高效液相色谱法分离和鉴定腐马素B1

腐马素是由串珠镰刀菌产生的真菌毒素,其中腐马素B1可引发马脑白质软化症等疾病。本文用高效液相色谱法从串珠镰刀菌玉米培养物中分离出了腐马紊B1并通过紫外光谱和快原子轰击质谱进行了鉴定。     

高效液相色谱法分离和鉴定腐马素B1

腐马素是由串珠镰刀菌产生的真菌毒素,其中腐马素Ｂ１可引发马脑白质软化症等疾病。本文用高效液相色谱法从忠珠镰刀菌玉米培养物中分离出了腐马素Ｂ１并通过紫外光谱和快原子轰击质谱进行了鉴定。     

腐马素的研究概况

腐马素（Fumonisins）是一组结构相关的镰刀菌毒素,现已确定这组毒素共有6种组分,分别为FB;、FB。、FB。、FB。、FA;foFA。l’,’］。其化学结构与动物的神经鞘氨醇很相似（图1）。F民是天然污染玉米样品或真菌培养物中的主要腐马素组分,其次是FB。,其它4种含量较少。FA;和FA。没有毒性。腐马素最初是由南非的Gelderblom等于1988年在研究马属动物霉玉米中毒症病因时,从串珠镰刀菌（Fu＄ariummonil和侧e）培养物中发现的［’］。此菌在世界上普遍存在,是对玉米污染严重的一种真菌。腐马素对不同动物可引起不同的病理反应…     

应用PCR技术检测串珠镰刀菌

串珠镰刀菌(Fusarium moniliform)是一种危害严重、在世界各地广泛流行的植物病原真菌,当前对串珠镰刀菌的鉴定主要根据其菌丝体及再生菌丝的形态结构学特征及染病作物的病害症状来进行鉴定。这些鉴定方法相对简单并在很大程度上依赖于经验,受主观因素影响较大。采用串珠镰刀菌种特异性的寡聚核苷酸为引物,运用PCR技术对串珠镶刀菌进行检测是一种快速可靠的检测鉴定方法,它无需病原菌的分离培养纯化,能从感病的玉米组织中直接实现对串珠镰刀菌的快速检测。经对霉变玉米样品和玉米穗腐病组织的检测,证明该方法是一种快建、有效的方法,具有重要的实际应用价值。     

青海豌豆根腐病病原菌种类及致病性的研究

豌豆极腐病是青海东部干旱地区豌豆生产上的一种新病害,近年来危害逐年加重,致使豌豆产量遭受严重损失。根据分离鉴定和致病性测定结果,青海豌豆根腐病病原真菌是由茄镰刀菌、尖孢镰刀菌、豌豆丝囊霉、根串珠霉、立枯丝核菌、腐霉、链孢粘帚霉等复合反染所引起的。经回接试验：镰刀菌和豌豆丝囊霉对豌豆具有较强的致病力;腐霉及其他病原菌则有加强腐烂作用。     

串珠镰刀菌和尖孢镰刀菌对玉蜀黍苗期侵染的接种试验

以土壤接种的方法测定了串珠镰刀菌(Fusarium moniliforme Sheld.) 3个菌株和尖孢镰刀菌(Fusarium oxysporum Schl．)9个菌株对玉蜀黍幼苗的侵染力。结果表明在试验菌株中存在强、中等、不稳定、弱至无四种致病力类型。串珠镰刀菌中的NF2109(分离白玉蜀黍杆腐病株)和哈医143(分离自玉蜀黍种粒),尖孢镰刀菌中的NF1800(分离自香蕉果柄)和NF109(分离自松林土壤)属于强致病力类型。尖孢镰刀菌中的NF033(分离自玉蜀黍种粒)、NF245(分离自苹果烂根)、哈医142(分离自玉蜀黍种粒)属于中等强度类型。其余5个试验菌株有2个不稳定,3个呈弱或无致病力类世。叙述了这些菌引起的玉米苗枯的症状,并认为对玉蜀黍苗期的侵染,串珠镰刀菌的危在较之尖孢镰刀菌为大,并就试验菌林的致病力讨论了有关尖孢镰刀菌号化型问题。     

拮抗枯草芽孢杆菌KC-5的分离鉴定及其发酵优化

为了筛选出对植物病原菌具有拮抗作用的生物防治细菌,采用平板对峙法从蔬菜根际土壤中分离获得一株对多种病原真菌具有抑制作用的枯草芽孢杆菌,并对抑制后的病原菌菌丝进行了观察,结果表明该菌株对尖孢镰刀菌、串珠镰刀菌、层出镰刀菌以及腐皮镰刀菌均具有抑菌活性。通过形态特征、生理生化特征及16S rRNA序列分析,将该菌株鉴定为枯草芽孢杆菌(Bacillus subtilis),并对其发酵培养基进行了优化。     

