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.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.     

Variation in Aggressiveness Components in the Hemileia vastatrix Population in Brazil

The Pucciniomycete fungus Hemileia vastatrix causes leaf rust on coffee trees. The pathogen is responsible for considerable yield losses in susceptible coffee cultivars if appropriate management strategies are not implemented. Rapid spread and epidemics of rust fungi are usually associated with the emergence of new races of the pathogen that overcome resistance or with the emergence of more aggressive populations of the pathogen. In Brazil, coffee production is dominated by susceptible cultivars of Coffea arabica and Coffea canephora. We assessed aggressiveness in 46 populations of H. vastatrix from Minas Gerais and Espírito Santo, two of the most important coffee‐producing states in Brazil. We observed a significant difference in the incubation period between the populations from Minas Gerais and Espírito Santo when 183 single‐pustule isolates were inoculated onto Catuaí Vermelho IAC 44, a susceptible C. arabica cultivar. Variation in aggressiveness components was observed between and within localities. Isolates with longer incubation periods also tended to have longer latent periods, although there was only a low correlation between these two aggressiveness components (r² = 0.34, P = 2.2 × 10^?16). Low‐sporulating isolates also had significantly longer incubation and latent periods. The H. vastatrix population from Minas Gerais and Espírito Santo is structured by the formation of groups of individuals with differential level of aggressiveness. Our results indicate that the variation in aggressiveness of the Brazilian H. vastatrix population may be associated with the geographic coffee‐producing areas.     

Sources of Resistance to Anthracnose in Traditional Common Bean Cultivars from Paraná, Brazil

Pathogenicity of physiologically distinct races of Colletotrichum lindemuthianum originating from Andean (races 7, 19 and 55) and Mesoamerican (races 9, 31, 65, 69, 73, 81, 89, 95 and 453) locations of the new world were evaluated on 26 landrace genotypes of common bean (Phaseolus vulgaris L.) from Paraná State, Brazil. Races 7 (Andean), 65, 73 and 89 (Mesoamerican) were the most pathogenic, while race 31 (Mesoamerican) was the least pathogenic. Most of the landrace genotypes evaluated (88%) were resistant to race 31, except Carioca 3, Preto 1 and Preto 2. In addition, about 50% of the landrace genotypes had resistance to races 9, 19, 55 and 453; and about 30% to races 7, 65, 69, 73, 81, 89 and 95. The resistance index, which measured the pathogenicity response averaged across all the physiologically distinct Andean and Mesoamerican races of C. lindemuthianum, of the landrace genotypes ranged from 8% to 83%. The most resistant cultivars were Carioca Pintado 1, Carioca Pintado 2, Jalo Vermelho and Jalo de Listras Pretas. In contrast, the most susceptible cultivars were Jalo Pardo, Jalo Pintado 1 and Bolinha that showed resistance only to the least pathogenic race 31. These results indicated that many of the common bean landrace cultivars evaluated have genes that could be useful in breeding programmes to enhance resistance to Andean and Mesoamerican races of C. lindemuthianum.     

Population Structure of Puccinia recondita in Western Europe During 1995, as Assessed by Variability in Pathogenicity and Molecular Markers

The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty‐five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Vegetative compatibility and genetic analysis of Colletotrichum lindemuthianum isolates from Brazil

The causal agent of common bean anthracnose, Colletotrichum lindemuthianum, has considerable genetic and pathogenic variability, which makes the development of resistant cultivars difficult. We examined variability within and between Brazilian pathotypes of C. lindemuthianum through the identification of vegetative compatibility groups (VCGs) and by RAPD analysis. Two hundred and ninety-five nit mutants were obtained from 47 isolates of various pathotypes of the fungus collected from different regions, host cultivars and years. In complementation tests, 45 VCGs were identified. Eighteen RAPD primers were employed in the molecular analyses, producing 111 polymorphic bands. Estimates of genetic similarities, determined from the Sorence-Dice coefficient, ranged from 0.42 to 0.97; the dendrogram obtained by cluster analysis revealed 18 groups of isolates. RAPD and VCG markers presented high genotypic diversity. The number of significant associations (P=0.05) between RAPD, VCG and pathogenicity markers ranged from 0 (VCG) to 80% (pathogenicity). The test of multilocus association (rd) for RAPD markers was significantly different from zero (P<0.001), suggesting linkage disequilibrium. However, the results for VCG markers show the presence of recombination mechanisms. In conclusion, RAPD markers and VCGs were useful for detecting genetic variability among isolates of C. lindemuthianum. We found considerable diversity among isolates from the same geographic origin within a short interval; this suggests rapid evolution. There is a need for further studies to elucidate the population structure of this pathogen in agro-ecosystems.     

