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.     

Effect of dsRNA-containing and dsRNA-free hypovirulent isolates of Fusarium oxysporum on severity of Fusarium seedling disease of soybean in naturally infested soil

In the sandy soils of eastern Virginia, soybean seedlings are colonized by hypovirulent and virulent isolates of Fusarium oxysporum and F. solani. Our objectives were to determine if prior inoculation of soybean seeds with hypovirulent F. oxysporum isolates reduced severity of seedling disease in naturally infested soil, and to determine if there was an association between the presence of dsRNA mycovirus and hypovirulence in isolates of F. oxysporum and F. solani from soybean plants. The presence of dsRNA was not associated with hypovirulence in F. oxysporum since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing F. oxysporum isolates, three were hypovirulent and three were virulent. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7 and 2.2 kb were detected in extracts of all six F. oxysporum isolates. No hypovirulent or dsRNA-containing of F. solani isolates were found. Prior inoculation of cv. Essex soybean seeds with conidia of dsRNA-free hypovirulent F. oxysporum isolates significantly (P < 0.05) reduced disease severity on cotyledons and hypocotyls, and increased the rate of seedling emergence in field soil, compared to control plants. No significant (P > 0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent F. oxysporum isolates in their effects on reducing disease severity. Hypovirulent isolates that colonize soybean tissues may play a role in reducing Fusarium seedling disease of soybean in natural soils.     

Cloning and characterization of the ribosomal RNA gene complex from the plant pathogen Verticillium albo-atrum

Abstract The DNA coding for the ribosomal RNA gene complex (rDNA) has been cloned from isolate 621P(PV1) of Verticillium albo-atrum which is pathogenic for hops ( Humulus lupulus ). The rDNA was mapped using a range of restriction enzymes. The functional units of the intergenic spacer (IGS), 18S, 5.8S and 25S regions were located by hybridization to specific gene probes from the rDNA complex of Aspergillus nidulans . The start points of the 18S and 5.8S regions were confirmed by partial sequencing. A genomic restriction enzyme map was found to be identical with the map of the cloned DNA. The rDNA repeat was 7.6 kb in length and this was used as an homologous probe to analyse the size of the repeat in 18 hop isolates of V. albo-atrum strains and in one isolate from alfalfa (Luc2). All of the isolates had a repeat size of 7.6 kb except for Luc2 where the rDNA complex was 8.4 kb.     

Vegetative compatibility relationships among weakly pathogenic isolates (pathotype E) ofVerticillium dahliae

Twenty-two isolates ofVerticillium dahliae, which were isolated from green soybeean (Glycine max), udo (Aralia cordata), horseradish (Cochlearia armoracia), sweetpea (Lathyrus odoratus), or a weed (Chenopodium album) were used in this study. Conidia and microsclerotia of these isolates were morphologically identical with those ofV. dahliae but did not coincide withV. longisporum. Pathogenicity tests showed that these isolates were of weak pathotype. Eleven of the 22 isolates, which were obtained from green soybean and udo, were pathogenic to green soybeans. Thus pathotype E was composed of two groups: ‘soybean pathotype’ which was pathogenic to green soybeans; and isolates nonpathogenic to green soybeans. The latter were defined as isolates of pathotype E in the narrow sense. Selected representativenit1 and NitM mutants of eachV. dahliae isolate were paired with VCGJ testers. Fourteen isolates ofV. dahliae (So1, So22, So23, So27, So28, So39, So40, So41, U54, U68, U69, U90, U95, and U115) showed complementary reactions with subgroups J1 and J3 and were assigned to subgroup J3. Isolate U108 was assigned to subgroup J2. Isolate HR1 was not compatible with any testers of VCGJ. With this exception, isolates of pathotype E in the narrow sense and those of ‘soybean pathotype’ were thus assigned to known VCGJ subgroups and did not form a unique group corresponding to their pathotype. ‘Soybean pathotype’ could not be distinguished among isolates of pathotype E by vegetative compatibility.     

