Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

Beneficial rhizospheric microorganisms mediated plant growth promotion and suppression of aflatoxigenic fungal and aflatoxin contamination in groundnut seeds

The potential of root‐colonising antagonistic microbial biocontrol agents was evaluated for their ability to improve plant growth and suppress aflatoxigenic fungal and aflatoxin contamination in groundnut. By considering root colonisation of groundnut seedlings, plant growth promotion and antagonism against aflatoxigenic Aspergillus flavus as preliminary criteria, eight rhizobacteria and nine Trichoderma spp. were selected and characterised for their beneficial traits. These strains gave varying results for IAA production, phosphate solubilisation, ACC deaminase, chitinase and siderophore production. Under laboratory and greenhouse conditions, these strains significantly (P < 0.05) suppressed seed‐borne and rhizospheric population of A. flavus and improved seed quality variables. However, cdELISA results revealed that none of the biocontrol strains were effective in reducing aflatoxin level in seed. Based on the overall performance, Pseudomonas fluorescens 2bpf, Bacillus sp. Bsp‐3/aM and Trichoderma atroviride UMDBT‐Dha.Tat8 were used for field trials in the form of talcum powder formulations. Under field conditions, biocontrol agents improved seedling emergence, plant biomass and pod yield. Seeds harvested from plots treated with biocontrol agents showed significant (P < 0.05) reduction in A. flavus infection and aflatoxin production after 6 months' storage. Use of microbial strains with multiple beneficial traits is advantageous in bioformulation development. Hence, in future, these formulations will play a major role as biofertilisers and biopesticides, which can reduce the usage of agrochemicals up to greater extents in groundnut production.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Distribution and characteristics of aflatoxin-producingAspergillus flavus in the soil in Kanagawa Prefecture,Central Japan

In Kanagawa Prefecture, located in central Japan, aflatoxin-producingAspergillus flavus was isolated in 4 (2.5%) of 160 field soil samples. In the 4 fields, whose soil contained aflatoxin-producingA. flavus, the annual average temperature of the sampling sites of the soil ranged from 13.8 to 15.1°C. Of all the isolated strains of aflatoxin-producingA. flavus, 4 strains, isolated from a single soil sample, produced large amounts of aflatoxin B₁ and B₂ when incubated in coconut agar, peanut agar, peanuts or trilaurin-added rice, although they did not produce aflatoxin when incubated in rice, yeast extract-sucrose broth or sucrose-low salts broth.     

Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®

Single nucleotide polymorphisms (SNPs) of genome sequences of eight Aspergillus flavus and seven Aspergillus oryzae strains were extracted with Mauve, a multiple-genome alignment programme. A phylogenetic analysis with sequences comprised of concatenated total SNPs by the unweighted pair group method with arithmetic mean (UPGMA) of MAFFT adequately separated them into three groups, A. flavus S-morphotype, A. flavus L-morphotype and A. oryzae. Divergence time inferred for A. flavus NRRL21882, the active agent of the biocontrol product Afla-Guard^®, and S-morphotype was about 5·1 mya. Another biocontrol strain, A. flavus AF36, diverged from aflatoxigenic L-morphotype about 2·6–3·0 mya. Despite the close relatedness of A. oryzae to A. flavus, A. oryzae strains likely evolved from aflatoxigenic Aspergillus aflatoxiformans (=A. parvisclerotigenus). A survey of A. flavus populations implies that prior Afla-Guard^® applications are associated with prevalence of NRRL21882-type isolates in Mississippi fields. In addition, a few NRRL21882 relatives were identified. A. flavus Og0222, a biocontrol ingredient of Aflasafe™, was verified as a NRRL21882-type strain, having identical sequence breakpoints that led to deletion of aflatoxin and cyclopiazonic acid gene clusters. A similar UPGMA analysis suggests that the occurrence of NRRL21882-type strains is a more recent event.     

Impact of a Streptomyces (AS1) strain and its metabolites on control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in stored peanuts

Six actinomycetes were isolated from peanuts in Egypt. Of these, a Streptomyces strain (AS1) was found in in vitro assays to inhibit directly or via secondary metabolites both germination and growth of Aspergillus flavus. Tests of the AS1 cells for direct control of A. flavus populations or aflatoxin B₁ (AFB₁) production on stored peanuts was unsuccessful over 14-day storage periods. However, crude extracts of AS1 metabolites at 50 and 100 ppm completely inhibited spore germination of conidia of A. flavus in vitro over 48 h. Comparison of solvents for extracting the metabolites showed that the ethyl acetate extract was most effective. This gave greater than 85% inhibition of mycelial growth at these concentrations at different water availabilities (water activity; a _w; 0.95, 0.92, and 0.89) and 25°C. Doses of 50, 200, and 500 ppm of AS1 metabolites significantly inhibited populations of A. flavus on stored peanuts at two water stress levels (0.90, 0.93 a _w) at 25°C over 14-day storage periods. The amounts of AFB₁ produced by A. flavus on peanuts stored at 0.90 a _w were significantly decreased by AS1 metabolites for only 7 days. However, at 0.93 a _w doses of 200 and 500 ppm significantly controlled AFB₁ accumulation in peanuts for 14 days.     

