中国小麦赤霉病菌优势种—禾谷镰刀菌产毒素能力的研究

试验测定了分离自中国小麦赤霉病常发生地区病麦穗上的４７个禾谷镰刀菌（Ｆｕｓａｒｉｕｍ ｇｒａｍｉｎｅａｒｕｍ）菌株的产毒素能力。结果表明,它们可以产生２５种包括 单端孢霉烯族化合物（Ｔｒｉｃｈｏｔｈｅｃｅｎｅｓ）、倍半萜类化合物（Ｓｅｓｑｕｉｔｅｒｐｅｎｅｓ）、赤霉烯酮（Ｚｅａｒａｌｅｎｏｎｅ）和丁烯羟酸内酯（Ｂｕｔｅｎｏｌｉｄｅ）等类的已知次生代谢物。这些菌株属于化学型Ｉ,其中,来自我国温暖麦区的     

禾谷镰刀菌降解毒素基因Tri101的克隆和序列分析

禾谷镰刀菌Tri101基因编码的单端孢酶烯3-O-乙酰转移酶可通过加乙酰基的形式使禾谷镰刀菌产生的单族毒素(如DON)转变为较低的毒性。本研究利用RT-PCR技术从禾谷镰刀菌0623中扩增并克隆了Tri101基因的cDNA片段,测序结果表明,Tri101基因核苷酸序列阅读框架全长1356bp(GenBank序列号:GQ907236),编码451个氨基酸的多肽,推测分子量为49.45kD,等电点为5.14。氨基酸序列同源性比对结果表明,它与Kimura报道的禾谷镰刀菌Tri101氨基酸序列同源性最高,为99.56%,与其它13种镰刀菌的Tri101氨基酸序列的同源性分别为97.91%-75.68%。系统进化树分析结果表明,Fusarium graminearium0623与Fusarium sporotrichioides属于同一进化枝且与Fusarium asiaticum有较近的亲缘关系,而与F.oxysporum、F.moniliforme、F.nygamai、F.nisikadoi和F.decemcellulare的亲缘关系较远。     

国产血竭与禾谷镰刀菌

血竭是我国传统中药。它始载于南北朝时代的（雷公炮炙论）,迄今已有1500余年的应用历史。由于它具有行瘀止痛、止血、生肌、敛疮之功效,主要用于外伤出血,溃疡不敛,跌打损伤,瘀滞作痛等症,据统计,全国中成药产品中含血竭者计有膏、丹、散、片及气雾剂等共十四种剂型60多个品种。例如：七里散、跌打丸、再造丸、狗皮育、阿魏化痞育等。中药血竭的基源来自于百合科、棕相科、豆科和大教科等4科5属的17种植物不同部位的树脂。但是长期以来,国内和国际币场上血竭主要来源于棕润科黄藤属和百合科龙血树属植物的树脂。如“皇冠牌”和“手…     

禾谷镰刀菌蛋白激酶研究进展

由禾谷镰刀菌引起的小麦赤霉病是小麦生产最重要的真菌病害之一,除了造成严重的产量损失外,其病原菌还会产生多种真菌毒素危害人畜健康。蛋白激酶在禾谷镰刀菌生长发育、植物侵染和胁迫应答等方面具有重要作用。综述了禾谷镰刀菌主要蛋白激酶在生物学功能和分子作用机制等方面的研究进展,并对未来禾谷镰刀菌蛋白激酶的研究趋势进行了展望,以期为今后禾谷镰刀菌蛋白激酶的研究与小麦赤霉病的防治提供理论参考。     

