Antifungal microbial strain, composition and use

Abstract

The invention relates to an atoxinogenic Aspergillus flavus strain suitable for use in biological control of aflatoxin-producing A.flavus, in particular in temperate, in particular in Central European climates. The invention also relates to a composition for biological control, that can be used in agriculture, and contains non-mycotoxin-producing, in particular non-aflatoxin-producing Aspergillus and chitosan. The invention further relates to the use of the atoxinogenic A.flavus strain and the composition containing the strain for plant protection.

Description

[0001] Antifungal microbial strain, composition and use

[0002] FIELD OF THE INVENTION

[0003] The invention relates to an atoxinogenic Aspergillus flavus strain suitable for use in biological control of aflatoxin-producing A. flavus, in particular in temperate, in particular in Central European climates. The invention also relates to a composition for biological control, that can be used in agriculture, which contains non-mycotoxin-producing, in particular non-aflatoxin-producing Aspergillus and chitosan; further the invention relates to the use of the atoxinogenic A. flavus strain and the composition containing the strain for plant protection.

[0004] PRIOR ART

[0005] Aspergillus flavus is an opportunistic pathogenic fungus that causes significant agricultural damage and poses a serious health risk through its toxin production. The fungus is present not only as a storage pest (e.g., a pest of stored oilseeds and com), but also as a parasite of field crops, such as corn. Infecting humans, A. flavus and the aflatoxin it produces, primarily aflatoxin Bl, can cause fatal disease. In addition to aflatoxins, A. flavus can also produce other mycotoxins, such as cyclopiazonic acid, aflatrem, leporin, aflavinin, and sterigmatocystin.

[0006] Several naturally occurring A. flavus strains do not produce aflatoxin, so they may be used as protection against aflatoxinogenic A. flavus strains, as they may be capable of competitive exclusion, partial occupation of the niche of aflatoxinogenic A. flavus strains to reduce their abundance, or even inhibition of aflatoxin biosynthesis.

[0007] In order for a non-aflatoxin-producing A. flavus strain to be used as a biological control in agriculture, it is necessary that it is viable in the given area, preferably exhibiting at least the same level of viability and virulence as the aflatoxinogenic A. flavus strains living in the given area, be genetically stable, be able to adapt to the climatic conditions of the given area even under rapidly changing, extreme conditions, and not produce other mycotoxins that pose a risk from an agricultural and health perspective.

[0008] Chitosan is increasingly used in agriculture: as a biodegradable fertilizer, a plant resistance enhancer, and a growth promoter. Its antifungal effects are also well known. Several studies (e.g. Sharif et al. The Multifunctional Role of Chitosan in Horticultural Crops; A Review. Molecules. 2018 Apr 10;23(4):872. doi: 10.3390 / molecules23040872; Abbas F. Shahadha et al 2023 IOP Conf. Ser.: Earth Environ. Sci. 1262 062015. Characterization Chitosan Nanoparticles Prepared from the Stalks of Agaricus bisporus Brown and its Effect on Inhibition of Aspergillus flavus and Reducing Aflatoxin Bl Production; Liu et al. Synthesis, Characterization,and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus. J Agric Food Chem. 2024 May 15;72(19): 11185-11194; Liu et al. Synergistic effect of acetic acid and chitosan against Aspergillus flavus, International Journal of Biological Macromolecules, Volume 281, Part 4, 2024,

[0009] 136548) have reported that chitosan inhibits growth and spores oi A. flavus, thus chitosan may be useful for protection against A. flavus.

[0010] BRIEF DESCRIPTION OF THE INVENTION

[0011] Atoxinogenic A. flavus strain designated AMK588-2 or a non-aflatoxin producing derivative thereof produced by culturing or genetic modification is provided, wherein AMK588-2 was deposited with the National Collection of Agricultural and Industrial Microorganisms, Somloi lit 14-16, 1118 Budapest, Hungary on the 10th of October 2024, and has the accession number NCAIM (P) F 001535.

[0012] The invention provides a composition comprising A. flavus belonging to the strain AMK588-2 or a non-aflatoxin producing derivative thereof produced by culturing or genetic modification, preferably A. flavus belonging to the strain AMK588-2. The composition preferably comprises an agriculturally acceptable carrier or excipient. Preferably, the agriculturally acceptable carrier is chitosan or a material comprising or consisting essentially of chitosan. Preferably the composition is for plant protection use. Preferably the use is for preventing or reducing mycotoxin contamination of a plant or plant part. Preferably the plant pest is a toxin, preferably aflatoxinproducing Aspergillus sp., preferably A. flavus. Preferably the composition comprises chitosan. Preferably the composition is formulated in chitosan granules.

[0013] The invention provides AMK588-2 or a non-aflatoxin producing derivative thereof produced by culturing or genetic modification, combined with chitosan. The invention provides AMK588-2 or a non-aflatoxin producing derivative thereof produced by culturing or genetic modification, embedded in chitosan. The invention provides a composition comprising a fungus belonging to the strain AMK588-2 or a non-aflatoxin producing derivative thereof produced by culturing or genetic modification and chitosan, or consists of A. flavus AMK588-2 strain and the following: chitosan or chitosan, lactic acid and polysorbate; or consists of the following: A. flavus, that is a non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification and chitosan; ox A. flavus, that is a non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification and chitosan, lactic acid and polysorbate.The invention further provides a composition comprising atoxinogenic, preferably non-aflato-xin producing Aspergillus, preferably Aspergillus flavus and chitosan. Preferably the composition is for plant protection use.

[0014] The invention provides the use of the composition against pests living on plants (plant pests). Preferably the use is for preventing or reducing mycotoxin contamination of a plant or plant part. Preferably, the plant pest is a toxin-producing, preferably aflatoxin-producing Aspergillus sp., preferably A. flavus.

[0015] Preferably, the composition is applied to the habitat of the plant.

[0016] The use of AMK588-2 or the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof for plant protection is provided. Preferably the use is a use against pests living on plants (plant pests). Preferably the plant pest is a toxin-producing, preferably an aflatoxin-producing (toxinogenic, aflatoxinogenic, respectively) Aspergillus sp., preferably A. flavus. Preferably the use is for preventing or reducing mycotoxin contamination, preferably aflatoxin contamination of a plant or plant part.

[0017] The use of AMK588-2 or a non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof for controlling an aflatoxin-producing Aspergillus sp., preferably A. flavus, wherein the use is not a therapeutic treatment of an animal (including humans).

