Use of sphingomonas yabuuchiae in the biological control of plant pathogens, and a novel strain of sphingomonas yabuuchiae

Abstract

The invention relates to a method for the treatment, prevention or containment of Fusarium fujikuroi infections in a plant, wherein the method comprises the application of live bacteria of the species Sphingomonas yabuuchiae to the seeds of the plant. The invention also relates to Sphingomonas yabuuchiae RS-ARS2 strain deposited at the Coleccion Espanola de Cultivos Tipo (CECT) under accession number CECT 31159 on 11 October 2024.

Description

[0001] Use of Sphingomonas yabuuchiae in the biological control of plant pathogens, and a novel strain of Sphingomonas yabuuchiae

[0002] Field of the invention

[0003] The present invention falls within the field of biological control methods against plant pathogens.

[0004] Background art

[0005] Plant diseases are one of the major challenges facing the agricultural sector worldwide. These are biotic stress factors caused mainly by fungi, oomycetes, bacteria, phytoplasma and viruses. From an epidemiological point of view, plant pathogens can reach the host using different methods of transmission, both in the field and post-harvest. The main types of transmission in the field are vertical transmission (i.e., transmission by seed or propagative material), through soil circulating solutions, and through the air.

[0006] Synthetic plant protection products have been the main method of containing plant diseases since the last century, as a result of the Green Revolution, leading to a significant increase in yields to meet the needs of an ever-growing world population. However, to date, due to a more careful risk assessment, many synthetic active ingredients in plant protection products are proving to be dangerous for the environment, since they are extremely persistent and capable of causing biomagnification phenomena in the food chain and the onset of resistance phenomena in pathogens. There are also risks for the human population and natural ecosystems, caused by the toxic effect that these synthetic active ingredients may have on off-target organisms.

[0007] The use of synthetic plant protection products, especially in the European Union, is therefore being drastically reduced, in order to promote environmental protection and consumer safety, under strong pressure from both legislation (Regulation (EC) No 1107 / 2009 and Directive 2009 / 128 / EC) and policy (New Green Deal and Farm-to-Fork Strategy).However, the reduction in the use of plant protection products must be compensated for by alternative plant protection technologies in order to contain production losses. These alternative technologies include biological control through the use of microorganisms that antagonize plant pathogens.

[0008] Rice (Oryza sativa L.) is one of the most important crops as it is the main staple food for half of the world's population. In Europe, Italy is the largest rice producer, with 218,420 ha of dedicated area and 1,236,960 tons of production in 2023. One of the main threats to rice production in Europe is the fungus Fusarium fujikuroi Nirenberg (teleomorph Gibberella fujikuroi (Sawada) Ito in Ito & K. Kimura), the causal agent of rice Fusarium wilt. Typical symptoms of this disease include chlorosis and abnormal internode elongation, due to overproduction of gibberellic acid, both fungal and plant-derived, during the biotrophic phase of infection. In later phases, this hemibiotrophic pathogen can cause crown rot or panicle sterility. Fusarium wilt is a monocyclic disease primarily transmitted by seed, using this organ for dispersal among fields, while sources of secondary inoculum are infected or dead plants. The white-pink mycelium of F. fujikuroi abundantly colonizes the crown during its necrotrophic phase, especially in the flowering phase, allowing the conidia to disperse within the field through water and wind to infect the flowers of new hosts and be transmitted vertically to the seeds. Yield losses due to Fusarium wilt are limited to the European Union area. However, by Implementing Directive 2012 / 1 / EU, the European Commission has set restrictive thresholds for plants infected with F. fujikuroi in the fields of rice seed producers, with the aim of limiting the spread of the disease. Although the most reliable conventional solution to prevent the onset of Fusarium wilt is chemical seed treatment, the risks to the environment and human health arising from the use of synthetic plant protection products have led the European Parliament to adopt measures aimed at prohibiting or restricting many active ingredients previously authorised under Regulation (EC) 1007 / 2009. Furthermore, the most recent Farm to Fork strategy, within the scope of the European Green deal, has set two key objectives for plant protection products, aiming to reduce the use and risk of synthetic plant protection products by 50%, as well as to reduce the use of the most hazardous plant protection products, referred to as "replacement candidates", by 50%. In this context, alternative solutions that safely and effectively reduce the presence and spread of F. fujikuroi in the field are especially necessary for seed producers in order to comply with the limitslaid down in European legislation.

