375-387. Figure 4. Huanglongbing microbiome disease model. Ginnan et al., 2018 Phytobiomes Journal, 2(2):64-70; Ginnan et al., 2020 Phytobiomes Journal, 4(4):375-387; Xi et al., 2022 Phytobiomes Journal, 6(4): 342-353; Ginnan et al., 2022 mBio, mBio 13:e00343-22; Kurbessoian et al., 2023 Microbiol Resour Announc 12:e00101-23.

[0023] Figures 5A-5E. Culture-dependent and -independent microbiome profile of HLB- affected citrus trees. Figure 5A. Exemplary scheme for establishing a citrus-associated microbe culture collection, including an in vitro agar diffusion inhibition bioassay for the identification of isolates with anti- / .. crescens properties. Figure 5B. Diversity within the culture-independent and culturable fractions of the bacteriomes of citrus leaves, stems, and roots. Plots illustrate the relative abundances of the bulk-cultured bacterial genera across leaf, stem, and root tissues (culture independent) compared to their cognate cultured bacterial communities derived from the same samples (culture dependent). Shades denote different genera with the most the 29 most abundant genera labeled. Figure 5C. Passage through culture medium produces diversity shifts in citrus-associated microbiota, (top and bottom) Violin plots illustrating Shannon’s alphadiversity index scores of the citrus bacteriome and its cultured counterparts, per tissue (top) and the citrus mycobiome and its cultured counterparts, per tissue (bottom). Diamonds represent the medians of each sample group. The cultured portion of the bacteriome represents 4.0% of the culture-independent taxa in the leaves, 5.4% of the culture-independent taxa in the stems, and 2.2% of the culture-independent taxa in the roots. The cultured mycobiome represents 16.7% of the culture-independent taxa in the leaves, 17.8% of the culture-independent taxa in the stems, and 7.6% of the culture-independent taxa in the roots. P values indicate the significance of the difference in alpha-diversity measures between culture-independent and culture-dependent samples per tissue, obtained via a Kruskal-Wallis with post hoc Dunn test, using Bonferroni correction (P < 0.05). Percent values indicate the proportions of culture-independent OTUs found in cultured microbiome samples. Figure 5D. Species richness in the citrus microbiome by tissue compartment. Figure 5E. Liberibacter crescens agar diffusion inhibition assay. (Figures 5Ei-5Eiv) Images of assay plates from the in vitro diffusion assay, showing uninhibited L. crescens BT-1 growth on a negative-control plate (Fig. 5Ei), a halo of / .. crescens BT-1 growth inhibition around a disk containing supernatant from C. cladosporioides (CF0052) (Fig. 5Eii), a halo of L. crescens BT-1 growth inhibition around a disk containing supernatant from E. nigrum (CB0051) (Fig. 5Eiii), and a halo of L. crescens BT-1 growth inhibition around a disk containing supernatant from Pantoea sp. isolate CB0072 (Fig. 5Eiv). Fifty microliters of MeOH was applied and evaporated off the disk prior to placement on the top agar. Blacutt et al., 2020, Appl Environ Microbiol. 86(8), e02883-19.

[0024] Figure 6. Mining the citrus microbiome. Using isolation techniques, genome sequencing and natural product characterization, we mine the citrus microbiome for anti- / .. crescens microbes and compounds.

[0025] Figure 7. In vitro antagonistic activity of 10 citrus bacterial isolates against Liberibacter crescens in agar diffusion bioassay. Boxplots show an average of three replicates.

[0026] Figure 8. Bacterial isolate CB729 identified as Bacillus safensis. Phylogenetic tree of Bacillus, constructed on SpeciesTree (v2.2.0) app of Kbase, using a set of 49 core, universal genes defined by COG (Clusters of Orthologous Groups) gene families. Bacillus safensis CB729 is highlighted.

[0027] Figures 9A-9C. Identification of amicoumacin in anti- / .. crescens fractions of CB729.

[0028] Figures 10A-10B. Figure 10A. Amicoumacin: antibiotic compounds with anti- / .. crescens activity. Park et al., 2016. Molecules. Figure 10B. Interaction of amicoumacin A with 30S subunit of the T. thermophilus 70S ribosome Shi et al., 2021. Nat Prod Res 35:5508-5512. Polikabov et al. 2014, Molecular Cell, 56(4):531-540.

[0029] Figure 11. Predicted secondary metabolite gene clusters of Bacillus safensis CB729. Bacillus safensis CB729 harbors the amicoumacin biosynthetic gene cluster (BGC).

[0030] Figures 12A-12B. Amicoumacin BGC comparison. Figure 12A. Gene cluster comparison of NRPS-PKS of Bacillus safensis CB729 to the characterized amicoumacin BGC of Bacillus subtilis subsp. inaquosorum KCTC 13429 and the zwittermicin A (ZmA) BGC of Bacillus cereus strain UW85. Clusters sequence similarity are indicated by the identity percentage bar on the bottom left. Figure. 12B. Proposed function and size of encoded proteins by the amicoumacin cluster of Bacillus safensis CB729 and adjacent open reading frames (ORF) and identity / similarity to the characterized amicoumacin gene cluster of Bacillus subtilis subsp. inaquosorum KCTC 13429.

[0031] Figure 13. Molecular networking reveals amicoumacin network in extracted metabolites of Bacillus safensis CB729.

[0032] Figure 14. Amicoumacin production and purification.

[0033] Figures 15A-15B. Amicoumacin A-enriched fractions are bioactive to CLAS. Figure 16. Molecular networking of amicoumacin-enriched bioactive samples.

[0034] Figure 17. Reconciling DNA-based studies with cognate culture collections.

[0035] Figures 18A-18G. Examination into whether HLB is a disease complex. HLB- associated Fusarium isolates.

[0036] Figures 19A-19C. Koch’s postulates with HLB-associated Fusarium isolates. Figure 19A. Methodology schematic. Figure 19B. Mean lesion lengths of Carrizo citrange, S-l citron, and Swingle citrumelo inoculated with F. oxysporum (F. oxy.), F. falciforme (F. falc.), F. solani (F. sol.) vs. a PDA control (Dunnett contrasts, p < ‘***’ 0.001; ‘**’ 0.01; 0.05; ‘NS’ not significant). Figure 19C. HLB-associated Fusarium strains isolated from roots are capable of causing disease in citrus. Root-associated F. oxysporum strains CF132 and CF159 caused highly significant (p < 0.001) lesions on Carrizo citrange, S-l citron, and Swingle citrumelo, while leaf- associated F. oxysporum strain CF141 only caused significant (p < 0.05) lesions on Carrizo. F. falciforme and F. solani also caused highly significant (p < 0.001) lesions on Carrizo, S-l, and Swingle. For each grouping, Carrizo is shown on the left, S-l in the middle, and Swingle on the right.

[0037] Figures 20A-20C. Koch’s postulates with HLB-associated Fusarium isolates. In Figure 20B, for each grouping, Sour is on the left, Swingle is in the middle, and Carrizo is on the right.

[0038] Figures 21A-21B. Citrus-associated Bacillus isolates inhibit citrus root pathogens. In Figure 21 A, for each grouping, Fusarium oxysporum is on the left, and Fusarium falciforme is on the right. In Figure 2 IB, for each grouping, Phytophthora nicotianae is on the left, and Phytophthora citrophthora is on the right.

[0039] Figures 22 A-22C. Targeted Metabolomics. Biologically active small molecules (e.g., cyclic lipopeptides) derived from the citrus microbiome. Blacutt et al., 2020, Appl Environ Microbiol. 86(8), e02883-19; and Lockner et al, 2020 Journal of Natural Products.

[0040] Figure 23. Scale-up.

[0041] Figures 24A-24D. Figure 24A. Prospector®, IsolationBio. Figure 24B. Micro-wells under the microscope. Figure 24C. Rezasurin (red fluorescence) converts to resorufin (green fluorescence) in the presence of respiring bacteria. Figure 24D. Fluoresecent microwells indicate growth.

[0042] Figure 25. Bacterial samples from Citrus rhizosphere demonstrating overgrowth.

[0043] Figure 26. Cartoon depicting phyllosphere and rhizosphere compartments of a plant. Figure 27. Image depicting Prospector® array being loaded with citrus tissue homegenate.

[0044] Figures 28A-28B. Figure 28 A. Example depiction of how growth on the array looks, with the top photo being 0 days post loading, and the bottom one being 7 days post loading. The wells that are brightly lit up green are indicative of metabolic oxidation of resazurin and signal active microbes in those microwells. Figure 28B. Image depicted computer output of a Prospector® array exhibiting growth after 7 days incubation.

[0045] Figures 29A-29B. Figure 29A. Bioassay against Liberibacter crescens. Figure 29B. High throughput inhibition assay. Schematic diagram of workflow using a plate replicator to assay 96 well plates against L. crescens.

[0046] Figures 30A-30B. Figure 30A. Image of a spotted plate with an isolate producing an inhibitory reaction towards L. crescens, circled. Figure 30B. Dual Microbial Assay. A non- inhibitory isolate (left), and an isolate producing a zone of inhibition (right) against L. crescens (visible as a white haze).

[0047] Figure 31. Preliminary inhibitory isolates. List of bacterial isolates that are inhibitory to L. crescens that were derived from the Prospector® workflow.

[0048] Figure 32. Scaling up: Bioassay guided fractionation.

[0049] Figure 33. High throughput bioassay that measures growth inhibition as a function of metabolic inhibition.

[0050] Figures 34A-34B. Assay validation using Kanamycin as positive control. Figure 34A. Metabolic dose response curve showing decreases in fluorescence as antibiotic concentration is increased. Figure 34B. Relationship expressed as a percent of inhibition when compared to untreated cells.

[0051] Figure 35. Pipeline in Action: obtain inhibitory phyllosphere isolate; grow it up as a large scall fermentation; extract its metabolites, and then fractionate the metabolome extract to separate out the molecules present.

[0052] Figure 36. Bioassay that allows for rapid and informative metabolome screening. Bioactivity across different fractions of the isolate’ s metabolome: at early non-polar fractions at the left; at a neutrally charged compound in the middle; and at a polar compound at the right. Kanamycin used as a positive control.

[0053] Figure 37. Genome Assembly statistics from a later assessment. Figures 38A-38B. Figure 38 A. Initial Phylogenetic tree generated using Species Tree and annotated using iTOL of closest related Bacillus species. Bacillus spp. identified in Example 5 are highlighted with an *. Bacillus bombysepticus isolate CB00676 was later determined to be Bacillus cereus as noted in Fig. 38B and Example 5. Figure 38B. Later phylogenetic tree of Bacillus, constructed on the SpeciesTree (v2.2.0) app of Kbase, using a set of 49 core, universal genes defined by COG (clusters of orthologous groups) gene families. The set of query genomes is inserted into curated multiple sequence alignment (MSA) for each COG family; the MSAs are concatenated, and a phylogenetic tree is reconstructed using FastTree2 (v2.1.10) (fastest setting) to infer approximately maximum likelihood. The bootstrap scores are shown on each node. Species marked in bold are described in Example 5 / Table 1 / Figure 37. The closely related genomes used to build the tree are labeled with the NCBI RefSeq species name and between brackets the GCF identifiers.

[0054] Figure 39. In vitro inhibition bioassay of CB729 fractions against Liberibacter crescens. N-acetyl-amicoumacin C was identified in a subsequent reversed-phase HPLC fractionation of fraction 6.

[0055] Figures 40A-40B. Featured-based molecular networking analysis of CB729 crude extract analyzed by LCMS / MS. Known compounds are labeled based on comparison with literature spectra. Amicoumacin A and amicoumacin B synthetic compounds were included as standards in this analysis. Nodes are shaded according to the metabolite origin, described in the legend box. Precursor ion m / z is inside the nodes and the edges width are based on cosine score.

[0056] Figure 41. Structures of amicoumacin compounds isolated from Bacillus safensis CB729 broth in this work.

[0057] Figures 42A-42B. Minimum inhibitory concentration of amicoumacin A (Fig. 42A) and amicoumacin B (Fig. 42B) to Liberibacter crescens (Lc). Growth of Lc was evaluated every 24 hours for 5 consecutive days. Points represent the average of six replicates per treatment.

[0058] Figure 43. CLas hairy root assay. Untreated (UT) and DMSO (0.5% and 1.0%) used to dissolve compounds were used as negative control and oxytetracycline (Oxy)-treated hairy roots (125 and 250 ppm) were used as positive control. Synthetic amicoumacin A (AmiA) and amicoumacin B (AmiB) were tested at 0.2 mg / ml, in addition to semi-purified amicoumacin A (AmiAP) from Bacillus safensis CB729 at 0.1 and 0.2 mg / ml. The bacterial titers were estimated by qPCR after 72 h of treatment with each sample and plotted relative to those of untreated samples (set to 100%). Error bars represent ± standard error of mean (n = 5). p-values were calculated by two-sample t test (one-tailed) relative to untreated samples. Samples that significantly reduced CLas titer compared to untreated were marked with an asterisk (*) and represent p-value < 0.01, two asterisks (**) represent p-value < 0.05.

[0059] Figure 44. Circular representation of Bacillus safensis genome for specific genome features. Circles display the following, from the inside out: (1) Negative GC skew (dark grey), (2) positive GC skew (light grey) and (3) GC content (black). Map generated with Proksee.

[0060] Figure 45.1H and13C NMR chemical shift comparisons for isolated N- acetylamicoumacin C with literature values.

[0061] Figure 46. LCMS data for isolated 7V-acetylamicoumacin C: UV254 (top), Total Ion Chromatogram (TIC, middle), and mass spectrum (bottom).

[0062] Figure 47.JH NMR spectrum (400 MHz, CDCI3) of isolated 7V-acetylamicoumacin C.

[0063] Figure 48.JH-13C HSQC spectrum (CDCI3) of isolated 7V-acetylamicoumacin C.

[0064] Figure 49.JH-13C HMBC spectrum (CDCI3) of isolated 7V-acetylamicoumacin C.

[0065] Figure 50. Entire featured-based molecular networking from Bacillus safensis CB729 crude extract generated by LCMS / MS. Nodes are shaded according to the metabolite origin: SYC media-produced metabolites, LCMS system blank-produced metabolites, CB729 broth produced metabolites, amicoumacin A standard, amicoumacin B standard. Precursor ion m / z is inside the nodes and the edges width are based on cosine score.

[0066] Figure 51.1H NMR chemical shift comparison for isolated amicoumacin A with literature values.

[0067] Figure 52. LCMS Chromatograms: TIC (top) and UV254 (bottom) for isolated amicoumacin A.

[0068] Figure 53. Mass spectrum (MS) for isolated amicoumacin A.

[0069] Figure 54. Tandem mass spectrum (MS / MS) for isolated amicoumacin A.

[0070] Figure 55.1H NMR spectrum (400 MHz, CD3OD) for isolated amicoumacin A.

[0071] Figures 56A-56B. (Fig. 56A) Extracted ion chromatogram of synthetic amicoumacin A. (Fig. 56B) MS2 compound analysis of synthetic amicoumacin A.

[0072] Figures 57A-57B. (Fig. 57A) Extracted ion chromatogram of synhtetic amicoumacin B. (Fig. 57B) MS2 compound analysis of synthetic amicoumacin B. Figures 58A-58B. CLas hairy root assay. Untreated (UT) and DMSO (0.1%, 0.5% and 1.0%) used to dissolve compounds were used as negative control and oxytetracycline (Oxy)- treated hairy roots (125 and 250 ppm) were used as positive control. (Fig. 58A) CB729 crude tested at doses 1, 5 and 10 mg / ml. (Fig. 58B) Amicoumacin A enriched fractions: Fraction 8, Fraction 9 (referenced on Figure 39), 729-peakl, 729-peak3 and 729-peak5. The three latter fractions resulted from another round of bioassay-guided fractionation from CB729 crude that displayed inhibition to Liberibacter crescens (results not shown). The bacterial titers were estimated by qPCR after 72 h of treatment with each sample and plotted relative to those of untreated samples (set to 100%). Error bars represent ± standard error of mean (n = 5). p-values were calculated by two-sample t test (one-tailed) relative to untreated samples. Samples that significantly reduced CLas titer compared to untreated were marked with an asterisk (*) and represent p-value < 0.01, two asterisks (**) represent p-value < 0.05.

[0073] Figure 59. CLas titer. The total titer of CLas was calculated by qPCR in citrus hairy roots that received treatments as indicated on the x-axis. Treatments were crude extracts of strains CB27, CB893 and CB909. CB27 and CB893 significantly reduced CLas titer at a concentration of 5 mg / ml as indicated by the asterisks.

[0074] Figure 60. Bacillus crudes against L. crescens. Crude extracts of Bacillus strains CB19, CB21, CB27, CB89, CB687, CB893, CB902, CB904, CB909, CB912 inhibited growth of L. crescens in vitro.

[0075] DETAILED DESCRIPTION

[0076] Described herein are certain microbes (e.g., isolated microbes), compounds and compositions that may be used to inhibit CLas growth and / or to treat CLas infections, such as Huanglongbing (HLB), also referred to as citrus greening disease. For example, Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and / or Bacillus subtilis, or an extract thereof, may be introduced to the plant to inhibit CLas growth and / or to treat a CLas infection. In certain embodiments, these organisms may be present in a composition, such as a bioinoculant composition. In certain embodiments, the microbe(s) is comprised in a whole cell broth composition. In certain embodiments, one or more of the microbial isolates are inactivated or present in an inactivated form or culture (e.g., a composition comprising an inactivated culture, such as heat-killed whole culture broth). Additionally, extracts from these organisms or compounds derived from these organisms (e.g., amicoumacin compounds or cyclic lipopeptides) may be used to inhibit CLas growth and / or treat CLas infections. In certain embodiments, the CLas inhibitory agent is provided as a total synthesis product.

[0077] Accordingly, certain embodiments of the invention provide a method of inhibiting Candidatus Liberibacter asiaticus (CLas) growth, treating a CLas infection, and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof.

[0078] In certain embodiments, the microbial isolate(s) is selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof.

[0079] In certain embodiments, the microbial isolate(s) is selected from the group consisting of Bacillus velezensis, Bacillus pumilus, and Bacillus cereus, or an extract thereof.

[0080] In certain embodiments, the microbial isolate(s) is selected from the group consisting of Bacillus velezensis and Bacillus pumilus, or an extract thereof.

[0081] In certain embodiments, the microbial isolate(s) is selected from the group consisting of Bacillus velezensis and Bacillus cereus, or an extract thereof.

[0082] In certain embodiments, a method described herein comprises introducing to the plant a combination of two or more types of microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof.

[0083] In certain embodiments, a combination of two or more types of microbes selected from Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof, is introduced to the plant.

[0084] In certain embodiments, a combination of two or more types of microbes selected from Bacillus velezensis, Bacillus pumilus, and Bacillus cereus, or an extract thereof, is introduced to the plant.

[0085] In certain embodiments, a combination of Bacillus velezensis and Bacillus pumilus, or an extract thereof, is introduced to the plant.

[0086] In certain embodiments, a combination of Bacillus velezensis and Bacillus cereus, or an extract thereof, is introduced to the plant. In certain embodiments, a combination of Bacillus safensis, or an extract thereof, and at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof, is introduced to the plant.

[0087] In certain embodiments, a method described herein comprises introducing to the plant a combination of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof.

[0088] In certain embodiments, the microbe or mixture of microbes is alive. In certain embodiments, the microbe or mixture of microbes is inactivated (e.g., heat-killed). In certain embodiments, the microbe or mixture of microbes is present in an inactivated culture (e.g., a heat-killed whole culture broth).

[0089] In certain embodiments, microbial extract(s) are introduced to the plant. In certain embodiments, one or more fractions of a microbial extract (e.g., a fraction described herein, such as a Bacillus safensis extract fraction as described herein) is introduced to the plant.

[0090] When more than one type of microbe / extract is introduced, the microbes / extracts may be introduced either simultaneously or sequentially. In certain embodiments, the microbes / extracts may be introduced simultaneously. In certain embodiments, a composition comprising two or more types of microbes / extracts are introduced. In certain embodiments, two or more types of microbes / extracts are introduced sequentially.

[0091] As described herein, in certain embodiments, a microbe(s) described herein or an extract from such a microbe(s) is present in a composition. In certain embodiments, the composition further comprises a carrier. In certain embodiments, the composition comprises Bacillus safensis, or an extract thereof. In certain embodiments, the composition comprises Bacillus velezensis, or an extract thereof. In certain embodiments, the composition comprises Bacillus pumilus, or an extract thereof. In certain embodiments, the composition comprises Bacillus cereus, or an extract thereof. In certain embodiments, the composition comprises Bacillus subtilis, or an extract thereof.

[0092] In certain embodiments, a composition comprises a combination of two or more types of microbes as described herein. In certain embodiments, the composition comprises a combination as described above. For example, in certain embodiments, the composition comprises two or more types of microbes selected from Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof. In certain embodiments, the composition comprises two or more types of microbes selected from Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract thereof. In certain embodiments, the composition comprises two or more types of microbes selected from Bacillus velezensis, Bacillus pumilus, and Bacillus cereus, or an extract thereof. In certain embodiments, the composition comprises Bacillus velezensis and Bacillus pumilus, or an extract thereof. In certain embodiments, the composition comprises Bacillus velezensis and Bacillus cereus, or an extract thereof. In certain embodiments, the composition comprises the Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis isolates as described in Table 1.

[0093] In certain embodiments, the composition comprises Bacillus safensis, or an extract thereof, and at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof, is introduced to the plant.

[0094] In certain embodiments, the composition comprises live microbe(s) described herein. In certain embodiments, the composition comprises an inactivated form or culture of a microbe described herein or mixture of microbes described herein, such as heat-killed whole culture broth.

