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     

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.     

Intraspecific variation of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG 3 isolates recovered from potato fields in Central Iran and South Australia

Pectic zymogram, RFLP and PCR analyses were used to characterize Rhizoctonia solani AG 3 isolates collected from diseased potatoes in South Australia. The pectic zymogram data were compared with those obtained for isolates collected from central Iran. Analyses of bands corresponding to pectin esterase and polygalacturonase revealed three zymogram subgroups (ZG) in AG 3. In addition to the previously reported ZG7 (here renamed ZG7-1), two new zymogram subgroups, ZG7-2 and ZG7-3, were identified. Of the 446 isolates tested, 50% of the South Australian and 46% of the Iranian isolates were ZG7-1. The majority of the isolates originating from stem and root cankers were ZG7-1, whereas most of the isolates designated ZG7-2 and ZG7-3 originated from tuber-borne sclerotia. Pathogenicity tests revealed that ZG7-1 generally produced fewer sclerotia and more severe cankers of underground parts of the potato plants than the other two ZGs. Two random DNA clones, one originating from an AG 3 isolate and the other from an AG 4 isolate, were used as probes for RFLP analyses of Australian isolates. The AG 3 probe, previously identified to be specific to this group, detected a high level of genetic diversity, with 11 genotypes identified amongst 50 isolates analysed. The low-copy AG 4 probe resolved three genotypes amongst 24 isolates. For 23 isolates analysed with both markers, the combined data distinguished a total of six genotypes and similarity analysis resolved the isolates into two main groups with 50% homology. PCR, using primers for the plant intron splice junction region (R1), also revealed variation. No obvious relationship among pectic zymogram groups, RFLP and PCR genotypes was observed.     

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 specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

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.     

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.     

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.     

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.     

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10?pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.     

Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia

A collection of isolates of Rhizoctonia solani anastomosis group (AG) 2 was examined for genetic diversity and pathogenicity. Anastomosis reactions classified the majority of isolates into the known subgroups of AG 2-1 and AG 2-2 but the classification of several isolates was ambiguous. Morphological characters were consistent with the species, with no discriminating characters existing between subgroups. Vertical PAGE of pectic enzymes enabled the separation of zymogram group (ZG) 5 and 6 within AG 2-1, but not the separation of ZG 4 and 10 within AG 2-2. PCR analysis using inter-simple sequence repeats (ISSR) and the intron-splice junction (ISJ) region supported the separation of ZG 5 and 6, while the AG 2-2 isolates were separated by geographic region. A comparison of distance matrices produced by the zymogram analysis and PCR indicated a strong correlation between the marker types. Pathogenicity studies suggested canola (Brassica napus) cultivars were most severely affected by AG 2-1, while cultivars of two species of medic (Medicago truncatula cv. Caliph and M. littoralis cv. Herald) were susceptible to both AG 2-1 and 2-2. The results indicate that AG 2 is a polyphyletic group in which the classification of subtypes is sometimes difficult. Further investigation of the population structure within Australia is required to determine the extent and origin of the observed diversity.     

Detection of Helminthosporium solani from soil and plant tissue with species-specific PCR primers

Two PCR primer pairs specific for Helminthosporium solani, which causes silver scurf on potato tubers, were designed from nucleotide sequences of the nuclear ribosomal internal transcribed spacer regions of H. solani. Both primer pairs amplified a single product with DNA from 48 North American and European isolates of H. solani, but not with DNA from 42 other fungi. Primers also amplified a single product with DNA extracted from silver scurf lesions on potato tubers and other plant tissue inoculated with spores of H. solani. Detection of the fungus in infested soil was only possible with nested PCR and after processing soil with a bead beater. Specific amplification of H. solani DNA can be used to study the saprophytic and pathogenic activity of this fungus in soil and plant tissue.     

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.     

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.     

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.     

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Development of DNA markers for identifying chrysanthemum cultivars generated by ion-beam irradiation

Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ (Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.     

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.     

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.