Cytochemical localization and ultrastructure of Aspergillus flavus in cottonseed

Cottonseeds having fluorescent fibers were harvested from fields in Arizona and examined utilizing light microscopy and transmission electron microscopy. The occurrence of fluorescent fibers indicated that seeds had been infected by Aspergillus flavus during development. Presence of A. flavus was verified by plating portions of seeds with fluorescent fibers. Hyphae, conidial heads, and conidia were identified readily in differentially-stained cotyledon tissue processed for light microscopy. Utilization of transmission electron microscopy permitted observations on lignified seed coats and cotyledons of mature cottonseeds. Hyphae were located throughout the cotyledon and in the nonlignified layers of the seed coat. The identification of hyphae in cross sections of vessel elements within the seed coat provided ultrastructural evidence supporting the hypothesis that A. flavus may enter seeds via the vascular tissue. Controls for the microscopy studies included observations on cottonseeds with no visual signs of infection and on laboratory-grown cultures of A. flavus. These observations demonstrated that the hyphae localized within fluorescent seeds had features characteristic of A. flavus and that fungal-like structures do not occur within uninfected seeds.     

Preharvest seed infection byAspergillus flavus group fungi and subsequent aflatoxin contamination in groundnuts in relation to soil types

Preharvest seed infection byAspergillus flavus and aflatoxin contamination in selected groundnut genotypes (fourA. flavus-resistant and fourA. flavus-susceptible) were examined in different soil types at several locations in India in 1985–1990. Undamaged mature pods were sampled at harvest and seed examined forA. flavus infection and aflatoxin content in two or more trials at ICRISAT Center on light sandy soils and red sandy loam soils (Alfisols), and on Vertisols, at Anantapur on light sandy soils, and at Dharwad and Parbhani on Vertisols. Rainy season trials (1985–1989) were all rainfed. Post-rainy season trials were irrigated; late-season drought stress (90 days after sowing (DAS) until harvest at 125 DAS) was imposed in the 1987/88 and 1989/90 seasons.A. flavus infection and aflatoxin contamination levels were much lower in seed of all genotypes from Vertisols than in seed from Alfisols across locations and seasons. Vertisols also had significantly lower populations ofA. flavus than Alfisols. There were no marked differences between light sandy soils and red sandy loam soils (Alfisols) in respect of seed infection byA. flavus and aflatoxin contamination. Significant interactions between genotypes and soil types were evident, especially in theA. flavus-susceptible genotypes. Irrespective of soil types,A. flavus-resistant genotypes showed lower levels of seed infection byA. flavus and other fungi than didA. flavus-susceptible genotypes. The significance of the low preharvest aflatoxin risk in groundnuts grown on Vertisols is highlighted.ICRISAT Journal Article No. JA 1122     

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Evaluation of bacteria and Trichoderma for biocontrol of pre-harvest seed infection by Aspergillus flavus in groundnut

Pre- and post harvest aflatoxin contamination of groundnut caused by Aspergillus flavus is a major problem in the semi-arid tropics. Fluorescent Pseudomonas, Bacillus and Trichoderma spp. potentially antagonistic to A. flavus were isolated from the geocarposphere (pod-zone) of groundnut and used successfully for the control of pre-harvest groundnut seed infection by A. flavus. In greenhouse and field experiments, inoculation of selected antagonistic strains on groundnut resulted in significant reduction of seed infection by A. flavus, and it also reduced >50% of the A. flavus populations (as cfu) in the geocarposphere of groundnut.     

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.     

Mycoflora and mycotoxin contamination of Roundup Ready soybean harvested in the Pampean Region, Argentina

