Encarnación Pérez-Artés

Identification of Pathogenic Races 0, 1B/C, 5, And 6 Of Fusarium Oxysporum F. Sp. Ciceris With Random Amplified Polymorphic DNA (RAPD)

Ninety-nine isolates of Fusarium oxysporum f. sp. ciceris (Foc), representative of the two pathotypes (yellowing and wilt) and the eight races described (races 0, 1A, 1B/C, 2, 3, 4, 5, and 6), were used in this study. Sixty isolates were analyzed by the RAPD technique using DNA bulks for each race and 40 primers. Bands presumably specific for a DNA bulk were identified and this specificity was confirmed by further RAPD analysis of individual isolates in each DNA bulk. Primers OPI-09, OPI-18, OPF-06, OPF-10, and OPF-12 generated RAPD marker bands for races 0, 1B/C, 2, 3, 4, 5, and 6. The reliability and utility of this procedure was validated in ‘blind trials’ using 39 new Foc isolates. Ten of the 39 isolates had already been typed to race by pathogenicity tests and 29 were typed both by pathogenicity and RAPD testing in this study. In these ‘blind trials’, we assigned the 39 new isolates to a race solely on the basis of their RAPD haplotype. Thus, we concluded that Foc races 0, 1B/C, 5, and 6 can be characterized by the RAPD markers. Cluster analysis of the RAPD data set resulted in three clusters of isolates within Foc. The yellowing isolates were grouped in two distinct clusters which correspond to races 0 and 1B/C. The wilt isolates constitute a third cluster that included races 1A, 2, 3, 4, 5, and 6. These results provide a means of studying the distribution of Foc races, to assist in the early detection of introduced race(s) and to facilitate the efficient deployment of available host resistance.

Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses

Severe Verticillium wilt of cotton in southern Spain is associated with the spread of a highly virulent, defoliating (D) pathotype of Verticillium dahliae. Eleven of the D and 15 of a mildly virulent, nondefoliating (ND) pathotype were analyzed by random amplified polymorphic DNA (RAPD) using the polymerase chain reaction (PCR). Six of 21 primers tested generated pathotype-associated RAPD bands. Another 21 V. dahliae isolates were compared in blind trials both by RAPD-PCR using the six selected primers and pathogenicity tests on cotton cultivars. There was a 100% correlation between pathotype characterization by each method. Unweighted paired group method with arithmetic averages cluster analysis was used to divide the 47 V. dahliae isolates into two clusters that correlated with the D or ND pathotypes. There was more diversity among ND isolates than among D isolates, these latter isolates being almost identical. ND- and D-associated RAPD bands of 2.0 and 1.0 kb, respectively, were cloned, sequenced, and used to design specific primers for the D and ND pathotypes. These pathotype-associated RAPD bands were present only in the genome of the pathotype from which they were amplified, as shown by Southern hybridization. The specific primers amplified only one DNA band of the expected size, and in the correct pathotype, when used for PCR with high annealing temperature. These specific primers successfully characterized V. dahliae cotton isolates from China and California as to D or ND pathotypes, thus demonstrating the validity and wide applicability of the results.

Comparative Study of Genetic Diversity and Pathogenicity Among Populations of Verticillium Dahliae from Cotton in Spain and Israel

Genetic diversity and phenotypic diversity in Verticillium dahliae populations on cotton were studied among 62 isolates from Spain and 49 isolates from Israel, using vegetative compatibility grouping (VCG), virulence and molecular assays. In Spain, defoliating V. dahliae isolates (D pathotype) belong to VCG1, and non-defoliating isolates (ND) belong to VCG2A (often associated with tomato) and VCG4B (often associated with potato). The D pathotype was not identified in Israel. The ND pathotype in Israel is comprised of VCG2B and VCG4B. Isolates in VCG2B and VCG4B ranged in virulence from weakly virulent to highly virulent. The highly virulent isolates induced either partial defoliation or no defoliation. Virulence characteristics varied with inoculation method and cotton cultivar. Highly virulent isolates from Israel were as virulent as D isolates from Spain under conditions conducive to severe disease. The D pathotype is pathologically and genetically homogeneous, whereas the ND pathotype is heterogeneous with respect to virulence, VCG, and molecular markers based on single-primer RAPD and on PCR primer pairs.

