Molecular and Pathogenic Variation Identified Among Isolates of <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Pathogenicity tests and inter simple sequence repeat (ISSR) molecular fingerprinting markers were utilized to analyze 24 Corynespora cassiicola isolates obtained from a lot of Hevea clones grown in most rubber nurseries and a few plantations in China. The C. cassiicola isolates were collected from Hainan and Yunnan provinces, China, from 2006 to 2008. The assay of 24 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypes PR 107, Dafeng 95, RRIM 600, and Reyan 7-33-97) indicated that 23 of the isolates were susceptible to RRIM 600, and were therefore considered race 1 except for CC-023. ISSR analysis grouped 24 C. cassiicola isolates into four clusters (A, B, C, and D). Unweighted pair-group method with arithmetic averaging (UPGMA) analysis based on Nei and Li's coefficient (calculated from the binary matrix data of 103 DNA fragments generated from 16 ISSR primers) indicated that cluster A included 19 isolates from Hainan and Yunnan (this cluster was further divided into two sub clusters (I, II), sub cluster II contained isolate CC-023); clusters B and C comprised of 1 isolates from Hainan, respectively; while cluster D encompassed 3 isolates from Hainan and Yunnan. Pathogenicity tests and ISSR analysis showed that there was no correlation between race structure, the geographical origin of the pathogen and their ISSR clusters because 23 of the isolates belonging to four distinct clusters were considered race 1 except for isolate CC-023. However, most of the isolates with different pathogenicity levels shared the same clades, and furthermore, the ISSR clusters and cology color had an exact correlation. These results should facilitate the development of rubber clones with enhanced resistance against all genetic clusters of C. cassiicola.     

Virulence Analysis and Oligonucleotide Fingerprinting to Detect Diversity Among Indian Isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis> Causing Chickpea Wilt

Virulence analysis of 64 isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from major chickpea growing states of India on 14 varieties, including 10 international differentials revealed that the isolates from each state were highly variable. Based on the reactions on international differentials, more than one race was found to be prevalent in every state. Majority of the isolates were not matched with the race specific reactions. Therefore, some of the cultivars, namely, GPF 2, DCP 92-3, and KWR 108 should be included as new differentials to obtain clear-cut differential responses. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and simple sequence repeat (SSR) markers were used to assess the genetic diversity of these isolates. Unweighted paired group method with arithmetic average (UPGMA) cluster analysis was used to divide the isolates into distinct clusters. The clusters generated by RAPD grouped all isolates into three categories at 25% genetic similarity and into two major categories at 30% genetic similarity. ISSR and SSR analyses also grouped all the isolates into two major categories. Majority of the isolates from Punjab and a few from Rajasthan were grouped in one category while the isolates from all other states were grouped in another suggesting the existence of diverse genetic populations of the pathogen at the same location. Some of the RAPD (OPM 6, OPI 9, P 17, OPN 4, OPF 1, P 17, P 21, and SC 1), ISSR (ISSR 7, ISSR 11, and ISSR 12) and SSR (MB 17) markers clearly distinguished area specific isolates.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

用RAPD和ISSR检测的橡胶树野生种质和栽培品种的遗传多样性

用19个RAPD引物和12个ISSR引物对14份野牛橡胶树种质和我国的37份栽培品种进行了遗传多样性分析。RAPD引物共产生132条带,多态性带占88．6％,相似系数变化范围在0．432—0．947。ISSR引物其产生101条带,多态性带占87．1％,相似系数为0．505—0．941。平均基因杂合度分析表明野生种质比栽培品种具有较高的遗传多样性。根据UPGMA法对51份材料进行聚类分析,结果表明,ISSR分析中所有材料可分为2类：第一类为野生种质,第二类为栽培品种：而RAPD分析中野牛种质和栽培品种不能被分为明显的两人类。虽然ISSR和RAPD的聚类分析结果存在差异,但对两种方法进行的相关分析表明,他们之间仍存在极显著相关性,相关系数为0．574。品种PR107、热研217等一些栽培品种可以通过特异带在51份供试材料中被区分开。这些结果可以对橡胶树的育种上作起到一定的指导作用,同时RAPD和ISSR技术也是进行橡胶树品种鉴定和遗传多样性研究的有效手段。     

Analysis of genetic and virulence diversity of Cylindrocarpon liriodendri and C. macrodidymum associated with black foot disease of grapevine

