小麦矮腥黑粉菌原生质体的制备与再生

本文优化了小麦矮腥黑粉菌原生质体的制备与再生条件。结果表明:小麦矮腥黑粉菌的冬孢子在土壤浸提液固体培养基中萌发后转接入T19液体培养基,培养45d,以菌丝作为酶解初始材料,用1.5%崩溃酶+1.5%溶壁酶+1.5%蜗牛酶复合酶液28℃酶解2.5h原生质体的获得率最高;以1.2mol/L的氯化钾为渗透压稳定剂,所得原生质体数量最多,且释放的原生质体能均匀分散分布,不聚集成堆;小麦矮腥黑粉菌的原生质体在TB3培养基上能长出较多的单菌落。     

小麦矮腥黑穗病菌对温度的敏感性

通过研究小麦矮腥黑穗病菌不同菌株冬孢子萌发对温度的敏感性,进一步明确不同TCK菌株冬孢子萌发对温度的反应,以便深入了解此病害发生和流行的成因.选取采自不同年份的45个TCK菌株,室内控制不同温度条件下对这些菌株的萌发过程及萌发特性进行观察记录,分析所得数据.5℃和10℃不同培养温度下,参试菌株的萌发率和萌发过程存在显著或极显著差异.同一采集年份不同菌株在相同培养温度下,萌发过程和萌发率有明显的差异,随着保存年限的增长,菌株冬孢子的萌发率会逐渐下降,但在常温下放置8年左右的部分菌株仍然具有萌发能力.不同的菌株萌发对温度确实具有不同的敏感性,此结果为防止该病害的传入、定殖、传播以及预测和防治决策提供了重要依据.     

小麦矮腥黑粉菌的鉴定及检测方法

由Tilletia controversa Kühn(TCK)引起的小麦矮腥黑穗病是重要的国际检疫性病害,也是我国对外的A类检疫对象。本文就该病原菌的形态学和生物学鉴定、分子检测方法进行了综述。展望了分子检测技术在病害检疫中的应用前景。     

多年生黑麦草中小麦矮腥黑穗病菌PCR 检测方法的建立

寄生于多年生黑麦草的Tilletia属腥黑粉菌共有4种,即小麦矮腥黑穗病菌Tilletia controversa（TCK）、黑麦草腥黑粉病菌T.lolii、T.vankyi、黑麦草粒腥黑穗病菌T.walkeri。本研究分析了黑麦草上冬孢子形态非常相似的3种腥黑粉菌的DNA序列差异,设计了TCK的特异引物,成功建立了TCK菌丝基因组DNA的特异PCR检测方法和冬孢子的套式特异PCR检测方法。     

电子辐照灭活小麦矮腥黑穗菌

电子射线辐照处理小麦矮腥黑穗菌冬孢子,5kGy的剂量可使冬孢子丧失萌发力失去活性.     

小麦矮腥黑粉菌及其近缘种的RPB2基因片段序列分析

以小麦矮腥黑粉菌（Tilletia controversa Kühn）及其近缘种小麦网腥黑粉菌［T. caries （DC.）Tul.］、小麦光腥黑粉菌(T. laevis Kühn)和其他6种黑粉菌的DNA为模板,用RNA聚合酶II的第2亚基RPB2基因的通用引物RPB2-740F/RPB2-1365R进行PCR扩增。结果表明,3种小麦腥黑粉菌均能扩增出617 bp大小的DNA片段,供试的其他6种黑粉菌没有任何扩增产物。利用DNAMAN软件进行序列分析结果表明,3种小麦腥黑粉菌的RPB2蛋白基因序列的相似性为99.08%,存在17个碱基的差异。利用RPB2基因的通用引物作为小麦腥黑粉菌的内置对照引物,与小麦矮腥黑粉菌的特异引物CQUTCK2/CQUTCK3相结合可提高小麦矮腥黑粉菌检测的准确性。     

