非脱羧勒克菌wt16对黄曲霉菌生长与产毒的抑制作用

黄曲霉菌及其毒素严重威胁农产品质量安全,本文旨在探寻非脱羧勒克菌对黄曲霉菌及其毒素污染防控效果。从湖北黄陂分离筛选出一株非脱梭勒克菌wt16,将其与黄曲霉菌在液体培养基中共培养后测定非脱羧勒克菌wt16菌株对黄曲霉菌生长及产毒的抑制率。结果表明,在沙氏液体培养基中,非脱羧勒克菌wt16能明显抑制黄曲霉菌的生长及产毒,对其菌丝生长的抑制率为77%~92%,对其产毒的抑制率为90%~96%。通过扫描电子显微镜观察发现,非脱羧勒克菌wt16能改变黄曲霉菌丝的形态,使得黄曲霉菌丝体由规则的球体聚集成不规则形状,单个菌丝会由细长型断裂成小截形态,菌丝表面也变得更为光滑;并且发现wt16在花生粉及未受机械损伤的花生颗粒上对黄曲霉菌的生长及产毒均表现出很强的抑制作用。进一步研究发现,非脱羧勒克菌wt16菌株发酵上清液中含有能抑制黄曲霉毒素合成的有效成分,且该发酵上清液的制备以培养4 d以上为最佳,培养温度为15~40 ℃。     

不同植物抑制剂对黄曲霉菌生长和产毒的影响

目的研究不同植物抑制剂对黄曲霉菌生长和产毒的影响。方法将产毒的黄曲霉菌接种在EMA培养基上,活化后分别培养在含不同浓度抑制剂的培养基中,在30℃的温度下恒温培养,通过观察抑菌圈直径的大小判断不同抑制剂对黄曲霉生长的影响,并将菌丝抽提使用酶标仪测定不同抑制条件下黄曲霉毒素含量。结果 0.2mg/mL的紫苏醛、烟草萜和香芹醇在第2d对黄曲霉菌生长抑制率分别为100%、67%和79%,第5d时分别降低到46%、63%和55%。柠檬醛、紫苏醛、香茅醇和芳樟醇处理组培养基中第5d时黄曲霉毒素含量很低,对照组中黄曲霉毒素含量是这4个处理组的2000倍以上,第6d时,6种植物抑制剂的抑制黄曲霉菌产毒能力都有所降低,但是0.2mg/mL的柠檬醛、紫苏醛、香茅醇、烟草萜和香芹醇仍具有较高的抑制黄曲霉菌产毒能力。结论柠檬醛、紫苏醛、烟草萜和香芹醇对黄曲霉菌的生长和产毒有着明显的抑制作用。     

黄曲霉菌株的筛选和产毒能力鉴定

本研究从发霉粮食中分离出数株黄曲霉菌菌株,并进行了形态学和分子生物学鉴定。为了探究分离菌株与黄曲霉标准菌株之间产毒能力的差异,通过对分离菌株和黄曲霉标准菌株进行发酵培养和HPLC测定,分析确定产毒能力。结果表明黄曲霉菌株之间产毒能力差异巨大：黄曲霉菌株3.4408产毒量很高,黄曲霉菌株HDWH产毒量很低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生四种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWS只产生黄曲霉毒素AFB1、AFB2。     

黄曲霉菌株的分离、鉴定及产毒能力分析

对几株从发霉粮食中分离出的黄曲霉菌菌株进行形态学和分子生物学鉴定,并进行发酵培养和产毒能力的HPLC测定。结果表明:试验分离菌株均为黄曲霉菌株且含有黄曲霉毒素产生的关键基因aflR;黄曲霉菌株之间产毒能力差异巨大:黄曲霉菌株3.4408产毒量最高,黄曲霉菌株HDWS产毒量最低,黄曲霉菌株3.2572甚至不产生黄曲霉毒素;产生黄曲霉毒素菌株中部分黄曲霉菌株产生4种黄曲霉毒素AFB1、AFB2、AFG1、AFG2,黄曲霉菌株HDWH只产生黄曲霉毒素AFB1、AFB2。     

香菇纤维素衍生物抑制黄曲霉菌产毒的研究

黄曲霉毒素具有诱导突变、抑制免疫和致癌作用。控制黄曲霉毒素污染一直是世界性难题,也是近年来的研究热点之一。实验研究了香菇纤维素衍生物GPX抑制黄曲霉菌产毒活性,初步探究了GPX抑制黄曲霉菌产毒的作用机制。结果表明,10、50μg/mL的GPX对黄曲霉菌产毒的抑制率分别达到70%、95%,而100、250、500、750、1000μg/mL的GPX对黄曲霉菌产毒的抑制率均可达到100%,抑制活性极其明显。GPX离体抗氧化能力很低,但是能够缓解黄曲霉菌丝的氧化胁迫。此外GPX促进了黄曲霉细胞内囊泡过早与大液泡的融合,该现象能够抑制黄曲霉毒素的产生。     

