| Agricultural residual wastes including husk ashes, the earthworm dung, refined cotton scraps and femented pine needles were screened to prepare optimal nursery substrates for cotton growth. Nursery substrate A05was picked out as the most favorable medium for next experiments. Three endophytic bacteria strains, Paenibacillus xylanilyticus MH-6, Paenibacillus polymyxa YUPP-1, and Bacillus subtilis MH-11were isolated from seedling stage, budding stage and bell stage, respectively. They were all confirmed with high activity to resist Verticillium dahliae Kleb. After the colonization dynamics of these endophytic bacteria were tested, the combination of three strains for inculating cotton seedlings showed that the disease resistance will be more durable and more efficiently. Based on above results, biocontrol bacteria group were mixed in A05nursery substrate with concentration of108cfu/g to raise cotton seedling. The results indicated that the nutrient soil combing with biocontrol bacteria should meet a great market demand. All results available were listed as follows:1) Studies on nursery substrate formulaThe high quality of formula can support not only strong cotton seedlings growth, but also the root carrying so much nursery substrate to improve transplanting survival rate. In this experiment, nursery substrates were prepared from plant ashes, the earthworm dung, refined cotton scraps and femented pine needles and Kudzuvine root residual fibre. The results could be listed as fellow:(1) The seedling growth ratio in A01, A02, A04, A05medium reached85%after three days with no Important difference. While the seedling growth ratio in A03medium containing refined cotton scraps was only75.4%;(2) The plant height in A01was20.32cm, which was the highest of all other mediums (p<0.05) relative to14.83cm,10.00cm,14.21cm and13.37cm in A02, A03, A04, A05medium, respectively);(3) The highest wet weight5.01g per seedling roots in A05medium was obtained, while the lowest wet weight was only2.00g per seedling roots in A03medium; it demonstrated that nursery substrate A05is optimal meidum for cotton seedling growth.2) Assay the characteristics of nursery substrateThe key components of the culture medium including the nitrogen, phosphorus, potassium, organic matter and pH, decided the medium performance. The results indicated that A01nursery substrate have the highest contents of the key components. But its pH was not for seedling growth. Contrast to the other nursery substrate, A05medium was moderate with pH7.1. While A03nursery substrate contained higher organic materials (312.29g/kg) when four volume of refined cotton scraps were added into medium, however, its pH reached7.88.3) Isolation and identification of endophytic antagonistic strains from cotton seedlingsStrains MH-6, YUPP-1and MH-11, with strong activity against Verticillium dahliae Kleb, were isolated from seedling stage, budding stage and bell stage, respectively. Diameters of clear zone against V. dahliae were10mm,15mm and17mm for strains MH-6, YUPP-1and MH-11.The16S rDNA sequence analysis showed that YUPP-1strain shared high homology of99%with P. xylanilyticus. this strain is proposed to be P. xylanilyticus. Through16S rDNA sequence alignment in GenBank database, MH-6strain shared98%homology with Paenibacillus polymyxa, while MH-11strain shared99%homology with Bacillus subtilis A23strains. Therefore, MH-6and MH-11strains were identified as P. polymyxa and B. subtilis.4) Colonization evaluation of endophytic biocontrol bacteria in cottonsThe colonization result showed that three bacteria of MH-6R, MH-11R and YUPP-1R could successfully infect cotton roots but the colonization number varied with the different development period of cottons, which was also verified that the bacterial concentration exerted a positive effect on the final colonization population number. The discovery still fited for Paenibacillus polymyxa MH-6R in the same way. The maximum colonization number was achived after40days of inoculation (original concentration was1×108cfu/strain and colonization concentration was1×105cfu/g). As cottons grew, the colonization number declined gradually with the final number declining to around1000colonies per gram of cotton microtubules in bell stage. The colonization number of YUPP-1R at the seedling stage was firstly less than1×104cfu, followed by the gradually increased colonization number. The maximum colonization number at the budding stage was achived after60days of inoculation, followed by a gradual decline to a constant colonization number at the belling stage (1×104cfu/g for1×108cfu/strain). The colonization number of Bacillus subtilis MH-11R at the seedling and budding stage was low, followed by a slow increase to the peak number at the belling stage after80days of inoculation. The final colonization number could be maintained at1×104cfu/g for1x108cfu/strains.5) Potting test and field test of biocontrol efficiencyIndoor potting results showed that the biocontrol effect for the single strain was listed as follows:P. xylanilyticus YUPP-1> Paenibacillus polymyxa MH-6> Bacillus subtilis MH-11when bio-control agents were used alone. The biocontrol effect was intensified when three strains were jointly utilized compared to the single stain. There was no diseased incidence of cotton plants at the seedling stage with the treatment of combined bacteria. The diseased incidence of the treated cottons was3.3%while the diseased incidence and the diseased index of the control cottons were up to95%and41respectively at the belling stage.Field experiments in2010showed that the biocontrol effect for the single strain in field tests was listed as follows:P. xylanilyticus YUPP-1> Paenibacillus polymyxa MH-6> Bacillus subtil. The biocontrol effect was also intensified when three strains were jointly utilized compared to the single stain. The diseased incidence of cottons at the budding and flowering stage was only5.2%, while the diseased incidence of the control counterparts at the same stage was33.5%. Similarly, the diseased incidence of cottons at the belling stage was only9.4%, while the diseased incidence of the control counterparts at the same stage was47.5%. The accessible results in2010and2011were similar. However, the overall diseased incidence of verticillium wilt in2011was much more serious than that in2010, which might be tightly attributed to local climatic conditions. The results in field trials were consistent with those in indoor trials with the discrepancy of the whole biocontrol efficiency between them.6) Population dynamics of the bio-control strain in A05mediumThe survival time course monitoring of three single strains in A05culture medium has revealed that the survival curve of three strains was alike. The original strain of1×106cfu/g was added into the intact culture medium, which went down by1-1.5magnitude after five days and then increased by one magnitude after ten days with a final concentraion until twenty days when the strain concentration dropped steeply. The control strain was kept at a low level (<60cfu/g).The survival time course monitoring of grouped strains in A05culture medium has also revealed that the original strain of1×106cfu/g was all added into the intact culture medium, where the concentration of MH-6and MH-11went down by1.5magnitude after five days and then maintained at a constant concentraion until twenty days when the strain concentration dropped steeply. However, the concentration of YUPP-1went down by1.5magnitude after five days and then increased by1.5magnitude after ten days with a final reduction until twenty days when the strain concentration still reached1.7×105cfu/g. The control strain was kept at a low level (<60cfu/g). Therefore, the proportion of three strains in the A05medium was10:1:1.7) Biocontrol test of the pathogen-resistant seedlings cultuted in strain-containing mediumsField trials at different places during two years have showed that the diseased incidence and disease index of the pathogen-resistant cottons varied with different locations while the two parameters in control cottons were higher. The diseased incidence of cottons at the flowering stage fluctuated between14%and21%while the diseased incidence of untreated cottons at the flowering stage fluctuated between45%and60%. The disease index of cottons at the flowering stage fluctuated between6.4and14.0while the diseased index of untreated cottons at the flowering stage fluctuated between35.0and41.0. The two parameters at the belling stage were similar to those at the flowering stage, of which the latter was more terrible. |
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