A bacillus complex bacterial agent for improving citrus iron deficiency chlorosis and application thereof

Abstract

The application discloses a kind of compound bacillus agent for improving citrus iron deficiency chlorosis and application thereof, the bacterium agent includes bacillus velezensis D006b and bacillus subtilis D004b;Bacillus velezensis D006b is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.32439, and the preservation date is October 31, 2024;Bacillus subtilis D004b is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.32438, and the preservation date is October 31, 2024.The compound bacterium agent of the application provides efficient and sustainable technical solutions for the microbial control of citrus iron deficiency chlorosis caused by alkaline soil through the iron supply function of two strains of iron carrier and the complementary cooperation of secondary metabolism function.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and in particular to a compound Bacillus agent for improving iron deficiency chlorosis in citrus and its application. Background Technology

[0002] Citrus is one of the most important economic fruit trees in southern my country, with the largest planting area and output in the world.

[0003] However, in the main citrus-producing areas of southern my country, the soils developed from limestone geology and purple sandstone and shale parent material have, through long-term rainwater leaching, released and accumulated calcium and magnesium in the topsoil, forming naturally alkaline soils (pH 7.5-8.5). In this alkaline environment, iron in the soil mainly exists as insoluble Fe³⁺ oxides or hydroxides, with extremely low bioavailability, leading to iron deficiency chlorosis (ICD) in citrus trees. Iron deficiency chlorosis severely affects citrus tree vigor, yield, and fruit quality, and has become a prominent problem restricting the healthy development of the citrus industry in alkaline soil areas of southern China.

[0004] Currently, domestic and international technologies for correcting iron deficiency chlorosis in citrus caused by alkaline soils focus on iron fertilizer improvement and physiological regulation. These include foliar spraying of ferrous sulfate, soil application of chelated iron fertilizers (such as EDDHA-Fe and EDTA-Fe), biochar, organic fertilizers, and acid regulators.

[0005] However, these methods have limitations such as short-lasting effects, the need for multiple applications, high costs, environmental pollution, or difficulty in precise control, making it difficult to meet the needs of the modern citrus industry for green, efficient, and sustainable development.

[0006] Biofortification technology has become a promising new field in recent years. Under iron deficiency stress, microorganisms synthesize and secrete substances that enhance iron production. 3+ It has extremely high affinity (affinity constant Kf can reach 10). 49 M -1 Small molecule iron carriers can chelate insoluble Fe in soil. 3+ Forming Fe that plants can directly absorb 3+ The iron carrier complex effectively improves iron nutrition in plants.

[0007] Bacillus spp. is one of the most widely studied and applied plant rhizosphere growth-promoting bacteria (PGPR). Bacillibactin is a catechol iron carrier produced by Bacillus, which can effectively chelate insoluble iron even in alkaline calcareous soils.

[0008] However, existing technologies lack functionally complementary compound Bacillus agents specifically designed for iron deficiency chlorosis in citrus grown in alkaline soils, which have been systematically screened and validated based on genomics and metabolomics.

[0009] Single strains often have limitations in terms of colonization ability, antibacterial spectrum, and diversity of metabolites, making it difficult to maintain stable effects in complex field environments.

[0010] Therefore, in order to solve the problems of the lack of long-lasting effect of existing chemical iron fertilizers and the limited function of single strains, a combination of Bacillus strains with complementary functions is provided, which has important theoretical and practical significance for the microbial control of iron deficiency chlorosis in citrus. Summary of the Invention

[0011] In view of this, the present invention provides a compound Bacillus agent for improving iron deficiency chlorosis in citrus and its application.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] A compound Bacillus agent for improving iron deficiency chlorosis in citrus, wherein the compound Bacillus agent comprises Bacillus belye D006b and Bacillus subtilis D004b; wherein,

[0014] The Bacillus belyssus D006b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32439 and deposit date of October 31, 2024.

[0015] The Bacillus subtilis D004b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32438 and deposit date of October 31, 2024.

[0016] Preferably, the ratio of viable bacteria of *Bacillus belyssus* D006b to *Bacillus subtilis* D004b is 1:2 to 2:1, and the total effective viable bacteria count is not less than 1×10⁻⁶. 8 CFU / mL.

[0017] Preferred,

[0018] The Bacillus belesi D006b has the following characteristics:

[0019] (1) The 16S rDNA sequence is shown in SEQ ID NO:1;

[0020] (2) The bacteria were identified as Bacillus velezensis by whole-genome sequencing and GTDB-Tk.

[0021] (3) Contains complete bacillibactin siderophore synthesis gene clusters, surfactin synthesis gene clusters, fengycin synthesis gene clusters, macrolactin H synthesis gene clusters, bacillaene synthesis gene clusters and difficidin synthesis gene clusters;

[0022] The Bacillus subtilis D004b has the following characteristics:

[0023] (1) The 16S rDNA sequence is shown in SEQ ID NO:2;

[0024] (2) The bacteria were identified as Bacillus subtilis by whole-genome sequencing and GTDB-Tk.

