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.

北部湾海洋真菌Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性研究

为获得具有生物活性的化合物,对一株分离于北部湾钉螺菌株Aspergillus fumigatus DL-p0m-g2的化学成分及药理活性进行研究。综合应用反相ODS(octadecylsilyl)柱色谱、半制备液相等多种色谱学方法进行分离纯化,根据化合物理化性质、质谱和核磁共振波谱学数据进行结构鉴定,并对分离得到的化合物进行细胞毒活性、抑菌活性和胆固醇转运蛋白NPC1L1(NPC1-like intracellular cholesterol transporter 1)蛋白结合等生物活性评价。实验共分离得到21个生物碱类化合物和1个甾体,分别鉴定为6-methoxyspirotryprostatin B(1)、spirotryprostatin A(2)、fumitremorgin C(3)、cyclotryprostatin A(4)、fumitremorgin B(5)、pseurotin A(6)、azaspirofuran A(7)、azaspirofuran B(8)、cephalimysin C(9)、cephalimysin B(10)、fumiquinazoline C(11)、fumiquinazoline B(12)、fumiquinazoline A(13)、fumiquinazoline D(14)、fumiquinazoline F(15)、tryprostatin B(16)、verruculogen(17)、chaetominine(18)、bisdethiobis(methylthio)glitoxin(19)、helvolic acid(20)、7-deacetylpyripyropene A(21)、terezine D(22)。其中化合物6对人肝癌细胞(Hep G2)、人肺癌细胞(A549)、人直肠癌细胞(HCT116)有一定的细胞毒活性;化合物1、3和20对金黄色葡萄球菌表现出抑菌活性;化合物14与NPC1L1蛋白具有较好的结合,显示其在降脂药物开发中的研究潜力。

红树老鼠簕内生真菌Aspergillus terreus GXIMD 03158次级代谢产物研究

文章对红树老鼠簕(Acanthus ilicifolius L.)内生真菌Aspergillus terreus GXIMD 03158的抑菌活性次级代谢产物进行研究。采用多种色谱技术对该菌次级代谢产物进行分离纯化,结合核磁氢谱、碳谱和高分辨率质谱数据综合解析鉴定单体化合物结构。从A.terreus GXIMD 03158代谢产物中共分离出10个化合物,经鉴定分别为dankasterones A(1),(14α,22E)-14-hydroxyergosta-4,7,22-triene-3,6-dione(2),steresterone B(3),ergosta-4,6,8(14),22-tetraen-3-one(4),herbarulide(5),demethylincisterol A3(6),isochaetochromin B1(7),isochaetochromin B2(8),(E)-7,9-二烯-11-羰基硬脂酸(9)和aspergilfuranone A(10)。并采用微量肉汤稀释法检测所有化合物对白色葡萄球菌(Staphylococcus albus)、金黄色葡萄球菌(Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)和副溶血弧菌(Vibrio parahaemolyticus)的最低抑菌浓度(minimal inhibitory concentrations,MIC),结果显示化合物6对S.albus、S.aureus和S.epidermidis的MIC分别为3.12、6.25和3.12μg·mL^(−1);化合物9对S.albus和S.aureus的MIC分别为1.56和3.12μg·mL^(−1)。

纳米银胁迫下黄曲霉(Aspergillus flavus)TL-F3的氧化应激响应及其吸附能力

为研究纳米银(AgNPs)胁迫下黄曲霉(Aspergillus flavus TL-F3)的氧化应激变化及其对AgNPs的吸附能力,通过序批式试验,探究不同浓度AgNPs对A.flavus TL-F3生长、细胞形态、胞外聚合物(EPS)分泌、抗氧化系统和吸附能力的影响。研究发现,AgNPs可抑制A.flavus TL-F3生长,且抑制效果与浓度正相关。AgNPs可致A.flavus TL-F3菌丝发育不完全,表面破损。与不加AgNPs的对照组相比,当AgNPs的质量浓度为80 mg·L^(-1)时,胞外聚合物含量升至237.55 mg·g^(-1),丙二醛含量、超氧化物歧化酶活性、还原性谷胱甘肽含量均显著(P<0.05)提高,而Na^(+)K^(+)-ATP酶活性显著(P<0.05)降低了50.37%。A.flavus TL-F3对AgNPs有一定的吸附能力,当AgNPs的质量浓度为1、30、50、80 mg·L^(-1)时,A.flavus TL-F3对AgNPs的吸附率可达68.54%以上。综上所述,A.flavus TL-F3可用于含Ag和AgNPs污染水体的修复。

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.

