| Verticillium wilt of cotton caused by fungal pathogen Verticillium dahliae Kleb is a severe disease that limits the development of the cotton industry in the world. While chemical control is challenged by environmental problems and field control practices such as crop rotation and disease-resistant cultivar adoption exists many limting factores, biological control becomes increasingly an important way in control of soilborne plant diseases. Previous reports have shown that arbarscular mycorrhizal fungi (AMF) and Bacillus vallismortis HJ-5have biological control effects against Verticillium wilt of cotton. However, little has been known the effect of dual inoculation of soil with AMF and the bacterium and their synergetic mechanisms. Greenhouse pot experiments were firstly conducted to study several AMF and the inoculation doses of HJ-5for dual inoculation. Three independent greenhouse pot experiments were conducted to study the effects of the combined application of Glomus versiforme (Gv) propagules and HJ-5fortified bio-organic fertilizer on control of Verticillium wilt of cotton. Green fluorescent protein (GFP) labeled strain (GHJ-5) of strain HJ-5was constructed by using green GFP labeling techniques and the ability of GHJ-5to colonize roots and rhizosphere soil of cotton was also studied. To identify the effects of HJ-5and Gv alone or both on root exudates during the incidence of the wilt disease, a split-root experiment was conducted in greenhouse. Additionally, a series of laboratory experiments were performed on observation of the interaction between the plant pathogen and the biocontrol agents. Results were listed as follows.(1) The primary screening experiments for selection of the best AMF strain and dosage of HJ-5showed that Gv was the suitable AMF strain and the proper innoclation dose of HJ-5was107CFU·g-1. The dual inoculation treatment with Gv and HJ-5had the lowest disease incidence of Verticillium wilt, which was reduced by26.71%as compared with other treatments, and the highest increase of plant biomass.(2) Inoculation of nursery soil with propagula of Gv and application of the HJ-5enhanced bio-organic fertilizer in the diseased pot soil achieved agreeable high effecenies in suppressing Verticillium wilt by three independent pot experiments. Combined treatment decreased the disease index of Verticillium wilt by65%and increased plant height, leaf area, shoot and root dry weigh by75%,44%,216%, and71%respectively, on the average of three trials as compared with the non-inoculation control. Dual inoculation also significantly increased N in shoot and root and P in shoot and root by83%and65%,49%and45%on the average, respectively, while decreased the number of phytopathogenic Verticillium dahliae in rhizosphere soil of cotton by83%on the average, as compared with the non-inoculation control. Interestingly, the colonization of cotton roots by Gv was not infected by inoculation with HJ-5.(3) Measurement from laboratory tests showed that the HJ-5was able to produce lipopeptide substances, growth-promoting substances indole acetic acid (IAA), siderophore, hydrogen cyanide (HCN) and ammonia, but not produce deaminase (ACC) and was unable to dissolve P in medium. Colonization, deformation and dissociation of hypha of pathogen V. dahliae Kleb by the green fluorescent protein labelled HJ-5(GHJ-5) was also obsverved, suggesting that HJ-5was an effective antagonist against the pathogen of Verticillium wilt of cotton.(4) Dual inoculation of soil with GHJ-5and Gv in the greenhouse experiment showed that the cotton plant biomass, root density, plant phosphorus content significantly as well as the number of colonization of the rhizosphere of GHJ-5increased as compared with single inoculation with GHJ-5or Gv, suggesting that synergistic effect existed in promotion of the growth of cotton and suppression of Verticillium wilt of cotton.(5) In root-splicing experiment, the co-inoculation with HJ-5and Gv reduced disease index by63.3%as compared with the control. Four phenolic acids were found in the cotton root exudates, and all the phenolic acids at low concentrations stimulated germination of V. dahliae spores, while higher concentrations were inhibitory. The phenolic acid concentrations in the root exudates decreased significantly with the application of HJ-5and Gv. The the exudation pattern of cotton roots by HJ-5, Gv and V. dahliae was systemically altered.(6) In the pot experiment, inoculation of Gv and HJ-5significantly increased the activities of plant defence enzymes such as polyphenoloxidase (PPO), phenylalanine ammonialyase (PAL) and chitinase, increased salicylic acid chlorophyll in plant leaves, and decreased callose in crown of cotton, and consequently relieved the symptoms of Verticillium wilt of cotton.In conclusion, the present study confirmed that co-inoculation of soil with AMF and rhizosphere bacteria had synergetic promotion of plant growth and resistance to phytopathogens. Abilities of Gv to facilitate plant uptake of nutrients and abilities of HJ-5to produce beneficial secondary metabolics against V. dahliae Kleb as well as successful colonization of soil and plant roots were essential to control Verticillium wilt of cotton. The alteration of the exudation pattern of cotton roots by HJ-5and Gv was related to plant systemic resistance to the phatopathogen. This was also supported by the enhanced activities of plant defence enzymes after inoculation of Gv and HJ-5. Results in this study would be useful in the developping new methods to better control cotton wilt disease. Further researches should be given to the molecular microbilogical aspects of the functions performed by bio-organic fertilizers in remediation of continuously cropping obstacles and in the promotion of plant growth and health exerted by soil functional microbes. |
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