| Virticillium wilt of cotton (Gossypium hirsutum L.) is one of the most destructive diseases of cotton in the world. The disease is caused by the cosmopolitan soilborne fungal pathogen Verticillium dahliae Kleb. (Vd) and can be controlled by some special bio-organic fertilizers (BIO) or amino acid fertilizer (AAF) applied into soils. Field and greenhouse experiments were conducted to evaluate effects of the BIO and AAF on disease control in Xingjiang. Mechanisms and soil microbial community diversity after application of BIO and AAF against Vd were explored by using biological and biochemical methods such as soil enzyme activity measurement, plate culture, green fluorescent protein (GFP) labelling, high performance liquid chromatography (HPLC) analysis, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Main results were showed as follows.Field experiment with application of amino acid fertilizer (AAF) was carried out in Shihezi, Xinjiang, China. The results showed that disease incidences of treatment with application of AAF were decreased by 2.9%～18.0% in 2006 and 4.2%～37.3% in 2007. Disease index were decreased by 9.4%～14.3% in 2006 and 11.9%～29.4% in 2007. Controlling percentage of the disease was 9.8%～50.3%. Fibre yields were increased by 8.9%～17.9% in 2006 and 6.8%～12.7% in 2007. Fibre quality analysis indicated that fibre quality was not significantly affected by application AAF in 2006, but fibre strength and elongation were significantly affected in 2007. AAF aplication increased numbers of soil fungi, actinomycetes, and bacteria as well as the ratio of bacteria to fungi. Numbers of bacteria in rhizosphere were significantly higher than those in bulk soil. Numbers of bacteria in rhizosphere of Xinluzao-8 (RS-8), which was sensitive to Vd, were less than those in rhizosphere of Xinluzao-12 (RS-12), which was resistant to Vd. Activities of catalase, catalase, alkaline phosphatase, urease and cellulase, urease and cellulase were significantly higher in rhizosphere soils than those in bulk soils. Thus, application of AAF had potential to increase activities of soil enzymes and contents of available nutrients either in rhizosphere or bulk soils but to decreased soil pH in rhizosphere soil when high application rate of the fertilizer was applied.Results of pots experiment showed that BIO No.1 was better than BIO No.2 in promoting cotton growth and reducing population of soil fungi. BIOs were applied into two soils, a health soil inoculated with Vd and a Vd-infected soil caused by continuous cotton cropping. Application of BIO decreased cotton Verticillium wilt incidence and disease index of Xinluzao-8 (XLZ-8, susceptible cultivar) and Xinluzao-12 (XLZ-12, resistant cultivar), increased biomass of cotton shoot. Cotrolling percentage of the Verticillium wilt by the application of BIOs were 28%～55%. Numbers of soil fungi, actinomycetes and bacteria of the BIO treatment were 2.7 times,0.9 times and 4.9 times higher than control for soil grown with XLZ-8, respectively; and no more,0.3 times and 1.8 times higher than control for soil grown with XLZ-12, respectively.Field experiments were conducted in soil that was inoculated with Vd about 5 years. Results showed that application of BIO and AAF decreased cotton Verticillium wilt disease index, enhanced controlling efficiencies by 49.5%and 29.8%, respectively, and increased yields by 66.4%and 47.1%, respectively. Compared with CK in three growth stages, application of the BIO and AAF increased soil invertase activity by 32%～35%and 26%～32%, polyphenol oxidase activity by 89%～155%and 60%～130%, protease activity by 317%～845%and 160%-344%, alkaline phosphatase activity by 19%～23%and 22%～43%, urease activity by 17%～28%and 14%～26%, respectively. There were no differences of catalase activity in the different treatments.Different application rates of BIO were applied into three soils which contained different organic matter(40.9 g·kg-1,18.9 g·kg-1,10.1 g·kg-1). According to cotton biomass and controlling efficiency to Virticillium wilt, application 10 g·kg-1 BIO to soil with higher content of organnic matter was a better choice,20～30 g·kg-1 for middle or lower content of organnic matter soil. Soil urease and invertase activity were increased with the increase of BIO application rates, but no significant changes were found in terms of protease and catalase activity; polyphenol oxidase activity was increased at the beginning then decreased with the increase of BIO application rates. High content of soil organic matter increased activities of protease, invertase and catalase, but decreased activity of polyphenol oxidase.Bio-organic fertilizers were