[0018] Preferably AMK588-2 or the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is combined with chitosan, preferably AMK588-2 or the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is used encapsulated into a material comprising or essentially cosisting of chitosan, preferably granules comprising or essentially cosisting of chitosan.

[0019] Preferably the control of the Aspergillus sp. is agricultural control.

[0020] Preferably AMK588-2 or the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is applied to a plant, crop or the soil of the plant. Preferably application to the plant is performed by injuring (e.g. by piercing), spraying, applying to the plant or the growing area (e.g. soil) of the plant.

[0021] Method for the prevention or reduction of the contamination of a plant or plant part, preferably fruit or seed with aflatoxin, the method comprising applying AMK588-2 or a non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof to the plant or plant part.

[0022] Preferably the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is applied to the plant or plant part in the vegetative or reproductive,preferably in the early reproductive phase of the plant.Preferably the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is delivered to the plant or plant part prior to an infection by an aflatoxin-producing Aspergillus sp.

[0023] Preferably the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof does not produce a secondary metabolite listed in table 1. Preferably the non-aflatoxin producing derivative of AMK588-2 produced by culturing or genetic modification thereof is atoxinogenic. Preferably the non-aflatoxin producing derivative of AMK588- 2 produced by culturing or genetic modification thereof is capable of colonizing a plant or plant part infected by A. flavus.

[0024] Preferably the plant is maize. Preferably the plant part is maize (corn) kernel.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figure 1. Inoculation was done by pricking the cobs (toxinogenic strain: AMK 4- 16 / IV). The experimental plots were not given any additional water supplementation other than rain during the experiment (A) or were given water supplementation (B). * p<0,l; **p<0,05; ***p<0,01. Figure 2. Colonization test on different maize hybrids. We inoculated toxinogenic Aspergillus flavus strain AMK 4-16-IV (gray bars) and strain AMK 588-2 (white bars).

[0027] Figure 3. Mold contamination of com kernels under different Aspergillus flavus treatments Figure 4. A) A. flavus AMK 588-2 strain in an electronmicroscopic picture (8000x); (B) Chitosan bead with the embedded A. flavus AMK 588-2 conidiospore chain, electronmicroscopic picture (lOOOx)

[0028] Figure 5. A. flavus colonies from the broken microcapsules on total count agar medium Figure 6. Average aflatoxin Bl levels in cobs that were inoculated with a toxigenic strain and to whose soil AMK 588-2 strain was applied as chitosan beads, based on data from one year (2024). **: P=0.01321, with 95% probability.

[0029] Figure 7. Effect of the use of microencapsulated atoxigenic A. flavus (AMK 588-2) under different conditions (irrigation, N) on mold counts and aflatoxin B 1 production in the R6 phase w / wo toxigenic A. flavus treatment. CT-control; TO- toxinogenic A. flavus in ear; IR-irrigated; Nl-non-irrigated; NB-no bead treatment; AB- atoxinogenic A. flavus microbeads on soil. Figure 8. Arbuscular mycorrhiza (AM) and total fungal PLFA content of R2 / R3 and R6 com developmental stages under different irrigation regimes and 120 kg / haN fertilisation. CT-control, TO-toxinogenic A. flavus inoculated in ear; Nl-non-irrigated, IR-irrigated, NB-no-beads, AB-atoxigenic A. flavus in chitosan beads.DETAILED DESCRIPTION OF THE INVENTION

[0030] The invention relates to an Aspergillus flavus strain (AMK588-2) isolated from Hungarian grain, in which the norA and omtA genes cannot be detected by conventional methods (e.g., the method described in the Examples section) (they carry mutations that prevent detection by conventional methods). Although the aflR gene can be detected by conventional methods in the AMK588-2 strain, the strain does not produce aflatoxin (especially aflatoxin Bl).

[0031] AMK588-2 does not produce any other known secondary metabolites that are harmful from an agricultural or health perspective on maize. The metabolites tested are presented in Table 1. Table 1. Secondary metabolites not produced by AMK588-2

[0032] compound name compound structure

[0033] a-cyclopiazonic C20H20N2O3

[0034] acid (CPA)

[0035] Izo- a -CPA C20H20N2O3

[0036] P-CPA C20H22N2O3

[0037] cAATrp (cycloC15H14N2O3

[0038] acetoacetol -L- tryptophan

[0039] a -CPA-imine C20H21N3O2

[0040] Pseuboydone E C19H20N2O3

[0041] 2-oxoCPA C20H20N2O4

[0042] Speradine A C21H22N2O4

[0043] 3-OH-speradine A C21H22N2O5

[0044] Speradine B C16H18N2O3

[0045] Speradine C C20H22N2O5

[0046] Speradine D C20H22N2O6

[0047] Speradine E C20H18N2O5

[0048] Speradine F C21H22N2O7

[0049] Speradine H C20H18N2O4

[0050] Speradine I C21H22N2O7

[0051] Aspergilline A C19H20N2O6

[0052] Aspergilline B C23H26N2O8

[0053] Aspergilline C C24H28N2O6

[0054] Aspergilline D C21H24N2O7

[0055] Aspergilline E C25H30N2O9

[0056] Cyclopiamide A C16H14N2O2

[0057] Cyclopiamide B C20H20N2O4

[0058] Cyclopiamide C C19H18N2O4

[0059] Cyclopiamide D C19H16N2O4

[0060] Cyclopiamide E C20H17N3O2

[0061] Cyclopiamide F C15H12N2O2

[0062] Cyclopiamide G C15H16N2O3

[0063] Cyclopiamide H C16H18N2O3

[0064]

[0065] Cyclopiamide J C22H24N2O7AMK588-2 is able to colonize field crops (e.g. maize) or stored crops (e.g. corn cobs, corn kernels) when artificially introduced. Its colonization ability is also significant in the presence of aflatoxin-producing A. flavus. In a field experiment, inoculation of com cobs with strain AMK588-2 reduced the average aflatoxin Bl contamination (Figure 1), i.e. the strain can be used in the control of aflatoxinogenic A. flavus.

[0066] Colonization by AMK588-2 did not reduce the protein and starch content of corn kernels, i.e. the use of the strain does not reduce the nutritional values of the crop, and thus its usability as food or feed.

[0067] A. flavus typically colonizes the plant through a wound on the epidermis, which poses a difficulty in the effective application of A. flavus strains for biological control.

[0068] Surprisingly, we found that AMK588-2, when applied in chitosan beads, exerted its effect against the aflatoxinogen A. flavus, i.e. despite the known antifungal effect of chitosan, it did not inhibit AMK588-2 spores and there was no need to injure the plant. As a result of applying AMK588-2 in chitosan beads to the maize growing area, aflatoxin Bl contamination was reduced even more significantly. The treatment with the combination of the strain and chitosan was particularly effective in dry growing areas, i.e. under conditions typical of the Central European region.

