Biocontrol of Aspergillus flavus Strains Isolated from Bambara Groundnut (Vigna subterranea (L.) Verdc.) Seeds Using Essential Oils of Lippia multiflora Moldenke, Ocimum americanum L. (Lamiaceae) and Eucalyptus cameldulensis Dehnh

In nature, plant extracts play a crucial role in defending plants against biotic and abiotic stressors. Moreover, the use of plant-based products, such as plant extracts, represents a promising alternative to synthetic fungicides, which pose potential health risks to consumers. In this study, the antifungal activity of the essential oils (EOs) of Lippia multiflora, Eucalyptus camaldulensis and Ocimum americanum was evaluated against two strains of Aspergillus flavus via the agar dilution method. These two Aspergillus flavus fungi was isolated from Bamabra groundnut seeds. Lippia multiflora essential oil (EO) showed the best results compared with the other oils, with a minimum inhibitory concentration (MIC) of 9000 μg∙mL−1. The MIC for Eucalyptus camaldulensis and Ocimum americanum EOs was 10,800 μg∙mL−1. In view of their antifungal properties, these EOs could be used to develop a new, safe antifungal agent for food preservation.

Geographical distribution of Aspergillus flavus in peanut harvest period in China

In order to grasp the distribution of Aspergillus flavus in the soil of peanut production areas in China,A.flavus biomarkers were tested on 555 soil samples from 37 sampling points in 17 provinces,peanut fields in four agroecological zones(Southern area,Yangtze River Basin,Northern area,Northeast area).The results showed that(1)the cultivation amount of A.flavus per gram of soil in the Yangtze River Basin is 1.30 times that of the southern area,1.56 times that of the northern area,and 6.20 times that of the northeast area,with obvious regional characteristics.(2)In the Yangtze River basin,the change of longitude in the east-west direction has no direct impact on the cultivation amount of A.flavus per gram of soil.(3)In the east coast,the A.flavus cultivated per gram of soil increased first and then decreased with the increase of latitude from south to north.(4)A.flavus can be isolated in the soil samples above 1000 m.Field pollution is an important source of aflatoxin contamination in peanut.The study on the distribution of A.flavus in soil in China could provide theoretical support for the early warning and prevention and control measures of aflatoxin contamination in peanut.

Optimization of Solid-State Fermentation with Lactobacillus brevis and Aspergillus oryzae for Trypsin Inhibitor Degradation in Soybean Meal

The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.

Research on Correlation between Synthesis of Stilbenes and Aflatoxin Accumulation Caused by Aspergillus flavus in Peanut Seeds

In peanuts, a mechanism of resistance to fungal infection is reported on the synthesis of stilbene phytoalexins, which are antibiotic, low molecular weight metabolites. The phytoalexin-associated response of different peanut varieties to the inoculation with A. flavus has not been investigated and might be useful for breed- ing resistant peanut cultivars. In this study, peanut varieties resistant or susceptible to aflatoxin accumulation caused by A. flavus were selected to compare the synthe- sis differences of the four main stilbenes （resveratrol, ε-viniferin, 6-viniferin and pterostilbene） and the activities of their resistant enzymes （PAL, POD, PPO）, and explore the relationship between stilbene synthesis and resistance to aflatoxin accu- mulation. The results indicated that the synthesis speed of stilbenes was related to the resistance to aflatoxin accumulation in peanuts seeds. The total content of the four stilbenes reached the highest value of 47.37 μg/g in the resistant cultivars （Qian huasheng 3） after inoculation by A. flavus （54 times of the control） on the 3nd day, while the total content of stilbenes in susceptible cultivars （Huayu 22） was 5.5 μg/g. The content of stilbenes and the activities of their resistant enzymes from the resistant cultivars were both higher than from the susceptible one. The four peanut varieties with relative higher stilbene contents and lower disease index and aflatoxin content were selected by using the stilbene content. Very significant negative corre- lation, with correlation coefficients （r） of -0.789, -0.851 and -0.850, was observed between the stilbene content with disease index, the content of aflatoxin B1 and the total content of aflatoxins, respectively. Therefore, the study suggested that the total content of stilbene compounds might serve as an important chemical index in peanut seeds after inoculation with A. flavus on the 3nd day, during screening and breeding of peanut varieties resistant to aflatoxin accumulation.

