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.     

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.     

Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi,Rhizoctonia solani in Wheat

Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS) production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT), 3,3''-diaminobenzidine (DAB) and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction.     

Molecular characterization of Rhizoctonia solani isolates the incitant of soybean root rot

Rhizoctonia solani isolates used in this investigation were identified as anastomosis-4 (AG-40), collected from different localities from Assiut governorate in Egypt. Pathogenicity test of seven isolates of R. solani was evaluated on soybean Giza 111 cultivar under greenhouse conditions. All tested isolates were able to infect soybean plants causing root rot with different degrees of severities, isolate No. 1, 2 and 3 showed significantly highest root rot severity, while isolate No. 5 gave the lowest percentage of root rot rating. The sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns were used to compare three isolates of R. solani. There are no variations among R. solani isolates except a few exceptions according to their protein patterns. DNA markers obtained from all isolates showed genetic similarity among different isolates obtained from different geographical regions barring few exceptions. Correlation between DNA patterns of R. solani isolates and their virulence was detected, but no correlation with anastomosis groups (AG).     

Molecular characterisation of an endornavirus from Rhizoctonia solani AG-3PT infecting potato

Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a soil-borne plant pathogenic fungus that has a broad host range, including potato. In this study, the double-stranded RNA (dsRNA) profiles were defined for 39 Rhizoctonia solani isolates representative of two different anastomosis groups (AGs) associated with black scurf of potato in New Zealand. A large dsRNA of c. 12 kb–18 kb was detected in each of the isolates, regardless of AG or virulence on potato. Characterisation of the large dsRNA from R. solani AG-3PT isolate RS002, using random amplification of total dsRNA and analyses of overlapping cDNA sequences, resulted in the assembly of a consensus sequence of 14 694 nt. A single, large open reading frame was identified on the positive strand of the assembled sequence encoding a putative polypeptide of at least 4893 amino acids, with a predicted molecular mass of 555.6 kDa. Conserved domains within this polypeptide included those for a viral methyltransferase, a viral RNA helicase 1 and an RNA-dependent RNA polymerase. The domains and their sequential organisation revealed the polyprotein was very similar to those encoded by dsRNA viruses of the genus Endornavirus, in the family Endornaviridae. This is the first report of an endornavirus in R. solani, and thus the putative virus is herein named Rhizoctonia solani endornavirus - RS002 (RsEV-RS002). Partial characterisation of the large dsRNAs in five additional AG-3PT isolates of R. solani also identified them as probable endornaviruses, suggesting this family of viruses is widespread in R. solani infecting potato. The ubiquitous nature of endornaviruses in this plant pathogen implies they may have an important, but yet uncharacterised, role in 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     

Cropping Systems and Cultural Practices Determine the Rhizoctonia Anastomosis Groups Associated with Brassica spp. in Vietnam

Ninety seven Rhizoctonia isolates were collected from different Brassica species with typical Rhizoctonia symptoms in different provinces of Vietnam. The isolates were identified using staining of nuclei and sequencing of the rDNA-ITS barcoding gene. The majority of the isolates were multinucleate R. solani and four isolates were binucleate Rhizoctonia belonging to anastomosis groups (AGs) AG-A and a new subgroup of A-F that we introduce here as AG-Fc on the basis of differences in rDNA-ITS sequence. The most prevalent multinucleate AG was AG 1-IA (45.4% of isolates), followed by AG 1-ID (17.5%), AG 1-IB (13.4%), AG 4-HGI (12.4%), AG 2-2 (5.2%), AG 7 (1.0%) and an unknown AG related to AG 1-IA and AG 1-IE that we introduce here as AG 1-IG (1.0%) on the basis of differences in rDNA-ITS sequence. AG 1-IA and AG 1-ID have not been reported before on Brassica spp. Pathogenicity tests revealed that isolates from all AGs, except AG-A, induced symptoms on detached leaves of several cabbage species. In in vitro tests on white cabbage and Chinese cabbage, both hosts were severely infected by AG 1-IB, AG 2-2, AG 4-HGI, AG 1-IG and AG-Fc isolates, while under greenhouse conditions, only AG 4-HGI, AG 2-2 and AG-Fc isolates could cause severe disease symptoms. The occurrence of the different AGs seems to be correlated with the cropping systems and cultural practices in different sampling areas suggesting that agricultural practices determine the AGs associated with Brassica plants in Vietnam.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Grouping of isolates in AG 2 ofRhizoctonia solani by total cellular fatty acid analysis