植酸对黄曲霉菌(Aspergillus flavus)糖化力的影响

该文通过添加不同浓度的植酸于黄曲霉菌(Aspergillus flavus)培养基中,用不同添加方式,于不同生长时刻测定黄曲霉菌糖化力,研究植酸对黄曲霉菌糖化力的影响.结果表明加入植酸后黄曲霉菌糖化力均有很大提高,可为工业生产带来可观的经济效益.植酸最佳作用浓度范围是0.20%～0.25%,发酵24h糖化力最大可提高44.32%.

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.

L-苹果酸产生菌黄曲霉Aspergillus flavus H-98发酵特性的研究

报道了Ｈ－９８菌株发酵Ｌ－苹果酸的特点，研究了碳源、氮源、温度、ＣａＣＯ３、金属离子、促进剂和抑制剂等因素对Ｈ－９８菌株苹果酸发酵的影响，并综合考察培养基配方对产酸的影响，确定了Ｈ－９８菌株的最佳发酵条件，其中最适碳源为１０％葡萄糖，最适氮源为０．２５％（ＮＨ４）２ＳＯ４，ＣａＣＯ３浓度为７％。生物素、丙氨酸和Ｍｎ２＋在一定浓度范围内能显著提高Ｈ－９８菌株的产酸水平，使该菌株的产酸超过５０ｇ／Ｌ。

植物核糖体抑制蛋白对黄曲霉(Aspergillus flavus)的抑制作用

测定了三种不同植物的核糖体抑制蛋白 (RIPs)———Momordin、Luffin和Saporin的抗真菌活性 .结果显示 ,Momordin有最强的抗黄曲霉活性 ,Luffin次之 ,而Saporin对黄曲霉的生长无影响 .所以 。

一种内生真菌Aspergillus flavus次生代谢产物研究

为了研究内生真菌Aspergillus flavus的次生代谢产物,利用硅胶、Sephadex LH-20、反相、中压、高效液相制备等多种色谱方法从内生真菌Aspergillus flavus发酵液的乙酸乙酯萃取部位中分离得到7个化合物,并通过1H NMR、13C NMR、ESI-MS等波谱技术鉴定其结构,依次为:对羟基苯乙酸(1),对羟基苯乙醇(2),环(D)-脯氨酸-(L)-苯丙氨酸(3),Nb-乙酰基色胺(4),(E)-4-(4-hydroxyphenyl) but-3-en-2-one(5),N-isobutylacetamide(6),琥珀酸乙酯(7)。所有化合物均为首次从该菌中分离得到。

一株产L-苹果酸黑曲霉菌株的诱变筛选及发酵培养基优化

为筛选一株产L-苹果酸能力强的黑曲霉菌株,通过紫外诱变与高浓度放线菌酮迭代诱变的方法,将野生型菌株诱变为一株产L-苹果酸能力强的黑曲霉菌株,并对其种子或发酵培养基成分进行优化。结果表明,诱变所得一株产L-苹果酸能力强的黑曲霉CGMCC NO.40550菌株,其摇瓶发酵时种子培养基最适葡萄糖浓度为60 g/L、最适氮源为(NH_4)_2SO_4 4.95 g/L,黑曲霉菌球形成最优转速220 r/min;发酵培养基最适葡萄糖和最适(NH_4)_2SO_4浓度分别为180 g/L和4.95 g/L,Cu SO_4·5H_2O为其生产苹果酸最佳微量元素,最适添加量为0.065 g/L。通过单因素实验的优化,优化后的培养基更有利于L-苹果酸的合成,发酵96 h时L-苹果酸产酸量达到18.15 g/L,显著提高了245%,L-苹果酸占总酸的百分比达到71.69%。本研究为L-苹果酸的工业化生产奠定基础。

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.

L—苹果酸产生菌Aspergillus flavus HLD—12产酸条件的研究

本文报道ＨＬＤ－１２菌株发酵Ｌ－苹果酸的特点．研究了碳源、氮源、温度、ＣａＣＯ３、金属离子和促进剂等因素对ＨＬＤ－１２菌株苹果酸发酵的影响，确定了ＨＬＤ－１２菌株的最佳产酸条件，其中最适碳源为８％的葡萄糖，最适氮源为０．１５％的（ＮＨ４）２ＳＯ４，ＣａＣＯ３浓度为７％。生物素和丙氨酸在一定浓度范围内能显著提高ＨＬＤ－１２菌株的产酸水平。

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.

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.