Isolates Of Pathotype Research Articles

Characterization of a Gene Identified in Pathotype 5 of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams' differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.

Genetic variability of Potato virus Y (PVY) and Tobacco etch virus (TEV) from naturally infected pepper fields in the Hatay region of Turkey

The genetic structure of Potato virus Y (PVY) and Tobacco etch virus (TEV) (Potyvirus) populations was investigated in pepper fields in two regions in Turkey. The diversity of PVY and TEV populations according to coat protein (CP) and VPg coding regions showed some similarity. All the isolates built a monophyletic group due to a single introduction event or multiple introductions of genetically similar isolates. All the isolates of both viruses showed evidence to the diversification for a long time. Based on VPg and CP sequences, all PVY isolates corresponded to clade C1. Turkish potyvirus isolates were only able to break the pvr21 resistance allele and therefore belonged to pathotype (0, 1). The Pvr4 dominant gene was found to be efficient and durable against PVY but not at all efficient against TEV. Consequently, the pvr22 resistance allele, efficient resistance against PVY and TEV pathotype (0, 1) isolates, would be the most suitable strategy to control potyviruses.

Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin. The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ∼10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates. Some of these mutations could explain their novel virulence phenotypes.

Studies on resistance phenotypes to Turnip mosaic virus in five species of Brassicaceae, and identification of a virus resistance gene in Brassica juncea

Studies were done to: (i) establish the occurrence of different types of Turnip mosaic virus (TuMV) resistance phenotypes in 69 Brassica juncea, 11 Camelina sativa, 11 B. oleracea, five B. rapa and three Raphanus sativus lines, and (ii) characterise a TuMV resistance gene in B. juncea. An isolate of TuMV pathotype 8 (WA-Ap1) was employed to inoculate plants, except in one instance where isolates of pathotypes 1 (isolate NSW-2) and 7 (isolate NSW-1) were also used. The resistance phenotypes found were O (extreme resistance), RN (localised hypersensitivity), R (resistance to systemic movement without necrosis), +N (systemic infection with some necrosis), +N 1 (mild variant of +N), +ND (systemic hypersensitivity and plant death), + (susceptibility), +st (severe stunting variant of +), RN/+ (systemic infection with necrosis limited to inoculated leaves), and RN/st/+ (severe variant of RN/+). Seven different resistance or susceptibility phenotypes were found in B. juncea (codes: +N, RN/+, RN/st/+, +ND, +N 1, + and +st), 22 lines developing only one phenotype and the remaining 47 segregating for 2–3 different phenotypes. Ten B. oleracea cultivars developed phenotype O alone, and one segregated for phenotypes R and O. One B. rapa cultivar produced uniform phenotype +, but four segregated for phenotypes +, RN, O, RN/+, +N, or +st. Two R. sativus cultivars developed phenotype O alone, and one segregated for phenotypes O and RN. All 11 C. sativa lines developed uniform phenotype +ND. Increasing temperature from 16 to 28 °C decreased the time lag between inoculation and symptom development without altering overall phenotypic responses in B. juncea, B. oleracea, B. rapa and C. sativa plants. When one line each of these four species were inoculated with two other TuMV isolates, NSW-1 and NSW-2 belonging to pathotypes 7 and 1 respectively, phenotypic responses remained the same in the B. juncea, B. oleracea and B. rapa lines, but pathotype 8 caused a different phenotypic response in C. sativa. The genetics of resistance to TuMV was studied in a cross between two B. juncea parents with uniform phenotypes, JM 06006 (+) and Oasis Cl (+ND). The results of four tests on F2 progeny plants and three types of control plants (JM 06006, Oasis Cl and mock-inoculated F2 progeny plants) were analysed for their responses to inoculation with TuMV isolate WA-Ap1. Segregation of F2 progeny plants from B. juncea Oasis Cl X JM 06006 fitted a 3:1 ratio (systemic necrosis: susceptibility) at an early stage of TuMV infection, and a 1:2:1 ratio at a late stage of infection +ND: +N: +. These findings show that a single incompletely dominant resistance gene, designated here as TuRBJU 01 (TuMV resistance in B rassica ju ncea 01), was responsible for systemic necrosis phenotypes +ND (homozygous) and +N (heterozygous) in B. juncea. B. juncea resistance gene TuRBJU 01, along with lines of B. oleracea and R. sativus that developed phenotype O uniformly, and of C. sativa that developed phenotype +ND uniformly are potentially valuable in breeding TuMV-resistant cultivars of these three species.