Mating type, pathotype and RAPDs analysis inDidymella rabiei, the agent of ascochyta blight of chickpea

Eleven pathotype groups (A-K), including five not previously reported, ofDidymella rabiei (anamorphAscochyta rabiei), representing isolates of the pathogen from Ascochyta blight-affected chickpeas mainly from India, Pakistan, Spain and the USA, were characterized using 44 single-spore isolates tested against seven differential chickpea lines. Of 48 isolates tested for mating type, 58% belonged to MAT 1-1 and 42% to MAT 1-2. Thirty-nineD. rabiei isolates, as well as two isolates ofAscochyta pisi and six isolates of unrelated fungi, were analyzed using Randomly Amplified Polymorphic DNAs (RAPDs) employing five primers (P2 at 40°C, and OPA3, OPC1, OPC11 and OPC20 at 35°C). Computer cluster analysis (UPGMA / NTSYS-PC) detected a relatively low level of polymorphism among all theD. rabiei isolates, although atca 7% dissimilarity,ca 10 RAPD groups [I-X] were demarcated, as well as subclustering within the larger groups. By the same criteria, the maximum dissimilarity for the whole population ofD. rabiei isolates wasca 13%. No correlation was found between different RAPD groups, pathotype, or mating type ofD. rabiei, although some evidence of clustering based on geographic origin was detected. The use of RAPDs enabled us to identify specific DNA fragments that may have a potential use as genetic markers in sexual crosses, but none which could be used as virulence markers.

Vegetative compatibility of cotton-defoliating Verticillium dahliae in Israel and its pathogenicity to various crop plants

Verticillium dahliae isolates recovered from a new focus of severe Verticillium wilt of cotton in the northeast of Israel were tested for vegetative compatibility using nitrate non-utilizing (nit) mutants and identified as VCG1, which is a new record in Israel. Other cotton isolates of V. dahliae from the northern and southern parts of the country were assigned to VCG2B and VCG4B, respectively. VCG1 isolates induced severe leaf symptoms, stunting and defoliation of cotton cv. Acala SJ-2, and thus were characterized as the cotton-defoliating (D) pathotype, whereas isolates of VCG2B and VCG4B were confirmed as the earlier described defoliating-like (DL) and non-defoliating (ND) pathotypes, respectively. This is the first record of the D-pathotype in Israel. The host range of representative isolates of each VCG-associated pathotype was investigated using a number of cultivated plants. Overall, the D isolates were more virulent than DL isolates on all tested host plants, but the order of hosts (from highly susceptible to resistant) was the same: okra (Hibiscus esculentus local cultivar), cotton (Gossypium hirsutum cv. Acala SJ2), watermelon (Citrullus lanatus cv. Crimson Sweet), safflower (Carthamus tinctorius cv. PI 251264), sunflower (Helianthus annuum cv. 2053), eggplant (Solanum melongena cv. Black Beauty), and tomato (Lycopersicon esculentum cv. Rehovot 13). The pattern of virulence of ND isolates differed from that of D and DL isolates, so that the former were highly virulent on eggplant but mildly virulent on cotton. Tomato was resistant to all cotton V. dahliae isolates tested. RAPD and specific PCR assays confirmed that the D isolates from Israel were similar to those originating from other countries.