小麦矮化腥黑穗病菌侵染能力的研究

中美专家合作在美国犹他州对小麦矮化腥黑穗病菌的侵染能力进行了研究。试验采用不含抗病基因的小麦品种Cheyanne,播种前用溴甲烷对试验小区进行了熏蒸。接种物为含有多种致病型的冬孢子悬浮液,采用土壤表面喷洒冬孢子悬浮液的方法进行接种,接种密度为每cm20、0.88、8.84、88.45、884.49、8844.89、88449个孢子。结果表明,接种密度为每cm20.88个孢子时即可产生0.21％的穗发病率。对试验数据进行分析后表明,小麦矮化腥黑穗病菌的侵染规律符合侵染概率理论,在本试验条件下,单个孢子的侵染概率为0.0002。     

基于ISSR标记开发的一种用于小麦矮腥黑穗病菌的分子鉴定方法

 小麦矮腥黑穗病菌(Tilletia controversa Kühn, 简称TCK)是小麦上的一种重要检疫性真菌。本研究利用内部简单重复序列(Inter-simple sequence repeat, ISSR)技术研究TCK及其近缘种的DNA多态性,开发了一种可靠而简单的方法用于TCK的分子鉴定。用ISSR引物P4从TCK中扩增出一条1 113 bp的特异性条带,据此设计了一对特异性引物TCKF/TCKR,在12个TCK菌株中均能扩增得到一条882 bp的特异性条带,而其他近缘种包括小麦网腥黑穗病菌(T. caries)和小麦光腥黑穗病菌(T. foetida)及相关黑粉菌的14个菌株均无扩增条带。用该特异性引物检测TCK的下限为25 μL反应体系中可检测到1 ng DNA模板。本研究开发的种特异性引物,可将TCK与其形态上相似的近缘种尤其是小麦网腥黑穗病菌准确区分开,本研究基于ISSR标记建立的小麦矮腥黑穗病菌的分子鉴定方法为腥黑粉菌的检疫提供了一种便捷的方法,是对现有分子鉴定方法的一个补充。     

小麦矮腥黑穗病菌的单孢接种侵染

采用进口美麦中截获的单个、经萌发的Ｔｉｌｌｅｔｉａ ｃｏｎｔｒｏｖｅｒｓａ Ｋｕｈｎ（ＴＣＫ）冬孢子人工接种创伤和未创伤的美国硬红冬小麦的胚芽鞘,每个胚芽鞘接种一个萌发ＴＣＫ冬孢子,接种后麦苗在５℃、１２ｈ光照和黑暗交替条件下接头２１或２９天,然后移植到离网室中的塑料周转箱中作常栽培管理,结果均得了ＴＣＫ病穗。这是单个萌发ＴＣ冬孢子引起小麦发病的首次报道。本试验结果表明,在自然条件下,当寄主和环境     

小麦矮腥黑穗病菌和普通腥黑穗病菌在几种灭活处理中的相关性分析

对小麦矮腥黑穗病菌和小麦普通腥黑穗菌(光腥和网腥)进行了60Co辐照、环氧乙烷熏蒸、直线加速器电子辐射等方法的灭活处理实验并进行了相关性分析.结果表明各处理对几种菌灭活效果基本一致.预示实验中的两种普通腥黑穗菌可做为矮腥黑穗菌的指示菌.     

温度对小麦矮腥黑穗病菌冬孢子萌发的影响

温度对小麦矮腥黑穗病菌冬孢子萌发影响的试验结果表明,TCK冬孢子在-2～12℃范围内都可以萌发,5℃为最佳萌发温度。同一分离菌冬孢子在不同温度处理的萌发特性和萌发率有差异;不同分离菌冬孢子在相同温度下的萌发特性和萌发率有很大差异。在5℃下5个分离菌中,冬孢子萌发起始时间最短的为15 d,最长为35 d,培养60 d时分离菌T_t1、T_t2、T_y、T_m和T_u冬孢子的萌发率分别为86.4%、23.9%、23.3%、44.3%和81.0%。根据分离菌T_t2不同温度下培养50 d时的萌发率,建立了萌发率(Y)与温度(X)的模型为Y=0.150 8 exp[-0.02949(X-4.957 6)]2,此结果为TCK的风险评估提供了基础数据。     

