Investigating variability and behaviour of Colletotrichum gloeosporioides strains from lesions of coffee blister spot

Coffee blister spot has been associated with species from the Colletotrichum genus, but there is no information on the variability of isolates present on leaf lesions. This study evaluated a population of Colletotrichum gloeosporioides strains from blister spot lesions in Coffea arabica. Colletotrichum spp. isolates were collected from blister spot lesions on leaves of coffee trees from Catuaí and Topázio cultivars (Coffea arabica). Monosporic cultures were obtained from colonies with sporulation. A pathogenicity test was carried out by inoculation of pathogens on the leaves of young coffee plants. C. gloeosporioides strains were characterized by morphologial, cytological and physiological analyses. The molecular analysis was carried out using Inter‐Retrotransposon Amplified Polymorphism (IRAP) markers. C. gloeosporioides strains showed no pathogenicity on coffee plants and presented a wide variability in all traits evaluated. The presence of sexual strains, formation of CATs (conidial anastomosis tubes) among conidial strains and high mycelial compatibility among strains observed suggest the occurrence of sexual and asexual recombination. The role of these C. gloeosporioides strains on the lesions of coffee plant leaves is unclear.     

Characterization of Colletotrichum kahawae Isolates Causing Coffee Berry Disease in Angola

Coffee Berry Disease, caused by Colletotrichum kahawae, is a major limitation for Arabica coffee cultivation in Africa and for which genetic control is only partially effective. As part of the effort to re‐launch coffee cultivation in Angola, our aim was to study the diversity of this pathogen and so contribute to more effective breeding for disease resistance. A collection of 30 C. kahawae isolates showed limited diversity in genetic and colony characters. However, some isolates are distinct, suggesting that breeding for disease resistance in Angola should be dependent on an adequate knowledge of the diversity of local and neighbouring C. kahawae isolates. Analysis of C. kahawae nrDNA nucleotide sequences showed distinct lineages clustering within the broad diversity of C. gloeosporioides, prompting further studies aimed at understanding the origin and pathogenic specialization of C. kahawae.     

Morphological and molecular identification of Colletotrichum species associated with cassava anthracnose in Thailand

The objective of this study was to identify the causal agent of anthracnose disease of cassava in Thailand. The study was carried out by collecting cassava samples with anthracnose symptoms from various planting areas including 10 districts of eight provinces in Thailand. One hundred and thirty‐six Colletotrichum samples were isolated from cassava anthracnose lesions on leaves, petioles and stems. Thirty‐eight single‐spore isolates were subsequently obtained and cultured on half potato dextrose agar for morphological and molecular characterizations. All 38 isolates were pathogenic with varying degrees of virulence when tested on detached leaves of Kasetsart 50, a susceptible cassava cultivar. Based on their growth habit, colony morphology, conidial morphology and the internal transcribed spacer sequences similarity to that of Colletotrichum accessions in the GenBank, one isolate was identified as C. capsici, one as C. lindemuthianum, two as C. aeschynomene, four as C. boninense and 28 C. gloeosporioides species complex. Geographically, the cosmopolitan C. gloeosporioides species complex was found in all regions, but other species were found only in particular regions. This is, so far, the first report of Colletotrichum complex species associated with cassava anthracnose in Thailand.     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Differential Organ Distribution,Pathogenicity and Benomyl Sensitivity of Colletotrichum spp. from Blackberry Plants in Northern Colombia

Blackberry anthracnose, caused by Colletotrichum spp., is an important disease of cultivated blackberry in the world. In Colombia, it is the number one limiting factor for commercial production. This study was conducted to determine the species of Colletotrichum infecting blackberry plants as well as the organ distribution, pathogenicity and response to benomyl of the isolated strains. Sixty isolates from stems (n = 20), thorns (n = 20) and inflorescences (n = 20) were identified as Colletotrichum acutatum and Colletotrichum gloeosporioides by a species‐specific polymerase chain reaction (PCR). Both Colletotrichum species were found in the same plant but on different organs. Colletotrichum gloeosporioides species predominated in thorn lesions (n = 16) and C. acutatum in stems (n = 15) and inflorescence (n = 15). Pathogenicity assays on detached blackberry organs demonstrated differences between the two species with an average period of lesion development of 8.7 days for C. gloeosporioides and 10.3 days for C. acutatum. Wound inoculated organs had 90% disease development compared to 17.5% in non‐wounded. All C. acutatum isolates (n = 34) were benomyl tolerant, whereas C. gloeosporioides isolates (n = 26) were 30.7% sensitive and 69.2% moderately tolerant. Phylogenetic analysis with ITS sequences of a subset of 18 strains showed that strains classified as C. gloeosporioides had 100% identity to Colletotrichum kahawae, which belongs to the C. gloeosporioides species complex, whereas C. acutatum strains clustered into two different groups, with high similarity to the A2 and the A4 molecular groups. These data demonstrate for the first time the differential distribution of both species complexes in blackberry plant organs and further clarifies the taxonomy of the strains.     

