利用同功酶分析丝核菌菌丝融合1群lA亚群(Rhizoctonia solani AG1-lA)的遗传多样性

利用系统聚类分析方法,通过对丝核菌菌丝融合一群Rhizoctonia solani (AG-1)的23个菌株在七个同功酶电泳所显示的50个表现型进行了遗传分析。结果显示,23个丝核菌菌株可聚类为三大类,他们分别代表了丝核菌菌丝融合一群中的三个亚群AG-I A,AG-IB,AG—IC;其中第一聚类又可进一步分为四个亚类。同功酶的分析证实了丝核菌菌丝融合1群中的三个亚群的分类概念是具有遗传背景的,并可作为丝核菌菌丝融合群之间及群内分类的重要手段。     

立枯丝核菌可溶性蛋白图谱研究II．用等电聚焦电泳比较各融合群及亚群

利用薄层(0.5mm)聚丙烯酰胺凝胶等电聚焦电泳对于pH3．5一10.0范围内立枯丝核菌(Rhizoctonia solani)AG-1～AG-5;各融合群及亚群共45个参试菌株的菌体可溶性蛋白进行了比较分析。参试菌株分别来自于日本、美国及国内鉴定的菌株。电泳结果表明：不同融合群或亚群的蛋白质图谱表现显著差异。AG-3、AG-4、AG-5各融合群和AG-IIA、IB、IC、AG-2-1、AG-2-2各亚群分别表现出各自的特征性图谱。以上结论与前篇(刘力、葛起新,1988)报道中的基本相符,且更为明确。针对试验结果,就可溶性蛋白等电聚焦电泳图谱与培养性状类型的比较以及不同菌培养时间对电泳结果的影响进行了分析讨论。     

新疆北疆棉田立枯丝核菌菌丝融合群及其营养亲和群研究

从新疆北疆棉区采集了典型的棉花立枯病病苗及棉田土标样686份,按常规分离方法分离得到399个分离物,从中鉴定出272个纯化的立枯丝核菌(Rhizotonia solaniKühn)菌株,经玻片吉姆萨氏染剂(Gimsa′sstain)染色程序观察细胞核数目,全部测试菌株均属于多核。用标准菌株,通过载玻片菌丝融合实验测定,将纯化的272个菌株划归为3个菌丝融合群:AG-2、AG-4和AG-5,分别占总菌株的6.24%、84.2%和1.1%。另有23个菌株不与任何标准菌株融合,占8.46%,说明新疆北疆棉田立枯丝核菌的优势菌系是多核丝核菌的AG-4融合群。通过从10种不同配方的培养基中筛选的效果好的麦芽蛋白胨(MPDA)配方培养基(Ⅱ)进行对峙培养,以建立标准菌株,将纯化获得的272个丝核菌菌株,划分为6个不同的营养亲和群,研究说明新疆北疆地区棉田立枯丝核菌各菌丝融合群内确有不同程度的分化。     

丝核菌的分类系统：现状及存在问题

以重要植物病原菌为特征的丝核菌是一类在土壤中广泛分布的丝状真菌,通常不产孢,以菌丝或菌核的形式存在,多样性非常丰富。本文基于国内外最新研究进展,对依据菌丝体的细胞核数目、菌丝融合、有性生殖和系统进化等方面的基本特征展开的丝核菌分类体系及分类现状进行了综述。基于菌丝的细胞核数目,丝核菌被分为单核、双核和多核丝核菌三大类群。自然界中单核丝核菌数量极少,多核和双核丝核菌在全球分布广泛,占丝核菌的绝大多数。基于菌丝融合试验的结果,目前多核丝核菌被分为13个菌丝融合群,双核丝核菌被分为18个菌丝融合群。部分融合群内又根据一些稳定的特征分了亚群,但亚群的建立标准并不统一。目前的分子系统学研究结果基本支持丝核菌的菌丝融合群及亚群的分类。基于部分有性世代被发现的菌株的形态特征,多核和双核丝核菌分别被鉴定为亡革菌属和角担菌属。此外,目前已有分属重要植物病原菌和兰科菌根菌类群的至少9个融合群或亚群的17个菌株完成了基因组测序,比较基因组学和线粒体组学开始在丝核菌分类和进化研究中发挥作用。丝核菌分类系统特殊且复杂,作者在文末提出了目前丝核菌分类学研究面临的问题和今后研究的趋势,期待更多的学者参与到这个重要菌...     