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.     

Selection of non-toxigenic strains of Aspergillus flavus for biocontrol of aflatoxins in maize in Thailand

Aflatoxin production in maize and peanuts remains a major public health problem, especially in developing countries. One promising method for combating aflatoxin formation is biocontrol using competitive exclusion, a management strategy currently being studied in maize crops in Thailand. It is important that the strains of Aspergillus flavus used in biocontrol be non-toxigenic and be incapable of reversion to toxigenicity. In the current study, 80 non-toxigenic strains of A. flavus, randomly selected from commercially produced dried maize samples from several sources in Thailand, were screened for their potential as biocontrol strains by examining the 24 aflatoxin biosynthesis genes, using a PCR assay. Assessment of the presence or absence of PCR products provides an indication of the function of pathway genes. Of the 80 strains, 78 showed no PCR products from one or more genes in the aflatoxin biosynthesis pathway. Twenty-three isolates showed only one failure, in the aflD (nor-1) gene, but most isolates failed to produce a PCR product for two or more genes. Nineteen isolates (24%) failed to show a PCR product in 10 or more genes. Altogether, 45 PCR product patterns were observed, usually common to only one or two isolates, indicating great diversity in the aflatoxin biosynthesis pathway in A. flavus isolates taken from dried Thai maize. Although the absence of a particular PCR product is not conclusive evidence that the particular gene is non-functional, the absence of several such PCR products provides reasonable evidence that the isolate in question will not revert to toxigenicity in the field.     

Potential for aflatoxin B1 and B2 production by Aspergillus flavus strains isolated from rice samples

In this study, we investigated the potential for aflatoxin B₁ (AFB₁) and B₂ (AFB₂) production in rice grain by 127 strains of Aspergillus flavus isolated from rice grains collected from China. These strains were inoculated onto rice grains and incubated at 28 °C for 21 days. AFB₁ and AFB₂ were extracted and quantified by high-performance liquid chromatography coupled with fluorescence detection. Among the tested strains, 37% produced AFB₁ and AFB₂ with levels ranging from 175 to 124 101 μg kg⁻¹ for AFB₁ and from not detected to 10 329 μg kg⁻¹ for AFB₂. The mean yields of these isolates were 5884 μg kg⁻¹ for AFB₁ and 1968 μg kg⁻¹ for AFB₂. Overall, most of the aflatoxigenic strains produced higher levels of AFB₁ than AFB₂ in rice. The obtained information is useful for assessing the risk of aflatoxin contamination in rice samples.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).     

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.     

Use of a granular bioplastic formulation for carrying conidia of a non-aflatoxigenic strain of Aspergillus flavus

Previous research demonstrated that aflatoxin contamination in corn is reduced by field application of wheat grains pre-inoculated with the non-aflatoxigenic Aspergillus flavus strain NRRL 30797. To facilitate field applications of this biocontrol isolate, a series of laboratory studies were conducted on the reliability and efficiency of replacing wheat grains with the novel bioplastic formulation Mater-Bi^® to serve as a carrier matrix to formulate this fungus. Mater-Bi^® granules were inoculated with a conidial suspension of NRRL 30797 to achieve a final cell density of approximately log 7 conidia/granule. Incubation of 20-g soil samples receiving a single Mater-Bi^® granule for 60-days resulted in log 4.2–5.3 propagules of A. flavus/g soil in microbiologically active and sterilized soil, respectively. Increasing the number of granules had no effect on the degree of soil colonization by the biocontrol fungus. In addition to the maintenance of rapid vegetative growth and colonization of soil samples, the bioplastic formulation was highly stable, indicating that Mater-Bi^® is a suitable substitute for biocontrol applications of A. flavus NRRL 30797.     

Effect of Inoculum Rate of Biological Control Agents on Preharvest Aflatoxin Contamination of Peanuts

Studies were conducted during 1994 and 1995 in the environmental control plot facility at the National Peanut Research Laboratory to determine the effect of different inoculum rates of biological control agents on preharvest aflatoxin contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants ofAspergillus flavusandAspergillus parasiticusthat were grown on rice for use as soil inoculum. Three replicate plots (4.0 × 5.5 m) were treated with 0, 2, 10, and 50 g/m of row (0, 20, 100, and 500 lb/acre, respectively) of an equal mixture of the color mutant-infested rice in 1994, and the same plots were retreated in 1995. Aflatoxin concentrations were determined by high performance liquid chromatographic analysis of all peanuts. Treatment means for total kernels in 1994 were 337.6, 73.7, 34.8, and 33.3 ppb for the 0, 2, 10, and 50 g/m treatments, respectively. Regression analysis indicated a trend toward lower aflatoxin concentrations with increasing rates of inoculum (R²= 0.40;P< 0.05). For the same repeated treatments in 1995 aflatoxin concentrations in total kernels averaged 718.3, 184.4, 35.9, and 0.4 ppb. Regression analysis revealed a stronger relationship between inoculum rate and aflatoxin concentrations (R²= 0.66;P< 0.05) in the second year of treatment. Compared with untreated controls, the 2, 10, and 50 g/m treatments produced respective reductions in aflatoxin of 74.3, 95.0, and 99.9% in the second year. The data indicated not only a treatment-related effect, but also that a higher degree of control might be achieved when plots or fields are retreated with biocontrol agents in subsequent years.     

Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir

An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B₁and B₂ in varying amounts. Aflatoxins G₁ and G₂ were not detected. All 25 of the investigated market samples were also found to be aflatoxin B₁ positive and the level of contamination ranged from 96 to 8164 g/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore,monitoring of aflatoxins in dry fruit slices of quincesis recommended for this region.This revised version was published online in October 2005 with corrections to the Cover Date.     

Recombination and lineage‐specific gene loss in the aflatoxin gene cluster of Aspergillus flavus

Aflatoxins produced by Aspergillus flavus are potent carcinogens that contaminate agricultural crops. Recent efforts to reduce aflatoxin concentrations in crops have focused on biological control using nonaflatoxigenic A. flavus strains AF36 (=NRRL 18543) and NRRL 21882 (the active component of afla‐guard^®). However, the evolutionary potential of these strains to remain nonaflatoxigenic in nature is unknown. To elucidate the underlying population processes that influence aflatoxigenicity, we examined patterns of linkage disequilibrium (LD) spanning 21 regions in the aflatoxin gene cluster of A. flavus. We show that recombination events are unevenly distributed across the cluster in A. flavus. Six distinct LD blocks separate late pathway genes aflE, aflM, aflN, aflG, aflL, aflI and aflO, and there is no discernable evidence of recombination among early pathway genes aflA, aflB, aflC, aflD, aflR and aflS. The discordance in phylogenies inferred for the aflW/aflX intergenic region and two noncluster regions, tryptophan synthase and acetamidase, is indicative of trans‐species evolution in the cluster. Additionally, polymorphisms in aflW/aflX divide A. flavus strains into two distinct clades, each harbouring only one of the two approved biocontrol strains. The clade with AF36 includes both aflatoxigenic and nonaflatoxigenic strains, whereas the clade with NRRL 21882 comprises only nonaflatoxigenic strains and includes all strains of A. flavus missing the entire gene cluster or with partial gene clusters. Our detection of LD blocks in partial clusters indicates that recombination may have played an important role in cluster disassembly, and multilocus coalescent analyses of cluster and noncluster regions indicate lineage‐specific gene loss in A. flavus. These results have important implications in assessing the stability of biocontrol strains in nature.     

Aflatoxin B1 producing potential of isolates of Aspergillus flavus Link ex Fries from cotton,maize and wheat

Twenty-one isolates ofAspergillus flavus Link ex Fries obtained from cotton, maize and wheat were screened for their ability to produce aflatoxins on two liquid media. Of these, sixteen isolates were toxigenic and produced only aflatoxin B₁ as assessed by bioassay on okra seedlings and TLC method. For screening isolates ofA. flavus for aflatoxin formation, 0.7 % YES+ Salt medium was found to be good as also for obtaining higher yields of the toxin. Isolates ofA. flavus produced aflatoxin B₁ ranging from 0.85 to 17.2 mg/50 ml. Maximum yield of aflatoxin was obtained when rice was used as the substrate in case of toxigenic isolates L-27 and C-9, and on maize in isolate M-11.     

Changes in moisture content,mycoflora and aflatoxin content of rice bran during storage

The changes in moisture content, storage mycoflora and aflatoxin B₁ (AFB₁) in bran from untreated or raw rice (Rr) and parboiled rice (Pbr) stored in small lots in polyethylene bags were studied at 15-day intervals up to 60 days, using five lots of each type of bran. Deterioration was more rapid with reference to all the three parameters, in Rr bran compared to Pbr bran, the former becoming completely overgrown and caked with fungi by the end of 60 days.Aspergillus flavus was the dominant fungus in Pbr bran, whereasA. candidus andTrichoderma viride were abundant in Rr bran. The frequency of incidence as well as concentration of AFB₁ increased with storage time in both types of bran, but the rate of increase as well as overall concentration were much higher in Rr bran. Thus raw rice bran is unsuitable for prolonged storage.Abbreviations AFB₁ aflatoxin B₁ - MC moisture content - Pbr parboiled rice - Rr raw rice     

Manganese Carbonate Precipitation Induced by Calcite-Forming Bacteria

Abstract

Several dissimilatory metal-reducing bacteria and a halophilic bacterium are able to induce manganese carbonate (rhodochrosite) precipitation. In this study, it was revealed that Ensifer adhaerens JCM 21105^T, Microbacterium testaceum JCM 1353^T, Pseudomonas protegens DSM 19095^T, and Rheinheimera texasensis DSM 17496^T, which are calcite-forming bacteria, were able to aerobically induce the precipitation of manganese carbonate crystals on an agar medium. In the case of all four strains, the principal morphology of the precipitated manganese carbonate crystals was that of micro-sized spheres, when they were aerobically cultivated over the entire surface of the agar medium at 28?°C for 7?days.