[0095] In certain embodiments, the composition is a bioinoculant composition, optionally comprising a carrier. For example, bacteria may be cultured and then mixed with carrier(s). In certain embodiments, the carriers act to support the viability of bioinoculant isolates. In certain embodiments, the carriers act as bulking materials to allow easy handling. Carriers commonly used for bioinoculation to disperse the microbial isolates are described in, e.g., US Patent Nos. 5,068,105; 7,097,830; 7,141,395 and 9,068,189. Some exemplary carriers include peat, perlite, vermiculite, charcoal, powdered sorghum grain, fermented press mud, sulphinated press mud, carbonation press mud, grain, maize meal, maize cob, compost, soil, rice husk, rice bran, wheat bran, cow dung and talc. Many other non-toxic and biologically inert substances of dried or granular nature are also capable of serving as carriers for a biocontrol agent. In certain embodiments, a bioinoculant composition may further comprise gums or sugars to improve adhesion.

[0096] In certain embodiments, a microbial extract or a combination of microbial extracts are introduced to a plant. In one embodiment, a composition described herein comprises a bacterial extract e.g., a bacterial supernatant filtrate or lysate filtrate). A bacterial extract can be prepared from a bacterial culture (e.g., supernatant filtrate or lysate filtrate). In certain embodiments, the bacterial extract is a crude extract, for example, microbial culture supernatant may be extracted with organic solvent, including but not limited to ethyl acetate, to obtain a crude extract. In certain embodiments, the extract(s) is present in a composition, such as a composition described herein. In certain embodiments, the extract is a crude extract or a fraction of an extract. The extract may be fractionated by chromatography or by using other preparative liquid chromatography methods known in the art. The crude extract or fractionated extract may be processed at any stage of the composition preparation to undergo any appropriate extraction, separation, purification, or formulation step(s).

[0097] Bacterial extracts can be formulated into a liquid or solid form. In one example, the extract(s) is formulated into a concentrated solution or dispersion for easy distribution and can be diluted in the field prior to use. Alternatively, the extract(s) is formulated into a ready-to-use solution or dispersion. In certain embodiments, the extract(s) is formulated as an aqueous solution. In certain embodiments, the extract(s) is formulated as an emulsion (e.g., oil-in-water emulsion). In certain embodiments, the extract(s) is formulated as powder.

[0098] Without wanting to be bound by theory, microbes described herein may act as a biocontrol agent to control plant disease, for example, by secreting compound(s) that act to inhibit or kill the disease-causing organism (z.e., CLas), and / or by occupying the ecological niche that would otherwise be available to the disease-causing organism. Certain embodiments of the present invention provide exemplary environmental bacterial strains and their secreted natural products as described herein, and biological control method for treating plant diseases of citrus, e.g., using microbes and their natural products isolated from the citrus microbiome to combat pathogens.

[0099] The inhibitory compounds, microbes and compositions described herein may also be used in combination with each other to enhance anti -CLas efficacy. In one embodiment, the compounds or compositions described herein can be introduced to a plant as a mixture. In certain embodiments, a composition comprising a compound described herein and a microbial isolate / extract is provided. In one embodiment, the compounds or compositions described herein can be introduced to a plant concurrently or sequentially. In certain embodiments, CLas growth is inhibited by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0100] In certain embodiments, the plant is a citrus plant. In certain embodiments, the citrus plant is an orange, lemon, grapefruit, pomelo or lime tree / shrub. In certain embodiments, the citrus plant is an orange tree.

[0101] In certain embodiments, the plant with HLB disease also has secondary root pathogen(s), such as fungal or oomycete pathogens. For example, the root of the plant with HLB may be infected with Phytophthora spp. and / or Fusarium spp. In certain embodiments, the compounds, microbes or compositions described herein may inhibit the secondary root pathogen(s), such as Phytophthora spp. and / or Fusarium spp (e.g., Fusarium solani, Fusarium oxysporum, Phytophthora nicotianae and / or Phytophthora citrophthora) . Accordingly, certain embodiments provide a method of inhibiting or treating a Phytophthora spp. and / or Fusarium spp. infection in a plant, comprising introducing to the plant 1) at least one microbial isolate(s) as described herein, or an extract thereof; 2) at least one compound as described herein, or a salt thereof; and / or 3) at least one composition as described herein.

[0102] In certain embodiments, at least one additional agent is introduced to the plant (e.g., an agent capable of inhibiting CLas growth and / or treating a CLas infection). In certain embodiments, the at least one additional agent is an antibiotic.

[0103] Certain Microbe Embodiments for Use in a Method Described Herein

[0104] In certain embodiments, the microbial isolate(s) is a microbe as described herein (including the Examples and Figures, such as in Example 5). In certain embodiments, the microbial isolate(s) is a microbe comprising a sequence described herein (e.g., a genomic sequence described herein, such as in Example 5).

[0105] Bacillus safensis

[0106] For example, in certain embodiments, the microbe is Bacillus safensis. Thus, in certain embodiments, a method described herein comprises introducing Bacillus safensis to the plant. In certain embodiments, Bacillus safensis, or an extract thereof, is introduced to the plant. Bacillus safensis species are known in the art and described herein. For example, this species includes Bacillus safensis strain FO-36b (see, e.g., NCBI RefSeq assembly GCF 000691165.1), as well as the additional Bacillus safensis strain described herein.

[0107] In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under an accession number described herein (e.g., NCBI RefSeq Accession No., SRA Accession No., or a WGS accession No.). For example, in certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973669.

[0108] In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIG010000001 - JAXKIG010000019). In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIG010000001-JAXKIG010000019). In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001 -JAXKIG010000019). In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001 -JAXKIGO 10000019). In certain embodiments, the Bacillus safensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001 -JAXKIGO 10000019). In certain embodiments, the Bacillus safensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001- JAXKIG010000019).

[0109] In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000691165.1. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000691165.1. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000691165.1. In certain embodiments, the Bacillus safensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000691165.1. In certain embodiments, the Bacillus safensis genome comprises the genome assembly sequences as provided under NCBI RefSeq Assembly GCF 000691165.1. In certain embodiments, the Bacillus safensis genome consists essentially of or consists of the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000691165.1.

[0110] In certain embodiments, the Bacillus safensis is a Bacillus safensis FO-36b strain.

[0111] In certain embodiments, the Bacillus safensis is isolate strain CB00729 as described herein. As used herein, the terms CB729 and CB00729 are used interchangeably.

[0112] Bacillus safensis CB729 / CB00729 is described herein, such as in Example 5, including its isolation, identification, and characteristics (see, e.g., Table 1 and Figures 37-38). In particular, the Bacillus safensis CB729 was sent by the University of California, Riverside Campus (900 University Ave, Riverside CA 92521), as a representative of and on behalf of The Regents of the University of California, to the American Type Culture Collection Depository (ATCC® Depository, 10801 University Boulevard, Manassas, Va. 20110 USA) on March 26, 2025 and its ATCC® Patent Deposit Designation is to be assigned. Accordingly, in certain embodiments, the Bacillus safensis is the Bacillus safensis isolate strain CB729 having an ATCC® Patent Deposit Designation that is to be assigned.

[0113] In certain embodiments, the microbe is not Bacillus safensis. Thus, in certain embodiments, the method does not comprise introducing Bacillus safensis, or an extract thereof, to the plant.

[0114] Bacillus velezensis

[0115] In certain embodiments, the microbe is Bacillus velezensis. Thus, in certain embodiments, a method described herein comprises introducing Bacillus velezensis to the plant. In certain embodiments, Bacillus velezensis, or an extract thereof, is introduced to the plant.

[0116] Bacillus velezensis species are known in the art and described herein. For example, this species includes Bacillus velezensis strain NRRL B-41580 (see, e.g., NCBI RefSeq assembly GCF 001461825.1), as well as the additional Bacillus velezensis strains described herein.

[0117] In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under an accession number described herein (e.g., NCBI RefSeq Accession No., SRA Accession No., or a WGS accession No.). For example, in certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674.

[0118] In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (JAXKIA010000001-JAXKIA010000021), J AXKIB 000000000 (JAXKIB010000001- JAXKIB010000021), JAXKIC000000000 (JAXKIC010000001-JAXKIC010000022), JAXKIF000000000 (JAXKIF010000001-JAXKIF010000014), JAXKU000000000 (JAXKU010000001-JAXKU010000040), JAXKIK000000000 (JAXKIK010000001- JAXKIK010000043), JAXKIL000000000 (JAXKIL010000001-JAXKIL010000034), or JAXKIM000000000 (JAXKIM010000001-JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (J AXKIA010000001-JAXKI AO 10000021), JAXKIB000000000 (JAXKIB010000001-JAXKIB010000021), JAXKIC000000000 (JAXKIC010000001- JAXKIC010000022), JAXKIF000000000 (JAXKIF010000001-JAXKIF010000014), JAXKU000000000 (JAXKU010000001-JAXKU010000040), JAXKIK000000000 (JAXKIK01 OOOOOO 1-JAXKIKO 10000043), JAXKILOOOOOOOOO (JAXKIL010000001- JAXKILO 10000034), or JAXKIM000000000 (JAXKIM01 OOOOOO 1-JAXKIMO 10000039). In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (JAXKIA01 OOOOOO 1-JAXKIA010000021), JAXKIB000000000 (JAXKIB010000001-JAXKIB010000021), JAXKIC000000000 (JAXKIC01 OOOOOO 1 - JAXKIC010000022), JAXKIF000000000 (JAXKIF01 OOOOOO 1 - JAXKIF010000014), JAXKIJ000000000 (JAXKIJO 1 OOOOOO 1 -JAXKIJO 10000040), JAXKIK000000000 (JAXKIK010000001-JAXKIKO 10000043), JAXKILOOOOOOOOO (JAXKIL01 OOOOOO 1-JAXKIL010000034), or JAXKIM000000000 (JAXKIM010000001- JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (JAXKIA010000001- JAXKIA010000021), JAXKIB 000000000 (JAXKIB01 OOOOOO 1-JAXKIB010000021), JAXKIC000000000 (JAXKIC01 OOOOOO 1-JAXKIC010000022), JAXKIF000000000 (JAXKIF01 OOOOOO 1-JAXKIF010000014), JAXKIJ000000000 (JAXKIJO 10000001- JAXKIJO 10000040), JAXKIK000000000 (JAXKIK010000001-JAXKIKO 10000043), JAXKILOOOOOOOOO (JAXKIL01 OOOOOO 1-JAXKIL010000034), or JAXKIM000000000 (JAXKIM010000001-JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIA000000000 (J AXKIA01 OOOOOO 1-JAXKI AO 10000021), JAXKIB000000000 (JAXKIB01 OOOOOO 1-JAXKIB010000021), JAXKIC000000000 (JAXKIC010000001- JAXKIC010000022), JAXKIF000000000 (JAXKIF01 OOOOOO 1-JAXKIF010000014), JAXKIJ000000000 (JAXKIJO 10000001 -JAXKIJO 10000040), JAXKIK000000000 (JAXKIK010000001-JAXKIKO 10000043), JAXKILOOOOOOOOO (JAXKIL010000001- JAXKIL010000034), or JAXKIM000000000 (JAXKIM01 OOOOOO 1-JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (JAXKIA01 OOOOOO 1-JAXKIA010000021), JAXKIB 000000000 (JAXKIB 010000001- JAXKIB010000021), JAXKIC000000000 (JAXKIC01 OOOOOO 1-JAXKIC010000022), JAXKIF000000000 (JAXKIF01 OOOOOO 1-JAXKIF010000014), JAXKIJ000000000 (JAXKU01 OOOOOO 1-JAXKUO 10000040), JAXKIK000000000 (JAXKIK010000001- JAXKIKO 10000043), JAXKIL000000000 (JAXKIL01 OOOOOO 1-JAXKILO 10000034), or JAXKIM000000000 (JAXKIM01 OOOOOO 1 -JAXKIM010000039).

[0119] In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 001461825.1. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 001461825.1. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 001461825.1. In certain embodiments, the Bacillus velezensis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 001461825.1. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under NCBI RefSeq Assembly GCF 001461825.1. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under NCBI RefSeq Assembly GCF 001461825.1.

[0120] In certain embodiments, Bacillus velezensis is Bacillus velezensis strain

[0121] NRRL B-41580.

[0122] In certain embodiments, the Bacillus velezensis is an isolate strain selected from the group consisting of CB00019, CB00021, CB00027, CB00687, CB00902, CB00904, CB00909, and CB00912 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00019 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00021 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00027 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00687 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00902 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00904 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00909 as described herein. In certain embodiments, the Bacillus velezensis is isolate strain CB00912 as described herein.

[0123] As used herein, the term CB00019 is used interchangeably with CB19; the term CB00021 is used interchangeably with CB21; the term CB00027 is used interchangeably with CB27, the term CB00687 is used interchangeably with CB687, the term CB00902 is used interchangeably with CB902; the term CB00904 is used interchangeably with CB904; the term CB00909 is used interchangeably with CB909; and the term CB00912 is used interchangeably with CB912.

[0124] Bacillus velezensis CB909 / CB00909 is described herein, such as in Example 5, including its isolation, identification, and characteristics (see, e.g., Table 1 and Figures 37-38). In particular, the Bacillus velezensis CB909 was sent by the University of California, Riverside Campus (900 University Ave, Riverside CA 92521), as a representative of and on behalf of The Regents of the University of California, to the American Type Culture Collection Depository (ATCC® Depository, 10801 University Boulevard, Manassas, Va. 20110 USA) on March 26, 2025 and its ATCC® Patent Deposit Designation is to be assigned. Accordingly, in certain embodiments, the Bacillus velezensis is the Bacillus velezensis isolate strain CB909 having an ATCC® Patent Deposit Designation that is to be assigned.

[0125] In certain embodiments, the microbe is not Bacillus velezensis. Thus, in certain embodiments, the method does not comprise introducing Bacillus velezensis, or an extract thereof, to the plant.

[0126] Bacillus pumilus

[0127] In certain embodiments, the microbe is Bacillus pumilus. Thus, in certain embodiments, a method described herein comprises introducing Bacillus pumilus to the plant. In certain embodiments, Bacillus pumilus, or an extract thereof, is introduced to the plant.

[0128] Bacillus pumilus species are known in the art and described herein. For example, this species includes Bacillus pumilus strain NCTC10337 (see, e.g., NCBI RefSeq assembly GCF 900186955.1), as well as the additional Bacillus pumilus strain described herein.

[0129] In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under an accession number described herein (e.g., NCBI RefSeq Accession No., SRA Accession No., or a WGS accession No.). For example, in certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973672.

[0130] In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKID000000000 (JAXKIDO 10000001-JAXKIDO 10000059). In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKID000000000 (JAXKIDO 10000001-JAXKIDO 10000059). In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKID000000000 (JAXKID010000001-JAXKIDO 10000059). In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIDOOOOOOOOO (JAXKIDO 10000001-JAXKIDO 10000059). In certain embodiments, the Bacillus pumilus genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKID000000000 (JAXKID010000001-JAXKID010000059). In certain embodiments, the Bacillus pumilus genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKID000000000 (JAXKID010000001- JAXKID010000059).

[0131] In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 900186955.1. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 900186955.1. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 900186955.1. In certain embodiments, the Bacillus pumilus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 900186955.1. In certain embodiments, the Bacillus pumilus genome comprises the genome assembly sequences as provided under NCBI RefSeq Assembly GCF 900186955.1. In certain embodiments, the Bacillus pumilus genome consists essentially of or consists of the genome assembly sequences provided under NCBI RefSeq Assembly GCF 900186955.1.

[0132] In certain embodiments, the Bacillus pumilus is a Bacillus pumilus isolate strain NCTC10337.

[0133] In certain embodiments, the Bacillus pumilus is isolate strain CB00069 as described herein. As used herein, the terms CB00069 and CB69 are used interchangeably.

[0134] In certain embodiments, the microbe is not Bacillus pumilus. Thus, in certain embodiments, the method does not comprise introducing Bacillus pumilus, or an extract thereof, to the plant. Bacillus cereus

[0135] In certain embodiments, the microbe is Bacillus cereus. Thus, in certain embodiments, a method described herein comprises introducing Bacillus cereus to the plant. In certain embodiments, Bacillus cereus, or an extract thereof, is introduced to the plant.

[0136] Bacillus cereus species are known in the art and described herein. For example, this species includes Bacillus cereus strain FORC087 (see, e.g., NCBI RefSeq assembly GCF_006384875.1) and Bacillus cereus strain UW85, as well as the additional Bacillus cereus strain described herein.

[0137] In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under an accession number described herein (e.g., NCBI RefSeq Accession No., SRA Accession No., or a WGS accession No.). For example, in certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973671.

[0138] In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIE000000000 (JAXKIE01 OOOOOO 1-JAXKIEO 10000087). In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIE000000000

[0139] (JAXKIEO 10000001-JAXKIEO 10000087). In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIE000000000

[0140] (JAXKIE010000001-JAXKIEO 10000087). In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIEOOOOOOOOO

[0141] (JAXKIEO 10000001-JAXKIEO 10000087). In certain embodiments, the Bacillus cereus genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIEOOOOOOOOO (JAXKIEO 10000001-JAXKIEO 10000087). In certain embodiments, the Bacillus cereus genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIEOOOOOOOOO (JAXKIEO 10000001- JAXKIE010000087).

[0142] In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 006384875.1. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 006384875.1. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 006384875.1. In certain embodiments, the Bacillus cereus genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 006384875.1. In certain embodiments, the Bacillus cereus genome comprises the genome assembly sequences as provided under NCBI RefSeq Assembly GCF 006384875.1. In certain embodiments, the Bacillus cereus genome consists essentially of or consists of the genome assembly sequences provided under NCBI RefSeq Assembly GCF 006384875.1.

[0143] In certain embodiments, Bacillus cereus is a Bacillus cereus isolate strain FORC087. In certain embodiments Bacillus cereus is Bacillus cereus isolate strain UW85.

[0144] In certain embodiments, the Bacillus cereus is isolate strain CB00676 as described herein. As used herein, the terms CB00676 and CB676 are used interchangeably.

[0145] In certain embodiments, the microbe is not Bacillus cereus. Thus, in certain embodiments, the method does not comprise introducing Bacillus cereus, or an extract thereof, to the plant.

[0146] Bacillus subtilis

[0147] In certain embodiments, the microbe is Bacillus subtilis. Thus, in certain embodiments, a method described herein comprises introducing Bacillus subtilis to the plant. In certain embodiments, Bacillus subtilis, or an extract thereof, is introduced to the plant.

[0148] Bacillus subtilis species are known in the art and described herein. For example, this species includes Bacillus subtilis subsp. subtilis str. 168 (see, e.g., NCBI RefSeq assembly GCF 000009045.1), as well as the additional Bacillus subtilis strain described herein.

[0149] In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under an accession number described herein (e.g., NCBI RefSeq Accession No., SRA Accession No., or a WGS accession No.). For example, in certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667. In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under SRA Accession No.

[0150] SRR26973668 or SRR26973667. In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667.

[0151] In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-JAXKIH010000036) or JAXKII000000000 (JAXKII010000001- JAXKII010000042). In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-

[0152] JAXKIH010000036) or JAXKII000000000 (JAXKII010000001-JAXKII010000042). In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-JAXKIH010000036) or JAXKII000000000 (JAXKII010000001-JAXKII010000042). In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-JAXKIH010000036) or JAXKII000000000 (JAXKII010000001- JAXKII010000042). In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-JAXKIH010000036) or JAXKII000000000 (JAXKII010000001- JAXKII010000042). In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 (JAXKIH010000001-JAXKIH010000036) or JAXKII000000000 (JAXKII010000001 -JAXKII010000042).

[0153] In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000009045.1. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF_000009045.1. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.5% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000009045.1. In certain embodiments, the Bacillus subtilis genome comprises a genomic sequence having at least about 99.9% sequence identity to the genome assembly sequences provided under NCBI RefSeq Assembly GCF 000009045.1. In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under NCBI RefSeq Assembly GCF_000009045.1. In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under NCBI RefSeq Assembly GCF_000009045.1.

[0154] In certain embodiments, Bacillus subtilis is Bacillus subtilis subsp. subtilis str. 168. In certain embodiments, the Bacillus subtilis is isolate strain CB00742 or CB00893 as described herein. In certain embodiments, the Bacillus subtilis is isolate strain CB00742 as described herein. In certain embodiments, the Bacillus subtilis is isolate strain CB00893 as described herein. As used herein, the terms CB00742 and CB742 are used interchangeably; and the terms CB00893 and CB893 are used interchangeably.

[0155] In certain embodiments, the microbe is not Bacillus subtilis. Thus, in certain embodiments, the method does not comprise introducing Bacillus subtilis, or an extract thereof, to the plant.

