Short communication. Telluric pathogens isolated from bean plants with collar and root rots in northwest Spain

Pathogens belonging to the disease complex responsible for bean collar and root rots in northwest Spain were identified and their pathogenic behaviour studied over a two year period (2004-2005). The potential fungal and oomycete pathogens Fusarium solani f. sp. phaseoli (Burkh.) W.C. Snyder & Hansen, Rhizoctonia solani Kühn, Pythium ultimum Trow, Pythium Group G, Fusarium avenaceum (Fr.) Sacc., F. culmorum W. G. Sm. (Sacc.), Botrytis cinerea Pers., Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotium rolfsii Sacc. were isolated from 419 bean (Phaseolus vulgaris L.) plants affected by collar or root rot. Fusarium solani f. sp. phaseoli was the most frequently isolated (found on 63.7% of the farms surveyed and isolated from 19.3% of the diseased plants), followed by R. solani and the Pythium species. Inoculating Phaseolus vulgaris cv. Musica and Zondra with 14 isolates of these fungi and oomycetes showed F. solani f. sp. phaseoli to be the most aggressive pathogen of the complex; F. avenaceum and F. culmorum were found not to be pathogenic for either cultivar. The results confirm that F. solani and R. solani are the main pathogens of the bean collar/root rot disease complex in northwest Spain, affecting crops in their early growth stages. The complex also includes P. ultimum and Pythium Group G. Additional key words: Fusarium solani f. sp. phaseoli, Rhizoctonia solani, Pythium, Phaseolus vulgaris.

