利用Fusarium poae制备T-2毒素的培养条件和提取方法

比较2种不同优化方法对分离纯化梨孢镰孢菌(Fusarium poae)产生的T-2毒素的效果,并得到高纯度的T-2毒素,解决国内T-2毒素产业化问题.在优化Fusarium poae产毒培养条件的基础上,对Burmeister的提取方法和Gregory培养基进一步优化以最大限度地提高T-2毒素的产率,从而得到高纯度并且...     

Production of T-2 toxin by a Fusarium resembling Fusarium poae

A Fusarium species with a micro morphology similar to F. poae and a metabolite profile resembling that of F. sporotrichioides has been identified. Like typical F. poae, the microconidia have a globose to pyriform shape, but the powdery appearance, especially on Czapek-Dox Iprodione Dichloran agar (CZID), less aerial mycelium and the lack of fruity odour on Potato Sucrose Agar (PSA) make it different from F. poae. The lack of macroconidia, polyphialides and chlamydospores differentiates it from F. sporotrichioides. All 18 isolates investigated, 15 Norwegian, two Austrian and one Dutch, produced T-2 toxin (25–400 μg/g) on PSA or Yeast Extract Sucrose agar (YES). In addition, neosolaniol, iso-neosolaniol, HT-2 toxin, 4- and 15-acetyl T-2 tetraol, T-2 triol and T-2 tetraol and4,15-diacetoxyscirpenol were formed in variable amounts. Neither nivalenol, 4- or 15-acetylnivalenolor 4,15-diacetylnivalenol were detected in any of the cultures, while these toxins were produced at least in small amounts by all the 12 typical F. poae isolates studied. The question of whether this Fusarium should be classified as F. poae or F. sporotrichioides or a separate taxon should be addressed. This revised version was published online in June 2006 with corrections to the Cover Date.     

一株产T-2毒素镰孢菌的分离、鉴定及其产毒条件的研究

【目的】从青海大骨节病区小麦麦穗中分离的内生真菌中筛选产T-2毒素的菌株,并研究影响其合成该毒素的条件。【方法】采用种子胚芽抑制试验和抑菌试验从分离所得的菌株中筛选产毒菌株;利用薄层层析和高效液相检测待测菌株产物,复筛出产T-2毒素的菌株。通过显微形态学观察及ITS序列分析对筛选出的菌株5-5m-1进行鉴定。应用单因素筛选方案研究固体培养时间、温度以及液体培养转速、初始p H等对其产T-2毒素的影响,并采用正交试验进一步优化。【结果】菌株5-5m-1的显微形态与梨孢镰孢菌(Fusarium poae)相似;ITS序列分析显示,该菌株与F.poae的相似度也较高。其产T-2毒素的最佳条件为:玉米固体培养基、日温25°C/夜温15°C、光暗交替。【结论】5-5m-1菌株为梨孢镰孢菌,培养条件对其产T-2毒素能力有很大影响。实验结果将为进一步研究T-2毒素产生的机制和防止真菌毒素污染提供参考。     

T-2 Toxin Production by Fusarium tricinctum on Solid Substrate

A method has been developed to produce and purify gram quantities of T-2 toxin [4beta, 15-diacetoxy-8alpha-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3alpha-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C.     

Fusarium tricinctum T-2 Toxin Inhibits Auxin-promoted Elongation in Soybean Hypocotyl

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, inhibited elongation of excised hypocotyl sections of Glycine max var. Hawkeye 63. Auxin-promoted elongation was inhibited more severely than was control elongation, and a 1 hour preincubation of 5 μm toxin prevented the induction of a faster rate of elongation by auxin. While the inhibition of elongation by cytokinin was similar to that of the toxin, the mode of action of the two compounds appeared to be different, i.e. their effects on elongation were additive, and only kinetin promoted radial enlargement. Toxin treatment did not diminish cytokinin-induced radial enlargement. The properties of the plasma membrane, as measured by electrolyte leakage, were not affected by the toxin.     

