Pichia anomala in biocontrol for apples: 20 years of fundamental research and practical applications.

Fungal pathogens such as Botrytis cinerea, Penicillium expansum and the Gloeosporioides group are mainly responsible for important economical losses of post-harvest apples. Application of biological control agents (BCAs) is an emerging alternative to synthetic fungicides. However, before becoming an economically feasible alternative to chemical control, BCAs have to satisfy different requirements related to biological, technological and toxicological properties. The different steps for a successful strategy of disease control (selection, production and formulation, study of mechanisms of action, ecological characterization, molecular monitoring, pilot efficacy trials, registration) are reviewed in this paper considering the antagonistic yeast Pichia anomala strain K. This strain was selected for its high and reliable antagonistic activity against B. cinerea and P. expansum on apples. The studies of mode of action and ecological fitness are emphasized because they can lead to a better efficacy of strain K. Recently advanced molecular techniques have contributed to improving knowledge on the modes of action. Thanks to the identification of genes involved in biocontrol properties, the genetic basis of action mechanisms can be understood. That approach was adopted for P. anomala strain K and led to the identification of genes coding for exo-ß-1,3-glucanases implicated in the efficacy. Based on that identification, a formulation involving ß-1,3-glucans was developed and applied with higher efficacy in controlled conditions. The importance of ecological characterisation is also highlighted in the context of pre-harvest application of P. anomala strain K. UV light, temperature and humidity were identified as major factors influencing the strain K population. A model taking into consideration temperature and humidity was developed and could be useful in deciding whether pre-harvest treatment is sufficient to allow fast colonization of wounds prior to the arrival of wound pathogens, or whether it is wise to apply further post-harvest treatment to increase the yeast population density. This summary presenting 20 years of work also paid attention to practical application of strain K and its integration with other methods of control.

Catecholamine biosynthesis pathway potentially involved in banana defense mechanisms to crown rot disease.

Variations in Cavendish bananas susceptibility to crown rot disease have been observed (Lassois et al., 2010a), but the molecular mechanisms underlying these quantitative host-pathogen relationships were still unknown. The present study was designed to compare gene expression between bananas (Musa acuminata, AAA, 'Grande-Naine') showing a high post-harvest susceptibility (S+) and bananas showing a low post-harvest susceptibility (S-) to crown rot disease. This comparison was performed between crowns (S+ and S-) collected one hour before standardized artificial inoculations with Colletotrichum musae. Fruit susceptibility was evaluated through lesion size on the crown 13 days later. Gene expression comparisons were performed with the cDNA-AFLP technique (Lassois et al., 2009). This revealed that a gene showing a strong homology with a dopamine-beta-monooxygenase (DoH) is differently expressed between S+ and S (Lassois et al., 2011). Furthermore, semi-quantitative real-time RT-PCR analyses between S+ and S- were applied to confirm the differential expression results for DoH obtained by cDNA-AFLP. Two biological replicates were tested. These semi-quantitative analyses were performed not only on tissues collected one hour before C. musae inoculation but also on crown tissues collected 13 days after inoculation. The real-time RT-PCR confirmed that DoH was upregulated in the S tissues collected at harvest, just before C. musae inoculation. This gene was also highly upregulated in the S- tissues collected 13 days after crown inoculation. Similar results were obtained for both biological replicates. Our results suggest that catecholamine's could play a role in banana defense mechanisms to crown rot disease.

Impact of rice-fish/shrimp co-culture on the N2O emission and NH3 volatilization in intensive aquaculture ponds.

How to reduce the gaseous nitrogen (N) pollution (N2O and NH3) of intensive aquaculture ponds to atmosphere has gained increasing attention for the sustainable development of aquaculture. In this study, we constructed a new rice-fish/shrimp co-culture system in aquaculture ponds by using a specially developed high-stalk rice variety, and performed a 2-year field experiment to investigate the effect of this system on the N2O and NH3 emissions from yellow catfish and freshwater shrimp ponds. The results showed that the mean emission factors of N2O and NH3 to the total N input in feed was 0.18% and 0.89% for catfish monoculture pond, and 2.46% and 13.45% for shrimp monoculture pond, respectively. Rice-fish/shrimp co-culture not only reduced the N2O and NH3 emission from rice platform of catfish and shrimp ponds, but also mitigated the N2O and NH3 emission from the ditch without rice planted. The total amount of N2O and NH3 were respectively mitigated by 85.6% and 26.0% for catfish pond, and by 108.3% and 22.6% for shrimp pond, as compared with that of monoculture ponds. Co-culture system was more effective on the mitigation of gaseous N loss in the catfish than shrimp ponds.

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).