Prolific production of sclerotia in soil by Rhizoctonia solani anastomosis group (AG) 11 pathogenic on lupin

Rhizoctonia solani anastomosis group (AG) 11 causes serious damping‐off and hypocotyl rot of narrow‐leafed lupins (Lupinus angustifolius) in the northern grain‐belt of Western Australia. R. solani AG‐11 produced abundant sclerotia in sand overlaid on potato dextrose agar. Sclerotia were produced in larger numbers in natural Lancelin sand than in Geraldton loamy sand collected from the northern grain‐belt of Western Australia. The majority of the sclerotia produced were in >250 to <500 μam size range. The germination levels of sclerotia in the first two cycles of drying and germination were not significantly different. Sclerotia still retained 50% germination after four such cycles, indicating that they may have the ability to withstand the climatic cycles of the Mediterranean environment of southwestern Western Australia. The radial growth of the mycelium from sclerotia, however, declined with each drying and germination cycle. Inoculum potential of the pathogen increased with the size of sclerotia resulting in more severe lupin hypocotyl rot with larger sclerotia. The number of sclerotia produced in soil increased with increasing density of lupin seedlings. The results also indicate that R. solani AG‐11 can produce sclerotia on infected plant tissues as well as in soil. This is the first report of the prolific production of sclerotia by AG‐11 and their significant role in infection of lupins in soil in Western Australia.     

Highly polymorphic in silico-derived microsatellite loci in the potato-infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes

Fourteen polymorphic microsatellite DNA markers derived from the draft genome sequence of Rhizoctonia solani anastomosis group 3 (AG-3), strain Rhs 1AP, were designed and characterized from the potato-infecting soil fungus R. solani AG-3. All loci were polymorphic in two field populations collected from Solanum tuberosum and S. phureja in the Colombian Andes. The total number of alleles per locus ranged from two to seven, while gene diversity (expected heterozygosity) varied from 0.11 to 0.81. Considering the variable levels of genetic diversity observed, these markers should be useful for population genetic analyses of this important dikaryotic fungal pathogen on a global scale.     

Highly polymorphic microsatellite loci in the rice- and maize-infecting fungal pathogen Rhizoctonia solani anastomosis group 1 IA

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.     

Polymorphism of genes coding for nuclear 18S rRNA indicates genetic distinctiveness of anastomosis group 10 from other groups in the Rhizoctonia solani species complex.

DNA polymorphism in the 18S nuclear rRNA gene region was investigated by using 11 restriction endonucleases for 161 isolates of 25 intraspecific groups (ISGs) representing 11 reported anastomosis groups (AGs) of Rhizoctonia solani. A PCR-based restriction mapping method in which enzymatically amplified DNA fragments and subfragments were digested with one or two restriction enzymes was employed. Four types of DNA restriction maps of this region were constructed for these 25 ISGs. Map type I of the 18S rDNA region was represented by isolates of a majority of R. solani ISGs. Map types II and III, represented by ISG 2E and 9 isolates and 5C isolates, respectively, differed from map I by the absence of one (map type II) or two (map type III) restriction sites. Map type IV, represented by ISG 10A and B (or AG 10) isolates, showed significant restriction site variations, with five enzymes in this region compared with those of the remaining ISGs or AGs. Ten of the 25 restriction sites in the 18S rRNA gene region were informative and selected for analysis. Previously reported restriction maps of the 5.8S rRNA gene region, including the internal transcribed spacers, were aligned with each other, and 12 informative restriction sites were identified. These data were used alone and in combination to evaluate group relationships. Analyses derived from these data sets by maximum parsimony and likelihood methods showed that AG 10 isolates were distinct and distantly related to the majority isolates of the other AGs of this species complex.     

