Antifungal Activities Of The Extracts From Curcuma Phaeocaulis Against Phoma Wasabiae

Wasabi(Eutrema wasabi Maxim) had been planted in China in 1990s later periods and taken along with the pathogen Phoma wasabiae Yokogi at the same time. Wasabi steak was the new plant disease in our country. The prevention against it was through agricultural and chemical measures since 1990s, but the effect of prevention was not obvious. Application of chemical and pesticides has brought entironment pollution, so biological-control for natural antagonistic organisms is a potential measure against plant diseases. One kind of traditional Chinese herb which could inhibit P. wasabiae Yokogi was screened and further study results were reported in this paper.Four Chinese herbs, including Scutellaria baicalensis Gerorgi, Belamcanda chinensis (L.) DC, Stemona japonica (Bl) Miq, Curcuma phaeocaulis Val, were extracted by different ways. During the extraction, water and 95% ethanol were used as solvents. Then those extracts were dissolved in water for the experiment in order to systematically investigate their fungistasis by growth rate method while P.wasabiae Yokogi was used as the pathogenic fungi. The antifungal activities of extraction by different solvents were quite different, and 95% ethanol extracts from C. phaeocaulis gave the best result. Results showed that the inhibition rate of 95% ethanol extracts from C. phaeocaulis was 67.08% on P. wasabiae Yokogi at the concentration of 1.0 mg/ml. But the extraction rate of C.phaeocaulis by 95% ethanol was only 4.42%. The 95% ethanol extracts of C.phaeocaulis were extracted by skellysolve B, acetic ether and methanol step by step and the fungistasis were tested further. Acetone was used as the solvent for the extracts. The results showed that skellysolve B extracts was the active component and it had 79.9% inhibiting rate on P. wasabiae Yokogi at the concentration of 1.0mg/ml with EC₅₀ being 0.3299 mg/ml, and 97.2% inhibiting rate on spore germination of P.wasabiae Yokogi at the concentration of 10.0 mg/ml. The chromogenic result of different solvent extracts from C. phaeocaulis indicated that curcumine was in the acetic ether extracts with no fungistasis.The chemical components of skellysolve B extracts of C. phaeocaulis had been studied systemically by GC-MS analysis. One hundred and eight peaks appeared in GC. Forty compounds were identified and quantified. The main contents (%) of the skellysolve B extracts of C. phaeocaulis were based on the percent total peak area of each component peak. The major compounds were Aristolene (23.91%) and 1, 2-Dehydrotestosterone (23.63%).In the antimicrobial spectrum test, the growth rate method was adopted to test the fungistasis on seven kinds of pathogenic fungi, including Fusarium graminearum, Sclerotinia sclerotiorum, Chaetomium olivaceum, Penicilium pallidum Smith, Mycogone peniciosa , Botrytis Cirerea, Verticillium dahliae Kled. The study showed that broad-spectrum antifungal active components were present in C. phaeocaulis. Mycelium growth declined when the concentration increased. The EC50 of extracts with skellysolve B of C. phaeocaulis to Fusarium graminearum, Sclerotinia sclerotiorum, Chaetomium olivaceum Cooket Ell, penicilium pallidum Smith, Mycogone peniciosa , Botrytis Cirerea, Verticillium dahliae Kled, were 0.2517 mg/ml, 0.2809 mg/ml, 0.8778 mg/ml, 0.2322 mg/ml, 0.7206 mg/ml, 0.1749 mg/ml, 0.4080 mg/ml, respectively. The skellysolve B extracts of C. phaeocaulis had 100% inhibition rate against Botrytis Cirerea at the concentration of 1.0 mg/ml. Its fungistasis on pathogenic fungi has potential pharmaceutical values, which provides experimental proof for the general application and research of C. phaeocaulis.The stability of skellysolve B extracts from C. phaeocaulis was studied by growth rate method against P. wasabiae Yokogi, Sclerotinia sclerotiorum, and Mycogone peniciosa . When the skellysolve B extracts was treated at 60℃for 10 minutes, or in pH 6.0～10.0, the fungistasis diminished distinctly, but the substance still had inhibitive effect on the pathogen. It is suggested that high temperature and alkalescence treatment should destroy the active components of skellysolve B extracts. Results indicated that the active compound was stable against UV but influenced by high temperature and alkalescence condition.After the treatment with skellysolve B extracts from C. phaeocaulis, the morphology and ultrastructure of P. wasabiae Yokogi and Sclerotinia sclerotiorum showed obvious changes, for example, their cell wall ruptured, intracellar components dissolved, mitochondria swollen and cristae dissolved. There was no change observed in morphology and ultrastructure of samples treated with water or acetone.