植物提取物抑制玉米中黄曲霉生长及产毒研究

通过液体培养 直接接触法比较植物提取物肉桂醛、柠檬醛和丁香酚对黄曲霉生长及产毒的抑制作用,选取抑制作用最强的肉桂醛应用到玉米中,研究了添加量、玉米水分含量和储藏温度对肉桂醛抑制黄曲霉生长及产毒的影响。结果表明：液体培养基中,肉桂醛、柠檬醛和丁香酚的最低杀菌浓度与各自完全抑制产毒浓度一致,分别为 100、500和500 μl/L。随着添加量的加大,肉桂醛对黄曲霉生长和产毒的抑制率逐渐升高,挥发浓度为96 μl/L时完全抑制黄曲霉生长,对黄曲霉毒素B1的抑制率为97.98％。在玉米水分为14%~40％时,肉桂醛对玉米黄曲霉污染的抑制率随水分含量的升高显著下降,14％时抑制率最高,为88.24%;对黄曲霉毒素B1始终保持较强的抑制作用,14％时产毒抑制率最高,为97.51％。储藏温度在20~37℃时,肉桂醛对黄曲霉污染的抑制率随温度的升高呈现先升高后降低的趋势,28℃黄曲霉污染抑制率最高,为85.67%;对黄曲霉毒素B1抑制率随温度的升高呈现先降低后升高又降低的趋势,20℃时抑制率最高,达到91.00%。     

薄荷精油对黄曲霉生长的抑制作用研究

目的 研究薄荷精油对黄曲霉生长及其毒素B1合成的抑制作用。方法 在培养基中加入不同体积浓度的薄荷精油,以不加薄荷精油作为对照,通过测定其对产毒黄曲霉的最小抑菌浓度(minimum inhibitory concentration, MIC)、孢子萌发、菌丝干重、菌落直径、微观结构及黄曲霉毒素B1合成量的影响,探究薄荷精油对黄曲霉的抑制效果。结果 薄荷精油对产毒黄曲霉的MIC为1.6μL/mL; 2.0 MIC薄荷精油处理对黄曲霉孢子萌发抑制率、黄曲霉菌丝干重抑制率及黄曲霉毒素B1合成抑制率均达到99%以上,且扫描电镜结果表明,薄荷精油会影响黄曲霉菌丝形态;同时,通过熏蒸处理,薄荷精油能够有效抑制花生中的霉菌增长,防止霉菌侵染花生。结论 薄荷精油对黄曲霉具有良好的抑制作用,在粮食储藏、食品防霉等方面具有进一步开发成安全防腐抑菌剂的潜力。     

山苍子精油抑制黄曲霉菌的生长和产毒作用研究

山苍子精油是一种纯天然植物精油,本文研究了其对黄曲霉生长、代谢和毒素产生的抑制作用,探讨了山苍子精油对黄曲霉菌的抑菌能力和作用机理。本研究将花生放置于自然环境染菌并分离纯化目标菌,采用形态学并结合ITS序列法进行菌株分类鉴定;结合抑菌圈、抑菌率和最低抑菌浓度(MIC)的测定探讨山苍子精油对黄曲霉菌的抑制能力;进行了山苍子精油影响黄曲霉孢子萌发率、生长曲线和黄曲霉毒素B1产生的实验研究;从细胞膜渗透性、细胞酶活性的变化探讨了山苍子精油抑制黄曲霉的作用机理。实验结果表明:从腐败花生中分离筛选出菌株HB₂,经ITS序列法鉴定为黄曲霉(Aspergillus flavus);黄曲霉素测定结果显示其含有黄曲霉素B1(AFB1),质量浓度为3.4×10³μg·kg^-1(纯湿菌体);抑菌圈随精油浓度的增大明显变大,对黄曲霉的最低抑菌体积分数(MIC)为0.800μL·mL^-1;孢子萌发率、牙管长度、黄曲霉菌体的生长量和AFB1的浓度随培养液中精油浓度的增大呈显著下降趋势,当山苍子精油浓度为0.100μL·mL     

