Department of Plant Pathology, UW-Madison, Madison 53706, Wisconsin, USA.

Background and objectives
Burkholderia cepacia AMMD and the rif-resistant derivative strain AMMDR1 are effective biocontrol agents against Pythium damping-off and Aphanomyces root rot of peas in field studies [1, 2]. We are studying the effects of the bacterium on several stages of the life cycle of these pathogens to clarify their relative importance and identify potential mechanisms of biocontrol. Pre-infection stages of the pathogens' life cycle at which interactions are studied include zoospore attraction, cyst germination and initiation of host infection. The post-infection stage examined is the mycelial growth of the fungus in the root. We are using a root-colonizing, in vitro antibiosis-deficient Tn5 mutant strain (B. cepacia 1324) to determine the importance of antibiosis in relation to other possible mechanisms of biocontrol (nutrient competition and induced systemic resistance).

Materials and methods
In vitro assays on pre-infection interactions test the effects of seed exudates on zoospores and cysts and demonstrate how these effects are modified by the presence of AMMDR1 and 1324 during exudation. The in situ assay on zoospore dispersal and infection consists of inoculation with Pythium zoospores at different distances from seeds coated with bacteria and monitoring the resulting disease incidence. The post-infection interaction assay involves monitoring lesion length and mycelial spread of Aphanomyces on and in roots that were coated with the biocontrol agent 1 day after pathogen inoculation.

Results and conclusions
Exudates of non-treated seeds were attractive to zoospores and stimulatory to encystment and germination of cysts. In contrast, exudates from AMMDR1-treated seeds lysed zoospores and inhibited cyst germination up to 100%. Exudates from 1324-coated seeds had similar effects on Aphanomyces zoospores and cysts as the exudates of non-treated seeds. However, exudates from 1324-coated seeds were less attractive to Pythium zoospores and resulted in a lower rate of encystment than exudates from non-treated seeds. The in situ dispersal assays showed a dramatic reduction (up to 95%) in disease incidence among AMMDR1-treated seeds at all inoculation distances, as compared to non-treated seeds. Strain 1324 failed to protect seeds when inoculated with zoospores at the 2 cm distance, but did confer some protection of seeds inoculated at the 4 cm distance. Root-lesion length and mycelial colonization were significantly reduced as compared to the pathogen-only control for AMMDR1-treated seedlings, but not for 1324-treated seedlings.

These results indicate that B.c. AMMDR1 displays biocontrol effects during at least two separate stages of the pathogens' life cycle. For both of these stages, the lack of protection in situ by the mutant strain indicates that antibiosis, and not nutrient competition, may be the major mechanism of antagonism. The short duration of the assays, and the observation that AMMDR1 reduces root lesions even when applied after inoculation with the pathogen, suggests that involvement of ISR at this stage in the plant development is unlikely.

1. Parke JL, Rand RE, Joy AE, King EB, 1991. Plant Disease 75, 987-92.
2. King EB, Parke JL, 1993. Plant Disease 77, 1185-88.