Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves and plants. Scanning electron microscopy revealed significant inhibition of Botrytis cinerea conidial germination in the presence of Pichia guilermondii, whereas Bacillus mycoides caused breakage and destruction of conidia. When both biocontrol agents were applied in a mixture, conidial destruction was more severe. The modes of action of each of the biocontrol agents were elucidated and the relative quantitative contribution of each mechanism to suppression of Botrytis cinerea was estimated using multiple regression with dummy variables. The improvement in control efficacy achieved by introducing one or more mechanisms at a time was calculated. Pichia guilermondii competed with Botrytis cinerea for glucose, sucrose, adenine, histidine, and folic acid. Viability of the yeast cells played a crucial role in suppression of Botrytis cinerea and they secreted an inhibitory compound that had an acropetal effect and was not volatile. Bacillus mycoides did not compete for any of the sugars, amino acids, or vitamins examined at a level that would affect Botrytis cinerea development. Viable cells and the compounds secreted by them contributed similarly to Botrytis cinerea suppression. The bacteria secreted volatile and non-volatile inhibitory compounds and activated the defense systems of the host. The nonvolatile compounds had both acropetal and basipetal effects. Mixture of Pichia guilermondii and Bacillus mycoides resulted in additive activity compared with their separate application. The combined activity was due to the summation of biocontrol mechanisms of both agents. This work provides a theoretical explanation for our previous findings of reduced disease control variability with a mixture of Pichia guilermondii and Bacillus mycoides.

Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves. The aims of the research were to determine whether the use of their combination would broaden the environmental conditions under which biological control is effective, and to test the hypothesis that it would reduce the variability of control efficacy under diverse conditions. Applied separately, the biocontrol agents significantly inhibited spore germination, lesion formation, and lesion development at most temperatures, relative humidities, and spray-timing combinations (temperatures: 10, 15, 20, 23, 25, and 30 degrees C; relative humidities: 78, 85, 96, and 100%; and spray-timings: 0, 4, and 7 days before inoculation). However, control efficacy was highly variable, and under certain combinations it was not adequate. Control efficacy achieved by the biocontrol agents applied separately ranged between 38 and 98% (mean 74%) and the coefficient of variation ranged from 9.7 to 75%. The mixture of Bacillus mycoides and Pichia guilermondii suppressed Botrytis cinerea effectively (80 to 99.8% control) under all conditions, and the coefficients of variation were as low as 0.4 to 9% in all cases. Thus, application of both biocontrol agents resulted in better suppression of Botrytis cinerea, and also reduced the variability of disease control. Application of more than one biocontrol agent is suggested as a reliable means of reducing the variability and increasing the reliability of biological control.

During avocado fruit ripening, decreasing levels of the flavonoid epicatechin have been reported to modulate the metabolism of preformed antifungal compounds and the activation of quiescent Colletotrichum gloeosporioides infections. Epicatechin levels decreased as well when C. gloeosporioides was grown in the presence of epicatechin in culture. Extracts of laccase enzyme obtained from decayed tissue and culture media fully metabolized the epicatechin substrate within 4 and 20 h, respectively. Purified laccase protein from C. gloeosporioides showed an apparent MW of 60,000, an isoelectric point at pH 3.9, and maximal epicatechin degradation at pH 5.6. Inhibitors of fungal laccase such as EDTA and thioglycolic acid reduced C. gloeosporioides symptom development when applied to ripening susceptible fruits. Isolates of C. gloeosporioides with reduced laccase activity and no capability to metabolize epicatechin showed reduced pathogenicity on ripening fruits. On the contrary, Mexican isolates with increasing capabilities to metabolize epicatechin showed early symptoms of disease in unripe fruits. Transcript levels of cglac1, encoding C. gloeosporioides laccase, were enhanced during fungal development in the presence of epicatechin at pH 6.0, where avocado fruits are susceptible to fungal attack. But transcript increase was not detected at pH 5.0, where the fruit is resistant to fungal attack. The present results suggest that biotransformation of epicatechin by C. gloeosporioides in ripening fruits is followed by the decline of the preformed antifungal diene compound, resulting in the activation of quiescent infections.

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