Biological Control Of Fusarium Wilt Of Cucumber With Trichoderma Harzianum Bioorganic Fertilizer And Its Mechanism

Cucumis sativus L. is a popular vegetable worldwide, and the plantation of cucumber accounts for 20% of the total vegetables in China, even 50% of the winter vegetable production in the major production region. As the agriculture production is industrialized and intensified,a series problem occurs resulting from the continuously cropped system.Fusarium wilt, a major disease in continuously cropped system, caused by soilborne pathogen Fusarium oxysporum f. sp.cucumerinum (FOC) is a main factor limiting the production of cucumber. The disease incidence can be 10-30%, and even high as 70%. On the other hand, agro-industrial wastes such as wastes from animal production, residues from food processing industry and crop straws are disposed any where, causing serious environmental pollution.Based on the mechanism of the continuous cropping disorder and from the viewpoint of alleviating the stress of continuous cropping by disease biocontrol and soil ecological biorestoration, the following studies were done: (1) population of F. oxysporum in cucumber continuously cropped soil, percentage of pathogenic strain of F. oxysporum,effects of soil environmental factors and the interaction of the factors on the proliferation of soil F. oxysporum, (2) isolation and identification of the antagonistic microorganisms to FOC and allelochemicals degradation by the antagonistic microorganisms, (3) the major allelochemicals exuded by cucumber plants in continuously cropped soil, the degradation of allelochemicals in the rhizosphere soil of cucumber plants, (4) separation and identification and determination of the antifungal activity of the secondary metabolite of the biocontrol agent, (5) effects of the inoculation of the biocontrol agent in the continuously cropped soil on the soil microbial community, (6) selection of the optimum formation of the biocontrol agent on biocontrol of Fusarium wilt, the production of bioorganic fertilizer with the biocontrol agent and antifungal metabolite by the solid-state fermentation of the agro-industrial waste with statistical method, effect of bioorganic fertilizer applied on the biocontrol of Fusarium wilt in the continuously cropped soil,and degradation of the phenolic allelochemicals, and restoration of the soil microbial community. Results obtained were listed as follow:Population of F. oxysporum in 5-year-old cucumber continuously cropped soil of Zhejiang province, Jinhuan city, was 2.02 × 103 CFU g-1 dry soil based on numeration on selective medium. 93.0% of soil F. oxysporum could infect cucumber susceptible cultivar"Xinxiafeng" and 74.6% of cucumber resistant cultivar "Jinchun No.4". The most pathogenic strain of F. oxysporum was identified as F. oxysporum f. sp. cucumerinum based on morphological and molecular identification and infection specialty of the pathogenic F.oxysporum. The direct or mutual effects of soil environmental factors of temperature, pH and water potential on soil F. oxysporum population detected by SYBR Green Real-Time PCR and analyzed by response surface methodology showed that the three factors significantly (p<0.05) affected soil F. oxysporum population. The predicted maximum soil F. oxysporum population could be at soil pH of 5.4, soil temperature of 27.4 ℃ and soil water potential of -8.1 kPa. This was accordance to the practical soil condition when Fusarium wilt broke out.One microorganism isolated with a capacity of both controlling FOC and degrading allelochemicals was identified as Trichoderma harzianum and named as SQR-T037.SQR-T037 could inhibited the growth of FOC by 91.4% as compared with the control, and could quickly degrade the allelochemicals both on mediums of each allelochemical as single carbon source, mixed allelochemicals as carbon source and nutritional medium added with mixed allelochemicals. No new phenolic allelochemicals was metabolized by SQR-T037 in the process of degrading the allelochemicals. SQR-T037 showed strong enzyme activity of chitinase, cellulase, β-1,3-glucanase, xylanase, laccase, pectinase and proteinase. The optimum conditions for the growth of SQR-T037 was PDA medium, pH 6.5 and 24 ℃ temperature. Medium of PDA, or corn flour and sucrose, PDA at pH 6.0 and temperature of 25 ℃ was optimum for the production of antifungal secondary metabolites of SQR-T037.Based on the analysis of mass spectrometer and HPLC,the allelochemicals cucumber present in cucumber continuously cropped soil and collected with Rhizobox were 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropionic acid and cinnamic acid. Three allelochemicals of 4-hydroxybenzoic acid, cinnamic acid and benzoic acid were detected in cucumber continuously cropped field with concentration of 1.6, 6.6 and 2.4 μg kg-1 dry soil, which quickly decreased to 38.9%, 35.3% and 53.0%, respectively,at 10 days after the cucumber seedlings harvested, and completely disappeared at 30 days after the cucumber seedlings harvested. The experiments were also