Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean

We isolated 99 yeast strains, including 40 red yeasts, from benthic animals and sediments collected from the deep-sea floor in various areas in the northwest Pacific Ocean. Comparing the yeast isolates from animals and sediments collected from shallow locations, the proportion of red yeasts differed considerably, comprising 81.5% and 10.6% of the isolates from animals and sediments, respectively. All of the red yeast isolates belonged to the genera Rhodotorula and Sporobolomyces. On the basis of morphological and physiological characteristics, the isolates were identified as R. aurantiaca, R. glutinis, R. minuta and R. mucilaginosa of the genus Rhodotorula, and S. salmonicolor and S. shibatanus of the genus Sporobolomyces. Only R. glutinis and R. mucilaginosa were isolated from sediments. All of the others were isolated from animal sources. Phylogenetic analyses based on internal transcribed spacer (ITS) regions and 5.8S rRNA gene sequences allowed us to establish the precise taxonomic placement of each of the isolates and thereby investigate the intraspecific relationships among the isolates. Twenty-two strains identified as members of R. glutinis, which showed a wide distribution in the deep-sea, and five isolates identified as R. minuta, which were isolated only from benthic animals, showed substantial heterogeneity within the species. The isolates phenotypically identified as Sporobolomyces species and R. mucilaginosa phylogenetically occupied the placements corresponding to these species. Some strains assigned to known species on the basis of phenotypic features should be regarded as new species as suggested by the results of molecular analysis.     

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).     

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.     

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.     

Morphological variability in Rhizoctonia solani isolates from different agro-ecological zones of West Bengal,India

Sheath blight caused by Rhizoctonia solani Kühn is one of the most important diseases of rice, resulting in significant yield loss in rice every year. The rice-based intensified cropping system, edapho-climatological and host variations make the disease problem more complicated. However, the incidence and severity of the disease differ from one location to other, one geographical area to other and even differs from country and region wise. The reasons for this disease severity have been attributed to the variation in host genotype, virulence of the pathogen, prevalence of congenial soil physico-chemical and plants’ surrounding environment and cultural practices. Sixty-seven number of isolates of R. solani from rice, 12 no. of R. solani isolates associated with maize, sugarcane, weeds, cabbage, pointed gourd, watermelon, potato, dolichos bean and aparajita were isolated from different agro-ecological region of West Bengal and three no. of isolates of R. oryzae-sativae obtained from Department of Plant Pathology, BCKV, were used in the present study. Cultural and morphological characteristics revealed considerable diversity among the R. solani isolates. Cultural and morphological analysis of WB isolates of rice has indicated that the diversity among the isolates does not correlated with their origin. On the basis of morphological characteristics, R. solani isolates could be easily separated from R. oryzae-sativae isolates. The no. of sclerotia, hyphal length, wt. of sclerotia and mycelial growth rate are the important morphological markers for differentiation of R. oryzae-sativae from R. solani isolates.     

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.     

Study on Rosellinia necatrix Isolates Causing White Root Rot Disease in Taiwan

White root rot is a serious soil‐borne disease of several woods and crops. Recently, white root rot of tea shrubs and ornamental trees has increasingly been observed in Taiwan. Thirty‐six isolates of white root rot pathogen, showing pear‐shape swellings adjacent to the hyphal septa, had been isolated from samples of white root rot collected from Taiwan for about 4 years. The pathogen isolates produced Dematophora anamorph. Conidia of the pathogen were one‐celled, hyaline, subglobal, with truncate base, 2.9–5.8 × 1.9–3.5 μm . Ascospore dimensions were in the range of 37.0–55.0 × 5.4–7.9 μm with a short, longitudinal and straight germ slit, which complied with Rosellinia necatrix. Based on molecular studies, the pathogen isolates collected from Taiwan except R701 were identified as R. nectarix. Isolate R701, which was relatively polymorphic in internal transcribed spacer DNA sequence than other isolates, was temporarily considered as R. necatrix‐related pathogenic Rosellinia spp. All the tea cuttings (Camellia sinensis) inoculated with isolates developed typical white root rot symptoms. Pathogenicity tests demonstrated the presence of variation in virulence among the Rosellina isolates. Most of the R. necatrix isolates originating from Acer morrisonense were less virulent than those that originated from other hosts. The pathogenic Rosellinia spp., isolate R701, was also highly virulent to both cultivars of tea cuttings.     

