| Citrus is the most important fruit in the world. During the process of postharvest store, transportation and distribution, a big lost has been caused by fungal pathogen. For citrus, the foremost pathogen is blue and green mould caused by Penicillium italicum Wehmer and Penicillium digitatum Sacc. By now, postharvest pathogens of citrus were controlled primarily by chemical fungicides. However, it seem urgency to develop alternative methods, because of fungicide residual, development of fungicide-resistant strains of pathogen, environmental pollution and human health threaten.Kloeckera apiculata 34-9 (KA) isolated from soil had been proved good for controlling the blue and green mould of citrus. In this study, the mode of action of K. apiculata was studied, including secreting antifungal compounds, inducing the resistance of citrus tissue, parasitism and competing for nutrients and space with fungal pathogen. And the results are as followed:1. Antifungal compound. Through (NH4)2SO4 precipitation, organic solvents extraction and cell-free zymotic fluid ultra-low temperature evaporation, we found that aether extraction had steady antifungal activity. For aether extraction, TLC, HPLC, GC-MS and LC-MS were applied for separation, purification and identification of antifungal compound. The result showed that the main antifungal compound in aether extraction was 2-phenylethanol (PEA). "In vitro",0.15% PEA can absolutely inhibit the growth of the blue and green mould; "In vivo ", through "Newhall Navel Orange" storing test, PEA can effectively decreased the rot rate amount to prochloraz (PCZ) and not obviously alter fruit quality (vitamin C, soluble solids content and titratable acid). In order to study the action mechanism of PEA, RNA-seq was applied in this part. Compared to control,1933 and 1909 genes were found with significant different expressions (|log2Ratio|≥1) in response to PEA treat 1 h and 3 h respectively. Through analysis we found that PEA can obviously inhibit:(1) DNA replication and repair, (2) RNA transcription and translation, (3) amino acids and amino acid-tRNA synthesize, (4) cells cycle. And PEA can also induce:(1) lipid acids and proteins metabolism, (2) phagosome, proteasome and peroxisome formation, ultimately induced fungal cells programmed cell death (PCD).2. Induced resistance in the citrus tissue. The enzymes assays result revealed that K. apiculata stimulate the activity of SOD, POD, CAT, P-1,3-glucanase and chitinase, production of H2O2 in the peel of citrus around of the wound. Similar result was found in aether extraction or PEA treatment. In genechip, K. apiculata treatment can induces a lot of resistance related genes expression, such as ethylene (ET), jasmonate (JA), abscisic acid (ABA), PR-protein, peroxidases, chitinase, P-1,3-glucanase and phenylanine/ phenylpropanodds related genes, meanwhile, the treatment inhibits lipid and cell wall metabolism related genes expression. In genechip,57.4% of the 803 genes with significant differences in expression in response to K. apiculata versus the control were also altered by treatment with aether extraction or PEA. Only 339 genes were affected under aether extraction treanment can be explained that the extent of aether extraction-responsive transcriptional changes varies depending upon the concentration of aether extraction. Those results showed that (1) K. apiculata induced the resistance of citrus tissue partly via excreting PEA or some other active compounds; (2) K. apiculata was likely to stimulate activity of SOD and product of H2O2 initially, and H2O2 as a global regulator can shift redox homeostasis in the citrus tissue and activate defense mechanisms (JA/ET signal pathway) in citrus tissue, such as the genes expression of PR proteins and phenypropanoid biosynthesis pathways.3. Parasitism and competition for space. In bead sprouts medium (BSM) medium, we found that K. apiculata flocculated strongly (79.2%). "In vivo", we found that K. apiculata can strongly adhered to P. italicum mycelium by "lectin". By adhering one cell to the other in citrus surface or wounds, K. apiculata can form a dense layer of cell embedded within an abundant extracelluler polymer substances (EPS), which created a mechanical barrier interposing between the wound surface and the pathogen, thus hamper infection by P. italicum or P. digitatum. These phenomenons implied that the mode of action of K. apiculata to control P. italicum and P. digitatum involved parasitism and competition for space.4. Without competition for nutrients of carbon and nitrogen source. Through analyzing carbon source of K. apiculata, P. italicum and P. digitatum, we found that K. apiculata only can use dextrose, fructose and cellobiose as carbon source, however, P. italicum and P. digitatum can use most of tested carbon source, such as sucrose. For nitrogen source, all of them can utilize most of tested nitrogen source, such as (NH4)2SO4·In citrus fruit, the main dissolubility sugars (main carbon source) are dextrose, fructose and sucrose, and available nitrogen source are amino acids, polypeptide and proteins. Among them, sucrose only can be used by P. italicum and P. digitatum, and a lot of optional nitrogen sources can be utilized by K. apiculata, P. italicum and P. digitatum. These results indicated that K. apiculata unlikely to compete with P. italicum and P. digitatum for nutrients of carbon and nitrogen source.In summary, the action mechanisms of K. apiculata against the green and blue mould may include excreting the antifungal compound, inducing the resistance of the citrus tissue, parasitism and competing with P. italicum and P. digitatum for space. |
PDF Full Text Request