Molecular differentiation of two races of Fusarium oxysporum special form cubense

Total cellular DNA was extracted from 28 isolates of Fusarium oxysporum from vascular wilt of banana. The DNA samples were digested with the restriction endonuclease Hae III, and separated in agarose gels. Two characteristic banding patterns were seen in ethidium bromide stained gels. The two patterns correlated to strains believed to be of pathogenic races 1 and 4. A minor variation to these was detected with isolates of race 1 from Tanzania, which gave a typical race 1 band pattern, with an additional band of approx. 10 kb.     

Specific PCR-based marker for detection of pathogenic groups of Fusarium oxysporum f. sp. cucumerinum in India

For the detection of Fusarium oxysporum f. sp. cucumerinum pathogenic groups, a specific PCR-based marker was developed. Specific random amplified polymorphic DNA (RAPD) markers which identified in four pathogenic groups I, II, III, and IV were cloned into P^Gem-Teasy vector. Cloned fragments were sequenced, and used for developing sequence characterized amplified regions (SCAR) primers for detection of pathogenic groups. F. oxysporum f. sp. cucumerinum isolates belonging to four pathogenic groups in India, cucumber nonpathogenic F. oxysporum, F. oxysporum f. sp. moniliforme and melonis, Fusarium udum, and isolate of Alternaria sp. were tested using developed specific primers. A single 1.320 kb, 770 bp, 1.119 kb, and 771 bp fragment were amplified from pathogenic group I, II, III, and IV isolates, respectively. Results showed the PCR based marker, which used in this research work, could detect up to 1 ng of fungal genomic DNA. The specific SCAR primers and PCR technique developed in this research easily detect and differentiate isolates of each F. oxysporum f. sp. cucumerinum pathogenic groups.     

Relatedness of Macrophomina phaseolina isolates from tallgrass prairie, maize, soybean and sorghum

Agricultural and wild ecosystems may interact through shared pathogens such as Macrophomina phaseolina , a generalist clonal fungus with more than 284 plant hosts that is likely to become more important under climate change scenarios of increased heat and drought stress. To evaluate the degree of subdivision in populations of M. phaseolina in Kansas agriculture and wildlands, we compared 143 isolates from maize fields adjacent to tallgrass prairie, nearby sorghum fields, widely dispersed soybean fields and isolates from eight plant species in tallgrass prairie. Isolate growth phenotypes were evaluated on a medium containing chlorate. Genetic characteristics were analysed based on amplified fragment length polymorphisms and the sequence of the rDNA-internal transcribed spacer (ITS) region. The average genetic similarity was 58% among isolates in the tallgrass prairie, 71% in the maize fields, 75% in the sorghum fields and 80% in the dispersed soybean fields. The isolates were divided into four clusters: one containing most of the isolates from maize and soybean, two others containing isolates from wild plants and sorghum, and a fourth containing a single isolate recovered from Solidago canadensis in the tallgrass prairie. Most of the sorghum isolates had the dense phenotype on media containing chlorate, while those from other hosts had either feathery or restricted phenotypes. These results suggest that the tallgrass prairie supports a more diverse population of M. phaseolina per area than do any of the crop species. Subpopulations show incomplete specialization by host. These results also suggest that inoculum produced in agriculture may influence tallgrass prairie communities, and conversely that different pathogen subpopulations in tallgrass prairie can interact there to generate 'hybrids' with novel genetic profiles and pathogenic capabilities.     

采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌

采用16S rRNA基因PCR扩增与限制性酶切片段多态性分析(RFLP)技术对选自弗氏中华根瘤菌(S.fredii)、大豆慢生根瘤菌(B.japonicum)和埃氏慢生根瘤菌(B.elkanii)的19株代表菌进行了比较分析,根据用3种限制性内切酶的RFLP分析结果,可将供试菌株分为S.fredii,B.japonicum, B.elkanii Ⅱ和B.elkanii Ⅱa等4种基因型。各类菌株之间没有交叉,因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S.fredii和B.japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA(IGS)的PCR-RFLP分析结果表明：S.fredii和B.japonicum菌株的IGS长度不同,所有供试S.fredii菌株的IGS为2.1 kb,而供试B.japonicum菌株则为2.0 kb。依据RFLP的差异,可将来自中国两个不同地区的S.fredii株区分为2个基因型,而来自中国东北黑龙江地区的19株B.japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP-PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。     