[0025] (3) Contains a complete bacillibactin siderophore synthesis gene cluster and at least 5 RiPP antimicrobial peptide gene clusters.

[0026] Preferably, the dosage form of the microbial agent is any one of liquid microbial agent, powder, granules or freeze-dried powder.

[0027] Preferably, the microbial agent further comprises 0.3%-1.0% w / v fermented vegetable waste water and 0.1%-0.5% w / v ferrous sulfate.

[0028] A compound Bacillus bio-organic fertilizer for improving iron deficiency chlorosis in citrus, comprising the compound Bacillus agent and an organic material carrier, the organic material carrier comprising the following components in parts by weight: 15-20 parts rapeseed cake, 40-50 parts wet citrus peel residue, 15-20 parts dry straw and 10-20 parts rice husk.

[0029] Preferably, the microbial bio-organic fertilizer is produced by composting and fermenting the compound Bacillus and the organic material carrier, wherein the total number of effective live bacteria is not less than 1×10⁻⁶. 8 CFU / g, organic matter content not less than 45%.

[0030] Application of compound Bacillus inoculant in improving iron deficiency chlorosis in citrus in alkaline soil.

[0031] Application of bio-organic fertilizer in improving iron deficiency chlorosis in citrus.

[0032] Preferably, the application includes at least one of the following:

[0033] Application methods include root drenching, drip irrigation, basal application, trench application, or hole application. For root drenching, apply 100-500 mL of a 50-300 times diluted bacterial solution per citrus seedling according to the formula described in claim 5. For mature trees, apply 5-20 L of a 50-300 times diluted bacterial solution around the drip line of the canopy. The bacterial solution concentration is 1×10⁻⁶. 8 CFU / mL; When applying as a base fertilizer or in a trench, apply 2-10 kg of the microbial organic fertilizer per citrus tree according to the formula described in claim 7.

[0034] The present invention achieves the following technical effects compared to the prior art:

[0035] (1) Significant advantages of functional combination: The combination of Bacillus subtilis D004b and Bacillus belyss D006b provided by this invention has a reasonable functional combination. Both strains were isolated from the rhizosphere of citrus orchards in alkaline soil. Both contain complete and 100% similar bacillibactin siderophore synthesis gene clusters. The siderophore functions are equivalent, which can provide double protection for crop iron supply. At the same time, the secondary metabolic functions of the two are clearly complementary: D006b has surfactin series lipopeptides as the dominant metabolites and has excellent rhizosphere colonization and biofilm formation capabilities. D004b has RiPP antimicrobial peptides as the dominant metabolites and can provide a richer antimicrobial spectrum and signaling molecule functions. The combined application of the two can produce a synergistic effect.

[0036] (2) Multiple growth-promoting and adaptability: Both strains have multiple growth-promoting functions, including detoxifying organic phosphorus, inorganic phosphorus, and potassium. While improving the iron nutrition of citrus, they can activate mineral nutrients such as phosphorus and potassium in the soil and comprehensively promote the growth and development of citrus. Moreover, the pH tolerance range of the two strains is 5-9, which can maintain stable activity in the alkaline soil of the south and adapt to the soil environment of the main citrus producing areas in southern my country.

[0037] (3) The iron nutrition improvement mechanism is clear and efficient: the iron nutrition of citrus is systematically improved through a multi-level mechanism of "side carrier chelation-phenolic acid reduction-polyamine regulation-rhizosphere colonization". Among them, D004b can produce isohydroxamic acid type side carrier candidate to chelate iron element, and at the same time generate phenolic acid reducing substances 4-hydroxycoumarin and polyamine spermine derivatives to participate in regulation; among them, D006b strain also produces catechol type side carrier bacillibactin, which also participates in the "side carrier chelation" link, and at the same time produces surfactantin lipopeptide to promote biofilm formation and rhizosphere colonization, and enhance the survival and continuous action of the strain in alkaline soil. The two together ensure the efficient absorption and utilization of iron.

[0038] (4) Excellent application effect and low industrialization cost: Both potted and field trials have verified the application effect of the present invention: the SPAD value of the composite treatment of autumn shoots reached 56.79, which was significantly better than that of each single treatment; in the field trial, the SPAD value of the composite microbial agent treatment of spring shoots increased by about 194% compared with the water control, the soluble solids increased from 10.8 to 13.6, and the solid-acid ratio increased from 13.99 to 19.81, which can significantly improve the symptoms of iron deficiency chlorosis in citrus, while improving fruit quality and promoting fruit development. It is applicable to a variety of citrus varieties that are prone to iron deficiency chlorosis, such as Jinqiu Sugar Orange, Newhall Navel Orange, Wenzhou Mandarin Orange, and Ponkan Orange; in addition, the preparation process of the microbial agent is simple, and agricultural waste such as citrus peel residue can be used as raw materials for the production of bio-organic fertilizer, which has a low production cost and is suitable for large-scale promotion and production. Attached Figure Description

[0039] Figure 1 This is a microscopic image of the colony morphology of strain D004b (Gram staining).

