Screening Of Bio-Control Antagonism And Its Functional Development For Control Soil-Borne Disease

Soil-borne disease, with large number of species, easily transmitted, great harm and hardly prevent, cause great effects on agriculture production. Chemical controls are effective against those diseases. However, repeated application of synthetic pesticides has resulted in a rapid increase in resistance, pesticide residue and environment pollution, so new control measure are needed. Recently, use of biocontrol microorganism to control soil-borne disease become research focuses because of its control effect and safety. Recent studies stimulated the investigation of properties of biocontrol microorganism and concluded that the enzyme activity of cellulase can be as screening index of biocontrol microorganism strain against soil-borne disease.In this paper, 236 strains of microorganisms with cellulase activity were isolated from soil samples, and their antifungal activity was systemic studied. The results showed that among these strains, the strain labeled F10-2 have higher antifungal activity. Then, the control efficacy strain F10-2 against soil-borne pathogen, strain identification, enzyme-producing character of cellulase, optimization of solid state fermentation, integrated utilization of plant pesticide residues degradation were researched systemically . The main results and conclusions were showed as follows:1. 236 strains of microorganisms with cellulase activity were isolated from soil samples, which dug from Taibai vegetable field of Shaanxi Province, Qingling mountain aera and rape field of Xinjiang uighur autonomous region, by using cellulose-congo red selective medium. There were 181 strains of actinomycetes, 64 strains of bacteria, 18 strains of fungi. The antifungal activities of those trains were tested against 7 soil-borne diseases by using method of plate test and mycelium growth rate method. Plate test showed that there are 84 strains of microorganisms with high antifungal activity, which inhibitory rate were greater than 60% against at least one plant pathogen, and possess 31.94% of the total separated microoganisms. Among them, actinomycetes strain was 62 strains (34.25% of all actinomycetes seperated), bacteria was 15 strains (23.44% of all bacteria separated), and fungi was 7 strains (38.89% of all fungi separated). Among 84 strains of microorganisms, mycelium growth rate method showed that inhibitory rates of fermentation broth of 16 strains (active strains) possess higher antifungal activity, and inhibitory rate of 5 strains of them were greater than 60% against 7 test soil-borne pathogen.Rape leaf test result showed that supernatant of fermentation broth and spore suspension (or bacterium suspension) of the above 16 active strains had control effect against Sclerotinia sclerotiorum. Among them, supernatant of fermentation broth of 11 strains, and spore suspension of 3 strains showed higher inhibitory rates, which control effects were greater than 60%. The strain labeled F10-2 have the best control effect, the fermentation broth and spore suspension possessed 85.29% and 93.4% control efficacy, respectively. Pot culture test showed fermentation broth of F10-2 strain had certain control effect against Phytophthora capsici and Verticillium dahliae. The protect effect and therapy effect were 73.82 % and 56.87 % against Phytophthora capsici respectively, and which were 65.21 % and 50.72 % against Verticillium dahliae respectively.2. Antagonistic culture test between F10-2 and Sclerotinia sclerotiorum showed the growth ratio of F10-2 was faster than that of Sclerotinia sclerotiorum. After the 3 days of antagonistic culture, colony of F10-2 began contact with, and then surrounded and covered Sclerotinia sclerotioru. Final, the growth of Sclerotinia sclerotiorum was inhibited, and the pathogen colony was wilt. The result showed the F10-2 had strong inhibitory action. The sprouting test of Sclerotinia sclerotiorum showed that surface of sclerotia was covered by turquoise spore of F10-2, and decayed after 7 days. All results showed spore liquid of F10-2 could control sprouting of sclerotia absolutely. Enzyme activity of ligninase, glucanase and protease were tested in 2 weeks, and results showed ligninase and glucanase had higher activities, while protease activity was too weak to identify. Glucanase arrived the highest activity after 8 days culture, reach to 11.2 U/mL. Ligninase activity arrived the highest activity at 6th day's culture, reach