Preliminary studies on resistance phenotypes to Turnip mosaic virus in Brassica napus and B. carinata from different continents and effects of temperature on their expression

Preliminary studies were undertaken to establish the occurrence of Turnip mosaic virus (TuMV) resistance phenotypes in 99 Brassica napus and 32 B. carinata accessions, breeding lines and cultivars mostly from Africa, Australia, the Indian sub-continent or China. An isolate of TuMV pathotype 8 (WA-Ap1) was used in most inoculations. The influence of temperature on expression of resistance phenotypes was determined. Those identified were O (extreme resistance), RN (localised hypersensitivity), R (resistance to systemic movement without necrosis), +N (systemic infection with some necrosis), + (susceptibility), and RN/+ (systemic infection with necrosis limited to inoculated leaves). In the initial glasshouse evaluations with B. napus, 18 lines developed phenotype O, 42 RN, two + (both from Australia), and 30 segregated for O and RN, two for R and RN, one for O and R, two for O, R and RN, one for O, RN and +N, and one for RN and +N. Phenotype +N only occurred in two lines from India. In the initial glasshouse evaluations with B. carinata, 14 lines developed O, two RN or R, and one +; the 13 remaining lines segregated for phenotypes O and R (12), or RN and R (1). In B. carinata, phenotype RN only occurred in African lines, and phenotype + only in a line from Pakistan. The 18 B. napus and 14 B. carinata lines that developed phenotype O uniformly were re-evaluated at low (16 and/or 18 °C) and high (25 °C) temperatures in the glasshouse three or two times, respectively, and again under controlled environment room conditions at 16 and at 28 °C. While in most lines phenotype O was replaced by other phenotypes or segregated with other phenotypes at the higher temperatures, it occurred uniformly regardless of temperature in Chinese B. napus line (06-6-3777) and three African B. carinata lines (IP 117, ST 18 and ST 50). Graft inoculations confirmed extreme resistance in these four lines. Other promising lines which displayed phenotype O in >75 % of inoculated plants included for B. napus Chinese lines 06-p71-1 and 06-p74-4 and French line Cresor, and for B. carinata African lines Mbeya Green, ML-EM-1, ML-EM-7 and ML-EM-8, and Australian lines P 195923.3 and 30200533. Five promising lines segregated for phenotype RN, B. napus Ding 110, Hyola 42, Fan 028, ZY 007 and Qu 1104. Chinese B. napus line Ding 110 developed phenotype RN uniformly at high (25 and 28 °C) temperatures. Thus, in plants developing phenotypes RN, R and +, low temperature retarded virus multiplication in inoculated leaves to below the level at which it can be identified using ELISA. No clear phenotypic differences attributable to TuMV pathotype were found in tests in which isolates from pathotypes 1, 7 and 8 were used. This highlights the need for future evaluations for TuMV resistance in Brassica germplasm to be done at higher temperatures. This study also highlights the need to identify additional genes responsible for the different resistance and susceptibility reactions found, especially in B. carinata. The four lines that developed phenotype O uniformly at all temperatures and also withstood graft-inoculation will be particularly valuable for developing new TuMV-resistant cultivars of oilseed and forage Brassicas.

Pathotype and genetic diversity amongst Indian isolates of Xanthomonas oryzae pv. oryzae.

A number of rice resistance genes, called Xa genes, have been identified that confer resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. An understanding of pathotype diversity within the target pathogen population is required for identifying the Xa genes that are to be deployed for development of resistant rice cultivars. Among 1024 isolates of Xoo collected from 20 different states of India, 11 major pathotypes were distinguished based on their reaction towards ten Xa genes (Xa1, Xa3, Xa4, xa5, Xa7, xa8, Xa10, Xa11, xa13, Xa21). Isolates belonging to pathotype III showing incompatible interaction towards xa8, xa13 and Xa21 and compatible interaction towards the rest of Xa genes formed the most frequent (41%) and widely distributed pathotype. The vast majority of the assayed Xoo isolates were incompatible with one or more Xa genes. Exceptionally, the isolates of pathotype XI were virulent on all Xa genes, but have restricted distribution. Considering the individual R-genes, Xa21 appeared as the most broadly effective, conferring resistance against 88 % of the isolates, followed in decreasing order by xa13 (84 %), xa8 (64 %), xa5 (30 %), Xa7 (17 %) and Xa4 (14 %). Fifty isolates representing all the eleven pathotypes were analyzed by southern hybridization to determine their genetic relatedness using the IS1112 repeat element of Xoo. Isolates belonging to pathotype XI were the most divergent. The results suggest that one RFLP haplotype that is widely distributed all over India and is represented in strains from five different pathotypes might be an ancestral haplotype. A rice line with xa5, xa13 and Xa21 resistance genes is resistant to all strains, including those belonging to pathotype XI. This three gene combination appears to be the most suitable Xa gene combination to be deployed in Indian rice cultivars.