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour‐clamped homogenous electric fields electrophoresis

Aims: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour‐clamped homogenous electric fields (CHEF). Methods and Results: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal‐related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. Conclusion: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. Significance and Impact of the Study: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.     

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.     

New insights into the evolutionary history of Plasmodium falciparum from mitochondrial genome sequence analyses of Indian isolates

Estimating genetic diversity and inferring the evolutionary history of Plasmodium falciparum could be helpful in understanding origin and spread of virulent and drug‐resistant forms of the malaria pathogen and therefore contribute to malaria control programme. Genetic diversity of the whole mitochondrial (mt) genome of P. falciparum sampled across the major distribution ranges had been reported, but no Indian P. falciparum isolate had been analysed so far, even though India is highly endemic to P. falciparum malaria. We have sequenced the whole mt genome of 44 Indian field isolates and utilized published data set of 96 genome sequences to present global genetic diversity and to revisit the evolutionary history of P. falciparum. Indian P. falciparum presents high genetic diversity with several characteristics of ancestral populations and shares many of the genetic features with African and to some extent Papua New Guinean (PNG) isolates. Similar to African isolates, Indian P. falciparum populations have maintained high effective population size and undergone rapid expansion in the past with oldest time to the most recent common ancestor (TMRCA). Interestingly, one of the four single nucleotide polymorphisms (SNPs) that differentiates P. falciparum from P. falciparum‐like isolates (infecting non‐human primates in Africa) was found to be segregating in five Indian P. falciparum isolates. This SNP was in tight linkage with other two novel SNPs that were found exclusively in these five Indian isolates. The results on the mt genome sequence analyses of Indian isolates on the whole add to the current understanding on the evolutionary history of P. falciparum.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

Neofusicoccum parvum and Diaporthe foeniculina associated with twig and shoot blight and branch canker of citrus in Greece

In a survey performed in Chania and Aetoloacarnania, Greece in years 2013–2014, fungal isolates causing twig and shoot blight and branch canker of citrus trees were morphologically characterized and identified by multiple gene sequence analysis. By sequencing the ITS‐5.8S rRNA, the elongation factor 1‐α (EF1‐α), the β‐tubulin and the RNA polymerase II subunit (Rpb2) genes, the isolates examined were associated with Diaporthe foeniculina (six isolates) and Neofusicoccum parvum (one isolate). All six D. foeniculina isolates showed slow colony growth rates (7.4 ± 3.2 mm/day), while the N. parvum isolate exhibited fast growth (41.6 mm/day). Koch's criteria were met after re‐isolation of D. foeniculina isolates from all inoculated Citrus spp. and N. parvum from inoculated C. reticulata “Ortanique” and after having developed symptoms similar to those detected on shoots and branches collected from citrus fields. Based on lesion length on detached C. medica “Lia Kritis” shoots, N. parvum caused long necrotic lesions (58 mm in length) in comparison with a length of 12–21 mm lesions caused by D. foeniculina isolates. Pathogenicity trials on nine Citrus spp., which had been inoculated with D. foeniculina and N. parvum, revealed different levels of susceptibility, indicating a host‐dependent infection effect, with Poncirus trifoliate × C. paradisi (“Citrumelo Swingle”) being the most resistant citrus genotype. Lack of host specificity suggests that their pathogen–host association could be attributed to ecological rather to co‐evolutionary factors. This work represents the first report, accompanied with pathogenicity tests, on botryosphaeriaceous and diaporthaceous pathogens associated with twig and shoot blight and branch canker of citrus in Greece.     

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole‐genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.     