The aim of the present work was to identify the pathogens associated with the disease complex responsible for bean collar and root rots in northwest Spain, and to evaluate their pathogenic behaviour.
During 2004 and 2005, 419 bean plants with collar or root rot symptoms were sampled from 58 farms in seven regions belonging to the provinces of A Coruña, Lugo, Ourense and Pontevedra (Table 1).The sampled plants had characteristic red streaks, dry brownish or soft humid rots along the base of the hypocotyls and collars, and showed discoloration and deterioration of the main taproot and lateral roots.Severely diseased roots were associated with visible symptoms on the aerial part of the plants, such as chlorosis, defoliation and stunting.Fragments of the collar and roots of affected plants were disinfected with 0.6% sodium hypochlorite for 4 min, washed with sterile distilled water for 1 min and then plated on potato dextrose agar (PDA) (Rapilly, 1968) at 22-24°C for fungal and oomycete isolation.Microscopic observations were made every 24 h over the period of one week.Fusarium, Pythium and Rhizoctonia isolates were classified according to Nelson et al. (1983), Van der Plaats-Niterink (1981) and Sneh et al. (1994) respectively.
Fusarium solani and R. solani were the most commonly isolated potential pathogens (Table 2).Fusarium solani f. sp.phaseoli has been reported by some authors as the most important pathogen of the disease complex in the USA (Maloy, 1959;Steadman et al., 1975), Lebanon (Davet et al., 1980), Brittany (France) (L'Échappe et al., 1988) and Spain (Berra and Arteaga, 1989;Sinobas et al., 1994).However, others did not consider this pathogen as the main pathogenic agent of the complex (Tello et al., 1985), and some have even failed to isolate this fungus from diseased plants (Luttrel and Garren, 1952;Rusuku et al., 1997).
In the present work, the incidence of Pythium was high (Table 2).This agrees with surveys performed in the USA (Hoch et al., 1975), Lebanon (Davet et al., 1980), France (L'Échappe et al., 1988) and Rwanda (Rusuku et al., 1997).However, only in the last of these countries is the Pythium group considered to be the main pathogenic group of the bean collar/root rot disease complex.Only two groups of Pythium species were found to be part of the complex in the present work: P. ultimum -reported as belonging to the complex in the USA (Drechsler, 1952;Hoch et al., 1975;Lumsden et al., 1976)-and Pythium Group G.This is the first report of Pythium Group G as a pathogen of bean in Spain.
Thielaviopsis basicola, which has been reported as a bean pathogen in the Basque region (Berra and Arteaga, 1989) and southern Spain (Tello et al., 1985), was not isolated in the present work.
It is important to note that several pathogens were frequently isolated simultaneously from the same sample (Table 2).This reinforces the hypothesis of the existence of a bean disease complex formed by several pathogens that can infect plants at the same time, which has been reported in other regions (Davet et al., 1980(Davet et al., , L'Échappe et al., 1988)).Further studies are required to determine which pathogens have synergic activity.
Rhizoctonia solani and Fusarium isolates were grown on PDA at 22-24°C for 7 days.Inocula were prepared by blending each isolate (from four Petri dishes) with 400 ml of sterile distilled water at low speed for 1 min.The suspensions were then adjusted to 105 propagules or macroconidia per ml using a Burker-Turk chamber.Each bean plant was inoculated at the two-leaf stage by dropping 10 ml of inoculum onto the collar using a sterile micropipette (Jones and Belmar, 1989;Schneider and Kelly, 2000).
Pythium isolates were inoculated at pre or post-emergence.The post-emergence inocula were prepared after growing each isolate on V8 juice agar (Erwin and Ribeiro, 1996) at 22-24°C for four days.Plants at the two-leaf stage were inoculated by 'seeding' pieces (10 mm diameter) of the isolate onto the collar (Moorman and Kim, 2004).Pre-emergence inoculation was performed according to Ricci et al. (1976).This consisted of placing four bean seeds on the surface of the oomycete culture and then plating both seeds and fungal culture onto a sterilized substrate in a plastic tray.Two cultures of the same strain and eight seeds of the same cultivar were included in each plastic tray and covered with sterile substrate.
The inoculated bean plants were grown on plastic trays in a glasshouse at 18-26°C during the months of July and August.The rooting substrate was a mixture of peat and sand (1:1, v:v) previously sterilised at 120°C for 45 min.The inoculation tests for Pythium spp., R. solani, F. solani and the other Fusarium spp.had a split plot design with randomised isolate subplots for the different cultivars.Three replications were undertaken per isolate-cultivar interaction.Each subplot included nine plants.In post-emergence inoculated plants, disease severity was determined according to Schneider and Kelly (2000) 30 days after inoculating the plants.The disease severity of plants inoculated with Pythium isolates at pre-emergence was determined using the method of Ricci et al. (1976) 15 days after the inoculation.Duncan's multiple range test was used to compare the means after transforming the disease severity values 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).
Table 3 shows that Fusarium solani f. sp.phaseoli was the most aggressive pathogen for both cultivars, followed by R. solani, P. ultimum and Pythium Group G.The Fusarium spp.isolates -F.avenaceum and F. culmorum-showed only very weak pathogenicity and arc sin /100 Y X = were therefore concluded not to be primary pathogens in this disease complex (Table 3).This situation differs from that described by other authors working with Fusarium species of the roseum group sensu Messiaen and Cassini (1968).Fusarium semitectum Berk & Rav. has been reported as the cause of considerable losses to bean seed production in Brazil under prolonged humid conditions (Dhingra and Muchovej, 1979).The Pythium species were significantly more aggressive when inoculated at pre-emergence, probably due to the early receptivity of the crop to these pathogens (Hall, 1991).
The present results confirm that F. solani and R. solani are the main pathogens of the bean collar/root rot disease complex in northwest Spain, a complex that also includes P. ultimum and Pythium Group G, affecting bean crops at early growth stages.

Table 1 .
Telluric pathogens from beans with collar and root rots in northwest Spain81 Farms surveyed and plants inspected in northwest Spain

Table 2 .
Potential telluric pathogens isolated from bean plants (Phaseolus vulgaris L.) with collar and/or root rots in northwest Spain

Table 3 .
Pathogenic behaviour of Pythium spp., Rhizoctonia solani, Fusarium solani and Fusarium spp.strains, isolated from bean plants (cv.Musica and Zondra) with collar and/or root rots