[0040] Figure 2 This is a microscopic image of the colony morphology of strain D006b (Gram staining).

[0041] Figure 3 This is a complete genome loop of strain D004b.

[0042] Figure 4 This is a complete genome loop of strain D006b.

[0043] Figure 5 Phylogenetic trees for strains D004b and D006b constructed based on their 16S rDNA sequences;

[0044] Wherein, a is a phylogenetic tree constructed based on the 16S rDNA sequence of strain D004b; b is a phylogenetic tree constructed based on the 16S rDNA sequence of strain D006b.

[0045] Figure 6 Comparative photographs of the spring shoot growth of citrus seedlings in different treatment groups in a pot experiment the following year;

[0046] Where a is CK1; b is CK2; c is D004b+D006b;

[0047] Figure 7 A bar chart comparing the SPAD values ​​of citrus leaves in different treatment groups during a field experiment;

[0048] Among them, a is the spring shoot SPAD (April) chart; b is the autumn shoot SPAD (October) chart;

[0049] Figure 8 A bar chart comparing the soluble solids-acid ratio of citrus fruits from different treatment groups in a field experiment. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] This invention discloses a compound Bacillus agent for improving iron deficiency chlorosis in citrus fruits. The compound Bacillus agent comprises Bacillus belyssus D006b and Bacillus subtilis D004b; wherein,

[0052] Bacillus belye D006b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32439 and deposit date of October 31, 2024.

[0053] Bacillus subtilis D004b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32438 and deposit date of October 31, 2024.

[0054] The ratio of viable counts of Bacillus vesiculosus D006b to Bacillus subtilis D004b was 1:2 to 2:1, and the total effective viable count was not less than 1×10⁻⁶. 8 CFU / mL.

[0055] Bacillus belyssus D006b has the following characteristics:

[0056] (1) The 16S rDNA sequence is shown in SEQ ID NO:1;

[0057] (2) The bacteria were identified as Bacillus velezensis by whole-genome sequencing and GTDB-Tk.

[0058] (3) Contains complete bacillibactin siderophore synthesis gene clusters, surfactin synthesis gene clusters, fengycin synthesis gene clusters, macrolactin H synthesis gene clusters, bacillaene synthesis gene clusters and difficidin synthesis gene clusters;

[0059] Bacillus subtilis D004b has the following characteristics:

[0060] (1) The 16S rDNA sequence is shown in SEQ ID NO:2;

[0061] (2) The bacteria were identified as Bacillus subtilis by whole-genome sequencing and GTDB-Tk.

[0062] (3) Contains a complete bacillibactin siderophore synthesis gene cluster and at least 5 RiPP antimicrobial peptide gene clusters.

[0063] The formulation of the microbial agent can be any one of liquid, powder, granules, or lyophilized powder.

[0064] The microbial agent also contains 0.3%-1.0% w / v fermented vegetable waste water and 0.1%-0.5% w / v ferrous sulfate.

[0065] The present invention also discloses a compound Bacillus bio-organic fertilizer for improving iron deficiency chlorosis in citrus, comprising compound Bacillus inoculant and an organic material carrier, the organic material carrier comprising the following components in parts by weight: 15-20 parts rapeseed cake, 40-50 parts wet citrus peel residue, 15-20 parts dry straw and 10-20 parts rice husk.

[0066] The microbial bio-organic fertilizer is made from the aforementioned compound Bacillus and the aforementioned organic material carrier through composting and fermentation, wherein the total number of effective live bacteria is not less than 1×10⁻⁶. 8 CFU / g, organic matter content not less than 45%.

[0067] This invention also discloses the application of compound Bacillus inoculant in improving iron deficiency chlorosis in citrus in alkaline soil.

[0068] This invention also discloses the application of bio-organic fertilizer in improving iron deficiency chlorosis in citrus.

[0069] Applications include at least one of the following methods:

[0070] Application methods include root drenching, drip irrigation, basal application, furrow application, or hole application. For root drenching, use a formula containing 0.3%-1.0% (w / v) fermented vegetable oil and 0.1%-0.5% (w / v) ferrous sulfate. Apply 100-500 mL of the diluted bacterial solution (50-300 times) per citrus seedling. For mature trees, apply 5-20 L of the diluted bacterial solution (50-300 times) around the drip line of the canopy. The bacterial solution concentration is 1×10⁻⁶. 8 CFU / mL; apply 2-10 kg of compound Bacillus bio-organic fertilizer per citrus tree when applying as a base fertilizer or in a trench.

[0071] Example 1: Isolation and identification of strains D004b and D006b

[0072] Rhizosphere soil samples were collected from healthy citrus plants in alkaline soil citrus orchards (soil pH 7.8-8.2) in Baojing County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. 10 g of rhizosphere soil was weighed and added to 90 mL of sterile physiological saline. The mixture was shaken at 30℃ and 180 rpm for 30 min to prepare a soil suspension. The suspension was then serially diluted 10-fold. -4 10 -5 10 -6 Each dilution (100 μL) was plated on NA medium plates and incubated at 30°C for 24–48 h. Single colonies with different morphological characteristics were picked and purified by streak plating multiple times, resulting in a total of 68 pure bacterial strains.