Estimation of Genetic Variation Among Verticillium Isolates using AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis was used to study genetic variation among 76 isolates of Verticillium. A dendrogram based on the AFLP data revealed three main groups. One group consisted of 35 European isolates derived from Brassica napus together with five Californian isolates taken from B. oleracea. This group displayed a high degree of genetic similarity and included three isolates earlier classified as Verticillium longisporum, indicating that all isolates in this group probably should be regarded as members of V. longisporum. V. dahliae isolates constituted the second group while the third group contained four V. albo-atrum isolates. In addition to these three groups, a cluster of six V. nigrescens isolates was observed. However, the genetic distances between the isolates of V. nigrescens were much higher than those between members in the other groups and the bootstrap value for the V. nigrescens cluster was subsequently low. Four isolates classified as V. tricorpus were highly diverse and did not cluster together. Analysis of molecular variance revealed that the isolates of V. longisporum were separated into four subgroups, based on geographic origin. The study furthermore shows that AFLP is a suitable method for studying population structure in Verticillium.     

土壤湿度对小麦矮腥黑穗病菌(TCK)冬孢子萌发的影响

通过土壤湿度对小麦矮腥黑穗病菌(Tilletiacontronversa Kühn,TCK)萌发率的影响试验研究表明,其冬孢子在土壤质量含水量为1%～28%(相对含水量3.57%～100%)范围内均可萌发,其适宜萌发的土壤质量含水量范围为10%～25%(相对含水量17.85%～89.3%),最佳土壤相对含水量范围在65%～75%之间;不同分离物在相同土壤湿度培养下,多数分离物冬孢子的萌发率之间差异不显著。根据分离菌T_t1和T_t2在不同土壤湿度下培养50d和60d的冬孢子萌发率,建立了TCK冬孢子萌发率与土壤相对含水量的关系模型,此结果为TCK的风险评估提供了基础数据。     

TaqMan-MGB探针在小麦印度腥黑穗病菌和黑麦草腥黑穗病菌鉴定上的应用

根据小麦印度腥黑穗病菌Tilletia indica和黑麦草腥黑穗病菌T.walkeri核糖体ITS序列设计了两对通用引物和两条特异性探针,建立了小麦印腥印度腥黑穗病菌Tilletia indica和黑麦草腥黑穗病菌T.walkeri的实时荧光PCR检测方法,检测的灵敏度为1个冬孢子.这种检测方法可以直接用于样品小麦印腥和黑麦草腥黑穗病菌冬孢子的快速检测,整个检测过程缩短至1天.     

High Genetic Similarity Among Populations of Phaeosphaeria nodorum Across Wheat Cultivars and Regions in Switzerland

ABSTRACT Phaeosphaeria nodorum was sampled from nine wheat fields across a 30-km transect representing three geographical regions in Switzerland to determine the scale of genetic differentiation among subpopulations. Three different wheat cultivars were sampled three times to determine whether differences in host genotype correlated with differences among corresponding pathogen populations. Seven restriction fragment length polymorphism (RFLP) loci and one DNA fingerprint were assayed for each of the 432 isolates in the collection. DNA fingerprints differentiated 426 unique genotypes. Though absolute differences were small, five RFLP loci exhibited significant differences in allele frequencies across the nine sub-populations. Gene diversity within all subpopulations was high (H(T) = 0.51), but only 3% of the total genetic variation was distributed among the nine subpopulations. When subpopulations were grouped according to geographical region or host cultivar, less than 1% of the genetic variation was distributed among groups, suggesting widespread gene flow and the absence of pathogen adaptation to specific wheat cultivars. Tests for gametic equilibrium within subpopulations and across the entire Swiss population supported the hypothesis of random mating.     

