Increased susceptibility and reduced phytoalexin accumulation in drought-stressed peanut kernels challenged with Aspergillus flavus.

Three genotypes of peanut (Arachis hypogaea L.), with ICG numbers 221, 1104, and 1326, were grown in three replicate plots and drought stressed during the last 58 days before harvest by withholding irrigation water. Within each plot there were eight levels of stress ranging from 1.1 to 25.9 cm of water. Kernels harvested from the plots were hydrated to 20% moisture and challenged with Aspergillus flavus. Fungal colonization, aflatoxin content, and phytoalexin accumulation were measured. Fungal colonization of non-drought-stressed kernels virtually ceased by 3 days after inoculation, when the phytoalexin concentration exceeded 50 micrograms/g (fresh weight) of kernels, but the aflatoxin concentration continued to rise exponentially for an additional day. When fungal colonization, aflatoxin production, and phytoalexin accumulation were measured 3 days after drought-stressed material was challenged, the following relationships were apparent. Fungal colonization was inversely related to water supply (r varied from -0.848 to -0.904, according to genotype), as was aflatoxin production (r varied from -0.876 to -0.912, according to genotype); the phytoalexin concentration was correlated with water supply when this exceeded 11 cm (r varied from 0.696 to 0.917, according to genotype). The results are discussed in terms of the critical role played by drought stress in predisposing peanuts to infection by A. flavus and the role of the impaired phytoalexin response in mediating this increased susceptibility.     

Circumstantial evidence for phytoalexin involvement in the resistance of peanuts to Aspergillus flavus

Three stilbene phytoalexins, elicited by slicing and incubating imbibed peanut kernels under aerobic conditions, inhibited spore germination and hyphal extension of Aspergillus flavus with ED50 values in the range 4.9-12.8 micrograms ml-1. Phytoalexin yield was dependent on cultivar, conditions and duration of incubation after slicing, and crop history. The yield of phytoalexin from ten cultivars studied, after slicing and incubating at 25 degrees C for 24 h, ranged from 28 to 935 micrograms per g fresh weight and was negatively correlated with dry kernel colonization by A. flavus [r = -0.868 when plotted as 1n (phytoalexin concn) against 1n (percentage peanut colonization)]. When the incubation period was extended to 96 h there was no such correlation. Reduced phytoalexin yields were obtained when sliced kernels of one cultivar studied were incubated in water or at 37 degrees C, and no phytoalexin was obtained when the slices were incubated under nitrogen gas or frozen before aerobic incubation. Drought stress during pod development in four cultivars studied reduced phytoalexin yields of sliced kernels incubated at 25 degrees C for 24 h by 17-65% compared with non-stressed controls.     

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.     

Effect of geocarposphere temperature on pre-harvest colonization of drought-stressed peanuts by Aspergillus flavus and subsequent aflatoxin contamination

Florunner peanuts grown in research plots were subjected to 5 soil temperature and moisture treatment regimes resulting in A. flavus infestation and subsequent aflatoxin contamination in drought-stressed peanuts. Treatments imposed beginning 85 days after planting were drought, drought with heated soil and 3 drought treatments with cooled soil. The incidence of A. flavus in drought-stressed, unshelled, sound mature kernels (SMK) decreased with decreases in the mean 5 cm deep soil temperature. The incidence of A. flavus was greater in inedible categories and in damaged kernels than in SMK. The mean, threshold, geocarposphere temperature required for aflatoxin development during the latter part of the peanut growth cycle was found to be between 25.7° C and 27° C.     

Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates,collected from peanut fields in China

Aims: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Methods and Results: Analysis of random‐amplified polymorphic DNA and microsatellite‐primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Conclusions: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Significance and Impact of the Study: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.     

Rhizosphere salinity and phytoalexin accumulation in soybean

Summary Rhizosphere salinity decreased the capacity of soybean to accumulate a pterocarpanoid phytoalexin (glyceollin) in the stem in response toPhytophthora megasperma var.sojae. Rapid (48h) accumulation was depressed by NaCl, Na₂SO₄, CaCl₂ and MgSO₄ applications. Time-course accumulations was slowed by applications. Time-course accumulation was slowed by application of 0.131M NaCl. Glyceollin accumulation was also reduced in plants subjected to a period of high salinity stress (0.177M NaCl, 72 h) after a period of nonsalinized growth. Calcium chloride completely suppressed glyceollin accumulation in normally-resistant plants but no susceptibility to the fungus was observed.     

