Study On Antifungal Active Substances Of Biocontrol Yeast 34-9 Of Citrus

Postharvest losses of Citrus may reach very high values due to postharvest diseases, and blue mold and green mold caused by Penicillium italicum and P. digitatum often account for a major part of the losses. Biological control of postharvest diseases by antagonistic microorganisms has been studied home and abroad, but only a few could be applied in practical. It's mainly because the effect of biocontrol agents is not as good as chemical fungicide and the fermentation technology of biocontrol agents is not so perfect. Therefore it's urgent to study the mechanism of biocontrol agents to enhance the antagonistic activity and improve the biological characteristics.The aim of this paper is to work out the mechanism of producing antifungal active substances of Kloeckera apiculata (34-9). It mainly included that the antifungal active substances were extracted, isolated and purified crudely, the antifungal spectra was detected, physiological and biochemical characteristics were studied, and the fermentation technology was investigated. The antagonistic activity was confirmed in vitro and in vivo. The main results were as follows:1. The extract process of the antifungal active substances was ascertained. It could not be precipitated by (NH₄)₂SO₄ at different degree of saturation but could be extracted by ethylether from inhibition zone and cell-free fermentation broth. Yeast cells could be effectual disrupted in suspension by microwave treatment. The antifungal active substances could also be extracted by ethylether from the solution of disrupted cells.2. The results of study on physiological and biochemical characteristics showed that the crude extract could inhibit the pathogenic fungi which cause postharvest diseases of Citrus, Vitis and Mango in common. It could resist to 121℃, 20min. Either trypase or Proteinase K could not destroy the activity of the crude extract. The stability of fungistasis could be kept at least in a month at 4℃. The production of antifungal active substances could be detected in the middle of log phase, and reached to a higher concentration at the end of log phase. There was no marked change after stationary phase. The result of color reactions showed that the crude extract was acidic, and had the positive reaction of phenols and flavonoid. The optimal developing agent (phene: ethylether=5:5) in TLC was found. Antifungal active substances which were purified was reclaimed at the spot where the Rf value was 0.78. The purity coefficient was detected by HPLC(Mobile phase ,45% v/v methanol; detector, UV 190nm-600nm). 3. The investigation of fermentation technology indicated that nature pH, 1% inoculum size, 50ml fermentation broth in 250ml shake flask, 24h ferment time were the optimal condition in which the yield of the antifungal active substances was highest. The crude extract after decade diluted could completely inhibite germination of P. italicum spores in vitro. P. digitatum could be inhibited significantly by the crude extract in water solution in vivo. It could preserve Citrus from decay over 96%. After 6 days in store, preservation of the crude extract was higher than the fermentation broth.