Assessment of some antagonists to fungal plant pathogens and development of methods for their large-scale cultivation

Wood fibre waste (WFW) of paper mill origin and composted fish waste were investigated for use as media for the cultivation or carriage of biological control agents active against plant pathogens, with a view to producing suppressive alternatives to chemical fungicides. Potential biological control agents were isolated from crop-soil or from the rhizosphere and rhizoplane of plants. Some 67 bacterial isolates of more than 100 cultures assessed were antagonistic to one or more fungal plant pathogens in vitro. Most antagonists were identified as Bacillus or Pseudomonas spp. Isolates of Pseudomonas corrugata, Bacillus megaterium, B. thuringiensis, B. polymyxa, B. pumilus, B. mojavensis, and Lysobacter antibioticus showing particular potential in a pot trial against Sclerotinia minor and S. sclerotiorum formed the focus of further developments as agents of biological control. The optimal growth conditions of the selected bacterial antagonists and of a fungal biological control Trichoderma sp. (Td22), were assessed in vitro prior to their cultivation in WFW- or fish waste compost, or in amended WFW without composting. All tested bacterial antagonists grew well at neutral pH and at temperatures between 25°C and 30°C. Td22 grew optimally at pH between 5.0 and 6.0 and at 25°C. This fungus had an advantage over the bacterial isolates of being cellulolytic, raising the possibility of its cultivation in cellulose-based materials, such as WFW. The selected antagonists were cultivated in composted WFW amended (20% w/w) with millet seed and enriched with ammonium nitrate-based mineral salts solution. The initial pH of the mix was adjusted to approximately neutral for the cultivation of the bacterial agents, no pH adjustment was needed for the cultivation of Td22 as the initial pH of the mix was around 4.5-5.0. All antagonists showed excellent growth response in this mix, reaching densities up to -10 10 colony forming units (cfu)/g dry weight mix after 14 days of incubation under sterile conditions. Lower population densities of between -10 7 and -10 9 cfu/g for Td22, or of between -10 8 and 10 9 cfu/g for the bacterial antagonists were achieved under non-sterile conditions. The efficacy of the suppressive mix in controlling S. minor was evaluated in pot trials. Td22 grown in this mix consistently protected lettuce plants from attack by S. minor. The degree of protection was correlated with the level of the suppressive mix amendment, with 100% protection being observed after four weeks at an amendment rate of 20% v/v (compost:soil mix). Soil inoculated with S. minor only (control treatment) showed 0% survival after the same interval. Pre-incubation of the fungus in the compost:soil mixture for four or more days prior to planting appeared to improve its disease control. Long-term storage of the Td22 suppressive mix was also demonstrated by the protection of pyrethrum plants from attack by S. minor following storage of the mix at ambient temperatures for 4.5 months. The efficacy of the selected bacterial antagonists in disease suppression was less consistent. Although the percentages of healthy seedlings/plants increased relative to controls following application of compost-grown bacterial antagonists (at a rate of 5% v/v), these increases were not statistically significant (p>0.05) in most cases. In an attempt to eliminate the need for sterilizing compost material used to cultivate biological control agents, the potential of utilizing the different optimal growth temperatures of normal compost biota at elevated temperature relative to those of biological control agents was investigated. Bacterial antagonists were inoculated into mature self-heating fish waste compost immediately following its rapid cooling from 52- 55C to ambient temperatures and supplementation with amendments favouring the antagonists. All antagonists reached high numbers after 14 days incubation, with two (P. corrugata and L. antibioticus) reaching -10 10 cfu/g dry weight. In both cases most of the indigenous compost microbiota were excluded, the antagonists being the dominant biota found following isolation. In a glasshouse trial, they also significantly (p<0.05) protected lettuce plants from attack by S. minor, the degree of protection ranging from 40 to 50% relative to the control treatments. When applied at the rate of 12.1 tonnes/hectare in a field trial, the effectiveness of these antagonists was comparable to that of a commercially available biological control agent (Companion) applied as a cell suspension, but was not significant statistically (p>0.05) compared to control treatments. The effectiveness of selected antagonists (Td22, P.corrugata, and L. antibioticus, prepared as cell suspensions) in protecting zucchini leaves from downy mildew incidence was also investigated, with a view to developing standardised 'compost teas' for the control of leaf pathogens. All but one antagonist (L. antibioticus) applied singly or in combination provided a significant (p<0.05) protection to the zucchini leaves at two weeks after the pathogen introduction, ranging from -22% (bacterial) to 83% (Td22) protection relative to the pathogen-only control. Co-inoculation of Td22 with P. corrugata, L. antibioticus, or a combination of Td22 with both bacteria resulted levels of control lower than those provided by Td22 alone. This could be attributed to the lower concentrations of the most active agent (Td22) and/or to the antagonism between the bacteria and fungus as noted in vitro. The survival or establishment of these antagonists on the leaf surface was very poor, resulting in declining levels of protection after two weeks, suggesting that re-application of these antagonists is needed to provide a reliable disease control. The molecular weights of two antimicrobial compounds produced by P. corrugata strain SAJ6 were determined to be 554 and 580. Other characteristics of these antibiotics were also elucidated, although determination of their structures was beyond the scope of this study. Siderophore activity by these compounds was eliminated from contention, since inhibition of S. minor was not affected by the presence of high levels of FeCI3 in growth medium.