Genetic diversity of legume yellow mosaic begomoviruses in Indonesia and Vietnam

High incidences of yellow mosaic symptoms were observed in soybean and yard‐long bean crops in Indonesia in 2009 and in mungbean crops in Vietnam in 2011. All five soybean and 20 yard‐long bean samples from Java, Indonesia, and 15 mungbean samples from Vietnam with symptoms tested positive for begomovirus infection by polymerase chain reaction (PCR) with primer pair PAL1v1978B/PAR1c715H. On the basis of collection location and the nucleotide sequence comparisons of the 1.5 kb begomoviral DNA‐A components amplified, a subset of samples comprising two soybean and six yard‐long bean isolates from Indonesia and five mungbean isolates from Vietnam were taken forward for more detailed examination. Sequence comparison and phylogenetic analysis of the full‐length sequences of all Indonesian and Vietnam isolates alongside other legume‐infecting begomoviruses revealed that all the isolates from Indonesia were Mungbean yellow mosaic India virus (MYMIV) strain‐A, and all from Vietnam were Mungbean yellow mosaic virus (MYMV) strain‐B. To the best of our knowledge, this is the first identification of MYMIV and MYMV associated with yellow mosaic of legumes in Indonesia and Vietnam, respectively. The epidemiological implications and potential consequences of the emergence of legume‐infecting begomoviruses on legume production in these areas of Southeast Asia are discussed.     

Analysis of double-stranded RNA and virus-like particles in trichothecene-producing strains of<Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Double-stranded RNAs (dsRNAs) have been found in two isolates of the plant pathogenic fungus Fusarium graminearum which produce trichothecene mycotoxins. The isolates 8.2 and 19.2 had dsRNAs in the size of about 2.0 kb and 6.0 kb, respectively, which were associated with capsid proteins and persisted within the cytoplasm of the infected host cells as encapsidated virus-like particles (VLPs). The dsRNAs contained in the VLP pellets were the same size as the dsRNA isolated in total nucleic acid preparations. In the VLP pellets the isolate 19.2 had a second dsRNA with the size of about 1.6 kb. After mycovirus purification one icosahedral particle of about 28 nm in diameter from the isolate 8.2 and two icosahedral particles of about 28 nm and 38 to 40 nm in diameter from the isolate 19.2 could be identified with electron microscopy. SDS-PAGE analysis of the VLPs from the isolate 8.2 revealed one major protein component of approximately 65 kDa, while the isolate 19.2 had two major protein bands at about 94 kDa and 105 kDa. Both isolates were studied for potential trichothecene production. Tox5 PCR showed a 658 bp fragment in each isolate. In addition, both strains were able to produce the trichothecenes deoxynivalenol (DON), the derivatives acetyl-DON (3-A-DON, 15-A-DON) and nivalenol (NIV) in vitro.     

Genetic differentiation of charcoal rot pathogen, Macrophomina phaseolina, into specific groups using URP-PCR

Forty isolates of Macrophomina phaseolina, a pathogen causing charcoal dry root rot of soybean, cotton, and chickpea, were genetically characterized with universal rice primers (URP; primers derived from DNA repeat sequences in the rice genome) using polymerase chain reaction (URP-PCR). Out of 12 URPs used in this study, 5 primers were effective in producing polymorphic fingerprint patterns from the DNA of M. phaseolina isolates. Three primers (URP-2F, URP-6R, and URP-30F) were quite informative and produced high levels of polymorphism among the isolates of M. phaseolina. Analysis of the entire fingerprint profiles using unweighted pair-group method with arithmetic averages (UPGMA) clearly differentiated M. phaseolina isolates obtained from soybean, cotton, and chickpea hosts into specific groups. In this study, we found for the first time transferability and use of PCR primers derived from plant genomes to generate host-specific fingerprint profiles of M. phaseolina, a broad host range plant pathogenic fungus. These results demonstrate that URPs are sensitive and technically simple to use for assaying genetic variability in M. phaseolina populations.     