Virulence and RAPD data—A tool to study the evolutionary trends of Colletotrichum lindemuthianum virulences in the North Western Himalayan region of India

Abstract

Bean anthracnose pathogen (Colletotrichum lindemuthianum) known to display high pathogenic variability, also explains the existence of large number of races in Himachal Pradesh. An evolution model based on virulence data of 29 C. lindemuthianum races and RAPD patterns revealed the existence of four evolutionary groups (EG I – EG IV) in Himachal Pradesh, accommodating 12, 14, 2 and 1 races, respectively. Some races viz., 935, 643, 529, 647 and 613, opted more than two evolutionary routes and races like 598, 707, 935, 631, 639, 615, 115 and 119 harbouring more than six virulence genes may pose a threat to bean cultivation in this part of the world as they can break many resistance genes present in the locally grown beans. However, two exotic accessions G 2333 and AB 136 resistant to all the Indian pathotypes could be exploited as resistance donors in developing anthracnose resistant cultivars suitable for cultivation in this region.     

Pathogenic Variability and Genetic Diversity of Phaeoisariopsis griseola Isolates from Two Counties in the State of Goias, Brazil

Angular leaf spot disease of common bean (Phaseolus vulgaris), caused by Phaeoisariopsis griseola, is one of the most important disease of this crop in Brazil. Control strategies for the disease include cultural practices, chemical control and genetic resistance. This pathogen is known to vary greatly in pathogenicity. For durable use of genetic resistance to control this disease, it is necessary to manage resistant cultivars by taking into account the population structure of P. griseola. Isolates of the pathogen from Goias, Brazil exhibited an important virulence polymorphism when inoculated on 12 differential cultivars. A total of 13 pathotypes was identified within a series of 96 isolates collected in Inhumas and Damolandia counties. Only pathotypes 63‐15, 63‐23, 63‐31 and 63‐63 were identified in both counties. Since all the isolates were capable of inducing disease in both Andean and Mesoamerican differential cultivars, they were considered to be of Mesoamerican origin. Random amplified polymorphic DNA (RAPD) analysis performed on the same 96 isolates revealed a great genetic diversity clustering the series into five groups at an Euclidean distance of 62.5%. Although the results did not show any clustering according to the isolate origin, it was possible to observe a tendency of the isolates to cluster in different groups according to their origin. No pathotype‐specific band was observed in the present study.     

Genetic and Pathogenic Variation Among Tobacco Black Shank Strains of Phytophthora parasitica var. nicotianae from the Main Tobacco Growing in China

Pathogenic and genetic variability among seven populations of Phytophthora parasitica var. nicotianae from individual tobacco fields (Yunnan, Shandong, Henan, Heilongjiang, Shanxi, Fujian and Sichuan provinces) were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses; 63 strains were isolated from different fields of seven tobacco growing regions, using tobacco cv. Hongda as a baiting host. Pathogenic variability was evaluated in greenhouse studies using five tobacco cultivars that have different levels of resistance to tobacco black shank; 75 and 73% of the strains were pathogenic on M3 and M4, 29 and 33% on M1 and M2, and 94% were pathogenic on M5, respectively. Disease severity incited by different strains varied significantly on individual tobacco cultivars. The percentage of strains pathogenic on different cultivars varied among locations. Genotypic variation among 63 strains was evaluated by RAPD analysis. Ten primers detected 89 polymorphic bands. Cluster and principal coordinates analysed cluster groups. the minor group contained 26 strains, and major group contained 37 strains. Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.34 within populations to 0.36 among all strains from all populations. Phytophthora parasitica var. nicotianae populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the seven locations.     