[0009] In order to achieve sustainable management of Fusarium wilt, breeding strategies have been proposed to obtain tolerant rice cultivars (Shakeel et al., 2023). However, only a limited number of resistant genotypes are currently available (Valente et al., 2017) and the selection of cultivars in Italy is strongly influenced by the market demand for "risotto" varieties. Further management solutions explored over time include physical and biological treatments of the seeds, such as thermotherapy (Forsberg et al., 2003), botanical and microbial biopesticides (Mongiano et al., 2021; Shakeel et al., 2023), or a combination of these methods (Matic et al., 2014). Most efforts, however, focus on selecting rice-associated microorganisms as potential biocontrol agents (BCAs) against F. fujikuroi. Microbial antagonists may rely on several strategies to suppress plant pathogens, particularly competition for space and nutrients, antibiosis, hyperparasitism and lytic enzyme production, host-induced resistance, or manipulation of the resident microbiota. A number of bacterial species (Hossain et al., 2016; Nawaz et al., 2022), yeasts (Matic et al., 2014) and fungi (Kato et al., 2012; Ng et al., 2015; Saito et al., 2021) have been shown to be able to reduce Fusarium wilt symptoms in vivo through different modes of action (MO As).

[0010] However, in most cases, an in vitro screening process using the classic dual culture method was predominantly used to select candidate antagonistic strains. Although in vitro assays are cost-effective and high-throughput, as they allow a large number of isolates to be examined, it is now well established that inhibition of the pathogen in vitro and reduction of the disease in vivo are rarely correlated. In addition, the fastest-growing strains in vitro and the most studied genera with antibiosis or competition MOAs, such as Bacillus, Pseudomonas, Trichoderma, and Clonostachys, tend to be preferentially selected, thus excluding potential BCAs whose antagonistic modes of action cannot be detected under these conditions, namely, induced resistance and microbiota manipulation.

[0011] It should also be added that antagonist microorganisms often exhibit inconsistent efficacy under field conditions, as they are sensitive to external abiotic stress factors that may hinder the colonization of the niche in which they are applied and the performance of their beneficial functions for the plant.Summary of the invention

[0012] In order to overcome these and other limitations of the prior art, the present inventors undertook a research project in which 135 fungal and bacterial rice endophytes were isolated from rice seeds and sprouts. Since, as indicated, in vitro screening rarely correlates with biocontrol efficacy in plants, each isolate was tested in vivo under controlled conditions.

[0013] This allowed the identification of isolates capable of significantly reducing the severity and incidence of Fusarium wilt in vivo and, in some cases, the preservation of fresh biomass production.

[0014] Using the methodology described above, the inventors found that isolates belonging to the bacterial species Sphingomonas yabuuchiae are able to efficiently colonize the host following seed treatment and prevent the onset of Fusarium wilt.

[0015] A first aspect of the present invention is therefore a method for the treatment, prevention, or containment of Fusarium fujikuroi infections in a plant as defined in the appended claims, which form an integral part of this specification.

[0016] The inventors also identified a novel strain of Sphingomonas yabuuchiae which proved particularly effective as an antagonist of F. fujikuroi in rice. This strain, designated as RS-ARS2, was deposited under the Budapest Treaty at the Coleccidn Espanola de Cultivos Tipo (CECT) under accession number CECT 31159 on 11 October 2024.

[0017] A further aspect of the present invention is therefore the aforementioned novel RS-ARS2 strain of Sphingomonas yabuuchiae, as well as a composition comprising the novel RS-ARS2 strain and a phytopharmaceutically acceptable carrier.

[0018] Detailed description of the invention

[0019] To the inventors' knowledge, this is the first report on the effectiveness of S. yabuuchiae inreducing rice Fusarium wilt. Microorganisms of the species S. yabuuchiae, and in particular strain RS-ARS2, are also suitable for use as a treatment against other rice diseases, such as rice blast (caused by Pyricularia oryzae), as they have also been shown to be effective in a different pathosystem, such as apple anthracnose. Other plants that are suitable for treatment with the aforementioned microorganisms are other cereals, such as wheat and maize, fruit crops, such as apples, and vegetable crops, such as lettuce and tomatoes.