Biocontrol potential of reducing aflatoxin contamination by an atoxigenic strain of Aspergillus flavus. isolated from peanut

An Aspergillus section/Zam isolate （ NAFFHB396） was isolated from a peanut kernel. It was identified as Aspergillus flavus based on morphology and molecular characteristics. It produced yellow to green - colored conidia on CYA medium and colonies with bright orange in color on AFPA medium. NAFFHB396 was grouped with A. flavus NRRL21882 and NRRL3357 by phylogenetic analysis of partial calmodulin sequence data. It was found that 12 genes were absent in aflatoxin gene cluster in NAFF- HB396. HPLC result further showed that it was an atoxigenic isolate. Co - inoculation of NAFFHB396 with a high aflatoxin producer AF2202 at the ratio of 1：1 both on CYA medium and peanut kernel resul-ted in reduction of aflatoxin production by 88.7% and 99. 8% respectively. These results suggested that the atoxigenic NAFFHB396 obtained in this study had a great potential to be a biocontrol agent to reduce aflatoxin contamination of peanut in China.

Effect of non-aflatoxigenic strains of Aspergillus flavus on aflatoxin contamination of pre-harvest peanuts in fields in China

Aflatoxin contamination of peanuts is one of the most concerns in peanut production in China.Applying nonaflatoxigenic Aspergillus flavus strains,based on competitive exclusion,has been proved to be a promising strategy to reduce aflatoxin contamination in pre-harvest peanuts.Two non-aflatoxigenic A.flavus strains collected in China,which have been proved effectively reducing aflatoxin in the laboratory,were mixed with high aflatoxin producer to the soil in peanut growing season.The two non-aflatoxigenic strains significantly(P<0.05)reduced aflatoxin contamination in peanut kernels under both normal and drought stresses in two fields.Compared to control,the total aflatoxin(sum of aflatoxin B1 and B2)was reduced 26.7–99.12%in field 1,and 84.96–99.33%in field 2.The aflatoxin was reduced 84.96–99.33%under drought stress in two fields.The present study indicated the non-aflatoxigenic A.flavus strains could be potential biocontrol agents for reducing aflatoxin contamination under field condition.

Genetic Analysis of the Resistance to Aspergillus flavus Infection in Maize (Zea mays L.)

Maize （Zea mays L.）, one of main crops in the world, is easily susceptible to Aspergillusflavus （Link： fr） infection, resulting huge loses worldwide. Breeding for A. flavus resistance has been proved an efficient way to solve the problem of aflatoxin contamination. Genetic analysis of the sources of resistance to A.flavus in maize is necessary for this purpose. The complete diallel crosses of 6 inbred lines with different resistance to A.flavus infection were implemented. Inoculation categorical data of each cross were analyzed with the additive-dominant and additive-dominant-epitasis genetic models. Results indicated some crosses fitted the 2 major genes with additive-dominant-epitasis genetic model. Others fitted the major gene and polygene mixed model. Moreover, the additive, dominant, and epitasis effects varied in crosses. The A.flavus resistance was controlled by both major gene and polygene.

Monitoring MVOC Profiles over Time from Isolates of Aspergillus flavus Using SPME GC-MS

Fungi produce a variety of microbial volatile organic compounds (MVOCs) during primary and secondary metabolism. The fungus, Aspergillus flavus, is a human, animal and plant pathogen which produces aflatoxin, one of the most carcinogenic substances known. In this study, MVOCs were analyzed using solid phase microextraction (SPME) combined with GCMS from two genetically different A. flavus strains, an aflatoxigenic strain, NRRL 3357, and a non-aflatoxigenic strain, NRRL 21882. A PDMS/CAR SPME fiber was used over 30 days to observe variations in MVOCs over time. The relative percentage of individual chemicals in several chemical classes (alcohols, aldehydes, esters, furans, hydrocarbons, ketones, and organic acids) was shown to change considerably during the varied fungal growth stages. This changing chemical profile reduces the likelihood of finding a single chemical that can be used consistently as a biomarker for fungal strain identification. In our study, discriminant analysis techniques were successfully conducted using all identified and quantified MVOCs enabling discrimination of the two A. flavus strains over the entire 30-day period. This study underscores the potential of using SPME GCMS coupled with multivariate analysis for fungi strain identification.

