Effects of soil moisture and temperature on preharvest invasion of peanuts by the Aspergillus flavus group and subsequent aflatoxin development.

Four soil temperature and moisture treatment regimens were imposed on Florunner peanuts 94 days after planting in experimental plots in 1980. At harvest (145 days after planting), the incidence of the Aspergillus flavus group and the aflatoxin concentration were greatest in damaged kernels. Extensive colonization of sound mature kernels (SMK) by the A. flavus group occurred with the drought stress treatment (56% kernels colonized); colonization was less in the irrigated plot (7%) and the drought stress plot with cooled soil (11%) and was intermediate in the irrigated plot with heated soil (26%). Aflatoxin was virtually absent from SMK with the last three treatments, but it was found at an average concentration of 244 ppb (ng/g) in drought-stressed SMK. Colonization of SMK by the A. flavus group and aflatoxin production were greater with hot dry conditions. Neither elevated temperature alone nor drought stress alone caused aflatoxin contamination in SMK. When the ratio of SMK colonized by A. flavus compared with A. niger was greater than 19:1, there was aflatoxin contamination, but there was none if this ratio was less than 9:1. Irrigation caused a higher incidence of A. niger than drought did. This may have prevented the aflatoxin contamination of undamaged peanuts.     

Influence of Fungicides and Irrigation Practice on Aflatoxin in Peanuts Before Digging

Peanuts grown under dryland conditions where drought stress occurred accumulated more aflatoxin before digging than peanuts grown under irrigation. Kernels became more susceptible to Aspergillus flavus and A. parasiticus invasion when the soil moisture in the pod zone approached levels at which moisture moved from the pod into the soil and the kernel moisture dropped below 31%. Isolation frequencies of these aspergilli from fresh-dug kernels were lowest in 1968 (maximum of 3%). In 1967 and 1969, maximum percentages of 100 and 74, respectively, were noted. Kernel infestation was correlated with degree of aflatoxin contamination. Dryland fresh-dug kernels contained a maximum of 35,800 parts per billion aflatoxin while a maximum of 50 parts per billion was detected in kernels from irrigated plots. In 1969 A. flavus infestation was as high as 59% in peanuts from irrigated plots; however, no aflatoxin was detected. Absence of aflatoxin in these samples is attributed to the higher kernel moisture content which reduced the aflatoxin-producing potential of A. flavus. Statistical analysis of the data revealed no significant differences in degree of fungal infestation, production levels, and grade factors between any fungicide treatments.     

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.     

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.     

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.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Color mutants of Aspergillus flavus and Aspergillus parasiticus in a study of preharvest invasion of peanuts.

A comparison of the invasion of flowers, aerial pegs, and kernels by wild-type and mutant strains of Aspergillus flavus or A. parasiticus along with aflatoxin analyses of kernels from different drought treatments have supported the hypothesis that preharvest contamination with aflatoxin originates mainly from the soil. Evidence in support of soil invasion as opposed to aerial invasion was the following. A greater percentage of invasion of kernels rather than flower or aerial pegs by either wild-type A. flavus or mutants. Significant invasion by an A. parasiticus color mutant occurred only in peanuts from soil supplemented with the mutant, whereas adjacent plants in close proximity but in untreated soil were only invaded by wild-type A. flavus or A. parasiticus. Aflatoxin data from drought-stressed, visibly undamaged peanut kernels showed that samples from soil not supplemented with a mutant strain contained a preponderance of aflatoxin B's (from wild-type A. flavus) whereas adjacent samples from mutant-supplemented soil contained a preponderance of B's plus G's (from wild-type and mutant A. parasiticus). Preliminary data from two air samplings showed an absence of propagules of A. flavus or A. parasiticus in air around the experimental facility.     

Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia.

