Microsatellite and Morphological Markers Reveal Genetic Variation within a Population of Sclerotinia sclerotiorum from Oilseed Rape in the Çanakkale Province of Turkey

The genetic variation among a population of Sclerotinia sclerotiorum collected from oilseed rape fields in the Çanakkale Province of Turkey was assessed using molecular and morphological markers. Seven microsatellite primer pairs (out of eight) revealed 32 clear polymorphic alleles among the 36 fungal isolates examined. An unweighted pair‐group mean analysis dendrogram was generated using the genetic distance matrix with the 32 microsatellite alleles. The level of similarity was as low as 15% between some isolates indicating a high level of genetic diversity within the fungal population; 23 distinct isolates were found (at a genotypic diversity level of 63%). Among the collection of 36 isolates, 19 mycelial compatibility groups (MCGs) were identified; 10 MCGs included at least two isolates. Molecular and morphological data suggest that most of the isolates within a single MCG were identical; however, the isolates belonging to the MCG2 and MCG4 had variable microsatellite haplotypes and were morphologically dissimilar. The data suggest that there is possibly a high rate of outcrossing as well as evolutionary potential within the population of the pathogen in oilseed rape fields. This is the first report demonstrating the genetic and morphological variation within a population of S. sclerotiorum in Turkey.     

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.     

Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Genetic Variation of Sclerotinia sclerotiorum from Multiple Crops in the North Central United States

Sclerotinia sclerotiorum is an important pathogen of numerous crops in the North Central region of the United States. The objective of this study was to examine the genetic diversity of 145 isolates of the pathogen from multiple hosts in the region. Mycelial compatibility groups (MCG) and microsatellite haplotypes were determined and analyzed for standard estimates of population genetic diversity and the importance of host and distance for genetic variation was examined. MCG tests indicated there were 49 different MCGs in the population and 52 unique microsatellite haplotypes were identified. There was an association between MCG and haplotype such that isolates belonging to the same MCG either shared identical haplotypes or differed at no more than 2 of the 12 polymorphic loci. For the majority of isolates, there was a one-to-one correspondence between MCG and haplotype. Eleven MCGs shared haplotypes. A single haplotype was found to be prevalent throughout the region. The majority of genetic variation in the isolate collection was found within rather than among host crops, suggesting little genetic divergence of S. sclerotiorum among hosts. There was only weak evidence of isolation by distance. Pairwise population comparisons among isolates from canola, dry bean, soybean and sunflower suggested that gene flow between host-populations is more common for some crops than others. Analysis of linkage disequilibrium in the isolates from the four major crops indicated primarily clonal reproduction, but also evidence of genetic recombination for isolates from canola and sunflower. Accordingly, genetic diversity was highest for populations from canola and sunflower. Distribution of microsatellite haplotypes across the study region strongly suggest that specific haplotypes of S. sclerotiorum are often found on multiple crops, movement of individual haplotypes among crops is common and host identity is not a barrier to gene flow for S. sclerotiorum in the north central United States.     

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.     

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.     

Use of RAPD and ISSR Markers in Detection of Genetic Variation and Population Structure among Fusarium oxysporum f. sp. ciceris Isolates on Chickpea in Turkey

Genetic variation among the isolates of Fusarium oxysporum f. sp. ciceris, the causal agent of chickpea wilt worldwide, was analysed using pathogenicity tests and molecular markers – random amplified polymorphic DNA (RAPD) and inter‐simple sequence repeat (ISSR) polymorphism. Hundred and eight isolates were obtained from diseased chickpea plants in 13 different provinces of Turkey, out of which 74 isolates were assessed using 30 arbitrary decamer primers and 20 ISSR primers. Unweighted pair‐grouped method by arithmetic average cluster analysis of RAPD, ISSR and RAPD + ISSR datasets provided a substantially similar discrimination among Turkish isolates and divided into three major groups. Group 1, 2 and 3 consisted of 41, 18 and 15 isolates, respectively. These methods revealed a considerable genetic variation among Turkish isolates, but no correlation with regard to the clustering of isolates from different geographic regions. Analysis of molecular variance confirmed that most genetic variability resulted from the differences among isolates within regions. Our results also indicated that the low‐genetic differentiation (F_ST) and high gene flow (Nm) among populations had a significant effect on the emergence and evolutionary development of F. oxysporum f. sp. ciceris. This is the first report on genetic diversity and population structure of F. oxysporum isolates on chickpea in Turkey.     