立枯丝核菌蛋白质电泳图谱研究I．融合群与图谱的相关性*

用来自日本和美国的立枯丝核菌8个融合群11个类群代表菌株进行可溶性蛋白质电泳,其结果表明,各融合群及亚群之间电泳图谱有显著差异,而同一类群菌株的电泳图谱则相似。分析来源于华东等地已鉴定的融合类群117个菌株的电泳图谱显示,同一融合群内菌株,虽然采集地区、寄主植物或致病力不同,其蛋白质图谱仍十分相似;而不同融合类群的菌株,即使在同一田块中同一种寄主植物上引起相似病害,其图谱也显示出明显差异。本文就上述可溶性蛋白质图谱显示的结果与其它研究者在血清学、DNA同源性．酯酶等生化水平上对融合群的研究结果进行了比较和探讨。     

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。     

ISOZYME ANALYSIS OF GENETIC DIVERSITY AMONG ISOLATES OF RHIZOCTONIA SOLANI ANASTOMOSIS GROUP 1-Ⅰ A(AG1-Ⅰ A)

利用系统聚类分析方法,通过对丝核菌菌丝融合一群Ｒｈｉｚｏｃｔｏｎｉａｓｏｌａｎｉ（ＡＧ１）的２３个菌株在七个同功酶电泳所显示的５０个表现型进行了遗传分析。结果显示,２３个丝核菌菌株可聚类为三大类,他们分别代表了丝核菌菌丝融合一群中的三个亚群ＡＧⅠＡ,ＡＧⅠＢ,ＡＧⅠＣ;其中第一聚类又可进一步分为四个亚类。同功酶的分析证实了丝核菌菌丝融合１群中的三个亚群的分类概念是具有遗传背景的,并可作为丝核菌菌丝融合群之间及群内分类的重要手段。     

立枯丝核菌蛋白质电泳图谱研究 Ⅰ.融合群与图谱的相关性

用来自日本和美国的立枯丝核菌8个融合群11个类群代表菌株进行可溶性蛋白质电泳,其结果表明,各融合群及亚群之间电泳图谱有显著差异,而同一类群菌株的电泳图谱则相似。分析来源于华东等地已鉴定的融合类群117个菌株的电泳图谱显示,同一融合群内菌株,虽然采集地区、寄主植物或致病力不同,其蛋白质图谱仍十分相似;而不同融合类群的菌株,即使在同一田块中同一种寄主植物上引起相似病害,其图谱也显示出明显差异。本文就上述可溶性蛋白质图谱显示的结果与其它研究者在血清学、DNA同源性.酯酶等生化水平上对融合群的研究结果进行了比较和探讨。     

丝核菌菌丝融合群种类,寄生专化性及与温度的关系

丝核菌（Ｒｈｉｚｏｃｔｏｎｉａｓｐｐ．）是最重要的土传植物病原菌之一,分布世界各地。１９２１年Ｍａｔｓｕｍｏｔｏ用菌丝融合群（ＡｎａｓｔｏｍｏｓｉｓＧｒｏｕｐ,简称ＡＧ）的方法来划分多核立枯丝核菌（Ｒ．ｓｏｌａｎｉ）在生理上特异的菌株［６］,Ｐａｒ...     

中国北方马铃薯黑痣病立枯丝核菌的融合群鉴定

从山东、甘肃、青海、内蒙古、河北和黑龙江6省采集马铃薯黑痣病标本300余份,分离获得251个立枯丝核菌Rhizoctonia solani菌株。融合群测定结果表明,这些菌株分别属于多核的丝核菌AG‐3、AG1‐IB、AG4‐HG‐Ⅰ、AG4‐HG‐Ⅱ、AG4‐HG‐Ⅲ、AG‐5和AG‐11融合群。其中AG‐3是优势致病群,占分离菌株总数的71.31%;其次是AG4‐HG‐Ⅰ,占15.14%;AG‐11融合群菌株是国内首次从罹病马铃薯植株上分离得到。从各融合群中选取代表性的菌株进行5.8S rDNA‐ITS区序列分析,结果表明,隶属不同融合群或亚群菌株的5.8S rDNA‐ITS区序列存在较大的差异,而相同融合群(亚群)不同菌株的序列具有较高一致性。     

玉蜀黍丝核菌的鉴定特征

在研究四川省玉米纹枯病菌系时,获得23个RhizoctoniazeaeVoorhees菌株。该菌菌丝形态与R.solaniKuhn相似;菌丝细胞含3个以上椭圆形细胞核;隔膜具桶孔,桶孔周围的隔膜肿体（septalswelling）与隔膜板垂直,两端不再膨大外延;菌丝生长较快,在PDA平板26℃下培养,每24小时菌落直径增长33．2～39．1mm;菌丝与苯酚相互作用后,着色较深,呈深褐色。菌核初期为白色或奶油色,成熟后为红褐色;常散生于基质内,少数着生在基质表面和培养皿的内壁上;球形或近球形,直径0.25～1．0mm;结构致密,无菌环和菌髓分化,表面较光滑。223个R.zeae菌株均与Oniki等建立的WaiteacircinataWAG－Z标准菌株发生融合,但与R.solani的融合群标准菌株均无融合反应。R.zeae对玉米的致病性较强,稍弱于玉米纹枯病主菌系R.solaniAG－1－IA,其病斑多半不连续,颜色较深,也可0侵染果穗引致穗腐并在苞叶上产生大量红褐至黑褐色的小菌核。     

