Nondefoliating Pathotypes Research Articles

Inheritance of resistance to Verticillium wilt (Verticillium dahliae) in cotton (Gossypium hirsutum L.)

Abstract Verticillium wilt, caused by Verticillium dahliae Kleb., is a major constraint to cotton production in almost all countries where cotton is cultivated. Developing new cotton cultivars resistant to Verticillium wilt is the most effective and feasible way to combat the problem. Little is known about the inheritance of resistance to Verticillium wilt of cotton, especially that caused by the defoliating (D) and nondefoliating (ND) pathotypes of the soil‐borne fungus V. dahliae. The objective of this study was to determine the inheritance of resistance in cotton against both pathotypes of V. dahliae. Crosses were made between the susceptible parent ‘Cukurova 1518’ and each of four resistant parents PAUM 401, PAUM 403, PAUM 405 and PAUM 406 to produce F2 generations in 2002 and F2:3 families in 2003. Disease responses of parent and progeny populations to the D and ND pathotypes were scored based on a scale of 0‐4 (0, resistant; 4, susceptible). F2 populations inoculated with the D pathotype showed a 3 : 1 (resistant : susceptible) plant segregation ratio. Tests of F2:3 families confirmed that resistance was controlled by a single dominant gene. In contrast, analysis of data from F2‐ and F2‐derived F3 families suggested that resistance to the ND pathotype is controlled by dominant alleles at two loci.

Simultaneous Detection of the Defoliating and Nondefoliating Verticillium dahliae Pathotypes in Infected Olive Plants by Duplex, Nested Polymerase Chain Reaction.

Pathogen-free certified planting material and accurate detection of Verticillium dahliae pathotypes infecting the plant are key components of successful management of Verticillium wilt of olive. Use of a nested-polymerase chain reaction (PCR) procedure developed in earlier studies for in planta detection of the defoliating (D) and nondefoliating (ND) V. dahliae pathotypes resulted in ambiguous detection of the pathogen in some cases, due to heterologous amplification of the D-associated marker in ND-infected olive plants. In the present study, an improved procedure was developed that eliminates ambiguity and reduces time and cost for detection of D and ND V. dahliae in olive. The improved procedure is based on the simultaneous amplification of both an ND- and a new D-specific marker by means of duplex, nested PCR. The procedure was effective in the rapid and unequivocal detection of the D and ND V. dahliae in both artificially inoculated, own-rooted olive plants and naturally infected adult olive trees of different cultivar, age, and growing conditions. Furthermore, the duplex, nested-PCR procedure detected simultaneously the D and ND pathotypes in adult olive trees naturally infected by both pathotypes and in young olive plants that were double-inoculated with D and ND isolates under controlled conditions.

Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR

An increasing incidence and distribution of verticillium wilt has occurred in the last few years in newly established olive orchards in southern Spain. This spread of the disease may result from use of Verticillium dahliae‐infected planting material. The early in planta detection of the pathogen would aid the implementation of certification schemes for pathogen‐free planting material. In this work, a nested polymerase chain reaction (PCR) method was developed for the in planta detection of the nondefoliating (ND) V. dahliae pathotype, aimed especially at nursery‐produced olive plants. For this purpose, specific primers were designed from the sequence of a 1958‐bp random amplified polymorphic DNA (RAPD) marker of ND V. dahliae, and a procedure for the extraction of PCR‐quality total genomic DNA from infected root and stem tissues of young olive plants was tested and further optimized. Nested PCR assays detected ND V. dahliae in 4‐ to 14‐month‐old artificially infected plants of three olive cultivars. The ND‐specific PCR product was not amplified from total genomic DNA extracted from olive plants infected with the defoliating V. dahliae pathotype. Detection of the ND pathotype was effective from the very earliest moments following artificial inoculation of olive plants with a V. dahliae conidial suspension. Also, detection was achieved in inoculated, though symptomless, olive plants as well as in plants that were symptomatic but became symptomless by 217 days after inoculation.