[0156] As described herein, a microbe described herein may produce compound(s) that act to inhibit or kill CLas and / or a secondary pathogen(s). For example, as described herein, the biosynthetic gene cluster (BGC) NRPS-PKS domain present in Bacillus safensis isolate CB00729 produces amicoumacin compound(s) that may be used to inhibit / treat CLas and / or a secondary pathogen(s). Accordingly, certain embodiments of the invention provide a method of inhibiting Candidatus Liberibacter asiaticus (CLas) growth, treating a CLas infection and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one microbe, or an extract thereof, that produces an amicoumacin(s) (e.g., amicoumacin B and / or A, as described below). In certain embodiments, the microbe comprises an amicoumacin BGC, or one or more genes thereof. In certain embodiments, the microbe is a recombinant microbe that has been engineered to comprise one or more exogenous genes from an amicoumacin BGC. Amicoumacin BGCs are known in the art and described herein (see, e.g., Li et al., Sci Rep 5, 9383 (2015), which is incorporated by reference herein in its entirety for all purposes). In certain embodiments, the amicoumacin BGC is a NRPS-PKS domain as described herein from Bacillus safensis. In certain embodiments, the microbe (e.g., recombinant microbe) comprises the amicoumacin BGC, or one or more genes thereof, from Bacillus safensis strain CB00729. For example, in certain embodiments, the microbe (e.g., recombinant microbe) comprises an amicoumacin BGC comprising a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity to a sequence located at positions 82126 to 127621 in GenBank accession no. GCA 036621555.1 (GenBank accession no. GCA 036621555.1 and its associated sequences are incorporated by reference herein in their entirety for all purposes). For example, in certain embodiments, the microbe (e.g., recombinant microbe) comprises:

[0157] (1) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 82126 to position 83325;

[0158] (2) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 83417 to position 87886;

[0159] (3) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 87883 to position 89424;

[0160] (4) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 89387 to position 90367;

[0161] (5) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 90364 to position 91074;

[0162] (6) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 91071 to position 91928;

[0163] (7) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 91943 to position 92995;

[0164] (8) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 93020 to position 93292;

[0165] (9) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 93279 to position 94421; (10) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 94441 to position 103530;

[0166] (11) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 103551 to position 106220;

[0167] (12) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 106213 to position 110715;

[0168] (13) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 110734 to position 117966;

[0169] (14) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 117959 to position 124369;

[0170] (15) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the nucleic acid sequence located at position 124362 to position 125369;

[0171] (16) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the complement of the nucleic acid sequence located at position 125410 to position 126816; and / or

[0172] (17) a nucleic acid sequence having at least about 85%, 90%, 95%, 99%, or 100% to the complement of the nucleic acid sequence located at position 126929 to position 127621, wherein the positions are relative to the sequence of Accession No. GCA_036621555.1.

[0173] In certain embodiments, the microbe (e.g., recombinant microbe) comprises:

[0174] (1) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:8;

[0175] (2) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:9;

[0176] (3) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 10;

[0177] (4) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 11;

[0178] (5) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 12;

[0179] (6) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13; (7) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 14;

[0180] (8) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 15; (9) a nucleic acid sequence encoding an amino acid sequence having at least about

[0181] 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 16;

[0182] (10) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 17;

[0183] (11) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 18;

[0184] (12) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 19;

[0185] (13) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:20; (14) a nucleic acid sequence encoding an amino acid sequence having at least about

[0186] 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:21;

[0187] (15) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:22;

[0188] (16) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:23; and / or

[0189] (17) a nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:24.

[0190] Table A. Sequence Information for NRPS-PKS domain from CB00729. Position numbering for each gene is relative to GCA_036621555.1.

[0191]

[0192] Certain Embodiments of Compounds for Use in a Method Described Herein

[0193] As described herein, a microbe described herein may produce compound(s) that act to inhibit or kill CLas and / or a secondary pathogen(s). Accordingly, certain embodiments provide the use of such compounds in a method described herein. In certain embodiments, such a compound may be derived from a microbial isolate described herein.

[0194] For example, certain embodiments of the invention provide a method of inhibiting Candidatus Liberibacter asiaticus (CLas) growth, treating a CLas infection and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound as described herein (including the Examples, such as in Example 1), or a salt thereof.

[0195] For example, certain embodiments provide a method of treating a Candidatus Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound selected from the group consisting of an amicoumacin compound, a bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or a salt thereof.

[0196] In certain embodiments, a method described herein comprises introducing two or more compounds selected from the group consisting of an amicoumacin compound, a bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or salts thereof.

[0197] In certain embodiments, a method described herein comprises introducing at least one amicoumacin compound, or a salt thereof. Amicoumacin compounds are known in the art and described herein (see, e.g., Tyurin et al., Studies in Natural Product Chemistry, 55:385-441 (2018), which is incorporated by reference herein in its entirety for all purposes). As used herein, an amicoumacin compound includes, e.g., amicoumacin A, amicoumacin B, amicoumacin C, N-acetylamicoumacin A, N-acetylamicoumacin B, N-acetylamicoumacin C, O- methylamicoumacin B, or O-methylamicoumacin C, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof).

[0198] In certain embodiments, the amicoumacin compound, or a salt thereof, is selected from the group consisting of amicoumacin A, amicoumacin B, amicoumacin C, N-acetylamicoumacin A, N-acetylamicoumacin B, N-acetylamicoumacin C, O-methylamicoumacin B, or O- methylamicoumacin C, or a salt thereof.

[0199] In certain embodiments, the compound is not an amicoumacin compound. In certain embodiments the compound is not a salt of an amicoumacin compound.

[0200] In certain embodiments, the compound is amicoumacin A, or a salt thereof. In certain embodiments, the compound is not amicoumacin A (e.g., or a salt thereof).

[0201] In certain embodiments, the compound is amicoumacin B. In certain embodiments, the compound is not amicoumacin B (e.g., or a salt thereof).

[0202] In certain embodiments, the compound is amicoumacin C. In certain embodiments, the compound is not amicoumacin C (e.g., or a salt thereof).

[0203] In certain embodiments, the compound is N-acetylamicoumacin A. In certain embodiments, the compound is not N-acetylamicoumacin A (e.g., or a salt thereof).

[0204] In certain embodiments, the compound is N-acetylamicoumacin B. In certain embodiments, the compound is not N-acetylamicoumacin B (e.g., or a salt thereof).

[0205] In certain embodiments, the compound is N-acetylamicoumacin C. In certain embodiments, the compound is not N-acetylamicoumacin C (e.g., or a salt thereof).

[0206] In certain embodiments, the compound is O-methylamicoumacin B or O- methylamicoumacin C, or a salt thereof.

[0207] In certain embodiments, the compound is amicoumacin A embodiment: or a salt thereof.

[0208] In certain embodiments, the compound is amicoumacin B: or a salt thereof.

[0209] In certain embodiments, the compound is amicoumacin C embodiment: or a salt thereof.

[0210] In certain embodiments, the compound is N-acetylamicoumacin A embodiment: salt thereof.

[0211] In certain embodiments, the compound is N-acetylamicoumacin B embodiment: or a salt thereof.

[0212] In certain embodiments, the compound is N-acetylamicoumacin C embodiment: or a salt thereof.

[0213] In certain embodiments, the compound is O-methylamicoumacin B embodiment: or a salt thereof.

[0214] In certain embodiments, the compound is O-methylamicoumacin C embodiment: , or a salt thereof.

[0215] In certain embodiments, a method described herein comprises introducing at least one bacilosarcin compound, or a salt thereof. Bacilosarcin compounds are known in the art and described herein (see, e.g., Azumi et al. 2008. Tetrahedron, 64(27):6420-6425, which is incorporated by reference herein in its entirety for all purposes). As used herein, a bacilosarcin compound includes, e.g., bacilosarcin A, bacilosarcin B, and bacilosarcin C, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof).

[0216] Thus, in certain embodiments, the bacilosarcin compound, or a salt thereof, is selected from the group consisting of bacilosarcin A, bacilosarcin B, and bacilosarcin C, or a salt thereof.

[0217] In certain embodiments, the compound is bacilosarcin A embodiment: or a salt thereof.

[0218] In certain embodiments, the compound is bacilosarcin B embodiment: or a salt thereof.

[0219] In certain embodiments, the compound is bacilosarcin C embodiment: or a salt thereof.

[0220] In certain embodiments, a method described herein comprises introducing at least one hetiamacin compound, or a salt thereof. Hetiamacin compounds are known in the art and described herein (see, e.g., Wang et al. 2020 Molecules, 25(19):4446, which is incorporated by reference herein in its entirety for all purposes). As used herein, a hetiamacin compound includes, e.g., hetiamacin A and hetiamacin C, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof).

[0221] Thus, in certain embodiments, the hetiamacin compound, or a salt thereof, is selected from the group consisting of hetiamacin A and hetiamacin C, or a salt thereof.

[0222] In certain embodiments, the compound is hetiamacin A embodiment: or a salt thereof.

[0223] In certain embodiments, the compound is hetiamacin C embodiment: or a salt thereof.

[0224] In certain embodiments, the compound is bacilosarcin A, bacilosarcin B, bacilosarcin C, hetiamacin A, or hetiamacin C, or a salt thereof.

[0225] In certain embodiments, a method described herein comprises introducing at least one cyclic lipopeptide compound, or a salt thereof. Cyclic lipopeptide compounds are known in the art and described herein (see, e.g., Schneider et al, 2014. Int J Med Microbiol, 304(l):37-43, which is incorporated by reference herein in its entirety for all purposes). As used herein, a cyclic lipopeptide compound includes, e.g., pumilacidin compounds and surfactin compounds, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof).

[0226] Thus, in certain embodiments, the cyclic lipopeptide compound, or a salt thereof, is selected from the group consisting of a pumilacidin compound and a surfactin compound, or a salt thereof.

[0227] For example, in certain embodiments, a method described herein comprises introducing at least one pumilacidin compound, or a salt thereof. Pumilacidin compounds are known in the art and described herein (see, e.g., Dasgupta et al, 2023, Archives in Microbiology, 205, 274, which is incorporated by reference herein in its entirety for all purposes). As used herein, a pumilacidin compound includes, e.g., pumilacidin C and pumilacidin E, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof). Thus, in certain embodiments, the pumilacidin compound, or a salt thereof, is selected from the group consisting of pumilacidin C and pumilacidin E, or a salt thereof.

[0228] In certain embodiments, the compound is pumilacidin C embodiment:

[0229] In certain embodiments, the compound is pumilacidin E embodiment: or a salt thereof.

[0230] In certain embodiments, a method described herein comprises introducing at least one surfactin compound, or a salt thereof. Surfactin compounds are known in the art and described herein (see, e.g., Zhen et al. 2023. AIMS Microbiology, 9(2): 195-217, which is incorporated by reference herein in its entirety for all purposes). As used herein, a surfactin compound includes, e.g., surfactin B, surfactin C and surfactin C13, as well as stereoisomers thereof (e.g., diastereomers and enantiomers thereof). Thus, in certain embodiments, the surfactin compound, or a salt thereof, is selected from the group consisting of surfactin B, surfactin C and surfactin C13, or a salt thereof.

[0231] In certain embodiments, the compound is surfactin B embodiment: or a salt thereof. In certain embodiments, the compound is surfactin C embodiment: or a salt thereof.

[0232] In certain embodiments, the compound is surfactin C13 embodiment: or a salt thereof.

[0233] In certain embodiments, one or more of the compounds are chemically synthesized. In certain other embodiments, the compound(s) described herein are isolated or purified from a natural product source (e.g., from a preparation derived from microbial culturing or fermentation). For example, compounds described above may be derived from Bacillus safensis (e.g., CB00729 isolate) or from a microbe that comprises / expresses an amicoumacin BGC (e.g., a NRPS-PKS domain derived from Bacillus safensis).

[0234] Certain embodiments also provide a composition comprising one or more of the compounds described herein (e.g., for use in method described herein). In certain embodiments, the composition further comprises a carrier. In certain embodiments, the composition comprises at least one amicoumacin compound, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof. In certain embodiments, the composition comprises amicoumacin B, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof. In certain embodiments, the composition comprises amicoumacin B, or a salt thereof, amicoumacin A, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof. In certain embodiments, the composition comprises amicoumacin B, or a salt thereof, amicoumacin A, or a salt thereof, amicoumacin C, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof. In certain embodiments, the composition comprises amicoumacin B, or a salt thereof, amicoumacin A, or a salt thereof, amicoumacin C, or a salt thereof, and at least one pumilacidin compound, or a salt thereof, and a surfactin compound, or a salt thereof. In certain embodiments, the composition comprises bacilosarcin C, or a salt thereof, N-acetyl- amicoumacin C, or a salt thereof, amicoumacin C, or a salt thereof, and pumilacidin E, or a salt thereof, (see, e.g., CB00729 extract fraction 729.S-SNP12 (R4); Figure 15B). In certain embodiments, the composition comprises N-acetyl-amicoumacin C, or a salt thereof, amicoumacin A, or a salt thereof, amicoumacin C, or a salt thereof, bacilosarcin B, or a salt thereof, and at least one surfactin, or a salt thereof, (see, e.g., CB00729 extract fraction 729.1- NPA (R5); Figure 15B). In certain embodiments, the composition comprises N-acetyl- amicoumacin A, or a salt thereof, N-acetyl-amicoumacin B, or a salt thereof, N-acetyl- amicoumacin C, or a salt thereof, amicoumacin A, or a salt thereof, amicoumacin B, or a salt thereof, amicoumacin C, or a salt thereof, bacilosarcin B, or a salt thereof, bacilosarcin C, or a salt thereof, hetiamacin A, or a salt thereof, hetiamacin C, or a salt thereof, at least one pumilacidin, or a salt thereof, and at least one surfactin, or a salt thereof, (see, e.g., CB00729 extract fraction 729.2-P3 (R9); Figure 15B). In certain embodiments, the composition is a microbial extract, or fraction thereof. For example, in certain embodiments, the composition is a Bacillus safensis extract, or a fraction thereof (e.g., a fraction as described herein, such as a CB00729 extract fraction 729.S-SNP12 (R4), CB00729 extract fraction 729.1-NPA (R5), or a CB00729 extract fraction 729.2-P3 (R9)). Certain embodiments also provide a Bacillus safensis extract fraction prepared by a method as described herein (e.g., a CB00729 extract fraction 729.S-SNP12, CB00729 extract fraction 729.1-NPA, or a CB00729 extract fraction 729.2-P3 prepared as described herein).

[0235] Certain Isolated Microbes and Compositions Thereof

[0236] Certain embodiments provide an isolated microbe as described herein (e.g., as described above for use in a method of the invention).

[0237] For example, certain embodiments provide an isolated Bacillus safensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669 or under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001 -JAXKIG010000019). In certain embodiments, the Bacillus safensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973669. In certain embodiments, the Bacillus safensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIG000000000 (JAXKIGO 10000001- JAXKIGO 10000019). In certain embodiments, the Bacillus safensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIG000000000 (JAXKIG010000001-JAXKIG010000019). In certain embodiments, the Bacillus safensis is CB729 as described herein. In certain embodiments, the Bacillus safensis is Bacillus safensis CB729 having an ATCC® Patent Deposit Designation to be assigned.

[0238] Certain embodiments provide an isolated microbe (e.g., a recombinant microbe) comprising an amicoumacin BGC, or one or more genes thereof, from the Bacillus safensis strain CB00729 (see, Table A). For example, certain embodiments provide a microbe comprising at least one nucleic acid sequence that has at least about 85%, 90%, 95%, 99%, or 100% to a nucleic acid sequence located at positions specified in Table A. Certain embodiments also provide a microbe comprising at least one nucleic acid sequence encoding an amino acid sequence having at least about 85%, 90%, 95%, 99%, or 100% sequence identity to a SEQ ID NO as specified in Table A.

[0239] Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678 or under WGS Accession No. JAXKIA000000000 (JAXKIA010000001-JAXKIA010000021). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973678. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973678. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIA000000000 (JAXKIA010000001-JAXKIA010000021). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIA000000000 (JAXKIA010000001-JAXKIA010000021). In certain embodiments, the Bacillus velezensis is an isolate strain CB00019. Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973677 or under WGS Accession No. JAXKIB000000000 (JAXKIB010000001-JAXKIB010000021). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973677. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973677. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIB000000000 (JAXKIB010000001-JAXKIB010000021). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIB000000000 (JAXKffiOl 0000001-JAXKffiO 10000021). In certain embodiments, the Bacillus velezensis is an isolate strain CB00021.

[0240] Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973673 or under WGS Accession No. JAXKIC000000000 (JAXKIC010000001 -JAXKIC010000022). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973673. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973673. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIC000000000 (JAXKIC010000001 -JAXKIC010000022). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIC000000000 (JAXKIC010000001-JAXKIC010000022). In certain embodiments, the Bacillus velezensis is an isolate strain CB00027. Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973670 or under WGS Accession No. JAXKIF000000000 (JAXKIF010000001-JAXKIF010000014). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973670. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973670. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIF000000000 (JAXKIF010000001-JAXKIF010000014). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIF000000000 (JAXKIF010000001-JAXKIF010000014). In certain embodiments, the Bacillus velezensis is an isolate strain CB00687.

[0241] Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973666 or under WGS Accession No. JAXKIJ000000000 (JAXKIJ010000001-JAXKIJ010000040). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973666. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973666. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIJ000000000 (JAXKIJ010000001-JAXKIJ010000040). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKU000000000 (JAXKIJ010000001-JAXKU010000040). In certain embodiments, the Bacillus velezensis is an isolate strain CB00902. Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973676 or under WGS Accession No. JAXKIK000000000 (JAXKIKO 10000001 -JAXKIK010000043). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973676. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973676. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIK000000000 (JAXKIKO 10000001 -JAXKIKO 10000043). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIK000000000 (JAXKIKO 10000001 -JAXKIKO 10000043). In certain embodiments, the Bacillus velezensis is an isolate strain CB00904.

[0242] Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973675 or under WGS Accession No. JAXKIL000000000 (JAXKIL010000001-JAXKILO 10000034). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973675. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973675. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIL000000000 (JAXKIL010000001-JAXKILO 10000034). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIL000000000 (JAXKIL010000001-JAXKILO 10000034). In certain embodiments, the Bacillus velezensis is an isolate strain CB00909. In certain embodiments, the Bacillus velezensis is Bacillus velezensis CB909 having an ATCC® Patent Deposit Designation to be assigned.

[0243] Certain embodiments provide an isolated Bacillus velezensis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973674 or under WGS Accession No. JAXKIM000000000 (JAXKIM010000001-JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973674. In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973674. In certain embodiments, the Bacillus velezensis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIM000000000 (JAXKIM010000001-JAXKIM010000039). In certain embodiments, the Bacillus velezensis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIM000000000 (JAXKIM010000001-JAXKIM010000039). In certain embodiments, the Bacillus velezensis is an isolate strain CB00912.

[0244] Certain embodiments provide an isolated Bacillus pumilus microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672 or under WGS Accession No. JAXKID000000000 (JAXKID010000001- JAXKID010000059). In certain embodiments, the Bacillus pumilus genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973672. In certain embodiments, the Bacillus pumilus genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKID000000000 (JAXKID010000001- JAXKID010000059). In certain embodiments, the Bacillus pumilus genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIDOOOOOOOOO (JAXKID01 OOOOOO 1-JAXKIDO 10000059). In certain embodiments, the Bacillus pumilus is an isolate strain CB00069.

[0245] Certain embodiments provide an isolated Bacillus cereus microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671 or under WGS Accession No JAXKIE000000000 (JAXKIE010000001- JAXKIE010000087). In certain embodiments, the Bacillus cereus genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973671. In certain embodiments, the Bacillus cereus genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIE000000000 (JAXKIE010000001- JAXKIE010000087). In certain embodiments, the Bacillus cereus genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIE000000000 (JAXKIE010000001-JAXKIE010000087). In certain embodiments, the Bacillus cereus is an isolate strain CB00676.

[0246] Certain embodiments provide an isolated Bacillus subtilis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or under WGS Accession No. JAXKIH000000000 (JAXKIH010000001- JAXKIH010000036). In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973668. In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973668. In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKIH000000000 (J AXKIH010000001- JAXKIH010000036). In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKIHOOOOOOOOO (JAXKIH01 OOOOOO 1-JAXKIH010000036). In certain embodiments, the Bacillus subtilis is an isolate strain CB00742.

[0247] Certain embodiments provide an isolated Bacillus subtilis microbe comprising a genome that comprises a genomic sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973667 or under WGS Accession No. JAXKII000000000 (JAXKII010000001- JAXKII010000042). In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under SRA Accession No. SRR26973667. In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under SRA Accession No. SRR26973667. In certain embodiments, the Bacillus subtilis genome comprises the genome assembly sequences as provided under WGS Accession No. JAXKII000000000 (JAXKII010000001- JAXKII010000042). In certain embodiments, the Bacillus subtilis genome consists essentially of or consists of the genome assembly sequences provided under WGS Accession No. JAXKII000000000 (JAXKII010000001-JAXKII010000042). In certain embodiments, the Bacillus subtilis is an isolate strain CB00893.

[0248] Certain embodiments also provide a composition described herein (e.g., as described herein for use in a method of the invention). For example, certain embodiments provide a composition comprising an isolated microbe as described herein, or an extract thereof. In certain embodiments, the composition comprises two or more microbes described herein, or extracts thereof.

[0249] Certain embodiments also provide a method of identifying a microbe that inhibits Liberibacter crescens from a plant microbiome sample, the method comprising contacting a test microbe from the plant microbiome sample with cultured Liberibacter crescens to provide a test culture sample, and identifying the test microbe as inhibitory when the amount of Liberibacter crescens growth is less than the growth of a corresponding control Liberibacter crescens culture sample that was not contacted with the test microbe, and wherein the identified test microbe is selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis. In certain embodiments, the identified test microbe that inhibits Liberibacter crescens further inhibits Candidates Liberibacter asiaticus (CLas).

[0250] In certain embodiments, method further comprises isolating the test microbe from the plant microbiome sample to provide an isolated test microbe. In certain embodiments, the method further comprises culturing the isolated test microbe to produce a culture of the isolated test microbe.

[0251] Certain embodiments provide a method of identifying a microbe from a plant microbiome sample that is capable of treating a Candidates Liberibacter asiaticus (CLas) infection in a plant, the method comprising contacting a test microbe from the plant microbiome sample with a plant, or a portion thereof, infected with Candidates Liberibacter asiaticus (CLas), and identifying the test microbe as being capable of treating a CLas infection when one or more symptoms of the infection improve and / or CLas titers decrease as compared to corresponding control plant that was not contacted with the test microbe, and wherein the identified test microbe is selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis.