Genetic Variation Among Vegetative Compatibility Groups of Fusarium oxysporum f. sp. cubense Analyzed by DNA Fingerprinting

ABSTRACT Genetic variation within a worldwide collection of 208 isolates of Fu-sarium oxysporum f. sp. cubense, representing physiological races 1, 2, 3, and 4 and the 20 reported vegetative compatibility groups (VCGs), was analyzed using modified DNA amplification fingerprinting. Also characterized were 133 isolates that did not belong to any of the reported VCGs of F. oxysporum f. sp. cubense including race 3 isolates from a Heliconia species and isolates from a symptomatic wild banana species growing in the jungle in peninsular Malaysia. The DNA fingerprint patterns were generally VCG specific, irrespective of geographic or host origin. A total of 33 different genotypes were identified within F. oxysporum f. sp. cu-bense; 19 genotypes were distinguished among the isolates that belonged to the 20 reported VCGs, and 14 new genotypes were identified among the isolates that did not belong to any of the existing VCGs. DNA fingerprinting analysis also allowed differentiation of nine clonal lineages within F. oxysporum f. sp. cubense. Five of these lineages each contained numerous closely related VCGs and genotypes, and the remaining four lineages each contained a single genotype. The genetic diversity and geographic distribution of several of these lineages of F. oxysporum f. sp. cubense suggests that they have coevolved with edible bananas and their wild diploid progenitors in Asia. DNA fingerprinting analysis of isolates from the wild pathosystem provides further evidence for the coevolution hypothesis. The genetic isolation and limited geographic distribution of four of the lineages of F. oxysporum f. sp. cubense suggests that the pathogen has also arisen independently, both within and outside of the center of origin of the host.     

Identification and Differentiation of Tilletia indica and T. walkeri Using the Polymerase Chain Reaction

ABSTRACT Karnal bunt of wheat, caused by Tilletia indica, was found in regions of the southwestern United States in 1996. Yield losses due to Karnal bunt are slight, and the greatest threat of Karnal bunt to the U.S. wheat industry is the loss of its export market. Many countries either prohibit or restrict wheat imports from countries with Karnal bunt. In 1997, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the recently discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3 kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was =3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mitochondrial DNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5' nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.     

Genetic Variation Among and Within Monilinia Species Causing Brown Rot of Stone and Pome Fruits

Nucleotide sequence analysis of the internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) divided the three brown rot pathogens Monilinia laxa, M. fructicola and M. fructigena into four distinct groups. Isolates of M. fructigena received from Japan, which varied by 5 base substitutions in the ITS region from the European M. fructigena isolates, formed the fourth group. Four of five Japanese isolates of M. fructicola tested varied from the New World isolates in that they did not possess a group-I intron in the small subunit (SSU) rDNA. RAPD-PCR data indicated that isolates of M. laxa varied but were randomly distributed worldwide; ITS data indicated no apparent distinction between those from Malus spp. and those from Prunus spp. M. fructigena similarly did not cluster according to geographic origin. In contrast, M. fructicola isolates tended to be clustered according to their origin; Japanese isolates of M. fructicola clustered together and showed similarity to some of the New Zealand isolates. Isolates from USA and Australia were more variable.     

Comparative Study of Genetic Diversity and Pathogenicity Among Populations of Verticillium Dahliae from Cotton in Spain and Israel

Genetic diversity and phenotypic diversity in Verticillium dahliae populations on cotton were studied among 62 isolates from Spain and 49 isolates from Israel, using vegetative compatibility grouping (VCG), virulence and molecular assays. In Spain, defoliating V. dahliae isolates (D pathotype) belong to VCG1, and non-defoliating isolates (ND) belong to VCG2A (often associated with tomato) and VCG4B (often associated with potato). The D pathotype was not identified in Israel. The ND pathotype in Israel is comprised of VCG2B and VCG4B. Isolates in VCG2B and VCG4B ranged in virulence from weakly virulent to highly virulent. The highly virulent isolates induced either partial defoliation or no defoliation. Virulence characteristics varied with inoculation method and cotton cultivar. Highly virulent isolates from Israel were as virulent as D isolates from Spain under conditions conducive to severe disease. The D pathotype is pathologically and genetically homogeneous, whereas the ND pathotype is heterogeneous with respect to virulence, VCG, and molecular markers based on single-primer RAPD and on PCR primer pairs.