茶多酚对黄曲霉生长及产毒能力的影响

基于氧化应激与黄曲霉毒素生物合成的持续相关性,研究了天然抗氧化剂茶多酚对黄曲霉生长及产毒的影响,并通过扫描电镜观察茶多酚对黄曲霉菌丝体形态的影响,旨在寻找一种安全、高效、环境友好型的抑菌剂来控制食品中黄曲霉毒素的污染。结果表明:茶多酚对黄曲霉菌落生长有一定抑制作用,在10 mg/m L浓度下,抑制率达到48.41%,使黄曲霉菌落生长速率为(5.57±0.16)mm/d,延滞期为(1.32±0.13)d;而茶多酚对黄曲霉产毒抑制效果显著,在1,5和10 mg/m L浓度下,茶多酚对AFB1生物合成的抑制率分别达到了60.15%,87.12%和97.48%;且茶多酚可严重破坏黄曲霉菌丝体的超微结构。因此,茶多酚可作为天然抑菌剂应用于食品与饲料中,控制黄曲霉毒素的污染。     

黄曲霉菌在蜜饯、水果罐头上生长及其产毒能力的试验

<正>黄曲霉菌能在花生玉米和其他许多农产品上产生黄曲霉毒素(AFT),引起人和动物的急慢性中毒,并具有明显的致癌力,国内外有关报导甚多,已为人们所熟知。一般食品在制备、储藏、运输和销售过程中,不可避免地受环境中的菌丛,甚至产毒菌株所污染。由于霉菌的生活力强,对高     

Cladal relatedness among Aspergillus oryzae isolates and Aspergillus flavus S and L morphotype isolates

Aspergillus flavus is the main etiological agent for aflatoxin contamination of crops. Its close relative, A. oryzae, does not produce aflatoxins and has been widely used to produce fermented foods. We compared the phylogeny of A. oryzae isolates and L- and S-type sclerotial isolates of A. flavus using single nucleotide polymorphisms in the omtA gene in the aflatoxin biosynthesis gene cluster and deletions in and distal to the norB-cypA intergenic region as phylogenetic signals. Aflatoxin-producing ability and sclerotial size also were weighted in the analysis. Like A. flavus, the A. oryzae isolates form a polyphyletic assemblage. A. oryzae isolates in one clade strikingly resemble an A. flavus subgroup of atoxigenic L-type isolates. All toxigenic S-type isolates closely resemble another subgroup of atoxigenic L-type isolates. Because atoxigenic S-type isolates are extremely rare, we hypothesize that loss of aflatoxin production in S-type isolates may occur concomitantly with a change to L-type sclerotia. All toxigenic L-type isolates, unlike A. oryzae, have a 1.0 kb deletion in the norB-cypA region. Although A. oryzae isolates, like S-type, have a 1.5 kb deletion in the norB-cypA region, none were cladally related to S-type A. flavus isolates. Our results show that A. flavus populations are genetically diverse. A. oryzae isolates may descend from certain atoxigenic L-type A. flavus isolates.     

Nonaflatoxigenic Aspergillus flavus TX9-8 competitively prevents aflatoxin accumulation by A. flavus isolates of large and small sclerotial morphotypes

Aflatoxins are a family of highly toxic and carcinogenic toxins produced by several Aspergillus species. Aflatoxin contamination of agricultural commodities both pre- and postharvest is a serious food safety issue and a significant economic concern. Using nonaflatoxigenic A. flavus isolates to competitively exclude toxigenic A. flavus isolates in agricultural fields has become an adopted approach to reduce aflatoxin contamination. From screening subgroups of nonaflatoxigenic A. flavus, we identified an A. flavus isolate, TX9-8, which competed well with three A. flavus isolates producing low, intermediate, and high levels of aflatoxins, respectively. TX9-8 has a defective polyketide synthase gene (pksA), which is necessary for aflatoxin biosynthesis. Co-inoculating TX9-8 at the same time with large sclerotial (L strain) A. flavus isolates at a ratio of 1:1 or 1:10 (TX9-8:toxigenic) prevented aflatoxin accumulation. The intervention of TX9-8 on small sclerotial (S strain) A. flavus isolates varied and depended on isolate and ratio of co-inoculation. At a ratio of 1:1 TX9-8 prevented aflatoxin accumulation by A. flavus CA28 and reduced aflatoxin accumulation 10-fold by A. flavus CA43. No decrease in aflatoxin accumulation was apparent when TX9-8 was inoculated 24 h after toxigenic L- or S strain A. flavus isolates started growing. The competitive effect likely is due to TX9-8 outgrowing toxigenic A. flavus isolates.     

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.     

Laboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents

Biocontrol by competitive inhibition using atoxigenic Aspergillus flavus strains has been shown to be an effective method for controlling aflatoxin production in peanuts, maize and cottonseed. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field. The study here described was conducted in order to evaluate the potential of the different atoxigenic A. flavus strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation when grown in mixed cultures together with atoxigenic strains; additionally, we developed a simple and inexpensive procedure that may be used to scale-up the screening process and to increase knowledge on the mechanisms interfering with mycotoxin production during co-infection.     