A total of 89 freshly harvested soybean seed samples (Roundup Ready [transgenic] soybean cultivars) from the 2010/2011 crop season were collected from five locations in the Northern Pampean Region II, Argentina. These samples were analyzed for internal mycoflora, toxin production of isolated fungi, and for a range of mycotoxins. Mycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs) and ochratoxin A (OTA) was done by HPLC-FLD (high performance liquid chromatography with postcolumn fluorescence derivatization), alternariol and alternariol monomethyl ether with HPLC-UV (HPLC with UV detection), trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were analyzed by GC-ECD (gas chromatography with electron capture detector). Fungal colonization was more frequently found for samples from América, Saladillo and Trenque Lauquen than for samples from General Villegas and Trenel; a total of 1,401 fungal isolates were obtained from the soybean seeds. The most commonly identified fungal genera were Alternaria, Sclerotinia, Chaetomium, Cladosporium, Aspergillus, Penicillium, Phomopsis and Fusarium. Alternaria alternata, A.tenuissima, Aspergillus flavus, Penicillium citrinum, Fusarium verticillioides and F.semitectum were the predominant toxigenic fungal species. Mycotoxin production was confirmed for several isolates of toxigenic species, including Aspergillus flavus, A. parasiticus, Alternaria alternata, A.tenuissima, Fusarium graminearum, F semitectum and F. verticillioides. In particular, the percentage of mycotoxigenic Alternaria alternata (100 %), A.tenuissima (95 %) and aflatoxigenic strains of A. flavus (57 %) were remarkably high. Although none of the mycotoxins, AFs, ZEA, FBs, trichothecenes and OTA, were directly detected in samples of soybean seeds, the frequent presence of toxigenic fungal species indicates the risk of multiple mycotoxin contamination.     

Relationship between Meloidogyne arenaria and Aflatoxin Contamination in Peanut

Damaged and developing kernels of peanut (Arachis hypogaea) are susceptible to colonization by fungi in the Aspergillus flavus group which, under certain conditions, produces aflatoxins prior to harvest. Our objective was to determine whether infection of peanut roots and pods by Meloidogyne arenaria increases aflatoxin contamination of the kernels when peanut is subjected to drought stress. The experiment was a completely randomized 2-x-2 factorial with 6 replicates/treatment. The treatment factors were nematodes (plus and minus M. arenaria) and fungus (plus and minus A. flavus inoculum). The experiment was conducted in 2001 and 2002 in microplots under an automatic rain-out shelter. In treatments where A. flavus inoculum was added, aflatoxin concentrations were high (> 1,000 ppb) and not affected by nematode infection; in treatments without added fungal inoculum, aflatoxin concentrations were greater (P ≤ 0.05) in kernels from nematode-infected plants (1,190 ppb) than in kernels from uninfected plants (79 ppb). There was also an increase in aflatoxin contamination of kernels with increasing pod galling (r² = 0.83 in 2001, r² = 0.43 in 2002; P ≤ 0.04). Colonization of kernels by A. flavus increased with increasing pod galling (r² = 0.18; P = 0.04) in 2001 but not in 2002. Root-knot nematodes may have a greater role in enhancing aflatoxin contamination of peanut when conditions are not optimal for growth and aflatoxin production by fungi in the A. flavus group.     

First report of secondary infection on Senna alata (L.) Roxb caused by two Aspergillus species

The present note is on secondary fungal infection in pods and flower buds of Senna alata (L.) Roxb caused by Aspergillus niger and A. flavus from the Puducherry region, India. Aspergillus niger was isolated more frequently from affected pods and flowers than A. flavus. Senna alata is a medicinally important plant and this is the first report of on fungal infection on this species.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Effect of delayed harvest on contamination of pearl millet grain with mycotoxin-producing fungi and mycotoxins

The response to delayed harvest of fungal and mycotoxin contamination of grain of the pearl millet hybrid HGM 100 was examined in 1992 and 1993. Samples of grain were assayed from seven plantings at locations near Tifton, Georgia, USA. Grain was harvested at 30, 40, and 50 days after anthesis and evaluated for infection byFusarium species andAspergillus flavus, and mycotoxin contamination. Mean isolation frequencies ofF. semitectum (35.6%) andF. chlamydosporum (17.2%) increased linearly with delayed harvest.Fusarium moniliforme andF. equisiti were infrequently isolated (<0.5%) and did not increase in the grain when harvest was delayed. Low mean concentrations of zearalenone (0.17 ppm), nivalenol (0.42 ppm), and deoxynivalenol (0.01 ppm) were detected but were not affected by delayed harvest. Isolation frequencies ofF. chlamydosporum andF. equiseti were correlated (P=0.07) with levels of nivalenol.Aspergillus flavus was not isolated from the grain, and aflatoxin concentrations averaged 1.9 ppb.     

