Short communication. Telluric pathogens isolated from blighted pepper (Capsicum annuum L.) plants in northwestern Spain

A survey of blighted pepper (Capsicum annuum L.) plants covering 120 farms in northwestern Spain was performed between 2001 and 2004 with the aim of identifying the main telluric pathogens associated with the disease in this part of the country. The following potential telluric pathogens were isolated from the 755 diseased plants inspected: Phytophthora capsici Leonian, P. nicotianae Breda de Haan, Verticillium dahliae Kleb., Rhizoctonia solani Kühn, Sclerotium rolfsii Sacc., Fusarium solani Mart. (Sacc.) and Botrytis cinerea Pers. R. solani was the most commonly isolated pathogen; this was detected on 38% of the farms and isolated from 16% of the plants analysed. Inoculation tests were performed with isolates of P. capsici, P. nicotianae and F. solani on C. annuum cv. Yolo Wonder. These confirmed P. nicotianae as a pepper pathogen, but with weaker pathogenic behaviour than P. capsici. F. solani was confirmed as a secondary pathogen.

The aim of the present work was to identify the fungal pathogens associated with blighted pepper plants in Galicia (northwestern Spain) and to assess their pathogenic behaviour.
A total of 755 adult pepper plants with symptoms of blight (brown-black discoloured collar and root rots causing permanent wilting and plant death; some of these symptoms may be associated with vascular browning) were sampled from 120 farms in 41 survey sessions between 2001 and 2004 (Table 1).These farms were located in the most important pepper producing regions of the provinces of A Coruña, Pontevedra and Ourense.Fragments of the collar of affected plants were disinfected with 0.6% sodium hypochlorite for 4 min and then plated on PDA (potato dextrose agar) (Rapilly, 1968) at 22-24°C for fungal isolation.Microscopic observations were made every 24 h for one week.Fusaria and Phytophthora isolates were classified according to Nelson et al. (1983) and Stamps et al. (1990) respectively.
Yolo Wonder pepper plants were inoculated with 10 fungal isolates, including four of P. capsici, two of P. nicotianae and four of F. solani, to study fungal and oomycete pathogenicity.
Phytophthora inocula were prepared after growing each isolate on V8 juice agar (Erwin and Ribeiro, 1996) at 22-24°C for 7 days.Each inoculum was prepared by seeding pieces of the isolate in sterile 1% potassium nitrate solution distributed in several Petri dishes (20 ml per Petri dish).This culture was grown under ultraviolet light at 24°C for seven days to stimulate sporangium formation.When abundant sporangia were formed, the potassium nitrate solution was replaced by sterile distilled water and the Petri dishes maintained at 5°C for 30 min, and then at 24°C for 3 h, to stimulate zoospore discharge.The zoospore suspension was then filtered through Whatman paper, vibrated for 1 min and adjusted to 20,000 zoospores per ml using a Burker chamber (Bartual et al., 1991).Each plant was inoculated at the 6-leaf stage by dropping 5 ml of the zoospore suspension onto the collar of each plant using a sterile micropipette (Gil Ortega et al., 1995).Fusaria isolates were grown on PDA (Rapilly, 1968) at 22-24°C for 7 days.Inocula were prepared by shaking 100 ml of sterile water per Petri dish with each isolate for 1 min and adjusting to 105 macroconidia per ml using a Burker chamber.Each plant was inoculated at the 6-leaf stage by dropping 10 ml of the macroconidia suspension onto the collar of the plants using a sterile micropipette.
Yolo Wonder pepper plants were grown on plastic trays in a glasshouse at 18 (night temperature) to 22°C.The rooting medium was a mixture of peat and sand (1:1, v v -1 ) previously sterilised at 120°C for 45 min.The inoculation tests had a completely randomised design with 3 replicates per isolate and 10 plants per unit and replicate.Disease severity for Phytophthora and F. solani was determined 28 days after the inoculation according to Kim and Hwang (1992) and Schneider and Kelly (2000) respectively.
Mean comparisons were made using Duncan¢s multiple range test after transforming the disease severity data as follows: where X is the disease index of each plant expressed as a percentage.All calculations were performed using SAS software v. 8.2 (SAS, 1999).
Rhizoctonia solani and F. solani were the most commonly isolated potential pathogens (Table 2).R. solani has previously been isolated from blighted pepper plants in Mexico (González-Pérez et al., 2004), Pakistan (Mushtaq and Hashmi, 1997), Australia (Stirling et al., 2004) and Spain (Tello, 1984;Pomar et al., 2001;Tello and Lacasa, 2004) but was not the predominant fungus in any of these surveys.Two Phytophthora species were also isolated, P. capsici and P. nicotianae, which confirms the results of previous studies performed in northwestern Spain (Andrés et al., 2003).The incidence of both species was similar, both in terms of the percentage of blighted plants infected and the percentage of affected farms.V. dahliae was detected on more farms than P. capsici, a result inconsistent with previous studies performed in this part of the country (Pomar et al., 2001) and indeed in other parts of Spain (Tello, 1984) (Table 2).
It is important to note that several pathogens usually affected the same pepper plant simultaneously (Table 2).This was particularly true for P. nicotianae and F. solani (of all positive samples only 7% were infected by P. nicotianae alone, and only 13% were infected by F. solani alone) (Table 2).
A number of differences were seen in the pathogenicity profiles of fungal species either not well known as pathogens of this crop or considered secondary pathogens.P. nicotianae caused typical collar rot symptoms similar to those observed in the field, plus very mild blight symptoms.Isolates of P. capsici (PA-1 and RO-4) produced intense blight symptoms causing plant death 28 days after inoculation.These results contrast with those previously described for P. nicotianae on a different pepper cultivar (cv.California).This may be due to the differences in virulence of this pathogen in Capsicum germplasm of different origin (Andrés et al., unpublished data).
Only two of the four F. solani isolates tested showed slight pathogenic behaviour (Table 3).The affected plants showed only small areas of rot at the base of the collar and did not develop clear blight symptoms.Such weak responses have previously been reported for this species in Spain (Palazón et al., 1978) and elsewhere (Messiaen et al., 1995).These results suggest that F. solani is a secondary pathogen that usually infects pepper plants already affected by some other pathogen or which are suffering abiotic stress (Gerlach and Nirenberg, 1982;Tello, 1984;Nuez et al., 1996).
The present results strongly suggest that P. capsici, V. dahliae, P. nicotianae and R. solani are involved in pepper blight in northwestern Spain.Whether F. solani, which was isolated from diseased pepper plants but found to have very weak pathogenic behaviour, can form part of a complex with other pathogens and thus increase the injuries produced, remains to be determined.

Table 1 .
Farms surveyed and plants inspected

Table 2 .
Potential telluric pathogens isolated from wilted pepper (Capsicum annuum L.) plants in northwestern Spain