黄淮海地区夏玉米纹枯病菌的融合群鉴定

从黄淮海地区采集玉米纹枯病标样250余份,分离得到176个丝核菌菌株。融合群测定及5.8SrDNA-ITS区序列分析结果表明,这些菌株分别属于多核丝核菌的AG1-IA、AG1-IB、AG4-HG-I、AG-5、WAG-Z融合群及双核丝核菌的AG-A、AG-Ba融合群。其中AG1-IA是优势融合群,占分离菌株总数的64.20%,其次是AG-Ba,占12.50%,再依次分别是WAG-Z(10.23%)、AGI-IB(5.11%)、AG-4-HG-I(3.98%)、AG-5(2.27%)和AG-A(1.70%)。其中AG-Ba融合群是国内首次在玉米上分离得到。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的菌株其5.8S rDNA-ITS区序列存在较大的差异,而相同融合群(或亚群)不同菌株之间其序列的一致性可高达97%-100%。     

中国东北地区玉米纹枯病菌的融合群鉴定

自东北三省采集玉米纹枯病标本300余份,分离获得286个丝核菌菌株。融合群测定及5.8S rDNA-ITS区序列分析结果表明,这些菌株分别属于多核丝核菌的AG1-IA、AG1-IB、AG1-IC、AG4-HG-I、AG4-HG-III、AG-5、WAG-Z群及双核丝核菌的AG-Ba群。其中AG1-IA是优势致病群,占分离菌株总数的38.46%,其次是WAG-Z和AG-5群,分别占26.92%及24.83%。AG4-HG-III群菌株是国内首次从罹病玉米植株上分离得到。自各融合群中选取代表性的菌株进行5.8     

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.     

Trials of direct detection and identification of Rhizoctonia solani AG 1 and AG 2 subgroups using specifically primed PCR analysis

Specifically primed polymerase chain reaction (PCR) analysis was used for direct detection and identification of Rhizoctonia solani isolates belonging to AG 1 subgroups (IA, IB, and IC) and AG 2 subgroups (2-1 and 2-2). A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract PCR templates. PCRspecific primer sets for each group were designed from sequences in the regions of the 28S ribosomal DNA of this fungus. The results of specifically primed PCR analysis showed that AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, and AG 2-2 primers sets contributed detection from the same AG isolates and could escape detection from different AG isolates at a high level of frequency. In this experiment, we suggested that our synthesized primer sets might provide a method for the direct detection and identification of AGs of R. solani.     

RFLP analysis of the PCR-amplified 28S rDNA inRhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.     

Trials of direct detection and identification of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG 1 and AG 2 subgroups using specifically primed PCR analysis

Specifically primed polymerase chain reaction (PCR) analysis was used for direct detection and identification of Rhizoctonia solani isolates belonging to AG 1 subgroups (IA, IB, and IC) and AG 2 subgroups (2-1 and 2-2). A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract PCR templates. PCR-specific primer sets for each group were designed from sequences in the regions of the 28S ribosomal DNA of this fungus. The results of specifically primed PCR analysis showed that AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, and AG 2-2 primers sets contributed detection from the same AG isolates and could escape detection from different AG isolates at a high level of frequency. In this experiment, we suggested that our synthesized primer sets might provide a method for the direct detection and identification of AGs of R. solani. Received: June 28, 2001 / Accepted: November 14, 2001     

中国北方棉花主产区立枯丝核菌的融合群鉴定

从山东、河北、河南三省采集棉花立枯病样品和土壤200余份,经分离获得198个丝核菌Rhizoctonia solani分离物。菌丝融合测定及5.8S rDNA-ITS区序列分析结果表明,这些分离物分别属于多核丝核菌的AG4-HG-I和AG4-HG-III融合群以及双核丝核菌的AG-A、AG-F、AG-Fb融合群。其中AG4-HG-I是优势融合类群,占分离物总数的88.38%,其次是AG4-HG-III,占10.10%,AG-A、AG-F、AG-Fb各仅有1株。其中双核丝核菌AG-A、AG-F和AG-Fb融     

Classification of <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. using rDNA-ITS sequence analysis supports the genetic basis of the classical anastomosis grouping