禾谷镰刀菌cAMP受体类GPCR生物信息学分析

禾谷镰刀菌(Fusarium graminearum)是引起小麦赤霉病的主要致病菌。G蛋白偶联受体(G protein coupled receptors,GPCRs)是一类重要的细胞表面受体,其介导的cAMP信号通路可能参与了禾谷镰刀菌的致病和毒素合成,因此分析cAMP受体类型的GPCRs蛋白的结构及其理化性质对了解GPCRs的功能及其与赤霉病致病的关系具有重要意义。本研究运用生物信息学方法,对禾谷镰刀菌全基因组序列中cAMP类GPCR基因进行了生物信息学分析。发现禾谷镰刀菌中存在5个典型的cAMP受体类型GPCRs:FgcAR1、FgcAR2、FgcAR3、FgcAR4和FgcAR5,均含有7个跨膜结构域,并定位于细胞膜上。除FgcAR1外,其余为疏水性蛋白。蛋白质二级结构分析表明,均含有大量α螺旋,比例在60%左右,FgcAR4和FgcAR5没有β转角,FgcAR1、FgcAR2和FgcAR3也只有较少比例的β转角。这些GPCRs中含有较多的Ser和Thr磷酸化位点。遗传分析表明,禾谷镰刀菌cAMP受体类型的GPCR蛋白与假禾谷镰刀菌及F.langsethiae同源性最高,亲缘关系最近。本研究明确了禾谷镰刀菌中cAMP类GPCR蛋白的理化性质、定位、二级结构、磷酸化位点及进化关系,为了解小麦赤霉病发病机制及以GPCRs为靶标的新型杀菌剂研发奠定了基础。     

小麦响应禾谷镰刀菌侵染的转录组学研究进展

由禾谷镰刀菌引起的小麦赤霉病直接为害作物穗部,不仅严重影响小麦产量,还可因为毒素污染问题威胁人畜健康。近年来对小麦与禾谷镰刀菌互作的转录组学研究带来了很多新见解,概述了小麦响应禾谷镰刀菌侵染的转录组学研究进展,主要比较了不同抗性品种、不同器官、不同籽粒发育时期的小麦穗部在禾谷镰刀菌侵染时的基因表达特征,总结了赤霉病感染时小麦的激素响应、信号传导、转录调控和防卫相关基因的表达规律,以期促进研究者对小麦响应禾谷镰刀菌侵染规律的理解。     

禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究

目的:旨在敲除禾谷镰刀菌Fusarium graminearum Fg PDE1基因,确定其缺失突变体表型,从而分析该基因的生物学功能。方法:应用Split-marker技术构建含有潮霉素基因敲除盒,通过PEG介导原生质体转化,PCR筛查抗潮霉素转化子以获得缺失突变体ΔFg PDE1,根据突变体表型变化及致病性的检测对Fg PDE1基因的功能进行分析。结果:采用Split-marker技术,成功构建了Fg PDE1基因敲除盒;PEG介导转化禾谷镰刀菌原生质体后成功获得转化子。经PCR筛查,得到3个PCR确认的敲除突变体;表型观察发现,ΔFg PDE1菌落的外型及菌落生长速度与野生型没有明显差异。孢子侵染西红柿果实实验证明:以西红柿为侵染宿主,相对于野生型,突变体致病性没有明显减弱;但突变体分生孢子产量显著下降。结论:Fg PDE1基因可能与禾谷镰刀菌分生孢子的形成有关。     

禾谷镰刀菌拮抗菌21-6的鉴定及其抑菌活性测定

【背景】禾谷镰刀菌（Fusarium graminearum）是一种危害小麦生产的重要病原真菌。【目的】筛选对禾谷镰刀菌具有拮抗活性的链霉菌菌株,为该病原的生物防治提供理论基础。【方法】采用稀释涂布法分离链霉菌,利用平板对峙法筛选高活性拮抗菌株;通过形态学、生理生化特征和16S rRNA基因序列分析确定其分类地位;采用生长速率法分析其发酵条件及无菌发酵液的稳定性;并测定该菌株的防病效果和抑菌谱。【结果】筛选到一株对禾谷镰刀菌具有较强抑制作用的链霉菌菌株21-6,抑菌率为75.2%±2.1%。根据形态学、生理生化特征及16S rRNA基因序列分析,将其鉴定为Streptomyces stelliscabiei。菌株21-6在pH为中性条件下的PDB培养基中培养5 d能够产生更好的抑菌效果。无菌发酵液能够抑制禾谷镰刀菌的菌丝生长和孢子萌发过程。无菌发酵液不受高温、胃蛋白酶、胰蛋白酶及蛋白酶K的影响,耐酸但对碱性条件敏感。发酵液对禾谷镰刀菌侵染小麦胚芽鞘具有抑制效果。菌株21-6具有聚酮合酶pks-Ⅰ和pks-Ⅱ基因。此外,该菌株对5种植物病原真菌均具有抑制效果。【结论】链霉菌菌株21-6对禾谷镰刀菌具有较好的生防潜力。     