[0069] AMK588-2 spores remain viable when embedded in chitosan. Viability was not negatively affected by storage in humid conditions. The storage period may be at least one year. After 1 year of storage at 4°C, AMK588-2 packaged in chitosan beads did not produce AFB1. AMK588-2 spores embedded in chitosan, when released into the plant environment (soil), are able to colonize the plant. The advantage of combining the fungus with chitosan (in addition to the effects of chitosan on plant growth and improving plant resistance) is that chitosan is consumed by animals (e.g. insects) and microorganisms living in the environment of the plant and thus can deliver the spores to different parts of the plant, they do not remain at the soil level. Chitosan (micro)beads applied to or into the soil degrade quickly (e.g. within a few weeks). The composition containing chitosan and A. flavus strain AMK588-2 applied to the soil of the plant did not cause significant changes in the chlorophyll content of the plant, nor in the protein content of the corn kernels. The total mold count of corn kernels was reduced, as was the aflatoxin production of toxigenic A. flavus inoculated into the com plant. The biocontrol effect of A. flavus strain AMK588-2 is particularly beneficial under dry (non-irrigated) conditions.

[0070] The term chitosan is used herein to refer to chitosan material, chitosan-containing material and material essentially consisting of chitosan. Chitosan, chitosan-containing material and materialessentially consisting of chitosan. are preferably materials suitable for packaging A. flavus, preferably AMK588-2 spores, in such a way that the packaged spores are suitable for agricultural use, for example for application to plant soil. The packaging material (which is chitosan, a material containing chitosan or consisting essentially of chitosan) allows A. flavus spores to escape from the composition at the site of application and colonize the plant. The terms chitosan and chitosan (micro)beads are also used to refer to a material that is, for example, prepared in a manner that can be prepared as described in the Examples section. The term chitosan bead can thus refer to, for example, a capsule or a particle of any shape. In the description, unless the context requires otherwise, a chitosan bead or particle is understood to mean a chitosan-based bead or particle, i.e. the main component of such a particle may be chitosan, but it may also contain other components necessary to maintain the viability of A. flavus or other components necessary or advantageous for the preparation of the particle. The chitosan-based particle may contain, in addition to chitosan, for example, a solvent (emulsifier), preferably an acidic solvent, such as lactic acid or acetic acid, a base (coagulant), such as NaOH, water, a crosslinking agent, technical or functional excipients, such as alginate, polysorbate or urea.

[0071] The terms atoxigenic, atoxicogenic, atoxinogenic A. flavus, unless otherwise clear from the context, refer to the fungus belonging to the A. flavus AMK588-2 strain.

[0072] EXAMPLES

[0073] Isolation and culture of Aspergillus species

[0074] Chloramphenicol-yeast extract-glucose agar was inoculated with randomly selected grain samples (maize and wheat) and grown for five days at 30 °C. Fungal colonies microscopically identified as Aspergillus spp. were collected and plated on malt extract agar (MEA) medium [20 g L'1glucose, 10 g L'1malate extract, 5 g L'1yeast extract, 15 g L'1agar].

[0075] Identification of Aspergillus species

[0076] To determine the species of the isolates, the ITS1-ITS4 (White et al., 1990) and cmd5-cmd6 (calmodulin) DNA segments were amplified by PCR (Hong et al., 2006 Novel Neo-sartorya species isolated from soil in Korea. International Journal of Systematic and Evolutio-nary Microbiology. 56: 477-486.)

[0077] Primers used:

[0078] ITS1 : 5-TCC GTA GGT GAA CCT GCG G-3

[0079] ITS4: 5-TCC TCC GCT TAT TGA TAT GC-3

[0080] cmd5: 5-GTC TCC GAG TAC AAG GAG GC-3

[0081] cmd6: 5-TCG CCG ATA GAG GTC ATA ACG TG-3After purification of the PCR product, the obtained DNA fragments were sequenced (BIOMI Kft.). The obtained data were analyzed using the MEGA 7 program and compared with the GenBank database (Benson et al., 2013. GenBank. Nucleic Acids Research 41 (Database issue): D36-42).

[0082] Three of the genes responsible for aflatoxin production in Aspergillus flavus were identified by performing a multiplex PCR. In this PCR, three primer pairs were used, the sequence of which was as follows (Varga et al., 2011. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Studies in Mycology. 69: 57-80.):

[0083] nori : 5-ACC GCT ACG CCGGCA CTC TCG GCA-3

[0084] nor2: 5-GTT GGC CGC CAG CTT CGA CAC AGC-3

[0085] aflR-R: 5-TGG KGC CGA CTC GAG GAA YGG GT-3

[0086] aflR-F: 5-GGG ATA GCT GTA CGA GTT GTG CCA-3

[0087] omtl : 5-GTG GAC GGA CCT AGT CCG ACA TCA C-3

[0088] omt2: 5-GTC GGC GCC ACG CAC TGG GTT GGG G-3

[0089] In strain AMK588-2, the norA and omtA genes were not detected, while the aflR gene was detectable by multiplex PCR.

[0090] Investigation of secondary metabolite production of isolates

[0091] Inoculation of corn kernels with Aspergillus flavus for in vitro aflatoxin Bl and secondary metabolite formation assay

[0092] We also examined the production of aflatoxin Bl and other secondary metabolites in the com hybrids, for which 50 g of com was treated with 70% ethanol and washed with distilled water, then placed in a sterile Erlenmeyer flask and inoculated with 500pl of 106 / ml spores. The incubation was carried out for 7 days at 30 °C in the dark.

[0093] Sample preparation for HPLC-FD measurement from corn kernels

[0094] 25 g of the inoculated and incubated corn was weighed, then 2.5 g of NaCl and 50 ml of 80% methanol were added. It was homogenized using a blender (Commercial Blender, Waring) at high speed for 3 minutes. Then the sample was filtered using pleated filter paper. 10 ml of the filtered sample was taken and diluted with 40 ml of distilled water. After stirring on a magnetic stirrer, 10 ml of this sample was passed through an Afla-BTM immunoaffinity column (Vicam). The column was then washed with 10 ml of distilled water. Aflatoxin Bl bound to the column was then eluted with 5 ml of HPLC grade methanol (Sigma-Aldrich). The sample was then evaporated in a Biichi Rotavapor R114 [Source: Vicam method (AflaB GN-MC9514-3 Rev B)].