Species diversity and phylogeography of Cornus kousa (Asian dogwood) captured by genomic and genic microsatellites

Cornus kousa (Asian dogwood), an East Asia native tree, is the most economically important species of the dogwood genus, owing to its desirable horticultural traits and ability to hybridize with North America‐native dogwoods. To assess the species genetic diversity and to better inform the ongoing and future breeding efforts, we assembled an herbarium and arboretum collection of 131 noncultivated C. kousa specimens. Genotyping and capillary electrophoresis analyses of our C. kousa collection with the newly developed genic and published nuclear genomic microsatellites permitted assessment of genetic diversity and evolutionary history of the species. Regardless of the microsatellite type used, the study yielded generally similar insights into the C. kousa diversity with subtle differences deriving from and underlining the marker used. The accrued evidence pointed to the species distinct genetic pools related to the plant country of origin. This can be helpful in the development of the commercial cultivars for this important ornamental crop with increased pyramided utility traits. Analyses of the C. kousa evolutionary history using the accrued genotyping datasets pointed to an unsampled ancestor population, possibly now extinct, as per the phylogeography of the region. To our knowledge, there are few studies utilizing the same gDNA collection to compare performance of genomic and genic microsatellites. This is the first detailed report on C. kousa species diversity and evolutionary history inference.     

Development of Pathogenicity and AFLP to Characterize Fusarium oxysporum f.sp. momordicae Isolates from Bitter Gourd in China

Bitter gourd (Momordica charantia L.) cultivated in China is regarded as an important vegetable crop and is of considerable economic importance. However, it is susceptible to fusarium wilt, which causes heavy economic losses. Forty‐eight isolates were isolated from diseased bitter gourd plants that displayed typical fusarium wilt symptoms. Based on the morphological features, the rDNA internal transcribed space (ITS) sequences, pathogenicity and host biotypes, all of the isolates tested were pathogenic to the susceptible bitter gourd plants species (cv. ‘Guinongke No. 2’) and were identified as Fusarium oxysporum f. sp. momordicae (FOM). Our results classified different isolates as slightly, moderately or highly virulent. Among the isolates tested, 43 isolates slightly infected bottle gourd (Lagenaria siceraria var. clavata), whereas they did not infect other species from the family Cucurbitaceae. Genetic diversity among 48 isolates was characterized using amplified fragment length polymorphism (AFLP) analysis. The number of bands amplified by each primer pairs ranged from 41 to 66, with sizes ranging from 200 to 500 bp. A total of 366 bands were observed, out of which 363 were polymorphic (99.14%). The Nei's genetic identity of the six geographical populations varied from 0.7362 to 0.9707. The mean Nei's gene diversity index (H = 0.2644) and the mean Shannon's information index (I = 0.4071) at species level were higher than ones at populations level, indicated that the variation within populations was greater than that among populations. The Nei's GST (0.5103) and gene flow (Nm = 0.4923) revealed that genetic differentiation was mainly among populations and few gene exchanges. The dendrogram obtained from AFLP marker showed that there was a good correlation between isolates from different geographical locations and their pathogenicity. The AFLP marker effectively distinguished the high virulent isolates from the less virulent isolates. The highly virulent isolates were distinctly separated in different clusters, which indicated a significantly high correlation with the geographical origin in the AFLP dendrogram. The pathogenicity and molecular marker analysis confirmed the presence of variation in virulence as well as genetic diversity among the FOM isolates studied.     

Genetic differentiation of Colletotrichum gloeosporioides and C. truncatum associated with Anthracnose disease of papaya (Carica papaya L.) and bell pepper (Capsium annuum L.) based on ITS PCR-RFLP fingerprinting

Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1–5.8S–ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment length polymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1–5.8S–ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.     

A novel approach to parasite population genetics: Experimental infection reveals geographic differentiation,recombination and host‐mediated population structure in Pasteuria ramosa,a bacterial parasite of Daphnia

The population structure of parasites is central to the ecology and evolution of host‐parasite systems. Here, we investigate the population genetics of Pasteuria ramosa, a bacterial parasite of Daphnia. We used natural P. ramosa spore banks from the sediments of two geographically well‐separated ponds to experimentally infect a panel of Daphnia magna host clones whose resistance phenotypes were previously known. In this way, we were able to assess the population structure of P. ramosa based on geography, host resistance phenotype and host genotype. Overall, genetic diversity of P. ramosa was high, and nearly all infected D. magna hosted more than one parasite haplotype. On the basis of the observation of recombinant haplotypes and relatively low levels of linkage disequilibrium, we conclude that P. ramosa engages in substantial recombination. Isolates were strongly differentiated by pond, indicating that gene flow is spatially restricted. Pasteuria ramosa isolates within one pond were segregated completely based on the resistance phenotype of the host—a result that, to our knowledge, has not been previously reported for a nonhuman parasite. To assess the comparability of experimental infections with natural P. ramosa isolates, we examined the population structure of naturally infected D. magna native to one of the two source ponds. We found that experimental and natural infections of the same host resistance phenotype from the same source pond were indistinguishable, indicating that experimental infections provide a means to representatively sample the diversity of P. ramosa while reducing the sampling bias often associated with studies of parasite epidemics. These results expand our knowledge of this model parasite, provide important context for the large existing body of research on this system and will guide the design of future studies of this host‐parasite system.     

