New sustainable approaches for control of postharvest diseases of citrus fruit
Injuries sustained by citrus fruit during harvest and postharvest handling and storage allow the entry of wound pathogens, including Penicillium digitatum Sacc (green mold) and P. italicum Wehmer (blue mold), the two major pathogens of citrus fruit being the causal agents of green and blue mold diseases, respectively. Control of these pathogens has classically been conducted with chemical fungicides, but a growing international concern over the often indiscriminate use of synthetic fungicides, the presence of residues in the food and in the environment, as well as the development of fungal resistance and the possible harmful effects on human health, make it necessary to undertake new approaches for disease control that minimize health and environmental risks. In this work we evaluated the effectiveness of inducers of disease resistance for postharvest control of green mold of citrus. The chemical inducers exerted direct antimicrobial activity against of P. digitatum, P. italicum and Geotrichum candidum when tested in vitro on PDA and on orange peel extract agar media, with differences related to their concentrations and challenged pathogens. The antimicrobial efficacy of BABA and chitosan was also confirmed by their ability to inhibit in vitro P. digitatum conidial germination. Moreover, BABA and chitosan significantly reduced disease incidence and severity when applied on citrus fruit. On the opposite, none of ASM concentrations conferred protection against P. digitatum. The induction of resistance of BABA compound was detected on lemon and grapefruit when P. digitatum was inoculated into wounds made at 1 cm from the original BABA-treated sites. Curative and preventive treatments with Pseudomonas syringae, Trichoderma atroviride and Wickerhamomyces anomalus biocontrol strains were evaluated alone and in different combined mixtures with hot water, sodium carbonate, sodium bicarbonate, ASM and chitosan. Combined applications of sodium bicarbonate, hot water and biocontrol agents efficiently controlled green mold when treatments were applied 24 and 48 hours before the pathogen inoculation. When sodium carbonate, hot water and biocontrol agents were co-inoculated with P. digitatum green mold was significantly controlled as well even if less effectively, whereas fruit inoculated 24h before treatments developed green mold, and no treatment managed to control significantly P. digitatum. We have analysed gene expression levels of chitinase, b-1,3-glucanase, phenylalanine ammonia lyase, gluthatione peroxidase in responses to curative and preventive biocontrol treatments. On the whole, CHI1, PAL and GPX1 over-expressions were observed within 2h after treatments, but only CHI1 over-expresion was observed for each treatment (curative and protectives). Quantitative RT-PCR technique showed that treatment with sodium bicarbonate, hot water and Pseudomonas syringae pv. syringae 48SR2 induced the highest increases in the levels of chitinase gene transcripts 48h after treatments and 2h after pathogen inoculation. P. syringae pv. syringae strains are able produce cyclic lipodepsipeptides correlated with the ability to control postharvest fungal pathogens. In this work treatment with sodium bicarbonate, hot water and Pss 48SR2 generated weak over-expression of syrB1 gene and high over-expression of sypA in citrus peel tissue 48h after teatment and in absence of P. digitatum. However, P. digitatum inoculation strongly induced syrB1 gene expression and, on the opposite, weakly induced sypA genes expression. Pathogen inoculation resulted to be strongly stimulatory only to syrB1 expression, suggesting that at least syrB1 gene is involved in biocontrol activity. These data indicate type of treatments that could be successfully used to control post-harvest fruit diseases and suggest that a signal produced by the pathogen is essential for triggering enhanced transcription levels of both defence-related and lipodepsipeptides synthetase genes.
Citrus fruit, postharvest decay, Penicillium digitatum, biocontrol agents, chemical elicitors, gene expression
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