Identification of Differentially Expressed Genes by cDNA-Amplified Fragment Length Polymorphism in the Biocontrol Agent Pichia anomala (Strain Kh5)

Abstract

cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was used to identify genes potentially involved in biological control, by strain Kh5 (Pichia anomala), of Botrytis cinerea, an important postharvest pathogen on apples. Strain Kh5 was grown in yeast nitrogen base (YNB) plus glucose (G medium) or YNB plus cell walls of B. cinerea (B medium). Thirty-five primer pairs were used in AFLP amplifications, resulting in a total of more than 2,450 bands derived from the mRNA of strain Kh5 grown in B medium. Eighty-six bands (3.5%) corresponded to genes upregulated in B medium compared with G medium. Of these 86 bands, 28 were selected, cloned, sequenced, and subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) to confirm their differential expression. An appropriate housekeeping gene, G2, was selected and used to normalize the results of RT-PCR. Eleven genes presented an increased gene expression in the presence of B. cinerea cell walls (expression ratio > 1). Statistical analysis showed a significant increase for 5 of these 11 genes. The overexpressed genes show homologies to yeast genes with various functions, including β-glucosidase, transmembrane transport, citrate synthase, and external amino acid sensing and transport. Some of these functions could be related to cell wall metabolism and potentially involved in mycoparasitic properties.Since the early 1970s, fruit losses due to postharvest diseases have reached up to 25% (23). Chemical treatments are still widely used to control diseases caused by postharvest pathogens, but the appearance of fungicide-resistant strains, the deregistration of fungicides, and public concerns about health and environmental impact may limit the future application of fungicides and increase the need to develop alternative protection methods such as biological control. In this context, the yeast Pichia anomala (E.C. Hansen) Kurtzman (strain K) has been isolated from the surface of apples (cv. Golden Delicious) and selected for its strong and reliable ability to antagonize Botrytis cinerea Pers.Fr., one of the most devastating pathogens of harvested apples (26). The antagonistic activity of P. anomala strain K was maintained during pre-and postharvest practical applications of industrial fermented cells (24).Understanding the mechanisms of action of a biocontrol agent is a prerequisite to its practical application, because this allows a more rational mode of production and a formulation enhancing its action. Various mechanisms, based mainly on competition for space and nutrients, mycoparasitism, antibiosis, and elicitation of plant defenses, are reported to contribute simultaneously or sequentially to the biocontrol properties of microorganisms (23). Jijakli et al. (27) suggested that competition for space and nutrient is involved in the biological control of B. cinerea by P. anomala strain K. In contrast, P. anomala strain K did not induce defense responses of the apple and did not produce detectable antibiotic or killer toxin (M. H. Jijakli, unpub...

cDNA-AFLP analysis of gene expression changes in apple trees induced by phytoplasma infection during compatible interaction

“…Different techniques can be employed for confirmation (to detect false-positive) of cDNA-AFLP results. qRT-PCR was privileged for its rapidity, simplicity, high sensitivity, low RNA quantity needed, and high reproducibility as intra-and interassay variation (Vandesompele et al 2002a;Massart and Jijakli 2005;Goossens et al 2005). In the current study, the expression of 24 (56%) out of 43 AFLPidentified genes [or 84 out of the 129 comparisons of fragment-samples] was reproducibly confirmed to be deregulated by phytoplasma using qRT-PCR.…”

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

“…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). Competition for nutrients is the main mode of action for strain O.…”

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).…”