串珠镰刀菌产生毒素条件研究

从培养基种类和培养方法等方面对串珠镰刀菌产生毒素条件进行了较系统的研究。结果表明串珠镰刀菌的最佳产生毒素条件为马铃薯 葡萄糖培养液、pH 91、2 h光暗交替、25℃、培养10 d。     

γ射线紫外线镰刀菌产生玉米赤霉烯酮毒素的影响

锭刀菌(Fusarium)经γ射线、紫外线辐射诱变后,接种于大米培养基中.经10℃低温产毒培养.采用薄层层析方法定性定量分析培养物中的镀刀菌毒素—玉米赤霉烯酮(zearalenone简称ZEA).结果表明:不同菌株对诱变剂的反应不同.产毒菌株NF5232、NF5946在较大剂量γ射线处理后,ZEA产量显著增加,但其产生的色素量下降.或者用紫外线处理,仍然产生ZEA.不产毒菌株NF6127、NF6123、NP6138和HD—002,经γ射线或紫外线处理后仍不产生ZEA.由此可知镰刀菌产生ZEA毒素的能力是相对稳定的,其产毒机制是由菌株本身的遗传特性决定的,一定剂量的γ射线可使低产毒菌株变为高产毒菌株.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China

A large number of isolates from the Fusarium graminearum clade representing all regions in China with a known history of Fusarium head blight (FHB) epidemics in wheat were assayed using PCR to ascertain their trichothecene mycotoxin chemotypes and associated phylogenetic species and geographical distribution. Of the 299 isolates assayed, 231 are from F. asiaticum species lineage 6, which produce deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON); deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON); and nivalenol and 4-acetylnivalenol (NIV) mycotoxins, with 3-AcDON being the predominant chemotype. Ninety-five percent of this species originated from the warmer regions where the annual average temperatures were above 15 °C, based on the climate data of 30 y during 1970–1999. However, 68 isolates within F. graminearum species lineage 7 consisted only of 15-AcDON producers, 59 % of which were from the cooler regions where the annual average temperatures were 15 °C or lower. Identification of a new subpopulation of 15-AcDON producers revealed a molecular distinction between F. graminearum and F. asiaticum that produce 15-AcDON. An 11-bp repeat is present in F. graminearum within their Tri7 gene sequences but is absent in F. asiaticum, which could be directly used for differentiating the two phylogenetic species of the F. graminearum clade.     

High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris

High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZαA with the Saccharomyces cerevisiae α-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)₆-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Fusarium nygamai, a potential bioherbicide for Striga hermonthica control in sorghum

Glasshouse trials were performed to investigate the control of the parasitic weed Striga hermonthica by Fusarium nygamai and the performance of the host plant sorghum (Sorghum bicolor) using different inoculum substrates and inoculum amounts of the fungus. Optimal constant and alternating temperatures for the growth of the fungus were 25°C and 30/20°C, respectively. Striga incidence was decreased up to 100% when the fungus was incorporated into the soil preplanting. Emerged Striga plants at different stages of growth up to the flowering stage were killed by the fungus when the fungus was applied postemergent. In root-chamber trials none of the Striga seeds germinated when 10 ml inoculum suspension of 8 × 10⁶ spores/ml of F. nygamai was applied on seeds of the parasitic weed sprinkled on the surface of filter paper. F. nygamai has potential as a bioherbicide for Striga control. Further studies regarding its performance under field conditions and its safety to the environment and humans should be assessed.     