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B₁?+?B₂ concentrations were significantly (p?<?0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B₁?+?B₂ reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B₁?+?B₂ concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.     

Spread of Aspergillus flavus and aflatoxin accumulation in postharvested maize treated with biocontrol products

Maize is a major staple crop and calorie source for many people living in Sub-Saharan Africa. In this region, Aspergillus flavus causes ear rot in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to reduce aflatoxin levels in maize grain at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the postharvest drying period, the time between harvest and the point when grain moisture is safe for storage. In Sub-Sahara Africa, this period often is extended by weather and the complexities of postharvest drying practices. Maize grain was collected from fields in Texas and North Carolina that were treated with commercial biocontrol products and untreated control fields. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 °C for 6 days. Although the initial number of kernels infected by fungal species was high in most samples, less than 24% of kernels were infected with Aspergillus flavus and aflatoxin levels were low (<4 ppb). Both toxigenic and atoxigenic strains grew and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields may have migrated to untreated fields. These results also indicate that the population of toxigenic A. flavus in the harvested grain will increase and produce aflatoxin during the drying period when moisture is high. Therefore, we conclude that preharvest biocontrol applications will not replace the need for better postharvest practices that reduce the drying time between harvest and storage.     

Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations

Contamination of corn, cotton, peanuts and tree nuts by aflatoxins is a severe economic burden for growers. A current biocontrol strategy is to use non-aflatoxigenic Aspergillus flavus strains to competitively exclude field toxigenic Aspergillus species. A. flavus K49 does not produce aflatoxins and cyclopiazonic acid (CPA) and is currently being tested in corn-growing fields in Mississippi. We found that its lack of production of aflatoxins and CPA resulted from single nucleotide mutations in the polyketide synthase gene and hybrid polyketide-nonribosomal peptide synthase gene, respectively. Furthermore, based on single nucleotide polymorphisms of the aflatoxin biosynthesis omtA gene and the CPA biosynthesis dmaT gene, we conclude that K49, AF36 and previously characterized TX9-8 form a biocontrol group. These isolates appear to be derived from recombinants of typical large and small sclerotial morphotype strains. This finding provides an easy way to select future biocontrol strains from the reservoir of non-aflatoxigenic populations in agricultural fields.     

Glufosinate-ammonium reduces growth and aflatoxin B1 production by Aspergillus flavus

The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammonium salt] was tested at concentrations from 2 to 2,000 g GA per ml for activity against growth and aflatoxin B1 (AFB) production by the mycotoxigenic fungus Aspergillus flavus Link:Fr. The highest concentration (2,000 microg GA per ml) reduced colony diameter of A. flavus strain AF13 by 80%. AFB1 production was inhibited by 90% at this concentration. Reduction in mycelial dry weight and AFB1 production in response to GA application ranged from 17.2 to 97.1% and from 39.1 to 90.1%, respectively. Of four concentrations tested, 2 microg GA per ml was weakly inhibitory. In the kernel screening assay, AFB1 production was inhibited 60 to 91% when kernels were preimmersed or immersed 5 days after incubation in 200 microg GA per ml. Both concentrations (2 and 200 microg GA per ml) reduced seed germination by 25 to 50%. Results indicate that GA has an inhibitory effect on growth and AFB1 production by A. flavus.     

Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize

Biological control of toxigenic Aspergillus flavus in maize through competitive displacement by non-aflatoxigenic strains was evaluated in a series of field studies. Four sets of experiments were conducted between 2007 and 2009 to assess the competitiveness of non-aflatoxigenic strains when challenged against toxigenic strains using a pin-bar inoculation technique. In three sets of experiments the non-aflatoxigenic strain K49 effectively displaced toxigenic strains at various concentrations or combinations. The fourth study compared the relative competitiveness of three non-aflatoxigenic strains (K49, NRRL 21882 from Afla-Guard?, and AF36) when challenged on maize against two aflatoxin- and cyclopiazonic acid (CPA)-producing strains (K54 and F3W4). These studies indicate that K49 and NRRL 21882 are superior to AF36 in reducing total aflatoxin contamination. Neither K49 nor NRRL 21882 produce CPA and when challenged with K54 and F3W4, CPA and aflatoxins were reduced by 84-97% and 83-98%, respectively. In contrast, AF36 reduced aflatoxins by 20% with F3W4 and 93% with K54 and showed no reduction in CPA with F3W4 and only a 62% reduction in CPA with K54. Because AF36 produces CPA, high levels of CPA accumulate when maize is inoculated with AF36 alone or in combination with F3W4 or K54. These results indicate that K49 may be equally effective as NRRL 21882 in reducing both aflatoxins and CPA in maize.