Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.     

Detection,identification and morphological characteristic of Macrophomina phaseolina: the charcoal rot disease pathogens isolated from infected plants in Northern Jordan

This is the first report about charcoal rot disease in Jordan. Twenty-five Macrophomina phaseolina isolates were collected from infected plants showing typical symptoms of charcoal rot disease. All of the 25 M. phaseolina isolates were pathogenic to cucumber plants under green house effect. The amplification of the isolated DNA from the 25 pathogenic fungal cultures using ITS specific primers (ITS 1?+?ITS 4) showed a single band of 580?bp. There was a significant variation of their mycelial linear growth rate on PDA medium. The 25 M. phaseolina isolates showed a wide heterogeneity in their mycelium colour, microsclerotia distribution, pycnidia formation and chlorate phenotypes. Based on the morphological characterisation, the 25 isolates were grouped into seven different groups as indicated in a dendrogram of their morphological variation. The overall means similarity matrix of the 25 M. phaseolina recovered isolates were 0.58. The means of similarity matrix of the 25 M. phaseolina was in between 0.83 and 0.14. The similarity coefficient between the 25 isolates varies between 0.27 and 1.0.     

Genetic diversity of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.     

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

Phylogeny,morphology and pathogenicity of Elsinoë ampelina,the causal agent of grapevine anthracnose in Brazil and Australia

Anthracnose caused by Elsinoë ampelina is one of the most important table grape diseases in humid regions in Brazil and Australia. The objective of this study was to characterize E. ampelina isolates from Brazil and Australia by means of phylogenetic analyses, morphological features and pathogenicity tests. Phylogenetic relationships among 35 isolates were determined based on a data set of internal transcribed spacer (ITS), histone H3 (HIS3) and elongation factor 1‐α (TEF) sequences. In phylogenetic tree analyses, using a combined ITS and TEF sequence alignment, all E. ampelina isolates were clustered together in a single well‐supported clade. In contrast to the absence of genetic variability within ITS and TEF sequences, HIS3 sequences showed 54 polymorphic sites. The haplotype network generated from HIS3 data set showed four distinct haplotypes. EA1 was the predominant haplotype including 29 isolates from both countries. High genetic variability was observed in two Brazilian isolates, haplotype EA4, which may have lost the intron region during species evolution. Colony colours differed between Brazilian and Australian isolates, but showed similar wrinkled colony texture, absence of spores, sparse‐to‐absent white aerial mycelium and slow growth (0.049–0.060 mm/day). Brazilian isolates produced conidia of 5.65 × 2.65 μm, larger than conidia from Australian isolates, which measured 5.14 × 2.30 μm. In pathogenicity tests, all nine Australian isolates inoculated were pathogenic on detached canes and potted vines of table grape.     

Genetic variation of Cordyceps sinensis, a fruit-body-producing entomopathogenic species from different geographical regions in China

Cordyceps sinensis is one of the most valuable medicinal fungi in the Orient. It is naturally distributed in the eastern extension area of the Qinghai-Tibet plateau, at an altitude over 4000 m high. In order to investigate genetic variation and evolutionary relationships of C. sinensis from different geographical regions, 17 isolates of C. sinensis were collected from different provinces and the complete sequences of rDNA ITS were determined. On the basis of 5.8S rDNA and ITS region analysis, it was clearly shown that the ITS sequences within C. sinensis are highly homologous regardless of geographical origin. The distance values between the sequences in this study were lower than 0.03. This implied that C. sinensis from different geographic regions are the same species; they are not different species or a species complex. The results also showed that distance values between C. sinensis and Hirsutella sinensis are of the same order as those within C. sinensis from different geographic regions. This confirmed our previous results that C. sinensis should only have H. sinensis as its asexual stage whatever the geographic region from which the samples were collected. An rDNA ITS clone library was established to obtain further evidence for the interpretation of the fungal community structure from C. sinensis and to confirm the accuracy of the taxonomic identities produced by directly sequencing the rDNA ITS region. The discrimination between intraspecies of C. sinensis might provide additional data for the authentication of medicinal material at the species or intraspecies level.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Heterogeneity in the ITS of the ribosomal DNA of <Emphasis Type="Italic">Pyrenophora graminea</Emphasis> isolates differing in xylanase and amylase production

Xylanase and amylase have gained increasing interest because of their various biotechnology applications. In this research, the restriction of PCR-amplified internal transcribed spacers (ITS) of ribosomal DNA (rDNA) was used to confirm the genetic variation among 22 isolates of Pyrenophora graminea differing in their xylanase and amylase production. The fingerprints generated from the six restriction digestions of the rDNA ITS region showed high levels of intraspecific variation within the P. graminea population. Neighbour-Joining diagram, based on Nei’s genetic distances, showed that isolates formed two phylogenetic groups. No apparent association could be observed between xylanase and amylase production and genetic diversity among the twenty-two isolates.     