Fed-batch production of 2-heptanone by Fusarium poae

Summary Natural 2-heptanone is a key component of various dairy flavours. The production of this compound from octanoic acid by Fusarium poae is described in this paper. The yield of the fermentation can be considerably increased by stripping the product from the outlet gas by adsorption on an Amberlite XAD-4 column.Offprint requests to: P. H. van der Schaft     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

封闭环境气载镰孢菌及其T-2毒素发生规律的研究进展

本文阐述了普遍存在的镰孢菌可增强封闭式环境中群体对疾病的易感性,并通过其次生代谢产物(如T-2毒素)对人和动物造成严重危害及预防和控制气载镰孢菌及T-2毒素危害的相关问题,包括气载真菌和毒素采集、毒素痕量富集技术、不同环境中的镰孢菌变化规律与产毒基因研究现状。提出研究封闭式环境中气载镰孢菌及T-2毒素发生规律研究的必要性。     

In Vitro Metabolism of T-2 Toxin

Incubation of T-2 toxin with the 9,000 x g supernatant fluid of both human and bovine liver homogenate resulted in conversion to a single, deacetylated product identified as HT-2 toxin. Metabolism is more rapid in human liver. HT-2 toxin was not produced when human plasma was the incubating medium nor was it produced by treatment of T-2 toxin with simulated gastric juice. T-2 toxin was stable in gastric juice for at least 1 h.     

Biosynthesis and preparation of T-2 Toxin,HT-2 Toxin and Neosolantol

Biosynthesis of trichothecenes by strains ofF. sporotriahioides KF 9 6 and KF 530 was performed on rice as a medium. Three toxins in significant amounts were produced, with yield: T-2 toxin 0.7g from 600g of drv culture, being the highest of the three metabolites, HT-2 toxin 0.06g an Neosolaniol 0.015g. Toxins were extracted with methanol from ground and defatted dry culture. Liquid/liquid partition, using chloroform/water saturated solution of sodium bicarbonate as a preliminary purification was applied. Silicagel and charcoal columns were used in further purification. Toxin were separated by preparative TLC and crystallized from methanol.     

低温环境中T-2毒素降解菌的分离鉴定及特性研究

离筛选低温环境中T-2毒素的降解菌并探明其生化特性,探究T-2毒素降解微生物生化特性异同点,为T-2毒素降解微生物的检验提供生化判断参考。针对暴露于－20 ℃低温环境中的低浓度T-2毒素标准品,采用LC-MS/MS定量分析T-2毒素残留量,利用营养琼脂培养基（NA）和马铃薯葡萄糖琼脂培养基（PDA）共分离出5株降解菌,16S rDNA结合生化鉴定结果为死谷芽胞杆菌（Bacillus vallismortis）、蜡状芽胞杆菌（Bacillus cereus）、阴沟肠杆菌（Enterobacter cloacae）、弯曲假单胞菌（Pseudomonas geniculata）和尼泊尔葡萄球菌（Staphylococcus nepalensis）。这些分离株在－20 ℃条件下对低浓度T-2毒素均有不同程度的降解能力,其中蜡状芽胞杆菌（Bacillus cereus）降解能力最强,降解率为91%。它们的理化特性具有很多相似性,主要表现在5株菌均不能利用D 塔格糖(dTAG）,对ELLMAN都表现出阴性;Bacillus vallismortis Bp1234-7、Pseudomonas geniculata Bp24-4、〖WTBX〗Staphylococcus nepalensis Bp124-5和Enterobacter cloacae Bp123-7都能分解D-葡萄糖,而Bacillus cereus Bp1234-8不能。     

Survey for Fusaria That Elaborate T-2 Toxin

Of the 136 strains of Fusarium examined, T-2 toxin was confirmed by thinlayer chromatography in 13 of the 21 extracts that inhibited either Rhodotorula rubra or Pencillium digitatum.     

Assay and Relationship of HT-2 Toxin and T-2 Toxin Formation in Liquid Culture

Both T-2 toxin and HT-2 toxin can be conveniently quantitated in crude extracts by using a combination of thin-layer chromatography and fluorodensitometry. This technique was used to follow the production of these toxins by liquid cultures of Fusarium poae (NRRL 3287). T-2 toxin was produced prior to HT-2 toxin and hexadeuterio-T-2 toxin was converted by the culture to trideuterio-HT-2 toxin.     

