Study On The Antagonistic Actinomycetes For Biocontrol Of Plasmodiophora Brassicae

Clubroot caused by Plasmodiophora brassicae is one of the most serious soil-borne plant diseases all over the world, which was first found in British Mediterranean west bank and southern Europe in 1737. In recent years, clubroot occurs at a spreading trend and now is widely distributed in Europe, North America, Japan and China. The hosts of P. brassicae are broad, including cruciferous vegetables, oilseed rape, radix isatidis, horseradish and cruciferous weeds. The occurrence and damage of clubroot is increasingly serious. Distorted roots with galls or clubs are the main symptoms of the disease. When the disease is severe, roots rot and even the whole plant dead, and clubroot will aggravate year after year until no harvests, leading to substantial yield losses. The average yield losses are 20%-30% and even above 60% in serious plots. Plasmodiophora brassicae is an obligate parasite, which was classified as protozoan and can survive in the soil for a long time, as a result, it is difficult to eradicate or prevent.At present, there are still no effective management strategies for controlling clubroot at home and abroad. In disease-resisted breeding, although few high resistant varieties were bred on Chinese cabbage such as Qiu-Lihuang, Chun-Fuhuang, there are no resistant materials for numerous cruciferous vegetables and oilseed crops. Chemical control has some effects, but also brings environmental pollution, pesticide residues, destroying the soil ecology environment, and many other adverse impacts. Due to the pathogens of roots diseases existence and harm roots of plants, and the pathogens of soil-borne diseases overwinter in the soil, so biological control have great potential for those plant diseases. Thus, conducting basic application research and bio-control practice has important theoretical and practical significance for biological control of clubroot, which provides a promising approach against P. brassicae. Study and development of antagonistic microbes is the important content of biological control. In bio-control microbial groups, actinomycetes are rich in resources, but also are the main producer of many secondary metabolites and biologically active substances. As a result, actinomycetes are important potential bio-control agents with bright development prospects. In order to search antagonistic actinomycetes with superior traits against P. brassicae, we isolated and screened actinomycetes from rhizosphere soil, root, stem and twigs of plants species. And then systematic studies of potential antagonistic actinomycetes were made, including identification, fermentation stability, antimicrobial spectrum, effects of enzyme on defense, colonization, optimization of fermentation conditions, preliminary separation and purification of active substances. The results are listed as follows:1. Isolation, screening and identification of antagonistic actinomycetes A total of 368 actinomycetes were isolated from rhizosphere soil, root, stem and twigs of plants species for clubroot control. The plant species included tea tree, Ormosia yaanensis, ginkgo tree, Chinese cabbage, and also oilseed rape. Results from germination rate investigation of P. brassicae resting spore after inoculation of isolated strains, there were 16 actinomycetes founded which could decrease the germination rates of P. brassicae. The control efficiencies of 16 antagonistic strains against clubroot in pot experiment were also studied. From these strains, antagonistic strains A316 and A10 were the most potential agents for biological control of clubroot.Adopting traditional classification, chemical classification and molecular classification, antagonistic strains A316 and A10 were identified. Based on the 16S rDNA sequence analysis, strain A316 was most closely related to Streptomyces griseoruber NBRC 12873 (99.9% similarity of 16S rDNA). Together with morphological, cultural and physio-biochemical characteristics, strain A316 was preliminary identified as S. griseoruber. While the genetic relationships between strain A10 and many sequences from GenBank database were closely related. According to morphological and cultural traits, physio-biochemical characteristics, A10 was affiliated to Griseofuscus of Streptomyces. The nucleotide sequences of A316 and A10 have been deposited in the GenBank database under accession numbers HQ335354 and HQ335355.2. Bio-control efficiency and antimicrobial spectrum of strains A316 and A10A316 was isolated from rhizosphere soil of Chinese cabbage, while A10 was from roots of Ormosia yaanensis in yaan city, Sichuan province. The resting spore germination inhibition rate of growth broths for strains A316 and A10 reached 77.58% and 72.60% respectively. The protection for clubroot conferred by the supernatant or by the mycelia was studied. Both the filtered supernatant and washed mycelia of strains A316 and A10 could decrease the germination rates of P. brassicae, so the active substances existed in supernatant and mycelia at the same time. The two strains had higher genetic stability, no obvious active degeneration phenomenon for ten generations.The control efficiencies of A316 and A10 against clubroot in pot experiment were 73.69%,70.37% on Chinese cabbage and 75.68%,71.65% on oilseed rape. The two antagonistic strains were also effective in field experiment, control efficiencies of A316 and A10 against clubroot in field plot