Genetic diversity analysis of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> causing stem rot in chickpea using RAPD,ITS-RFLP,ITS sequencing and mycelial compatibility grouping

Sclerotinia sclerotiorum is one of the most devastating soil-inhabiting fungal plant pathogens infecting various crop plants including chickpea. Genetic diversity of 24 isolates of S. sclerotiorum representing 10 different states of India was determined by different molecular markers and mycelial compatibility grouping (MCG). The majority of the isolates showed more than 90% genetic similarity. Unweighted paired group method with arithmetic average cluster analysis of DNA profiles generated by 21 RAPD primers grouped the isolates into seven categories showing high magnitude of genetic homogeneity and showed partial correlation with geographical origin of the isolates. Identical ITS-RFLP profiles were generated in all the isolates. Limited variability was observed among the nucleotide sequences of ITS region of the isolates. The phylogenetic tree generated from bootstrap neighbor-joining analysis indicated that 50% of Indian populations were distinct and grouped separately. The isolates were variable in mycelial compatibility and they were grouped into seven MCGs, namely, MCG A, MCG B, MCG C, MCG D, MCG E, MCG F and MCG G.     

Genetic Diversity of <Emphasis Type="Italic">Cercospora kikuchii</Emphasis> Isolates From Soybean Cultured in Argentina as Revealed by Molecular Markers and Cercosporin Production

Leaf blight and purple seed, caused by the fungal pathogen Cercospora kikuchii (Matsumoto & Tomoyasu) M. W. Gardner are very important diseases of soybean (Glycine max L. Merr.) in Argentina. The aims of this work were: (a) to confirm and to assess the genetic variability among C. kikuchii isolates collected from different soybean growing areas in Santa Fe province using inter simple sequence repeats (ISSR) markers and sequence information from the internal transcribed spacer (ITS) region of rDNA and (b) to analyze the cercosporin production of the regional C. kikuchi isolates in order to assess whether there was any relationship between the molecular profiles and the toxin production. Isolates from different regions in Santa Fe province were studied. The sequence of the ITS regions showed high similarity (99–100%) to the GenBank sequences of C. kikuchii BRCK179 (accession number AY633838). The ISSR markers clustered all the isolates into many groups and cercosporin content was highly variable among isolates. No relationship was observed between ITS region, ISSR groups and origin or cercosporin content. The high degree of genetic variability and cercosporin production among isolates compared in this study characterizes a diverse population of C. kikuchii in the region.     

Genetic variation and population structure of the carpet shell clam <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> along the Tunisian coast inferred from mtDNA and ITS1 sequence analysis

Surveys of allozyme polymorphisms in the carpet shell clam Ruditapes decussatus have revealed sharp genetic differentiation of populations. Analysis of population structure in this species has now been extended to include nuclear and mitochondrial genes. A partial sequence of a mitochondrial COI gene and of the internal transcribed spacer region (ITS-1) were used to study haplotype distribution, the pattern of gene flow, and population genetic structure of R. decussatus. The samples were collected from twelve populations from the eastern and western Mediterranean coasts of Tunisia, one from Concarneau and one from Thau. A total of twenty and twenty-one haplotypes were detected in the examined COI and ITS1 regions respectively. The study revealed higher levels of genetic diversity for ITS1 compared to COI. The analysis of haplotype frequency distribution and molecular variation indicated that the majority of the genetic variation was distributed within populations (93% and 86% for COI and ITS1 respectively). No significant differentiation was found among eastern and western groups on either side of the Siculo-Tunisian strait. However, distinct and significant clinal changes in haplotypes frequencies between eastern and western samples were found at the most frequent COI haplotype and at three out of five major ITS1 haplotypes. These results suggest the relative importance of historical processes and contemporary hydrodynamic features on the observed patterns of genetic structure.     

Molecular tools separate harmful algal bloom species, Karenia mikimotoi, from different geographical regions into distinct sub-groups

Karenia mikimotoi is a toxic dinoflagellate that is known to form extensive populations in the Eastern North Atlantic and Pacific Oceans that that impacts significantly on recreation and fisheries industries in these areas. Attempts to resolve the complex taxonomy within this “species” have been hampered by the requirement for fine-scale morphological analyses and by the lack of suitable genetic markers. Here we report the use of a novel combination of primer sets designed to facilitate amplification of the rDNA LSU and ITS, and the rbcL genes that can be used to discriminate between K. mikimotoi isolates originating from different geographical regions. We show that isolates from Europe and New Zealand are more closely related to each other than either is to isolates from Japan. Specific PCR-based primers were designed to amplify a region of the rbcL gene for subsequent high resolution analysis of the PCR amplicon melting temperatures. This innovative technique allows us to rapidly discriminate K. mikimotoi from distinct geographic localities and we propose the separation of K. mikimotoi into two distinct sub-species.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Molecular characterization of isolates of Beauveria bassiana obtained from overwintering and summer populations of Sunn Pest (Eurygaster integriceps)