Pathogenicity and its alternation with both morphological and genetic variation in Plasmopara halstedii (sunflower downy mildew)

Morphological, pathogenic and genetic variation was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of several races using five singlezoosporangium isolates per pathogen isolate. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Genetic relationships were detected between the single zoosporangium isolates using 12 expressed sequence tags (EST)-derived markers. Analysis of the five single zoosporangium isolates for P. halstedii isolates showed variability within pathogen isolates for all aggressiveness criteria, but not for all pathogen isolates. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than the isolate of races 7xx. All pathogen isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria. There was no intra-genetic variation for all pathogen isolates, but it was observed an important genetic variation between single zoosporangium isolates of all races. No correlation was detected between pathogenicity traits and EST genotypes.     

Development of new molecular markers for the <Emphasis Type="Italic">Colletotrichum</Emphasis> genus using <Emphasis Type="Italic">RetroCl1</Emphasis> sequences

A nonautonomous element of 624 bp, called RetroCl1 (Retroelement Colletotrichum lindemuthianum 1), was identified in the plant pathogenic fungus Colletotrichum lindemuthianum. RetroCl1 contains terminal direct repeats (223 bp) that are surrounded by CTAGT sequences. It has a short internal domain of 178 bp and shows characteristics of terminal-repeat retrotransposon in miniature (TRIM) family. We used RetroCl1 sequence to develop molecular markers for the Colletotrichum genus. IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) markers were used to analyze the genetic diversity of C. lindemuthianum. Fifty-four isolates belonging to different races were used. A total of 45 loci were amplified. The Nei index showed significant differences among the populations divided according to race, indicating that they are structured according to pathotype. No clear correlation between IRAP and REMAP markers with pathogenic characterization was found. C. lindemuthianum has high genetic diversity, and the analysis of molecular variance showed that 51% of variability is found among the populations of different races. The markers were also tested in different Colletotrichum species. In every case, multiple bands were amplified, indicating that these markers can be successfully used in different species belonging to the Colletotrichum genus.     

Genetic characterization of cassava (Manihot esculenta) landraces in Brazil assessed with simple sequence repeats

Based on nine microsatellite loci, the aim of this study was to appraise the genetic diversity of 42 cassava (Manihot esculenta) landraces from selected regions in Brazil, and examine how this variety is distributed according to origin in several municipalities in the states of Minas Gerais, São Paulo, Mato Grosso do Sul, Amazonas and Mato Grosso. High diversity values were found among the five above-mentioned regions, with 3.3 alleles per locus on an average, a high percentage of polymorphic loci varying from 88.8% to 100%, an average of 0.265 for observed heterozygosity and 0.570 for gene diversity. Most genetic diversity was concentrated within the regions themselves (H_S = 0.52). Cluster analysis and principal component based scatter plotting showed greater similarity among landraces from São Paulo, Mato Grosso do Sul and Amazonas, whereas those from Minas Gerais were clustered into a sub-group within this group. The plants from Mato Grosso, mostly collected in the municipality of General Carneiro, provided the highest differentiation. The migration of human populations is one among the possible reasons for this closer resemblance or greater disparity among plants from the various regions.     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.     

Pathogenic and molecular variation support the presence of genetically distinct clonal lineages in Australian populations of Puccinia graminis f. sp. avenae

Previous studies of the causal agent of stem rust of oats (Puccinia graminis f. sp. avenae; P. g. avenae) in Australia have demonstrated a high level of pathogenic variability. In this work, the pathotypic structure of the Australian P. g. avenae population in 1999 was investigated, as well as the pathotypic and genetic diversity of a collection of 26 Australian isolates representing a 25-year period (1971–1996). In the 1999 sample, 16 races belonging to six international standard races were identified from 97 isolates, with standard race 94 predominant in all regions. Race 94+Pg-13,Pg-Sa,Pg-a, detected in southern New South Wales (sNSW) and northern New South Wales (nNSW), was virulent on all of the differential genotypes used. Detailed analyses of pathogenicity and AFLP variability among 26 isolates collected from 1971–1996 revealed that isolates of standard race 94 collected in 1999 were genetically distinct from other Australian races of P. g. avenae. This evidence, along with data from annual pathogenicity surveys, suggests that the group to which standard race 94 belongs appeared during the late 1980s, and that it increased in frequency to dominate P. g. avenae pathogen populations throughout Australia from 1992 onward. The existence of groups of P. g. avenae isolates in Australia that differ in pathogenicity and AFLP phenotype suggests that current populations have evolved from a number of isolates of the fungus that differ in their genetic backgrounds, which may have originated from independent introductions or from asexual hybridisational events.     