Morphological and pathological variations of rice sheath blight inciting south Indian Rhizoctonia solani isolates

Frequent assessment of pathogen diversity is one of the most important criteria in designing disease management programmes. A study on diversity of field isolates of Rhizoctonia solani from sheath blight-infected rice fields of south India has been carried out. A total of 236 R. solani isolates were obtained from 45 locations in the surveyed area. Sclerotial features such as colour, size and shape and distribution pattern were varying among isolates. However, no other morphological features found to differ among isolates. Majority of the R. solani isolates were fast growers as they attained complete mycelial growth within 2 days in a 90-mm Petri plate and the emergence of sclerotial structures was seen even in 4 days of incubation. Selected 10 R. solani isolates exhibited considerable variations in pathogenicity on three different rice cultivars. Vellai ponni was found to be the most susceptible rice cultivar to all the field isolates of R. solani.     

Detection,Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

A species‐specific Polymerase Chain Reaction (sPCR) method was developed to identify and detect isolates of Ralstonia solanacearum, the cause of bacterial wilt disease in chilli. PCR primers for R. solanacearum were identified by alignment of hrpB gene sequences and selection of sequences specific for R. solanacearum at their 3′ ends. The primers were shown to be specific for R. solanacearum, as no PCR product was obtained when genomic DNA from other bacterial species including closely related Ralstonia species, were used as test species. Lone pair of primers (RshrpBF and RshrpBR) was designed using hrpB gene sequence, unique to R. solanacearum which amplified a predicted PCR product of 810 bp from 20 different isolates. Phylogenetic analysis was also attempted to understand the evolutionary divergence of Indian R. solanacearum isolates. Based on phylogenetic analysis, Indian isolates showed homology with the standard reference isolates from other countries but, interestingly, one new isolate showed complete evolutionary divergence by forming an out‐group.     

Field and Laboratory Studies on some Photosynthetic Bacteria in Aswan High Dam Lake. II. Isolation and Identification of Purple Non-sulfur Bacteria (Rhodospirillaceae)

485 isolates were recorded from different localities between summer 1984 and spring 1985. These isolates comprise five different genera with eight different species of purple non-sulfur bacteria- Rhodopseudomonas was the most common genus at all sites and was represented by three species. R. acidophila, R. blastica, R. palustris. Rhodomicrobium was the second in frequency and was represented by R. vannielli only. This species was only recorded for khor sites. Rhodocyclus was represented by two species, R. gelatinosus and R. tenuis. Rhodobacter and Rhodospirillum were represented by one species each, Rhodobacter capsulatus and Rhodospirillum rubrum. Khor sites were richer in number of isolates and genera of purple non-sulphur bacteria than the main body of AHDL. Some species were more common in winter and spring while others predominate in summer and autumn.     

Identification and pathogenicity of Rhizoctonia species isolated from bean and soybean plants in Samsun,Turkey

A total of 434 isolates of Rhizoctonia belonging to 10 anastomosis groups were obtained from the roots and rhizosphere soils of bean and soybean plants grown in Samsun, Turkey. AG-4 was found to be the most common group on bean and soybean plants and AG-5, AG-6, binucleate AG-A, AG-B and R. zeae were other groups isolated from the both plant species. AG-1, AG-7 and AG-K from bean and AG-E from soybean were other groups obtained in the study. The pathogenicity tests on bean and soybean seedlings showed that the highest disease severities were caused by AG-4 isolates, whereas AG-1 and AG-6 isolates were moderately pathogenic. Binucleate Rhizoctonia AG-B isolates were also moderately pathogenic, while other binucleate Rhizoctonia were found to be weakly pathogenic. Rhizoctonia zeae isolates caused moderate disease symptoms on bean, but soybean plants were slightly affected by this group of isolates. This is the first reported observation of R. solani AG-6 and AG-7 and binucleate Rhizoctonia AG-B on bean, and R. solani AG-5 and AG-6 and binucleate Rhizoctonia AG-A, AG-B and AG-E on soybean, in Turkey.     