[0020] The treatment of plants with microorganisms of the species S. yabuuchiae, in particular strain RS-ARS2, can be carried out using any known methodology, the selection of which depends in particular on the type of plant to be treated. For example, live bacteria of the species S. yabuuchiae, preferably the RS-ARS2 strain, are applied to the plant as a whole (e.g., as in the case of lettuce) or to the soil surrounding it, or to parts of the plant, or still to fruits (e.g., as in the case of apples) or seeds. A preferred method of application in the case of rice is seed treatment, a procedure that comprises directly immersing rice seeds in a suspension of the microorganisms or spraying the suspension onto the seeds, followed by drying the seeds.

[0021] In a preferred embodiment of the invention, microorganisms of the species S. yabuuchiae, in particular the RS-ARS2 strain, are lyophilised using lyophilisation methodologies known per se, which generally include the use of cryopreservatives, to obtain a preferably solid lyophilised preparation, with the aim of ensuring a longer shelf-life without requiring refrigeration of the product, but only storage in a dry place. The solid lyophilised preparation is preferably obtained by combining the lyophilised microorganism with a solid support, such as rice-processing residues, bentonite or calcium carbonate, and / or with additives to ensure adhesion to the seeds, such as the adhesive agents carboxymethylcellulose, alginates and chitosan, but also the rice processing residues themselves. Before use, the lyophilised preparation is reconstituted by adding water, thus obtaining a suspension of reconstituted live bacteria with a pH in the range of 6-7 and a concentration preferably in the range of 107'8CFU ml’1.

[0022] The following examples show that, of all strains of fungal and bacterial endophyte strains isolated from rice, the strain RS-ARS2, belonging to the species Sphingomonas yabuuchiae, was the most effective in reducing disease severity and incidence, while allowing the plantto maintain its vigour even in the presence of the pathogen. The examples also show that the RS-ARS2 strain has a poor ability to produce substances with antimicrobial activity in vitro, which is an additional advantage since the production of antimicrobial substances could pose a hazard to off-target organisms. For this reason, it is believed - without wishing to be bound by any theory - that the mechanism of action of the RS-ARS2 strain requires the presence of the plant host to be carried out, so it could have an effect of competition for space and / or nutrients, induction of plant resistance or manipulation of the resident microbiota for the benefit of the host.

[0023] A further advantage is linked to the method of application of the microorganism: treating the seed prevents the onset of the disease, protecting the plant and leading to the production of a plant biomass statistically similar to the healthy control. Existing solutions for disease containment are synthetic plant protection products, which however, if applied indiscriminately, in addition to environmental and human health risks, can lead to the development of resistance in pathogens, making the active ingredient ineffective against the biotic adversity. The use of live microorganisms as antagonists in the containment of plant diseases does not present the risk of acquiring resistance as, unlike synthetic plant protection products, antagonists do not have a specific molecular site as the target of their antagonist activity, but often use different mechanisms of action.

[0024] The ability of the RS-ARS2 strain to colonize the internal tissues of rice provides versatility and ease of application and use, both for seed companies and, potentially, for farmers themselves.

[0025] The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims. In the examples, reference is made to the appended figures wherein:

[0026] Figure 1 is a bar graph showing the results of the first in vivo mass screening of rice endophytes against F. fujikuroi (partial data). Each bar represents the average of 2 tests, in which each treatment was divided into 3 biological replicates, each consisting of 10 plants. Error bars represent standard errors. Different letters indicate a significant difference (P <0.05) between treatments according to Tukey's test.

[0027] Figure 2 is a bar graph showing F. fujikuroi in vitro growth inhibition by selected endophytes, assessed using the dual culture (dark grey) and sandwich (light grey) methods. Each bar represents the average of 2 tests, in which each treatment consisted of 5 biological replicates. Error bars represent standard errors. Different letters indicate a significant difference (P < 0.05) between treatments according to the Games-Howell test.

[0028] Figure 3 is a boxplot showing the in vivo antagonism efficacy of selected endophytes against F. fujikuroi, assessed on the basis of Fusarium wilt severity (a) and incidence (b), and of the total fresh rice plant biomass (c). Each boxplot represents the median, the first and third quartiles, the minimum and maximum of the data obtained from 2 tests, in which each treatment was divided into 6 biological replicates, each consisting of 30 plants. Different letters indicate a significant difference (P < 0.05) between treatments according to Tukey's test.