[0095] Sample preparation for HPLC-MS and HPLC-FD measurements from nutrient agar cornIn this process, the fungus was placed in a Stomacher bag together with the nutrient agar in the Petri dish, then 10 ml of chloroform was added, and the sample was homogenized with a Masticator IUL Instruments Stomacher homogenizer for 2 minutes.

[0096] Then 10 ml of chloroform was added again and homogenized again using a Stomacher for 1 minute. The sample was filtered through filter paper into a round-bottom flask and then evaporated in a Biichi Rotavapor R114 apparatus under vacuum at 50 °C. The mobile phase, which was a 45:55 mixture of methanol: water, was then added, 2 ml of which was added to the evaporated sample. The sample was then filtered using a syringe filter (Millex-GV 0.22 pm Filter, Merck).

[0097] For the HPLC-MS measurement, corn inoculated and incubated as described in section 2.1.3.1 was treated in the same way after grounding (Fente et al., 2001. New additive for culture media for rapid identification of aflatoxin-producing Aspergillus strains. Applied and Environmental Microbiology. 67: 4858-4862.).

[0098] HPLC-FD measurement

[0099] The HPLC instrument included a Merck Hitachi AS-4000 Intelligent Autosampler and a Merck Hitachi L-6200A Intelligent pump. Detection was performed using a fluorescent detector with an extinction of 360 nm and an emission of 440 nm. A Genesis C18 column was used, which was 4.6 x 150 mm in size and had a particle diameter of 4 pm. The injection volume was 20 pl and the flow rate was 1 ml min-1. The eluent used was methanol: water, 45:55 [Vicam method (AflaB GN-MC9514-3 Rev B9],

[0100] HPLC-MS measurement

[0101] MV 251 corn hybrid was used in the experiment.

[0102] The HPLC-MS equipment included of a Waters Separations module (2695) and a Waters 2996 Photodiode Ar-ray detector. The metabolites were separated on a Zorbax SB-C18, 4.6x75 mm, 3.5 pm column. The flow rate was 0.5 ml min'1, the elution time was 60 min and the temperature was 40°C. UV detection was performed at 225 nm. The methanol: water gradient was as follows (Table 2). Mass spectra were recorded on a Bruker microTOF-Q mass spectrometer equipped with an Atmospheric Pressure Photoionization (APPI) ion source. The ion source temperature was 390°C. The results were analyzed using Bruker Daltonics DataAnalysis software.

[0103] Table 2. Elution gradient used during HPLC-MS analysis

[0104] Time (min) MeOH (%) H2O (%)

[0105] 0 10 90

[0106] 50 80 20

[0107]

[0108] 54 80 20

[0109] 54,1 10 90

[0110] 60 10 90

[0111]

[0112] During our studies, we identified several secondary metabolites based on the publication of Uka et al. (Unravelling the diversity of the cyclopiazonic acid family of mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS. Toxins. 9: 35. 2017).

[0113] AMK588-2 did not produce aflatoxin and did not produce any of the tested secondary metabolites (Table 1) on corn kernels.

[0114] Stress testing of strain AMK588-2

[0115] The fungal strain was also tested on com agar. To prepare the medium, 15 g of corn flour was added to 1 liter of water, then boiled for half an hour, filtered through gauze and sterilized after adding 20 g L-l agar.

[0116] Stress tests were also performed, during which the growth of the fungal strain, sclerotia formation, spore formation and pigmentation were examined on malt agar and corn extract agar media. For the stress tests, 0; 1; 1.5; 2 and 2.5 M NaCl concentrations; 0; 0.05; 0.1; 0.5 and 0.75 g / 1 SDS (sodium dodecyl sulfate) concentrations and 100-300 pg / ml Congo red were used. All culture media used were purchased from VWR International Kft. (Debrecen, Hungary) or Scharlab Hungary Kft. (Debrecen, Hungary). All chemicals and reagents used were of analytical or chromatographic grade and were purchased from VWR International (Debrecen, Hungary) and Merck Life Science Ltd. (Budapest, Hungary). Ultra-pure water was obtained from an Elix Millipore purification system (Merck Millipore, Darmstadt, Germany).

[0117] AMK588-2 is optimally grown on malt agar at 30°C for 5-7 days, producing small, yellowish-green sclerotia.

[0118] Resistance to stress on com agar: 100-300pg / ml Congo red did not change the morphology of the fungus, 2M NaCl inhibited spore formation, 0.5 g / 1 SDS inhibited spore formation. On malt agar: 0.5 g / 1 SDS resulted in less vegetative growth, 0.75g / l SDS resulted in the appearance of sclerotia, 2M NaCl caused smaller conidiophores.

[0119] Corn kernel colonization study

[0120] Toxinogenic (previously identified) and atoxinogenic (AMK588-2) strains of A. flavus were inoculated onto kernels from different maize hybrids (MV251 (FAO 280), DEKALB440 (FAO 330), BC442, DKC5031 (FAO 430), L6, ARSANO O). Ten kernels were treated with 70% ethanol and washed with distilled water in a Petri dish and inoculated with 10 pl of 106spores / ml / kernel of A. flavus, in triplicate. A The growth of the fungus on the kernels wasexamined. Incubation was performed in the dark for 7 days at 30 °C. The results were evaluated using a two-sample t-test.

[0121] AMK588-2 was able to colonize all hybrids. The colonization ability of the toxinogenic and atoxinogenic strains did not differ from each other in any hybrid (Figure 2).

[0122] Field experiment

[0123] . The experiment was conducted during the 2020 / 2024 growing season on Hungarian chernozem soil at the Experimental Station of the Agricultural Research Center of the University of Debrecen. A complex small -plot field experiment was conducted, where the SY Orpheus (FAO 370-390) hybrid was evaluated. The plot treatments were two irrigation levels. Two water supplies were used: I (irrigated) represented the optimal water supply plots, where the evapotranspiration loss (ET 100%) was replaced; in the non -irrigated plots (NI), the plants were grown under rain-fed conditions. Evapotranspiration loss was determined from daily weather data from a meteorological station near the experiments using the method described by Shuttleworth and Wallace (1985. Evaporation from sparse crops-an energy combination theory. Quarterly Journal of the Royal Meteorological Society, 111(469), pp.839-855.). The irrigation water requirement was calculated based on actual evapotranspiration as described by Allen et al. (1998. Crop evapotranspiration-Guidelines for computing crop water requirements-FAO Irrigation and drainage paper 56. Fao, Rome, 300(9), p.D05109.). Each plot (100 m2) contained 12 rows, with a row spacing of 76 cm and a plant spacing of 20 cm. The seeds were sown by hand.