[0073] Siderogenic strains were screened using the CAS plate assay: purified strains were inoculated onto CAS plates and incubated at 30°C for 48-72 h. The diameter of the orange-yellow discoloration zone around the colony was observed and measured. Initial screening yielded 8 strains with distinct orange-yellow discoloration zones. Further screening resulted in two strains of Bacillus with the strongest siderogenic capacity and the highest ratio of discoloration zone diameter to colony diameter, designated D004b and D006b.

[0074] On NA medium, D004b colonies are flat or slightly raised, off-white to slightly yellow, with a rough, dry surface and obvious wrinkles. They do not produce mucus, and the edges are wavy or curly. Gram-positive, the bacteria are straight rods with blunt, rounded ends, often arranged in chains, and produce spores (elliptical, located in the center or at one end of the cell; the cell does not swell after spore formation), consistent with the typical morphological characteristics of Bacillus subtilis. 16S rDNA sequencing yielded a 1387 bp sequence (SEQ ID NO:2), and BLAST alignment showed greater than 99% homology with Bacillus subtilis.

[0075] D006b colonies on NA medium are raised, milky white, with a wrinkled and dry surface, do not produce mucus, and have irregular edges. They are Gram-positive, elongated oval in shape, and produce spores. 16S rDNA sequencing yielded a 1394 bp sequence (SEQ ID NO:1), and BLAST alignment showed greater than 99% homology with *Bacillus velezensis*.

[0076] Physiological and biochemical characteristics of the two strains: positive for VP test, negative for indole test, capable of hydrolyzing starch, casein and gelatin, capable of hydrolyzing tyrosine, tolerant of 10% NaCl (no growth in 15% NaCl), capable of growing at 50℃ (no growth at 60℃), capable of utilizing citrate, and capable of aerobic and facultative anaerobic growth.

[0077] Strain preservation information:

[0078] Bacillus velezensis D006b was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32439.

[0079] Bacillus subtilis D004b was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32438.

[0080] Example 2: Whole genome sequencing and analysis of strains D004b and D006b

[0081] High-quality genomic DNA was extracted from strains D004b and D006b, and paired-end 150 bp whole-genome sequencing was performed using the Illumina NovaSeq platform. After quality control filtering, the sequencing data were assembled using SPAdes, and genome integrity was assessed using CheckM.

[0082] The D004b whole genome assembly results showed a complete circular DNA molecule with a size of 4,207,695 bp and a GC content of 43.50%. GTDB-Tk v2.4.0 classification identified it as *Bacillus subtilis*, with an ANI of 99.98% (AF=1.0) compared to the reference genome GCF_000009045.1. A total of 4,299 ORFs were predicted, with a total coding region length of 3,701,910 bp, a coding density of 1.022 genes / kb, and a coding region proportion of 87.98%.

[0083] The D006b whole genome assembly results showed a complete circular DNA molecule with a size of 4,021,070 bp and a GC content of 46.43%. GTDB-Tk classification identified it as *Bacillus velezensis*, with an ANI of 97.68% (AF=0.921) compared to the reference genome GCF_001461825.1. A total of 3,935 ORFs were predicted, with a total coding region length of 3,568,911 bp, a coding density of 0.979 genes / kb, and a coding region proportion of 88.76%.

[0084] Example 3: Evaluation of fertility-promoting potential

[0085] A systematic evaluation of the growth-promoting potential of the two strains was conducted:

[0086] (1) Siderophore production capacity: CAS plate test showed that both strains had strong siderophore production capacity (++), and obvious orange-yellow discoloration rings were produced around the colonies.

[0087] (2) Phosphorus solubility: Both strains produced obvious clear zones (++) on both organic phosphorus and inorganic phosphorus (PKO) media, showing strong ability to solubilize organic and inorganic phosphorus.

[0088] (3) Potassium solubilization ability: Both strains showed potassium solubilization ability (++) on potassium bacteria culture medium.

[0089] (4) IAA production capacity: No IAA production was detected in either strain in LB medium containing L-tryptophan (-).

[0090] (5) pH tolerance: Both strains can grow normally in LB liquid medium with pH 5.0-9.0, showing a wide pH tolerance.

[0091] The above results indicate that, in addition to their core siderophore function, D004b and D006b also possess multiple growth-promoting functions such as phosphorus solubilization and potassium solubilization, and can maintain their activity over a wide range of soil pH.

[0092] Example 4: Analysis of antiSMASH secondary metabolite BGC

[0093] BGC analysis of secondary metabolites of D004b and D006b was performed on the whole genome using antiSMASH v7.1.0.