[0029] Figure 4 is a bar graph containing the results of the efficacy of RS-ARS2 against apple anthracnose causative agent (partial data). Each treatment was divided into 5 biological replicates, each consisting of 1 apple inoculated at 3 different sites. Error bars represent standard errors. Different letters indicate a significant difference (P < 0.05) between treatments according to Tukey's test.

[0030] Examples

[0031] 1. Materials and Methods

[0032] 1.1 Endophyte isolation

[0033] Seeds from 24 experimental and commercial Italian rice lines, provided by CREA-DC (Vercelli, Italy) and the seed company Sa.Pi.Se. Coop. Agricola (Vercelli, Italy), were used for endophyte isolation.Of these, 5 genotypes (Oceano, Unico, S 18042, S 19048, S 19067-1) were grown in the greenhouse for 4 weeks for sampling and isolation from rice aerial tissues. One gram of seed or aerial tissue (pool of leaves and stems) was used. The aerial organs were further cut into smaller pieces (3-5 cm) and collected into 50 mL Falcon tubes for surface sterilization. The seeds were washed with 30 ml of 0.1% Tween 20 in a Falcon tube, which was then placed on an orbital shaker at 100 rpm for 1 minute; the suspension was then discarded and the seeds were immersed in 30 ml of 70% ethanol in the same tube and stirred for 2 min, followed by 2% sodium hypochlorite for 10 min, 0.1% Tween 20 for 1 minute, 70% ethanol for 2 min and, finally, they were rinsed 5 times with sterile deionized water (SDW), at least 30 s per wash. The leaf and stem pools were surface sterilized with 0.1% Tween 20 for 30 s, 70% ethanol for 30 s, 2% sodium hypochlorite for 1 min, 0.1% Tween 20 for 30 s, 70% ethanol for 30 s and rinsed 5 times with SDW. To confirm the absence of epiphytic microorganisms, 100 pl of the final wash water from each sample was plated onto Luria-Bertani Agar (LBA, Merck, Germany) and Potato Dextrose Agar (PDA, VWR, Italy) in triplicate and incubated at room temperature (22 ± 2°C) for 7 days.

[0034] The surface sterilized tissues were ground with a sterile mortar and pestle in 9 ml of sterile G-strcngth Ringer solution (Merck). The homogenized suspension was serially diluted and 100 pl of each dilution was plated onto LBA and Reasoner's 2A Agar (R2A, VWR) in triplicate for isolation and total microbial counts, as described in Walitang et al. (2017). All plates were incubated at room temperature for up to 14 days and microbial counts were performed after 7 days. Individual colonies of unique morphotypes were streaked onto LBA to obtain pure cultures. Isolates were grown in LB broth under stirring (100 rpm) at room temperature for 2 days and stored at -80°C in 30% glycerol.

[0035] 1.2 In vivo screening to assess the effectiveness of biological control

[0036] Mass screening of the endophyte collection, aimed at selecting potential BCAs, was initially performed by evaluating the reduction of disease symptoms in vivo. The preparation of the bacterial inoculum and the treatment of the seeds were performed by following the protocol of Walitang et al. (2017), with some changes. Individual colonies were transferred to 30 ml of LB and PDB, respectively, in flasks and incubated at room temperature on an orbitalshaker (100 rpm). After 2 days, the culture broth was removed by centrifugation at 6,000 x g for 10 min and the cells were resuspended in sterile Ringer solution added with 0.5% sodium carboxymethylcellulose (CMC-Na, Merck). The bacterial inoculum was adjusted to a concentration of ODeoo = 0.5 (~ 107-108CFU ml’1) using a spectrophotometer. Rice seeds of the Galileo cultivar, susceptible to Fusarium wilt, were sterilized by immersion in 2% sodium hypochlorite for 30 min under stirring, followed by 5 washes with SDW. The seeds were then soaked in the microbial suspension under stirring (100 rpm) at room temperature for 2 hours. The treated seeds were dried on sterile blotting paper under a laminar hood for 30-60 min.

