Short Communication: Virulence of different isolates causing verticillium wilt of pepper in the Padrón region

A survey of verticillium wilt of pepper was carried out in farms of the Padron region, including both greenhouses andfields. Pepper plants cv. Padron showing wilt symptoms were collected in three successive sampling sessions performedfrom May to October 1997. A total number of 79 isolates of Verticillium dahliae Kleb were obtained. The high frequencyof farms with plants infected with V. dahliae, especially at the end of the crop season, was related to a high persistenceof the fungus during the whole of the 1990s. Twenty-two isolates were tested for pathogenicity, and all of them were ableto induce wilting in cv. Padron pepper plants. Dry weight and stem length data of inoculated plants showed that there wasa high variability in the virulence of the isolates tested, and significant differences were observed among them.

Samples were taken from a total of 12 farms (9 greenhouses and 3 open-air farms) in three successive sampling periods, from the beginning of May to the beginning of October 1997 (Table 1). In each farm, 10 Padrón pepper plants with symptoms of the disease were collected.
For isolation of the fungi, samples were taken from different parts of the plants corresponding to the base of the stem, the neck and base of the root. The surface of the fragments was disinfected with sodium hypochlorite at 0.5% for 5 minutes and inoculated onto plates with PDA medium (Potato Dextrose Agar). Verticillium determination was done according to Smith's (1965) and Isaac's (1967) criteria and the following aspects were studied: presence of microsclerotia, presence and characteristics of microconidia and conidiophores, and cardinal temperatures. Two numbers were assigned to the different isolates: the first referred to the farm and the second to the sampled plant number.
The presence of Verticillium dahliae was detected in all the sampling sessions (Table 1) and a total of 79 isolates were obtained in the three sampling sessions performed. No other species of Verticillium was detected.
Symptoms corresponding to V. dahliae attack were scarce in the first sampling session and the main ones were partial wilting and dwarfism. These became more severe in the second and third sampling session and were accompanied later by other symptoms such as defoliation and vascular necrosis.
In the third sampling session (September and October), coinciding with the end of production, there was an increase in the number of farms in which V. dahliae was detected. This increase could be related with temperature since this factor is closely related to the incidence of verticillium wilt (Kendrik and Middleton, 1959). The temperatures reached in this season in the Padrón greenhouses are around 24ºC, which is optimum for Verticillium growth and development of the disease according to Kendrik and Middleton (1959) and Barriuso et al. (1992).
In a previous survey carried out in 1991in and 1992in (Saavedra, 1993, the presence of V. dahliae was detected in farms 1, 2, 3, 4 and 7, permitting us to deduce that these farms had verticillium wilt almost constantly over this decade. This persistence of V. dahliae in the Padrón farms is because the farmers did not solarise the soil to destroy the microsclerotia and also due to the repetition of pepper crops during several years in the same plot, increasing the infectious potential. In general, all reported cases refer to microsclerotia as the main form in which the fungus is conserved in the soil. Isaac (1967) indicated a survival of 6 to 14 years of these organs in normal crop rotations and up to 4 years in the total absence of hosts in the rotation, with the potential to germinate when the environmental conditions are more favourable (Pegg, 1974).
A total of 22 of the isolates obtained (30%) were studied to determine their pathogenicity and degree of virulence. This percentage was chosen taking into account the number of isolates per farm. Hence, in farms with the greatest number of isolates (9 or more), 3 were studied and in the rest of the farms 1 or 2.  Pathogenicity experiments were carried out on 16 day-old plants of the Padrón pepper variety in the phenological stage of 4 true leaves. The plants, in groups of 10, were inoculated with the different isolates of V. dahliae, according to the method of Tello et al. (1991), that consists in dipping the roots in an «inoculum unit» for 20 min. This «inoculum unit» was obtained by milling a PDA petri dish, totally covered with the mycelia from the isolate to be inoculated, in 400 ml of distilled water. Another group of 10 plants was treated with sterile water instead of the inoculum and corresponded to the control group. After carrying out the inoculation, the plants were transplanted to 10 well-trays with a sterilised substrate composed of potting soil and vermiculite in a ratio of 2:1 (v/v). The experiments were conducted in a chamber with controlled temperature (25ºC) and photoperiod (16 hours light and 8 hours darkness).
Plants were checked regularly for symptoms produced by each of the isolates of V. dahliae, and the stem length of inoculated plants was recorded over 30 days. After this period, different organs were separated from the plants (roots, hypocotyl, epicotyl and leaves) and the dry weight was determined.
The data were studied by one way analysis of variance (ANOVA) and Duncan's test with a significance of 5%. The data were transformed by the logarithmic function in base 10 prior to their analysis. The statistical analysis was done with the help of the Statgraphics Plus 4.0 software for Windows.
In all cases, V. dahliae was reisolated from experimentally inoculated plants that had developed symptoms of the disease. Table 2 shows that the epicotyl and the leaves were the organs most affected by the disease followed by the hypocotyl and the root. On the other hand, there was considerable variability in the virulence of the isolates, with significant differences between some of these. The least and most virulent isolates were clearly 2-9 and 1-3, respectively. Isolates with an intermediate virulence could be found between these two extremes. Another important point is that, in some cases, there was a great variability between the isolates from the same farm. For example, from farms 2 and 10, extremely high virulent isolates were obtained (2-5 and 10-8) and also ones with an especial low virulence (2-9 and 10-10).
The measurements of stem growth of the plants inoculated with isolates 1-3 and 2-9 conf irm the dry weight results. Figure 1 shows clear differences in the growth of pepper plants inoculated with the most virulent isolate, 1-3, and the least virulent isolate, 2-9. The plants inoculated with isolate 1-3 hardly exceeded 40 mm in length at the end of the experiment while plants inoculated with 2-9 reached almost 60 mm, and the control plants reached a height of 70 mm. This slower growth of the plants inoculated with isolate 1-3 was the consequence of an early appearance of symptoms of the disease, finally reflected in a smaller dry weight than that of plants inoculated with isolate 2-9 (Table 2). It is known that Verticillium can infect a wide range of organisms. More specifically, the hosts of V. dahliae include crops of cotton, tomato, pepper, aubergine, potato, mint and several cucurbitaceous species, ornamental woody shrubs and fruit trees (Heale, 1988;Bejarano-Alcázar et al, 1996;Tsror et al., 2000). Possibly, the isolates of V. dahliae with the least virulence are not specific to pepper. In fact, isolate 4-3 came from a farm in which pepper and tomato plants were grown together and this strain could be more specific to tomato than to pepper or could be a strain with little host specif icity. However, several cases have been described of strains of V. dahliae specific to pepper (Douira et al., 1995;Riley and Bosland, 1997). The most virulent group of isolates tested, headed by 1-3, could, therefore, correspond to isolates specific to pep-per. In any case, in order to assign host specificity to an isolate or a group of isolates, or even the possible establishment of races of V. dahliae among these isolates would require further experiments to be conducted that include other host plants and different pepper cultivars.