A Simple PCR-based Method for the Detection of the Chickpea-wilt Pathogen Fusarium oxysporum f.sp. ciceris in Artificial and Natural Soils

A fast and simple polymerase chain reaction method has been developed for the detection of Fusarium oxysporum f.sp. ciceris, the causal agent of Fusarium wilt of chickpea, in natural and artificial soils. The method involves the disruption of fungal biomass by grinding dry soil, using its abrasive properties, in the presence of skimmed milk powder. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction, the DNA is suitable for direct PCR amplification without a precipitation step. For the efficient detection of small amounts of DNA extracted from soil, a two step amplification with nested primers was used. The dilution step reduced the effect of Taq-polymerase inhibitors. The specificity of the amplification, and consequently the yield of specific product, was increased by the use of a modified ‘touch down’ process during the annealing step. The method has been applied to the specific detection of wilt-inducing isolates of the pathogen in a variety of natural and artificial soils. The amplification was improved by the use of increased concentrations of skimmed milk powder in soils with high organic or clay contents.

Identification of Pratylenchus thornei, the cereal and legume root-lesion nematode, based on SCAR-PCR and satellite DNA

Two different molecular tools for the diagnosis of the cereal and legume root-lesion nematode Pratylenchus thornei were developed. A randomly amplified DNA (RAPD) fragment specific to P. thornei was identified. After sequencing the fragment, longer primers were designed that complement the terminal sequences of the RAPD fragment, and this pair of specific primers was used to amplify the sequence-characterized amplified region (SCAR). Using the developed pair of SCAR primers, the SCAR fragment specific to P. thornei was easily amplified with DNA extracts obtained from different life stages of the nematode. The described SCAR-PCR-based assay has the potential to be optimized for routine practical diagnostic tests. In addition, the use of a species-specific satellite DNA sequence to distinguish P. thornei from other Pratylenchus spp. is discussed.

Molecular analysis of Spanish populations of Fusarium oxysporum f. sp. dianthi demonstrates a high genetic diversity and identifies virulence groups in races 1 and 2 of the pathogen

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi (Fod), is the most important carnation disease worldwide. The knowledge of the diversity of the soil population of the pathogen is essential for the choice of suitable resistant cultivars. We examined the genetic diversity of Fod isolates collected during the period 1998–2008, originating from soils and carnation plants in the most important growing areas in Spain. Additionally, we have included some Fod isolates from Italy as a reference. Random amplified polymorphic DNA (RAPD) fragments generated by single-primer PCR were used to compare the relationship between isolates. UPGMA analysis of the RAPD data separated Fod isolates into three clusters (A, B, and C), and this distribution was more related to aggressiveness than to the race of the isolates. The results obtained in PCR amplifications using specific primers for race 1 and race 2, and SCAR primers developed in this work, correlated with the molecular groups previously determined from the RAPD analysis, and provided new molecular markers for the precise identification of the isolates. Results from successive pathogenicity tests showed that molecular differences between isolates of the same race corresponded with differences in aggressiveness. Isolates of races 1 and 2 in cluster A (R1I and R2I isolates) and cluster C (R1-type isolates) were all highly aggressive, whereas isolates of races 1 and 2 in cluster B (R1II and R2II isolates) showed a low aggressiveness profile. The usefulness of the molecular markers described in this study has been proved in double-blind tests with Fod isolates collected in 2008. Results from this work indicate a change in the composition of the Spanish Fod population over time, and this temporal variation could be related to the continuous change in the commercial carnation cultivars used by growers. This is the first report of genetic diversity among Fod isolates in the same race.

Detection of the defoliating and nondefoliating pathotypes of Verticillium dahliae in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.

The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum

We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium albo-atrum. We determined that its larger segment (dsRNA1) was 1747 base pairs in length and potentially encoded an RNA-dependent RNA polymerase of 539 amino acids, whereas the smaller segment (dsRNA2) was 1517 base pairs long and was predicted to encode a capsid protein of 435 amino acids. Homology searches and phylogenetic analysis confirmed that, as expected from its dsRNA banding profile, the identified virus was a new member of the family Partitiviridae, and we have therefore designated it V. albo-atrumpartitivirus 1 (VaaPV1). This is the first report of a mycovirus identified in V. albo-atrum.