Genetic variability of green citrus aphid populations from Tunisia,assessed by RAPD markers and mitochondrial DNA sequences

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova ) showed that the genetic structure was not associated with geographic location or year of collection (P = 0.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (P < 0.01). Multidimensional scaling analysis indicated that the distribution of genetic variability was significantly influenced by the host‐plant with no evidence of spatial differentiation. Based on 20 barcode sequences of the mitochondrial cytochrome‐c oxidase subunit I (COI) gene, we revealed the occurrence of two haplotypes in association with the host‐plant. Results reported here suggest the occurrence of a limited gene flow between A. spiraecola populations from Rosaceae and Rutaceae and, therefore, a possible host‐race status that could be considered in the development of an integrated controlling strategy.     

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.     

Genetic Diversity and Pathogenic Variability of Colletotrichum truncatum Causing Anthracnose of Pepper in Malaysia

Colletotrichum truncatum was initially described from pepper and has been reported to infect 180 host genera in 55 plant families worldwide. Samples were collected from pepper plants showing typical anthracnose symptoms. Diseased samples after isolation were identified as C. truncatum based on morphological characters and ITS‐rDNA and β‐tubulin sequence data. Intersimple sequence repeat (ISSR) markers were used to estimate genetic diversity in C. truncatum from Malaysia. A set of 3 ISSR primers revealed a total 26 allele from the amplified products. Cluster analysis with UPGMA method clustered C. truncatum isolates into two main groups, which differed with a distance of 0.64. However, the genetic diversity of C. truncatum isolates showed correlation between genetic and geographical distribution, but it failed to reveal a relationship between clustering and pathogenic variability. Phylogenetic analyses discriminated the C. truncatum isolates from other reference Colletotrichum species derived from GenBank. Among the morphological characters, shape, colour of colony and growth rate in culture were partially correlated with the ISSR and phylogenetic grouping. Pathogenicity tests revealed that C. truncatum isolates were causal agents for pepper anthracnose. In the cross‐inoculation assays, C. truncatum isolates were able to produce anthracnose symptoms on tomato, eggplant, onion, lettuce and cabbage. A pathogenicity and cross‐inoculation studies indicated the potential of C. truncatum for virulence and dominancy on plant resistance.     

Genetic diversity of <Emphasis Type="Italic">Rhynchosporium secalis</Emphasis> in Tunisia as revealed by pathotype,AFLP, and microsatellite analyses

Genetic variability among 122 Rhynchosporium secalis isolates collected from barley in three regions of Tunisia was investigated using host differentials, amplified fragment length polymorphism (AFLP), and microsatellite markers. The isolates were collected from a widely grown scald-susceptible barley cultivar Rihane and a range of local landrace cultivars in geographically distinct regions with different agroclimatic conditions. Pathotypic diversity (the proportion of unique pathotypes) was high in R. secalis populations from the high (100% diversity), moderate (95%), and low (100%) rainfall areas of Tunisia, and from both Rihane (which is the sole variety grown in the high rainfall region) and local landraces (which predominate in the low rainfall area). This may reflect a general adaptability for aggressiveness and suggests that the widely grown cultivar Rihane has exerted little or no selection pressure on the pathogen population since its release in 1983. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from Rihane, local landraces, and different agro-ecological zones (GD = 0.96–0.99). There was low genetic differentiation among pathogen populations from different host populations (G _ST ≤ 0.08, θ ≤ 0.12) and agro-ecological zones (G _ST ≤ 0.05, θ ≤ 0.04), which may be partly explained by gene flow due to the movement of infected stubble around the country. There was no correlation (r = 0.06, P = 0.39) between virulence phenotype and AFLP haplotype. A phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on host, pathotype, or agro-ecological region. The implications of these findings for R. secalis evolutionary potential and scald-resistance breeding in Tunisia are discussed.