Identification of anastomosis group of Rhizoctonia solani, the causal agent of seed rot and damping-off of bean in Iran

Bean is one of the major crops in Iran. Seed rot and damping-off caused by Rhizoctonia solani is the most important disease of bean. In this research, infected roots and seedlings of beans were collected from different fields of Tehran Province. The samples were sterilized with 10% sodium hypochloride (5% stock) and incubated on PDA surface in petri-dishes. The purified fungi kept on filter paper and identified, pathogenicity test of R. solani was carried out on 2 cultivars of bean (red bean cv. Naz and white bean cv. Dehghan) and it determined. For identification of the anastomosis groups, the discs of cultured media with 5 mm. diameter of standard AG placed on one side of microscopic slides covered with water agar (2%) of 1 mm. thick and the isolates of the fungus on another side of slide about 2 cm away from each other. Experiment carried out in 4 replications. The cultures were incubated in 25 +/- 1 degrees C incubator for 24 hours, then the mycelial contact stained with lactophenol, cotton blue and hyphal anastomosis looked for under the light microscope with 10 x 40 and 10 x 100 magnifications. As a result, anastomosis groups: AG4, AG4HGII, AG2-2-2B and AG6 determined, frequency of these groups were 64, 18, 2, 16%, respectively. The group AG6 and subgroups AG4HGII and AG2-2-2B are introduced as new anastomosis groups on bean in Iran.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^? RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^? RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA

A method based on restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA was developed for the rapid characterization of large populations of Rhizoctonia solani at the anastomosis group (AG) level. The restriction maps of the internal transcribed spacers (ITS) sequences were compared for 219 isolates of R. solani belonging to AG-1 to AG-12 and AG-BI, representing diverse geographic and host range origins. Four discriminant restriction enzymes (MseI, AvaII, HincII, and MunI) resolved 40 restriction fragment length polymorphism (RFLP) types among the 219 ITS sequences of R. solani. Each RFLP type could be assigned to a single AG except for two RFLP types, which were common to two AG. A fifth enzyme allowed the discrimination of AG-6 and AG-12. In addition, the combination of four enzymes allowed the discrimination of subsets within AG-1, AG-2, AG-3, and AG-4. The efficiency of the typing method was confirmed by analyzing PCR-amplified ITS sequences of 30 reference strains. Furthermore, the PCR-RFLP method was used to characterize at the AG level 307 isolates of R. solani originating from ten sugar beet fields exhibiting patches of diseased plants in France. The PCR-based procedure described in this paper provides a rapid method for AG typing in R. solani.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

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.

     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.     

Unique DNA plasmid pRS64 associated with chromosomal DNAs of the plant pathogenic fungus Rhizoctonia solani.

Unique DNA sequences homologous to the linear DNA plasmid pRS64 were investigated in chromosomal DNAs of isolates belonging to anastomosis group 4 (AG-4) of the plant pathogenic fungus Rhizoctonia solani. Chromosome-sized DNAs of isolates RI-64 and 1271 of AG-4 were separated into six bands by orthogonal-field-alternation gel electrophoresis and hybridized to a cloned segment of pRS64. A small chromosome-sized DNA band of approximately 1.1 Mb carried the sequences homologous to pRS64 DNA. Sequences homologous to pRS64 were also maintained within the chromosomal DNA of isolate 127.1 of AG-4 which does not possess the plasmid. The plasmid showed no homology to the mitochondrial DNA of isolate 1271. The possibility that the linear plasmid pRS64 may act as a transposable genetic element is discussed.     

Study of the aggressiveness of Rhizoctonia solani isolates

This paper presents an in vitro test to screen the pathogenicity of different Rhizoctonia solani isolates on a host range. The level of aggressivity of the different isolates was different for several host plants tested. There were significant differences between the crops and the isolates tested. In general, the disease level was higher on beans, lettuce and cabbage. In carrot and rye grass the level of infection was lower for the isolates of R. solani tested. The potato isolates of R. solani were less aggressive than the isolates coming from maize, fodder beet and sugar beet. The R. solani isolates were also biochemically characterized by pectic zymograms: the isolates Rs0401 (from maize) and Rs0504 (from sugar beet) belong both to the anastomosis group AG2-2.