Aflatoxin contamination caused by natural fungal infection of preharvest corn

Summary Aflatoxin contamination of developing corn (Zea mays L.) kernels caused by natural infection byAspergillus flavus Link ex Fries was studied in hybrids developed for the U.S. corn belt and for the southern U.S. and grown at diverse locations in 1977. Planting dates were staggered to examine the effect of crop maturity on infection by the toxin-producing fungus. A broad range of toxin values was observed at harvest; some levels exceeded the highest that had been previously recorded in corn. The highest concentration of aflatoxin B₁ detected was 8030 ppb. Levels of toxin differed significantly among planting dates in Florida and Georgia; the second planting date at these locations contained the highest toxin levels. Elevated concentrations of toxin were characteristic of kernel samples from southern locations and southeast Missouri; at these locations samples from hybrids developed for the south had significantly lower levels of toxin than hybrids developed for the corn belt. Ears with heavy insect damage had higher toxin levels than ears with less evidence of insect attack.Mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

The occurrence of Aspergillus flavus in vegetative tissue of cotton plants and its relation to seed infection

Twenty-seven mature cotton bolls with Aspergillus flavus Link colonies naturally occurring on the surface of the boll or lint were collected in the field in Arizona along with their subtending stems and peduncles. Bolls inoculated through the carpel wall 30 days after anthesis were allowed to mature in the field and were collected in the same manner. The seed and stem and peduncle sections of each boll were surface-sterilized, plated on agar media and observed for A. flavus. Seventy-eight percent of the naturally contaminated bolls with A. flavus in the seed also had the fungus in the stem and peduncle, whereas only 31% of the naturally contaminated bolls with no A. flavus in the seed had the fungus in the stem or peduncle. This difference was significant (P=0.0125), indicating a positive relationship between seed infection and stem and peduncle infection. All of the bolls inoculated through the carpel wall had A. flavus in the seed, but only 11% of the stem and peduncle sections were infected, indicating that the fungus does not readily grow downward from the boll into the supporting stem or peduncle.This unidirectional pattern of movement (upward) was further substantiated in greenhouse experiments where cotton seedlings were inoculated at the cotyledonary leaf scar with A. flavus and plants were sequentially harvested, surface sterilized and plated. Aspergillus flavus was isolated from the cotyledonary leaf scar, flower buds, developing bolls, and stem sections in the upper portion of the plant. It was never isolated from roots or stem sections below the cotyledonary node, again indicating that the fungus does not readily move downward through the plant.     

Mycobiota of ground red pepper and their aflatoxigenic potential

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 10³ CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03?2.08 μg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88?909.53 μg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.     

Studies on the seed-borne phases of dark leaf spot Alternaria brassicicola and grey leaf spot Alternaria brassicaeof brassicas

Tests in Britain on samples of basic and commercial Brassica oleracea seed between 1976 and 1978 showed that many lots were infected with Alternaria brassicicola. A. brassicae was uncommon in basic seed in these years and in commercial seed harvested in 1976 and 1977 but was frequent in seed harvested in 1978. Most affected seeds were contaminated by surface-borne spores and mycelium of A. brassicicola but many were internally infected by the fungus situated within the seed-coat and in some seeds in the embryo tissues. Superficial contamination by the fungus declined rapidly after 2 yr in cabbage seeds stored at 10 °C, 50% r.h. but internal infection persisted for up to 12 yr. In some samples, internal infection was commonly associated with small shrivelled seeds. Surface contaminated and internally infected seeds transmitted the disease but seedling infection was more closely correlated with the latter.     

Quantitative trait locus mapping of resistance to Aspergillus flavus infection using a recombinant inbred line population in maize

