| Cucumber (Cucumis sativus Linn) is one of the main greenhouse vegetables which widely distributes around the world. The soil-born disease of cucumber Rhizoctonia damping-off is mainly caused by Rhizoctonia solani, which occurs around the world with different degree and causes a lot of economic loss every year. The control of the disease is a world wide problem. Traditional chemical and farming control methods result in serious environmental, foot safety problems and time-and labor-consuming, thus can not be successfully applied in agricultural production. Biological control of soil-born disease is an efficient and environmentally friendly way and is receiving increased attention.In the present study, we isolated and screened Trichoderma harzianum SQR-T37(T37) and Bacillus pumilus SQR-N43(N43) which showed highly antagonistic effect against Rhizoctonia solani, the pathogen of damping-off disease. The two antagonistic strains and their bio-organic fertilizers were used in pot experiments to assess their control efficiencies of cucumber damping-off disease. The populations of pathogen and antagonistic strains and microbial communities in the rhizosphere soils after applied with antagonistic strains and their bio-organic fertilizers were also determined. Besides, the abilities of colonization in cucumber rhizosphere and the mechanisms of antagonism against the pathogen of the antagonists were studied. The influences of plant root exudates on the antagonistic strains were also investigated.The main results obtained were listed as follows:1. In this study, Rhizoctonia solani Q1was verified as the pathogen of cucumber damping-off disease using Koch’s postulates. More than400bacterial strains were isolated from rhizosphere soil of cucumber plants, and the dual culture method was used to screen bacterial antagonists against R. solani. Sixteen strains of bacterial antagonists were isolated and their inhabiting zone against R. solani ranged from0.8cm to1.9cm. Three strains (N33, N34and N43) with inhabiting zone more than1.6cm could produce volatile antibiotics and soluble antibiotics. Based on the results of morphologic characteristics, physiological and biochemical properties and phylogenetic analysis of16S rDNA, the three strains (Genbank accession number HM439651, GQ465936and GQ465935, respectively) were identified as Pseudomonas fluorescens, Bacillus pumilus and Bacillus pumilus, respectively. Antagonists of B. pumilus SQR-N43and Trichoderma harzianum SQR-T37were further chosen for following experiments.2. Pot Experiments were performed to assess the promotional efficiency on the growth of cucumber and in vivo disease-control efficiency of the antagonistic microbes and bio-organic fertilizers (BIOs). Results indicated that compared with control, Bacillus pumilus SQR-N43and Trichoderma harzianum SQR-T37increased the dry weight of cucumber shoot by38.6%and55.9%, respectively. The antagonists N43and T37could control damping-off disease of cucumber; further more, the control efficiency of T37increased during the second growth cycle by25.0%as compared with the first growth cycle. Besides, the control efficiencies of BIO products (68.2%and81.8%, respectively) were higher than those applied with antagonist N43or T37solely (22.7%and27.3%, respectively). The population of R. solani in the rhizosphere soil of cucumber was decreased from106ITS copies g-1soil to104ITS copies g-1soil in the presence of the antagonists. Twenty days after incubation, the population of T. harzianum was107ITS copies g-1soil in the BIO treatment, which was much higher than that in the treatment only T37was applied (105ITS copies g-1soil). Significant differences in the number of CFUs and the percentage of spores of N43were recorded between the N treatment (2.20×107CFU g’1of soil and79%, respectively) and the BIO treatment (1.67×108CFU g-1of soil and52%, respectively). The antagonists N43and T37and BIOs decreased the number of harmful fungal species, such as Rhizoctonia solani, Setosphaeria sp. and Lecythophora, and increased the population of bacteria and the number of bacterial species. As a conclusion, the antagonistic microbes and BIOs could promote the growth of cucumber, prevent the damping-off disease of cucumber, decrease the population of the pathogen in the rhizosphere