Reaction of Lines of the Rapid Cycling Brassica Collection and Arabidopsis thaliana to Four Pathotypes of Plasmodiophora brassicae.

The clubroot reaction of five Rapid Cycling Brassica Collection (RCBC) lines (Brassica carinata, B. juncea, B. napus, B. oleracea, and B. rapa) and 84 lines of Arabidopsis thaliana to pathotypes 2, 3, 5, and 6 of Plasmodiophora brassicae (as classified on William's system) was assessed. Also, the reaction of the Arabidopsis lines to a single-spore isolate of each of pathotypes 3 and 6 was compared with that of a field isolate. Seedlings were inoculated with resting spores of P. brassicae, maintained at 25 and 20°C (day and night, respectively), and assessed for clubroot incidence and severity at 6 weeks after inoculation. Several lines of A. thaliana and RCBC exhibited a differential response to pathotype but none of the lines were immune. Among the RCBC lines, B. napus was resistant to all of the pathotypes; B. oleracea was resistant to pathotypes 2, 3, and 5; B. carinata and B. rapa were resistant to pathotypes 2 and 5; and B. juncea was susceptible to pathotypes 5 and 6 and had an intermediate response to pathotypes 2 and 3. Line Ct-1 of A. thaliana was highly resistant to pathotype 2, Pu2-23 was highly resistant to pathotype 5, and Ws-2 and Sorbo were highly resistant to pathotype 6. These results indicate that the lines of RCBC and A. thaliana have potential for use as model crops for a wide range of studies on clubroot, and could be used to differentiate these four pathotypes of P. brassicae. The reaction of the RCBC lines to pathotype 6 was highly correlated with response under field conditions but the reaction to the single-spore isolates of pathotypes 3 and 6 was not strongly correlated with reaction to the field collections in the Arabidopsis lines.

Variation in aggressiveness is detected among Puccinia triticina isolates of the same pathotype and clonal lineage in the adult plant stage

Puccinia triticina reproduces asexually in France and thus individual genotype is the unit of selection. A strong link has been observed between genotype identities (as assessed by microsatellite markers) and pathotypes (pools of individuals with the same combination of qualitative virulence factors). Here, we tested whether differences in quantitative traits of aggressiveness could be detected within those clonal lineages by comparing isolates of identical pathotype and microsatellite profile. Pairs of isolates belonging to different pathotypes were compared for their latent period, lesion size and spore production capacity on adult plants under greenhouse conditions, with a high number of replicates. Isolates of the same pathotype showed remarkably similar values for the measured traits, except in three situations: differences were obtained within two pathotypes for latent period and within one pathotype for sporulation capacity. One of these differences was tested again and confirmed. This indicates that the average aggressiveness level of a leaf rust pathotype may increase without any change in its virulence factors or microsatellite profile.

Effect of temperature on conidial germination, mycelial growth and aggressiveness of the defoliating and nondefoliating pathotypes of Verticillium dahliae from cotton in China