[0037] The highly virulent A5 strain of F.fujikuroi (TUCC00000714) deposited at Turin University Culture Collection (TUCC) was grown on PDA at room temperature for 7 days. Conidia were collected using 0.5% CMC-Na Ringer solution by scraping the aerial mycelium, subsequently the suspension was filtered through 2 layers of sterile gauze to remove the mycelium fragments, and the final concentration was adjusted to 105ml’1conidia using a Burker chamber. Two days after treatment with the endophyte, the rice seeds were inoculated with the pathogen by immersing them in the conidia suspension under stirring for 30 min, followed by drying on sterile filter paper under a laminar hood for 30-60 min. Three controls were included: I) a healthy control, without inoculation of the pathogen; II) an inoculated control, inoculated with F. fujikuro', III) a chemical control, treated with fludioxonil (20% active ingredient, treatment by immersion under stirring at 100 rpm for 1 h) after inoculation of the pathogen.

[0038] Sowing was carried out in a substrate consisting of 60% peat and 40% sand in plastic trays containing 30 plants divided into 3 rows. For each treatment, 30 seeds were divided into 3 biological replicates and sown in 3 different trays in a randomized block design. The trays were placed in the greenhouse under controlled conditions (photoperiod: 12 hours, temperature: 27 ± 5°C day / 22 ± 5°C night) and, after germination, disease severity (SD) was assessed weekly using the following scale (Piombo et al. 2020): 0, healthy plant; 1, small size, chlorotic leaves; 2, significant dwarfism, significant yellowing, internode elongation; 3, crown necrosis; 4, dead or non-germinated plant. The germination rate and total fresh biomass were recorded for each biological replicate at the end of the assay, when at leasthalf of the inoculated control plants were dead (4 weeks after germination). Each experiment was carried out twice.

[0039] 1.3 Molecular identification

[0040] Endophytic isolates that significantly reduced the severity of Fusarium wilt in vivo were molecularly identified to exclude potential plant pathogens on economically relevant crops.

[0041] Bacterial endophytes were grown at room temperature in 30 ml of LB on a shaker for 2 days, and the biomass was obtained by removing the culture broth by centrifugation at 6,000 x g for 10 min. Total bacterial DNA was extracted using MasterPure™ Complete DNA & RNA Purification Kit (Biosearch Technologies, USA) according to the manufacturer's instructions. The partial bacterial marker gene 16S was amplified with the universal primers FD1 (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1) and RD1 (5'-GTTACCTTGTTACGACTT-3', SEQ ID NO:2) (Weisburg et al., 1991).

[0042] PCR amplifications were performed in a total volume of 25 pL containing: 2.5 pL of 10X buffer, 0.5 pL of MgCh, 0.5 pL of dNTPs (10 mM), 0.5 pL of each primer (10 mM), 0.2 pL of Taq DNA polymerase (Qiagen) and 10 ng of sample DNA. Thermal cycles were set up as follows: an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 60 s, and a final extension step at 72°C for 5 min. PCR products were sent for sequencing in both directions at Macrogen Europe (Milan, Italy). Consensus sequences were obtained with the DNA Baser program (Heracle Biosoft S.R.L., Arges, Romania). After low-quality region trimming and manual correction, the sequences were pre-compared to those deposited in public datasets (National Center for Biotechnology Information, NCBI) using the BLASTn program and subsequently aligned with CLUSTALW using the Molecular Evolutionary Genetics Analysis (MEGA6) software. To perform phylogenetic analysis with the Maximum Likelihood (ML) algorithm or the Neighbor-Joining (NJ) method, the most suitable nucleotide model was determined with MEGA version 6 and a phylogenetic tree was constructed with the same program. NCBI reference sequences (RefSeq) were used for the phylogeny of the 16S region.1.4 In vitro characterization

[0043] Promising BCAs identified at the molecular level underwent an additional selection step based on their growth rate at 37°C to exclude potential opportunistic human pathogens, as described in Latz et al. (2020), with some changes. Each strain was grown on LBA and incubated at 25°C and 37°C to assess the preferred growth temperature. The plates were checked daily for 5 days and strains with a faster growth rate at 37°C were discarded.