[0252] In certain embodiments, the method further comprises isolating the test microbe from the plant microbiome sample to provide an isolated test microbe. In certain embodiments, the method further comprises culturing the isolated test microbe to produce a culture of the isolated test microbe.

[0253] In certain embodiments, the plant microbiome sample is derived from a citrus tree infected with CLas.

[0254] In certain embodiments, the identified test microbe is selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis. In certain embodiments, in a method described herein, the identified test microbe is selected from the group consisting of Bacillus velezensis, Bacillus pumilus, and Bacillus cereus. In certain embodiments, the identified test microbe is no Bacillus safensis. In certain embodiments, the identified test microbe is not Bacillus subtilis. In certain embodiments, the identified test microbe is not Bacillus pumilus.

[0255] In certain embodiments, the identified test microbe is selected from the group consisting of Bacillus safensis isolate strain CB00729, Bacillus velezensis isolate strain CB00019, Bacillus velezensis isolate strain CB00021, Bacillus velezensis isolate strain CB00027, Bacillus velezensis isolate strain CB00687, Bacillus velezensis isolate strain CB00902, Bacillus velezensis isolate strain CB00904, Bacillus velezensis isolate strain CB00909, Bacillus velezensis isolate strain CB00912, Bacillus pumilus isolate strain CB00069, Bacillus cereus isolate strain CB00676, Bacillus subtilis isolate strain CB00742 and Bacillus subtilis isolate strain CB00893. In certain embodiments, the identified test microbe is no Bacillus safensis isolate strain CB00729.

[0256] Certain embodiments provide a microbe identified and isolated by a method described herein. Certain embodiments also provide a culture of a an isolated and identified microbe produced by a method described herein.

[0257] Certain Formulation Embodiments

[0258] In certain embodiments, the compounds, compositions and / or microbial extracts comprising anti-CLas compounds may be formulated for plant application / introduction. Common agrochemical formulations are well known in the field, and include liquid and solid formulations. Exemplary formulations comprising active agents include gel, aqueous or oilbased solutions, dispersions, suspensions or emulsions, such as those described in US Patent Nos 5,139,152; 6,403,529; 6,878,674; 7,094,831; 7,109,267 and 9,706,771. In certain embodiments, the compound(s) may be present in a liquid formulation, which may be administered or sprayed onto a plant or agricultural medium using, e.g., ground / aerial spraying. In other examples, the active agent(s) may be formulated in pellet or tablet formulations. Such formulations may be capable of rapid break-up in water using minimal or no agitation while providing fine dispersions of the active ingredient (see, e.g., US Patent Nos. 5,180,587 and 7,550,156). Suitable additives or excipients which may be present in the formulations include organic solvents, solubilizers, emulsifiers, surfactants, dispersants, preservatives, colorants, fillers, diluents, binders, glidants, lubricants, disintegrants, anti adherents, lubricants, sorbents, coatings, wetting agents, penetrants and vehicles. Well known additives, excipients and agrochemical formulations are described in US Patent No 6,602,823 and the aforementioned US Patents. Certain Kit Embodiments

[0259] Certain embodiments provide a kit comprising: 1) at least one microbial isolate described herein, or extract thereof, and / or at least one compound as described herein, or a salt thereof; 2) packaging material; and 3) instructions to introduce to a plant the microbial isolate / extract and / or compound to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant and / or to treat Huanglongbing (HLB) in the plant.

[0260] For example, certain embodiments provide a kit comprising:

[0261] 1) at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus sublUis. or an extract(s) thereof;

[0262] 2) packaging material; and

[0263] 3) instructions to introduce to a plant the microbial isolate / extract to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant and / or to treat Huanglongbing (HLB) in the plant.

[0264] Certain embodiments also provide a kit comprising:

[0265] 1) at least one compound selected from the group consisting of an amicoumacin compound, bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or a salt thereof;

[0266] 2) packaging material; and

[0267] 3) instructions to introduce to a plant the compound to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant or to treat Huanglongbing (HLB) in the plant.

[0268] Certain embodiments provide a kit comprising:

[0269] 1) a composition comprising at least one amicoumacin compound, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof.

[0270] 2) packaging material; and

[0271] 3) instructions to introduce to a plant the composition to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant or to treat Huanglongbing (HLB) in the plant. In certain embodiments, the composition is a microbial extract, such as a Bacillus safensis extract (or a fraction thereof). Certain Embodiments

[0272] Embodiment 1. A method of treating a Candidates Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus vietnamensis, Bacillus pumilus, Bacillus altitedinis, Bacillus bombysepticus, and Bacillus subtilis, or an extract thereof.

[0273] Embodiment 2. The method of embodiment 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus vietnamensis, Bacillus pumilus, Bacillus altitedinis, Bacillus bombysepticus and Bacillus subtilis, or an extract thereof.

[0274] Embodiment 3. The method of embodiment 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus vietnamensis, Bacillus pumilus, Bacillus altitedinis, and Bacillus bombysepticus, or an extract thereof.

[0275] Embodiment 4. The method of embodiment 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus vietnamensis, Bacillus altitedinis, and Bacillus bombysepticus, or an extract thereof.

[0276] Embodiment 5. The method of any one of embodiments 1-4, wherein the method does not comprise introducing to the plant Bacillus safensis or an extract thereof.

[0277] Embodiment 6. The method of any one of embodiments 3-4, further comprising introducing to the plant Bacillus safensis or an extract thereof.

[0278] Embodiment 7. A method of treating a Candidates Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound selected from the group consisting of amicoumacin A, amicoumacin B, amicoumacin C, N-acetylamicoumacin A, N-acetylamicoumacin B, N- acetylamicoumacin C, O-methylamicoumacin B, O-methylamicoumacin C, bacilosarcin A, bacilosarcin B, bacilosarcin C, hetiamacin A, hetiamacin C, pumilacidin C, pumilacidin E, surfactin B, surfactin C, and surfactin Cl 3, or a salt thereof.

[0279] Embodiment 8. The method of embodiment 7, comprising introducing to the plant at least one compound selected from the group consisting of O-methylamicoumacin B, O- methylamicoumacin C, pumilacidin C, pumilacidin E, surfactin B, surfactin C, and surfactin Cl 3, or a salt thereof.

[0280] Embodiment 9. The method of embodiment 7, comprising introducing to the plant at least one compound selected from the group consisting of pumilacidin C, pumilacidin E, surfactin B, surfactin C, and surfactin Cl 3, or a salt thereof.

[0281] Embodiment 10. A method of treating a Candidates Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound (e.g., an amicoumacin compound, a pumilacidin compound, or a surfactin compound), or microbe or an extract thereof as described herein.

[0282] Embodiment 11. A composition (e.g., a bioinoculant composition useful for treating HLB) as described herein.

[0283] Embodiment 12. The composition, comprising at least one microbial isolate(s) selected from the group according to any one of embodiments 1-4, or an extract thereof.

[0284] Certain Definitions

[0285] The invention encompasses isolated or substantially purified microbes and microbial compositions. In the context of the present invention, an "isolated" or "purified" microbe is a microbe that exists apart from its native environment. Similarly, an “isolate” is a microbe(s) that has been isolated.

[0286] The terms “inhibit” and “inhibition” may refer to, e.g., a reduction in the growth rate of bacteria (e.g., CLas or L. ere see ns), reduced or limited expansi on / growth of a bacterial colony, reduced bacterial viability, or cell or organism death.

[0287] The terms "introduce" and "introduction" refers to contacting a plant, or a portion thereof, either directly or indirectly with an agent e.g., a compound, microbe or composition described herein). For example, an agent may be directly applied to the plant, or a portion thereof e.g., seed, seedling, leaf, stem or root) and / or indirectly applied to the surrounding ecosystem adjacent to the plant e.g., water, air or soil, such as a planting bed). In certain embodiments, the plant or the surrounding ecosystem is sprayed with the agent. In certain embodiments, the plant or a portion thereof is coated with the agent e.g., a rootstock is dipped in the agent). In certain embodiments, the agent is administered or delivered to the plant or the surrounding ecosystem e.g., via injection). In certain embodiments, an agent may be applied to a plant or seed through the use of a suitable coating mechanism or binder prior to the seeds or plants being sold into commerce for planting. The process of coating seeds and plants is also generally well known to those skilled in the art. For example, the agent may be mixed with a porous, chemically inert granular carrier as described by U.S. Pat. No. 4,875,921. In certain embodiments, the agent is introduced to the plant or its surroundings to increase its present concentration (e.g., to increase the concentration of the agent to be higher than a preexisting naturally occurring level or concentration of the agent, if any, in the plant or its surrounding that previously failed to be inhibitory).

[0288] The term “bioinoculant” refers to a population of live cells of single or multiple organisms present in a viable form, which can be introduced to a plant to inhibit pathogenic microbe growth (e.g., bacterial growth) or promote plant growth / productivity.

[0289] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder in a plant. For purposes of this invention, beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival of the plant as compared to expected survival if not receiving treatment. Plants in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0290] The phrase "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0291] Accession numbers associated with sequence data as used herein refer to the first version. Additionally, certain accession numbers (e.g., WGS Accession Numbers) may reference the master record for the whole genome sequencing project. Such a number includes all the sub-Accession Numbers associated with the master record and the sequence data associated with each sub-Accession Number. For example, WGS Accession No. JAXKIAOOOOOOOOO is the master record for a whole genome shotgun sequencing project for Bacillus velezensis strain CB00019, and therefore, includes all sequence data associated with the master record, including WGS Accession Nos. JAXKIA010000001-JAXKIA010000021, which are accession numbers / sequence data for each of the 21 contigs from this project.

[0292] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the invention can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0293] It will be appreciated by those skilled in the art that certain compounds described herein have a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[0294] When a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities.

[0295] When a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted. In one embodiment, the compound may be at least 51% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95 the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted.

[0296] The invention will now be illustrated by the following non-limiting Examples.

[0297] Example 1. Identification of antimicrobial compounds from the citrus microbiome with potential to manage HLB disease.

[0298] Huanglongbing (HLB) is a devastating citrus disease, associated with the Gram-negative, phloem-limited and unculturable bacterium, Candidatus Liberibacter asiaticus (CLas), and transmitted by the psyllid, Diaphorina citri. Secondary root pathogens, such as Phytophthora and Fusarium spp., are linked to accelerated HLB tree decline. In previous work, we investigated the citrus-associated microbiome profile of HLB -impacted trees, which generated a citrus microbe culture collection. Bacillus isolates from our culture collection that showed in vitro inhibitory activity against Liberibacter crescens (Lc), have been screened against two species of Phytophthora and Fusarium spp. in in vitro assays. Isolates that showed inhibition of Lc and Phytophthora and Fusarium spp. have been selected for metabolite profiling using LC- MS. Mass spectrometry analysis revealed the presence of amicoumacins in inhibitory fractions to Lc; for example, the known antibiotic amicoumacin A was observed in an isolate identified as Bacillus safensis. MS / MS-based molecular networking analysis of the crude extract revealed a network of amicoumacins and related compounds, as well as a pumilacidin / surfactin network. In addition, samples containing amicoumacin A showed significant inhibitory activity to CLas in an in vivo assay. Purification of amicoumacin A is still ongoing and bioassays with purified amicoumacin A compound will reveal its potential use in HLB-affected plants. We identified several Bacillus spp. inhibitory to citrus root pathogens Phytophthora spp. and Fusarium spp. in dual-microbial assays. Further investigation of the crude extract of these isolates and comparison with the predicted BGCs will aid in the identification of target compounds to be obtained either via purification or heterologous expression. We are building upon our previous findings and discovering the chemistry and functional diversity of the citrus microbiome, identifying potential natural products and bioinoculants that can be used to manage HLB as a disease complex.

[0299] In summary, as described in Example 1, we are mining the citrus associated microbiome to find antimicrobial compounds with potential to suppress Candidatus Liberibacter asiaticus (CLas), the HLB associated pathogen, and secondary citrus root pathogens that are known to aggravate root decline in HLB trees. We found one antimicrobial compound, amicoumacin A, produced by a bacterial isolate that showed inhibitory activity to Liberibacter crescens and CLas and we identified several Bacillus spp. inhibitory to citrus root pathogens Phytophthora spp. and Fusarium spp. We are building upon our previous findings and discovering potential natural products and bioinoculants that can be used to manage HLB as a disease complex.

[0300] Introduction

[0301] The plant microbiome comprises all the microorganisms (bacteria, fungi, protists, nematodes and viruses) associated with the host in the different plant compartments (phyllopshere, endosphere and rhizosphere) (see, Figure 1). These microorganisms can form complex associations with plants and have important roles in promoting the plant productivity and health. For example, the plant microbiome benefits the host in many aspects: promotes plant growth, nutrient acquisition, abiotic stress tolerance, and disease suppression (Gao et al., 2021). Beneficial microbes contribute to disease suppression by priming the plant immune system, excreting antibiotic compounds, and competing resources with pathogen (Gao et al., 2021). Pathogen invasion can disturb the plant-microbiome interactions by negatively affecting the relative abundances of keystone species leading to the destabilization of microbial communities (Agler et al. 2016, Ginnan et al., 2020, Zhang et al., 2021).

[0302] HLB is a century-old disease that has been present in the US for the last two decades and is a threat to citrus production worldwide. Currently, there is no effective long-term treatment available. One of the main reasons for that is due to the complexity of this disease. The HLB- associated pathogen, Candidatus Liberibacter asiaticus, resides in the phloem vessel and induces an immune-mediated response in the host, that leads to nutrient depletion and eventually tree death. The vector spreads quickly and is already present in most of the citrus producing area in the US. HLB management requires integrating prevention with control. Current management strategies include, e.g., ACP control, antibiotics, enhancing plant nutrition, and removal of symptomatic trees (see, Figure 2). However, there is a need for new chemicals or biological solutions for long-term and more sustainable management strategies.

[0303] Microbial community diversity and composition are affected by HLB disease severity (see, Figure 3 and 4). Loss of keystone taxa is speculated to create dysbiosis (e.g., favoring the increase of secondary pathogens and saprophytes). Microbes enriched in the early to moderate stages of disease include known plant growth promoting and biocontrol agents. In the late stage, there is a decline of potentially beneficial fungi, combined with the increase of secondary fungal pathogenic species, which can contribute to root decline.

[0304] Accordingly, described herein are a series of experiments designed to mine the citrus microbiome and to identify biologicals and antimicrobial compounds having anti- / .. crescens or anti-CLas properties (Figure 5A-5E, 6). In particular, these experiments were designed to address, e.g., the following questions. 1) What other isolates are inhibitory to L. crescens or CLas? 2) What are the compounds responsible for inhibition? 3) Are these compounds also inhibitory to CLas?

[0305] Results

[0306] Initial screening of a citrus microbial culture collection was performed based on in vitro antagonistic activity to Liberibacter crescens (Lc) (Blacutt et al., 2020). From a subset of isolates with inhibitory activity to L. crescens, CB729 (isolated from leaf tissue) presented high activity and was selected for further investigation (Figure 7). Isolate CB729 was identified as Bacillus safensis by WGS and phylogenetic placement in species tree (Figure 8). Bacillus safensis is a gram-positive, spore-forming, aerobic bacterium (Lateef et al., 2015). Based on previous descriptions, Bacillus safensis may be applicable as an industrial enzymes producer, PGPB, biocontrol agent, probiotic, or for bioremediation (Lateef et al., 2015).

[0307] Initial bioassay-guided fractionation of the crude extract of CB729 resulted in 7 active fractions to Lc (Figure 9A). LC-MS / MS analysis revealed the presence of amicoumacin compounds in the active fractions (Figure 9B). NMR data revealed N-acetyl-amicoumacin C as the major compound in fraction P6D. This compound was used as an internal standard in subsequent LC-MS / MS runs to assist in the identification of other amicoumacins (Figure 9C).

[0308] Amicoumacins were originally isolated from Bacillus subtilis (Itoh et al., 1981) and belong to a class of natural products, dihydroisocoumarin (Figure 10A) (Tyurin et al., 2018). They have been described to display antibacterial, antifungal, anticancer, and anti-inflammatory activities (Park et al., 2016). Additionally, they have also been described to be effective against plant-pathogens (Tyurin et al., 2018); however, it is a novel class of compounds against CLas. The mechanism of action of these compounds is based on protein synthesis inhibition (Figure 10B) (Shi et al., 2020, Maksimova et al., 2021).

[0309] Predicted secondary metabolite biosynthetic gene clusters (BGC) analysis in antiSMASH revealed 11 BGC (Figure 11). The hybrid NRPS-PKS domain was identified to be the most similar to the zwittermicin A (ZmA) cluster of Bacillus cereus. Gene cluster comparison revealed the NRPS-PKS cluster of B. safensis CB729 to be highly similar to the characterized amicoumacin BGC of Bacillus subtilis subsp. inaquosorum KCTC 13429 and had 17 genes (including 2 orfs), resulting in 45.2 kb (Figures 12A-12B). Eleven amicoumacin derivative compounds were identified, including amicoumacin A, B and C (Figure 13). A surfactin network, including surfactin B and C and pumilacidin C, E, were identified by spectral library match (Figure 13). Amicoumacin production and purification steps are described in Figure 14.

[0310] Amicoumacin A-enriched fractions were evaluated using a high throughput CLas infected citrus hairy root assay (Figure 15 A). Amicoumacin A-enriched fractions significantly reduced CLas titer (R4 (150 ppm), R5 (79 ppm) and R9 (175 ppm)) (Figure 15B). These data showed that it is possible that screened compounds to Lc can lead to compounds that suppress CLas in vivo. It is also noted that the active samples contain cyclic lipopeptides that may also be contributing to bioactivity. Molecular networking of amicoumacin-enriched bioactive samples is shown in Figure 16. In summary, amicoumacin, an antimicrobial compound, has been identified in anti- / .. crescens and CLas fractions of Bacillus safensis CB729, based on structure determination and semi-purified fractions. Amicoumacin-enriched fractions decreased CLas titer in infected tissue in hairy root assays. Expression of the amicoumacin BGC and / or large-scale purification may be performed to obtain enough pure material for whole-plant bioassays. As described herein, potential natural products and bioinoculants that can be used to manage HLB may be identified by investigating the chemistry and functional diversity of the citrus microbiome.

[0311] Materials and Methods

[0312] Methods similar to those as described in Example 6 were used to generate the data.

[0313] Example 2. Understanding the citrus pathobiome to mitigate Huanglongbing as a disease complex.

[0314] Using high throughput, DNA-based sequencing technologies, we derived an HLB disease ecology model that indicates as HLB severity increases, the root microbiome becomes enriched in soil-borne pathogens that include fungal taxa (Fusarium) and oomycete (Phytophthora) root pathogens. Fibrous root decline is a significant symptom of HLB that exacerbates tree decline. Our working hypothesis is that trees with HLB ultimately succumb to a complex of pathogens that include the primary pathogen (Candidatus Liberibacter asiaticus), but also secondary soil-borne fungal / oomycete parasites that attack the root compartment of CLas- weakened trees. We are conducting functional microbiome studies to test this hypothesis. We have completed Koch’s postulates with a number of HLB -associated Fusarium spp. and determined they are pathogenic on several common citrus rootstocks. Moreover, using a combination of high throughput, automated culturomics and natural product chemistry, we are conducting experiments to empirically identify a consortia of biologicals (bioinoculants and antimicrobial natural products) derived from the native citrus microbiome that target the primary pathogen, CLas, and / or secondary pathogens, Fusarium and Phytophthora. The overall project goal is to develop HLB mitigation strategies that work with the citrus microbiome, rather than against it and support root health that feeds back into canopy health.

[0315] In summary, as described in Example 2, the overall project goal is to leverage the knowledge we have learned about how the citrus microbiome changes under increasing HLB severity. Using these data we are developing a consortia of microbes that will support root and canopy health by treating HLB as complex of pathogens.

[0316] Results

[0317] Through the examination of the citrus holosystem and Huanglongbing (HLB), we sought to further understand disease antagonists and disease facilitators, e.g., investigating what changes occur as disease severity increases and what microbial differences might affect rate of tree decline (see, e.g., Figure 4). For example, as HLB disease severity increases, a decline in putatively beneficial microbes was observed and an increase in putatively parasitic microbes was observed.

[0318] Figure 17 shows a schematic for culture-dependent and culture-independent methods.

[0319] Figures 18A-18G show HLB-associated Fusarium isolates. Further experimentation described below focused on whether they cause disease in citrus and whether they contribute to fibrous root decline associated with HLB.

[0320] Figure 19A shows a schematic of lesion length assay for Fusarium isolates in citrus. Figure 19B shows the statistical significance of lesion lengths on citrus stems as compared to an uninoculated control, asterisks indicate significanc . Figure 19C shows the mean lesion length of Carrizo citrange, Swingle citrumelo, and S-l citron seedlings six weeks post-inoculation.

[0321] Figure 20A shows plate images of colony morphology of citrus-associated Fusarium. Figure 20B shows plant biomass of citrus seedlings inoculated of citrus-associated Fusarium. Figure 20C shows images of citrus seedlings inoculated of citrus-associated Fusarium.

[0322] Figure 21 A shows inhibition of Bacillus cell-free supernatants to citrus-associated Fusarium. Figure 2 IB shows inhibition of Bacillus cell-free supernatants to citrus-associated Phytophthora.