experiment were 65.91%,60.25% on Chinese cabbage and 67.92%,61.32% on oilseed rape. Strains A316 and A10 could still increase production of Chinese cabbage to some extent, yield increase rate per mu were 96.1% and 64.9%.Inhibitory activity test in vitro showed that the two antagonistic strains had inhibition on many pathogenic fungi and bacteria to some extent, such as Exserohilum turcicum, Bipolaris maydis, Curvularia lunata, Fusarium oxysporum, Bacillus subtilis and so on. And the antimicrobial spectrum of A10 was wider than A316. There were no positive connection between filtered fermentation broth and strains itself for antimicrobial activity.3. Fermentation stability of strains A316 and A10 The active substances fermented by A316 and A10 were stable in ultraviolet, neutral or acidic conditions, which were also stable to proteinase K. When pH value was 11, the inhibition rate of P. brassicae treated with fermentation broths of strains A316 and A10 slumped 28.06% and 32.73%. Fermentation broths were processed for 30 min and 60 min respectively in 100℃, the inhibition rate fell only 9.71% and 13.31% for strain A316, while 27.69% and 32.73% for strain A10. So the fermentation products of A316 had thermal stability, and A10 only stabilized in low and medium temperature. The fermentation broths of A316 were stable when storing in low temperature, but the storage stability for fermentation broths of A10 was worse than A316. After placed at 4℃and root temperature respectively for 22 d, the inhibition rate fell 14.08% and 28.16% for strain A316, while 25.27% and 36.82% for strain A10.4. Effects of strains A316 and A10 on activities of defense enzymes in Chinese cabbage The changes of defense enzymes activities in Chinese cabbage were detected. Treated with P. brassicae and fermentation broths of bio-control actinomycetes in the meanwhile, enzyme activities of PAL, POD and PPO in the leaves of Chinese cabbage increased rapidly with trend of "earlier raised and later decreased". For strain A316, the maximum activities of PAL, POD and PPO reached in 7 d (36.87 U/g·h),5 d (141.5 U/g·min) and 5 d (28.97 U/g·min) after treatment, which increased by 42.63%,40.45%, 53.69% respectively compared to only inoculation with P. brassicae. While for strain A10, the maximum activities of PAL, POD and PPO reached in 9 d (33.4 U/g·h),7 d (133.65 U/g·min) and 7 d (26.14 U/g·min) after treatment, which increased by 29.21%,32.66%, 38.67% respectively compared to only inoculation with P. brassicae.5. Colonization of strains A316 and A10 Using conventional antibiotics notation, the colonization for rifampicin-resistant mutants of A316 and A10 in soil and Chinese cabbage were determined, both using natural soil and sterilized soil. Two strains had strong colonization ability in soil and plant body. In natural soil, the largest colonization quantity of strain A316 in soil and roots, stems, leaves of Chinese cabbage appeared in 14 d (6.79×106 cfu/g soil),17 d (4.33×104cfu/g root),17 d (2.82×103cfu/g stem),24 d (3.8×102 cfu/g leaf) respectively, while the largest colonization quantity of strain A10 appeared in 21 d (9.23×106 cfu/g soil),17 d (9.06×103 cfu/g root),17 d (3.23×102 cfu/g stem) and 24 d (0.981×102 cfu/g leaf); In sterilized soil, the largest colonization quantity of strain A316 in soil and roots, stems, leaves of Chinese cabbage appeared in 14 d (2.36×106 cfu/g soil),24 d (1.77×104cfu/g root),24 d (7.53×102cfu/g stem),24 d (2.71×102cfu/g leaf) respectively, while the largest colonization quantity of strain A10 appeared in 21 d (5.4×106 cfu/g soil), 17 d (3.79×103 cfu/g root),24 d (2.23×102cfu/g stem) and 24 d (0.851×102cfu/g leaf). The colonization ability of A10 was better than A316 in soil, but worse in plant body. The detectable populations of two strains in sterilized soil were lower than natural soil, and the vary range of populations in sterilized soil were larger than natural soil. Numbers of populations for A316 and A10 in roots were the most, stems and leaves followed.6. Optimization of fermentation for strains A316 and A10 The fermentation media and culture conditions of actinomycetes A316 and A10 were optimized by orthogonal experiment and single factor analysis, respectively. The optimal combination for strain A316 were 1% glucose,1% soybean meal,0.5% yeast extract, culture time of seed liquid 36 hours, inoculation volume 8%, initial pH 7, liquid medium volume 40%, temperature 28℃, rotated speed of shaker 180 r/min, fermentation time 5 days. While optimal combination for strain A10 were 1.5% glucose,1% soybean meal,0.3% peptone, culture time of seed liquid 60 hours, inoculation volume 10%, initial pH 7.5, liquid medium volume 30%, temperature 28℃, rotated speed of shaker 180 r/min, fermentation time 6 days.7. Preliminary separation and purification of active substances from strain A316 against Plasmodiophora brassicae With the help of solvent extract, thin layer chromatography, and silica gel chromatography, purified compounds with antimicrobial activities against P. brassicae were isolated from the fermentation products of Streptomyces griseoruber strain A316. According to gas chromatograph-mass spectrometer extrapolating, there were mainly two substances in the active component. The matching degree between compoundⅠand 2,2'-Methylenebis (6-tert-butyl-4-methylphenol) could reach 97.84%. CompoundⅡhad larger polarity, contained lively hydrogen, and had matching degree of 74.28% with Phthalic acid, mono-(2-ethylhexyl) ester.