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     

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.     

Identification and Pathogenicity of Rhizoctonia spp. Causing Wirestem of Betula nigra in China

During July 2004, wirestem was frequently observed on the seedlings of Betula nigra at Dehong district in Yunnan Province, China. Isolates of Rhizoctonia spp. consistently obtained from their diseased leaves, roots and stems were identified as belonging to binucleate Rhizoctonia anastomosis groups (AG) AG‐P and AG‐R, and R. solani AG‐I IB and AG‐4 HG‐I, based on cultural characteristics, nuclear staining, anastomisis reaction and analysis of their ITS rDNA region. The percentage of recovery of AG‐P, AG‐1, AG‐R and AG‐4 was 48%, 39%, 8% and 3%, respectively. This is the first report of wirestem of red birch cause by binucleate Rhizoctonia AG‐P and AG‐R, and R. solani AG‐1 IB and AG‐4 HG‐I in China.     

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.     

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.     

Impact of Continuous Cropping of Lettuce on the Disease Dynamics of Bottom Rot and Genotypic Diversity of Rhizoctonia solani AG 1‐IB

The impact of continuous cropping of lettuce on the disease dynamics of bottom rot and genotypic diversity of the causal pathogen Rhizoctonia solani AG 1‐IB was studied over 3 years with two crops per year within a field naturally infested with R. solani the pathogen. This field had not had lettuce cultivated in it for 7 years. The disease incidence (DI) and disease severity (DS) were assessed at each harvest and mapped. Surprisingly, a high DI was already observed in the first crop of year one of this field study. In addition, the pathogen was also found to be evenly distributed. Severely infected plants occurred mainly in patches, and the position varied between crops. A significant increase in DS was already observed in the second year, and both temperature conditions and continuous cropping influenced the DS on average over time. Rhizoctonia isolates were randomly collected from the first crop in 1999 and the sixth crop in 2001. The genotypic diversity within the subgroup of R. solani AG 1‐IB was analysed by BOX‐PCR genomic fingerprinting and the aggressiveness of isolates by bioassay. The fingerprints revealed a high level of genotypic diversity within the AG 1‐IB field population. However, continuous cropping was found not to have an impact on genotypic diversity and aggressiveness.     

Genetic Diversity of Thanatephorus cucumeris Infecting Tomato in Iran

The necrotrophic fungus Thanatephorus cucumeris (anamorph Rhizoctonia solani) is among the most important soil‐borne pathogens which causes tomato foot and root rot worldwide. We investigated virulence and genetic relationships among and within different taxonomic groups of R. solani from the tomato‐growing regions in the north‐east of Iran. Characterization of R. solani taxonomic groups revealed that, of 56 isolates, four were AG‐2‐1, 16 were AG‐3 PT, 21 were AG‐4 HG‐I and 15 were AG‐4 HG‐II. Because interprimer binding site (iPBS), which is based on amplification of retrotransposons, is known as novel and powerful DNA fingerprinting technology, we selected four iPBS primers, which can detect polymorphisms of tomato foot root and root rot pathogen, for investigating genotypic variability of the isolates. The iPBS analyses separated various taxonomic groups of R. solani and showed great diversity among the isolates, demonstrating that the R. solani isolates obtained from tomato were not a clonal population. Crop rotation strategies and geographic location seem to be important factors affecting genetic structure of the isolates. Pathogenicity tests on tomato cultivar ‘Mobil’ showed significant differences in the virulence of various isolates. The overall results indicated that isolates of AG‐3 and AG‐4 were more virulent than AG‐2‐1. There was no significant correlation between genetic diversity and virulence of the isolates. This is the first report of R. solani AG‐4 HG‐II, causing tomato foot and root rot. Also, our research is the first in assessment of genetic diversity in fungal populations using iPBS molecular markers.     

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.     

Chemotaxonomy of fungi in the Rhizoctonia solani species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.