[0323] Figure 22A depicts a natural product discovery pipeline. Figure 22B shows compounds that have been identified to be antagonistic to L. crescens. Figure 22C shows a molecular network describing cyclic lipopetides found in fractions that are inhibitory to L. crescens

[0324] Figure 23 shows a depiction of scale-up pipeline of bacterial strains and natural products for testing in whole citrus trees Certain Materials and Methods

[0325] Isolation of Fusarium spp. from citrus in Florida and California. 18 strains of

[0326] Fusarium spp. were isolated to purity from 11 samples of leaf, stem, and root tissue of Florida citrus trees with low to severe HLB severity, as previously described, or from roots of Schaub rough lemon (Citrus jambhiri) and Mexican lime (Citrus aurantifolia) located in the UC Riverside Agricultural Operations, Riverside, California, in June 2021 (Ginnan et al. 2020, Phytobiomes Journal, 4(4):375-387). California trees showed signs of decline including leaf chlorosis, defoliation, and dieback typical of Fusarium Dry Root Rot. Roots were collected and stored on ice prior to processing, then rinsed and surface sterilized for 60s in 10% sodium hypochlorite, 60s in 70% EtOH, and 60s in sterile distilled water. Approximately 1 cm sections were plated on malachite green agar (MGA), a / ’Z / .w / / / / 77-selective media. Single spore cultures of both California and Florida isolates with morphology consistent with Fusarium were grown on potato dextrose agar (PDA). Conidial suspensions in glycerol of these single-conidium isolates were then placed in long-term storage at -80°C.

[0327] Genomic characterization of Fusarium isolates. The 18 Fusarium-W isolates were collected from tissues of Florida and California citrus. DNA was extracted from mycelial tissue; the genomes were sequenced, assembled, annotated, and assigned taxonomic identification as previously described.

[0328] Morphological characterization of select Fusarium isolates. The morphological characteristics of selected isolates were described. Colony morphology and pigmentation were described for cultures grown on Potato Dextrose Agar (PDA) for 7-10 days at room temperature (~23°C) and exposed to diurnal (14 / 10) UV light. Chlamydospores, micro- and macroconidia, and phialides were described from cultures grown in the same conditions for 2-4 weeks on Carnation Leaf Agar (CLA). Micro- and macroconidia were examined in situ using a Zeiss Axioskop 2 microscope, and with a water slide mount on a Zeiss Axio Observer 5 inverted microscope. A total of 30 micro- and macroconidia per isolate were photographed using a Zeiss Axiocam 503 Mono microscope camera and Zen Blue 2.5 software v.2.5.75.6, and the length and width were measured using Fiji / ImageJ software (Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682 (2012).). Mean conidia dimensions and standard deviation were calculated.

[0329] Quantifying virulence of Fusarium isolates via stem inoculation. Three root isolates and one leaf isolate were selected for pathogenicity testing via stem inoculation: HLB-associated F. oxysporum (CF132, CF141, and CF159) and falciforme CF175, and California isolate F. solani CF177, which was included in the second experimental repetition as a positive control. Randomized stem inoculations (n = 10) were performed on two different dates as follows: A PDA plug from an actively growing 5-7-day-old culture was placed into a 4 mm2wound on the trunk of a healthy 4-8-month old citrus plant and wrapped with Parafilm® (American National Can, Chicago, IL, USA). Sterile PDA was used for the control. Plants were placed in moist greenhouse conditions at an average temperature of 23.4°C for six weeks before the bark at the inoculation site was peeled back and lesion length (vascular discoloration) measured with an INSIZE 1110-150A electronic caliper. The experiment was repeated on two different dates (n = 20 total).

[0330] Data distribution was analyzed and lesion length estimated for each cultivar by fitting a generalized linear mixed model with Template Model Builder in R. The ‘glmmTMB’ function with a gamma distribution was used to examine interaction effects of cultivar and inoculum. The model included cultivar and inoculum as fixed effects, with experimental repetition as a random effect to account for random variance. The boxplot significant difference labels were derived by fitting the model to one cultivar at a time and applying the ‘emmeans’ function in R.

[0331] To fulfill Koch’s postulates, tissue from the lesion margin was surface sterilized for 60s in 10% sodium hypochlorite, 60s in 70% EtOH, and 60s in sterile distilled water, and plated on the Fusarium-selective medium Malachite Green Agar (MGA). Colonies were morphologically identified, and representative colonies (three per cultivar / isolate combination) were sub-cultured onto MGA. DNA was extracted and amplified using primers targeting the rRNA internal transcribed spacer (ITS) region using ITS1 (TCC GTA GGT GAA CCT GCG G (SEQ ID NO: 1)) and ITS4 (TCC TCC GCT TAT TGA TAT GC (SEQ ID NO:2)) primers and the translation elongation factor 1-a (TEF) coding region using primers EF1-194F (AGC TCA GCG GCT TCC TAT TG (SEQ ID NO:3)) and EF1-660R (GAC TCT GGC AAG TCG ACC AC (SEQ ID NO:4)). Sanger sequencing was performed by Retrogen, Inc., San Diego, CA. Sequences were trimmed and aligned with the genomes of the original Fusarium spp. used as inoculum using Geneious Prime® (2023.2.1).

[0332] Pathogenicity of Fusarium isolates on Carrizo citrange and Swingle citrumelo roots. Fusarium isolates that caused stem lesions were then evaluated for their ability to cause root decline. Cultures of solani CF177, F. oxysporum CF159, and F. falciforme CF175 were grown on PDA. 1 L flasks containing 100 g long-grain white rice and 72 mL ultrapure water were autoclaved on two consecutive days prior to use. Twelve 7 -mm diameter plugs of actively growing culture were transferred into each flask, shaken well, and placed in a 25°C incubator for 7-14 days until rice grains were fully colonized. Control treatments received rice inoculated with sterile PDA only. The colonized rice was then incorporated into the planting medium of uniform 4-month old citrus seedlings in 983 mL D60L Deepot™ cells (Steuwe & Sons, Inc., Tangent, OR, USA) at a rate of 6 g L'1substrate. The planting medium was steam-sterilized coco coir amended with Osmocote® Smart Release® Plant Food Plus at the recommended rate of 1.5 Tbsp gal'1planting medium. Three experimental repetitions were performed for each isolate and the control on Carrizo citrange and Swingle citrumelo rootstocks, with eight biological replicates (n = 24 total) per cultivar / treatment combination.

[0333] Seedlings were placed in a greenhouse (24°C, 73% RH), and foliar disease symptoms were observed for 4 months. Plants were assessed weekly using a 1-5 visual disease rating (VDR) scale where 1 = healthy plants, 2 = 5-25% chlorosis / vascular discoloration, 3 = 26-50% chlorosis / vascular discoloration, 4 = wilting with 51-75% chlorosis / vascular discoloration, 5 = dead / dying plants with wilting and 76-100% chlorosis / vascular discoloration.

[0334] After 4 months, plant heights were recorded. Stems were cut 25 mm above the soil line, and roots were separated from the soil and gently washed. Root VDR was recorded as percent of roots exhibiting symptoms including reddish-brown or greyish discoloration, stunting, and reduced root volume. Six 2-cm long pieces of symptomatic root were placed into 15 mL conical centrifuge tubes and stored at 4°C prior to culture. Fresh root and shoot mass were recorded, and tissues were dried in a 105°C oven for 24 hours prior to recording dry root and shoot mass. 2-cm pieces of root tissue were surface sterilized as described above. Ends were trimmed, and five 1- cm pieces per plant plated on MGA and incubated at 25°C for 2-5 days. Cultures were identified morphologically and percent of roots infected recorded. To fulfill Koch’s postulates, three representative colonies per treatment were subcultured onto MGA. DNA was extracted from the subcultures, amplified, sequenced, and identified as described above.

[0335] Statistical analyses including ANOVA were performed using R version 4.3.3 (2024-02- 29). In addition to the response variables of root VDR, shoot VDR, root percent infection, final root length, root area, and shoot area, days to germination, root fresh mass (FR), root dry mass (DR), shoot fresh mass (FC), and shoot dry mass (DC), the following were calculated. Root dry

[0336] 1X 100 matter content (DMC) was calculated as FR , and shoot dry matter content was calculated

[0337] BSxioo SB as FC . Root: shoot dry ratio (r:s dry) was calculated as DC , and root: shoot fresh ratio (r:s

[0338] 1 1< fresh) was calculated as FC (Bray, J. R. Root production and the estimation of net productivity. Can. J. Bot. 41, 65-72 (1963).

[0339] The experiment was repeated on three different dates, and for the majority of the analysis this structure was accounted for using linear mixed-effects models (LMMs) or generalized linear mixed-effects models (GLMMs) implemented in the R packages Tme4’ and ‘glmmTMB’. Each of the response variables was considered separately to determine which distribution family was most appropriate, and whether including cultivar and inoculum as fixed effects and experimental repetition as a random effect improved model outcomes.

[0340] Example 3. High Throughput Culturomics Yielded Potential New Tools in the Fight Against Huanglongbing

[0341] The community of microorganisms that live in and on plants, termed the microbiome, plays a pivotal but not yet fully defined role in plant health. Our ability to understand these roles is limited by our understanding of the microorganisms that make up these communities. Looking to move beyond DNA based inferences, the field of culturomics seeks to improve our ability to isolate microbiome members from complex environments and understand their contributions to their host. Previous studies have indicated that the citrus microbiome may have a distinct role in Huanglongbing development, and that certain microbiome members possessed the capability to inhibit the growth of L. crescens. To fully understand the disease suppressing capabilities of the citrus microbiome, we established a pipeline to isolate and screen citrus associated bacteria from field grown trees against Liberibacter crescens, the closest culturable relative to the associated causal agent of HLB. Utilizing automated high throughput microbial cultivation array technology, made possible by the Prospector® from Isolation Bio™, we were able to increase the diversity of our previously established citrus plant associated microbial culture collection. We captured several bacterial isolates that produce inhibitory compounds that could be promising microbiome informed management tools, in the form of bio-inoculants or natural products, to help ameliorate the existential threat Huanglongbing poses to citrus growing regions.

[0342] In summary, as described in Example 3, utilizing new microbial isolation technology, we enriched our pre-existing citrus associated bacterial collection with an automated and high throughput workflow. We identified several bacterial isolates, native to the Citrus microbiome, that produce compounds inhibitory to L. crescens or CLas, that could be developed into bioinoculants or natural products for the treatment of HLB.

[0343] Introduction

[0344] As HLB becomes endemic to US citrus growing regions, solutions and strategies for its management remain lacking. Current options remain exceedingly limited and are available only under emergency EPA registrations. For example, oxytetracycline, which is a broad spectrum and medically relevant antibiotic, is the only tool most growers have at their disposal. With limited options, the threat of developing resistant pathogen populations increases (Batuman et al.). Accordingly, there is a need for new narrow spectrum and agriculturally appropriate tools for growers to manage HLB.

[0345] In medicine, anti-microbials often come from microbes. For example, 70-80% of antibacterial drugs currently in use are derived from microbes, such as penicillin, streptomycin, tetracycline, erythromycin, vancomycin, etc. (Newman and Cragg, 2016). Examples of other drugs that were discovered in microorganisms include: antifungals (nystatin, cycloheximide, amphotericin B), antivirals (acyclovir), anti-parasitics (ivermectin), and anti-cancer agents (doxorubicin). Microbially derived compounds also are a major player in agro-chemicals. For example, agricultural bactericides from microbes, include, e.g., oxytetracycline (Streptomyces rimosus). Kasugamycin (Strptomyces kasugaensis) and agricultural fungicides derived from microbes, include, e.g., Strobilurins (Strobilurus tenacellus) and Natamycin (Streptomyces natalensis).

[0346] Natural products are tools from the natural world and can be foundational. Beyond use as purified natural products, microbial secondary metabolites can be studied and further derived to become fully synthetic compounds that are further optimized for efficacy. For example, discovery of the structure of oxytetracycline led to further derivatization and yielded doxycycline, one of the most widely used and effective antibiotics to date. Strobilurins were further derivatized into azoxystrobin, and are the second largest group of agricultural fungicides in use.

[0347] The microbiome of citrus trees plays a dynamic role in HLB disease development (Ginnan et al. 2020, Phytobiomes Journal, 4(4):375-387). Previous research indicates that the citrus microbiome is a potential reservoir of resources that could be used to create solutions to the problem HLB has created (Blacutt, et al., Applied and Environmental Microbiology 86, no. 8 (2020): e02883-19). Described herein are methods to isolate and identify these resources and to identify microbially mediated solutions to this disease (e.g., bio-inoculants and natural products). In particular, such methods may include the use of culturomics to further mine the citrus microbiome. Culturomics refers to high throughput and system informed methodologies that are used to efficiently recover bacteria from their environments and also helps to understand and deconvolute hosts and their microbiomes. It involves robust sampling and culturing methods to accurately sample the host’s microbiome.

[0348] Results

[0349] As described herein, the Prospector® from Isolation Bio™, which is an automated laboratory workfol ow device that allows for high-throughput cultivation of bacteria from environmental samples, was used for microbial isolation from Citrus tissue (Figure. 24A). The Prospector® uses etched microscope slides, termed Arrays, to create microwells that are able to isolate just a few cells at a time (Figure 24B). The samples are loaded in media with a color changing dye, resazurin, that changes in color in response to cellular respiration (Figure 24C). Wells that change color also change in green fluorescence, and the machine has a camera that detects this, indicating which wells have growing cells in them. It then picks up the contents of these wells with a sterile pin and transfers them to 96 well plates for downstream use. This process greatly reduces the chance of cocultures without the need for multiple subcultures. In particular, the Prospector’s® arrays allow bacteria to be separated, cell by cell, into their own micro well, and prevent bacteria from out competing each other on a Petri dish. Some bacteria grow faster than others and can eclipse and overgrow their neighbors (see, e.g., Figure 25), leading to biases in microbial culture collections, for bacteria that grow well on petri dishes. The use of the Prospector® addresses this issue. Samples are obtained from the above ground parts of the tree, the phyllosphere, and the below ground parts of the tree, the rhizosphere (Figure 26). Collected plant tissue is then processed to generate high potency microbial extracts. Serial dilutions of microbial extracts are loaded in a vacuum chamber that allows the bacterial suspension to proliferate into the microwells present on the arrays (Figure 27). The arrays are incubated at 28°C for 7 days and then imaged, wherein green fluorescence is the result of metabolic oxidation of resazurin and signals active microbes in those microwells (Figure 28A). A heat map is generated via a computer program based on the green fluorescence and specific wells are then selected for aseptic transfer into 96 well plates for downstream characterization / screening (Figure 28B).

[0350] A bioassay, using the general workflow described above, was set up against Liberibacter crescens. Candidatus Liberibacter asiaticus (CLas) has still yet to be obtained in pure culture and in-vitro bioassays for antibiosis screening must use the closest culturable relative to CLas, Liberibacter crescens (Jain et al., 2019). A previous agar-based assay was adapted to be more high-throughput and to allow the screening of entire 96 well plates at once (Figures 29A-29B). In particular, the adapted assay uses a plate replicator to transfer liquid cultures of single isolates obtained from the Prospector workflow to a 96 well plate shaped petri dish containing agar incoluated with L. crescens, which allows the bacteria to grow alongside the L. crescens (Figure 29B). The petri dishes are then examined to identify antibiosis, which is indicated by a clearing of L. crescens from around the isolate producing an inhibitory interaction (Figure 30A).This phenotype is then repeated using an adapted dual microbial assay to confirm this inhibition is robust and repeatable (Figure 30B).

[0351] This pipeline generated an inhibitory isolate library comprising several different genera, wherein for many of these genera, multiple strains that show inhibitory activity were identified (Figure 31). Paired genome and metabolome datasets then guide downstream compound identification.

[0352] In order to identify the compound or compounds responsible for the experimentally observed antibiosis, a process called bioassay guided fractionation is performed (Figure 32). In particular, this process involves growing large scale fermentations of inhibitory isolates, extracting their metabolites and concentrating them, and then utilizing flash column chromatography to separate the metabolites present based on their polarity. These fractions are then assayed against L. crescens and this process is repeated until active fractions only containing a few potentially inhibitory compounds are obtained.

[0353] To address time constraints associated with the very slow growth of L. cresecens, a high throughput bioassay to screen fractions for inhibition was developed. The workflow uses a resazurin based bioassay with L. crescens, so that large numbers of metabolome fractions can be rapidly screened without having to wait weeks at a time (Figure 33; see also, Figure 24C for resazurin conversion). Specifically, this assay is done in a 96 well footprint, and combines the desired treatment or fractions with growth media, L. crescens and dye. As the L. crescens undergoes basic metabolic functions, the resazurin present in the dye is reduced into the compound resorufin. The more respiration that is occurring, the more resorufin is generated. How much resorufin has been generated in the sample can be measured with a plate reader, measuring the fluorescence at a specific wavelength. The treated samples can be compared with a control(s) and growth inhibition can be observed as a function of metabolic inhibition. To validate this assay, the antibiotic Kanamycin was used a positive control, wherein a metabolic dose response curve showed decreases in fluorescence as antibiotic concentration increased (Figures 34A-34B).

[0354] The overall workflow of the pipeline is depicted in Figure 35, wherein an inhibitory phyllosphere isolate was processed / evaluated. The resazurin bioassay described above was then used to screen each individual metabolome fraction for activity. Bioactivity across different fractions of the isolate’ s metabolome was observed (Figure 36). Inhibitory compounds are then identified and further tested. Specifically, inhibitory fractions are sent to the Metabolomics core at UCR for Liquid Chromatography -Mass Spectrometry, wherein resulting spectra are interpreted by software such as GNPS to identify compounds based on mass, column retention time, and fragmentation. Additionally, purified or synthesized versions of these molecules will be tested in in planta assays against CLas.

[0355] Materials and Methods

[0356] We harvested tissue from a field grown citrus tree, utilized a multi-step cell liberation protocol, and further purified extracts using a Nycodenz gradient. Citrus phyllosphere tissue was collected from the Parent Washington Navel accession from the Givaudan Variety Collections at UC Riverside (Riverside, CA, USA). Leaf and stem tissue was collected from the entire circumference of the tree, in a continuous perimeter, using sterilized clippers and placed into gallon zip lock bags, placed on ice and transported back to the lab for processing. Microbial extracts were purified and concentrated from large volumes of tissue slurry. To accomplish this, 160g of leaves and segments of their cognate stems were separated into two 80g aliquots, and placed in sterile beakers. The tissue was chopped and macerated well with sterilized clippers in 250ml of sterile cold IX PBS + 0.1% Tween 20 + 0.1% sodium pyrophosphate. The beakers were sealed with sterile aluminum foil and were alternately shaken in a shaker incubator and gently sonicated in a water bath sonicator with five-minute intervals for each. After three intervals of shaking and sonication, the beakers were then gently shaken one final time for 40 minutes on a rotary shaker. The resulting leaf and wash slurry was filtered through cheese cloth into a sterile IL bottle, sealed and allowed to settle overnight at 4°C. The next day, the supernatant was collected and centrifuged at 5,000 x g for 30 minutes, at 4°C. The supernatant was discarded, and the pellet was resuspended in 20mL IX PBS + 0.1% Tween. This resuspension was then filtered through a 40pm cell strainer, followed by a 15pm cell strainer. The filtrate was split equally into two lOmL aliquots, and each aliquot was overlay ed onto a Nycodenz® (Accurate Chemical, New York, USA) gradient in a 50mL ultracentrifuge tube that consisted of 15ml of 80% Nycodenz at the bottom, lOmL of 60% in the middle, and 5mL of 40% on the top. The filtrate overlay and gradient was centrifuged at 10,000 x g for 45 minutes, at 4°C, in a swing bucket rotor. Upon centrifugation, visible layers formed at each step of the gradient. All layers above the 80% step were collected, diluted to a final volume of 100ml in IX PBS and centrifuged at 5,000 x g for 30 minutes, at 4°C. The resulting pellet was resuspended in 2.2 mL of R2A broth (HiMedia Laboratories, PA, USA) amended with the 25 pg / mL natamycin and O.lmM resazurin. In order to ensure appropriate cell density, a miniaturized dilution to extinction assay was employed. A 200 pL aliquot of the extract was used for serial dilutions in a 96 well plate in R2A + 25 pg / mL natamycin and O.lmM resazurin. Resazurin served as an indicator of the presence of viable bacterial cells, and dilutions with living cells would turn pink, and the last dilution that turned pink was interpreted as the dilution of extinction. Ten pL of this aliquot was also spread plated on R2A agar + 25 pg / mL natamycin to ensure morphological diversity within the sample.

[0357] Prospector Workflow. Two milliliters of the cell suspension was loaded onto the array in a biosafety cabinet using the array loading chamber according to manufacturer instructions (IsolationB io, Inc.) Cell suspensions were incubated on the array for 5 minutes. The remaining suspension was removed and the array was sealed. Arrays were imaged immediately after loading, as well as after 4-5 days of incubation. The wells with cellular growth (as indicated by fluorescence of the resazurin dye) were transferred into 96 well plates containing 200pL R2A broth amended with 25 pg / mL Natamycin and 0. ImM resazurin following the manufacturer’s instructions. The 96 well plates were incubated for at least three days before having wells with living cells transferred to deep well plates, where glycerol was added to a final concentration of 15%, and the isolates were stored at -80°C.

