D. Friel

Separate and combined disruptions of two exo-beta-1,3-glucanase genes decrease the efficiency of Pichia anomala (strain K) biocontrol against Botrytis cinerea on apple.

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.

Genetic characterization of the yeast Pichia anomala (strain K), an antagonist of postharvest diseases of apple

Aims:  To obtain information about the genomic organization of Pichia anomala (strain K) and about its genomic diversity at species and intraspecies level.

Cloning and sequence analysis of the TRP1 gene encoding the phosphoribosyl anthranilate isomerase from Pichia anomala (strain K)

Pichia anomala (strain K) is an efficient biocontrol agent against post‐harvest diseases affecting apples. To study the role of strain K genes in biocontrol activity, it is useful to identify selectable markers on which to base a gene disruption strategy. The Pichia anomala TRP1 gene (PaTRP1) was isolated by complementation of the multi‐auxotrophic S. cerevisiae strain FY‐1679‐18b. DNA sequence analysis revealed the presence of a 699 bp ORF encoding a 233 amino acid protein showing the typical conserved structure of proteins of the phosphoribosyl anthranilate isomerase (PRAI) family. Codon analysis revealed a high number of unused codons. Downstream from PaTRP1 was found the 3′ extremity of a gene highly similar to the IPP1 gene (coding for the inorganic pyrophosphatase). In addition, a sequence of the 5′ extremity of the insert is highly similar to a fragment of the S. cerevisiae PRP9 gene, coding for a spliceosome‐associated protein. The nucleotide sequence has been deposited in the Genbank database under Accession No. AY198188. Copyright