[0044] 1.4.1 F. fujikuroi growth inhibition

[0045] The selected endophytic strains were tested in vitro for their antagonistic mode of action against F. fujikuroi using the dual culture method, to check for the presence of agar-diffusible antimicrobial compounds (Spadaro et al., 2002), and the sandwich method, to check for the production of volatile antimicrobials (Sipiczki, 2023). In the dual culture assay, a mycelium- colonized PDA disk was cut with a sterile corer (6 mm diameter) from a 7-day-old F. fujikuroi colony and transferred to a 90 mm PDA plate, 30 mm from the edge. Bacterial endophytes were collected with a 10 pl loop from a suspension with ODeoo adjusted to 0.3 (~ 107CFU ml’1) and streaked on the opposite side of the plate, 30 mm from the edge.

[0046] In the sandwich method, F. fujikuroi was inoculated at the centre of a 90 mm PDA plate as a mycelium-colonized PDA disk. Endophytic strains were inoculated onto an independent PDA plate by spread plating 10 pl of bacterial suspension with ODeoo adjusted to 0.3. The plate containing F. fujikuroi was combined with the endophyte plate and sealed with parafilm, allowing the microorganisms to share the same atmosphere.

[0047] For both experiments, control plates containing the pathogen alone and 10 pl LBA were included. For both assays, 5 replicates were used, and the plates were incubated at 22°C for 5 days before measuring the radius of the pathogen colonies (in cm). In vitro F. fujikuroi inhibition was calculated as a percentage of the pathogen colony size reduction caused by the endophytes compared to control plates. In vitro antagonistic activity against F. fujikuroi was tested twice for each method.1.4.2 Temperature and osmotic stress tolerance

[0048] The selected promising strains were tested for thermal and osmotic stress tolerance in vitro. Tolerance to different temperatures (4, 10, 22, 28 and 37°C) was assessed on LBA plates. Osmotic stress tolerance was tested as described by Jones et al. (2015), by adding the following KC1 concentrations to the substrate: none (-0.1 MPa), 10.06 g L-1(-1.1 MPa), 17.9 g L’1(-1.9 MPa), 28.44 g L’1(-3.0 MPa) and 36.5 g L’1(-3.9 MPa), where MPa is the measurement of the osmotic potential. Bacterial endophytes were inoculated onto LBA plates by dripping 10 pl spots taken from a microbial suspension with ODeoo adjusted to 0.3 and serial dilutions thereof (10'-10 ) in triplicate on the same plate. After 5 days of incubation, the assessment was performed by counting the number of dilutions that fully colonized the inoculum spot in at least 2 technical replicates. The incubation temperature was set to 25°C for all strains and 5 replicates were used for each condition in both assays. Each experiment was carried out twice.

[0049] 1.5 In vivo biological control efficacy of the selected strains

[0050] The selected endophytic strains were tested in vivo to confirm their antagonism efficacy against F. fujikuroi. Treatment of seeds with rice endophytes and inoculation of the pathogen were performed as described above. Bacterial growth curves in LB broth were assessed to adjust the inoculum to 107CFU ml’1, corresponding to ODeoo = 0.3 for all strains. Each treatment was divided into 6 biological replicates of 30 seeds each, for a total of 180 plants. A randomized block design was adopted. In addition, disease incidence was extrapolated from disease severity data as described in Li et al. (2018), considering as F.fujikuroi-infected plants only those displaying symptoms with a disease score of 2 or higher. The efficacy of the selected strains was tested twice.

[0051] 1.6 Efficacy of biological control against apple anthracnose

[0052] RS-ARS2 was grown in 30 mL of LB under stirring at room temperature. After 24 hours, the cells were harvested by centrifugation at 4000xg for 10 minutes, washed andresuspended in sterile Ringer solution. The suspension was adjusted to ODeoo = 1. Colletotrichum acutatum SC strain CVG 2459, isolated from apples with anthracnose symptoms and stored in the DISAFA collection, was grown on PDA for 15 days at 25°C. Conidia were collected in 10 mL of a 0.1% Tween-20 solution, and the suspension was filtered through four layers of sterile gauze. Quantification was carried out in a Burker chamber, and the suspension was adjusted to IxlO5conidia / mL.