[0358] High throughput zone of inhibition bioassays. We developed a high throughput zone of inhibition assay by adapting a previously described assay in Blacutt, et al., Applied and Environmental Microbiology 86, no. 8 (2020): e02883-19. In brief, 4-day old Liberibacter crescens (ODeoomn -0.27) was inoculated into molten mBM7 medium top agar (0.8% agar) cooled to 60°C, to a final concentration of 10% and overlay ed on a Nunc™ OmniTray™ (Thermo Fisher Scientific, Waltham, Massachusetts) containing 20mL of solid mBM7 media. After the agar solidified, the plates were incubated at 28°C for 2 days. Meanwhile, 96 well formatted cryopreserved stocks were replica inoculated into 96 well plates containing 200 pL of R2A broth. The isolates were propagated at 28°C at 180rpm for 2 days. Following this, liquid cultures from the 96 well plate were replica plated onto the Omni Tray using a 96-pin plate replicator. The co-inoculated Omni Trays were incubated at 28°C for 4-5 days, until L. crescens growth was observed. Isolates with clear zones of inhibition of L. crescens growth were considered antagonistic to L. crescens and moved forward in the screening pipeline. When many inhibitory isolates were observed, making observation difficult by clearing large sections of the Omni Tray, this process was repeated, assaying only one column of the 96 well plate at a time, in a standard petri dish, following the same workflow, but on 20mL plates with 3.5mL of top agar.

[0359] Isolates that showed inhibition in the high throughput assay were tested again individually to confirm the inhibition phenotypes in an adapted assay from Blacutt, et al., Applied and Environmental Microbiology 86, no. 8 (2020): e02883-19, using live microbes instead of extracts on discs, and allowing the L. crescens to grow three days on the plates before challenging. In some instances mixed cultures were present in the Prospector® output plates, these were sub-cultured to purity before being re-screened as individual isolates. Each plate contained three technical replicates, and this assay was repeated three times for each isolate. An isolate that produced inhibitory halos two out of the three times it was assayed was considered inhibitory and was moved forward in the pipeline to screen for production of inhibitory secreted metabolites.

[0360] Bulk Taxonomic Identification of Isolates. To identify the taxonomy of the bacteria derived from the Prospector output, we used a plate replicator to replicate 96 well plates of isolates in cry opreservation into 96 well plates with 200pL R2A and propagated at 28°C with 150rpms of shaking. To generate large amounts of microbial biomass, plates of cultures were subcultured to two deep well 96 well plates after 2 days of incubation. Deep well plates were sealed with gas permeable film and incubated for 3 days. These cells were pelleted at 2,000 x g for 15 minutes, the supernatants discarded and the cell pellets resuspended in a total of 800 pL of Qiagen DNeasy Power Soil Pro Lysis buffer (QIAGEN, Hilden, Germany). The resuspended cells were transferred to a Qiagen PowerBead Pro plate, and homogenized in a Tissue Lyser II (QIAGEN, Hilden, Germany), for 5 minutes at 30 Hz, twice. Following homogenization, the plates were centrifuged at 300 x g for 15 minutes and 480-600 pL of the supernatant was transferred into another deep 96 well plate and loaded into an automated DNA extraction workflow within the QIAcube with the DNeasy 96 PowerSoil Pro QIAcube HT Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.

[0361] DNA was pooled into one sample for each plate, with each well contributing 500ng of DNA. DNA pools were assessed for quality with a spectrophotometer and an agarose gel (1% agarose, 90V for 40 minutes), and purified using Zymo’s Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) if necessary.

[0362] Pooled DNA samples were submitted to SeqCenter (Pittsburgh, PA, USA) for 16S based amplicon sequencing. Samples were prepared using Zymo Research’s Quick-16S kit with phased primers targeting the V3 / V4 regions of the 16S gene. The specific primer sequences for the region used were 341f (CCTACGGGDGGCWGCAGCCTAYGGGGYGCWGCAG (SEQ ID NO:5)) and 806r (GACTACNVGGGTMTCTAATCC (SEQ ID NO:6)).

[0363] Following library clean up and normalization, samples were sequenced on a Pl or P2 600cyc NextSeq2000 Flowcell to generate 2x301bp paired end (PE) reads. Quality control and adapter trimming was performed with bcl-convertl (v4.2.4). Primer-dimer sequences identified as PCR artifacts were filtered from the generated FASTQ files based on the following criteria: read length > 15Obp, PolyN strings < 10 sequential Ns; PolyG strings < 150 sequential Gs.

[0364] Sequences were imported to Qiime2 for analysis. (Bolyen, et al., Nature Biotechnology 37, no. 8 (2019): 852-57) Primer sequences were removed using Qiime2’s cutadapt (Martin, Marcel. EMBnet. Journal 17, no. 1 (2011): 10-12) plugin using the following degenerate primer queries: Forward Trim: CCTAYGGGNBGCWGCAG (SEQ ID NO:7), Reverse Trim: GACTACNVGGGTMTCTAATCC (SEQ ID NO:6). Sequences were denoised using Qiime2’s dada2 plugin (Callahan, et al., Nature Methods 13, no. 7 (2016): 581-83). Denoised sequences were assigned operational taxonomic units (OTUs) using the Silva 138 99% OTUs full-length sequence database and the VSEARCH (Rognes, et al., Peer J 4 (2016): e2584) utility within Qiime2’s feature-classifier plugin. OTUs were then collapsed to their lowest taxonomic units, and their counts were converted to reflect their relative frequency within a sample.

[0365] Example 4. Pathogenic Potential of HLB-Associated Fusarium Species

[0366] Summary

[0367] Fusarium spp. isolated from roots, leaves, and stems of HLB -impacted citrus were screened for pathogenicity, and those originating from roots were found to be pathogenic. Fusarium spp. increase in roots as HLB disease progresses, and may speed fibrous root decline in infected trees. Understanding interactions between HLB and other pathogens will enable the development of management strategies that include all contributors to HLB-related citrus decline.

[0368] Introduction

[0369] Fibrous root loss is an important and understudied symptom of HLB-related citrus decline (Johnson, et al., Association cM Candidates Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms. Plant Pathol. 63, 290-298 (2014); Graham, et al., Presymptomatic Fibrous Root Decline in Citrus Trees Caused by Huanglongbing and Potential Interaction with Phytophthora spp. Plant Dis. 97, 1195-1199 (2013)). We observed that as HLB disease severity increased, so did the relative abundance of Fusarium spp. in roots (Ginnan, N. A. et al. Disease-Induced Microbial Shifts in Citrus Indicate Microbiome-Derived Responses to Huanglongbing Across the Disease Severity Spectrum. Phytobiomes Journal 4, 375-387 (2020)). Fusarium spp. are associated with dry root rot in citrus, and trees weakened by other stressors can be more susceptible to infection.

[0370] Methods

[0371] To begin testing the hypothesis that Candidatus Liberibacter asiaticus (CLas) infection increases susceptibility to root invasion by soilborne pathogens, we isolated 18 strains of Fusarium from the roots, leaves, and stems of citrus under high and low HLB pressure and sequenced their genomes (Kurbessoian, T. et al. Genome sequence and assembly of 18 Fusarium isolates from Florida citrus under high huanglongbing disease pressure and California citrus under low huanglongbing disease pressure. Microbiol. Resour. Announc. 12, e0010123 (2023)). By inoculating the stems of Carrizo citrange, Swingle citrumelo, and S-l citron, we screened representative isolates for their pathogenic potential by completing Koch’s postulates (see, Figure 19 A).

[0372] Results

[0373] Root-associated isolates from the Fusarium solani and Fusarium oxysporum species complexes were pathogenic on Carrizo citrange, S-l citron, and Swingle citrumelo, but oxysporum and other Fusarium spp. isolated from leaves and stems were not (see, Figures 19B- 19C).

[0374] Conclusions

[0375] Fusarium spp. isolated from the roots of HLB -impacted citrus are capable of causing significant disease, in contrast with those isolated from other parts of the tree. This supports the hypothesis that Candidatus Liberibacter asiaticus (CLas) infection increases susceptibility to root invasion by soilborne pathogens, although further research is still needed. Example 5. Thirteen draft genome assemblies of Bacillus spp. derived from the citrus microbiome

[0376] Abstract

[0377] We report the draft genome assembly, annotation and phylogenetic placement of 13 Bacillus spp. isolated from citrus groves under high (Florida) or low (California) Huanglongbing (HLB) disease pressure.

[0378] Announcement

[0379] Bacillus is a well-studied bacterial genus of Gram-positive and spore-forming bacteria, commonly soil-borne and plant-associated [1-3], Many Bacillus species produce a range of secondary metabolites and volatile organic compounds that are beneficial to plants through various mechanisms, such as pathogen suppression or plant growth promotion [4-7] . Genome availability is the initial step in unlocking the beneficial metabolic potential of plant-associated Bacillus spp. by enabling the identification of bioactive compound-producing gene clusters. Here we announce the draft genome assemblies of thirteen Bacillus spp. isolated from citrus trees in areas of high (Florida) or low (California) Huanglongbing (HLB) disease pressure. Sampling of plant material, isolation and genus-level identification were initiated as a component of a bioprospecting effort designed to generate a citrus-associated microbial repository that can be mined for antimicrobial bioactivity against the associated causal agent of HLB, Candidatus Liberibacter asiaticus and HLB -associated secondary pathogens, such as Phytophthora spp. and Fusarium spp. [8, 23], In particular, leaves, stems, and roots were collected from citrus orchards in Florida and California, detailed in Table 1 and Figure 37. Samples were macerated with IX PBS and propagated on tryptic soy agar (TSA) and potato dextrose agar with 0.1 g / liter tetracycline hydrochloride (PDA) and incubated at 28°C for 4 days. The microbial consortia on each plate were scraped after adding 1 mL of IX PBS to generate bulk culture tubes. Isolates were recovered from bulk culture tubes on TSA and PDA and incubated at 28°C for up to 5 days. Single colonies of each bacterial isolate were streaked to purity, and cryostocks of each isolate were stored in 15% glycerol at -80°C.

[0380] DNA was extracted from a single colony grown overnight at 28°C in tryptic soy broth (TSB), using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, Wisconsin, USA), following manufacturer’s instructions for Gram-positive bacteria. Library preparation and sequencing were performed by SeqCenter (Pittsburgh, Pennsylvania, USA). Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10 bp unique dual indexing (UDI) indices, and sequenced on an Illumina NovaSeq 6000, producing 2x151 bp reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v4.0.3) [9], The total of paired-end raw reads recovered ranged from 2,560,758 to 13,949,264. Sequence analysis from quality control to annotation was performed on the KBase web service

[0381]

[0010] and the publicly available narrative containing all analyses and data can be found at https: / / narrative.kbase.us / narrative / 157793 and https: / / doi.org / 10.25982 / 157793.280 / 2368552

[0382]

[0011] , Default parameters were used except where otherwise noted. Quality of raw reads were assessed using FastQC (vO.12.1)

[0012] and quality control was performed using JGI RQCFilter pipeline BBTools (v38.22)

[0013] and PRINSEQ (v0.20.4)

[0014] , Genomes were assembled using SPAdes (v3.15.3)

[0015] with k-mer sizes set for 21, 33, 55, 77, 91, 111. The quality of assemblies was assessed with QUAST (v4.4)

[0016] and CheckM (vl.0.18)

[0017] , An initial assessment demonstrated that the number of contigs in the resulting assemblies ranged from 14 to 87 and the N50 value ranged from 164,068 to 1,085,110. A later assessment demonstrated that the number of contigs in the resulting assemblies ranged from 17 to 98 and the N50 value ranged from 151,282 to 1,085,110. The completeness of the genomes was initially determined to be between 99.38 to 99.59 and later determined to be between 97.28 and 99.41; and the GC content percentage was initially determined to range from 34.95 to 46.26 and was later determined to range from 34.95 to 46.50. The genomes were annotated using Prokka (vl.14.5)

[0018] , revealing the largest genome size of 5,551,588 bp from Bacillus cereus, and the smallest genome size of 3,677,118 bp from Bacillus safensis. It is also noted that the Bacillus cereus isolate was initially identified as Bacillus bombyspticus, but further analysis indicated that it was indeed Bacillus cereus. The number of predicted genes among the genomes was initially determined to range from 3,742 to 5,644 and a later analysis determined the range to be from 3,763 to 5,644 (Table 1 and Figure 37). Taxonomic identification was performed on GTDB-Tk (v2.3.2)

[0019] utilizing FastANI

[0020] , A Bacillus phylogenetic tree with closely related species was constructed using FastTree2 (v2.1.11)

[0021] through SpeciesTree (v2.2.0) on KBase and annotated on iTOL (v6.8.1)

[0022] (Figure 38B (initial construction shown in Figure 38A)). Table 1. Genome assembly statistics from an initial assessment. Genome assembly statistics from a later assessment are provided in Figure 37.

[0383] *This isolate was initially identified as Bacillus bombyspticus and later determined to be

[0384] Bacillus cereus.

[0385] Supplementary Table 1.

[0386] *This isolate was initially identified as Bacillus bombyspticus and later determined to be

[0387] Bacillus cereus.

[0388] Data availability statement The accession number for the raw reads and whole-genome sequences of the isolates are described in Table 1 and Figure 37. The Sequence Read Archive and genomic data has been deposited in GenBank BioProject under no. PRJNA1046128.

[0389] Documents Cited in Example 5 1. Andric S, Meyer T, Ongena M. 2020. Bacillus Responses to Pl ant- Associated Fungal and Bacterial Communities. Frontiers in Microbiology 11 : 1350.

[0390] 2. Caulier et al., 2018. Versatile Antagonistic Activities of Soil-Borne Bacillus spp. and Pseudomonas spp. against Phytophthora infestans and Other Potato Pathogens. Front Microbiol 9: 143. 3. Andric et al., 2023. Plant-associated Bacillus mobilizes its secondary metabolites upon perception of the siderophore pyochelin produced by a Pseudomonas competitor. ISME J 17:263-275.

[0391] 4. Radhakrishnan et al., 2017. Bacillus'. A Biological Tool for Crop Improvement through Bio-Molecular Changes in Adverse Environments. Frontiers in Physiology 8:667.

[0392] 5. Chaabouni et al., 2012. Secondary Metabolites of Bacillus'. Potentials in Biotechnology, p. 347-366. In Sansinenea, E (ed.), Bacillus thuringiensis Biotechnology. Springer Netherlands, Dordrecht.

[0393] 6. Sansinenea E, Ortiz A. 2011. Secondary metabolites of soil Bacillus spp. Biotechnol Lett 33: 1523-1538.

[0394] 7. Mondol et al., 2013. Diversity of Secondary Metabolites from Marine Bacillus Species: Chemistry and Biological Activity. Marine Drugs 11 :2846-2872.

[0395] 8. Blacutt et al., 2020. An In Vitro Pipeline for Screening and Selection of Citrus- Associated Microbiota with Potential Anti-“ Candidatus Liberibacter asiaticus” Properties. Appl Environ Microbiol 86:e02883-19.

[0396] 9. BCL Convert. https: / / support- docs.illumina.com / SW / BCL_Convert / Content / SW / FrontPages / BCL_Convert.htm. Retrieved 2 November 2023.

[0397] 10. Arkin et al., 2018. KBase: The United States Department of Energy Systems Biology Knowledgebase. Nat Biotechnol 36:566-569.

[0398] 11. Vieira, et al., 2024. 13 draft genomes of Bacillus from HLB-impacted citrus trees. Kbase narr. https: / / doi.org / 10.25982 / 157793.280 / 2368552.

[0399] 12.BibSonomy. 2024. FASTQC. A quality control tool for high throughput sequence data. Available from: https: / / www.bibsonomy.org / bibtex / f230a919c34360709aa298734d63dca3. Retrieved 20 May 2024.

[0400] 13. Bushnell B. 2014. BBMap: a fast, accurate, splice-aware aligner. LBNL-7065E. Berkeley, CA (United States) Lawrence Berkeley National Lab. (LBNL)

[0401] 14. Schmieder R, Edwards R. 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863-864. https: / / doi.org / 10.1093 / bioinformatics / btr026 15.Bankevich, et al., 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477.

[0402] 16. Gurevich et al., 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072-1075.

[0403] 17. Parks, et al., 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25: 1043-1055.

[0404] 18.Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069.

[0405] 19.Chaumeil, et al., 2020. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics 36: 1925-1927.

[0406] 20. Jain, et al., 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114.

[0407] 21. Price, et al., 2010. FastTree 2 - approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490.

[0408] 22. Letunic I, Bork P. 2021. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research 49:W293-W296.

[0409] 23. Ginnan et al., 2020. Disease-Induced Microbial Shifts in Citrus Indicate Microbiome- Derived Responses to Huanglongbing Across the Disease Severity Spectrum. Phytobiomes Journal 4:375-387.

[0410] Example 6. Amicoumacins produced by the native citrus microbiome isolate, Bacillus safensis, inhibit the Huanglongbing-associated bacterial pathogen, Candidatus Liberibacter asiaticus

[0411] Abstract

[0412] Huanglongbing (HLB) is a devastating citrus disease associated with the Gram-negative, phloem-limited, and unculturable bacterium Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid, Diaphorina citri. Despite extensive research, effective, long-term, and sustainable solutions for managing HLB remain elusive. Oxytetracycline (OTC) is currently used as an emergency measure, but there is an urgent need for alternative compounds to complement or replace OTC. In this study, we identified amicoumacin B, an antimicrobial compound produced by Bacillus safensis CB729, a bacterium isolated from the citrus microbiome, and demonstrated its ability to suppress CLas. Genome mining of Bacillus safensis CB729, combined with analytical natural product chemistry, revealed the presence of amicoumacins and related derivatives in fractions inhibitory to Liberibacter crescens, a culturable surrogate for CLas. We tested commercially available synthetic amicoumacins A and B, along with a 7>. .sz / / c / / .s / .s-derived amicoumacin mixture, against L. crescens and CLas. We determined the minimum inhibitory concentrations (MICs) of amicoumacin A (1.25 pg / mL) and amicoumacin B (10 pg / mL) against L. crescens. Furthermore, amicoumacin B and the amicoumacin mixture significantly reduced CLas populations in vivo. In conclusion, this study highlights the potential of amicoumacins as a promising group of natural products for the management of HLB, offering valuable insights for the development of novel and sustainable disease control strategies.

[0413] Introduction

[0414] Citrus is a major subtropical and tropical fruit crop with high nutritional and economic value, largely cultivated in China, Brazil, the United States, the European Union, Mexico, and Egypt (Zhong, Nicolosi, 2020; USDA, 2024). The current global orange production is forecast to be 48.8 million tons (USDA, 2024). Nevertheless, citrus production and sustainability have been impacted and threatened worldwide by Huanglongbing (HLB), associated with the unculturable phloem-limited bacteria, Candidatus Liberibacter spp. (Bove et al. 2006). In the United States, the prevalent associated pathogen is Candidatus Liberibacter asiaticus (CLas) and is vectored by the Asian Citrus Psyllid (ACP), Diaphorina cilri. (Halbert, Manjunath, 2004, Bove et al. 2006). HLB causes severe disruption of photosynthate transport due to phloem blockage mediated by deposition of callose in the phloem cells. (Achor et al., 2019). This disruption results in the typical HLB symptoms that include accumulation of starch in the leaves yellow shoots, thinning of the canopy, premature fruit drop, reduced fruit quality, root mass loss, and significant yield reduction and eventually leads to tree death (Bassanezi et al., 2011; McCollum, Baldwin, 2015, Achor et al., 2019). HLB has been present in the U.S. for almost two decades and was first detected in Florida in 2005 (Bove et al. 2006) and became endemic in 2013. HLB has since drastically altered the landscape of the U.S. citrus industry by causing a significant reduction in citrus acreage and yield (Li et al., 2020; Graham et al., 2020).

[0415] Significant progress has been made in developing sustainable solutions for HLB, including development of HLB-resistant scion and rootstock citrus varieties (Wang et al., 2016; Curtolo et al., 2020; Soares et al., 2020), nutritional enhancement programs, and the use of hormones and plant-immunity inducers (Ma et al., 2022; Makam et al., 2023; Bassanezi et al., 2024). Additionally, novel chemistries for vector control (Boina & Bloomquist, 2015) and antimicrobial compounds have been explored (Huang et al., 2020; Gardner et al., 2020; Dominguez et al., 2023). Despite these advances, no effective long-term and sustainable solutions are currently available for growers. Currently, the use of antibiotics to combat Huanglongbing (HLB) has increased in Florida as an emergency response. Initially, oxytetracycline and streptomycin were approved for foliar application on HLB-affected trees. However, research has shown that trunk injection is the most effective method for delivering these compounds directly to the tree's vascular system (Hu et al., 2017; Vincent et al., 2022; Archer et al., 2022, 2023). In 2022, Florida approved the commercial application of oxytetracycline (OTC) via trunk injection, and it has since been widely implemented. This method has been shown to enhance fruit yield and quality, improve overall tree health (Hu et al., 2017; Archer et al., 2022, 2023) Despite the immediate effectiveness of OTC in managing HLB, there is growing concern among scientists and growers about the potential development of antibiotic-resistant CLas in citrus orchards under high HLB pressure. This underscores the urgent need to identify alternative compounds that can be used in rotation with or as substitutes for OTC for medium- to long-term management (de Gracia Coquerel et al., 2023).

[0416] Plant microbiomes play a crucial role in enhancing plant health, productivity, and disease resistance (Trivedi et al., 2020). Specifically, microbes can produce a unique array of bioactive compounds that can benefit the plant host and can be a valuable resource for bioprospecting of microbially-produced antimicrobial natural products. In a prior bioprospecting effort, we generated a citrus-associated microbial collection from groves under high (Florida) and low (California) HLB pressure (Blacutt et al., 2020). Notably, Bacillus species were the most abundant isolates in our citrus microbial culture collection, constituting 37.4% of isolates from leaves, 34.7% from budwoods, and 28.5% from roots (Blacutt et al., 2020). Bacillus species are commonly found in plant-associated microbiomes, including soil, the rhizosphere, and the plant endosphere (Bokhari et al., 2019, Saxena et al., 2019; Lahlali et al. 2022). Bacillus spp. produce an array of secondary metabolites with antibacterial and antifungal properties (Miljakovic et al., 2020). Additionally, Bacillus species compete for niches and nutrients with pathogens and can induce systemic resistance in plants (Zhang et al., 2023). As a result, several commercially available products have emerged based on beneficial strains of Bacillus, such as Bacillus velezensis, B. amiloliquefaciens, B. subtilis, B. pumilus, B.cereus, B. megaterium and B. licheniformis (Mazzola et al., 2017; Rabbee et al., 2019; Miljakovic et al., 2020; Lahlali et al., 2022).