carried out to detect the capability of SQR-T037 to decrease the concentration of allelochemicals of cucumber root exudates and decomposition of plant residues in the cucumber continuously cropped soil.The results showed that SQR-T037 could decrease the concentration of allelochemicals from cucumber root exudates in the rhizosphere soil, and the decreased percentages of 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropanoic acid and cinnamic acid were 88.8%, 90.0%, 100%, 94.9%, 100% and 100%, respectively.SQR-T037 could completely degrade the allelochemicals from the decomposition of root residues, except for at the 40 days after cucumber plants were harvested, concentrations of allelochemicals of 4-hydroxybenzoic acid, cinnamic acid and benzoic acid were 15.9%,2.9% and 33.5% of control treatment, respectively. The population of soil F. oxysporum increased as the root residues decomposed, and SQR-T037 decreased the population of F. oxysporum consistently. SQR-T037 could decrease concentration of allelochemicals in the cucumber continuously cropped soil and thus decreased disease index of Fusarium wilt and increased dry weight of cucumber seedlings.Antifungal chemicals from SQR-T037 which could be used effectively against FOC were purified by fraction separation and HPLC, and identified using both mass spectrometry and nuclear magnetic resonance spectroscopy, and determined antifungal activity against FOC in vivo and in vitro. The antifungal chemical was identified as 6-pentyl-a-pyrone (6PAP). An in vitro assay of a series of 6PAP concentrations (50, 150,250, 350 and 450 mg L"1) in medium showed that the antifungal activity increased as the concentration increased. At 350 mg L-1 of 6PAP, 73.7% of mycelia growth and 79.6% of conidia germination was inhibited as compared with the control. Additionally,at a 6PAP concentration of 150 mg L-1, sporulation and fusaric acid production (mg g-1 dry mycelia)of FOC were decreased by 88.0% and 52.7%, respectively. Furthermore, 6PAP produced a significant effect on Fusarium wilt control in cucumber continuously cropped soil in pot experiments. Compared to control treatment, 6PAP, at concentration of 350 and 450 mg kg-1 soil, decreased populations of soil F. oxysporum by 41.2% and 83.2%, respectively. In the 350 mg kg-1 soil 6PAP treatment, disease incidence was under 15.0%, while 68.3-80.0% disease incidence occurred in the control. Adding 350 mg kg-1 soil 6PAP could increase the dry weight of the cucumber plant by 37.5-48.1%. 6PAP demonstrated antifungal activity against FOC and could control Fusarium wilt in cucumber continuously cropped soil.Cucumber continuous cropping caused deleterious effect on soil microbial community by root exudates,which simplified microbial community structure,increased soilborne pathogens and degraded soil microbial function. The aim of this study was to detect the effects of inoculation with SQR-T037 on the rhizosphere soil community structure. The experiment carried out in Rhizobox showed that, compared with control treatment,inoculation of SQR-T037 differentiated community structures while analyzed PCR-DGGE fingerprints by both pairwise similarity and cluster analysis. Compared with control treatment, inoculation of SQR-T037 significantly increased microbial diversity(Shannon-Weaver index) values by 17.1% and 41.2% in bacteria and fungi, respectively.Inoculating SQR-T037 increased culturable microbes of fungi and bacteria, and decreased the population of pathogenic microorganism group of F. oxysporum. Also, decrease of allelochemical concentration and disease incidence of Fusarium wilt and increase of dry weight of cucumber seedlings were found in SQR-T037 treatment. The results suggested that SQR-T037 could improve the health of cucumber continuously cropped soil by diversifying the soil microbial community.Pot experiments were carried out over two growing periods to assay the biocontrol efficacy and rhizosphere colonization of SQR-T037 applied as SQR-T037 conidia suspension (TCS),SQR-T03 7 conidia suspension blended with organic fertilizer (TBF) or SQR-T037 fermented organic fertilizer (TFF). Each formulation had three levels of T.harzianum numbers. In the two experiments, the disease incidence decreased with the increase of SQR-T037 number added to soils. The TFF treatment consistently exhibited the lowest disease incidence at same amendment rate of SQR-T037 and 0-8.9%, 25.6%-78.9%and 4.4%-50.0% of disease incidence were found in TFF, TCS and TBF treatment,respectively. Soils treated with TFF showed the highest SQR-T037 population detected by TaqMan Real-Time PCR in rhizosphere and bulk soil. Decrease of F. oxysporum population in both bulk and rhizosphere soils occurred in the treatment SQR-T037 at 105 and 106 CFU g-1 soil rate. The TFF treatment at the SQR-T037 rate of 103 CFU g-1 soil significantly (p<0.05) increased SQR-T037 population within the rhizoplane, but no effect was found on F. oxysporum population, when compared to TCS and TBF. Among the different biocontrol formulations TCS (106), TBF (106) and TBF (106), organic fertilizer based formulations, TBF (106) and TFF (106) showed higher (p<0.05) capacity on inducing disease resistance; TFF (106) treatment exhibited higher (p<0.05) activity of chitinase andβ-1,3-glucanse than TBF (106). Generally, TFF treatments were superior to TCS and TBF treatments on disease control by sustaining colonization of SQR-T037 and decreasing F.oxysporum abundance in the rhizosphere soil and inducing of higher disease resistance. We propose that TFF treatment at SQR-T037 rate of 107 CFU g-1 (i.e. 105 CFU g-1 soil after applied to soil) was the best formulation for controlling Fusarium wilt of cucumber.Therefore, agro-industrial wastes, such as cattle dung, vinegar-production residue and rice straw, were solid-state fermented by inoculation with SQR-T037 for production of bioorganic fertilizers (BIO) containing SQR-T037 and 6PAP which had a high biocontrol capacity against Fusarium wilt of cucumber. Variations in the number of fermentation days,the temperature of fermentation, the inoculum content and the ratio of vinegar-production residue added demonstrated significant effects on the SQR-T037 biomass and the yield of 6PAP,vased on the 26-2 fractional factorial design of the experiment. These four significant factors were subsequently optimized using a central composite design. Three optimum conditions were obtained by response surface analysis and confirmed by validation experiments to produce the maximum SQR-T037 biomass and 6PAP yield. Fermented BIO production with actual values of 8.46 Log10 ITS copies g-1 dry weight of SQR-T037 and 1291.73 mg kg-1 dry weight of 6PAP, which could have highest (p<0.05) biocontrol efficacy of Fusarium wilt of cucumber in continuously cropped soil, was achieved from 36.7 days fermentation at 25.9 ℃, with a substrate composition of 7.6% inoculum, 41.0%vinegar-production residue, 20.0% rice straw and 39.0% cattle dung. The BIO produced offered a high efficacy method for the biocontrol of cucumber Fusarium wilt in continuously cropped soil and a value-added utilization for agro-industrial waste. The effects of the different application methods of the BIO,applied in nursery soils (N treatment), or in both nursery soils and transplanted soils (N+T treatment), on the biocontrol efficacy of Fusarium wilt and restoration of the soil microbial community were studied. The results showed that the two application ways, N treatment and N+T treatment could decrease Fusarium wilt, the percentage disease indexes of which were 25.4% and 15.1%, respectively, while the control was 72.2%. Biocontrol efficacy of N+T treatment was significantly (p<0.05) higher than N treatment. The dry weights of the treatments showed same trends as the biocontrol efficacy. The two application ways could increase SQR-T037 population both in rhizosphere soil and rhizoplane. The N+T treatment showed higher (p<0.05) efficacy than N treatment in increasing population of SQR-T037 in the rhizosphere and rhizoplane. The two application ways significantly (p<0.05) deceased F.oxysporum population as compared with control, and N+T treatment showed higher efficacy (p<0.05). The two application ways could induce disease resistance that enzyme activity of chitinase, β-1,3-glucanase and polyphenol oxidase were significantly higher(p<0.05) than control at the 8 and 30 days after the transplanting of the seedlings. Moreover,at the 35 days after transplanting, the N+T treatment showed higher (p<0.05) enzyme activity of chitinase and β-1,3-glucanase than N treatment. The two application ways could increase microbial diversity of soil. Based on the analysis of DGGE,Shannon-Wiener indexes of soil Trichoderma spp., fungi and bacteria of N+T treatment were highest among all the treatments, including the Uncropped soil. Cluster analysis of the DGGE profiles of the Trichoderma spp.,fungi and bacteria showed that the microbial communities of Trichoderma spp. and bacteria of N+T treatment were close to Uncropped soil, and fungal community of N+T treatment was diversified by slightly changged. Assay of soil culturable microbial populations showed that N+T treatment significantly (p<0.05) increased populations of Trichoderma spp., fungi and bacteria. Three phenolic acids of 4-hydroxybenzoic acid,cinnamic acid and benzoic acid were detected in cucumber rhizosphere soil in control treatment with concentration of 7.54、1.79 and 14.00 μg kg-1 dry soil, respectively. None of the phenolic acids was detected in the BIO applied treatments.Application of BIO both in seedling nursery and in transplanted soils could control the Fusarium wilt and restore the microbial community of cucumber continuously cropped soil.The effects of SQR-T037 BIO on the disease control and production of cucumber in continuously cropped soil in the field experiment showed that the BIO could decrease disease incidence of Fusarium wilt by 81.2% (disease incidence of the control was 18.6%,and SQR-T037 treatment was 3.5%), and increase yield by 20.7%. SQR-T037 BIO could alleviate the stress of the continuous cropping disorder of cucumber.