Pathogenic Variability of Isolates of Pseudocercospora griseola, the Cause of Common Bean Angular Leaf Spot, and its Implications for Resistance Breeding

The pathogenic variability was evaluated of 48 Pseudocercospora griseola isolates collected in the State of Minas Gerais, Brazil. Isolates were inoculated to a set of 12 international differential cultivars in a greenhouse. Ten pathotypes (55-15, 63-7, 63-15, 63-23, 63-25, 63-27, 63-31, 63-47, 63-55 and 63-63) were identified, showing the great pathogenic variability of this fungus in Minas Gerais State. Pathotypes 55-15, 63-15, 63-25 and 63-27 had not previously been reported in the State. Of the 48 isolates, all except pathotype 55-1547 induced a compatible reaction with all cultivars from the Andean group. Isolates were highly pathogenic in both Andean and Mesoamerican cultivars, thus being classified as Mesoamerican pathotypes. Pathotype 63-63 was the most widespread, and overcame the resistance genes present in all differential cultivars.     

Morphological, Pathological and Molecular Variability in Colletotrichum capsici, the Cause of Fruit Rot of Chillies in the Subtropical Region of North-western India

Fruit rot of chillies (Capsicum annuum L.), caused by Colletotrichum capsici under tropical and subtropical conditions, results in qualitative and quantitative yield losses. Based on variation in cultural and morphological traits of C. capsici populations, 37 isolates were categorized into five groups designated, respectively, as Cc‐I, Cc‐II, Cc‐III, Cc‐IV and Cc‐V. In culture, most of the isolates produced cottony, fluffy or suppressed colonies. However, no significant differences were noticed in shape and size of conidia. The reaction of the 37 isolates on an indigenously developed differential set of Capsicum cultivars indicated the existence of different virulences in Himachal Pradesh (HP) chilli populations. Fifteen pathotypes of the pathogen were characterized from various chilli‐growing regions of HP. Pathotype CCP‐1 was most virulent and attacked all the differential cultivars. The genetic relationship between five morphological groups recognized within C. capsici was investigated using random amplified polymorphic DNA (RAPD) analysis. Molecular polymorphism generated by RAPD confirmed the variation in virulences of C. capsici and different isolates were grouped into five clusters. However, four isolates (Cc‐5, Cc‐33, Cc‐29 and Cc‐37) exhibited identical RAPD haplotypes. The pathological and RAPD grouping of isolates suggested no correlation among the test isolates.     

Genetic diversity of Puccinia kuehnii,the causal agent of orange rust of sugarcane,from Brazil

The use of resistant genotypes is the preferred method to control orange rust of sugarcane (Saccharum spp) caused by Puccinia kuehnii. This approach has been adopted in Brazil but outbreaks of the disease on previously resistant varieties showed that the efficacy of this method is limited and requires a better understanding of pathogen diversity. Nevertheless, adequate molecular markers for examining pathogen diversity at population level are not available, which limits the success of orange rust control by genetic resistance. Therefore, two independent investigations were conducted to examine genetic diversity of P. kuehnii from São Paulo state, the most important sugarcane growing state of Brazil. First, simple-sequence repeat (SSR) markers were developed in the present work and genotypic diversity of orange rust isolates from different locations investigated. Second, phenotypic diversity was examined by the single-pustule inoculation technique on P. kuehnii isolates retrieved from three susceptible commercial sugarcane cultivars. A total of 96 SSR markers were generated and tested for this species. Subsequently, 29 isolates of P. kuehnii were fingerprinted with nine SSR markers to estimate the genotypic diversity by neighbour-joining and 3D principal coordinates. The 29 isolates of the pathogen clustered into four main groups, which were identified by three SSR markers (NPRL_PK_108a, NPRL_PK_162_spka and NPRL_PK_221_spka). Phenotypic data at 21 days after the single-pustule inoculation showed that P. kuehnii from highly susceptible commercial cultivars harboured a small proportion of variants capable of causing disease on resistant cultivars. A differential reaction was demonstrated for the most virulent variant in a repeated experiment confirming the existence of races within P. kuehnii in Brazil.     

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.