Shiro Kuninaga

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Abstract Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

Characterization of AG-13, a Newly Reported Anastomosis Group of Rhizoctonia solani

ABSTRACT Rhizoctonia solani anastomosis group (AG)-13 was collected from diseased roots of field grown cotton plants in Georgia in the United States. Isolates of AG-13 did not anastomose with tester isolates of AG-1 through AG-12. Mycelium of all isolates of AG-13 were light brown but darkened as cultures aged. All isolates produced aerial mycelium. Concentric rings were visible after 3 to 4 days of growth but disappeared as cultures aged and darkened. Individual sclerotia were up to 1.5 mm in diameter, similar in color to the mycelium, and generally embedded in the agar. Clumps of sclerotia up to 5 mm in diameter were produced on the agar surface. All attempts to induce basidiospore production were unsuccessful. The 5.8S region of the rDNA from isolates of AG-13 was identical in length and sequence to isolates of all other AGs of R. solani. Length and sequence of the internal transcribed spacer (ITS) regions of rDNA from isolates of AG-13 were unique among AGs of R. solani. Similarity between AG-13 and other AGs of R. solani ranged from 68 to 85% for ITS region 1 and 85 to 95% for ITS region 2. Selected isolates of AG-13 caused minor or no damage to barley, cauliflower, cotton, lettuce, potato, and radish in laboratory or greenhouse studies.

Comparison of rDNA-ITS Sequences between Potato and Tobacco Strains in Rhizoctonia solani AG-3

Genetic diversity among 51 isolates of Rhizoctonia solani AG-3, representing potato and tobacco populations, was inferred from the sequences of the internal transcribed spacer (ITS) and 5.8S ribosomal RNA (rRNA) gene. The 5.8S rDNA sequence was completely conserved not only in AG-3, but across all the AG isolates examined, whereas the rDNA-ITS sequence was found to be variable among the isolates. The nucleotide sequence similarity in the ITS 1 region was high (96-100%) for isolates within each of the two populations, but was 91-92% for isolates from different populations. The AG-3 isolates had 56 to 91% sequence similarities in the ITS 1 region with R. solani isolates of the other AGs. Phylogenetic analysis based on the ITS-5.8S rDNA sequence data indicated that the different populations in AG-3 are distantly related to each other. Genetic divergence between the two populations was also supported by the results of DNA-DNA hybridization studies. This study suggests that AG-3 consists of two genetically isolated groups corresponding to separate subgroups: AG-3 PT (potato type) and AG-3 TB (tobacco type). Specific primer sets for the detection of the two AG-3 subgroups were developed from the aligned rDNA-ITS sequences.

Impact of Fumigation with Metam Sodium upon Soil Microbial Community Structure in Two Japanese Soils

Abstract The effects of fumigation with sodium methyl dithiocarbamate (metam sodium) on the microbial community structure and function in 2 soils were investigated using a variety of techniques. In both soils ca. 50% and 90% of the populations of total and culturable bacteria, respectively, were killed by fumigation, with recovery to levels prevailing in control soils 26 d after cessation of fumigation. The size of the ammonium and nitrite oxidiser populations was reduced by up to 4 orders of magnitude by fumigation, with the latter showing a slight recovery 105 d later. There were substantial changes in the C-utilisation (Biolog GN) profiles in the fumigated soils even 105 d later. The number and pattern of amplified 16S ribosomal DNA restriction analysis (ARDRA) fragments was changed by fumigation, and there was a shift in the %G+C profile toward a greater proportion of lower %G+C classes in treated soils. It appeared that DNA released from killed cells remained for some time after fumigation, and maske...

Phylogenetic analysis of Rhizoctonia solani subgroups associated with web blight symptoms on common bean based on ITS-5.8S rDNA

Sixty-eight Rhizoctonia solani isolates (31 AG-1, 37 of AG-2-2) associated with web blight (WB) of common bean, Phaseolus vulgaris, were examined for sequence variations in the ITS-5.8S rDNA region. The isolates were collected in bean-growing lowland and mountainous regions in Central and South America. Sequences of these isolates were aligned with other known R. solani sequences from the NCBI GenBank and distance and parsimony analysis were used to obtain phylogenetic trees. WB isolates of AG-1 formed two clades separated from known AG-1 subgroups. WB isolates of AG-2-2 formed one clade separated from known AG-2-2 subgroups. Other isolates belonged to AG-1 IA and AG-1 IB. Based on phylogenetic analysis, we confirmed that at least five genetically different subgroups incite WB of common beans. Three new subgroups of R. solani have been identified and designated as AG-1 IE, AG-1 IF and AG-2-2 WB. DNA sequences of these isolates provided needed information to design taxon-specific primers that can be employed in ecological/epidemiological studies and seed health tests.