to 7.5 U/mL. The bioassay under the room temperature showed that supernatant of fermentation broth of F 10-2 had higher inhibitory activity than spore suspension against Sclerotinia sclerotiorum. Pot culture test showed percentage of the disease leaves was 4.75%, and the control effect was 86.81% treated by fermentation broth. The inhibitory ratio was decreased following the concentration reduce of fermentation broth. Under the concentration of 10 times'dilution, the control effect was 39.44% only. Series of spore concentration treating of F10-2 spore showed the lowest percentage of the disease leaves was 6.67%, and the control effect was 95.56 % after treatment by 107 spores per micro litter.3. According to identification method of Pitt Penicillium, combined with molecular biology method and alignment analyses, the F10-2 strain was identified as Penicillium wakmanii.4. Enzyme activity test of cellulase liquid showed that endoglucanase had the highest enzyme activity, followed by exoglucanase, and activity ofβ-glucosidase was the lowest. At the 4th, 6th and 8th day of fermentation, enzyme activities of endoglucanase, exoglucanase andβ-glucosidase were up to the maximum, which was 8.79 U/mL,4.30 U/mL and 1.71 U/mL, reseparately. According the producing curve of xylanase of F10-2, the quantity of enzyme was rising rapidly after 2 days'culture. The enzyme activity was trend to stability between 6 and 8 days'culture. At the 6th day of culture, activity of xylanase reached to maximum value of 2.21 U/mL. Cellulase characters of F10-2 indicated that the optimum pH was 5～6, and the optimum temperature was 55℃for enzyme action. Enzyme stability decreased when the temperature was higher than 60℃.5. In order to solve the secondary pollution of plant extract residue, 7 plant pesticide draffs were used as fermentation substrate and the optimized fermenting conditions were determined by using single factor method and the response surface method. The results showed that chinaberry residues can be as optimum fermentation substrate, and the optimum composition of the fermentation medium (g/L) and conditions: chinaberry residues 8 g, bran 2 g, peptone 0.14 g, KH2PO4 0.04 g, initial pH 6.2, water ration 2:1. After optimization, ,the cellulose activity of strain F 10-2 was 6.47 U·g-1, which improved by 46·38% than original culture conditions.6. Chinaberry residues was used as fermentation substrate, and excrete character of cellulose and change of draff in structure after degradation were explored. The result showed that Penicillium wakmanii (F10-2) can grow in bark draff of chinaberry residues, and produce cellulose degrading enzyme. The scanning electron microscope and infrared spectrum analysis showed that the draff become loosen and have arisen interspace, the relative chemical functional group change, Degradating rate of F10-2 was 42.7%, 33.96% and 24.62% against cellulose, hemicellulose and lignin, reseparately. Those indicated that the degradating of bark draff was as a result of joint action of cellulase, hemicellulase, lignin peroxidase, manganese peroxidase.The fermentation production of chinaberry residues after 7 day fermentation was extract with water (Product:Water=1:10 in quality), and the cellulase activity can reach to 4.08 U/mL, and spore concentration can reach to 4.3×107 per micro litter. The fermentation liquid were formulated by simply formulation study, the liquid possesses excellent antifungal activities, the 2 fold dilution of fermentation liquid had control effect of 91.20% against Sclerotinia sclerotiorum, is higher than original fermentation liquid.The comprehensive analysis results indicated that separated F10-2 strain can culture cellulose as unique carbon source, and even high efficiently utilize plant pesticide draff to ferment and produce high cellulose activity formulation. Those finding provided a new approach to eliminate secondary pollution of plant pesticide draff and integrated utilization of plant draff. The enzyme activity of F10-2 strain fermentation is higher and enzyme system have completed function, can be as excellent cellulase producing strain. The formulation of the strain fermentation can control Sclerotinia sclerotiorum and other important soil-borne disease efficiently by nutrition competition and mycelium growth inhibition. Furthermore, direct utilization of bio-control strain of cellulase producing for prevention and cure of important soil-boron disease is a preliminary attempt under the guide of IPM and IPP theory.