Three defoliating (D) pathotype isolates and five nondefoliating (ND) pathotype isolates of Verticillium dahliae from cotton in central China were tested for adaptation to various temperatures in conidial germination on water agar (10–33°C), mycelial growth on potato dextrose agar (10–33°C) and infection of cotton seedlings (Gossypium hirsutum cvs. ‘E Mian 24’ and ‘Yin Rui 361’) (25–33°C). Results showed that the D-pathotype isolates adapted better than the ND-pathotype isolates to 30°C for conidial germination and mycelial growth, although the isolates of the two pathotypes had the same optimum temperature of 25°C. Under day/night temperatures of 25/25, 27/27 and 30/25°C for 20 and 25 days, the D-pathotype isolates induced the defoliation syndrome on seedlings of the two upland cotton cultivars, whereas the ND-pathotype isolates did not induce defoliation syndrome. The values for the areas under the disease progress curve (AUDPC) and the vascular discoloration index (VDI) were higher for the D-pathotype isolates than those for the ND-pathotype isolates in each temperature treatment. Under 30/30 or 33/27°C, at least two of the three D-pathotype isolates still had higher AUDPC values and/or VDI values than all the ND-pathotype isolates on E Mian 24. Therefore, the D-pathotype isolates appear more aggressive than the ND-pathotype isolates in infection of cotton. Results also showed that the cotton cultivar Yin Rui 361 was more tolerant than the cotton cultivar E Mian 24 to infection by both pathotypes of V. dahliae. This study suggests that the D-pathotype isolates can well adapt to high temperature and heavily infect cotton under 25–30°C, and these features might be responsible for the rapid spread of this pathotype in central China.

Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper: support for the matching allele model in eIF4E resistance–potyvirus interactions

The pathogenic properties of Potato virus Y (PVY; genus Potyvirus, family Potyviridae) isolates collected in naturally infected pepper (Capsicum annuum) fields in Tunisia were evaluated against recessive resistance alleles at the pvr2 locus of pepper. Two pathotypes were observed. Pathotype (0,1,3) isolates were able to infect plants carrying the susceptibility allele pvr2+, together with pvr21/pvr21 and pvr23/pvr23 plants, but not pvr22/pvr22 plants. Pathotype (0) isolates were only able to infect pvr2+/pvr2+ plants. On the other hand, sequence data and phylogenetic analyses revealed three major groups of isolates, each characterized by particular amino acid residues in the central part of the VPg, the pathogenicity factor towards pvr2. Correspondence between pathogenic properties and phylogeny suggested a single evolutionary step for pathogenicity towards the pvr21 and pvr23 resistances, possibly under the selective pressure of pvr21. Indeed, 23% of the pepper plants in this area were shown to carry the pvr21 resistance, while pvr23 was not detected. The data suggested that pathogenicity towards pvr21 and pvr23 were not costly for PVY to infect susceptible pepper genotypes and supported the matching allele model for pepper–PVY interactions.

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.

Pathotypes and Genetic Relationship of Worldwide Collections of Elsinoë spp. Causing Scab Diseases of Citrus

Two scab diseases are recognized currently on citrus: citrus scab, caused by Elsinoë fawcettii, and sweet orange scab, caused by E. australis. Because the two species cannot be reliably distinguished by morphological or cultural characteristics, host range and molecular methods must be used to identify isolates. Four pathotypes of E. fawcettii and two of E. australis have been described to date based on host range. The host specificity and genetic relationships among 76 isolates from Argentina, Australia, Brazil, Korea, New Zealand, and the United States were investigated. Based on pathogenicity tests on eight differential hosts, 61 isolates were identified as E. fawcettii and 15 as E. australis. Of 61 isolates of E. fawcettii, 24 isolates were identified as the Florida broad host range (FBHR) pathotype, 7 as the Florida narrow host range (FNHR) pathotype, 10 as the Tryon's pathotype, and 3 as the "Lemon" pathotype. Two new pathotypes, the "Jingeul" and the satsuma, rough lemon, grape-fruit, clementine (SRGC), are described, and four isolates did not fit into any of the known pathotypes of E. fawcettii. Of the 15 isolates of E. australis from Argentina and Brazil, 9 belonged to the sweet orange pathotype and 6 from Korea to the natsudaidai pathotype. E. fawcettii and E. australis were clearly distinguishable among groups by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assays and the E. fawcettii group was divided into three subgroups, A-1, A-2, and A-3. The A-1 group was composed of the FBHR, FNHR, and SRGC pathotypes; some Lemon pathotypes; and the uncertain isolates. The A-2 subgroup included all of the Tryon's pathotype isolates and one of the three Lemon pathotype isolates and the A-3 group contained the Jingeul pathotype isolates. E. australis was differentiated into two groups: B-1, the natsudaidai pathotype isolates, and B-2, the sweet orange pathotype isolates. Isolates of E. fawcettii and E. australis were clearly distinguishable by sequence analysis of the internal transcribed spacer (ITS) region and the translation elongation factor 1 alpha (TEF) gene. There were also fixed nucleotide differences in the ITS and TEF genes that distinguished subgroups separated by RAPD-PCR within species. We confirmed two species of Elsinoë, two pathotypes of E. australis, and at least six pathotypes of E. fawcettii and described their distribution in the countries included in this study.