The Breeding of a Pigment Mutant Strain of Steroid Hydroxylation Aspergillus Flavus by Low Energy Ion Implantation

In the process of the fermentation of steroid C11α-hydroxylgenation strain Aspergillus flavus AF-ANo208, a red pigment is derived, which will affect the isolation and purification of the target product. Low energy ion beam implantation is a new tool for breeding excellent mutant strains. In this study, the ion beam implantation experiments were performed by infusing two different ions： argon ion （Ar^＋） and nitrogen ion （N^＋）. The results showed that the optimal ion implantation was N^＋ with an optimum dose of 2.08- 1015 ions/cm^2, with which the mutant strain AF-ANm16 that produced no red pigment was obtained. The strain had high genetic stability and kept the strong capacity of C11α-hydroxylgenation, which could be utilized in industrial fermentation. The differences between the original strain and the mutant strain at a molecular level were analyzed by randomly amplified polymorphic DNA （RAPD）. The results indicated that the frequency of variation was 7.00%, which would establish the basis of application investigation into the breeding of pigment mutant strains by low energy ion implantation.

Vanillin and its derivatives,potential promising antifungal agents,inhibit Aspergillus flavus spores via destroying the integrity of cell membrane rather than cell wall

To study the structure-antifungal activity of vanillin against Aspergillus flavus(A.flavus),the susceptibilities of A.flavus to vanillin and its isomers(o-vanillin,2-hydroxy-4-methoxybenzaldehyde(HMB),2-hydroxy-5-methoxybenzaldehyde)and the possible antifungal mechanisms have been investigated.All the four volatile aldehydes could inhibit the germination of spores,and the minimum inhibitory concentrations(MICs)of them were in this order:vanillin(200μg/mL),o-vanillin(100μg/mL),2-hydroxy-5-methoxybenzaldehyde(100μg/mL),HMB(70μg/mL).The minimum fungicidal concentrations(MFCs)of them were in this order:vanillin(240μg/mL),o-vanillin(160μg/mL),HMB(140μg/mL),2-hydroxy-5-methoxybenzaldehyde(140μg/mL).Spore size was arrested at 0 h with the treatment of the four volatile aldehydes.Effects of the four volatile aldehydes on the cell wall and cell membrane integrity of A flavus were observed by calcofluor white(CW)staining and propidium iodide(PI)staining.The results showed that HMB exerted the strongest antifungal and fungicidal effects on the growth of A.flavus.The four volatile aldehydes had little influence on cell wall integrity after 3-hour treatment,however,they could strongly damage the cell membrane integrity.All the four volatile aldehydes could effectively prevent the growth of A.flavus on peanut seeds.The antifungal mechanisms of the four volatile aldehydes provide theoretical foundations for their development of new antifungal agents.

Antifungal properties and mechanisms of three volatile aldehydes(octanal,nonanal and decanal)on Aspergillus flavus

The harmof Aspergillus flavus(A.flavus)and aflatoxin is a severe food safety problemworldwide,which causes huge economic losses every year.Therefore,it is urgent to control the growth of A.flavus and the biosynthesis of aflatoxin.Plant-derived natural compounds are superior to synthetic fungicide in inhibiting the growth of A.flavus benefiting from their high safety to the environment,humans and stock,and low cost.This study aimed to evaluate the antifungal effects and potential antifungal mechanisms of three plant-derived compounds(octanal,nonanal and decanal)against A.flavus.We determined the minimum inhibitory concentrations(MICs)and action mechanism of the three volatile aldehydes on A.flavus and also performed calcofluor white(CW)staining for visualizing the distribution of septa.Cell respiration metabolism and the pathogenicity on maize kernels were also carried out to evaluate the efficacy of the three volatile aldehydes on the growth of A.flavus.The results showed that the three volatile aldehydes could inhibit the germination of spores and mycelial growth of A.flavus,the MICs on spores and mycelia were:octanal(1.0 and 0.5μL/mL),nonanal(0.5 and 2.0μL/mL),and decanal(1 and 5μL/mL).The three volatile aldehydes could strongly damage the integrity of both the cell wall and the cell membrane of A.flavus.Meanwhile,they could decrease the content of total lipid and inhibit respiration metabolism of A.flavus cell.Results of in vitro antifungal test showed that all the three volatile aldehydes could effectively prevent the growth of A.flavus on maize kernels.The study revealed that octanal,nonanal and decanal could effectively inhibit the growth of A.flavus both in culture medium and on maize kernels to different extent.The results confirmed that the plant-derived compounds could be developed into promising antifungal agents applied in the preservation of grains.This study provides a theoretical basis for the research and application of potential antifungal agents.

