Study On Molluscicidal Activity Of Rhizosphere Fungi SL-30 From Phytolacca Acinosa Roxb. Against Oncomelania Hupensis

Snail (Oncomelania hupensis) is the only intermediate host of Schistosoma japonicum, therefore one of the effective preventive methods against schistosomiasis is the control of snails to break the life cycle of the parasite. However, the dilemma has been emerging, such as high cost of synthesis, acute and potential toxicity to non-target organisms, deleterious effects on the environment, and so on. The paper aimed to screen molluscicidal microorganisms against Oncomelania hupensis from the rhizosphere of plant, to search effective and safe microbial molluscicidal ingredient, to exploit the rational way of application in situ, and to provide a supplementary method of chemical drugs. The main results was as follows,1,Microorganisms were isolated from fourteen kinds of molluscicidal medicinal plant, finally a fungi strain named as SL-30 exhibiting high molluscicidal activity was obtained from the rhizosphere of Phytolacca acinosa Roxb.. The 4% exocellular broth of SL-30 could kill 100% of snails with submerged period of 24 h, and was safe to fish and shrimp. The exocellular broth of SL-30 could resist heat and illumination to some extent. Strain SL-30 was finally identified as Aspergillus fumigitus based on the colony morphology, conidial head morphology and rDNA-ITS sequence.2,Soluble amylum, peptone and initial pH were decided as important affectois by single factor analysis and Plackett-Burman design. Combined with response surface methodology the optimal fermentation conditions were determined as follows, soluble amylum 16.40 g/L, peptone 3.76 g/L, KH2PO4 0.5 g/L, MgSO40.25 g/L, inoculum size 0.5%, broth volume 50 ml/250 ml, initial pH 3, culture temperature 28℃, and after six batches cultivation, the predicted values were verified by validation experiments.3,Based on the tests of system separation combining molluscicidal activity, The diethyl ether polar fraction from SL-30's exocellular broth exhibited a significant molluscicidal activity against Oncomelannia hupensis, from which the molluscicidal ingredient (MI) was obtained by silica gel column chromatography, with the LC50 and LC90 values of 0.101 mg/L and 0.355 mg/L for 24 h,0.062 mg/L and 0.121 mg/L for 48 h,0.022 mg/L and 0.066 mg/L for 72 h, respectively. The MI was determined as gliotoxin by HPLC,IFR,LC/MS/MS,1H-NMR,13C-NMR,1H-1H COSY and 1H-13C HSQC analysis. The MI was safe to Brachydanio rerio and Rana limnochris under effective molluscicidal concentration, and was low toxicity to Eisenia fetida and soil microorganism.4,The result of morphology of the cells of Snail with Hoechst 33342 dealed with the MI showed that inducing apoptosis was not the main way of the MI to leading to intoxation and death of snails.5,Observed with light microscope and TEM, it was obtained that the tissue form and ultrastructure of snail was changed by the MI. Normal tissue form and cell structure were the structural basis of normal vital movement of organism, if not, it would lead to abnormality of cell function and metabolism of snail.6,Analyse with PAGE, it was showed that the activity and composition of EST isozyme, and deintoxication of snail was influenced by the MI. It would lead to intoxation and death of snail when exceeding the deintoxication capability.7,Determination of enzyme activity showed that, when treated with the MI, the activities of MDH, Na+K+-ATPase and Mg2+-ATPase were enhanced, the CHE activity was weakened, the activities of AKP and GPT were enhanced in the beginning and then weakened, the activity of SDH was weakened in the beginning and then enhanced, and the LDH activity was enhanced in ephalopodium and was weakened in liver. Accordingly, abnormality and derangement of energy metabolism and physiologic function were one of the important reasons of death of snail.8,Results of acute toxicity showed, the conidium of strain SL-30 was safe to waters, fish and shrimp, and was low toxicity to acute toxicity of KM mice by mouth, of SD mice per cutem, and of SD mice by inhalation. After strain SL-30 was inoculated in non-sterilized non-rhizosphere soil, molluscicidal activity appeared in the 20th d, and disappeared in the 40th d. Nevertheless, after the mutant strain Nysr-2 was inoculated in rhizosphere soil, molluscicidal activity remained exist in 90th d, and Nysr-2 could colonized stabily in rhizosphere soil in 60 d. it was thus evident that strain SL-30 could present molluscicidal activity in soil to some extent, and maintained longer period in rhizosphere soil. Accordingly, it was worth further studing that strain SL-30 was appllied in the snail-breeding beach environment of river and lake to present molluscicidal activity as alive thalline.