[0124] The control plants were not inoculated with A.flavus, the. A.flavus strain that produced aflatoxin in a previous study was used as the toxinogenic strain, and the AMK588-2 strain was used as the atoxinogenic strain.

[0125] Artificial contamination of maize plants with A. flavus via puncture channel

[0126] The fungal isolate used in the experiment was cultured in Petri dishes and spore suspensions (106ml-1) were prepared. The plants were treated via a puncture channel on August 12, 2020, August 2, 2021 and August 4, 2022 at the R2 phenol ogical stage, as well as on July 20, 2023, June 17, 2024 (chitosan beads) and July 16, 2024.

[0127] The amount of fungus applied via the puncture channel was 2.5* 105spores.

[0128] Microencapsulation of strain AMK588-2 in chitosan

[0129] 1.5 g of chitosan was weighed on an analytical scale. After that, 100 ml of 2% lactic acid was added and stirred at 55 °C in order to obtain a homogeneous solution. The addition of lactic acid also helped the dissolution, as it contributed to providing the acidic pH necessary for the dissolution of chitosan. Then 0.2 g of TWEEN 20 was added, which allowed for better emulsionformation, and the mixing was again carried out at 55 °C for 1 hour. In the next step, 1.9 ml of the cell suspension to be packaged, Aspergillus flavus fungal spores, were added to the solution at a concentration of 108spores / ml. Using a Pharmacia LKB Pump P-1 peristaltic pump, the prepared solution was added dropwise to 300 ml of 2 M sodium hydroxide (NaOH) to form beads (Sang-suwan et al., 2016. Effect of chitosan beads incorporated with lavender or red thyme essential oils in inhibiting Botrytis cinerea and their application in strawberry packaging system. LWT - Food Science and Technology 74 (2016) 14e20). The prepared beads were filtered by vacuum filtration, then washed with sterile distilled water to remove excess NaOH from their surface. They were then transferred to a Petri dish. Finally, they were dried at room temperature for 5 hours to remove residual moisture.

[0130] In 2025, spore encapsulation in chitosan was prepared with ~1.2 x 10A2 spores / g microcapsule (AB1), as in 2024, and without spores involved (EB).

[0131] Viability of A. flavus AMK588 in chitosan beads

[0132] Calculated viable spore number was 1.2*102spores / g microbeads (Figure 4).

[0133] Stability of spore viability of A. flavus AMK588 in microbeads under long storage Microencapsulated A. flavus AMK588 was stored in a wet state at 4°C ± 2°C. At the time of production and after 313 days, 100 pl of the suspension was placed on total count agar medium after mechanical breaking of the wet beads (with a mortar in buffered peptone water). The plates were incubated at 30 °C ± 2 °C in the dark for 7 days.

[0134] Results: Wet storage did not significantly affect the viability of the embedded spores, and no contamination was detected (Figure 5).

[0135] Plant treatment with a composition containing A. flavus strain AMK588-2 fungus and chitosan 3.16 g of beads were applied to the base of the maize plant (GEIX1770 hybrid) on the soil before inoculation with the toxinogenic Aspergillus flavus fungus. In each plot, chitosan beads were applied on the base of 3 plants, and were covered with soil to eliminate UV-induced damage.

[0136] Chitosan microbeads ’ stability in soil

[0137] 200 g of agricultural soil was weighed into planting pots, and 1-2 g of wet microcapsules containing A. flavus AMK588-2 were placed on the soil surface, then covered with the same soil to a depth of 1-2 cm. The experiment was also done in vivo under field conditions.

[0138] Result: After 3 weeks, a few beads were visible in a shrivelled form, although they were difficult to separate from the soil particles. In the field, the microcapsules were also undetected after 2-3 weeks.Between 2024 and 2025, the micro-plot experiment was done according to Molnar et al. (The effect of environmental factors on mold counts and AFB 1 toxin production by Aspergillus fl a-vus in maize. Toxins, 15(3), p.227, 2023) and Mwalugha et al. (Irrigation, nitrogen supplementation, and climatic conditions affect resistance to Aspergillus flavus stress in maize. Agriculture, 15(7), p.767., 2025). GEIX1770 maize hybrid (Syngenta) was selected for the study. A Completely Randomised Design (CRD) was employed to investigate the physiological changes in the hybrid resulting from toxinogenic A. flavus contamination and chitosan-embedded atox-inogenic A. flavus treatment. The study examined key response variables, including mycotoxin levels, mold count, starch, protein, total polyphenols, and kernel numbers per ear length. The explanatory variables included inoculation (A. / Tmv / .s-inoculated vs control), irrigation (irrigated vs non-irrigated), and fertiliser application rates (60, 120, and 180 kg / haN (N regimes)). Each treatment had three replicates. The plots consisted of 4 rows, each 5 m long and 3 m wide. The row spacing was 76 cm, and the plant spacing was 20 cm. Seeds were sown manually in midApril each year. Treatments were randomly assigned across plots, ensuring each plot received a unique combination of inoculation, irrigation, and fertiliser treatments. Data was collected and analysed using analysis of variance (ANOVA) to compare means across treatment groups and determine the effects of the experimental factors on com physiology and mycotoxin contamination (Mwalugha et al., 2025).

[0139] In chitosan-embedded A. flavus AMK588-2 treatment, 3 g beads were applied per plant in the R2 phenological stage. The toxinogenic A. flavus isolate used in the microplot experiment was cultured on petri dishes, and spore suspensions (106ml-1) were produced. Treatment of com ears was carried out via a puncture channel (50pL) in the R2 / R3 phenological stage after 25-29 days of atoxinogenic A. flavus treatment.

[0140] In 2025, empty microbeads (EB) were further applied under 120 kg / haN fertilisation and without toxinogenic A. flavus inoculation in ears.

[0141] Chlorophyll content of corn plants (treatment with chitosan-embedded (beads) A. flavus AMK588-2 on soil with or without toxigenic A. flavus inoculation in ears)

[0142] The handheld chlorophyll content index (CCI) meter (model MC-100, Apogee Instruments, Logan, UT, USA) was used to estimate leaf chlorophyll content. In the case of SPAD measurements, it is important to consider irradiance, leaf water status, and the time of measurement. To eliminate such possible errors, the measurement time in our experiment was always between 10 and 12 am. In the case of corn, the Apogee MC-100 chlorophyll meter is well-suited, as the leaves are thin and the leaf surface is smooth. The chlorophyll meter utilises the transmittance ratio at 653 nm and 931 nm to determine the relative chlorophyll content. The ratio is termedCCI, and the MC-100 converts CCI to SPAD units. The measurements were performed once a week, starting from 10:00 a.m., for 5 weeks after inoculation. The measurements were taken on the leaves above and next to the ears. In each replication, five measurements were taken along the entire length of the leaf surface on the five designated plants. The measurement area was 63.6 mm2.No significant alterations in corn chlorophyll were detected under the different treatments.