立枯丝核菌可溶性蛋白图谱研究 Ⅱ.用等电聚焦电泳比较各融合群及亚群

利用薄层(0.5mm)聚丙烯酰胺凝胶等电聚焦电泳对于pH3.5-10.0范围内立枯丝核菌(Rhizoctonia solani)AG-1～AG-5各融合群及亚群共45个参试菌株的菌体可溶性蛋白进行了比较分析。参试菌株分别来自于日本、美国及国内鉴定的菌株。电泳结果表明:不同融合群或亚群的蛋白质图谱表现显著差异。AG-3、AG-4、A G-5各融合群和AG-1IA、IB,IC、AG-2-1、AG-2-2各亚群分别表现出各自的特征性图谱。以上结论与前篇(刘力、葛起新,1988)报道中的基本相符,且更为明确。针对试验结果,就可溶性蛋白等电聚焦电泳图谱与培养性状类型的比较以及不同菌培养时间对电泳结果的影响进行了分析讨论。     

立枯丝核菌第一融合群的遗传分化*

利用12个随机引物对来自云南省不同地理环境的立枯丝核菌RhizoctoniasolaniKühn第一融合群(AG-1)的13个菌株进行遗传分化关系的研究。结果表明受试菌株被标记的DNA谱带多态性检测率为100%,即受试菌株间几乎无同质的DNA谱带。利用UPGMA法构建分子系统树分析发现,遗传距离0.5将其划分为9个遗传聚类组,遗传距离0.86将其划分为6个遗传聚类组,而遗传距离0.95可将其划分为3个遗传聚类组。结果表明受试AG1融合群的13个菌株具明显的遗传分化。     

Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA

A universally primed (UP)-PCR cross hybridization assay was developed for rapid identification of isolates of Rhizoctonia solani into the correct anastomosis group (AG). Twenty-one AG tester isolates belonging to 11 AGs of R. solani were amplified with a single UP primer which generated multiple PCR fragments for each isolate. The amplified products were spotted onto a filter, immobilized and used for cross hybridization against amplification products from the different isolates. Isolates within AG subgroups cross hybridize strongly, whereas between different AGs little or no cross hybridization occurs. Sixteen Rhizoctonia isolates from diseased sugar beets and potatoes were identified using the assay. The results were supported by restriction fragment length polymorphism analysis of the ITS1-5.8S-ITS2 region of the nuclear encoded ribosomal DNA. Through standardization and use of quick non-radioactive labeling techniques, the UP-PCR cross hybridization assay has potential for routine use by modern DNA chip technology.     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

SUITABILITY OF INTERGENIC SPACER OR INTERNAL TRANSCRIBED SPACER MICROSATELLITE-PRIMED PCR FOR THE IDENTIFICATION OF RHIZOCTONIA SOLANI AND SOME PHYTOFUNGI

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS / ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS / ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments.

PRACTICAL APPLICATIONS

Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and R. solani in agriculturally important plant diseases.     

Esterase isoenzyme variation in the genus Saprolegnia, with particular reference to the fish-pathogenic S. diclina-parasitica complex

Esterase isoenzyme patterns determined by slab gel electrophoresis were compared for nearly 60 Saprolegnia isolates, particularly from the S. diclina-parasitica complex. Consistent differences were found between S. diclina and S. parasitica isolates, with the latter being characterized by having between one and five very fast moving esterase bands that were generally absent from the former. A range of asexual (unidentifiable) isolates, taken from the vicinity of fish hatcheries or from fish lesions, were also examined and their esterase isoenzymes shown to be similar to those of S. parasitica, although usually showing fewer bands. The use of esterase isoenzymes for screening potential fish pathogenic isolates of Saprolegnia is briefly compared with results obtained using oogonium morphology or cyst ornamentation, and the relative merits of each method are discussed. It is proposed that, on the basis of their distinctive cyst coat ornamentation (bundles of long 'boathooks') and esterase isoenzymes, fish lesion isolates can be distinguished from saprophytic isolates, and that analysis of isoenzymes should provide a useful method for the screening of potential pathogenic isolates.