Inter-simple sequence repeat (ISSR) analysis was used to investigate the genetic diversity of 87 Cylindrocarpon liriodendri and C. macrodidymum isolates, the causal agents of black foot disease of grapevine. The four ISSR primers (GT)₇, (CCA)₅, (CGA)₅ and (TCG)₅, were able to provide reproducible and polymorphic DNA fingerprint patterns and detected relevant genetic diversity in C. macrodidymum. The cluster analysis of ISSR data showed 21 different genotypes that were grouped in seven ISSR groups, from which two corresponded to C. liriodendri (G1 and G2) and five to C. macrodidymum (G3-G7). Nineteen isolates selected from the seven ISSR groups were inoculated in grapevine seedlings obtained from cv. ‘Tempranillo’. The pathogenicity tests detected virulence diversity in C. macrodidymum. The isolates belonging to ISSR groups G6 and G7 were significantly more virulent than the other C. macrodidymum and C. liriodendri isolates.     

Assessment of vegetative compatibility of race-2 tomato wilt isolates ofVerticillium dahliae in Japan

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.     

Intraspecific Diversity within Diaporthe Helianthi: Evidence from Rdna Intergenic Spacer (IGS) Sequence Analysis

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas.     

Laboratory evaluation of the virulence of Beauveria bassiana isolates to the sorghum shoot borer Chilo partellus Swinhoe (Lepidoptera: Pyralidae) and their characterization by RAPD-PCR

Beauveria bassiana has long been used as a mycopesticide. It has a wide host range; isolates have been reported to differ in host range and virulence to a given insect species. Identification of a molecular marker linked to a virulent phenotype to a target pest would be useful in screening for isolates effective against it. Twenty B. bassiana isolates were tested for their virulence to the second instar larvae of Chilo partellus Swinhoe in laboratory bioassays and their DNA fingerprints were generated by RAPD-PCR. Three arbitrary categories of aggressiveness were chosen; isolates that caused >70%, between 70 and 40% and <40% larval mortality were grouped as highly, medium and less aggressive types, respectively. In the random amplified polymorphic DNA (RAPD) analysis a 30% variability was observed among the isolates; which clustered into three major groups. The groups based on virulence rating did not match with the RAPD clusters. One of the highly aggressive isolates clustered with less aggressive isolates in one cluster and the other grouped along with the medium aggressive isolates in a different cluster. The B. bassiana isolates were classified phenotypically based on the taxonomic order of the original insect host and the climatic zone (tropical/temperate) from which they were isolated. No correlation between the aggressiveness of the isolate and the relatedness of the original insect host to the tested insect was observed; both the highly aggressive isolates were from coleopteran insects. A correlation was found between the RAPD grouping and the phenotypic classification of the isolates. All the lepidopteran isolates grouped into one major cluster, most sub clusters were constituted by isolates from the same climatic zone.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.     

Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa

Studies were conducted tosearch for fungal strains with potentialpathogenicity against Diabrotica speciosa(Germar) (Coleoptera: Chrysomelidae).Among sixteen fungal isolates screenedthe most virulent was a Beauveria bassiana(Balsamo) Vuillemin isolate (FHD13) thatcaused 70% mortality of D. speciosathird instar larvae. The LC₅₀ value ofB. bassiana isolate FHD13 was3.48 × 10¹⁰ conidia/ml.Different temperatures (4, 17 and 26 °C)and vegetable oils (corn, sunflower and canola)used for storage did not significantly affectviability of conidia. A pathogenicity trialagainst D. speciosa larvae performed withthe corn oil formulation (1 × 10⁸ conidia/mlof oil) caused 65% of mortality.     

A comparative study of Phytophthora meadii isolates from rubber (Hevea brasiliensis) plantations in Sri Lanka

Thirty-three Phytophthora meadii isolates were obtained from different Hevea brasiliensisclones grown in different climatic regions in Sri Lanka. Growth, morphology and pathogenicity of the isolates were compared. Growth and pathogenicity levels varied among the isolates. Some isolates obtained from moderately susceptible rubber clones were highly pathogenic, compared to isolates obtained from resistant clones. Highly pathogenic isolates produced a higher number of sporangia on agar at 27 ± 2°C. It was impossible to group isolates according to the clone or the region from where they were obtained.This revised version was published online in October 2005 with corrections to the Cover Date.     