[0143] Sample preparation

[0144] Samples were collected from the plots from the marked inoculated and control cobs. After cleaning the cobs, the obtained corn samples were dried in a drying oven (LABORMIM Kft., Budapest, Hungary) at 54 °C ± 1 to constant weight. As a next step, the corn samples were crushed and ground.

[0145] Determining the total mold count

[0146] The dried kernels were ground and collected in sterile Stomacher homogenizer bags, suspended in a 1 :9 buffered peptone water (BPW) solution (Scharlab, Barcelona, Spain), and homogenized with a Stomacher Masticator homogenizer (IUL Instruments, Barcelona, Spain) for 2 min, repeated twice. As a next step, decimal dilutions were made on the suspensions and total mold counts were determined on CYG agar (Scharlab, Barcelona, Spain) using the pour plate method. The inoculated solid agar medium was incubated for five days at 25 °C to determine the mold count. Each inoculation was performed in triplicate.

[0147] Figure 3 shows that the AMK588-2 strain was able to infect the plant both alone and when inoculated together with an aflatoxin-producing strain (50% AMK588-2 strain and 50% aflato-xinogenic strain).

[0148] The success of the treatment with the different strains is demonstrated by the fact that the number of colony-forming units in the control treatment ranged from 3.3 x 104CFU g-1 to 4.7 x 106CFU g-1, and in the case of artificial infection it ranged from 4.5 x 105CFU g-1 to 1.7 x 107CFU g-1.

[0149] Detection of Aflatoxin Bl by HPLC-FD

[0150] All HPLC measurements were performed on a Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) HPLC system. The dried samples (25 g) were homogenized with 2.5 g sodium chloride (VWR) and 50 ml 80% methanol (HPLC, Sigma-Aldrich, St. Louis, MO, USA) under high-speed stirring. The extract was diluted 1:4 with 40 ml distilled water. The homogenized sample was filtered into round-bottom flasks using filter paper (Macherey-Nagel). The diluted extract was filtered and 10 ml was loaded onto the aflatoxin immunoaffinity column (VLCAM AflaTest WB HPLC Columns, Weber Consulting Kft., God, Hungary). The column waswashed with 10 ml of distilled water, the toxin was eluted with methanol (5 ml) and evaporated in Rotavapor R114 (Buchi). After adding 1 ml of mobile phase (methanol: water, 45:55), the solute was filtered through a Millex-GV 0.22 pm filter (Merck-Millipore) and applied to the HPLC. A Phenom enex (Torrance, CA, USA) RP-C18 column (150 * 4.6 mm, 5 pm) with a Romer UV derivatization unit (Romer Labs Ltd., Tulin, Austria) and an ex360 nm, em440 nm fluorescence detector were used, with methanol: water (45:55) as eluent. Biopure Aflatoxin Mix 1 standard solution (Romer Labs, Tulin, Austria) was applied to the column.

[0151] Figure 1 shows that under field conditions, in the case of toxinogenic and AMK588-2 strains introduced into the plant via the injection channel, the average aflatoxin Bl contamination in maize inoculated with the AMK588-2 strain decreased.

[0152] By pre-inoculating the soil with a toxinogenic strain in chitosan beads, the toxinogenic inoculation of the cobs in the non-irrigated area showed a significant (83%, p=0.01321) difference from the samples without bead treatment (Figure 6).

[0153] Corn content testing

[0154] The protein content was determined by the Kjeldahl method according to the MSZ EN ISO 5983-2:2009 standard, while the starch content was determined by polarimetry according to the standard methods of MSZ 6830-18:1988 (withdrawn standard) Chapter 2.

[0155] The use of strains had no significant effect on protein or starch content. The presence of aflatoxin and protein content covariation were typical. We found a small but significant difference in the protein content under chitosan microbead treatment.

[0156] Table 3 Effect of chitosan-microencapsulated atoxigenic AMK588-2 A. flavus on different corn physiological parameters, mold counts and aflatoxin B 1 production in ears in the R6 developmental stage in 2024. One-way ANOVA.

[0157] Treatment Protein Starch Total polyphenol AFB1 Mold count (m / m% DW) (m / m% DW) (mg GAE / lOOg DW) (Rg / kg) (logio CFU / g) CT-NI-NB 12.99 ± 0.02 c 60.04 ± 0.19 d 218.28 ± 1.28 be 0.17 ± 0.11 b 6.61 ± 0.17b CT-NI-AB1 9.97 ± 0.10 f 64.42 ± 0.21 a 200.82 ± 6.39 c 0.06 ± 0.02 b 4.90 ± 0.47 e AT-IR-NB 12.93 ± 0.03 c 61.38± 0.17bc 229.50 ± 4.06 be 0.04 ± 0.02 b 6.54 ± 0.08 be AT-NI-NB 13.22 ± 0.03 c 58.72 ± 0.22 e 221.25 ± 0.68 be 0.05 ± 0.00 b 6.49 ± 0.07 be CT-IR-NB 14.47 ± 0.11 a 53.39± 0.13 f 243.16 ± 3.07 b 0.01 ± 0.01 b 5.71 ± 0.40 cd CT-IR-AB1 14.00 ± 0.18 b 52.54 ± 0.14 f 239.24 ± 4.49 b 5.70 ± 5.63 b 5.45 ± 0.28 de TO-NI-NB 12.26 ± 0.09 d 60.57 ± 0.28 cd 206.38 ± 12.09 c 212.92 ± 39.85 ab 6.48 ± 0.06 be TO-NI-AB1 11.15 ± 0.05 e 62.25 ± 0.21 b 228.71 ± 2.03 be 30.69 ± 19.95 b 6.34 ± 0.02 be TO-IR-NB 13.94 ± 0.03 b 48.98 ± 0.14 h 244.11 ± 4.73 b 251.45 ± 186.49 a 7.47 ± 0.12 a TO-IR-AB1 13.77 ± 0.05 b 50.58 ± 0.10 g 291.14 ± 7.6.3 a 152.32 ± 56.87 ab 7.10 ± 0.27 ab

[0158]

[0159] CT-control; AT-atoxinogenic A. flavus in ear; TO- toxinogenic A. flavus in ear; IR-irrigated; Nl-non-irrigated; NB-no bead treatment; AB1- atoxinogenic A. flavus on soil. Values given as means ± SE; Values followed by different letters within columns are significantly different at p < 0.05 Duncan’s multiple range test (DMRT). Highest values are colored in grey.