Sclerotia Formation and Toxin Production in Large Sclerotial Aspergillus flavus Isolates from Kenya

We studied the relationship between sclerotia formation and aflatoxin production by Aspergillusflavus strains isolated from maize kernels from Nandi County. Isolates recovered from maize kernels were tested for their ability to form sclerotia on different growth media. PCR analysis was done on the isolates to detect 2 structural genes, aflD and aflQ, involved in aflatoxin biosynthesis pathway. Positive A. flavus isolates for one or both genes were grown on Yeast Extract Sucrose Agar medium and aflatoxins quantified using LCMSMS. All the isolates formed large sclerotia and their formation was influenced by media type but could not be related to amount of aflatoxins produced both in vivo and in vitro. Though sclerotia are perennating structures and so contribute to survival index of a fungus, their initiation is regulated by external factors though ability to form is genetic. This brings ambiguity of their presence or abundance as a measure of toxicity.

Genetic diversity of atoxigenic Aspergillus flavus isolates from peanut kernels in China

Aflatoxin contamination in peanuts is a major threat to public health, especially in tropical and subtropical regions of the world. Recently, the use of atoxigenic Aspergillus flavus strains as biological control agents prove to be effective in reducing aflatoxin contamination in crops. A total of 208 atoxigenic A. flavus isolates, collected from peanut kernels, were grouped into 7 deletion patterns by quadruplex PCR products of nor-1, ver-1, aflR and omtA genes. 49 SNPs, found in 1254 bp fragment of omtA gene, showed genetic variation of omtA among different A. flavus isolates. These isolates were assigned to either MAT1-1 type or MAT1-2 type with primers for each MAT locus. Thus, rich genetic diversity was found in the atoxigenic A. flavus isolates of peanut in China. The results indicated that quadruplex PCR would be an effective method for rapid screening of atoxigenic isolates with gene deletion in aflatoxin biosynthetic cluster.

Peanut aflatoxin contamination and soil Aspergillus flavus isolates in different climate regions

Peanuts are important oil and food sources worldwide and can be easily contaminated by highly toxic aflatoxins （AFs） , which often exist in peanuts and related foodstuff. In this experiment, a total of 12 A. flavus strains were isolated from soil collected from 7 cities of China and the aflatoxin con-tamination levels in peanuts in these areas were detected to find out the relationship between aflatoxin contamination level and amount of soil isolates in the same region, as well as the correlation between cli-mate type and peanut aflatoxin contamination level. In this study, we found that in high - temperature and moist areas, toxin producing strains were easier to be isolated and the aflatoxin contamination level in peanuts was higher. We also studied the association between the aflR expression determined by fluorogen- ic quantitative PCR and the aflatoxin production. The results showed all 3 atoxigenic isolates had the same cycle threshold 21. 38, suggesting that atoxigenic strains had the same expression of aflR, which was the key gene of aflatoxin production.

The Metabolic Responses of Aspergillus flavus to N-Acetylcysteine,Ascorbate,and H2O2

Aflatoxin, the secondary metabolite of Aspergillus flavus and A. parasiticus, is the most toxic product in nature. In this study, N-acetylcysteine （NAC）, ascorbate, and H2O2 were used to ascertain their effects on fungal metabolic response of A. flavus. The results demonstrated that NAC did not affect fungal growth, but inhibited the aflatoxin Bt production, with the concomitant sporulation reduction. NAC increased the ratio of reduced glutathione （GSH） and oxidized glutathione （GSSG）, but decreased the activity of glutathione reductase （GR）. Ascorbate had similar effect on fungal growth, sporulation, and GR activity, but GSH/GSSG and total glutathione （tGSH, including GSH and GSSG） were significantly increased. H2O2 at high concentration （5 mM） inhibited fungal growth, but the aflatoxin production was increased. At the same time, it reduced GR activity and enhanced tGSH. Though reductive agents had different effects on GSH metabolism, reductive conditions inhibited aflatoxin production and sporulation without any effect on fungal growth. The results in this report confirmed that the relationship between oxidative stress and aflatoxin production is theoretically important in controlling aflatoxin contamination.

Inhibitory Effect of Garlic, Clove and Carrot on Growth of Aspergillus Flavus and Aflatoxin Production

The inhibitory effeet of crude extracts of garlic, clove and carrot at concentrations of 20,000, 40,000,60,000, 80,000 and 100,000 μg/mL on growth of Aspergillus and aflatoxin production in rice was investigated. Theresults showed that garlic, clove and carro could inhibit the growth of A. jlavus and aflatoxin production. Garlic and cloveat 100,000 μg/mL. highly reduced the level of anatoxin from 5.94 to 0. 15 and 0.06 μg/g respectively whereas carrot at20,000μg/mL reduced the most level of aflatoxin from 5.94 to 0.03μg/g. Garlic, clove and carrot at 100,000μg/mL also inhibited the mould groWth. The most effective herb for inhibiting the growth of A. flavus and anatoxin production was garlic.