soil, and improve the rhizosphere microbial community. Among of them, the BIOs had the best efficiency.3. For clarification of the mechanisms of the antagonism of Trichoderma harzianum SQR-T37and Bacillus pumilus SQR-N43against Rhizoctonia solani Q1, the effect of two antagonistic strains on the pathogen mycelium was microscopically observed and the activities of chitinase and β-1,3-glucanase produced by T37and production of antibiotic by N43were determined. Result showed that the mycelia of T37coiled themselves round and penetrated into the mycelia of pathogen, causing leakage of cytoplasm and crimple of pathogen cell walls. Both fresh and dried cell walls of the pathogen could induce the production of chitinase (9.49and10.80nkat ml-1crude enzyme, respectively) and β-1,3-glucanase (37.57and43.18nkat ml-1crude enzyme, respectively). N43produced antibiotic substances towards R. solani Ql at logarithmic growth phase. The antibiotics caused enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1mycelia. The activities of the antibiotics had the highest value when the concentration of NaCl in the medium was1%(w/v).70%saturation of ammonium sulfate made complete precipitation of the antibiotics in culture broth. When treated with protease K and trypsase, the activities of antibiotics were decreased by79%and53%, respectively, compared with control. The antibiotics were sensitive to high temperature and were alkaline stable. The molecular weights of the substances were between500-1000Da. The control efficiencies of the antibiotics and cell suspension of N43on damping-off disease had no significant difference. As a conclusion, the mechanism of antagonism of T37against R. solani Q1was mycoparasitism. N43produced oligopeptides which had inhibitory effect on R. solani Q1.4. In this study, green fluorescent protein tagged Bacillus pumilus SQR-N43strain (GFP-N43) was constructed and abilities of both GFP-N43and Trichoderma harzianum SQR-T37to colonize roots and the rhizosphere soil of cucumber were determined. Results showed that constructed GFP-N43strain could emit green fluorescent. When grew up to89generations,43.3%of population of the strain still contained plasmid pHAPⅡ. The inhibitory effect of GFP-N43against Rhizoctonia solani Q1was identical with that of N43strain. In the treatments inoculated with107and108GFP-N43CFU g-1of soil, the presence of GFP-N43through colonization and formation of biofilm on tips and elongation zones of plant roots could ensure the protection of the hosts from the pathogen. Quantitative measurement showed that in the treatments of10and10GFP-N43CFU g-1soil, the numbers of GFP-N43strain decreased to4.6×106CFU g-1soil and1.1×107CFU g-1soil, respectively, at14days after inoculation (DAI). However, in the treatments inoculated with105and106GFP-N43CFU g-1soil, the populations of GFP-N43strain had upward trends in the period from0to14DAI, and GFP-N43strain at14DAI were3.55and2.09times higher than those at0DAI, respectively. The hyphae of T37strain coiled round cucumber roots and formed protection layer on cucumber root surfaces.5. The organic acids in cucumber root exudates were identified by high performance liquid chromatography (HPLC). Chemotaxis of B. pumilus SQR-N43towards organic acids, utilization of organic acids by T. harzianum SQR-T37strain and the influences of organic acids on T37conidial germination and mycelial growth were also determined. Results indicated that oxalic acid, malic acid, acetic acid, citric acid and succinic acid were the mainly organic acids in cucumber root exudates. N43exhibited positive, concentration-dependent chemotactic behavior towards malic acid and citric acid with a relative chemotaxis index3.1and2.3, respectively. Except for succinic acid, the organic acids had different degrees of promotional effects on T37conidial germination. The largest number of germinated conidia which increased by39%, compared with control, was obtained with30μg ml-1oxalic acid. The five organic acids had various degree of promotional effect on T37mycelial growth. The largest diameter of T37colony, which was1.1times wider than that in control, was obtained with80μg ml-1malic acid. When supplied with the five organic acids, T37strain had a preference for acetic acid, oxalic acid and malic acid. |
PDF Full Text Request