applied in field at rate of 1500 kg·hm-2 in different locations of Xinjiang. Results showed that application of BIO reduced disease incidence and disease index and increased disease controlling effectivness by 20%～79%compared to CK. In BIO treatments, cotton yield incresed about 4.9%～21.4%and chlorophyll SPAD value of cotton leaf were increased. In flowering period, application of BIO increased the content of soil available nitrogen, phosphorous and potassium by 47.4%,35.6%and 5.5%, repectively, in XLZ-8 growing soils, and by 20.4%,45.6%and 2.6%, respectively, in XLZ-12 growing soil.GFP labelled Bacillus subtilis ZJ-6 could colonize cotton root. Most of ZJ-6 cells were on the hair roots of older lateral roots, some were on taproot, but few were on calyptrogen. Numbers of ZJ-6 were higher in the treatment, where ZJ-6 was inoculated one day before Vd was inoculated, than in the treatment, where Vd was inoculated one day before ZJ-6 was inoculated. No difference of colonization rates was observed between the inoculation in cotyledon unwinded period and the inoculation in 4 leaves period. Few of ZJ-6 could colonize cotton root 30 days after inoculation. The optimum temperature to colonize soil for the antagonists was in the range from 15℃to 35℃, while that for Vd was in 25℃. When soil field water capacity was maintained at 50%, the functional antagonists could colonize soils at the highest density. There was not obvious biocontrolling efficiency when the two antagonists were mixed with OF without secondary solid fermetation and immediately applied into soil. This indicated that proper organic carriers and nutrients were needed for the functional antagonists to suvive after applied into soils. Antagonists in the BIO with secondary solid fermentation easily colonized soil and functioned well with high biocontrol efficiency.Soil microbial community was significantly influenced by the application of BIO. Soil actinomycetes were increased with increase of BIO application rates in the three soils which contained different organic matter, but soil fungi and bacteria numbers were increased at the beginning and then decreased, and proper rates of BIO applied were 10 g·kg-1 for high organic matter soil and 20 g·kg-1 for middle or low level of organic matter soil. PCR-DGGE analysis showed that application of BIO decreased fungal bands when BIO application rates were increased. Fungal diversity of the soil with middle level of organic matter was higher than that of the soil with high or low organic matter. Fungal structrue indices of the soil with high organic matter were decreased with increase of BIO application rate, but the indices of the soil with middle or low organic matter were increased at the beginning then decreased, and maximum indices of the rates were 20～30 g·kg-1 for middle organic matter soil and 10～20 g·kg-1 for low level of organic matter soil. Application of BIO or the mixture of Bacillus antagonists and organic fertilizer increased bacteria community diversity, maintained a stable bacterial and fungal structrue indices, and led soil microbial diversity to a healthy status.Growth of Vd in plate was inhibited by concentrated protein from ZJ-6 fermentation liquids. Cotton root exudates were detected by HPLC. The species and amount of phenolic acids from the root exudates of susceptible cultivar XLZ-8 in 4 leaves period were higher than those in cotyledon unwinded period. Inoculating plant with Vd in cotyledon unwinded period increased contents of gallic acid,4-hydrobenzoic acid and 4-hydroxybenzaldehyde. However, content or ratio of cinnamic acid was increased when plant was inoculated with Vd in 4 leaves period. The species and amount of phenolic acids were decreased when plants were inoculated with ZJ-6 either in cotyledon unwinded period or in 4 leaves period. Inoculation with ZJ-6 reduced some amino acids in root exudates, which stimulated germination of Vd spores and mycelium growth of Vd, and increased arginine in root exudates, which inhibited Vd growth.In conclusion, antagonists in BIO efficiently colonized cotton roots, decreased autointoxicants such as phenolic acids and amino acids from cotton root exudates. Application of BIO improved soil microbial community diversity and thus increased activities of soil enzymes. Application of BIO also increased soil available nutrients and cotton leaf chlorophyll. The cotton yields in different soils were significantly improved by application of BIO via successful biocontrol of the Virticillium wilt. Application of BIO is one of best means to remediate continuously cropping obstacles and is worthy to be extended. |
PDF Full Text Request