[0160] The highest protein and starch content was measured in control plant kernels. The biocontrol effect was lower under irrigated conditions (TO-IR-AB1 to TO-IR-NB: 30-40%; TO-NI-AB1 to TO-NI-NB: 60-90% Aflatoxin Bl reduction).

[0161] Table 4 Total effect of usage of microencapsulated atoxigenic AMK588-2 A. flavus under any conditions (irrigation, N) in 2024 on the R6 stage w / wo toxigenic A. flavus treatment. Independent t-test.

[0162] Parameters No beads (NB) Beads (AB) Kernel number per ear length 27.12 ± 0.41 a 26.44 ± 0.95 a Protein (m / m% DW) 13.30 ± 0.74 a ** 12.22 ± 0.36 b ** Starch (m / m% DW) 57.18 ± 0.76 a 57.47 ± 1.24 a Total polyphenols (mg GAE / lOOg DW) 227.11 ± 3.14 a 239.98 ± 7.29 a AFB1 (ug / kg) 77.44 ± 37.74 a 47.19 ± 22.66 a Log mold count 6.55 ± 0.14 a 5.95 ± 0.28 a

[0163]

[0164] Values are given as means ± SE; Values in the groups followed by different letters within rows are significantly different at p < 0.05 t-test; ** p < 0.01 t-test.

[0165] Mold counts in corn kernels were reduced with atoxigenic AMK588-2 A. flavus chitosan-mic-robead treatment. The most significant decrease was observed with toxigenic fungal inoculation of the ear under non-irrigated conditions (Table 3). Aflatoxin Bl concentrations correlated well with these findings. Mold viability affects secondary metabolite production. Atoxigenic AMK588-2 A. flavus strain applied to the soil affected mold counts in corn kernels indirectly (Table 8), supposedly through changing plant physiology.

[0166] Table 5: Effect of usage of microencapsulated atoxigenic AMK588-2 A. flavus under any conditions in 2025 in R6 developmental stages altogether (60-180 kg / ha N). Independent t-test. Values are given as means ± SE; Values in the groups followed by different letters within rows are significantly different at p < 0.05 t-test.

[0167] Parameters No beads Empty beads AB

[0168] controls

[0169] Kernel number per ear length 24.53 ± 0.51 a 23.70 ± 0.70 ab 24.81 ± 0.45 a Protein (m / m% DW) 12.70 ± 0.09 b 12.31 ± 0.04 c 13.08 ± 0.07 a Starch (m / m% DW) 51.14 ± 0.16 b 51.75 ± 0.08 a 52.15 ± 0.19 a

[0170]

[0171] Total polyphenols (mg GAE / lOOg 264.60 ± 3.37 a 246.56 ± 4.60 b 264.78 ± 2.98 a DW)

[0172] AFB1 (pg / kg) 85.51 ± 38.28 a 1.14 ± 0.35 b 60.20 ± 28.05 a Log mould count 6.04 ± 0.12 a 5.96 ± 0.18 a 5.93 ± 0.14 a

[0173]

[0174] CT-control; AT-atoxinogenic A. flavus in ear; TO- toxinogenic A. flavus in ear; IR-irrigated; Nl-non-irrigated; NB-no bead treatment; EB-beads with no embedded spores; AB- 1.2 * 10A2 spores / g micro-capsule.

[0175] Table 6: Effect of usage of microencapsulated atoxigenic AMK588-2 A. flavus under any conditions in 2025 on R2 / 3, R4 / 5 and R6 developmental stages altogether (120 kg / ha N). Independent t-test. Values are given as means ± SE; Values in the groups followed by different letters within rows are significantly different at p < 0.05 t-test; ** p < 0.01 t-test. Highest values are marked in grey.

[0176] Parameters No beads Empty beads AB

[0177] controls

[0178] Kernel number per ear length 25.60 ± 0.44 a 24.00 ± 0.65 b 23.80 ± 0.42 b Protein (m / m% DW) 13.29 ± 0.27 a 12.82 ± 0.28 a 13.40 ± 0.20 a Starch (m / m% DW) 40.09 ± 2.49 a 41.93 ± 2.97 a 42.53 ± 1.75 a Total polyphenols (mg GAE / lOOg 367.53 ± 30.19 a 346.68 ± 40.57 b 359.08 ± 26.48 b DW)

[0179] AFB1 (pg / kg) 79.59 ± .38.8.3 a 0.66 ± 0.42 b .39.57 ± 27.64 a Log mold count 4.13 ± 00.60 a 3.22 ± 1.00 a 4.35 ± 0.52 a

[0180]

[0181] Values given as means ± SE; Values followed by different letters within rows are significantly different at p < 0.05 Duncan’s multiple range test (DMRT); AB - Atoxigenic A. flavus beads. Chitosan itself (empty microbeads) did not induce polyphenol synthesis or nutrient alterations (Table 5 and Table 6).

[0182] The effect of chitosan-embedded atoxigenic AMK588-2 A. flavus on soil microbiome on the basis of fatty acid composition

[0183] During com cultivation, soil samples for microbiome analysis were collected at different com devel op-mental stages within a 20 cm radius around the corn stalks (n=3 per plant). The samples were homogenised, dried, and PLFA (phospholipid fatty acid) extraction of soil samples was carried out using a modified Bligh & Dyer method (chloroform-methanol-buffer) using an internal standard (C19:0) (UD FAFSEM). The lipid fraction was measured by GC-FID after conversion to methyl esters and analysed with the MIDI SherlockTM PLFA Software Package on a Shimadzu GC -2010 / 2030 (Shimadzu) (SD < 3%).Table 7: Microcapsules' effect on soil microbiome from 120 kg N / ha plots (a) in R2 / R3 corn developmental stage; (b) in R6 corn developmental stage. PLFA content in nmol / g soil is presented. CT-control, TO-toxinogenic A.flavus inoculated in ear; Nl-non-irrigated, IR-irriga- ted, NB-no-beads, EB-no A. flavus embedded in microbeads; AB-atoxigenic AMK588-2 A. flavus in chitosan beads.