[0053] Cv. Gala apples were disinfected with 1% sodium hypochlorite for 2 minutes, rinsed with tap water and, once dry, pierced with a sterile tip in the equatorial region (3 mm depth; three wounds per fruit). Treatment was performed by pipetting 20 ml of bacterial cell suspension into the wounds. After 3 hours, 20 ml of conidial pathogen suspension was pipetted into the wounds. Two controls were included. The chemical control was represented by fruits inoculated with the pathogen and treated with thiabendazole (Tecto® SC, 19.7% active ingredient). Inoculated and untreated apples were used as an inoculated control. Once dried, the apples were randomly packaged in plastic trays and stored at 25 °C for 15 days. Five fruits per treatment were used (15 inoculation sites). Disease severity was assessed by measuring the diameter of each developed rot along the two perpendicular axes, which was subsequently averaged.

[0054] 1.7 Statistical analysis

[0055] Data on disease severity on rice were converted into percentage values as described in Matic et al. (2014), obtaining the weighted average disease severity percentage for each biological replicate based on the frequency of each disease score in the empirical scale. In the screening tests, strains leading to a statistically significant reduction in disease severity were considered as antagonists only when the effect was significant in both tests. All statistical analyses were performed in the R environment (version 4.3.1). Shapiro-Wilk and Levene tests were performed to check for data normality and homoscedasticity before performing parametric tests. When the analysis of variance was statistically significant (P < 0.05), Tukey's HSD test was used for pairwise comparisons among treatments. The data for F. fujikuroi in vitro growth inhibition were heteroscedastic; therefore, Welch's ANOVA was performed, followed by Games-Howell post-hoc test.2. Results

[0056] 2.1 Rice endophyte isolation and in vivo screening

[0057] One hundred and thirty-five rice endophytes were isolated from rice seeds, leaves, and stems. Total microbial counts were performed for each sample, obtaining microbial concentrations ranging from logio 5.84 to 8.02 CFU g'1in seeds, and from logio 3.42 to 5.63 CFU g'1in aerial organs. In total, 99 endophytes were isolated from seeds and 36 from leaves and stems.

[0058] Each isolate was tested in two greenhouse biological control assays, each consisting of three replicates of 10 plants. Each test verified the efficacy of 45 isolates. No significant differences in germination rate were observed.

[0059] Partial data from this mass screening are shown in Fig. 1, which shows that RS-1852.2 strain was not able to reduce disease symptoms, while the RS -MIS 3 and RS-APO3 strains were selected in this first screening step together with RS-ARS2 because they were able to significantly reduce disease symptoms (P < 0.05).

[0060] 2.2 Molecular identification and growth at 37°C

[0061] Of the 11 bacterial isolates identified, most genera included potential human opportunistic pathogens rather than off-target agricultural crops, which led to the exclusion of all selected bacterial strains with a higher growth rate at 37°C, with the exception of four strains. Maximum Likelihood phylogeny using 16S sequences led to the identification of strain RS-ARS2 as Sphingomonas yabuuchiae (bootstrap 98%) and strain RS-APO3 as Microbacterium testaceum (bootstrap 97%). Instead, based on Neighbor Joining phylogeny, strain RS-MIS3 was identified as Methylobacterium oryzae. The RS-1852.2 bacterial strain was not further investigated because it was not effective in mass screening.

[0062] 2.3 In vitro antagonismThe selected strains were tested in vitro to assess their antagonistic potential against F. fujikuroi. The results are shown in Figure 2, where the control consisted of capsules inoculated with the pathogen and in the absence of antagonistic bacteria. The figure shows that strain RS-MIS3 did not cause changes in the in vitro growth of the pathogen; strain RS- ARS2 caused significant (P < 0.05) F. fujikuroi growth inhibition mainly through the production of volatile antimicrobial metabolites; strain RS-APO3 caused a negative effect on the growth of the pathogen, through the production of metabolites diffusible across agar substrate and volatile metabolites. In all cases, however, the inhibitory effect was significantly lower than that observed in vivo, suggesting that the direct, metabolite-mediated antimicrobial effect of the antagonists is secondary to other mechanisms of action involved in the antagonistic activity.

[0063] 2.4 Temperature and osmotic stress tolerance

[0064] The selected BCAs were grown at different temperatures and salt concentrations to assess their tolerance to abiotic stresses.

[0065] None of the selected endophytes were able to proliferate at 37°C.