Total-cellular fatty acid compositions of 34 isolates ofRhizoctonia solani belonging to intraspecific groups (ISGs) of anastomosis group (AG) 2, i.e., AG 2-1, AG 2-2 IIIB (mat rush), AG 2-2 IV (sugar beet), AG 2-2 LP (turfgrass), and AG 2–3 (soybean), were compared. The major fatty acids identified were palmitic, stearic, and oleic acids. Principal component analysis based on the percentage composition of total cellular fatty acids revealed consistently low variability among isolates of a single ISG of AG 2. Average linkage cluster analysis showed that isolates obtained from turfgrass representing a newly proposed group, AG 2-2 LP, were differentiated from other AG 2 ISGs. Isolates of another newly proposed group AG 2–3, from diseased soybean were also closely related to AG 2-1 and AG 2-2 IIIB but distinguishable from the AG 2-1 and AG 2-2 LP isolates by the average linkage cluster analysis. These results suggested that the percentage composition of total-cellular fatty acids is a distinct characteristic for the five ISGs belonging to AG 2, and fatty acid analysis is useful for the differentiation and characterization of these ISGs of AG 2 inR. solani.     

Characterization of Rhizoctonia solani Isolates from Tobacco Fields Related to Anastomosis Groups 2-1 and BI (AG 2-1 and AG BI)

Tobacco has been reported to be infected by Rhizoctonia solani isolates belonging to anastomosis groups 1 through to 5. Ten pathogenic isolates of the fungus were collected from tobacco fields in Italy and France that anastomosed in high frequencies with AG BI tester isolates and in low frequencies with tester isolates of all described subgroups of AG2, although morphology and thiamine requirement of the isolates were similar to AG 2-1. Biomolecular evaluations by means of electrophoresis of polygalacturonase isozymes and RFLPs of ribosomal DNA internal transcribed spacers were carried out. The isolates shared a common pectic zymogram, distinct from those of AG BI and AG 2-subgroups, while RFLPs of rDNA-ITS evidenced a limited genetic variation within the homogeneous group and a closer similarity to AG 2-1. As far as priority is due to the anastomosis behaviour, the isolates should be ascribed to AG BI. However, tobacco isolates differ from tester strains of the known AG BI in their morphology, thiamine requirement, pathogenicity and biomolecular features. In addition they do not anastomose with both AG 3 and AG 6. Therefore they may represent a new subgroup.     

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.     

First Report of Rhizoctonia solani AG‐7 on Cotton in Egypt

Eighty‐two isolates of Rhizoctonia solani were recorded from roots of naturally‐infected seedlings of the Egyptian cotton (Gossypium barbadense L.). Anastomosis groups (AGs) of the isolates were determined by using 13 different AGs testers. Three (3.7%) of the isolates were identified as R. solani AG7, while the remaining isolates were belonging to the AG 2‐1, AG4 and AG5. The identification of the three isolates was based on the frequency of the C2 reaction with the AG7 tester isolate. No fusion was observed between AG7 and isolates representing the other 13 AGs. Colonies of AG7 isolates grown on potato dextrose agar (PDA), malt yeast agar (MYA) and melt peptone agar (MPA) were brown to dark brown with aerial mycelium and sclerotia. The isolates had pitted sclerotial clusters and brownish exudates after 21 days of culturing on PDA, but without clear zonation. Pathogenicity test under greenhouse conditions revealed that AG7 caused the common symptoms of damping–off, which included seed rot, lesions on the hypocotyls and root rot.     