Population structure ofSclerotium rolfsii in peanut fields

Sclerotium rolfsii isolates from peanut fields in Ibaraki were classified into mycelial compatibility groups (MCGs) based on the barrage zone formation. A total of 132 isolates collected from four fields within a 120 m radius in 1994 comprised four MCGs; MCG A (71 isolates), B (34 isolates), C (26 isolates) and D (one isolate). Fields 1 and 2 were occupied exclusively by MCG A. MCG A also predominated in field 3. In field 4, MCGs A, B and C were dominant. Population structure in 3 additional fields was determined in 1997. All 11 isolates from Field 5, which was 400 m distant from field 1, belonged to MCG C. A total of 42 isolates from fields 6 and 7, 2.5 km distant from other fields and 100 m distant from each other, were all MCG A. These results suggested that the population structure ofS. rolfsii was simple. RAPD fingerprintings showed that most isolates of the same MCG were clonal.     

Heterokaryons and parasexual recombinants of Cryphonectria parasitica in two clonal populations in southeastern Europe

Evidence for parasexuality in natural populations of haploid fungi requires the demonstration of diploids or heterokaryons and recombinant genotypes in the absence of sex. We studied clonal populations of the chestnut blight fungus, Cryphonectria parasitica, in southeastern Europe and found evidence of parasexuality in two locations. In Osoj, Macedonia, we found one isolate (Os05-66) that had two alleles at six codominant loci, giving a haplotype that was a composite of two clones in this population. Six single-conidial isolates from Os05-66 had two alleles at some loci, suggesting partial diploidy or aneuploidy, and we found four recombinant haplotypes among single-conidial isolates from hyphal-tip isolates of the same isolate. In Teano, Italy, we found two heterokaryon isolates that were partial composites of two dominant clones. Single-conidial isolates from hyphal-tip isolates had recombinant haplotypes. These results provide evidence that is consistent with the hypothesis of parasexuality in C. parasitica in Europe, similar to an earlier report in a natural population in the USA.     

The molecular diversity of different isolates of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Bals.) Vuill. as assessed using intermicrosatellites (ISSR<Subscript>s</Subscript>)

Inter-microsatellite PCR (ISSR-PCR) markers were used to identify and to examine the genetic diversity of eleven Beauveria bassiana isolates with different geographic origins. The variability and the phylogenetic relationships between the eleven strains were analyzed using 172 ISSR-PCR markers. A high level of polymorphism (near 80%) was found using these molecular markers. Seven different isolates showed exclusive bands, and ISSR primer 873 was able to distinguish between all the strains. The dendrogram obtained with these markers is robust and in agreement with the geographical origins of the strains. All the isolates from the Caribbean region were grouped together in a cluster, while the other isolates grouped in the other cluster. The similarity exhibited between the two clusters was less than 50%. This value of homology shows the high genetic variability detected between the isolates from the Caribbean region and the other isolates. ISSR-PCR markers provide a quick, reliable and highly informative system for DNA fingerprinting, and allowed the identification of the different B. bassiana isolates studied.     

Electrophoretic karyotypes of <Emphasis Type="Italic">C. neoformans</Emphasis> serotype A recovered from Thai Patients with AIDS

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)₄. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.     

Intra-isolate Heterogeneity and Reproducibility of PCR-based Genotyping of Cryptosporidium parvum Using the β-tubulin Gene

Cryptosporidium parvum is a common contaminant in surface waters and presents significant problems for the water industry, public health and agriculture. Consequently, ascertaining the contaminating source of waterborne oocysts is of paramount importance. Based on currently available information, isolates of C. parvum can be differentiated into at least two genotypes using polymorphic genetic markers: genotype 1, to date isolated almost exclusively from humans, and genotype 2 isolates from humans and many other animals. Differentiation into these two genotypes has been based on either restriction fragment length polymorphisms or sequencing of PCR amplified gene fragments. The objective of this study was to evaluate the reproducibility of genotyping methods using a single isolate of C. parvum. A 620 bp fragment of the C. parvum -tubulin gene, generated by PCR from multiple aliquots of a single preparation of oocysts of the Iowa isolate, was sequenced. Significant sequence heterogeneity was detected within this single isolate; there was more sequence variation between clones originating from the Iowa isolate (up to 0.9 %) than between individual clones originating from different isolates of C. parvum. Over 6 % of the -tubulin gene sequence positions (38 out of 620 bp) were variable when comparing multiple clones from the one isolate. The results indicated that while the various procedures used for genotyping isolates may introduce some sequence errors, the Iowa isolate used for this investigation appeared to be composed of multiple sub-genotypes. While none of the sequence variations resulted in clones of the Iowa isolate (genotype 2) being mis-identified as genotype 1, the results have important implications if minor sequence variations are to be used for subtyping isolates and drawing conclusions regarding the origin of, or relationships between, C. parvum oocysts in water and the community.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.