[0094] A total of 14 BGCs were detected in the D004b genome (see Table 1). Among them, the bacillibactin siderophore gene cluster (Region 9, NRP-metallophore type, 3,253,392-3,305,202 bp, 100% similarity) is a complete siderophore synthesis gene cluster. In addition, D004b also contains 5 RiPP-like antimicrobial peptide gene clusters: sporulation killing factor (Region 1, 100%), sublancin 168 (Region 7, 100%), subtilosin A (Region 11, 100%), RRE-containing (Region 13), and epipeptide (Region 14, 10% similarity to thailanstatin A).

[0095] Table 1: BGC analysis of secondary metabolites from the whole genome of strain D004b

[0096] A total of 13 BGCs were detected in the D006b genome (see Table 2). Among them, the bacillibactin siderophore gene cluster (Region 12, NRP-metallophore type, 3,098,485-3,150,275 bp, 100% similarity) is also a complete siderophore synthesis gene cluster. D006b is rich in lipopeptide and polyketide BGCs: surfactantin (Region 1, 82%), fengycin (Region 8, 100%), macrolactin H (Region 6, 100%), bacillaene (Region 7, 100%), difficidin (Region 11, 100%), and bacilysin (Region 13, 100%).

[0097] Table 2: BGC analysis of secondary metabolites from strain D006b across the entire genome.

[0098]

[0099] The analysis results showed that the two strains were equivalent in bacillibactin siderophore function (both were 100%), while they were clearly complementary in other secondary metabolic functions: D006b was dominated by lipopeptides and polyketides, while D004b was dominated by RiPP antimicrobial peptides.

[0100] Example 5: LC-MS analysis of metabolites in fermentation broth

[0101] D004b and D006b were inoculated into NB liquid medium, respectively. D004b was incubated at 37℃ and D006b at 30℃ with shaking at 180 rpm for 48 h. The fermentation broth was filtered through a 0.22 μm sterile filter membrane to obtain 50 mL of sterile fermentation filtrate, which was then frozen at -80℃ and vacuum dried. After being dissolved in 2 mL of sterile water, it was analyzed by LC-MS. Chromatographic conditions: The column was a Kromasil ODS reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A) and water (B), with a gradient elution program: 0–20 min, 10% A → 80% A; 20.01–30 min, 80% A → 100% A; 30.01–40 min, 100% A; flow rate 1.0 mL / min; column temperature 25℃; detection wavelength 254 nm. Molecular weight was determined by high-resolution mass spectrometry (HRMS).

[0102] Analysis of the D006b fermentation broth revealed that the surfactin homologues (C14-C18, MW 1007-1067 Da) were the dominant components, with a total abundance of approximately 25% Vol. The specific abundance distribution was as follows: C14-surfactin (MW 1007.65 Da, RT 32.801 min) 8.22% Vol; C15-surfactin (MW 1021.66 Da, RT 34.502 min) 6.79% Vol; C16-surfactin (MW 1035.68 Da, RT 35.263 min) 5.29% Vol; and other surfactin homologues (C17-C18 series and isomers) totaled approximately 4.7% Vol. The 14 Da stepwise mass difference (difference in the -CH2- fatty acid chain) of the surfactin series is a typical mass spectrometric feature of cyclic lipopeptides. Other major components, due to the complexity of some compound signals, are characterized by high-resolution mass numbers as follows: MW 1044.54 Da (RT 17.501 min, 4.29% Vol), C25H46N8O11 (MW 634.33 Da, RT 5.778 min, 3.12% Vol), MW 1030.53 Da (RT 16.438 min, 2.70% Vol), C48H75N21O3 (MW 993.64 Da, RT 31.489 min, 2.08% Vol), MW 1021.67 Da (RT 34.198 min, 1.93% Vol), C27H46N8O11 (MW 658.33 Da, RT 7.438 min, 1.79% Vol), etc.

[0103] Analysis results of the D004b fermentation broth: The main components were peptides and aromatic small molecule metabolites. The main components and their abundance are shown in Table 3.

[0104] Table 3: LC-MS analysis results of major components in the fermentation broth of strain D004b (relative abundance ≥0.5%)

[0105] No large molecular weight lipopeptides (MW>1000 Da) such as surfactantin or fengycin were detected in the fermentation broth of D004b. Its metabolite profile was characterized by small molecular weight peptides of the RiPP class, which was consistent with the results of the five RiPP gene clusters in the genomic analysis.

[0106] Based on the LC-MS analysis results of the D004b fermentation broth, the mechanism by which its metabolites improve iron deficiency chlorosis in fruit trees can be summarized in three points:

[0107] First, the siderophore components directly enhance the bioavailability of iron. C31H51N13O8 and C29H51N13O10 possess typical isohydroxamic acid-type siderophore structures, which can efficiently chelate ferric iron in alkaline soils to form soluble complexes. C9H8O3 (4-hydroxycoumarin) reduces Fe... 3+ For Fe 2+ Enhance siderophore activity.

[0108] Secondly, the metabolic components of the strain activate the iron absorption regulation system of fruit trees. Spermine derivative C12H31N7 and tryptophan amide C11H14N4O2 can induce the expression of the iron transport protein gene IRT1 in the roots of fruit trees and enhance the activity of iron chelate reductase FRO2, ensuring efficient iron absorption in alkaline soil environments.