串珠镰刀菌胶孢变种的玉米培养物对雏鸭毒性的病理学观察

<正> 串珠镰刀菌胶孢变种是玉米等谷物上的常见病原菌之一,该菌可产生对动物有害的毒素——串珠镰刀菌素(moniliformin)(Buck et al.,1979:Burmeister et al.,1979;Cole et al.,1973)。最近的研究发现,串珠镰刀菌胶孢变种的玉米培养物还可引起实验性马属动物脑白质软化症(Equine leucoence-phalomalacia)(辛德颐,1987),更引起了人们对该菌的重视。为了进一步研究串珠镰刀菌胶孢变种的毒性,特别是为将来研究其引起马属动物脑白质软化症的致病毒素及其机理提供依据,本实验应用曾引起实验性马属动物脑白质软化症的串珠镰刀菌胶孢变种LBFMV 14的玉米培养物(辛德颐,1987),按不同的比例与雏鸭饲料混合,饲喂1日龄北京鸭,观察其中毒死亡情况和病理变化。     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

串珠镰刀菌对棉花枯萎病的交互保护作用研究

试验证明,人工给棉花接种串珠镰刀菌,可对棉花枯萎病产生交互保护作用;采用五氯硝基苯拌种诱发自然串珠镰刀菌,同样可达到上述目的;在进行交互保护作用的同时,棉花不会因该菌的作用,而出现大量死苗和影响寄主生长发育的现象。另外还观察到,串珠镰刀菌和棉花枯萎菌之间具有互为抑制作用,抑制力强弱,取决于二者先后间隔接种时间的早晚;除两种菌等量分开接种,在培养皿中心产生拮抗带外,二者等量和倍量混合接种,均长出单一的串珠镰刀菌。     

Production of fumonisins by Fusarium moniliforme and Fusarium proliferatum isolates associated with equine leukoencephalomalacia and a pulmonary edema syndrome in swine.

Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.     

Production of fumonisins by Fusarium moniliforme and Fusarium proliferatum isolates associated with equine leukoencephalomalacia and a pulmonary edema syndrome in swine

Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.     

Survey of fumonisin production by Fusarium species

Fumonisins B1 (FB1) and B2 (FB2), two structurally related mycotoxins with cancer-promoting activity, were recently isolated from corn cultures of Fusarium moniliforme MRC 826. These toxins have been reported to be produced also by isolates of F. proliferatum. Contamination of foods and feeds by F. moniliforme has been associated with human esophageal cancer risk, and FB1 has been shown to be the causative agent of the neurotoxic disease leukoencephalomalacia in horses. Because of the toxicological importance of the fumonisins, the potential to produce FB1 and FB2 was determined in a study of 40 toxic Fusarium isolates representing 27 taxa in 9 of the 12 sections of Fusarium, as well as two recently described species not yet classified into sections. With the exception of one isolate of F. nygamai, fumonisin production was restricted to isolates of F. moniliforme and F. proliferatum, in the section Liseola. The F. nygamai isolate produced 605 micrograms of FB1 g-1 and 530 micrograms of FB2 g-1, and the identity of the toxins was confirmed by capillary gas chromatography-mass spectrometry. This is the first report of the production of the fumonisins by F. nygamai.     

Fum3p, a 2-ketoglutarate-dependent dioxygenase required for C-5 hydroxylation of fumonisins in Fusarium verticillioides

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species. The B series fumonisins, FB(1), FB(2), FB(3), and FB(4), are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae. Yeast cells expressing the Fum3p converted FB(3) to FB(1), indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe(2+), ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.     

Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatun (Matsushima) Nirenberg culture material

Fumonisins, a group of mycotoxins produced by the ubiquitous fungi Fusarium moniliforme and F. proliferatum, were first identified about eight years ago. They have been shown to cause a variety of health effects in animals, including epidemiological evidence of esophageal cancer in humans. Cattle are less sensitive to ill effects than horses and swine. Fumonisins are common contaminants of low quality grain fed to cattle. Culture material containing fumonisins (FB1, FB2, and FB3) was mixed into the total diet and fed for 14 days to two midlactation Jersey cows to determine if fumonisins are excreted in milk. The dietary equivalent of fumonisin was approximately 75 ppm and the two cows consumed an average of 3 mg fumonisin Bl /kg body weight (b wt)/day. Fumonisins were not detected in any of the milk samples by two analytical laboratories using methods with a sensitivity of 5 ng/ml. Except for transient diarrhea at the beginning of the contaminant feeding period and an increase in serum cholesterol, clinical and hematologie changes were not observed in the animals. The appearance or carry over of fumonisins from feed to milk in dairy cows does not appear to be significant and likely not a hazard or food safety concern for humans.     