Conidial movement of nontoxigenic Aspergillus flavus and A. parasiticus in peanut fields following application to soil

The use of nontoxigenic strains of Aspergillus flavus and A. parasiticus in biological control effectively reduces aflatoxin in peanuts when conidium-producing inoculum is applied to the soil surface. In this study, the movement of conidia in soil was examined following natural rainfall and controlled precipitation from a sprinkler irrigation system. Conidia of nontoxigenic A. flavus and A. parasiticus remained near the soil surface despite repeated rainfall and varying amounts of applied water from irrigation. In addition, rainfall washed the conidia along the peanut furrows for up to 100 meters downstream from the experimental plot boundary. The dispersal gradient was otherwise very steep upstream along the furrows and in directions perpendicular to the peanut rows. The retention of biocontrol conidia in the upper soil layers is likely important in reducing aflatoxin contamination of peanuts and aerial crops such as corn and cottonseed. This revised version was published online in August 2006 with corrections to the Cover Date.     

土壤类型与作物基因型对花生籽实镉积累的影响

以花生主产区的棕壤和潮土为供试土壤,以种植面积最大的白沙1016、花育22和湛油27基因型花生(Arachis hypogaea)为供试作物,采用不添加Cd(对照)和添加Cd(1.5 mg· kg-1)处理进行盆栽试验,研究土壤类型和作物基因型对花生籽实Cd积累的影响.结果表明:两种土壤对照处理的3种基因型花生籽实Cd含量均低于国家食品安全标准,而Cd处理下均高于食品安全标准.同种土壤Cd处理3种基因型花生籽实Cd含量显著高于对照,不同基因型间表现为湛油27＞白沙1016＞花育22,棕壤花生籽实Cd含量及总量均高于潮土.对照处理3种基因型花生籽实Cd生物富集系数均大于1.0,Cd处理下多小于1.0,表明花生籽实对土壤中Cd的累积能力较强,土壤Cd含量进一步增加时,其生物富集能力降低.     

A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

S im , T.S., T eo , T heresa & S im , T.F. 1985. A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus. Journal of Applied Bacteriology 59 , 29–34.
All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/ A. parasiticus , of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

A note on the screening of dried shrimps, shrimp paste and raw groundnut kernels for aflatoxin-producing Aspergillus flavus

All of 33 samples of dried shrimps, shrimp paste, peanut butter and raw groundnut kernels were contaminated with fungi. Aspergillus and Penicillium spp. were the predominant types in dried shrimps and raw groundnut kernels but no Aspergillus spp. were present in peanut butter or shrimp paste samples. Among 81 Aspergillus isolates obtained from dried shrimps and raw groundnut kernels, 10 were A. flavus/A. parasiticus, of which five were potential aflatoxin-producing A. flavus strains. No aflatoxins were detected in the food samples although some were visibly mouldy and some had high mould counts. The occurrence of aflatoxin-producing strains of A. flavus in dried shrimps and raw groundnut kernels warrants further investigation of these foods and their products as potentially significant sources of aflatoxins.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous phylogenetic studies of A. flavus have shown that it consists of two subgroups, called groups I and II, and morphological studies indicated that it consists of two morphological groups based on sclerotium size, called "S" and "L." The industrially important non-aflatoxin-producing fungus A. oryzae is nested within group I. Three different gene regions, including part of a gene involved in aflatoxin biosynthesis (omt12), were sequenced in 33 S and L strains of A. flavus collected from various regions around the world, along with three isolates of A. oryzae and two isolates of A. parasiticus that were used as outgroups. The production of B and G aflatoxins and cyclopiazonic acid was analyzed in the A. flavus isolates, and each isolate was identified as "S" or "L" based on sclerotium size. Phylogenetic analysis of all three genes confirmed the inference that group I and group II represent a deep divergence within A. flavus. Most group I strains produced B aflatoxins to some degree, and none produced G aflatoxins. Four of six group II strains produced both B and G aflatoxins. All group II isolates were of the "S" sclerotium phenotype, whereas group I strains consisted of both "S" and "L" isolates. Based on the omt12 gene region, phylogenetic structure in sclerotium phenotype and aflatoxin production was evident within group I. Some non-aflatoxin-producing isolates of group I had an omt12 allele that was identical to that found in isolates of A. oryzae.     

Aspergillus flavus Infection and Aflatoxin Production in Fig Fruits

Immature fig fruits did not support colonization and aflatoxin production by Aspergillus flavus Lk. but became susceptible when ripe. While sun-drying on the tree, fruits were particularly vulnerable to fungal infection and colonization. Aflatoxin accumulation equaled levels frequently reported for such seeds as peanuts and cereal grains.     

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.