Characterization of Colletotrichum lindemuthianum Isolates from the State of Minas Gerais, Brazil

Anthracnose disease of common bean (Phaseolus vulgaris), caused by Colletotrichum lindemuthianum, is responsible for extensive yield losses worldwide. This pathogen is known to vary greatly in its pathogenicity. Control strategies include chemical control and, mainly, the development of resistant cultivars, taking into account the population structure of C. lindemuthianum. The objective of this study was to investigate the pathogenic and genetic diversity and population structure among C. lindemuthianum isolates collected in Minas Gerais state, Brazil. When these isolates were inoculated on 12 differential cultivars, a total of 10 races were identified within a series of 48 isolates collected in Minas Gerais, Brazil. Races 65, 81 and 73 were the most frequent races and occurred in most of the regions. This study also detected race 337, which had not been reported previously in the literature. Random amplified polymorphic DNA (RAPD) analysis performed on the same 48 isolates revealed great genetic diversity, clustering the series into five groups at a maximum similarity value of 89.6%. There was no clear relationship between the loci sampled by RAPD markers and the pathogenic characterization. Analysis of molecular variance showed that 96.06% of the variability was contained within regions and 3.94% among regions, indicating a high exchange of genetic material among the regions of the State. Most of the variability was detected within races (75.24%). The pathogenicity and RAPD assays corroborated the broad genetic diversity of the pathogen and the results have been useful in breeding for resistance to anthracnose.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

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 Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

Mycelial compatibility in Valsa malicola,the causal agent of canker disease in Iran

Mycelial compatibility of 62 isolates of Valsa malicola from different hosts and areas of Iran were investigated on potato dextrose agar (PDA) and oat meal agar (OMA). Four mycelial compatibility groups (MCGs) on PDA, 1–4, including three single membered (1–3) and a 58 membered (4) were identified. However, 8 MCGs, 1–8, consisting of 6 single membered (1–3, 5, 6 and 8), a 6 membered (4) and a 50 membered (7) were identified on OMA. On PDA, the number of groups and the time to achieve results were less than on OMA as well as the barrage zones were clearer on PDA than OMA. There was no correlation between groups and host or geographical origins of the isolates. The low number of identified MCGs on both culture media revealed low genetic diversity of investigated isolates of V. malicola.     

Infra-specific diversity of Colletotrichum truncatum associated with chilli anthracnose in India based on microsatellite marker analysis

Colletotrichum truncatum is a fungal species associated with anthracnose disease in many economically important crops within the plant families Fabaceae and Solanaceae. Understanding the degree of genetic diversity within C. truncatum population will provide insights into the ability of this species to evolve in response to environmental conditions, and thus be helpful in designing effective control strategies for this pathogen. In this study, microsatellite markers from 27 loci were used to investigate the genetic diversity and population structure among 99 isolates of C. truncatum from India. All the loci (100%) were polymorphic and a total of 140 different alleles were amplified. Six distinct groups were obtained based on unweighted pair group method with arithmetical average cluster analysis. The isolates belonging to Group V showed the highest level of genetic diversity and a broad host range. Analysis of molecular variance analysis showed that the variation occurs mostly within groups. Microsatellite markers-based genetic diversity estimation revealed high diversity among C. truncatum isolates from India.     

Molecular diversity of Fusarium oxysporum causing rot diseases of vanilla in south India

Incidence of root, stem and beans rot of vanilla (Vanilla planifolia Andrews) caused by Fusarium oxysporum Schlecht was surveyed in vanilla growing areas of south India during December 2008. The incidence of the disease varied from 1 to 100% in different locations. A total of 60 isolates of F. oxysporum were obtained from diseased samples, and nine morphologically different isolates were taken for molecular characterization using Randomly Amplified Polymorphic DNA (RAPD) markers to study the genetic variability if any, among them. PCR amplification of total genomic DNA with random oligonucleotide primers generated unique banding patterns depending upon primers and isolates. Nine oligonucleotide primers were selected for the RAPD assays, which resulted in 384 bands for nine isolates of F. oxysporum. The number of bands obtained was entered into a NTSYS and the results showed that the variability among the pathogen isolates was moderate. The nine isolates studied were grouped into single major cluster at 0.66 similarity index. Hence, it is inferred that F. oxysporum infecting vanilla in south India consists of a single clonal lineage with a moderate level of genetic diversification.     

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.     

Biological and molecular variability of Sarocladium oryzae, the sheath rot pathogen of rice (Oryza sativa L.)