Phylogenetic relationships of Riemerella anatipestifer serovars and related taxa and an evaluation of specific PCR tests reported for R. anatipestifer

Aims: The aims of the present investigation were to characterize and identify serovars of Riemerella anatipestifer and Riemerella‐like isolates genetically and to test the specificity of PCR tests reported for the identification of R. anatipestifer. Methods and Results: A total of 50 isolates from poultry tentatively classified with Riemerella anatipestifer were characterized genetically by partial sequencing of rpoB and by nearly full sequencing of the 16S rRNA gene for selected isolates. The results obtained were compared with the data from 13 reference strains by phylogenetic analysis. A total of 41 isolates were identified as R. anatipestifer, three as Wautersiella falsenii like, a single isolate as Pelistega europaea, while five isolates were classified as new, unnamed taxa. None of the reported PCR tests for identification of R. anatipestifer were found specific. Conclusions: Characterization of R. anatipestifer and related bacteria by traditional methods is often inconclusive because of inconsistent reactions and phenotypic diversity. For the same reason, gene sequencing and phylogenetic analysis are essential to allow proper classification and identification as demonstrated in the present study. Significance and impact of the Study: The present investigations demonstrated that isolates of R. anatipestifer are often misidentified, and that new serovars should not be accepted unless they have been properly characterized by relevant genetic methods such as gene sequencing. In addition, we showed that the published PCR tests are not specific for this species. Finally, two new taxa were outlined, the final taxonomic positions of which remain to be identified.     

Phylotype classification of Ralstonia solanacearum biovar 1 strains isolated from banana (Musa spp) in Malaysia

During 2011–2012, 15 bacterial isolates were obtained from wilting banana plants from seven locations in Malaysia. Characterisation of the Malaysian isolates was determined by biovar determination, pathogenicity test, phylotype-specific multiplex PCR (Pmx-PCR) and endoglucanase (egl) gene amplification. Based on the genotype, phenotype and pathogenic characteristics, all isolates were identified as Ralstonia solanacearum. Pmx- and egl-PCRs indicated that all isolates belong to phylotype II of Ralstonia species complex hierarchical classification. The neighbour joining phylogenetic tree of egl sequences also verified the results where the isolates were all clustered into phylotype II, together with the reference sequences strains, UW070 and UW162. Therefore, the results of our study may provide a better understanding on the taxonomy of R. solanacearum species occupying banana plantations in Malaysia. This study is indeed the first report of phylotype II classification of R. solanacearum biovar 1 strains isolated from banana plants in Malaysia.     

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.     

Vector specificity of barley yellow dwarf virus serotypes and variants in southwestern Idaho

Plants with symptoms of barley yellow dwarf virus (BYDV) obtained in infection feeding assays of aphids collected in the field in Idaho between 1986 and 1988 were tested for virus transmissibility by possible aphid vectors. Isolates obtained during 1987–1988 were also tested with a range of polyclonal antisera which distinguished PAV, MAV, SGV, RPV and RMV serotypes. In 1989 some Idaho (ID) BYDV isolates, maintained as standards for comparison, were serotyped and tested for aphid transmissibility, using 11 species of aphids. There was not always the expected correspondence between serotype and vector specificity for ID isolates. For isolates obtained from field-collected Rhopalosiphum padi, vector transmissibility and serotype corresponded with previous reports; however, 44% of isolates which were serotyped as RMV were also transmissible by species other than Rhopalosiphum maidis. Similarly, the transmissibility of the ID laboratory standards did not always conform to the reported vector specificity of serotypes. The laboratory ID-MAV culture was transmitted by Metopolophium dirhodum and Myzus persicae as well as by Sitobion avenae. The laboratory ID-SGV culture was transmitted by R. padi and 5. avenae as well as by Schizaphis graminum. The ID-RPV culture was transmitted by S. graminum and Rhopalosiphum insertum as well as R. padi. Both of two laboratory ID-RMV cultures were transmissible by R. insertum and R. padi transmitted one of them. The results indicate that, for isolates collected in Idaho, vector specificity cannot be assumed from their serotypes.     

