Biocontrol of Stemphylium vesicarium and Pleospora allii on Pear by Bacillus subtilis and Trichoderma spp.: Preventative and Curative Effects on Inoculum Production

Trials under controlled and field conditions were conducted to establish the effect of strategies of application of biological control agents (BCAs) in the reduction of Stemphylium vesicarium and Pleospora allii inoculum production on pear leaf debris. Six BCAs based on different strains of Trichoderma spp. (Tr1, Tr2) and Bacillus subtilis (Bs1, Bs2, Bs3 and Bs4) were evaluated. Two strategies were tested in controlled experiments: application before (preventative strategy) or after (curative strategy) pear leaf debris colonization by S. vesicarium, evaluating the growth inhibition and sporulation of S. vesicarium and the pseudothecia production of P. allii. When the BCAs were applied preventatively, the efficacy of treatments based on B. subtilis was higher than those based on Trichoderma spp. in controlling the pathogen colonization, but that of controlling the inoculum production of S. vesicarium and P. allii was similar. However, when the BCAs were applied curatively, Trichoderma based products were more effective. In field trials, Trichoderma spp. Tr1 and B. subtlilis Bs1 produced a consistent 45–50% decrease in the number of S. vesicarium conidia trapped compared to the non-treated control. We conclude that Bacillus subtilis Bs1 and Trichoderma spp. Tr1 and Tr2 can be expected to reduce fungal inoculum during the pear vegetative period by at least 45–50%. Additionally, Trichoderma spp. Tr1 and Tr2 have the potential to reduce the fungal overwintering inoculum by 80% to 90%.

Optimizing inoculum production methods for infesting soil with Phytophthora species

Inoculum production is an important part of conducting research with soilborne Phytophthora species. One common method is to incubate Phytophthora cultures in nutrient-amended vermiculite. However, inoculum levels often vary among batches of inoculum even when production methods remain the same, and incubation typically takes ≥ 6 weeks, increasing risks for delayed experiments if the resulting inoculum level is too low. A more reliable and rapid method is needed for future studies. Experiments were conducted to: (1) determine inoculum levels of P. cinnamomi and P. plurivora after incubation in V8 juice-amended vermiculite (standard method); (2) evaluate how inoculum viability was affected by air drying; (3) develop a modified method that takes less time to produce a vermiculite-based inoculum; and (4) evaluate the effect of storage on inoculum viability. Results showed that the standard method produced inoculum levels from 716 to 1808 colony forming units/g and that drying to < 78% moisture content significantly reduced viability. The modified method used 2-week-old Phytophthora cultures to infest vermiculite at 80% moisture content and produced inoculum levels from 214 to 525 CFU/g. Storage for >1 day generally reduced inoculum viability. Although inoculum levels from the modified method were lower than the standard method, inoculum levels for each isolate were more consistent between trials and the modified method was 6 to 8 weeks faster. Production with the modified method can also be easily scaled up by infesting a greater volume of vermiculite with additional cultures of Phytophthora. These results are important because they help explain variability in soilborne Phytophthora inoculum production and storage, and provide a new method for producing inoculum more quickly.

From laboratory to field: applying the Fo47 biocontrol strain in potato fields

Endophytic microbes conferring biocontrol are an eco-friendly alternative to control diseases in crops. Unfortunately, the use of endophytes to control diseases is not yet widespread as their application in agricultural settings is challenging and the outcome variable. Translating strains that perform well under laboratory conditions to the field poses several challenges. One is large scale inoculum production in a cost-effective manner. Here, we developed a framework to scale up inoculum production of Fusarium oxysporum 47 (Fo47), assess inoculum viability and its performance in the field and effects on potato yield and performance. The Fo47 endophyte is a well-described biocontrol agent, isolated from disease suppressive soils in the 1980’s. Using mung bean medium, we could routinely produce ≈7 × 108 spores/mL. Using 60 mL of 107 spores/mL per tuber we could re-isolate the fungus 79 days after application from 60 to 70% of the inoculated plants in a large-scale potato field trial (Clenze, Germany). Furthermore, this protocol can be used to assess Fo47 biocontrol potential under field conditions. The presence of the fungus did not negatively affect plant yield or starch production and did not increase susceptibility to endemic pathogens.