Aflatoxin contamination of maize (Zea mays L.) grain caused by Aspergillus flavus is a serious health hazard to animals and humans. Development of maize varieties resistant to A. flavus infection and/or aflatoxin production can reduce this contamination. This study was conducted to identify quantitative trait loci (QTL) associated with resistance to A. flavus infection. A recombinant inbred line population was developed derived from RA, a maize inbred line resistant to A. flavus infection, and M53, a susceptible inbred line. After inoculation with A. flavus under controlled conditions, the kernels from each plant line grown in three different environments were evaluated for infection level. Categorical inoculation data were collected for each plant line based on the percentage of the kernel surface covered by fungal conidia. Significant genotypic variation in infection level was observed in all environments. Based on a genetic map containing 916 polymorphic simple sequence repeat and single nucleotide polymorphism markers, the resistance QTL were initially analyzed by composite interval mapping (CIM) separately for each environment. One QTL in bin 5.03 was detected in all environments, and seven other QTL were identified in one environment. Next, a mixed model based on CIM (MCIM) was employed for QTL analysis using data from the three environments simultaneously. Significant epistasis and epistasis × environment interaction to A. flavus infection were revealed. The QTL in bin 5.03 was repeatedly detected by the MCIM. This QTL explained the largest phenotypic variation among all of the detected QTL and could be considered as a major QTL for use in breeding for A. flavus resistance.     

The Degree of Susceptibility of Nectary-Inoculated Cotton Flowers and Bolls to Subsequent Seed Infection by Aspergillus flavus Is Determined at or before Anthesis

In greenhouse and field studies, cotton (Gossypium hirsutum L.) flowers were inoculated with Aspergillus flavus at the involucral nectaries. Bolls developing from early-season flowers had significantly higher percentages of A. flavus-infected seed than did bolls from flowers formed later in the season. Seeds from bolls inoculated 2 weeks after anthesis had the same infection levels as those from flowers inoculated at anthesis. These results indicate that early-season flowers are predisposed to A. flavus infection and that the degree of susceptibility at anthesis is retained through early boll development.     

Peanut Pod Invasion by Aspergillus flavus in the Presence of Meloidogyne hapla

''Argentine'', ''Early Runner'' and ''Florigiant'' peanut cultivars were grown in methyl bromide treated soil in field microplots inoculated with: (i) Aspergillus flavus or (ii) A. flavus + Meloidogyne hapla. Nematode infection produced heavy root galling and light pod galling equally on all cultivars. A. flavus, A. niger, Cephalosporium spp., Colletotrichum sp., Curvularia spp., Fusarium spp., Penicillium spp. and Trichoderma viride were isolated from shells and kernels. A significantly greater incidence and density of A. flavus was obtained from kernels of plants inoculated with both organisms than from kernels of plants receiving only the fungus. Differences were not significant, however, for incidence and density of A. flavus in shells or for the total of all fungal propagules in shells and kernels. Shells of ''Early Runner'' contained significantly greater incidence and density of A. flavus than the other two cultivars; also, kernels of this cultivar contained more fungal propagules than kernels of ''Argentine.'' A significantly larger number of total fungi was isolated from kernels of ''Argentine'' than from ''Florigiant.'' Aflatoxins were found only in two shell samples and not in kernels.     

Changes in secondary metabolites and fiber quality of cotton (Gossypium hirsutum) seed under consecutive water stress and in silico analysis of cellulose synthase and xyloglucan endotransglucosylase

Global warming has led to severe drought conditions. The selection of plant varieties that can withstand drought and produce increased yields are of utmost importance. In the current study, secondary metabolites, seed trait and fiber characteristic of cottonseeds (Gossypium hirsutum) exposed to double and third water stress exposure was investigated. Total phenol and tannin content in W₁S₃₃ increased significantly after third water stress exposure. Accumulation of wax was enhanced in seeds of W₃S₃₃ and W₃S₃₄ that were subjected to third water stress. Fiber quality parameters decreased when cottonseeds were rainfed. High irrigation resulted in fragile and delicate fiber. Seeds grown under 66% FC irrigation saved water and produced seeds that had the potential of producing high quality fibers. In silico analysis was performed on cellulose synthase A (CesA) and xyloglucan endotransglycosylase (XET) enzymes present in Gossypium hirsutum. The intracellular locations of the CesA and XET1 enzymes are the plasma membrane and cell wall, respectively. Proline is conserved in the C-terminal of the CesA enzyme and plays an important role in enzyme functionality. This study provides a better understanding as to the mechanisms by which the plant can tolerate and combat water stress conditions as well as reduce water consumption. In order to grow cotton seeds with desirable morphometric characteristics and optimal fibers under water stress exposure and in dry areas, it is better to use seeds that are irrigated under optimal irrigation conditions, ie 66% FC.     

Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 °C. Aspergillus was the most frequent genus and it emerged in 87–100% of the samples contributing 70–98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B₁ and B₂. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.