Natural variation of ascospore and conidial germination by Fusarium verticillioides and other Fusarium species

Fusarium verticillioides and other Fusarium species were examined for their spore germination phenotypes. In general, germinating spores of F. verticillioides formed germ tubes that immediately penetrated into agar. Such invasive germination was the predominant growth phenotype among 22 examined field isolates of F. verticillioides from a broad range hosts and locations. However, two of the field isolates were unique in that they formed conidial germ tubes and hyphae that grew along the surface of agar before penetration eventually occurred. Conidia of 22 other Fusarium species were assessed for their germination phenotypes, and only some strains of F. annulatum, F. fujikuroi, F. globosum, F. nygamai, and F. pseudoanthophilum had the surface germination phenotype (21 % of the strains assessed). Sexual crosses and segregation analyses involving one of the F. verticillioides surface germination strains, NRRL 25059, indicated a single locus, designated SIG1 (surface vs. invasive germination), controlled the germ tube growth phenotypes exhibited by both conidia and ascospores. Perfect correlation was observed between an ascospore germination phenotype and the germination phenotype of the conidia produced from the resulting ascospore-derived colony. Recombination data suggested SIG1 was linked (7 % recombination frequency) to FPH1, a recently described locus necessary for enteroblastic conidiogenesis. Corn seedling blight assays indicated surface germinating strains of F. verticillioides were less virulent than invasively germinating strains. Assays also indicated pathogenicity segregated independently of the FPH1 locus. Invasive germination is proposed as the dominant form of spore germination among Fusarium species. Furthermore, conidia were not necessary for corn seedling disease development, but invasive germination may have enhanced the virulence of conidiating strains.     

Physiological variation among native and exotic winter annual plants associated with microbiotic crusts in the Mojave Desert

An experiment was established in 1992 in eastern Ontario, Canada to determine the effects of crop rotation (continuous maize, soybean-maize and alfalfa-maize) and nitrogen (N) amendment [0, 100 and 200 kg N ha^–1 of fertilizer (NH₄NO₃), and 50 and 100 Mg ha^–1 (wet wt.) each of stockpiled and rotted dairy manure] on maize production and soil properties. From 1997 to 1999, an additional study was added to the experiment to investigate treatment effects on the susceptibility of maize hybrids to gibberella ear rot. A moderately resistant and a susceptible hybrid were planted in each plot and inoculated with a macroconidial suspension of Fusarium graminearum by both the silk channel injection and the kernel-wound techniques. At harvest, ears were rated for the severity of disease symptoms and harvested kernels were analyzed for the mycotoxin deoxynivalenol (DON). The greatest number of significant N effects were found in the continuous maize treatments and with the susceptible hybrid. Most N amendments decreased both disease severity and DON accumulation in the susceptible hybrid. The most consistent effect was a decrease in disease severity with 100 kg N ha^–1 fertilizer and an increase in disease severity with the higher rate of 200 kg N ha^–1. This study is the first to report on the effects of soil N amendments on gibberella ear rot susceptibility.     

Deoxynivalenol and contaminant mycoflora in freshly harvested Argentinian wheat in 1993

A mycological survey was carried out on wheat heads from the main production area of Argentina. The isolation frequency and relative density of species from genus Fusarium and dematiaceous fungi were calculated. F graminearum was the predominant Fusarium species; similar to that observed in the USA and Canada. An analysis of deoxynivalenol (DON) natural contamination also was performed on a limited number (44) of samples. DON contamination levels in positive samples ranged from 0.2 to 30 ppm. A stepwise regression procedure showed that, among the species analysed, F. graminearum relative density was related to the DON contamination level and that other prevalent fungi did not influence or modify that relationship.