[0066] Regarding tolerance to simulated osmotic stress, the strain that tolerated the highest concentration of KC1 in the substrate was RS-APO3 (Af. testaceum), proliferating up to -3.9 MPa. Strain RS-ARS2 OS'. yabuuchiae) tolerated up to -1.9 MPa, while RS-MIS3 (Methylobacterium oryzae) failed to grow even at the lowest osmotic stress level.

[0067] 2.5 Confirmation of in vivo biological control efficacy

[0068] The selected strains were tested in vivo to confirm their antagonism efficacy against F. fujikuroi. The results obtained are shown in Figure 3 (partial data).

[0069] The figure shows that RS-MIS3 was the least effective in containing disease symptoms, although it still had a significant effect (P < 0.05); however, this strain resulted in an increase in total biomass compared to the inoculated control. Strain RS-APO3 enabled effectivecontainment of the pathogen but failed to prevent the loss of fresh biomass linked to F. fujikuroi infection. Only the S. yabuuchiae strain (RS-ARS2) demonstrated the best performance in reducing disease severity and incidence, while also allowing the plant to maintain its vigour even in the presence of the pathogen.

[0070] 2.6 Biological control efficacy on apples against Colletotrichum acutatum

[0071] The RS-ARS2 bacterial strain was tested in a different pathosystem to assess a possible broadening of its antagonist spectrum of action. Partial data are shown in Fig. 4, which shows that the RS-ARS2 strain was able to significantly reduce the diameter of rot caused by the pathogen C. acutatum (P < 0.05).

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Claims

CLAIMS1. A method for the treatment, prevention or containment of infection by Fusarium fujikuroi in a plant, the method comprising the application of live bacteria of the species Sphingomonas yabuuchiae to the whole plant, parts thereof or its surrounding environment, or to the fruits or seeds of the plant.

2. The method according to claim 1, the method comprising the application of live bacteria of the species Sphingomonas yabuuchiae to the seeds of the plant.

3. The method according to claim 2, wherein said application comprises immersing the seeds of the plant in a suspension of live bacteria of the species Sphingomonas yabuuchiae or spraying a suspension of live bacteria of the species Sphingomonas yabuuchiae on the seeds of the plant, followed by drying the seeds.

4. The method according to any of claims 1 to 3, wherein the plant is selected from cereals, horticultural crops and fruit crops.

5. The method according to claim 4, wherein the plant is selected from rice, wheat, maize, lettuce, tomato and apple.

6. The method according to any of claims 1 to 5, wherein the live bacteria of the species Sphingomonas yabuuchiae belong to the strain RS-ARS2 deposited at the Coleccidn Espanola de Cultivos Tipo (CECT) under accession number CECT 31159 on 11 October 2024.

7. RS-ARS2 strain of Sphingomonas yabuuchiae deposited at the Coleccidn Espanola de Cultivos Tipo (CECT) under accession number CECT 31159 on 11 October 2024.

8. Use of a lyophilised preparation of bacteria of the species Sphingomonas yabuuchiae in the treatment, prevention or containment of Fusarium fujikuroi infection in a plant, wherein the use comprises reconstituting the preparation in water to obtain a reconstitutedsuspension of live bacteria and applying the reconstituted suspension of live bacteria to the whole plant, parts thereof or its surrounding environment, or to the fruits or seeds of the plant.

9. The use according to claim 8, wherein the reconstituted suspension of live bacteria has a pH value in the range of 6-7 and a concentration of live bacteria in the range of 107'8CFU ml’1.

10. The use according to claim 8 or 9, wherein the lyophilised preparation of bacteria of the species Sphingomonas yabuuchiae also comprises a solid support and / or an adhesive agent.

11. The use according to claim 10, wherein the solid support is selected from the group consisting of rice processing residues, bentonite, calcium carbonate and combinations thereof, and wherein the adhesive agent is selected from carboxymethylcellulose, alginates, chitosan and combinations thereof.

12. The use according to any of claims 8 to 11, wherein the lyophilised bacteria of the species Sphingomonas yabuuchiae belong to the isolate RS-ARS2 deposited at the Coleccidn Espanola de Cultivos Tipo (CECT) under accession number CECT 31159 on 11 October 2024.

13. The use according to any of claims 8 to 12, wherein the plant is selected from cereals, horticultural crops and fruit crops.

14. The use according to claim 13, wherein the plant is selected from rice, wheat, maize, lettuce, tomato and apple.

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