[0109] Thirdly, there is a synergistic effect mechanism of rhizosphere microecology. The siderophore component C32H56N14O11 can regulate the rhizosphere microbial community, enrich siderophore-producing bacteria, promote soil organic carbon accumulation, optimize carbon metabolism pathways to support siderophore regeneration and cycling, and indirectly enhance the iron nutrient supply of fruit trees by continuously releasing bioavailable iron.

[0110] Example 6: Pot experiment to verify the effect of the strain on improving iron deficiency chlorosis in citrus.

[0111] Pot experiments were conducted in a greenhouse. The tested citrus seedlings were one-year-old Jinqiu sugar mandarin orange seedlings grafted in March 2024, and the potting substrate was an alkaline substrate (pH 8.0). Five treatments were set up: CK1 (water control), CK2 (commercially available Bacillus subtilis control), D004b treatment (D004b bacterial solution, 100 mL per plant for root irrigation), D006b treatment (D006b bacterial solution, 100 mL per plant for root irrigation), and D004b+D006b treatment (equal volumes of the two bacteria mixed, 100 mL per plant for root irrigation). Root irrigation was performed three times, on July 30th, August 30th, and September 20th. Each treatment was replicated 10 times.

[0112] The SPAD values ​​of leaves were measured using SPAD-502Plus in October 2025 (autumn shoot stage) and April 2026 (spring shoot stage). The results showed:

[0113] SPAD values ​​of spring shoots (April 2026): CK1 group: 5.99±0.88 e; CK2 group: 15.47±0.32 d; D004b group: 22.65±0.25 c; D006b group: 23.43±0.71 b; D004b+D006b group: 25.44±0.15 a. The D004b+D006b treatment was the best, and all three inoculant treatments were significantly higher than the control (P<0.05).

[0114] SPAD values ​​of autumn shoots (October 2025): CK1 7.43±1.67 e (note), CK2 32.51±1.46 d, D004b 52.20±1.97 b, D006b 50.58±1.03 c, D004b+D006b 56.79±0.28 a. The D004b+D006b combined treatment was significantly higher than that of each single-strain treatment (P<0.05).

[0115] In April 2026, observations of the following spring shoots revealed that the shoots treated with added microbial agents were noticeably green, while the shoots in the untreated group showed yellowing. This indicates that the addition of microbial agents can reshape the rhizosphere microbiome and continuously improve alkaline soil.

[0116] Example 7: Field trial to verify the practical application effect of compound microbial agent

[0117] A field trial was conducted in a citrus orchard in Bier Town, Baojing County, Xiangxi Prefecture, Hunan Province. The tested citrus variety was Newhall navel orange. Three treatments were set up: CK1 (water control), CK2 (organic fertilizer control), and D004b+D006b treatment (routine management + D004b+D006b compound microbial agent + 0.5% fermented vegetable waste water + 0.2% ferrous sulfate + organic fertilizer). It should be noted that the pot experiment has already verified that the compound microbial agent is more effective than single-strain application; this field trial focuses on verifying the overall effect of the compound microbial agent in the field.

[0118] SPAD values ​​of spring shoots (April): CK1 7.76±1.35 e, CK2 15.81±0.37 d, D004b+D006b 22.78±0.24 c, the treatment group was about 194% higher than the water control.

[0119] SPAD values ​​of autumn shoots (October): CK1 46.75±9.02 c, CK2 46.02±1.28 c, D004b+D006b 52.71±1.75 b.

[0120] SPAD values ​​of old leaves (October): CK1 36.15±3.28 d, CK2 45.99±2.13 c, D004b+D006b 61.11±4.23 a.

[0121] Fruit soluble solids: CK1 10.8±0.3c, CK2 12.1±0.6b, D004b+D006b 13.6±0.4a.

[0122] Fruit glutamate-acid ratio: CK1 13.99±0.34c, CK2 17.99±0.26b, D004b+D006b 19.81±0.35a.

[0123] Note: The SPAD value of autumn shoots in field CK2 (organic fertilizer) (46.02) was close to that of CK1 (46.75), but the difference in spring shoots was significant (15.81 vs 7.76). This indicates that fermented vegetable compost can provide some nutritional improvement during the rapid growth period of spring shoots, but the effect is not lasting and fades by the autumn shoot stage. This is consistent with the characteristic of fermented vegetable compost, which mainly provides organic matter and basic nutrients but lacks sustained iron activation capacity.

[0124] Example 8: Preparation of compound Bacillus liquid inoculant

[0125] (1) Inoculate Bacillus belye D006b into LB liquid medium and incubate at 30℃ and 180 rpm for 24-36 h until the viable count is ≥1×10⁻⁶. 9 CFU / mL was used to obtain the fermentation broth for D006b.

[0126] (2) Inoculate Bacillus subtilis D004b into LB liquid medium and incubate at 37℃ and 180 rpm for 24-36 h until the viable count is ≥1×10⁻⁶. 9 CFU / mL was used to obtain the fermentation broth for D004b.