     

Reappearance of Cucumber mosaic virus Isolates Belonging to Subgroup IB in Tomato Plants in North-eastern Spain

A disease characterized by symptomless leaves and fruit decolouration, loss of consistency and mild deformation on ripening was detected in tomato fields in north‐eastern Spain during 2003 and 2004. DAS‐ELISA analysis revealed the presence of the Cucumber mosaic virus (CMV) in all diseased plants. CMV isolates were characterized by polyacrylamide gel electrophoresis (PAGE) analysis of double‐stranded RNAs (dsRNAs) and nucleotide sequence analysis, and compared with some CMV isolates belonging to different subgroups used as controls. A total of 12 isolates obtained from the infected tomato plants selected for this study gave the same electrophoresis pattern for the three genomic dsRNAs, which was different to the patterns showed by the CMV isolates collected in the same region a few years ago. The identity of the complete nucleotide sequence of one of these CMV isolates and the partial sequence of other five isolates compared to the Tfn strain from Italy and the BAR92/1 isolate from tomato collected in Barcelona in 1992 was higher than 99%, both belonging to subgroup IB of CMV. The CMV isolates of this subgroup found in eastern Spain in previous studies were not detected after 1996. The nucleotide sequences of two isolates that were chosen as representatives of the CMV isolates more frequently detected in previous years revealed that they belonged to the CMV subgroups IA. The origin and the possible causes of reappearance of CMV IB isolates in north‐eastern Spain are discussed.     

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.     

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.     

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.     

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).     

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.     

Rice varieties with resistance to multiple races of Magnaporthe oryzae offer opportunities to manage rice blast in Australia

Rice blast caused by Magnaporthe oryzae is the most destructive disease of rice worldwide. Development of resistant varieties is considered as the most cost‐effective and sustainable way to manage rice blast. However, there remains a lack of knowledge about the resistance of rice varieties to blast disease in Australia. This study was conducted to determine if there was any resistance existing among the rice varieties grown in Australia to M. oryzae isolates from this country that belong to different races. There was a resistant reaction of the variety SHZ‐2 to all the five races of IA‐1, IA‐3, IA‐63, IB‐3 and IB‐59, with a percent disease index (%DI) less than 40. Varieties NTR587, BR‐IRGA‐409, Ceysvoni and Rikuto Norin 20 showed a resistant reaction to races IA‐3, IA‐63, IB‐3 and IB‐59; and the variety Kyeema exhibited a resistant reaction to races IA‐3, IB‐3 and IB‐59. For the races IA‐1 and IB‐59 with more than one isolate, varieties with differential disease reactions across different isolates belonging to the same race were also revealed: five varieties, Langi, Opus, Sherpa, Viet 1 and Topaz, exhibited differential disease reactions to the three IA‐1 isolates; 10 varieties showed differential disease reactions to the four IB‐59 isolates; in addition, the varieties that had differential disease reactions to the IA‐1 isolates also exhibited differential disease reactions to the IB‐59 isolates of race. This study provides valuable resistance sources for breeding programmes to develop rice varieties with resistance to multiple races of M. oryzae in Australia.     

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.     

Rapid detection ofRhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.     

Chemotaxonomy of fungi in the <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> species complex performing GC/MS metabolite profiling

Hyphal anastomosis testing and molecular methods have been the primary criteria employed to understand the evolutionary and taxonomic relationships of the soil-borne fungal plant pathogen Rhizoctonia solani species complex. In this study, a metabolomics-based approach for characterizing and identifying isolates of R. solani using gas chromatography/mass spectrometry (GC/MS) metabolite profiling and footprinting was developed. Multivariate and hierarchical cluster analyses of GC/MS data provided resolution of isolates belonging to anastomosis groups (AGs) 1–6, 9, and 10 of R. solani. Clustering of R. solani AG-3 isolates, based on host origin, was also observed and attributed to metabolite-biomarkers belonging to amino, carboxylic and fatty acids. The chemotaxonomic approach using metabolomics is a high-throughput methodology that complements existing molecular approaches for the taxonomic investigation of Rhizoctonia isolates and monitoring of fungal metabolism.     

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.