A New α-Cyclopiazonic Acid Alkaloid Identified from the Weizhou Island Coral-Derived Fungus Aspergillus flavus GXIMD 02503

A new oxygenated tricyclic cyclopiazonic acid(CPA)alkaloid,asperorydine Q(1),along with seven known compounds,namely,asperorydines O(2)and J(3),speradine H(4),cyclopiamides A(5)and H(6),saadamysin(7),and pyrazinemethanol(8),were isolated from the coral-associated Aspergillus flavus GXIMD 02503.The structures were elucidated by physicochemical properties and comprehensive spectroscopic data analysis.Compounds 1−5 and 7−8 exhibited potent inhibition of lipopolysaccharide(LPS)-induced nuclear factor-κB(NF-κB)with the IC50 values ranging from 6.5 to 21.8μmol L^(−1).In addition,the most potent one,pyrazinemethanol(8),dose-dependently suppressed receptor activator of NF-κB ligand(RANKL)-induced osteoclast differentiation without obvious cytotoxicity in bone marrow macrophages cells(BMMCs),suggesting it is a promising lead compound for the treatment of osteolytic diseases.

In vitro activity of neem oil [Azadirachta indica A. Juss (Meliaceae)] on Aspergillus flavus growth, sporulation, viability of spores, morphology and Aflatoxins B1 and B2 production

The effectiveness of neem (Azadiracta indica) oil on the growth, morphology, sporulation, viability of spores, aflatoxin B1 and B2 production by A. flavus on Yeast Extract-Sucrose medium was determined. Neem oil inhibited the fungal growth (i.e. mycelia dry weight, diameter of colony and growth rate) on solid media at concentrations from 0.5 to 5.0% v/v, although it significantly increased sporulation in the same conditions. Spores obtained from cultures grown without neem oil reduced germination when incubated in a neem oil supplemented medium. Colonies grown on solid media and in submerged cultures in the presence of neem oil exhibited morphological alterations, including granular cytoplasm, atypical hyphae branching pattern, abnormal and undifferentiated conidiophores. High Performance Liquid Chromatography was used to measure aflatoxins. In submerged cultures, neem oil at concentrations from 0.5 to 4.0% v/v caused approximately 95% inhibition in Aflatoxin B1 and B2. On other hand, these conditions failed to suppress fungal growth. Current research emphasized that neem oil was not fungistatic or fungicidal, but exhibited anti-aflatoxigenic activity.

Construction and Application of a High-throughput Method for Identifying Maize Resistance to Aspergillus flavus in the Field

In order to establish a fast and effective high-throughput method for identifying maize resistance to Aspergillus flavus infection in the field,5 conidial concentrations and 3 inoculation methods were compared and optimized. A correlation model between disease levels and aflatoxin B1 accumulation was established. The results showed that inoculating maize inbred lines with the optimal conidial concentration of 1.0×10^6 c/mL could accurately show the different resistance levels of maize to A.flavus infection,and the spiral quantitative inoculation technique could effectively avoid the defect of calculation error caused by commonly used methods. The positive correlation model of resistance level and aflatoxin B1 accumulation could effectively predict the capability of maize being resistant to A.flavus. 8 resistant and 9 medium resistant maize inbred lines of the RIL population were identified.The spiral quantitative identification method realized the high-throughput identification of maize inbred lines resistant to A.flavus infection in the field,and the results have shown high accuracy,stability and repeatability.

Evaluation of SRAP markers efficiency in genetic diversity of Aspergillus flavus from peanut-cropped soils in China

In order to evaluation the efficiency of SRAP markers on genetic diversity of Aspergillus flavus,we screened out 17sets of primer pairs which could produce clear and reproducible SRAP bands from 150 SRAP primer pairs.The size of SRAP fragments ranged from 120 to 2100 bp.Primer pair Me10/Em9 produced the maximum number of polymorphic bands(12 bands),while Me8/Em13 produced the fewest number of polymorphic bands(only 1).Through analysis genetic diversity ability of different sets of primer pairs,the set of 12 primer pairs was selected for SRAP genetic marker of A.flavus.Cluster analysis was performed based on the genetic similarity coefficients,which ranged from 0.53 to 0.89.A dendrogram assembled using the unweighted pair-group method with arithmetic averages grouped A.flavus samples into 5 main clusters.The results suggested that SRAP marker is a useful molecular technology for the diversity of A.flavus from peanut soils in China.