Function of Genes Encoding Acyl-CoA Synthetase and Enoyl-CoA Hydratase for Host-Selective ACT-Toxin Biosynthesis in the Tangerine Pathotype of Alternaria alternata

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.

Application of pathogen surveys, disease nurseries and varietal resistance characteristics in an IPM approach for the control of wheat yellow rust

The present paper presents the rationale for the use of pathogen surveys, inoculated and non-inoculated disease nurseries and varietal resistance characteristics in an integrated approach to control wheat yellow rust in Denmark. The non-inoculated disease observation plots, which gave valuable information about yellow rust at the year, site and variety level, served as the primary sample source for the pathogen survey revealing pathogen virulence dynamics. This survey was also the main source for isolates of new pathotypes, a prerequisite for the assessment of the resistance characteristics of varieties and breeding lines in inoculated nurseries, and the postulation of race-specific resistance genes. A simple grouping of varieties into four categories with respect to resistance to the current yellow rust population proved robust, and this grouping was used as a determinant in a web-based decision support system for pesticide applications in cereals, Crop Protection On-line (CPO). The interplay between the different research and survey activities in the integrated pest management (IPM) approach demonstrated the need for a coherent and long-term involvement at all stages from plant breeding to the official variety approval system, extension service and research in disease epidemiology and resistance genetics.

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of Verticillium dahliae

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)5, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.

Adaptation and competition ofCochliobolus sativusisolates on barley

The competitive and adaptive capacity of Cochliobolus sativus were determined using the two major pathotypes in populations on barley in Syria Pt1 and Pt4, which differed widely in their virulence. Following greenhouse co-inoculation with the two pathotypes, Pt4 frequency average increased from the first to the last of four infection cycles, but decreased for Pt1. The number of lesions caused by Pt4 was individually higher than their mixture on both barley cultivars, Bowman and WI 2291. Moreover, the number of lesions was increased over the four cycles on both cultivars. The results suggest that isolates of different pathotypes would adapt at different rates in a mixed environment due to competitive advantage imposed by their virulence properties.

Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus.

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers

ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.

A fitness cost for Turnip mosaic virus to overcome host resistance

The relative fitness of the Turnip mosaic virus (TuMV) isolate UK 1 was compared with that of two other wildtype isolates CZE 1 and CDN 1. The isolates CZE 1 and CDN 1 are able to overcome the effect of the resistance gene TuRB01 and at least three other resistance sources that are effective against UK 1. Comparisons were also made between the fitness of UK 1 and a recombinant virus with a single nucleotide change (v35Tunos +5570 A>G) conferring the ability to overcome TuRB01 resistance. Co-inoculation experiments were carried out where pairs of isolates were serially passaged over 5 months in a plant line possessing no known resistance genes in order to examine the relative fitness of the isolates. In each case, UK 1 dominated the mixture in the susceptible host background. It out-competed CZE 1 and v35Tunos +5570 A>G within four passages, and CDN 1 after one passage. The greater fitness of UK 1 suggests that there may be a fitness cost to TuMV overcoming resistance genes of brassica crops. This may shed some light on the frequency of naturally occurring isolates, in that pathotype 1 isolates are found much more frequently than isolates of other pathotypes. Implications for the deployment of TuRB01 are discussed.

Specific Detection of Tomato Pathotype of Verticillium dahliae by PCR Assays

Klebahn is the causal agent of tomato wilt disease. Isolates of V. dahliae can be classified based on pathogenicity to tomato, but the pathotypes are indistinguishable in morphology. We designed PCR primers for specific detection of isolates pathogenic to tomato (tomato pathotype) from the sequences of a pathotype-specific gene, vdt1. With the primer pair Tg5/Tc3, a PCR product (approximately 3.2 kb) specific to tomato pathotype was amplified from the genomic DNA of isolates. Using the primer pair, a tomato pathotype isolate was specifically detected from hypocotyls of inoculated tomato and eggplant. On the other hand, no amplification was observed from non-tomato pathotype isolates of V. dahliae, some other wilt pathogens of tomato and a healthy host plant. Therefore, the primer pair can be useful for pathotype-specific detection of V. dahliae as well as for diagnosis of wilt disease of tomato plant.