[0127] (3) Mix the fermentation broth of D006b and D004b at a volume ratio of 1:1, and adjust the total number of viable bacteria to ≥1×10⁻⁶ with sterile water or phosphate buffer (pH 7.0). 8 The CFU / mL concentration yields a compound Bacillus liquid inoculum. 0.5-2% (w / v) of glycerol or trehalose can also be added as a preservative, if needed.

[0128] Example 9: Preparation of Compound Bacillus Powder

[0129] (1) Prepare D006b fermentation broth and D004b fermentation broth according to the method in Example 8.

[0130] (2) After mixing the two fermentation liquids in a volume ratio of 1:1, add 5-10% of the total weight of the fermentation liquid as carrier material (the carrier material is selected from one or more of light calcium carbonate, diatomaceous earth, maltodextrin or corn starch) and stir evenly.

[0131] (3) The powder is dried using spray drying (inlet air temperature 160-180℃, outlet air temperature 70-85℃) or freeze drying (pre-freezing at -50℃ for 4 h, followed by vacuum drying for 24 h), and then pulverized through an 80-100 mesh sieve to obtain the compound Bacillus powder. The total number of viable bacteria in the obtained powder is ≥1×10⁻⁶. 10 CFU / g.

[0132] Example 10: Preparation of a compound microbial agent containing fermented rapeseed water and ferrous sulfate

[0133] The compound Bacillus liquid bacterial agent prepared in Example 8 (total effective viable bacteria count ≥1×10⁻⁶) 8 The bacterial agent (CFU / mL) is mixed with fermented rapeseed meal water and ferrous sulfate in a certain proportion to make the final solution contain 80-95% (v / v) of the original bacterial agent, 0.3%-1.0% (w / v) of the fermented rapeseed meal water, and 0.1%-0.5% (w / v) of ferrous sulfate; the preferred ratio is 0.5% (w / v) of fermented rapeseed meal water and 0.2% (w / v) of ferrous sulfate. The mixture is thoroughly mixed to obtain the final product. The fermented rapeseed meal water is a water-soluble extract of rapeseed meal after natural fermentation, rich in organic acids and microbial metabolites, which can synergistically enhance the soil iron activation effect with the bacterial agent. This formula has the dual function of providing iron from microbial iron carriers and supplementing inorganic iron sources, and is suitable for rapid correction and long-term improvement of iron deficiency chlorosis in citrus under alkaline soil conditions.

[0134] Example 11: Preparation of Compound Bacillus Bio-organic Fertilizer

[0135] (1) Preparation of organic materials: By weight, take 18 parts of rapeseed cake, 45 parts of wet citrus peel residue (moisture content about 80%), 18 parts of dry straw (crushed to 1-3 cm), and 19 parts of rice husk, mix them evenly, adjust the carbon-nitrogen ratio to about 28:1, and add water to adjust the moisture content to 55-65%.

[0136] (2) Inoculation with bacterial agent: Prepare compound Bacillus liquid bacterial agent (total effective viable bacteria count ≥ 1 × 10⁻⁶) according to the method in Example 8. 8 (CFU / mL), inoculate 5-10% of the total weight of organic materials into the material in step (1), and mix thoroughly.

[0137] (3) Composting fermentation: After inoculation, the material is piled into windrows 1.0-1.5 m high, covered with plastic film or straw mats to retain moisture, and turned over every 3-5 days. The pile temperature is controlled at 50-65℃, and fermentation is carried out for 25-30 days until the material is fully decomposed (dark brown in color, loose in texture, odorless, and with an earthy aroma), thus obtaining compound Bacillus bio-organic fertilizer. The total number of effective live bacteria in the finished product is ≥1×10⁻⁶. 8 CFU / g, organic matter content ≥45%.

[0138] Example 12: Field application method of compound Bacillus inoculant on citrus

[0139] Apply compound Bacillus inoculant before the spring shoots of citrus (late February to early March) and before the autumn shoots (late July to early August).

[0140] Application method: Prepare a compound Bacillus liquid inoculant containing fermented vegetable waste water and ferrous sulfate according to Example 10 (total effective viable bacteria count ≥ 1 × 10⁻⁶).8 After diluting the bacterial solution (CFU / mL) 50-300 times, apply 5-20 L of the diluted solution to each citrus tree along the drip line of the canopy, which is equivalent to applying 50-200 mL of the original bacterial solution per tree.

[0141] Application method of bio-organic fertilizer: Apply 3-8 kg of compound Bacillus bio-organic fertilizer (adjust according to tree age and vigor) to each citrus tree by basal application or trench application. Apply the fertilizer in a trench inside the drip line of the tree canopy to a depth of 20-30 cm. Cover with soil and water promptly after application.