Fusarium species and fusarium mycotoxins in cereals from West Romania: preliminary survey

Fungal contamination of plant products is an important risk factor for health, because of the high mycotoxin potential deriving from these contaminations with multiple effects: hepatic toxicity, teratogenic, mutagenic and carcinogenic. The contamination of cereals with mycotoxins has been a serious problem in Balkan communities. Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy). The trichothecenes are of particular concern because they are ubiquitous found in wheat, corn and barley throughout the world. Fumonisins have been isolated from certain Fusarium species of which FB1, FB2 and FB3 are the major ones produced in naturally contaminated foods.These mycotoxins are produced on cereal grains infected by Fusarium while being grown in-the-field. The aim of this study is to evaluate the presence of the Fusarium species in cereals from West side of Romania and to determinate the concentrations of deoxynivalenol (DON) and fumonisine (F1+F2). Identification of Fusarium species was done using the total number of fungal species determination method. The level of mycotoxins was determined with the immune-enzymatic method ELISA. 27 cereal samples from rural households in three counties in West Romania were analysed.     

FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

A bidomain nonribosomal peptide synthetase encoded by FUM14 catalyzes the formation of tricarballylic esters in the biosynthesis of fumonisins

Fumonisins are a group of polyketide-derived mycotoxins produced by Fusarium verticillioides, a filamentous fungus infecting corn and contaminating food and feeds. Fumonisins contain two tricarballylic esters that are critical for toxicity. Here, we present genetic and biochemical data for the esterification mechanism. FUM14 in F. verticillioides has been deleted by homologous recombination, and the resultant mutant lost the ability to produce fumonisins. Two new metabolites, HFB(3) and HFB(4), which are biosynthetic precursors of fumonisins lacking the tricarballylic esters, were detected in the mutant. The results suggest that FUM14 is required for the esterification of fumonisins. FUM14 was predicted to encode a nonribosomal peptide synthetase (NRPS) containing two domains, peptidyl carrier protein and condensation domain. Both the intact Fum14p and the condensation domain have been expressed in Escherichia coli and purified for activity assays. Fum14p was able to convert HFB(3) and HFB(4) to the tricarballylic esters-containing fumonisins, FB(3) and FB(4), respectively, when incubated with tricarballylic thioester of N-acetylcysteamine. In addition, the condensation domain was able to convert HFB(1) to FB(1). These data provide direct evidence for the role of Fum14p in the esterification of fumonisins. More interestingly, the results are the first example of an NRPS condensation domain catalyzing a C-O bond (ester) formation, instead of the typical C-N bond (amide) formation in nonribosomal peptides. The understanding of the esterification mechanism provides useful knowledge for mycotoxin reduction and elimination. The study also provides new insight into the reactions catalyzed by NRPS.     

Fumonisin- and AAL-Toxin-Induced Disruption of Sphingolipid Metabolism with Accumulation of Free Sphingoid Bases

Fumonisins (FB) and AAL-toxin are sphingoid-like compounds produced by several species of fungi associated with plant diseases. In animal cells, both fumonisins produced by Fusarium moniliforme and AAL-toxin produced by Alternaria alternata f. sp. lycopersici inhibit ceramide synthesis, an early biochemical event in the animal diseases associated with consumption of F. moniliforme-contaminated corn. In duckweed (Lemna pausicostata Heglem. 6746), tomato plants (Lycopersicon esculentum Mill), and tobacco callus (Nicotiana tabacum cv Wisconsin), pure FB1 or AAL-toxin caused a marked elevation of phytosphingosine and sphinganine, sphingoid bases normally present in low concentrations. The relative increases were quite different in the three plant systems. Nonetheless, disruption of sphingolipid metabolism was clearly a common feature in plants exposed to FB1 or AAL-toxin. Resistant varieties of tomato (Asc/Asc) were much less sensitive to toxin-induced increases in free sphinganine. Because free sphingoid bases are precursors to plant "ceramides," their accumulation suggests that the primary biochemical lesion is inhibition of de novo ceramide synthesis and reacylation of free sphingoid bases. Thus, in plants the disease symptoms associated with A. alternata and F. moniliforme infection may be due to disruption of sphingolipid metabolism.