Separate and Combined Disruptions of Two Exo-β-1,3-Glucanase Genes Decrease the Efficiency of Pichia anomala (Strain K) Biocontrol Against Botrytis cinerea on Apple

Abstract

The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-beta-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-beta-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-beta-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-beta-1,3-glucanase to biological control may be masked by other modes of action, such as competition.

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

“…on apple have been investigated. Competition for nutrients and implication of exo-ß-1,3-glucanases have been highlighted (Friel et al, 2007). Furthermore several genes are also suspected to play a role in the antagonistic activity of strain K (Massart and Jijakli, 2006).…”

Control of Apple Blue Mold by the Antagonistic YeastPichia anomalaStrain K: Screening of UV Protectants for Preharvest Application

“…Strain K was reported to be effective against P. expansum, B. cinerea, and other postharvest pathogens of fruit (26,32). Its mechanism of action in controlling B. cinerea on apple includes competition for nutrients and mycoparasitism (14,25,26,33). Several monitoring systems have been developed to track the population dynamics of strain K on apple fruit (9,37).…”

Non-Saccharomyces Killer Toxins: Possible Biocontrol Agents Against Brettanomyces in Wine?

“…Exo-glucanase activity has been detected in killer toxinproducing yeast species of W. anomalus, P. membranifaciens, W. saturnus, P. anomala strain K and Candida oleophila (Jijakli & Lepoivre, 1998;Masih & Paul, 2002;Bar-Shimon et al, 2004;İzgü & Altinbay, 2004;İzgü et al, 2006;Friel et al, 2007;Wang et al, 2007;Xu et al, 2011;Guo et al, 2012). Three killer strains of W. anomalus (BCU24, BS91 and BCA15) exhibited killer activity against a S. cerevisiae wild type strain, while mutants deficient in β-1,6-glucan were resistant to the toxins of the strains.…”

“…P. anomala strain K is an efficient and reliable antagonist of B. cinerea and Penicillium expansum in apples. The disruption of P. anomala's exo-glucanase genes PaEXG1 and PaEXG2 resulted in reduced efficiency -8% from 71% in the biocontrol of B. cinerea in apples (Friel et al, 2007). Growth of B. cinerea in the presence of P. membranifaciens resulted in extensive damage to the fungal cell wall, with complete rupture and fragmentation of the hyphal filaments of B. cinerea.…”