Comparative study of the yield of T-2 toxic produced by Fusarium poae,F. sporotrichioides and F. sporotrichioides var. tricinctum strains from different sources

Eleven strains of Fusarium poae and F. sporotrichioides from the U.S.S.R. and 7 strains of these species and one of F. sporotrichioides var. tricinctum from U.S.A. and France have been compared as to their capacity to yield T-2 toxin. The presence of this toxin was confirmed by thin layer chromatography, gas liquid chromatography and mass spectroscopy. The strains which came from the U.S.S.R. and were originally involved in causing Alimentary Toxic Aleukia (ATA), produced several times more T-2 toxin than the others. We assume that the ability of the U.S.S.R. strains to produce much more T-toxin is due to the different ecological conditions under which they were first isolated. The remarkable maintenance of so high a level of toxin production over more than 30 years of culturing is worthy of note.This work was partly supported by the Joint Research Fund of the Hebrew University and Hadassah.     

Improved Skin Test for Detection of T-2 Toxin

A modified rat skin test based on dermatitic properties of trichothecenes is described which is quick, convenient, and sensitive to 0.05 mug of T-2 toxin.     

Isolation of 15-acetoxyscirpendiol from culture ofFusarium poae on corn

The main toxic metabolites of a strain ofFusarium poae, isolated from oats, were diacetoxyscirpenol (DAS) and a monoacetoxyscirpendiol. An accurate¹H and 13C NMR analysis allowed to identify the monoacetoxyscirpendiol as the 15-acetoxy-3α, 4 β - di hydroxy -12,13 - epoxy trichothec - 9 - ene (15-acetoxyscirpendiol). This is the first report on the production of 15 - acetoxyscirpendiol byFusarium poae.     

T-2 toxin production by Fusarium acuminatum isolated from oats and barley

Oats grain from South Africa was frequently found to be infested by toxic strains of Fusarium acuminatum, as was one barley sample. All 11 toxic strains tested produced T-2 toxin (0.8 to 2,600 mg/kg), and 6 of 11 strains produced diacetoxyscirpenol (0.6 to 8.4 mg/kg). This is the first record of T-2 toxin-producing Fusarium isolates from Africa and of the production of large amounts of T-2 toxin at relatively high (25 degrees C) temperatures.     

Characterization of Fusarium poae Microsatellite Markers on Strains from Switzerland and Other Countries

Fusarium poae is a pathogen of increasing importance within the disease complex Fusarium head blight (FHB). Eleven microsatellite markers were developed, and 72 F. poae strains from Switzerland and other countries were used to assess the level of marker polymorphism. The number of alleles for each of the markers ranged from 4 to 15, and the average gene diversity was 0.62, ranging from 0.25 to 0.84. Using these novel markers, 44 genotypes could be differentiated among all F. poae strains. Two genotypes were represented by nine and ten strains, respectively, deriving from distinct geographic areas within Switzerland and indicating a potential selection advantage. Four markers were F. poae‐specific, whereas seven markers also yielded amplification products in one to four strains of five other Fusarium species. Of the latter, five markers revealed F. poae‐specific allele size ranges. Hence, these microsatellite markers could be used both for FHB species differentiation and for intra‐specific distinction of F. poae strains.     

对虾肠道中T-2毒素降解菌的分离纯化与鉴定

从新鲜健康的凡纳滨对虾肠道中分离筛选和鉴定降解T-2毒素微生物,为日后进一步研究T-2毒素(T-2)降解菌株的相关特性及其分布规律提供参考。取新鲜健康的凡纳滨对虾肠道,通过对该肠道菌群进行定性定量分析;从T-2与对虾肠道共培养液分离纯化得到6株优势菌株;将6株优势菌株分别和T-2在胰蛋白胨大豆(TSB)肉汤中进行共培养,采用LC-MS/MS法检测该培养液前后T-2含量,筛选得到T-2降解菌株,并用微生物鉴定仪VITEK2和16S rRNA鉴定目标菌株。TSB肉汤中检测T-2的标准曲线线性良好(R=0.999 5),T-2加标回收率为91.5%~108.4%,检测限为0.01 ng/m L,相对标准偏差为0.0%~8.4%和精密度为94.5%~107%。对虾肠道中的蜡状芽胞杆菌(Bacillus cereus)和阴沟肠杆菌(Enterobacter cloacae)对T-2毒素降解率最高,分别达到91.8%和78.8%。