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.     

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.     

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.     

Factors Influencing Aflatoxin Accumulation in Peanut Kernels and the Associated Mycoflora

Accumulation of aflatoxin in Spanish peanut kernel samples from different geographical areas in Texas during 1966, as detected by the thin-layer chromatographic method, was relatively low. Analysis of samples obtained from growers using artificial drying equipment (forced air and supplemental heat), when windrow conditions were unfavorable for rapid drying, suggests that this practice reduces the possibility of aflatoxin accumulation. In general, peanuts harvested from land planted to peanuts the previous year were more highly infested with fungi and contained more aflatoxin than peanuts grown on land planted with rye, oats, melons, or potatoes the previous year. Aflatoxin incidence tended to decrease from south to north Texas. These findings verify previous research observations that moist tropical climates are conducive to fungal infestation and aflatoxin accumulation. Detection of aflatoxin in sound mature kernels (kernels screened for minimal size) indicates that the practice of screening for removal of small immature kernels and removal of obviously damaged kernels does not completely eliminate aflatoxin contamination.     

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.     

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.     

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.     

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.     

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-producing Potential of Isolates of the Aspergillus flavus-oryzae Group from Peanuts (Arachis hypogaea)

Seventy-eight samples of farmer stock peanuts, representing peanuts grown in nine different geographical areas during 1964, were assayed for aflatoxin and examined for associated microflora. Only two samples contained more than 50 ppb of aflatoxin. Infestation by members of the Aspergillus flavus-oryzae group varied from 35 to 100% of the kernels per area and from 1 to 100% of the kernels per sample. Aflatoxin production by individual isolates ranged from 0 to 349,143 ppb under the test conditions employed. In general, the isolates produced 8 to 10 times more B₁ than B₂, and no isolate producing aflatoxins G₁ or G₂ was found. The importance of proper postharvest handling of peanuts is emphasized by the prevalence of isolates of A. flavus-oryzae capable of producing aflatoxins on farmers stock peanuts.     

Uptake and partitioning of cadmium by cultivars of peanut (Arachis hypogaea L.)

Cadmium has been found to accumulate in peanut (Arachis hypogaea) kernels to levels exceeding the current maximum permitted concentration in Australia of 0.1 mg kg^-1. Little is known of the mechanisms of Cd uptake into kernels by cultivars of peanut, so the aims of the experiments reported here were to determine if Cd is absorbed directly through the pod wall or via the main root system, and if differences exist between cultivars in this respect. Split-pot soil and sand/nutrient solution experiments were performed with two cultivars of peanut (cv. NC7 and Streeton) known to accumulate Cd to different levels in the kernel. The growth medium was separated into pod and root zones with Cd concentrations in each zone varied. In confirmation of previous field trial results, cv. NC7 had higher concentrations of Cd in kernels, given the same Cd levels in the external medium (solution or soil). Despite total Cd uptake by cv. NC7 being similar to cv. Streeton, cv. NC7 appeared to retain more Cd in the roots and translocate less Cd to shoots. Results from both soil and sand/solution culture indicated that the dominant path of Cd uptake by peanut was via the main root system, with direct pod uptake contributing less than 5% of the total Cd in the kernel. There was little difference between cultivars in this characteristic. This indicates that unlike Ca nutrition of peanuts, agronomic techniques to manage Cd uptake will require modification of soil to the full depth of root exploration, rather than just the surface strata where pods develop. Cadmium concentrations in testa were up to an order of magnitude higher than in the kernel, indicating that blanching of kernels would be effective in reducing Cd in the marketed product. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aflatoxin contamination of developing corn kernels

Preharvest of corn and its contamination with aflatoxin is a serious problem. Some environmental and cultural factors responsible for infection and subsequent aflatoxin production were investigated in this study. Stage of growth and location of kernels on corn ears were found to be one of the important factors in the process of kernel infection with A. flavus & A. parasiticus. The results showed positive correlation between the stage of growth and kernel infection. Treatment of corn with aflatoxin reduced germination, protein and total nitrogen contents. Total and reducing soluble sugar was increase in corn kernels as response to infection. Sucrose and protein content were reduced in case of both pathogens. Shoot system length, seeding fresh weigh and seedling dry weigh was also affected. Both pathogens induced reduction of starch content. Healthy corn seedlings treated with aflatoxin solution were badly affected. Their leaves became yellow then, turned brown with further incubation. Moreover, their total chlorophyll and protein contents showed pronounced decrease. On the other hand, total phenolic compounds were increased. Histopathological studies indicated that A. flavus & A. parasiticus could colonize corn silks and invade developing kernels. Germination of A. flavus spores was occurred and hyphae spread rapidly across the silk, producing extensive growth and lateral branching. Conidiophores and conidia had formed in and on the corn silk. Temperature and relative humidity greatly influenced the growth of A. flavus & A. parasiticus and aflatoxin production.     

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.     

Evaluation of biological control formulations to reduce aflatoxin contamination in peanuts

A two-year study was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of Aspergillus flavus and Aspergillus parasiticus to reduce preharvest aflatoxin contamination of peanuts. Formulations included: (1) solid-state fermented rice; (2) fungal conidia encapsulated in an extrusion product termed Pesta; (3) conidia encapsulated in pregelatinized corn flour granules. Formulations were applied to peanut plots in 1996 and reapplied to the same plots in 1997 in a randomized design with four replications, including untreated controls. Analysis of soils for A. flavus and A. parasiticus showed that a large soil population of the nontoxigenic strains resulted from all formulations. In the first year, the percentage of kernels infected by wild-type A. flavus and A. parasiticus was significantly reduced in plots treated with rice and corn flour granules, but it was reduced only in the rice-treated plots in year two. There were no significant differences in total infection of kernels by all strains of A. flavus and A. parasiticus in either year. Aflatoxin concentrations in peanuts were significantly reduced in year two by all formulation treatments with an average reduction of 92%. Reductions were also noted for all formulation treatments in year one (average 86%), but they were not statistically significant because of wide variation in the aflatoxin concentrations in the untreated controls. Each of the formulations tested, therefore, was effective in delivering competitive levels of nontoxigenic strains of A. flavus and A. parasiticus to soil and in reducing subsequent aflatoxin contamination of peanuts.     

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.