Serological and biological characterisation of isolates of barley yellow dwarf virus in Sweden in 1983

In 1983, cereal plants showing symptoms of barley yellow dwarf virus (BYDV), collected from 15 localities in Sweden, were tested for BYDV using enzyme-linked immunosorbent assay (ELISA). Antisera against two Swedish isolates of BYDV were used, a mild isolate (27/77) transmitted specifically by Sitobion avenae and a severe one (39/78) transmitted mainly by Rhopalosiphum padi. No virus was detected in 57 of 607 plants of oats and barley tested. Of the 550 plants in which virus was detected, 366 were infected with viruses similar to isolate 27/77, 116 with viruses similar to 39/78 and the remaining 68 reacted strongly with both antisera. When tested, the latter isolates were shown to be mixtures. Thirty-nine selected samples were also tested with antisera against the USA isolates RPV, RMV, MAV and PAV, and for transmission by S. avenae and R. padi. Twenty-six of these samples were transmitted specifically by S. avenae, one was transmitted only by R. padi and the remaining 12 samples were shown to be infected with a mixture of an S. avenae-specific isolate and one transmitted mainly by R. padi. Antisera against PAV and MAV each detected all isolates tested and the results were very similar to those with the antisera to the 39/78 and 27/77 isolates, respectively. None of the field isolates reacted with antisera against RMV or RPV. It was concluded that 1983 was an epidemic year for BYDV in Sweden and that isolates specifically transmitted by S. avenae predominated. Symptoms of infection by these isolates on oat plants ranged from mild to severe.     

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas withPinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas ofPinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza ofQuercus myrsinaefolia were tested for the ability to form mycorrhizas withP. densiflora seedlings in glass tubes. Ten isolates from sporocarps ofHebeloma sp.,Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae andR. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including ofCenococcum geophilum, R. mariae andR. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.Contribution No. 122, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Long‐term preservation of a collection of Rhizoctonia solani using cryogenic storage

Rhizoctonia solani is an important plant pathogen for a number of crops and maintaining an extensive collection of reference isolates is important in understanding relationships of this pathogen with multiple hosts. Current long‐term storage methods typically call for frequent transfer increasing the risk of changes in morphological, physiological or virulence characteristics. Cryopreservation using storage in liquid nitrogen (LN) was evaluated to examine the potential for storage of a R. solani culture collection containing 106 isolates (primarily from sugar beet). Cultures were stored on autoclaved barley grains in the vapour phase of LN. After 60 days, 5 years and 10 years in storage, all isolates were tested for viability by calculating the percentage of barley grains from which R. solani mycelia grew. Five years after initial storage, all isolates except one had no change in viability. After 10 years in storage, 67 of 106 isolates had no significant decrease in viability, 39 of 106 isolates had a significant decrease in viability but only 9 isolates had less than 10% growth, with 4 having no growth. A subset of isolates stored for 10 years were tested for pathogenicity on a susceptible (FC901) and resistant (FC703) sugar beet germplasm. All isolates tested maintained approximately the same level of virulence that they had prior to storage on both germplasms. This indicates that cryogenic methods are suitable for the preservation or storage of R. solani culture collections, although efficacy may vary with individual isolates.     

Phenotypic variation of Rhizoctonia oryzae in sheath disease of rice in Myanmar

Similarity in the cultural characteristics among closely related species of Rhizoctonia creates confusion and uncertainity in diagnosis. The present research was conducted to study the existence of phenotypic groups among isolates of R. oryzae in Myanmar. It was aimed to study the variation in phenotypic and molecular profiles among some isolates of R. oryzae and R. zeae. We found the occurrence of two distinct phenotypic groups of R. oryzae and a group of R. zeae. Ribosomal DNA-ITS sequencing was conducted and the resulting dendrogram agreed with those of the morphological grouping. A genetic distance of 0.064–0.072 wts was found between the R. oryzae and R. zeae groups. A pairwise distance of 0.014 and 0.022 was found between the RO1 and RO2 groups of R. oryzae. Our research revealed the existence of two distinct phenotypes in the isolates of R. oryzae collected from rice sheath in Myanmar and their differentiating features with R. zeae.