Aim:  To examine whether isolates of the entomopathogenic fungus Beauveria bassiana are more closely associated to their summer hosts compared with overwintering hosts, with recently developed molecular tools based on mitochondrial regions. Methods and Results:  Primers for the traditional ITS1‐5·8S‐ITS2 region and two mitochondrial intergenic regions, namely, nad3‐atp9 and atp6‐rns, were used. All amplified products were sequenced, aligned and Neighbour‐Joining (NJ), parsimony and Bayesian phylogenetic inference analyses were performed. The isolates examined were grouped with very good support into three distinct groups, two of them showed geographical correlation, but no clear association to their host. Conclusions:  The mitochondrial intergenic regions used were more informative than the nuclear ITS1‐5·8S‐ITS2 sequences. The sequence variability observed, that allowed the phylogenetic placement of the isolates into distinct groups, depended on the geographical origin of the isolates and can be exploited for designing group‐specific and isolate‐specific primers for their genetic fingerprinting. No clear associations with summer Sunn Pest populations were observed. Significance and Impact of the Study:  Studies on the genetic variability of biocontrol agents like B. bassiana are indispensable for the development of molecular tools for their future monitoring.     

Population Evidence of Cryptic Species and Geographical Structure in the Cosmopolitan Ectomycorrhizal Fungus, Tricholoma scalpturatum

Tricholoma scalpturatum is an ectomycorrhizal fungus that forms symbioses with roots of diverse trees and shrubs. It is commonly encountered in a wide range of habitats, across temperate ecosystems. A previous study has revealed a high genetic diversity at a local scale, and ruderal abilities. To examine genetic structure at a large geographical scale, a total of 164 basidiocarps were collected from 30 populations located in Western Europe, from Spain to Scandinavia. These samples were analyzed by three molecular methods with different levels of resolution: inter-simple sequence repeats (ISSRs), restriction fragment length polymorphisms (RFLPs) in the rDNA internal transcribed spacer (ITS), and ITS sequence analysis. Considerable genetic variation was found, and the morphospecies was separated into two genetic groups that were distinct from each other. The ISSR data and the relatively low percentage value (96%) of shared sequence polymorphisms in the ITS between isolates from the two groups, strongly suggest cryptic species and long-lasting separation. No geographical exclusion was detected for these two widely distributed taxa. However, high estimates of population differentiation were observed in each group, including between populations less than a few kilometers apart. This result provides evidence for limited gene flow and/or founding effects. It also indicates that T. scalpturatum does not constitute a random mating population, and the hypothesis of endemism cannot be excluded for this cosmopolitan wind-dispersed fungus.     

Detection of double stranded RNA in phytopathogenic <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> causing charcoal rot in <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>

One hundred one isolates of Macrophomina phaseolina from various hosts and eco-geographical locations were employed for elucidating relationships among genetic diversity and virulence. Highly pathogenic, moderately pathogenic, and hypovirulent cluster bean specific isolates were identified. In order to correlate respective phenotypes of plant pathogenic fungus multiple and complex patterns of dsRNA elements were analyzed. Double-stranded ribonucleic acids (dsRNA) are ubiquitous in all major groups and most of them have vast potential as biological control agents for fungi. Rate of virulence and its further association could ascertain by host plant and their fungal genotypes. Variability of the fungal genotypes decides the link between the complexity of dsRNA with different variants and the change in virulence pattern. Double-stranded RNA was identified in approximately 21.7% of M. phaseolina isolates from charcoal rot infected cluster bean varieties. After recurrent laboratory transfer on culture media, the preponderance of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to cluster bean. Macrophomina has successfully showed diversified and reproducible banding profile in dsRNA containing/free isolates. This is the first report of hypovirulence and detection of dsRNA in Macrophomina phaseolina isolates of cluster bean origin.     

Restriction analysis of the amplified ribosomal DNA spacers ITS1 and ITS2 of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> isolates

The purpose of this study was to examine the genotypic variability of Bipolaris sorokiniana by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using rDNA. Fifty B. sorokiniana isolates from Brazil and other countries, one Bipolaris oryzae and six Drechslera teres isolates were used. The intragenic spacer regions (ITS1 and ITS2) were the regions used for characterization of isolates. The amplification products for both ITS regions, showed two DNA fragments for all isolates. Two B. sorokiniana isolates presented an intraspecific variability showing a third fragment for the ITS1 region. The dendrograms generated with PCR-RFLP data showed intra- and inter-specific groups. The dendrograms showed that most of Brazilian isolates clustered together forming groups between them, and this behavior was repeated with most isolates from other countries. The dendrograms did not enable the separation of B. sorokiniana isolates by their geographic origin or host type. These results suggest the occurrence of gene flow between different populations of the fungus isolated in geographically distant regions and lends cogency to the occurrence of gene flow between species.