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar)and bovine trypsin

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.

The first report of tomato foot rot caused by Rhizoctonia solani AG-3 PT and AG-2-Nt and its host range and molecular characterization.

Foot rot of mature tomato plants was found in four cities of Hokkaido, Japan, from 2004 to 2007. Six of eight isolates obtained from damaged tissues were identified as Rhizoctonia solani anastomosis group (AG)-3, and the remaining two isolates belonged to AG-2-1. We compared these isolates with nine reference isolates including the different subgroups in AG-3 (PT, TB and TM) and AG-2-Nt (pathogen of tobacco leaf spot) within AG-2-1 in terms of pathogenicity to tomato, tobacco and potato. All eight isolates caused foot rot on tomato. The six AG-3 isolates caused stem rot on young potato plants. While, all reference isolates of AG-3 PT causing stem rot of young potato plants incited foot rot on tomato. The two AG-2-1 isolates and an AG-2-Nt reference isolate caused severe leaf spot on tobacco leaves. The sequences of rDNA- ITS region and rDNA-IGS1 region of the AG-3 isolates showed high similarity to that of AG-3 PT isolates. Phylogenetic tree based on ITS and IGS1 regions of rDNA indicated that the AG-2-1 isolates from tomato formed a single clade with AG-2-Nt isolates and that they were separate from Japanese AG-2-1 isolates (culture type II). Pathogenicity tests and DNA sequence evaluation of the causal fungi revealed that the present isolates of AG-3 and AG-2-1 belonged to AG-3 PT and AG-2-Nt, respectively. This is the first report of tomato foot rot caused by R. solani in Japan.

First report of white leaf rot on Chinese chives caused by Rhizoctonia solani AG-2-1

Delayed sprouting and white rot of leaf tips were found on Chinese chives in a greenhouse in Hokkaido, Japan, in the spring of 2006 and 2007. The causal fungus was identified as Rhizoctonia solani anastomosis group (AG)-2-1 and discriminated from the leaf rot pathogen R. solani AG-4 HG-I in terms of pathogenicity. Symptoms and the time of year that the disease occurs also apparently differ for the two pathogens. This is the first report of white leaf rot on Chinese chive caused by R. solani AG-2-1.

Genetic relationships in Pseudocercosporella herpotrichoides determined from DNA relatedness

Isolates of Pseudocercosporella herpotrichoides , causal agent of the eyespot disease of cereals, are divided into two groups based on colony morphology on PDA: fast-growing, even-edged (FE) and slow-growing, feathery or uneven-edged (SF). This study with nDNA reassociation kinetics was undertaken to determine the genetic relationships among isolates in the groups. Within each group, DNA relatedness was high (82% or higher) among isolates from diverse geographical origins including Japan, Europe, North America, South Africa and New Zealand. Between the FE and SF groups, low DNA relatedness values were observed, ranging from 24 to 34%. Both groups of P. herpotrichoides showed little DNA relatedness (0 to 4%) with isolates of P. anguioides and P. aestiva . This study suggests that P. herpotrichoides consists of two genomic species, which are genetically isolated groups based on the FE, SF classification system.

First report of leaf sheath rot of Welsh onion caused by nine taxa of Rhizoctonia spp. and characteristics of the pathogens

From 2007 to 2013, a disease of Welsh onion, causing leaf sheath rot and concomitant death of outer leaves was found in 20 fields in Hokkaido, Japan. We obtained 20 Rhizoctonia isolates from diseased tissues and identified them based on the number of nuclei, hyphal fusion reactions, and molecular techniques using specific PCR primers and sequence of the rDNA-ITS region. The 20 isolates consisted of 16 multinucleate and four binucleate isolates. Of the multinucleate isolates, five were found to be so far unknown and designated here as Rhizoctonia solani AG-4 hybrid subgroup between HG-I and HG-II. Others were identified as AG-1 IB (three isolates), AG-2-2 IIIB (two isolates), AG-4 HG-I (two isolates), AG-1 IC (one isolate), AG-2-1 (one isolate), AG-4 HG-II (one isolate) and AG-5 (one isolate). All four binucleate isolates were binucleate Rhizoctonia AG-U. Original symptoms were reproduced on all plants inoculated with these isolates. Thus, we revealed that as many as nine taxa of Rhizoctonia spp. were associated with the disease. This is the first report of leaf sheath rot of Welsh onion caused by Rhizoctonia spp.