| Soilless cultivation develops rapidly in horticulture because it can alleviate continuous cropping obstacles such as soil-borne diseases. The common approach to eradicate obstacles currently is to replace the substrate. So look for new substrate, which can suppress soil-borne diseases and overcome continuous cropping obstacles has been become the important issue of horticulture. Vinegar residue substrate is a new horticultural substrate made from waste residue produced by the vinegar industry. And the results of current research showed that the continuous cropping obstacles did not appear when continuous planting tomatoes or cucumbers for3years. In order to study whether the vinegar residue substrate has suppressive effects on soil-borne diseases, what is the mechanism? This paper studied the suppressive effects of mixed substrates of vinegar residue mixed with peat and vermiculite in different proportions to wilt caused by Fusarium oxysporum and damping off caused by Rhizoctonia solani of cucumber in pot experiment under greenhouse, especially deeply researched the antagonistic microorganisms in vinegar residue substrate. These rearches primary proved the reasons that the vinegar residue substrate can avoid continuous cropping diseases such as wilt, and isolated23antagonistic microorganisms, which provide scientific basis for using of vinegar residue substrate efficient and developing the new substrate with suppressive effects to soil-borne diseases eventually. The main conclusions of this study areas follows:1. Mixed the vinegar residue substrate with peat and vermiculite in different proportions, their suppressiveness to Fusarium oxysporum wilt were studied in pot experiment under greenhouse. These results showed that:①comparison with pure peat, vinegar residue substrate used alone or mixed with peat or vermiculite all had a certain inhibitory effect on cucumber wilt. The ability to suppress diseases of different mixed vinegar residue substrate followed the order:vinegar residue substrate:peat: vermiculite (3:1:1)> pure vinegar residue substrate> vinegar residue substrate peat:vermiculite (6:1:1)> vinegar residue substrate:peat:vermiculite (1:1:1)> vinegar residue substrate:peat (1:1)(ratios are by volume);②The results of Pearson correlation analysis of cucumber wilt disease index between the physicochemical property and the number of microorganisms found that there was significant positive correlation between wilt disease index and C/N (r=0.4788)、bulk density(r=0.6994) respectively; there was a significant negative correlation between cucumber wilt disease index and total porosity (r=-0.6463)、aeration porosity (r=-0.6463_、pH(r=-0.5708) and EC (r=-0.7766) of substrate; there was a significant negative correlation between cucumber wilt disease index and the number of Bacillus spp.(r=-0.5783) in substrate before planting、the number of bacteriar (=-0.5795) and Bacillus spp.(r=-0.386) after planting;③Using the disease index、height、stem diameter、shoot dry weight、root dry weight as an evaluation object to make a principal component analysis of growth of seedlings, the results of comprehensive evaluation found that under inoculation with the pathogen conditions, The synthesis score of seedlings in different mixed vinegar residue substrate followed the order:vinegar residue substrate:peat:vermiculite (3:1:1)> pure vinegar residue substrate> vinegar residue substrate:peat:vermiculite (6:1:1)> vinegar residue substrate:peat:vermiculite (1:1:1)> vinegar residue substrate:peat (1:1)(ratios are by volume).2. Mixed the vinegar residue substrate with peat and vermiculite in different proportions, their suppressiveness to Rhizoctonia solani damping-off were studied in pot experiment under greenhouse. These results showed that:①comparison with pure peat, vinegar residue substrate used alone or mixed with peat or vermiculite all did not have inhibitory effect on cucumber damping-off;②The results of Pearson correlation analysis of disease index values between the physicochemical property(bulk density, total porosity, aeration porosity, water-holding porosity, aeration porosity/water-holding porosity, pH, EC, total carbon, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, available potassium and C/N) and the number of microorganisms (fungi, bacteria, Bacillus spp.) found that there was no significant correlation.3. In the plate experimental, each of the non-sterilized, filter-sterilized and heat-sterilized extracts collected from vinegar residue substrate were added to the PDA medium to study their inhibitory effect on mycelial growth of Fusarium oxysporum f.sp. Cucumerinum and Rhizoctonia solani. These results showed that filter-sterilized and heat-sterilized extracts collected from vinegar residue substrate all had no inhibitory effect on mycelial growth of two pathogenic fungi tested, non-sterilized extracts had significant inhibitory effect compared with the control, the inhibitory ratio to Fusarium oxysporum f. sp. Cucumerinum and Rhizoctonia solani were65.16%and40.67%repectilvy.4. Using an improved dilution plate method isolated antagonistic bacteria from vinegar residue substrate; using16S rDNA gene sequencing to identify them; using a dual plate assay to test their broad antifungal inhibitory activity; using plate medium to test their hydrolytic enzymes by cleared zone; some growth-promoting features also were tested, such as mineral phosphate solubilisation activity was determined on dicalcium phosphate plates; the nitrogen fixation ability of the isolates was tested according to the observation of a pellicle formed near the surface of the NFb semi-solid N-free media; the production of indole-3-acetic acid (IAA) by Salkowski colorimetric technique; Five pre-screened prominent antagonistic bacteria in vitro studies were further evaluated individually for their antagonistic potential against disease and plant growth promotion potential under greenhouse conditions. These results showed that: Using the improved method rapidly isolated18Bacillus spp. and1Paenibacillus sp.; All isolated19antagonistic bacteria exhibited broad-spectrum antagonism towards the tested8fungi; Paenibacillus sp. showed antifungal activity against Gram-negative and Gram-positive bacteria; All of the selected19strains exhibited cellulase activity,11strains secreted three hydro lytic enzymes(cellulose, chitinase, protease),13strains (68%) chitinase activity,18strains (95%) protease activity respectively, all of the strains exhibited at least two enzyme activities;18strains (95%) were siderophore producer, no strains phosphatase activity, small strains showed nitrogen-fixing and producing IAA ability, all of strains showed one of the growth-promoting features, four out of five selected strains were found both to be effective in controlling wilt and damping-off disease and four strains showed strong growth-promoting activities for cucumber seedlings under greenhouse conditions.5. Using the traditional dilution plate method, isolated antagonistic fungi from vinegar residue substrate; using18S rDNA gene sequencing to identify them; using cellophane membranes and divided plate method to test their volatile and non-volatile inhibitors; testing phosphate solubilisation activity by Ca3(PO4)2dicalcium phosphate plates; testing the production of indole-3-acetic acid (IAA) by Salkowski colorimetric technique. These results showed that:four antagonistic fungi were isolated from vinegar residue substrate and the four fungus all belonged to the genus Trichoderma; All the four fungus can produce volatile and non-volatile metabolites that may inhibit the growth of R. solani; All the four Trichoderma have cellulase and chitinase activity, Three out of four can secrete siderophore; three out of four were IAA producer, one out of four strains had phosphatase activity. |
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