The effect of corn and peanut cultivation on the proportion of Aspergillus flavus to A. parasiticus in soil was examined. Soil populations were monitored in three fields during three different years in southwestern Georgia. Each field was planted in both peanuts and corn, and soil was sampled within plots for each crop. A. flavus and A. parasiticus were present in similar proportions in plots from all fields at the beginning of the growing season. A. terreus, A. niger, and A. fumigatus were the other dominant aspergilli in soil. Fields A and B did not show drought stress in peanut or corn plants, and soil populations of A. flavus and A. parasiticus remained stable during the course of the year. In field C, drought stress in corn plants with associated A. flavus infection and aflatoxin contamination greatly increased soil populations of A. flavus relative to A. parasiticus upon dispersal of corn debris to the soil surface by a combine harvester. Colonization of organic debris after it has been added to the soil may maintain soil populations of A. parasiticus despite lower crop infection.     

Effect of soil temperature and drought on peanut pod and stem temperatures relative to Aspergillus flavus invasion and aflatoxin contamination

Peanut stem and pod temperatures of plants growing in irrigated, drought, drought-heated soil, and drought-cooled soil treatments were determined near the end of the growing season. Mean soil temperatures of the treatments during this period were 21.5°, 25.5°, 30° and 20 °C, respectively. Peanut stem temperatures in all drought treatments reached a maximum of ca. 40 °C and for 6–7 h each day were as much as 10 °C warmer than irrigated peanut stems. Pod temperatures in drought-heated soil and drought treatments were ca. 34 °C and 30 °C, respectively, for several hours each day. As pod temperatures approached the optimum for A. flavus growth (ca. 35 °C), the proportion of kernels colonized and aflatoxin concentrations increased. Increased plant temperature without accompanying pod temperature increases (drought-cooled soil) resulted in colonization percentages and aflatoxin concentrations only slightly higher than those of the irrigated peanuts.     

Colonization of wounded peanut seeds by soil fungi: selectivity for species from Aspergillus section Flavi

Soil is a source of primary inoculum for Aspergillus flavus and A. parasiticus, fungi that produce highly carcinogenic aflatoxins in peanuts. Aflatoxigenic fungi commonly invade peanut seeds during maturation, and the highest concentrations of aflatoxins are found in damaged seeds. A laboratory procedure was developed in which viable peanut seeds were wounded and inoculated with field soil containing natural populations of fungi, then incubated under different conditions of seed water activity and temperature. Densities of Aspergillus section Flavi in soil used for inoculating seeds were low relative to the total numbers of filamentous fungi (<1%). Aspergillus species from section Flavi present in soil included A. flavus morphotypes L and S strains, A. parasiticus, A. caelatus, A. tamarii and A. alliaceus. Wounding was required for high incidences of fungal colonization; viability of wounded seeds had little effect on colonization by Aspergillus species. Peanut seeds were colonized by section Flavi species as well as A. niger over broad ranges of water activity (0.82-0.98) and temperature (15-37 C), and the highest incidences of seed colonization occurred at water activities of 0.92-0.96 at 22-37 C. A. parasiticus colonized peanut seeds at lower temperatures than A. flavus, and cool soil temperatures relative to temperatures of aerial crop fruits might explain why A. parasiticus is found mostly in peanuts. Other fungi, dominated by the genera Penicillium, Fusarium and Clonostachys, colonized seeds primarily at water activities and temperatures suboptimal for section Flavi species and A. niger. Eupenicillium ochrosalmoneum frequently sporulated on the conidial heads of section Flavi species and showed specificity for these fungi. The inoculation of wounded viable peanut seeds with soil containing natural populations of fungi provides a model system for studying the infection process, the interactions among fungi and those factors important in aflatoxin formation.     

Factors influencing the inhibition of aflatoxin production in corn by Aspergillus niger

Aspergillus niger, a mold commonly associated with Aspergillus flavus in damaged corn, interferes with the production of aflatoxin when grown with A. flavus on autoclaved corn. The pH of corn-meal disks was adjusted using NaOH-HCl, citric acid-sodium citrate, or a water extract of A. niger fermented corn. Aflatoxin formation was completely inhibited below pH 2.8-3.0, irrespective of the system used for pH adjustment. When grown in association with A. flavus NRRL 6432 on autoclaved corn kernels, A. niger NRRL 6411 lowered substrate pH sufficiently to suppress aflatoxin production. The biodegradation of aflatoxin B1 or its conversion to aflatoxin B2a were eliminated as potential mechanisms by which A. niger reduces aflatoxin contamination. A water extract of corn kernels fermented with A. niger caused an additional inhibition of aflatoxin formation apart from the effects of pH.     

