Studies On Mechanisms Of Disease Resistance And Growth Promoting Effect Of Biocontrol Streptomyces On Verticillium Wilt Of Cotton

Verticillium wilt of cotton, caused by Verticillium dahliae Kleb., is a serious soil-bornedisease that limits cotton production in China. Finding effective disease control approaches isthe first priority to solve the problem of sustainable cotton production. Conventionalmanagement options for Verticillium wilt of cotton include the development of partiallyresistant cultivars, tillage management, and the application of chemical fungicides. However,there was no significant progress in breeding for disease resistance, and other managementoptions are ineffective for controlling Verticillium wilt of cotton. Exploring new ways ofcontrolling this disease has become the current problems to be solved. Microbial biocontrolagents have been proposed as a more effective and environmentally friendly means ofcontrolling soil-borne diseases. Actinomycetes are widely used in control of soil-bornediseases for their production of various antibiotics, extracellular enzymes and survivablespores. However, few specialized studies have been conducted about the biocontrolmechanism and efficacy of actinomycetes against Verticillium wilt of cotton. This study wasconducted from the perspective of soil microbial ecological restoration. The authorsinvestigated the occurrence of cotton Verticillium wilt, screened and identified antagonisticactinomycetes against V. dahliae, evaluated biocontrol and growth promoting effects ofselected actinomycete strains, and finally explored mechanisms of interaction amongpathogenic fungi, biocontrol actinomycetes and cotton plants. Biocontrol actinomycetes withhigh efficacy and stability were obtained for control of cotton Verticillium wilt andmanagement cotton continuous cropping obstacles. Results provided a scientific basis forfuture commercial production and practical application of biocontrol actinomycete agents.The results and conclusions are listed below:1. A total of712antagonistic actinomycete were previously isolated from croprhizosphere in Xinjiang, Qinghai, Shaanxi, Tibet and Heilongjiang, primarily in northwest ofChina. Of these,11actinomycete isolates with high inhibitory activity against V. dahliaewere selected by stepwise screening using the agar diffusion method and mycelium growthrate measurement. The selected actinomycete isolates efficiently inhibited V. dahliae growth, conidia production, and germination, and utilized cotton self-toxic phenolic acids （i.e.,p-hydroxybenzoic acid, ferulic acid and gallic acid） as the sole carbon source. Morphologicaland physiological tests, cluster analysis, combined with16S rRNA-based molecularidentification indicate that these11isolates are members of the genus Streptomycescyaneofuscatus, S. kanamyceticu, S. rochei, S. flavotricini and S. fradiae, respectively.2. The11Streptomyces isolates showed certain tolerance to NaCl and survived in thepresence of <15g·L^-1content. There were few changes in colony morphology andantagonistic activity of these Streptomyces isolates with <7g·L^-1NaCl. This result indicatesthat the tested Streptomyces can be used in the salt soil of Xinjiang. The mycelia growth rate,spores production and antagonistic activity of the11Streptomyces isolate grown with3g·L^-1NaCl were better than that with0.5g·L^-1NaCl Gause No.1agar medium.3. With V. dahliae mycelium as the sole carbon source, the11biocontrol Streptomycesproduced6extracellular fungal cell-wall degrading enzymes, i.e., chitinase,β^-1,3-glucosidase, β-glucosidase, cellulose, polyphenol oxidas and protease. Maximumenzyme activities were observed in extracellular hydrolases fermentation of the Streptomycessupplied with10g·L^-1fungi mycelium at28℃for7d. The Streptomyce crude enzymeinduced disintegration of V. dahliae mycelia and hypha. All tested Streptomyces isolates werecapable of twining with V. dahliae hypha, leading to fungal cell wall lysis. The Streptomycesisolates produced antifungal active substances with V. dahliae mycelium as the sole carbonand nitrogen source. When supplied with20g·L^-1fungi mycelium, the5-fold diluted fermentfiltrate of isolate B49showed maximum inhibition rate against V. dahliae,95.7%.4. The influence of salinity （concentration and type） and pH on the microsclerotiaformation in V. dahliae was evaluated by determination of the mycelium growth rate usinglaboratory culture medium. The colony growth of V. dahliae was inhibited at acidic andalkaline pH, with optimal colony growth observed at pH7.0. The microsclerotia formation ofV. dahliae was enhanced at alkaline pH, and the microselerotia area was increased by22.6%at pH8.0compared with that at pH7.0. The mycelium growth and microsclerotia formationof V. dahliae was affected by salinity concentration. With an increasing concentration ofNaCl in the culture medium, the colony growth and mycelium area of