[0417] Through mining our citrus-associated microbial collection for isolates with anti- / .. crescens properties, using the surrogate species for CLas, Liberibacter crescens (Jain et al.,

[0418] 2019), we identified a Bacillus sp. particularly effective to inhibit Lc in bioactive fractions. Here we describe the identification and isolation of amicoumacins produced by Bacillus safensis CB729, an isolate from the citrus microbiome, and its ability to suppress in vitro and in vivo the HLB-associated pathogen, CLas.

[0419] Results

[0420] Bacillus safensis CB729 harbors the amicoumacin biosynthetic gene cluster.

[0421] We initiated the screening of citrus-associated microbial collection by assessing the in vitro antagonistic activity of various microbial isolates against L. crescens (Lc) (Blacutt et al.,

[0422] 2020). From a subset of 10 isolates that demonstrated inhibitory activity against Lc, isolate CB729 exhibited particularly strong inhibition (Figure 7), leading to its selection for further investigation. Whole genome sequencing confirmed isolate CB729 belongs to the B. safensis species, and its phylogenetic placement is illustrated in the species tree (Figure 8). The draft genome assembly of . safensis (GCA 036621555.1) is described in detail in Campos Vieira et al., (2024). In summary, the genome assembly resulted in 22 contigs, accounting for a total size of 3,677,118 bp, N50 of 871,191 bp and GC content of 41.50%. Genome annotation analysis identified 3,763 predicted genes and 3,654 protein-coding sequences (CDS) and taxonomy was confirmed with 97.4% of average nucleotide identity (ANI) (Table 2, Figure 44). Table 2. - Genome assembly statistics of Bacillus safensis (GenBank accession no: GCA_036621555.1).

[0423] Features Value

[0424] Genome topology Circular

[0425] Genome Size (bp) 3,677,118

[0426] N50 (bp) 871,191

[0427] Number of contigs 22

[0428] GC content (%) 41.50

[0429] Number of predicted genes 3,763

[0430] Protein coding sequences (CDS) 3,654

[0431] Predicted secondary metabolite biosynthetic gene cluster (BGC) analysis in antiSMASH revealed 11 BGC with annotations to known clusters in the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) database, including to the bacillibactin, fengycin, bottromycin, zwittermicin A, lichenysin, bacilysin and schizokinen clusters (Figure 11). The hybrid non-ribosomal peptide synthase (NRPS) polyketide synthase (PKS) domain (NRPS-PKS) was identified to be the most similar to the zwittermicin A (ZmA) cluster of B. cereus. Subsequently, gene sequence alignment of the NRPS-PKS cluster to the characterized amicoumacin cluster of Bs\. subtilis subsp. inaquosorum KCTC 13429 (Yi et al., 2014, Systematic and Applied Microbiology, 37(2):95-99; Li et al., 2020) was performed. The analysis revealed a moderate identity (46%-80%) and a strong similarity (86%-100%) between clusters (Figure 12A), corroborating findings by Terekhov et al. (2018) and Baranova et al. (2022). Figure 12B presents the proteins encoded by the NRPS-PKS cluster of B. safensis CB729, along with their proposed functions and their similarities to the characterized amicoumacin gene cluster in 7>. subtilis subsp. inaquosorum KCTC 13429. These findings strongly suggested that the amicoumacin gene cluster is present in CB729. Anti-Z. crescens fractions of B. safenesis CB729 contain amicoumacins.

[0432] Initial bioassay-guided fractionation of the crude extract of CB729 subjected to normal phase medium-pressure liquid chromatography (NP-MPLC) resulted in 9 fractions, 7 of which were inhibitory to Z. crescens (Figure 39). Liquid chromatography-mass spectrometry (LC-MS) analysis revealed a family of compounds with similar masses around m / z 400-500 and a distinctive UV chromophore in many of the fractions. Reversed-phase HPLC of fraction 6 gave four subfractions (6a-6d), one of which (fraction 6d) was identified as pure / f-acetyl- amicoumacin C based on an [M-H]' peak in the LCMS at m / z 447.1, and comparison of NMR data with those previously reported (Park et al., 2016, Figure 45, Figures 46-49). Although the isolated A-acetylamicoumacin C was inactive against Z. crescens, its structural relationship to known antibiotic amicoumacins led us to explore the extract further.

[0433] Molecular networking reveals amicoumacins in crude extracts of B. safensis CB729.

[0434] To explore the full metabolite profile of CB729 extract, we performed feature-based molecular networking analysis of the CB729 crude extract. Molecular networking is a tool that permits the rapid annotation of known metabolites, or of families of related metabolites, based on their fragmentation Mass Spectrometry (MS) spectra. In the obtained feature-based molecular network (FBMN), 20656 individual MS / MS spectra were organized into 655 nodes and 1174 edges (Figure 50). The network was then filtered to remove nodes corresponding to SYC media- produced metabolites and LC / MS system blank-produced metabolites (methanol only), thereby reducing the size of the network to 115 nodes and 245 edges. The inclusion of MS / MS data of synthetic amicoumacins A and B as standards in the FBMN analysis aided in the identification of an amicoumacin molecular family (Figure 40). The amicoumacin molecular family consisted of 35 nodes and 83 edges ranging from m / z 410.1896 to 522.2486. Based on the molecular formulas predicted from the precursor ion m / z values and MS / MS fragmentation pattern, 7 amicoumacin related compounds were identified, including amicoumacins A, B and D, N- acetylamicoumacin C, hetiamacin F and bacilosarcins A and B (Table 3). Table 3. Annotated compound information from FBMN analysis.

[0435] Compound name m / z Base peak Retention time Cluster index amicoumacin A 424.2082 250.1436 4.11 1764 amicoumacin B 425.1915 250.1434 4.22 2070 amicoumacin D 421.1990 250.1430 5.38 4941 hetiamacin F 448.2073 390.1538 4.24 2010

[0436] A-acetylamicoumacin C 449.1894 449.1917 5.53 5283 bacilosarcin A 492.2354 474.2235 4.49 2637 bacilosarcin B 494.2487 494.2499 4.25 1852

[0437] Scaled-up production and purification of amicoumacin A.

[0438] Following the detection of amicoumacins in the crude extract of CB729, the bacterial fermentation conditions were optimized using SYC medium (Terekhov et al., 2020) for isolation of amicoumacin A. SYC contains mainly sucrose, yeast extract and calcium carbonate and it has been associated with improvement of amicoumacin production, in combination with abundant aeration and shorter incubation period (Terekhov et al., 2020). The A-acetylation of amicoumacin A during fermentation has been described as a strategy employed by the producer bacteria to reduce its toxicity (Li et al., 2015; Park et al., 2016). To prevent this, we grew CB729 in 12 L of SYC in the presence of HP-20 solid-phase extraction resin to absorb amicoumacin A as it is produced and sequester it away from any A-acetylase enzymes produced by the bacteria. We subjected the crude extract to reversed-phase flash chromatography followed by RP-HPLC to give amicoumacin A, B and C (Figure 41). The structures were confirmed by LCMS and comparison of the 'H NMR spectrum with that reported in the literature (Park et al., 2016, Figures 51-55). Unfortunately, we observed rapid, nonenzymatic conversion of the amicoumacin A purified sample to a mixture of amicoumacins, composed primarily of amicoumacin C, in addition to amicoumacin B and other derivatives. This transformation could be observed after just a couple of hours in a prepared LCMS sample, and seemed to be accelerated by trace amounts of trifluoroacetic acid that had been added to the HPLC eluent in order to get baseline separation of the amicoumacins. Thus, due to the limited amount of isolated amicoumacin A, we decided to perform downstream bioassay analysis with synthetic commercially available amicoumacins A and B, included as standards in the LCMS analysis and molecular networking. MS2spectra of in-house purified amicoumacin A and synthetic amicoumacins A and B can be found in Supplementary Materials (Figures 56-57).

[0439] Minimal inhibitory concentrations of amicoumacins A and B against L. crescens.

[0440] Amicoumacin A has been reported to be highly effective against several Gram-positive and Gram-negative bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA) (Hashimoto et al., 2007, Berrue et al., 2009), various Bacillus strains and others (Pinchuk et al., 2001, Li et al., 2012). We examined the effect of commercially available amicoumacin A and amicoumacin B, against L. crescens, the surrogate bacteria for the HLB- associated pathogen to determine the lowest dose at which no bacteria growth is observed, known as the minimum inhibitory concentration (MIC). Amicoumacin A showed strong inhibition to L. crescens growth in vitro over 5 consecutive days, with a MIC of 1.25 pg / mL (Figure 42). This concentration was similar to the previously reported MIC (1.25 pg / mL) for the Gram-negative shrimp pathogens belonging to Vibrio spp. (Wang et al., 2020). Furthermore, amicoumacin B was less inhibitory to L. crescens than amicoumacin A. The MIC value for amicoumacin B was at 10 pg / mL, although there was moderate inhibition at 5 pg / mL.

[0441] Amicoumacin B and amicoumacin mixture are inhibitory to CLas.

[0442] To validate the effectiveness of amicoumacins A and B to CLas, we conducted a citrus CLas hairy root assay, using CLas-infected citrus roots in a rapid and efficient high throughput anti-CLas test (Irigoyen et al., 2020). We included the synthetic amicoumacins A and B and the amicoumacin mixture from B. safensis CB729 in different doses. We observed a significant (p < 0.05) reduction in CLas titer with amicoumacin B at 0.2 mg / ml, but not with amicoumacin A at the same concentration. We also observed a significant (p < 0.05) reduction in titer from AmiAP (amicoumacin mixture) at 0.1 mg / ml (Figure 43). Together, these results indicate the inhibitory effect of amicoumacins towards CLas.

[0443] Discussion

[0444] As part of a large-scale anti- / .. crescens bioprospecting effort from the citrus microbiome, we determined that crude extracts of cell-free supernatants collected from B. safensis CB729 were highly inhibitory to / .. crescens, the culturable surrogate for CLas (Blacutt et al., 2020). Predictive secondary metabolite analyses based on genome-wide comparisons of known biosynthetic gene clusters suggested that production of amicoumacins may be largely responsible for the inhibitory activity of CB729. Indeed, bioassay-guided fractionation of a crude extract of CB729 coupled with LC-MS led to the identification of amicoumacins and their derivatives within the fractions with antimicrobial activity against L. crescens. Further fractionation resulted in the isolation of / ' / -acetylamicoumacin C, whose structure was confirmed via NMR. We then used the structure of the isolated / ' / -acetylamicoumacin C coupled with molecular networking analysis to facilitate the identification of other amicoumacins A, B and other derivatives present in the B. safensis CB729 crude extract. Amicoumacins are a small group of natural products known for their broad antimicrobial properties against clinically- relevant human pathogens, such as Helicobacter pylori (Pinchuk et al., 2001) and methicillin- resistant Staphylococcus aureus (MRSA). Their mode of action is to inhibit protein synthesis by stabilizing the interaction between the 16S rRNA and mRNA (Polikanov et al. 2014, Molecular Cell, 56(4):531-540). Amicoumacins, including amicoumacin B, can also disrupt the quorumsensing system of Chromobacterium violaceum ATCC 12472 (Shi et al., 2021). In addition, they also have antiulcer and anticancer activities (Itoh et al., 1981, Prokhorova et al., 2016, Sci Rep 6, 27720). Bacillus is the main genus of bacteria to produce amicoumacins (Tyurin et al., 2018), but members of other genera, such as Nocardia, Xenorhabdus and Streptomyces can produce amicoumacins and their derivatives (McInerney et al., 1991; Sun et al., 2009; Rajan and Kannabiran, 2014). Amicoumacin A, is recognized as the most bioactive member of this group (Park et al., 2016; Tyurin et al., 2018; Wang et a., 2020).

[0445] Amicoumacin A-enriched fractions had significant inhibitory activity against L. crescens and CLas so we initially sought to generate sufficient quantities of isolated amicoumacin A by large scale fermentation of B. safensis CB729 to test against L. crescens and in downstream CLas inhibition assays using the citrus hairy root assay (Irigoyen et al., 2020). Using two rounds of RP-HPLC, we successfully isolated amicoumacins A, B, and C from spent B. safensis CB729 broth. However, during this process we observed the spontaneous cyclization of amicoumacin A into amicoumacin C or hydrolysis to amicoumacin B or other derivatives, which hindered our ability to test purified amicoumacin A isolated directly from the B. safensis CB729 strain in the downstream bioassays. The low stability of amicoumacin A and its conversion by cyclization or hydrolysis into less active derivatives, such as amicoumacin C and B has been reported in the bacterial systems B. subtilis and Staphylococcus aureus ((Itoh et al., 1981; Park et al., 2016; Terekhov et al., 2018). Specifically, amicoumacins B and C were inactive against S. aureus and B. subtilis, while amicoumacin A was highly active against these and other bacteria (Hashimoto et al., 2007, Li et al., 2015). While amides are usually quite stable to hydrolysis, one hydroxy group in the structure of amicoumacin A is poised for an intramolecular esterification reaction to form the y-lactone amicoumacin C, which in turn is susceptible to hydrolysis to amicoumacin B. Other examples in which an intramolecular-catalyzed hydrolysis mechanism accounts for hydrolysis of amides even in mild conditions include the hydrolysis of TV-acylated peptide derivatives (Samaritoni et al., 2014), and the classic Edman degradation method for sequencing peptides (Edman, 1956). This non-enzymatic transformation appeared to proceed more rapidly in the presence of trace amounts of trifluoroacetic acid that were added to the HPLC eluent to achieve baseline separation of the amicoumacins.

[0446] Because of the rapid conversion of amicoumacin A isolated from B. safensis CB729 , we tested commercially available synthetic amicoumacins A and B against L. crescens to obtain the MICs for these compounds against L. crescens. Amicoumacin A was more effective against L. crescens in the in vitro assay with an MIC of 1.25 pg / ml, whereas the MIC value for amicoumacin B was 10 pg / mL. Interestingly, in vivo, in the citrus hairy root assay we observed essentially the opposite where synthetic amicoumacin B was significantly inhibitory to CLas and synthetic amicoumacin A was not significantly inhibitory. We observed conversion of amicoumacin A isolated from B. safensis under mild acid conditions inherent to the HPLC preparations and we speculate that the stability of amicoumacin A may be particularly vulnerable to the pH of the plant environment that can oscillate from acid to alkaline conditions depending on developmental stage and spatial location within the plant (apoplast vs. cytosol). Interestingly, the amicoumacin mixture that contained amicoumacins A and B isolated from B. safensis CB729 was inhibitory to CLas in vivo. This suggests that amicoumacin B is more bioactive against CLas than amicoumacin A in the citrus hairy root assay.

[0447] B. safensis has been identified as a plant growth promoting bacterium and a biocontrol agent in other systems (Lateef et al., 2015; Romero-Severson et al., 2021; Chebotar et al., 2023; Altimira et al., 2024). Here we describe the anti- / .. crescens and anti-CLas activity of B. safensis CB729 mined from the HLB-impacted citrus microbiome. The next steps of this collective work are to evaluate amicoumacins for anti-CLas activity within citrus trees. Our overall goal is to determine how the citrus microbiome interfaces with the CLas pathogen and eventually to understand the impact of microbial community composition on HLB outcomes. In the long term, these findings will lay the foundation for the development of sustainable plant disease mitigation strategies for commercial citriculture.

[0448] Summary

[0449] For two decades, the citrus industry has been severely impacted by Huanglongbing (HLB), a devastating disease caused by Candidates Liberibacter asiaticus (CLas) and transmitted by the Asian citrus psyllid Diaphorina citri). Despite extensive research, effective, long-term, and sustainable solutions remain unavailable for growers. Currently, medically relevant antibiotics, such as oxytetracycline (OTC) are used as an emergency response to combat HLB in Florida, the most affected citrus-producing state in the U.S. This underscores the urgent need for alternative treatments that can be used in rotation or as replacements for OTC. Here, we present amicoumacins, a group of bioactive secondary metabolites with antibiotic properties. We identified amicoumacin B and its derivatives from the culture broth of a Bacillus safensis isolate native to citrus and demonstrated their ability to inhibit L. crescens and reduce CLas populations in citrus tissue. This study highlights how microbial discovery can lead to identification of antimicrobial compounds with potential applications in plant disease management.

[0450] Materials and methods

[0451] Bacterial isolates and growth conditions. Bacillus safensis CB729 was isolated from the leaf tissue of citrus trees in Florida, USA, under high HLB disease pressure. Sampling of plant material, bacterial isolation and genus-level identification were initiated as a component of a bioprospecting effort designed to generate a citrus-associated microbial repository, and is described in detail by Blacutt et al (2020). B. safensis CB729 was recovered from glycerol stocks and cultivated in tryptic soy agar (TSA) at 28°C for 3 days and propagated in liquid culture of tryptic soy broth (TSB), incubated at 28°C at 180 rpm.

[0452] Whole genome sequencing, assembly and annotation. The whole genome sequencing, assembly and annotation of Bacillus safensis CB729 is described in Campos Vieira et al., 2024. Briefly, DNA was extracted from a single colony grown overnight at 28°C in TSB, using Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, Wisconsin, USA), following the protocol for Gram-positive bacteria. Library preparation and sequencing were performed by SeqCenter (Pittsburgh, Pennsylvania, USA). Sequence analysis from quality control to annotation was performed on the KBase web service, and the publicly available narrative containing analyses and data can be found at doi.org / 10.25982 / 157793.280 / 2368552. Default parameters were used, except where noted. Quality of raw reads was assessed using FastQC (vO.12.1), and quality control was performed using JGI RQCFilter pipeline BBTools (v38.22) and PRINSEQ (v0.20.4). Genome assembly was performed on SPAdes (v3.15.3) (with k-mer sizes set for 21, 33, 55, 77, 91, and 111, and quality of assembly was assessed with QUAST (v4.4) and CheckM (vl.0.18). The genome was annotated using Prokka (vl.14.5) and taxonomic identification was performed on GTDB-Tk (v2.3.2). The phylogenetic tree was generated with Species Tree (v2.2.0) and annotated on iTOL (v6.8.1). Identification of biosynthetic gene clusters was performed with antiSMASH v7.0 (Blin et al., 2023). Gene cluster comparison analysis was performed on the clinker entry point of Cagecat (Van del belt et al., 2023).

[0453] Isolation of secondary metabolites of B. safensis CB729. B. safensis CB729 crude extract was initially produced by inoculating a single-colony of CB729 in A21 media (20 g / L D- glucose, 5 g / L yeast extract, Ig / L K2HPO4, 0.5 g / L MgSO4:7 H2O, 0.5 g / L KC1, 1.6 mg / L CuSO4, 1.2 mg / L Fe2(SO4)3, 0.4 mg / L MnSCU) (Hmidet et al., 2017). The culture was incubated at 28°C, 180 rpm for 72h. The fermentation broth was centrifuged at 10,000 g for 10 min and the supernatant was extracted three times by liquidliquid partitioning with 2 volumes of ethyl acetate. The organic layer was pooled and concentrated on a rotary evaporator (Rotavapor, R- 200, BUCHI, Flawil, Switzerland), yielding a crude extract of 164.2 mg. The crude extract was loaded into RediSep Rf Gold 12 g HP Silica Column (Teledyne, ISCO, Nebraska, USA) and subjected to normal phase high-performance liquid chromatography (NP-HPLC) on a CombiFlash EZ-Prep (Teledyne, ISCO, Nebraska, USA) with a gradient elution (20%-100% hexane:ethyl acetate, 10%-20% dichloromethane:methanol) over 35 min, flow rate: 22 mL / min. All fractions were collected and tested against L. crescens in an established inhibition bioassay.

[0454] High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) analysis.) Fraction 6 from flash chromatography was subjected to reverse-phase HPLC (Prominence-i LC-2030C liquid chromatograph equipped with a diode-array detector; Shimadzu Scientific Instruments) on a Luna C18(2) semi -preparative column (5pm X 10 mm X 250 mm) with gradient elution (45% to 60% acetonitrile: water over 8 minutes; ramped to 100% over 5 minutes) to give pure 7V-acetylamicoumacin C (0.8 mg, tR = 10.2 min). Liquid chromatographyelectrospray ionization mass spectrometry (LC-ESIMS) was performed on an HPLC system (Agilent, Model 1260 Infinity) that was equipped with a degasser, binary pump, autosampler, and diode array detector, coupled to a QToF device (Agilent, Model 6530 Accurate-Mass QToF) with an ESI source. ID ' H NMR and 2D (^H COSY,JH-13C HSQC, andJH-13C HMBC) NMR spectra were obtained using a JEOL ECS spectrometer (400 MHz for 'H and 100 MHz for13C) using CDCh from Cambridge Isotope Laboratories, Inc., and referenced to tetramethylsilane (TMS).