[0142] After application, observe the changes in leaf color of citrus leaves regularly, measure the SPAD value of leaves during the spring and autumn shoot stages, and measure yield and quality indicators during fruit ripening. The citrus varieties mentioned include, but are not limited to, Jinqiu Sugar Orange, Newhall Navel Orange, Wenzhou Mandarin Orange, Ponkan, and other citrus varieties that are prone to iron deficiency chlorosis.

[0143] The compound Bacillus inoculant provided by this invention uses Bacillus subtilis D004b and Bacillus belyss D006b as its core active ingredients. Both strains contain complete bacillibactin siderophore synthesis gene clusters, which can efficiently chelate insoluble iron in alkaline soils, while also possessing multiple growth-promoting functions such as phosphorus and potassium solubilization. Both pot experiments and field trials have confirmed the significant improvement effect of this invention on iron deficiency chlorosis in citrus, as well as its significant increase in soluble solids and the acid-to-solids ratio of the fruit. The preparation process of this inoculant is simple, and the bio-organic fertilizer uses agricultural waste such as citrus peel residue as raw materials, resulting in low production costs and suitability for large-scale production. This invention can be widely applied in citrus-producing areas with alkaline soils in southern China, providing an efficient, inexpensive, and sustainable microbial solution for the green prevention and control of iron deficiency chlorosis in citrus, and has good industrial applicability.

[0144] in,

[0145] SEQ ID NO:1:

[0146]

[0147] SEQ ID NO:2:

[0148]

[0149] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A compound Bacillus agent for improving iron deficiency chlorosis in citrus, characterized in that, The compound Bacillus agent contains Bacillus belyss D006b and Bacillus subtilis D004b; wherein... The Bacillus belyssus D006b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32439 and deposit date of October 31, 2024. The Bacillus subtilis D004b is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32438 and deposit date of October 31, 2024.

2. The compound Bacillus agent for improving iron deficiency chlorosis in citrus according to claim 1, characterized in that, The ratio of viable bacteria of *Bacillus belyssus* D006b to *Bacillus subtilis* D004b is 1:2 to 2:1, and the total effective viable bacteria count is not less than 1×10⁻⁶. 8 CFU / mL.

3. The compound Bacillus agent for improving iron deficiency chlorosis in citrus according to claim 1, characterized in that, The Bacillus belesi D006b has the following characteristics: (1) The 16S rDNA sequence is shown in SEQ ID NO:1; (2) The bacteria were identified as Bacillus velezensis by whole-genome sequencing and GTDB-Tk. (3) Contains complete bacillibactin siderophore synthesis gene clusters, surfactin synthesis gene clusters, fengycin synthesis gene clusters, macrolactin H synthesis gene clusters, bacillaene synthesis gene clusters and difficidin synthesis gene clusters; The Bacillus subtilis D004b has the following characteristics: (1) The 16S rDNA sequence is shown in SEQ ID NO:2; (2) The bacteria were identified as Bacillus subtilis by whole-genome sequencing and GTDB-Tk. (3) Contains a complete bacillibactin siderophore synthesis gene cluster and at least 5 RiPP antimicrobial peptide gene clusters.

4. The compound Bacillus agent for improving iron deficiency chlorosis in citrus according to claim 1, characterized in that, The formulation of the microbial agent is any one of liquid microbial agent, powder, granules or lyophilized powder.

5. The compound Bacillus agent for improving iron deficiency chlorosis in citrus according to claim 1, characterized in that, The microbial agent also contains 0.3%-1.0% w / v fermented vegetable waste water and 0.1%-0.5% w / v ferrous sulfate.

6. A compound Bacillus bio-organic fertilizer for improving iron deficiency chlorosis in citrus, characterized in that, The compound Bacillus agent comprising any one of claims 1 to 5, and an organic material carrier comprising the following components in parts by weight: 15-20 parts rapeseed meal, 40-50 parts wet citrus peel residue, 15-20 parts dry straw, and 10-20 parts rice husk.

7. The compound Bacillus bio-organic fertilizer for improving iron deficiency chlorosis in citrus according to claim 6, characterized in that, The microbial bio-organic fertilizer is produced by composting and fermenting the compound Bacillus and the organic material carrier, wherein the total number of effective live bacteria is not less than 1×10⁻⁶. 8 CFU / g, organic matter content not less than 45%.

8. The application of the compound Bacillus agent according to any one of claims 1 to 5 in improving iron deficiency chlorosis in citrus in alkaline soil.

9. The application of the bio-organic fertilizer according to any one of claims 6 or 7 in improving iron deficiency chlorosis in citrus.

10. The application according to claim 8 or 9, characterized in that, The application includes at least one of the following methods: Application methods include root drenching, drip irrigation, basal application, trench application, or hole application. For root drenching, apply 100-500 mL of a 50-300 times diluted bacterial solution per citrus seedling according to the formula described in claim 5. For mature trees, apply 5-20 L of a 50-300 times diluted bacterial solution around the drip line of the canopy. The bacterial solution concentration is 1×10⁻⁶. 8 CFU / mL; When applying as a base fertilizer or in a trench, apply 2-10 kg of the microbial organic fertilizer per citrus tree according to the formula described in claim 7.

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