Detection of molecular variation in the insect pathogenic fungus Metarhizium using RAPD-PCR

Abstract DNA polymorphism among isolates of the insect pathogenic fungus Metarhizium anisopliae and M. flavoviride was investigated by RAPD-PCR. DNA fragments of between 0.3 and 2.7 kb were obtained using eight 10-mer PCR primers of arbitrary nucleotide sequence, and each isolate differed in the size and number of RAPD products, indicating considerable polymorphism. Isolate-specific RAPD fingerprints were used to calculate relative genetic similarity; this differentiated isolates into two major groups, separating nine of the ten isolates of M. anisopliae from the two of M. flavoviride . However, an Australian M. anisopliae isolated from an Orthopteran host exhibited a higher degree of genetic similarity to the M. flavoviride group. M. anisopliae isolates were further segregated into three subgroups which were loosely related to their geographical origins. although considerable polymorphism was observed within these groups. There was no apparent association between genotype and original insect host.     

大豆疫霉菌的分离与鉴定

采用叶碟诱捕法从2007年进口的美国大豆携带的土壤和2006年从黑龙江感病大豆田采集的土壤中分离出2株疫霉菌菌株,并对病原菌进行了形态特征、致病性、分子检测。结果表明:形态观察为疫霉属真菌;接种大豆后出现典型的大豆疫病症状;采用大豆疫霉的特异性引物PCR检测,2个菌株均能扩增出分子量为330 bp的特异性条带。结合形态、致病性测定和分子检测,2株病菌鉴定为大豆疫霉菌(Phytophthora sojaeKauf-mann et Gerdemann)。     

Biological Control of Phytophthora Root Rots on Alfalfa and Soybean with Streptomyces

A collection of 53 antibiotic-producing Streptomyces isolated from soils from Minnesota, Nebraska, and Washington were evaluated for their ability to inhibit plant pathogenic Phytophthora medicaginis and Phytophthora sojae in vitro. Eight isolates having the greatest pathogen-inhibitory capabilities were subsequently tested for their ability to control Phytophthora root rots on alfalfa and soybean in sterilized vermiculite and naturally infested field soil. The Streptomyces isolates tested significantly reduced root rot severity in alfalfa and soybean caused by P. medicaginis and P. sojae, respectively (P < 0.05). On alfalfa, isolates varied in their effect on plant disease severity, percentage dead plants, and plant biomass in the presence of the pathogen. The same eight isolates of Streptomyces were also tested for inhibitory activities against each other and against three strains of Bradyrhizobium japonicum and two strains of Sinorhizobium meliloti isolated from soybean and alfalfa, respectively. Streptomyces isolates clustered into two major compatibility groups: isolates within the same group were noninhibitory toward one another in vitro. The compatibility groups corresponded with groupings obtained based upon inhibition of B. japonicum and S. meliloti strains.     

Detection of a large linear plasmid in Borrelia spielmanii isolate

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.     

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.     

Genetic variance of Trichomonas vaginalis isolates by Southern hybridization

In the present study, genomic DNAs were purified from Korean isolates (KT8, KT6, KT-Kim and KT-Lee) and foreign strains (CDC85, IR78 and NYH 286) of Trichomonas vaginalis, and hybridized with a probe based on the repetitive sequence cloned from T. vaginalis to observe the genetic differences. By Southern hybridization, all isolates of T. vaginalis except the NYH286 strain had 11 bands. Therefore all isolates examined were distinguishable into 3 groups according to their banding patterns; i) KT8, KT6 and KT-Kim isolates had 11 identical bands such as 1 kb, 1.2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb and 6.0 kb. ii) The metronidazole-resistant IR78 strain had the same bands as KT-Lee isolate at bands of 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb and 6.0 kb. Bands of CDC85, metronidazole-resistant strain, were similar to those of IR78 and KT-Lee, except that 3.2 kb replaced 2.9 kb. iii) NYH286 particularly had 12 bands and band patterns were similar to IR78 with a few exceptions as follows: i) 6.2 kb in place of 6.0 kb, ii) 2.0 kb and 2.2 kb instead of 2.1 kb. Through the results obtained, genetic variance of T. vaginalis isolates was demonstrated by Southern hybridization.     