Currently, rDNA-ITS sequence analysis seems to be the most appropriate method for comprehensive classification of Rhizoctonia spp. Our previous review article was concerned with detailed analysis of multinucleate Rhizoctonia (MNR), and the current review complements the previous one with detailed analysis of binucleate Rhizoctonia (BNR) (teleomorphs: Ceratobasidium spp. and Tulasnella spp.) and uninucleate Rhizoctonia (UNR) (teleomorph: C. bicorne). Data of all the appropriate BNR and UNR accumulated in GenBank were analyzed together in neighbor-joining (NJ) trees supplemented with percent sequence similarity within and among the anastomosis groups (AGs) and subgroups. Generally, the clusters of the isolate sequences supported the genetic basis for the AG based on hyphal fusion anastomosis. Comprehensive interrelationships among all the currently available MNR, BNR, and UNR groups and subgroups in GenBank were subsequently analyzed in NJ and maximum-parsimony (MP) trees, showing the genetic relatedness among the different groups and indicating possible bridging groups between MNR, BNR, and UNR. The review also indicates serious inaccuracies in designation of sequences of some isolates deposited in GenBank. Several additional teleomorph genera with Rhizoctonia spp. anamorphs have also been reported in the literature. However, as they have not been intensively studied, there were no available data on their rDNA-ITS sequences that could be included in this review.     

Characterization and Sensitivity to Fungicides of Rhizoctonia spp. Recovered from Potato Plants in Bolu,Turkey

Isolates of Rhizoctonia spp. associated with stem canker and black scurf disease of potato were examined for their anastomosis group, sequence variations in the ITS‐5.8S rDNA region, pathogenicity and sensitivity to fungicides. A total of 92 isolates were obtained from diseased tuber, stolon and sprouts of the potato plants, collected from five districts of Bolu province, Turkey. Based on the anastomosis group and the similarity of the nucleotide sequence of the ITS‐5.8S rDNA, most of the isolates (81.5%) were identified as AG 3 PT. Other isolates belonged to AG 2‐1 (1.08%), AG 2‐2 IV (1.08%), AG 4 HG II (8.07%), AG 5 (2.17%), binucleate Rhizoctonia AG A (1.08%) and AG K (4.35%). Pathogenicity tests showed that isolates of AG 3 PT, AG 4 HG II and AG 5 caused similar degrees of disease severity on 45‐day‐old potato seedlings, whereas AG 2‐1 was moderately virulent. AG 2‐2 IV and binucleate Rhizoctonia spp. were weakly pathogenic or non‐pathogenic on potato seedlings. In this study, anastomosis groups of Rhizoctonia spp. isolates associated with potato in Turkey were characterized for the first time using molecular techniques and classified at the level of subgroups. Furthermore, the effect of selected fungicides was evaluated on disease development caused by soil‐borne inoculums of different anastomosis groups (AGs). Flutolanil and Bacillus subtilis QST 713 were found to be most effective against the Rhizoctonia isolates tested. These results revealed significant differences among the fungicides on disease development resulted from the different AGs.     

Rhizoctonia spp. Causing Root and Hypocotyl Rot in Phaseolus vulgaris in Cuba

Sixty isolates of Rhizoctonia spp. were obtained from Cuban bean fields during the period 2004–2007. Isolates were characterized with different techniques, including nuclei staining, pectic zymogram, PCR–RFLP analysis of the rDNA–ITS region and sequencing of the rDNA–ITS region. The majority of the isolates were identified as multinucleate Rhizoctonia solani isolates, representing two different anastomosis groups (AGs), AG 2‐2 WB and AG 4 HGI; the remaining isolates were binucleate Rhizoctonia isolates and belonged to AG F and AG A. AG 4 HGI isolates were equally distributed in all soil types; AG 2‐2 isolates were more frequently isolated from cambisols, whereas AG F isolates were related to calcisols. Pathogenicity experiments in vitro and in the greenhouse, revealed that binucleate isolates only caused root rot, whereas R. solani isolates were able to cause root rot and hypocotyl rot. Furthermore, differences in virulence level were observed between R. solani and binucleate isolates and among different AGs. Isolates of R. solani AG 4 HGI and R. solani AG 2‐2 WB were the most aggressive, binucleate isolates of AG F were intermediate aggressive, whereas a binucleate isolate of AG A was weakly aggressive. In contrast with other reports about R. solani in bean, web blight symptoms were never observed during this study.     

Interactions between cauliflower and Rhizoctonia anastomosis groups with different levels of aggressiveness

Background  

The soil borne fungus Rhizoctonia is one of the most important plant pathogenic fungi, with a wide host range and worldwide distribution. In cauliflower (Brassica oleracea var. botrytis), several anastomosis groups (AGs) including both multinucleate R. solani and binucleate Rhizoctonia species have been identified showing different levels of aggressiveness. The infection and colonization process of Rhizoctonia during pathogenic interactions is well described. In contrast, insights into processes during interactions with weak aggressive or non-pathogenic isolates are limited. In this study the interaction of cauliflower with seven R. solani AGs and one binucleate Rhizoctonia AG differing in aggressiveness, was compared. Using microscopic and histopathological techniques, the early steps of the infection process, the colonization process and several host responses were studied.     

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...     

Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers

AIMS: The aim of this study was to develop a specific and sensitive identification method for Rhizoctonia solani AG 1-IB isolates based on phylogenetic relationships of R. solani AG-1 subgroups using rDNA-internal transcribed spacer (rDNA-ITS) sequence analysis. METHODS AND RESULTS: A neighbour-joining tree analysis of 40 rDNA-ITS sequences demonstrated that R. solani AG-1 isolates cluster separately in six subgroups IA, IB, IC, ID, IE and IF. A molecular marker was generated from a random amplified polymorphic DNA fragment (RAPD). After conversion into a sequence-characterized amplified region (SCAR), a specific primer set for identification of subgroup AG 1-IB was designed for use in a polymerase chain reaction (PCR). The primer pair amplified a single DNA product of 324 bp. CONCLUSIONS: R. solani AG-1 subgroups were discriminated by sequence analysis of the ITS region. The designed SCAR primer pair allowed an unequivocal and rapid detection of R. solani AG 1-IB in plant and soil samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence analysis of the rDNA-ITS region can be used for differentiation of subgroups within AG-1. The use of the developed SCAR primer set allowed a reliable and fast identification of R. solani AG 1-IB and provides a powerful tool for disease diagnosis.     

Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens.     

Variability in the Sensitivity of Rhizoctonia solani Anastomosis Groups to Fungicides

Tolclofos-methyl, iprodione and cyproconazole, among the eleven fungicides tested in vitro, gave consistently strong inhibition against all ten anastomosis groups (AGs) of Rhizoctonia solani. Carboxin, furmecyclox, thiabendazole, fenpropimorph and vinclozolin also inhibited all AGs but with wide variations in toxicity levels (EC90 values). Pencycuron showed strong activity against four AGs but was ineffective against the other six AGs. Generally, R. solani AGs were insensitive to fenarimol and imazalil. Tolclofos-methyl strongly inhibited 23 AG2-1 and 20 AG4 rapeseed/canola R. solani isolates from different locations in Saskatchewan, Alberta and Manitoba. The same isolates were also sensitive to iprodione, cyproconazole and carboxin. All AG4 canola isolates were insensitive to pencycuron (EC90 > 500 mg/l) while AG2-1 isolates showed highly variable levels of sensitivity with EC90s ranging from 0.5 to 220 mg/l. Tolclofos-methyl, applied to Brassica napus (canola) cv. Westar seed at 1 g a.i./kg, provided 75—100 % control of seedling damping-off in pots infested with AG2-1 or AG4 isolates. In parallel experiments, pencycuron (1 g a.i./kg seed) failed to control damping-off by AG4 canola isolates and gave variable disease control against AG2-1 isolates.     

Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun,Turkey

A total of 434 isolates of Rhizoctonia belonging to 10 anastomosis groups were obtained from the roots and rhizosphere soils of bean and soybean plants grown in Samsun, Turkey. AG-4 was found to be the most common group on bean and soybean plants and AG-5, AG-6, binucleate AG-A, AG-B and R. zeae were other groups isolated from the both plant species. AG-1, AG-7 and AG-K from bean and AG-E from soybean were other groups obtained in the study. The pathogenicity tests on bean and soybean seedlings showed that the highest disease severities were caused by AG-4 isolates, whereas AG-1 and AG-6 isolates were moderately pathogenic. Binucleate Rhizoctonia AG-B isolates were also moderately pathogenic, while other binucleate Rhizoctonia were found to be weakly pathogenic. Rhizoctonia zeae isolates caused moderate disease symptoms on bean, but soybean plants were slightly affected by this group of isolates. This is the first reported observation of R. solani AG-6 and AG-7 and binucleate Rhizoctonia AG-B on bean, and R. solani AG-5 and AG-6 and binucleate Rhizoctonia AG-A, AG-B and AG-E on soybean, in Turkey.     

山东省玉米纹枯菌融合群类型及遗传多样性

从山东省14个县市区采集的玉米纹枯病标本上分离获得103个玉米纹枯菌菌株。核荧光染色确定菌丝细胞核的数目,以及利用配对培养法确定不同菌株细胞是否融合。结果表明这些菌株分别属于多核丝核菌的AG-1-IA、AG-1-IB、AG-1-IC、AG-3、AG-4-HG-I、AG-5和WAG-Z融合群和双核丝核菌的AG-Ba融合群,其中AG-1-IA类型菌株数量占菌株总数的60.19%,为优势融合群。通过inter-simple sequence repeats（ISSR）标记技术进行菌株的遗传多样性分析,获得45个ISSR分子标记,其中91.1%的片段具有多态性,表明种群间存在丰富的遗传多样性。UPGMA聚类分析将103个菌株分成6个遗传聚类群,遗传聚类群的菌株组成说明遗传群组的划分与菌株的地理来源和菌株融合群类型均存在一定的相关性。