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.     

Interrelationship of kernel water activity,soil temperature,maturity, and phytoalexin production in preharvest aflatoxin contamination of drought-stressed peanuts

Samples of Florunner peanuts were collected throughout a period of late-season drought stress with mean geocarposphere temperatures of 29 and 25 °C, and determinations of maturity, kernel water activity (a_w), percent moisture, capacity for phytoalexin production, and aflatoxin contamination were made. Results showed an association between the loss of the capacity of kernels to produce phytoalexins and the appearance of aflatoxin contamination. Kernel a_w appeared to be the most important factor controlling the capacity of kernels to produce phytoalexins. Mature peanuts possessed additional resistance to contamination that could not be attributed solely to phytoalexin production. Kernel moisture loss was accelerated in the 29 °C treatment compared to the 25 °C treatment, and data indicated that the higher soil temperature also favored growth and aflatoxin production by Aspergillus flavus in peanuts susceptible to contamination.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Root vs pod infection by root-knot nematodes on aflatoxin contamination of peanut

Aflatoxins are potent carcinogens produced by some Aspergillus spp. Infection of peanut (Arachis hypogaea) by root-knot nematodes (Meloidogyne arenaria) can lead to an increase in aflatoxin contamination of kernels when the plants are subjected to drought stress during pod maturation. It is not clear whether the increased aflatoxin contamination is primarily due to greater invasion of the galled pods by toxigenic Aspergillus spp. or whether root galling is also involved. Our objective was to determine the contribution of root and pod galling caused by root-knot nematodes to the increase in aflatoxin contamination in peanut. Two greenhouse experiments were conducted in which pods and roots were physically separated. Pod set was restricted to soil-filled pans (41 cm dia. x 10 cm depth), while the roots grew underneath the pan into a pot. The experiments had a factorial arrangement of treatments: pod zone with and without nematodes, and root zone with and without nematodes. In Experiment 1, 5000 eggs of M. arenaria were added to the root zone14 days after planting (DAP) and 8000 eggs were added to the pod zone 60 and 80 DAP. In Experiment 2, 3000 eggs were added to the root zone 30 DAP and 8000 eggs were added to the pod zone every week starting 60 DAP. The four treatment combinations were replicated 10 to 13 times. Conidia of Aspergillus flavus/A. parasiticus was added to the soil surface (pods zone) at mid bloom. Plants were subjected to drought stress 40 days before harvest. In Experiment 1, adding nematodes to the pod zone had no effect on aflatoxin concentrations in the peanut kernel. However, the lack of an effect may have been to due to the low occurrence of galling on the hulls. In pots where nematodes were added to the root zone, 50 to 80% of the root system was galled. Adding nematodes to the root zone increased aflatoxin concentrations in the peanut kernels from 34 ppb in the control to 71 ppb. In Experiment 2, there was heavy pod galling with galls present on 53% of the pods. Adding nematodes to the pod zone increased aflatoxin concentrations in the kernels from 19 ppb in the control to 572 ppb. Based on the results of the two experiments, it appears that infection of either the roots or pods by M. arenaria can lead to greater aflatoxin contamination of peanut kernels.     

Mean geocarposphere temperatures that induce preharvest aflatoxin contamination of peanuts under drought stress

Apparently undamaged peanuts grown under environmental stress in the form of drought and heat become contaminated with Aspergillus flavus and aflatoxin in the soil prior to harvest. The upper mean temperature limit for aflatoxin contamination in undamaged peanut kernels grown under drought stress the latter 4–6 weeks of the growing season was between 29.6–31.3°C. The lower limit was between 25.7–26.3°C. That is, peanuts grown under drought stress with a mean geocarposphere temperature of 29.6°C were highly contaminated while those at 31.3°C were not contaminated. Likewise, those grown under drought stress with a mean geocarposphere temperature of 25.7°C were not contaminated while those subjected to a mean geocarposphere temperature of 26.0°C resulted in some categories becoming contaminated. Increasing the mean temperature up to 29.6°C caused increasing amounts of contamination.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.     