Clonality of Vibrio anguillarum strains isolated from fish from the Scandinavian countries, Sweden, Finland and Denmark

In order to investigate whether outbreaks of vibriosis in the Baltic region were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarum isolates from Finland (n = 156), Sweden (n = 88) and Denmark (n = 47) were studied with respect to serogroup, ribotype, plasmid content, and biochemical phenotypes as expressed with the PhenePlate (PhP) typing system. For comparison, 54 V. anguillarum serogroup O1 from other countries worldwide were included. Most isolates from Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30). Four Finnish isolates cross-reacted strongly with antisera against two new serogroups VaNT2 and VaNT4, whereas two strains were non-typeable. The serogroup O1 isolates displayed ten different ribotype patterns, whereas the other strains were considerably more diverse with respect to ribotypes. Most of the O1 isolates carried the 67 kb virulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid. Additional plasmids with molecular weights of 63, 76, 135 or 260-290 kb were found in single O1 isolates. With few exceptions, strains of serogroup O2 either had no plasmids or carried one or two small plasmids. PhenePlate typing revealed considerable diversity within the species, serogroup O1 being the most homogeneous. A few PhP types were dominant, whereas other types were observed only in one to four isolates. The prevalence of the different types changed significantly from one year to another but in Finland, one clonal lineage became increasingly important from 1992 (20% of isolates) to 1996 (80%). Remaining clones were mostly restricted to specific geographic areas. By cluster analysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmark belonged to two clusters, and most of the strains from Southern Europe fell into two other, distinct clusters. Most isolates from the UK, North America, Chile and Tasmania grouped together in a distinct cluster. For the typing of V. anguillarum, O-serotyping should be the primary method. For isolates belonging to serogroups other than O1, plasmid profiling in combination with ribotyping gives a very good discrimination between strains, whereas for serogroup O1, another method is required. It is concluded that PhP typing is a tool that provides a good discrimination between O1 isolates.     

Production of Cytokinin-like Substances by Cylindrocarpon destructans (Zins.) Scholt. Isolates Pathogenic and Nonpathogenic to Fir (Abies alba Mill.) seedlings

The production of cytokinin-like substances by different strains of Cylindrocarpon destructans either pathogenic or non pathogenic to fir (Abies alba Mill.) was studied. The cytokinins were extracted from the fungal culture media and separated by means of column chromatography. The biological activities in different fractions were assayed by callus growth of soybean (Glycine max Merill var. Acme). The amount of these substances was calculated on the basis of fresh weight of callus obtained from cultures containing authentic cytokinin. Cytokinin-like substances were detected in 5 of 6 pathogenic isolates tested and in 2 of 5 non-pathogenic isolates. On the basis of their occurrence in particular column fractions, most of the substances seem to be identical or similar to zeatin and zeatin riboside. In addition the fungi studied produced other, not identified cytokinin-like substances. The synthesis of Ri P can not be excluded. However the method applied did not allow to separate Ri P from zeatin and zeatin riboside.     

First report of <Emphasis Type="Italic">Verticillium tricorpus</Emphasis> isolated from potato tubers in Japan

In 1998, Verticillium sp. (CE98Vt1 and CE98Vt2) were isolated from discolored vascular structures of potato tubers sold at a market in Chiba Prefecture. These isolates were identified as Verticillium tricorpus on the basis of cultural and morphological characteristics and PCR diagnosis. This observed vascular discoloration of the potato tuber was demonstrated in three cultivars (Touya, Toyoshiro, and Waseshiro) among eight cultivars by inoculation to seedlings. External and internal symptoms of these isolates were not distinct in potato plants. The virulence of these isolates to potato was very low as compared with Verticillium dahliae. These two isolates were not pathogenic to Chinese cabbage, eggplant, green pepper, larkspur, parsley, snapdragon, soybean, tobacco, and tomato. This is the first report of V. tricorpus from potato in Japan.