[0455] Molecular networking. A molecular network was created with the Feature-Based Molecular Networking (FBMN) workflow (Nothias et al., 2020) on GNPS2 (https: / / gnps2.org, Wang et al., 2016). The mass spectrometry data were first converted to open format files (mzML) using DataConnect, available from the Waters MicroApps Website. The files were then processed with MZMINE 3.9.0 (Schmid et al., 2023) and the results were exported to GNPS2 for FBMN analysis. The data was filtered by removing all MS / MS fragment ions within + / - 17 Da of the precursor m / z. MS / MS spectra were window-filtered by choosing only the top 6 fragment ions in the + / - 50 Da window throughout the spectrum. The precursor ion mass tolerance was set to 0.02 Da and the MS / MS fragment ion tolerance to 0.02 Da. A molecular network was then created in which edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each other's respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to 100, and the lowest-scoring edges were removed from molecular families until the molecular family size was below this threshold. The spectra in the network were then searched against GNPS spectral libraries (Wang et al., 2016, Horai et al., 2010). The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks. The DEREPLICATOR was used to annotate MS / MS spectra (Mohimani et al., 2018). The molecular networks were visualized using Cytoscape software v. 3.9.1 (Shannon et al., 2003).

[0456] Large-scale production and purification of amicoumacins. Experimentation with different growth conditions revealed high amicoumacin yield from B. safensis CB729 growth in SYC medium (containing 40 g / L sucrose, 5 g / L yeast extract, 4 g / L CaCCL, 1.5 g / L K2HPO4, 2 g / L glucose, 2 g / L NaCl, 1.5 g / L MgSO4, 2 g / L (NH4)2SO4, 0.01 g / L FeSO4, 0.01 g / L MnCh) (Terekhov et al., 2020) A single colony was grown overnight in TSB at 28°C and 1 mL of the overnight culture was used to inoculate each of 12 X IL of fresh SYC medium in 2.5 L Ultra Yield flasks (Thomson) containing Diaion HP -20 resin (20g / L) resin, and incubated at 28°C, 180 rpm for 48h. The resin was collected using cheesecloth and rinsed with deionized water. The resin was transferred to a beaker, covered with acetone and stirred for 1 hour, and the mixture filtered using cheesecloth to give an acetone extract. The resin beads were extracted a second time with acetone, the extracts combined, and the volume reduced on a rotary evaporator (Rotavapor, R-210, BUCHI, Flawil, Switzerland) to leave a concentrated residue. The residue (200 mL) was loaded onto a RediSep Rf C-18 SPE Cartridge (Teledyne). ISCO, Nebraska, USA), rinsed with deionized water, and subjected to flash column chromatography on a CombiFlash Rf+ LC system (Teledyne ISCO) using a RediSep Gold C18 Reversed Phase Column (30 g, Teledyne ISCO) with gradient elution (50% aqueous methanol up to 100% methanol, with isocratic pauses at each peak; total run time 32.6 min) at a flow rate of 35 mL / min. Fraction E (267 mg) eluted at 72% aqueous methanol and exhibited NMR signals consistent with the amicoumacins. This fraction was subjected to reverse-phase HPLC (Prominence-i LC-2030C liquid chromatograph equipped with a diode-array detector; Shimadzu Scientific Instruments) on a Luna C18(2) semi -preparative column (5pm X 10 mm X 250 mm) with isocratic elution (20% aqueous acetonitrile containing 0.01% trifluoroacetic acid) to give amicoumacins A (34.0 mg, tR = 7.4 min), B (6.2 mg, tR = 10.0 min), and C (1.2 mg, tR = 11.4 min).

[0457] Mass spectrometry analysis. LC-MS / MS analysis of CB729 crude extract and fractions was performed at the UC Riverside Metabolomics Core Facility as described previously (Rothman et al., 2019) with minor modifications. Briefly, analysis was performed on a Synapt G2-Si quadrupole time-of-flight mass spectrometer (Waters, Milford, MA, USA) coupled to an Lclass UPLC system (Waters). Separations were carried out on a CSH phenyl-hexyl column (2.1 x 100 mm, 1.7 pM) (Waters). The mobile phases were (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The flow rate was 250 pL / min and the column was held at 40°C. The injection volume was 1 pL. The gradient was as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 40% B; 24 min, 100% B; 26.5 min, 100% B; 27 min, 1% B; 30 min, 1% B. The MS scan range was (50 to 1200 or 1600 / z) with a 100 ms scan time. MS / MS was acquired in data dependent fashion. Source and desolvation temperatures were 150°C and 600°C, respectively. Desolvation gas was set to 600 L / hr and cone gas to 0 L / hr. All gases were nitrogen except the collision gas, which was argon. Capillary voltage was 1 kV in positive ion mode. Leucine enkephalin was infused and used for mass correction. Waters raw files (.raw) were converted to standard output format (mzML) via Waters microapp, DataConnect (https: / / microapps.on- demand . waters . com / ) .

[0458] Liberibacter crescens inhibition bioassay. B. safensis CB729 crude extract and steppurification fractions were tested against Liberibacter crescens in an agar diffusion inhibition bioassay (Blacutt et al., 2020). Briefly, crude extract or fractions were resuspended in 100% MeOH , 15 pl applied to sterile paper discs (Becton, Dickinson, Franklin Lakes, NJ) and allowed to dry in a biosafety cabinet. Liberibacter crescens was cultivated in mPM7 (2 g / L a- ketoglutarate, 10 g / L aces buffer, 3.75 g / L potassium hydroxide, 1 g / L methyl-P-cyclodextrin) liquid medium at 28°C, 150 rpm for up to 4 days. mPM7 top agar (0.8% agar) was prepared, cooled to 60°C, and amended with a 4-day L. crescens liquid culture at 10% of the top agar volume. The amended top agar was dispensed to evenly coat previously prepared mpM7 agar plates, after which sample-loaded filter discs were placed. Plates were incubated for 7 days at 28°C for the development of clear zones of inhibition. The diameter of the zones of inhibition was measured (cm) in triplicates for each treatment.

[0459] Minimum inhibitory concentration of amicoumacin A and B against Liberibacter crescens. The minimum inhibitory concentration was calculated for amicoumacin A and B in liquid bBM7 + 1.0 mPc as described previously in Brandenburg et al., 2020. In brief, amicoumacin A and B were individually dissolved in methanol and added to a 96-well plate. Methanol evaporated in a biosafety cabinet overnight. The following day, 150 pL of sterile bBM7 + 1.0 mPc was added to the wells, along with 50 pL of 4-day-old liquid culture (OD600 nm ~0.27) of L. crescens. The plate was incubated at 28 °C at 150 rpm. Absorbance was read at 600 nm using an Infinite 200 Pro plate reader (Tecan Group Ltd., Switzerland) daily for 5 days. The lowest dose at which no growth was observed was recorded as the MIC. Each treatment had six technical replicates.

[0460] Citrus CLas-hairy root assay. The CLas-hairy root assay was performed using HLB- confirmed sour orange citrus tissues (Citrus x aurantium L.). Briefly, citrus budwood tissues were transformed with Rhizobium rhizogenes to induce hairy roots (Irigoyen et al., 2020). The presence of CLas in the emerging hairy roots was verified by quantitative PCR (qPCR) using primers specific to the CLas ribonucleotide reductase P-subunit (nrdB) encoding gene (Zhang et al. 2016). Next, CLas-citrus hairy roots were collected, surface-sterilized with 70% ethanol and 1% bleach, and transferred into multi-well plates with Gamborg’s B-5 medium containing 1% sucrose. Synthetic amicoumacin A (AmiA) and amicoumacin B (AmiB) were tested at 0.2 mg / ml, in addition to amicoumacin mixture (AmiAP) from Bacillus safensis CB729 at 0.1 and 0.2 mg / ml. Treatments were vacuum infiltrated and incubated at 25°C in the dark for 72 hours and each treatment included five biological replicates. Oxytetracycline hydrochloride (OTC) treatment was used as a positive control alongside untreated or mock (DMSO) negative controls. After 72 hours, the hairy root tissue was treated with propidium monoazide (PMAxx, Biotium) dye to inactivate DNA from dead CLas. Total DNA was extracted, and viable bacterial titers were measured by qPCR using primers specific to the CLas (nrdB) (Zhang et al. 2016). Raw CLas Ct values were further normalized to an endogenous citrus housing gene, the glyceraldehyde-3 -phosphate dehydrogenase 2 (GAPC2) gene (Mafra et al., 2012; Irigoyen et al., 2020), and the relative levels were compared to untreated control.

[0461] Certain Documents

[0462] Itoh et al. 1982. Chemical Structures of Amicoumacins Produced by Bacillus pumilus. Agricultural and Biological Chemistry 46:2659-2665.

[0463] Samaritoni, et al. 2014. Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides. J Org Chem 79:3140-3151.

[0464] 2015 Meeting | Quorum sensing controls Candidatus Liberibacter asiaticus interactions with host plant and insect vector. www.apsnet.org / meetings / Documents / 2015_meeting_abstracts / aps2015abP204.htm#:~:text=Th e%20typical%20LuxR%20bacterial%20%22quorum%20sensing%22%20or,the%20absence%20 of%201uxI%20to%20produce%20AHL. Retrieved 10 January 2025.

[0465] Shi et al. 2021. Amicoumacins from a desert bacterium: quorum sensing inhibitor against Chromobacterium violaceum. Nat Prod Res 35:5508-5512.

[0466] Leelasuphakul, et al. 2008. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit. Postharvest Biology and Technology 48: 113-121. McInerney, et al. 1991. Biologically active metabolites from Xenorhabdus spp., Part 1. Dithiolopyrrolone derivatives with antibiotic activity. J Nat Prod 54:774-784.

[0467] Rajan BM, Kannabiran K. 2014. Extraction and Identification of Antibacterial Secondary Metabolites from Marine Streptomyces sp. VITBRK2. Int J Mol Cell Med 3: 130-137.

[0468] Sun, et al. 2009. Nocardia jinanensis sp. nov., an amicoumacin B-producing actinomycete. Int J Syst Evol Microbiol 59:417-420.

[0469] Prokhorova, et al. 2016. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome. Sci Rep 6:27720.

[0470] Itoh, et al., 1981. Amicoumacin-A, a new antibiotic with strong antiinflammatory and antiulcer activity. J Antibiot (Tokyo) 34:611-613.

[0471] Park, et al. 2016. Activating and Attenuating the Amicoumacin Antibiotics. Molecules 21 :824.

[0472] Tyurin, et al. 2018. Chapter 12 - Amicoumacins and Related Compounds: Chemistry and Biology, p. 385-441. / / / Atta-ur-Rahman (ed.), Studies in Natural Products Chemistry. Elsevier.

[0473] Pinchuk, et al. 2001. In Vitro Anti- Helicobacter pylori Activity of the Probiotic Strain Bacillus subtilis 3 Is Due to Secretion of Antibiotics. Antimicrob Agents Chemother 45:3156-3161.

[0474] Li, et al. 2012. Five New Amicoumacins Isolated from a Marine-Derived Bacterium Bacillus subtilis. Marine Drugs 10:319-328.

[0475] Berrue, et al. 2009. Newly isolated marine Bacillus pumilus (SP21): A source of novel lipoamides and other antimicrobial agents. Pure and Applied Chemistry 81 : 1027-1031.

[0476] Hashimoto, et al. 2007. Isolation of 8'-Phosphate Ester Derivatives of Amicoumacins: Structure-activity Relationship of Hydroxy Amino Acid Moiety. J Antibiot 60:752-756.

[0477] Terekhov, et al. 2018. Ultrahigh-throughput functional profiling of microbiota communities. Proc Natl Acad Sci USA 115:9551-9556.

[0478] Balouiri, et al. 2016. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal 6:71-79.

[0479] Wang, et al. 2020. Hetiamacin E and F, New Amicoumacin Antibiotics from Bacillus subtilis PJS Using MS / MS-Based Molecular Networking. Molecules 25:4446.

[0480] Yang, et al. 2021. Genome Mining, Heterologous Expression, Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost- Associated Bacillus subtilis fmb60. Molecules 26: 1892. Tsukaguchi, et al. 2019. Unified Total Synthesis of Hetiamacins A-D. Eur J Org Chem 2019:6110-6116.

[0481] Wang, et al. 2020. Mechanism of the Potential Therapeutic Candidate Bacillus subtilis BSXE-1601 Against Shrimp Pathogenic Vibrios and Multifunctional Metabolites Biosynthetic Capability of the Strain as Predicted by Genome Analysis. Front Microbiol 11 :581802.

[0482] Terekhov, et al. 2020. Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin. Antibiotics 9: 157.

[0483] Mohimani, et al. 2018. Dereplication of microbial metabolites through database search of mass spectra. Nat Commun 9:4035.

[0484] Horai, et al. 2010. MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom 45:703-714.

[0485] Wang, et al. 2016. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat Biotechnol 34:828-837.

[0486] Nothias, et al. 2020. Feature-based molecular networking in the GNPS analysis environment. Nat Methods 17:905-908.

[0487] Schmid, et al. 2023. Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nat Biotechnol 41 :447-449.

[0488] Shannon, et al. 2003. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res 13:2498-2504.

[0489] Example 7.

[0490] Using methods similar to those described in Examples 1 and 6, further inhibition studies were performed in vitro against L. crescens and in vivo against CLas using the hairy root assay. As shown in Figure 59, the positive control oxytetracycline (OTC) significantly reduced CLas titers compared to untreated (higher DCt and / or normalized CLas Ct). Crude extract from CB27 (5mg / mL) and CB893 (5mg / mL) also showed significant activity (p < 0.05). Figure 60 shows inhibition of L. crescens by Bacillus strains CB19, CB21, CB27, CB89, CB87, CB893, CB902, CB904, CB909, and CB912.

[0491] All publications, accession numbers and associated sequences (see, e.g., Example 5, Table 1, Figure 37 and Supplementary Table 1), patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. For example, the information and sequences associated with GenBank BioProject No. PRJNA1046128 are incorporated by reference herein in their entirety, for all purposes. Additionally, Campos Vieira et al., “Thirteen draft genome assemblies of Bacillus spp. isolated from HLB-impacted citrus trees”, Plant Microbiology, vol. 13, issue 10, pages 1-5 (2024) is incorporated by reference in its entirety for all purposes. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a Candidatus Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus and Bacillus subtilis, or an extract thereof.

2. The method of claim 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus and Bacillus subtilis, or an extract thereof.

3. The method of claim 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus pumilus, and Bacillus cereus, or an extract thereof.

4. The method of claim 1, comprising introducing to the plant at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis and Bacillus cereus, or an extract thereof.

5. The method of claim 1, comprising introducing Bacillus safensis, or an extract thereof, to the plant.

6. The method of claim 5, wherein the Bacillus safensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973669 or WGS Accession No. JAXKIG000000000.

7. The method of claim 5, wherein the Bacillus safensis is isolate strain Bacillus safensis CB729.

8. The method of claim 1, wherein the method does not comprise introducing to the plant Bacillus safensis, or an extract thereof.

9. The method of any one of claims 1 and 5-8, comprising introducing Bacillus velezensis, or an extract thereof, to the plant.

10. The method of claim 9, wherein the Bacillus velezensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973678, SRR26973677, SRR26973673, SRR26973670, SRR26973666, SRR26973676, SRR26973675, or SRR26973674.

11. The method of claim 9, wherein the Bacillus velezensis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIA000000000, JAXKIB000000000, JAXKIC000000000, JAXKIF000000000, JAXKIJ000000000, JAXKIK000000000, JAXKIL000000000, or JAXKIM000000000.

12. The method of claim 9, wherein the Bacillus velezensis is an isolate strain selected from the group consisting of CB19, CB21, CB27, CB687, CB902, CB904, CB909, and CB912.

13. The method of claim 12, wherein the Bacillus velezensis is isolate strain Bacillus velezensis CB909.

14. The method of any one of claims 1 and 5-13, comprising introducing Bacillus pumilus, or an extract thereof, to the plant.

15. The method of claim 14, wherein the Bacillus pumilus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973672 or JAXKID000000000.

16. The method of claim 14, wherein the Bacillus pumilus is isolate strain CB69.

17. The method of any one of claims 1 and 5-16, comprising introducing Bacillus cereus, or an extract thereof, to the plant.

18. The method of claim 17, wherein the Bacillus cereus genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973671 or JAXKIE000000000.

19. The method of claim 17, wherein the Bacillus cereus is isolate strain CB676.

20. The method of any one of claims 1 and 5-19, comprising introducing Bacillus subtilis, or an extract thereof, to the plant.

21. The method of claim 20, wherein the Bacillus subtilis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under SRA Accession No. SRR26973668 or SRR26973667.

22. The method of claim 20, wherein the Bacillus subtilis genome comprises a genomic sequence having at least about 99% sequence identity to the genome assembly sequences provided under WGS Accession No. JAXKIH000000000 or JAXKII000000000.

23. The method of claim 20, wherein the Bacillus subtilis is isolate strain CB742 or CB893.

24. The method of any one of claims 1 and 5-23, comprising introducing to the plant a combination of two or more types of microbial isolates selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof.

25. The method of claim 24, comprising introducing to the plant a combination of Bacillus safensis, or an extract thereof, and at least one microbial isolate(s) selected from the group consisting of Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or extract(s) thereof.

26. The method of any one of claims 1-25, wherein microbial extract(s) are introduced to the plant.

27. An isolated microbe as described in any one of claims 6-7, 10-13, 15-16, 18-19, or 20- 23.

28. The isolated microbe of claim 27, which is Bacillus safensis isolate strain Bacillus safensis CB729.

29. The isolated microbe of claim 27, which is Bacillus velezensis isolate strain Bacillus velezensis CB909.

30. A composition comprising at least one microbe as described in any one of claims 27-29, or an extract(s) thereof.

31. A kit comprising:1) at least one microbial isolate(s) selected from the group consisting of Bacillus safensis, Bacillus velezensis, Bacillus pumilus, Bacillus cereus, and Bacillus subtilis, or an extract(s) thereof;2) packaging material; and3) instructions to introduce to a plant the microbial isolate / extract to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant and / or to treat Huanglongbing (HLB) in the plant.

32. A method of treating a Candidatus Liberibacter asiaticus (CLas) infection in a plant and / or treating Huanglongbing (HLB) in a plant, comprising introducing to the plant at least one compound selected from the group consisting of an amicoumacin compound, bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or a salt thereof.

33. The method of claim 32, comprising administering two or more compounds selected from the group consisting of an amicoumacin compound, bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or salts thereof.

34. The method of claim 32 or 33, comprising introducing to the plant at least one amicoumacin compound, or a salt thereof.

35. The method of claim 34, wherein the amicoumacin compound, or salt thereof, is selected from the group consisting of amicoumacin A, amicoumacin B, amicoumacin C, N- acetylamicoumacin A, N-acetyl amicoumacin B, N-acetylamicoumacin C, O- methylamicoumacin B, and O-methylamicoumacin C, or a salt thereof.

36. The method of claim 35, wherein the at least one amicoumacin compound, or salt thereof, is O-methylamicoumacin B or O-methylamicoumacin C, or a salt thereof.

37. The method of claim 32, wherein the at least one compound is not an amicoumacin compound, or a salt thereof.

38. The method of any one of claims 32-37, comprising introducing to the plant at least one bacilosarcin compound, or a salt thereof.

39. The method of claim 38, wherein the least one bacilosarcin compound, or a salt thereof, is selected from the group consisting of bacilosarcin A, bacilosarcin B, and bacilosarcin C, or a salt thereof.

40. The method of any one of claims 32-39, comprising introducing to the plant at least one hetiamacin compound, or a salt thereof.

41. The method of claim 40, wherein the at least one hetiamacin compound, or a salt thereof, is selected from the group consisting of hetiamacin A and hetiamacin C, or a salt thereof.

42. The method of any one of claims 32-41, comprising introducing to the plant at least one cyclic lipopeptide compound, or a salt thereof.

43. The method of claim 42, wherein the at least one cyclic lipopeptide compound, or a salt thereof, is selected from the group consisting of a pumilacidin compound and a surfactin compound, or a salt thereof.

44. The method of claim 43, comprising introducing to the plant at least one pumilacidin compound, or a salt thereof.

45. The method of claim 44, wherein the at least one pumilacidin compound, or a salt thereof, is selected from the group consisting of pumilacidin C and pumilacidin E, or a salt thereof.

46. The method of any one of claims 43-45, comprising introducing to the plant at least one surfactin compound, or a salt thereof.

47. The method of claim 46, wherein the at least one surfactin compound, or a salt thereof, is selected from the group consisting of surfactin B, surfactin C, and surfactin C13, or a salt thereof.

48. The method of claim 33, wherein the two or more compounds comprise amicoumacin B, or a salt thereof, amicoumacin A, or a salt thereof, and a cyclic lipopeptide, or a salt thereof.

49. The method of any one of claims 32-48, wherein the at least one compound is present in a composition.

50. The method of claim 49, wherein the composition is a microbial extract, optionally, wherein the microbial extract is a Bacillus safensis extract.

51. The method of any one of claims 1 -26 and 32-50, which further treats a Phytophthora spp. and / or Fusarium spp. infection in the plant.

52. A kit comprising:1) at least one compound selected from the group consisting of an amicoumacin compound, bacilosarcin compound, a hetiamacin compound and a cyclic lipopeptide compound, or a salt thereof;2) packaging material; and3) instructions to introduce to a plant the compound to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant or to treat Huanglongbing (HLB) in the plant.

53. A composition comprising at least one amicoumacin compound, or a salt thereof, and at least one cyclic lipopeptide compound, or a salt thereof.

54. The composition of claim 53, wherein the at least one amicoumacin compound is amicoumacin B and / or amicoumacin A.

55. The composition of claim 53 or 54, which is a microbial extract.

56. The composition of claim 55, which is a Bacillus safensis extract.

57. A kit comprising:1) a composition as described in any one of claims 53-56;2) packaging material; and3) instructions to introduce to a plant the composition to treat a Candidatus Liberibacter asiaticus (CLas) infection in the plant or to treat Huanglongbing (HLB) in the plant.I l l

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