Advances in molecular interactions on the Rhizoctonia solani-sugar beet pathosystem

Rhizoctonia solani is a soilborne pathogen with a broad host range. An anastomosis group (AG) system based on hyphal fusions has been established to distinguish between different R. solani subgroups in this species complex. Members of the AG2-2IIIB subgroup can cause serious problems in sugar beet production, resulting in Rhizoctonia root and crown rot. In this review, we summarize the current molecular advances in the R. solani sugar beet pathosystem. The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb, larger than any of the R. solani AGs sequenced to date. The genome of AG2-2IIIB has been predicted to harbor 11,897 protein-encoding genes, including a high number of carbohydrate-active enzymes (CAZymes). The highest number of CAZymes was observed for polysaccharide lyase family 1 (PL-1), glycoside hydrolase family 43 (GH-43), and carbohydrate esterase family 12 (CE-12). Eleven single-effector candidates were predicted based on AG2-2IIIB genome data. The RsLysM, RsRlpA, and RsCRP1 genes were highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola and model plant species demonstrated their involvement in virulence. However, despite the progress achieved thus far on the molecular interactions in this pathosystem, many aspects remain to be elucidated, including the development of efficient transformation systems, important for functional studies, and the silencing of undesirable traits in the sugar beet crop.     

Rhizoctonia solani AG 11 isolated for the first time from sugar beet in Poland

Two isolates of Rhizoctonia solani AG11 were isolated from sugar beet seedlings from South-west Poland. Both isolates gave C2 reactions in anastomose pairings with the tester isolates of AG11. The membership of both isolates to AG11 was confirmed by analysis of pectic isozyme profiles, and by verification that the internal transcribed spacer sequences of both isolates matched the references in the GenBank database. Both AG11 isolates formed white-beige to creamy-colored mycelium with wide concentric zonation. One of them formed light-colored sclerotia. The average daily rate of hyphal growth at 21 °C was 22.8 mm and 22.6 mm on PDA. They were mildly pathogenic to sugar beet seedlings due to the mycelial and secondary metabolites’ activity. The sensitivity to fungicides typically used in sugar beet protection was different for each isolate; one of them (isolate ID11) was less sensitive to thiram than the other (isolate ID3). This article discusses the worldwide occurrence of R. solani AG11, expands the currently known host range, shows its broad world distribution in regions of moderate climate, and confirms the isolates’ low frequency.     

Genetic Structure and Aggressiveness of Rhizoctonia solani AG1‐IA,the Cause of Sheath Blight of Rice in Southern China

One hundred and eighty isolates of Rhizoctonia solani AG1‐IA, the causal agent of rice sheath blight, were obtained from six locations in southern China. The genetic structure of R. solani isolates was investigated using random amplified polymorphic DNA (RAPD) markers, and a considerable genetic variation among R. solani isolates was observed. Most of the genetic diversity was distributed within populations, rather than among them. The distribution pattern of the genetic variation of R. solani appears to be the result of high gene flow (Nm) and low‐genetic differentiation among populations. The aggressiveness of R. solani was visually assessed by rice seedlings of five different cultivars in the glasshouse. All isolates tested were found to induce significantly different levels of disease severity, reflecting considerable variation in aggressiveness. The isolates were divided into highly virulent, moderately virulent and weakly virulent groups, and the moderately virulent isolates were dominant in R. solani population. No significant correlation was observed among the genetic similarity, pathogenic aggressiveness and geographical origins of the isolates. Information obtained from this study may be useful for breeding for improved resistance to sheath blight.     

Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2 % in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1–2 mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1 mm). Majority of R. solani isolates showed <8 μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6 %) of AG3 was the highest followed by AG2–3 (20.9 %) and AG5 (17.4 %). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3 %). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.     

Characterization of Rhizoctonia solani AG 4HG-III Causing Stem Canker and Wirestem on Green Amaranth and Chinese Amaranth

During December 2003, stem canker and wirestem were observed on the stems of green amaranth (Amaranthus viridis) and Chinese amaranth (Amaranthus tricolor) in greenhouses at Ximao district in Yunnnan Province, China. Isolates of Rhizoctonia solani obtained from the two amaranths with stem canker and wirestem, were identical to anastomosis group (AG)‐4. The isolates from diseased plant showed high virulence on young seedlings of two amaranths. Results of sequence analysis of 5.8s rDNA‐ITS of Chinese isolates showed 99–100% sequence similarity with AG‐4HG‐III tester isolates. When compared with other subgroups of AG‐4, Chinese isolates showed similarity levels of 94%. This is the first report of stem canker and wirestem of Green amaranth and Chinese amaranth caused by AG‐4HG‐III and AG‐4HG‐III in China.