Vegetative Compatibility Groups of Verticillium dahliae in Israel: Their Distribution and Association with Pathogenicity

A collection of 565 isolates of Verticillium dahliae, recovered between 1992 and 1997 from 13 host plant species and soil at 47 sites in Israel, was tested for vegetative compatibility using nitrate-nonutilizing (nit) mutants. Three vegetative compatibility groups (VCGs) were found and identified as VCG2A (28 isolates), VCG2B (158 isolates), and VCG4B (378 isolates) by using international reference strains. One isolate was heterokaryon self-incompatible. Of the VCG2B isolates, 92% were recovered from the northern part of Israel and 90% of VCG4B isolates were recovered from the south, with some overlap in the central region. Isolates of the minor group VCG2A were geographically scattered among the two major VCGs. Isolates of the same VCG resembled one another more than isolates from different VCGs based on colony and microsclerotial morphology, temperature responses, and, partially, pathogenicity. Different pathotypes were defined among 60 isolates tested, using cotton (cv. Acala SJ-2) and eggplant (cv. Black Beauty) as differentials. All isolates in VCG2A and 86% of the isolates in VCG4B, irrespective of their origin, induced weak to moderate symptoms on cotton and moderate to severe symptoms on eggplant and were similar to the previously described cotton nondefoliating patho-type. In contrast, all cotton isolates in VCG2B caused severe foliar symptoms, stunting, and often death, but little or no defoliation of inoculated cotton plants. These were defined as a cotton defoliating-like pathotype and induced only weak to moderate symptoms on eggplant. We concluded that vegetative compatibility grouping of V. dahliae in Israel is closely associated with specific pathogenicity and other phenotypic traits.

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.

Etiology, Importance, and Distribution of Verticillium Wilt of Cotton in Southern Spain

Surveys for Verticillium wilt in 142 cotton fields in 1981 to 1983 and 1985 indicated that the disease is widespread in the Guadalquivir Valley, southern Spain, where it occurs in 80.0 to 82.5% of the fields. Verticillium wilt was most prevalent in the upper valley in the 1981 to 1983 surveys and in the lower valley in the 1985 survey. Analyses of dry soil samples collected in spring 1985 by means of an Andersen sampler detected Verticillium dahliae propagules in 35.1% of the fields surveyed. Inoculum density in soil was much higher in the lower valley (average 37.1 CFU/g) than in the upper and central areas of the valley (average 4.7 CFU/g). The increasing importance of the disease in the lower valley corresponds to the spread of a defoliating pathotype of V. dahliae in this area during the early 1980s. Two groups of isolates, including 100 mild nondefoliating and 90 severe defoliating, were distinguished among 191 isolates by means of morphological, physiological, and pathogenicity tests. The remaining isolate was identified as nondefoliating with intermediate virulence. All defoliating isolates were obtained from the lower valley, while nondefoliating isolates were widespread. Isolates of the defoliating pathotype produced elongated and rounded microsclerotia on water agar, were able to grow on sanguinarine-amended potato dextrose agar, and fluoresced under UV light. Isolates of the nondefoliating pathotype formed only rounded microsclerotia on water agar, were inhibited greatly on sanguinarine-amended PDA, and did not fluoresce under UV light. Furthermore, the defoliating isolates had an optimum temperature for in vitro growth of 24 to 27°C compared with 21 to 24°C for the nondefoliating isolates.

Influence of Inoculum Density of Defoliating and Nondefoliating Pathotypes ofVerticillium dahliaeon Epidemics of Verticillium Wilt of Cotton in Southern Spain

Linear regression analysis of data transformed according to several mathematical models showed no significant correlation between the inoculum density of Verticillium dahliae determined at sowing time in randomly chosen cotton fields and the incidence of plants with vascular discoloration, the incidence of plants with foliar symptoms, or a disease intensity index for Verticillium wilt of cotton calculated at the end of the crop season. Epidemic development of Verticillium wilt in cotton was investigated in field plots naturally infested with different inoculum densities of the defoliating and nondefoliating pathotypes of V. dahliae in 1986 and 1987. The straight line model adequately described the increase in the incidence of plants showing foliar symptoms and the disease intensity index over the accumulated physiological time from sowing. For similar levels of initial inoculum, the defoliating pathotype of V dahliae caused an earlier epidemic onset, a higher rate of increase in the incidence of foliar symptoms and the disease intensity index over physiological time, and a higher final amount of both disease measurements than the nondefoliating pathotype. Also, for the two disease measurements, the rate of progress increased with the initial inoculum density of the defoliating pathotype up to a threshold of 24 to 44 CFU/g of dry soil in 1986 and 44 to 75 CFU/g of dry soil in 1987. Differences in severity among Verticillium wilt epidemics were described more appropriately by the descriptive parameters of the increase in the disease intensity index over physiological time than by the corresponding parameters of the increase in the incidence of foliar symptoms.

Differentiation ofVerticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton

Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains ofVerticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars ofGossypium hirsutum, G. barbadense andG. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cotton-defoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data fromnit pairings indicated that the sub-populations ofV. dahliae (VCGs) may not be completely isolated genetically.