Biological control of Aphanomyces euteiches root rot of pea with spore-forming bacteria

Abstract

Spore-forming bacteria were investigated as potential biological control agents of the pea root rot fungus, Aphanomyces euteiches (Oomycete). Isolations of the bacteria were made both directly from field soils (558) and from the rhizosphere of peas grown in soil (146). When the 704 isolates were screened for inhibition of A. euteiches mycelial expansion in vitro, a low frequency of suppressiveness was observed in isolates taken directly from field-soil (0.05%), whilst 19% of the bacterial isolates taken from the rhizosphere were suppressive.

Mycelial suppressive isolates were subsequently screened for inhibition of zoospore germination and germ tube growth. The seven isolates Bacillus cereus 15•80, Paenibacillus polymyxa 18•25, P. polymyxa 18•94, unidentified bacterium PB 45, Paenibacillus macerans PT 1, Bacillus pumilus PT 10 and Bacillus subtilis PT 69, inhibited growth by 50% or more. Bacterial isolates were identified using carbohydrate fermentation profiles and, for Bacillus mycoides MW 27 and P. polymyxa 18•25, on their 16S rRNA gene sequences.

The seven suppressive isolates identified, and an additional five isolates which had appeared promising in an earlier study (unidentified bacterium MW 9b, B. cereus MW 10, B. pumilus MW 12, B. pumilus MW 18, B. mycoides MW 27), were tested for disease control in glasshouse trials. Bacillus cereus 15•80 and Apron C70SD significantly (∝=0.05) reduced visual root rot disease in the initial trial. However, the presence of other root rot pathogens, causing disease with symptoms similar to Aphanomyces root rot, confounded the results. In a subsequent trial, counts were made of oospore numbers per gram of root tissue which allowed specific distinction of A. euteiches disease. Several species of bacteria were able to reduce oospores and root rot symptoms. Paenibacillus polymyxa 18•25 was the most effective, reducing oospores from 450 to 160 gram⁻¹ of root. Apron C70SD reduced overall root disease but had no effect on oospore counts.

On the basis of both in vitro and glasshouse trial results, B. subtilis PT 69, B. pumilus MW 18 and PT 10, B. mycoides MW 27, B. cereus 15•80, P. polymyxa 18•25 and 18•94 were selected for field testing. In the first trial, no significant differences (∝=0.05) were found between nil and each of the other treatments. In part, this was due to low overall disease pressure (disease score of 1.5 in untreated) and inadequate separation I resolution of treatment means (all bacterial treatment means were 1.0). Bacillus mycoides MW 27 and P. polymyxa 18•25 were selected for further field trials. Bacillus mycoides MW 27 significantly (∝=0.05) increased the mean pod weight per plant from 2.39 g (nil) to 2.71 g, and the average plant root weight from 0.81 g to 0.95 g. No benefits were found by applying the two isolates together as opposed to individually, nor was there a benefit by combining them with Apron C70SD.

Seed coat, prill and granule formulations of B. mycoides MW 27 were evaluated in a field trial. As a seed coat formulation, the isolate significantly (∝=0.05) increased plot stand by about 10%. In a prill formulation, B. mycoides MW 27 had no effect on plot stand (early damping-off disease), but significantly (∝=0.05) increased the number of pods per plant and average pod weight per plant. Apron C70SD controlled damping-off disease but had no effect on root rot disease.

Bacillus pumilus PT 10, B. mycoides MW 27, P. polymyxa 18•25 and fluquinconazole were tested as seed treatments for control of another root disease, take-all of wheat. Bacillus pumilus PT 10 significantly (∝=0.05) increased plant yield by 77%. Bacillus mycoides MW 27 had similar efficacy as fluquinconazole, increasing yield by approximately 10% (not significant at ∝=0.05). As a seed coat treatment, B. mycoides MW 27 also effectively controlled damping-off disease.

Paenibacillus polymyxa 18•25 suppressed a wide range of plant pathogenic fungi in vitro, possibly through production of the cell wall-degrading enzymes chitinase or cellulase, or an antibiotic compound which the bacterium was found to produce. Bacillus mycoides MW 27 was slightly suppressive to only two pathogenic fungi in vitro, and did not degrade chitin or cellulose.

The soil bacterium Bacillus mycoides MW 27 has excellent potential for development as a biological control agent of Aphanomyces root rot of pea. A formulation of the bacterium could be marketed on both its ability to increase yields under Aphanomyces disease pressure, and to limit oospore development, thereby limiting disease in continuous cropping situations.