Sheath rot disease of rice caused by Sarocladium oryzae (Sawada) (=Acrocylindrium oryzae, Sawada) has become an important production constraint in all rice-growing countries. Pathogenicity, phytotoxic metabolites, and random amplified polymorphic DNA (RAPD) markers were used to assess the level of genetic variability of S. oryzae derived from rice cultivars, CR1018, IR36, and IR50, of different locations in North East and South India. Variability in pathogenicity, phytotoxic metabolite production, and DNA polymorphisms was detected among S. oryzae isolates. Results indicated that S. oryzae isolates produced both cerulenin and helvolic acid at concentrations 0.3–0.62 and 0.9–4.8 μg mL⁻¹ of culture filtrate, respectively. Isolates that produce higher concentration of helvolic acid induced a high percent incidence of sheath rot disease. Oligonucleotide primers, GF and MR, generated either a simple (up to 2 bands) or complex (up to 6 bands) RAPD pattern. According to their level of similarity, S. oryzae isolates from North East and South India were grouped separately into two major clusters and 13 genotypes. Molecular- and pathogenicity-based classifications were not correlated, but a high level of genetic variability within S. oryzae isolates was identified. The molecular variability of S. oryzae isolates will be an important consideration in breeding programs to develop durable resistance for sheath rot disease.     

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.     

Virulence and Molecular Diversity of Venturia inaequalis in Commercial Apple Growing Regions in Kashmir

Seventy‐one isolates of Venturia inaequalis collected from commercial apple growing areas of Kashmir were characterized on international differential apple hosts and analyzed by Random Amplified Polymorphic Microsatellites (RAMS), PCR–RFLP and sequencing of rDNA for elucidation of variability. Virulence analysis on a differential set categorized them into four pathogenic races, viz. (0), (1), (2) and (1,2) in the first time comprehensive molecular analysis of this in India and especially from Jammu and Kashmir, a north‐western Himalayan state of India. Race groups (0), (1), (2) and (1,2) contained isolates from diverse areas without specificity to any geographical zone or region. Cluster analysis of the RAMS and PCR–RFLP revealed a high genotypic diversity within V. inaequalis isolates. Three major clusters were obtained and the isolates could not be categorized on the basis of either their geographical distribution or the cultivar from which they were isolated. amova analysis of pathogen populations at regional or race level revealed high diversity within the populations. Pairwise F_ST comparisons between the populations revealed less genetic differentiation, thereby indicating existence of frequent gene flow in Kashmir. The 24 rDNA sequences of V. inaequalis showed high haplotype diversity of 0.938 and 0.40 nucleotide diversity. Again clustering at regional or race level detected greater part of variability within groups than among groups, thereby indicating high diversity in V. inaequalis populations in Kashmir valley.     

Genotypic characteristics in populations of Sclerotinia sclerotiorum from New York State,USA

White mould, caused by the fungus Sclerotinia sclerotiorum, is one of the most destructive diseases of beans globally. In New York State, USA, white mould causes substantial losses in soybean, snap, dry and succulent baby lima beans, which are grown successively in intensive crop rotations. Management strategies for white mould in these crops are reliant upon the prophylactic use of fungicides. No complementary information on the genetic structure of the populations of S. sclerotiorum in New York State, USA is available. Twenty isolates of S. sclerotiorum were collected from symptomatic bean plants within each of 10 fields across New York State, USA in 2014. Eight microsatellite (SSR) markers were used to characterise the genotypic diversity of the hyphal‐tipped isolates. Twenty‐four multilocus genotypes (MLGs) were detected within the population but one MLG was most prevalent. Although STRUCTURE analysis identified two subpopulations, these subpopulations were not associated with geographic location, suggesting no spatial structure to the population. In addition, the pathogen populations were predominantly clonal, with some evidence of infrequent outcrossing. These findings may assist in understanding the durability of management strategies for white mould and support the selection of representative isolates for host resistance screening for pathogen populations in the sampling area.     

Comparative genetic analysis of quantitative traits in sunflower (Helianthus annuus L.)

One hundred and fifty F₂–F₃ families from a cross between two inbred sunflower lines FU and PAZ2 were used to map quantitative trait loci (QTL) for resistance to white rot (Sclerotinia sclerotiorum) attacks of terminal buds and capitula, and black stem (Phoma macdonaldii). A genetic linkage map of 18 linkage groups with 216 molecular markers spanning 1,937 cM was constructed. Disease resistances were measured in field experiments for S. sclerotiorum and under controlled conditions for P. macdonaldii. For resistance to S. sclerotiorum terminal bud attack, seven QTL were identified, each explaining less than 10% of phenotypic variance. For capitulum attack by this parasite, there were four QTL (each explaining up to 20% of variation) and for P. macdonaldii resistance, four QTL were identified, each having effects of up to 16%. The S. sclerotiorum capitulum resistance QTL were compared with those reported previously and it was concluded that resistance to this disease is governed by a considerable number of QTL, located on almost all the sunflower linkage groups.