[0184] a) Component Group CT-NI-NB CT-NI-AB TO-NI-AB CT-IR-NB CT-IR-AB TO-IR-AB Am Fungi (16:lw5) 1909 11.32 6.25 15.50 15.54 6.39 Fungi (C18:2w6) 5.13 3.54 3.57 8.08 3.74 3.07 Gram-negative bacteria iili 35.34 33.33 34.57 34.42 30.42 Gram-positive bacteria 47.12 47.92 42.40 45.16 45.62 38.48 General FAME 35.40 30.92 30.15 38.93 32.07 27.65 Total PLFA 174.94 156.04 143.34 171.43 159.00 130.83 GP / GN 1.41 1.55 1.48 1.47 1.51 1.47 Fungi / Bacteria 0.27 0.17 0.13 0.29 0.23 0.13 Gneg stress* 2.95 2.78 2.95 2.66 2.45 2.42

[0185]

[0186] *Gneg stress: C17-C19 fatty acids are degraded to cyclopropyl fatty acids, meaning a stress effect.

[0187] b) Component CT-NI- CT-NI- TO-NI- CT-IR- CT-IR- TO-IR- CT-NI- CT-IR- Group NB AB AB NB AB AB EB EB Am Fungi 15.54 14.12 18.22 9.83 26.34 10.99 14.51 14.38 (16:lw5)

[0188] Fungi (C18:2w6) 4.46 2.77 4.85 2.61 4.27 805 1.98 2.50 Gram-negative 29.43 27.17 40 15 28.54 36.00 34.86 22.23 23.72 bacteria

[0189] Gram-positive 63.68 34.75 45.55 31.37 38.58 41.64 29.81 30.78 bacteria

[0190] General FAME 28.31 22.32 30.71 21.22 26.14 24.17 28.16 30.35 Total PLFA 154.63 126.41 175.74 111.37 155.02 145.48 115.88 124.02 GP / GN 1.85 1.54 1.33 1.18 1.12 1.28 1.62 1.57 Fungi / Bacteria 0.25 0.26 0.26 0.20 0.39 0.24 0.30 0.30 Gneg stress 8.88 9.83 10.14 13.04 13.30 10.52 9.81 8.12

[0191]

[0192] Under a low samp e size, statistical analysis was not avai able. However, during the R2 / R3 corn development stage, chitosan's antifungal effect against arbuscular mycorrhizal (AM) fungi and other fungi was demonstrated. The atoxigenic AMK588-2 A. flavus treatment increased the antifungal effect, and under non-irrigated conditions, the anti-fungal effect was more prominent. At the R6 developmental stage, these detected effects were not observable, thereby strengthening the positive, temporary effect of chitosan plus atoxigenic A. flavus on the fungalcommunity. Meanwhile, visible differences in bacterial PLFA were not detected. Gneg stress increases from R2 / R3 to R6, marking the increasing stress on the microbiome.

[0193] Mould counts in corn kernels, irrigation, fertilizer use

[0194] Table 8: Correlation analyses of explanatory and response variables. Treatments cover irrigation, microbeads and N regimes. Inoculation covers toxigenic and atoxigenic inoculations.

[0195] Explanatory variables Response variables

[0196] Variables InocuIrrigaSoil beTreTotal polyN Protein Starch AFB1 lation tion ads atment phenols

[0197] Irrigation 0.000

[0198] Soil beads -0.327 * 0.000

[0199] -0.313

[0200] Treatment -0.186 0.142

[0201] *

[0202] N 0.000 0.000 0.000 0.000

[0203] 0.708 -0.394

[0204] Protein 0.047 -0.258 * 0.037

[0205] ** **

[0206] -0.758 -0.821

[0207] Starch 0.109 0.028 -0.046 0.008

[0208] ** **

[0209] Total poly0.633 0.574 -0.713

[0210] 0.068 0.232 0.019

[0211] phenols ** 0.048 ** **

[0212] 0.509

[0213] AFB1 0.128 0.127 -0.114 0.133 -0.340 0.143

[0214] ** 0.034

[0215] Log mold -0.369 -0.385 0.471

[0216] 0.495 ** 0.179 0.312 0.398 * 0.394 * count * 0.007 * **

[0217]

[0218] * correlation significant at 0.05 level; ** correlation significant at 0.01 level.

[0219] Statistical analysis

[0220] Analysis of variance (ANOVA) was performed using SPSS 28 statistical software, and differences between means were compared using Duncan's multiple range test and independent sample T-test at a significance level of p<0.05. Linear and polynomial Pearson correlations were used to explain the variation in fungal abundance and aflatoxin accumulation. Data for mold count (CFU g-1) were log-transformed, while data for aflatoxin content (AFB1) were Intransformed before statistical analyses to reduce the variance of the original data (Clewer and Scarisbrick, 2013). The distribution of the transformed data was normal.

Claims

CLAIMS1. Fungus belonging to Aspergillus flavus AMK588-2 strain, wherein the A. flavus AMK588-2 strain was deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM) on the 10th of October 2024, with the accession number NCAIM (P) F 001535.

2. Aspergillus flavus fungus, which is a non-aflatoxin producing derivative of the fungus belonging to A. flavus AMK588-2 according to claim 1, produced by culturing or genetic modification thereof.

3. A composition comprising the A. flavus fungus according to claim 1 and a carrier or excipient acceptable for plant protection use.

4. The composition according to claim 3, wherein the carrier comprises chitosan.

5. Use of the fungus belonging to Aspergillus flavus AMK588-2 strain according to claim 1 or the composition according to claim 3 or 4 against pests living on a plant or plant part.

6. The use according to claim 5 for preventing or reducing aflatoxin contamination of a plant or plant part.

7. The use according to claim 5 or 6, wherein the pest living on a plant or plant part is a toxin producing Aspergillus flavus.

8. Use of the fungus belonging to the Aspergillus flavus strain AMK588-2 according to claim 1 or the composition according to claim 3 for preventing or reducing mycotoxin contamination of a plant or plant part caused by Aspergillus lavus, wherein the fungus belonging to the A. flavus strain AMK588-2 or the composition is applied to the plant or plant part.

9. Use of the composition according to claim 4 for preventing or reducing mycotoxin contamination of a plant caused by Aspergillus flavus, wherein the composition is applied to the soil of the plant.

10. Composition comprising non-atoxinogenic or non-aflatoxin producing Aspergillus flavus and a carrier comprising chitosan.

11. Use of the composition according to claim 10 for preventing or reducing mycotoxin contamination of a plant caused by Aspergillus flavus, wherein the composition is applied to the soil of the plant.

12. The composition according to claim 3, 4 or 10 or the use according to any one of claims 5-9 and 11, wherein the A. flavus is formulated in chitosan-based granules.

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