V. dahliae wereinhibited and decreased, respectively, whereas the formation of microsclerotia was enhanced.The microselerotia area in the culture medium with10g·L^-1NaCl was increased by40.7%compared with that in the medium without NaCl. The growth of V. dahliae was affected bydifferent salinity types. With an increasing concentration of chloride （i.e., NaCl, KCl） orsulfate （i.e., Na₂SO₄, MgSO₄） salts in the culture medium, the microsclerotia formation of V.dahliae was accelerated, whereas the mycelium growth was enhanced in the presence of CaCl2, and the microsclerotia formation of V. dahlia was inhibited at concentrations wasgreater than7g·L^-1in culture medium. The mycelium growth, microsclerotia formation andgermination of V. dahlia were significantly inhibited by culture filtrates of11Streptomycesisolates.5-fold-diluted ferment filtrates of Streptomyces isolates B49and D184displayedcompletely inhibitory effects on microsclerotia formation of V. dahlia with100%ofinhibitory rate. The mutant strains of V. dahliae was obtained from these3Streptomycesferment filtrate treatments, which produced no microsclerotia. The mutants were not reversedto the wild type after transferred5times in the PDA medium containing no Streptomycesferment filtrate. Its germination rate was38.3%at168h in the PDA medium containing5-fold-diluted ferment filtrate of Streptomyces D184.5. Under greenhouse conditions, the application of a powdered Streptomyces biocontrolagent to soil at the time of sowing considerably enhanced the plant defense-related enzymeactivities as well as the ortho-dihydric phenol and lignin contents in cottons after6-weekcultivation. When treated with V. dahliae phytotoxin, the defense-related responses of cottonwere accelerated and strengthened in Streptomyces-inoculated cottons relative tonon-inoculated cottons within24h. The changes in the defense-related responses furthersuppressed the accumulation of malondialdehyde （MDA）, and reduced the loss of rootactivity, leaf total chlorophyll, and leaf water contents during V. dahliae phytotoxin treatment.The pathogenic effect of V. dahliae phytotoxin on Streptomyces-inoculated cottons waseventually weakened, and the biocontrol efficacy was68.2%at72h.6. When inoculated individually or mixed with V. dahliae, the biocontrol Streptomyceseffectively improved the induced disease resistance in cotton plants during different growthstages. The Streptomyces treatment considerably enhanced the activities of peroxidase （POD）,polyphenol oxidase （PPO） and phenylalanine ammonialyase （PAL） in cotton leaf and rootwhile increasing the tannin, ortho-dihydric phenol, lignin and free proline contents,decreasing the accumulation of malondialdehyde （MDA） in cotton leaf, and reducing the lossof root activity and leaf water contents.7. The selected Streptomyces isolates were prepared as powdered biocontrol agents forapplication through seed coating or soil inoculation. The ability of the powdered biocontrolStreptomyces agents showed excellent and stable efficacies in control of Verticillium wiltduring the seedling stage in wilt-related pot and field experiments. The disease severity andthe area under the disease progress curve （AUDPC） of cotton Verticillium wilt were bothreduced. Among the tested Streptomyces isolates, X4showed the best biocontrol effect, withbiocontrol efficacies up to59.3%,65.8%and51.4%during the seedling stage in wilt-relatedpot and field experiments, respectively. 8. The selected Streptomyces isolates produced indoleacetic acid （IAA） up to47.56and56.19μg·mL^-1in ferment filtrate at17d. Treatment with Streptomyces ferment filtratepromoted the seed germination and the growth of cotton hypocotyl and radicle. In pot andfield experiments, inoculation of Streptomyces considerably increased plant height, rhizomediameter, total biomass, above-ground weight, root weight, and boll weight of cotton, andprompted the greenness and photosynthesis of cotton leaf. Among the tested Streptomycesisolates, X4showed the most significant growth promoting effect, increasing the plant height,fresh weight of total plant, root and boll, and net photosynthetic rate by24.4%,62.4%,107.7%,45.1%and31.9%, respectively.9. The selected biocontrol Streptomyces survived in the rhizosphere and rhizoplane soilsof cotton. In pot and field experiments, the abundance of the isolate X4in the rhizospheresoil of cotton was up to2.85×10⁶and2.44×10⁶CFU g^-1dry soil, respectively. The biocontrolStreptomyces improved the microbial ecology of cotton rhizosphere and rhizoplane soils bydecreasing soil fungi and increasing soil bacteria and actinomyces, leading to the transition ofmicrobial populations from “fungal-type” to “bacteria-type”. When inoculated by mixingwith V. dahliae, the isolate X4increased the abundance of bacteria and actinomycespopulation in rhizosphere soil of cotton by40.6%and32.1%, respectively, while decreasingthe fungi population by64.6%.