Drought Stress and Preharvest Aflatoxin Contamination in Agricultural Commodity: Genetics, Genomics and Proteomics

Throughout the world, aflatoxin contamination is considered one of the most serious food safety issues concerning health. Chronic problems with preharvest aflatoxin contamination occur in the southern US, and are particularly troublesome in corn, peanut, cottonseed, and tree nuts. Drought stress is a major factor to contribute to preharvest afiatoxin contamination. Recent studies have demonstrated higher concentration of defense or stress-related proteins in corn kernels of resistant genotypes compared with susceptible genotypes, suggesting that preharvest field condition (drought or not drought) influences gene expression differently In different genotypes resulting in different levels of "end products": PR(pathogenesis-related) proteins in the mature kernels. Because of the complexity of Aspergillus-plant interactions, better understanding of the mechanisms of genetic resistance will be needed using genomics and proteomics for crop improvement. Genetic Improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy. Proteomic comparisons of corn kernel proteins between resistant or susceptible genotypes to Aspergillus flavus infection have identified stress-related proteins along with antifungal proteins as associated with kernel resistance. Gene expression studies in developing corn kernels are In agreement with the proteomic studies that defense-related genes could be upregulated or downregulated by abiotic stresses.     

Deciphering drought‐induced metabolic responses and regulation in developing maize kernels

Drought stress conditions decrease maize growth and yield, and aggravate preharvest aflatoxin contamination. While several studies have been performed on mature kernels responding to drought stress, the metabolic profiles of developing kernels are not as well characterized, particularly in germplasm with contrasting resistance to both drought and mycotoxin contamination. Here, following screening for drought tolerance, a drought‐sensitive line, B73, and a drought‐tolerant line, Lo964, were selected and stressed beginning at 14 days after pollination. Developing kernels were sampled 7 and 14 days after drought induction (DAI) from both stressed and irrigated plants. Comparative biochemical and metabolomic analyses profiled 409 differentially accumulated metabolites. Multivariate statistics and pathway analyses showed that drought stress induced an accumulation of simple sugars and polyunsaturated fatty acids and a decrease in amines, polyamines and dipeptides in B73. Conversely, sphingolipid, sterol, phenylpropanoid and dipeptide metabolites accumulated in Lo964 under drought stress. Drought stress also resulted in the greater accumulation of reactive oxygen species (ROS) and aflatoxin in kernels of B73 in comparison with Lo964 implying a correlation in their production. Overall, field drought treatments disordered a cascade of normal metabolic programming during development of maize kernels and subsequently caused oxidative stress. The glutathione and urea cycles along with the metabolism of carbohydrates and lipids for osmoprotection, membrane maintenance and antioxidant protection were central among the drought stress responses observed in developing kernels. These results also provide novel targets to enhance host drought tolerance and disease resistance through the use of biotechnologies such as transgenics and genome editing.     

Aspergillus flavus and other mycoflora of groundnut kernels in Israel and the absence of aflatoxin

More than 300 groundnut (peanut) samples collected from different regions of Israel were examined by ELISA for aflatoxin contamination. Samples were designated for export, local consumption or for sowing. None of the samples were contaminated with the toxin. However, when kernels were kept at high humidity (RH?99%), aflatoxin could be frequently detected seven days after incubation and the toxin was not uniformly distributed among kernels.Aspergillus niger, A flavus, Penicillium citrinum andP pinophilum were the dominant fungi and no differences were observed among cultivars. Almost half of the commercial samples examined were devoid ofA flavus. Other fungi identified wereA tamaril, A amstelodami, P rubrum, Rhizoctonia solani, Macrophomina phaseolina, Rhizopus spp., Sclerotium rolfsll, Fusarium andAlternaria spp; the two last ones comprising a group of low incidence. Although groundnut samples that containA flavus—infected kernels are moderately common, the local climate and agrotechniques In use in Israel are not conducive to aflatoxin accumulation. Nevertheless infected kernels may become a threat to health if stored under inadequate conditions.