十字花科作物根肿病生防放线菌研究
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摘要
十字花科作物根肿病是由芸薹根肿菌(Plasmodiophora brassicae Woron)侵染引起的严重世界性土传植物病害。1737年在英国地中海西岸和欧洲南部发现该病,近年来,呈蔓延之势,现已广泛分布于欧洲、北美、日本及中国。根肿病菌寄主范围较为广泛,不仅危害十字花科蔬菜、油菜、板蓝根和山葵,还可寄生十字花科杂草。根肿病的发生和危害日趋严重,该病危害植株根部,严重时引起根部腐烂乃至全株枯死,一旦发生,病害逐年加重,直至绝产绝收,造成重大经济损失,平均产量损失达20%-30%,严重田块直接产量损失达60%以上。芸薹根肿菌现归类于原生动物界,专性寄生,可在土壤中长期存活,难以根除。
目前,国内外对根肿病尚无有效的防治方法。在抗病育种方面,虽已育成白菜高抗品种(如秋利皇、春福皇等),但对众多的十字花科蔬菜和油料作物尚无抗病品种；化学药剂防治虽有一定效果,但同时也带来环境污染、农药残留、破坏土壤微生态环境等诸多不利影响。由于根及根茎病害的病原物存在和危害根部,且土传病害的病原菌在土壤中越冬,故对此类病害进行生物防治有极大的潜力。因而,开展根肿病生物防治的应用基础研究和生防实践,对根肿病的生物防治具重要的理论及实践意义,从而为控制该病提供了一条有希望的途径。生防微生物的研究和开发是生物防治的重要内容,在生防微生物类群中,放线菌资源丰富,而且是微生物代谢产物中具有生物活性物质的主要产生菌,具开发应用前景。为获得能有效防治十字花科作物根肿病且性状优良的生防放线菌,本研究从根际土壤及植株体内分离并筛选放线菌入手,开展了生防放线菌的防效测定、菌株的鉴定、发酵液稳定性、抗菌谱、对防御酶系的影响、定殖状况、发酵条件的优化、活性物质的初步分离纯化及鉴定等方面的研究,取得了以下结果：
1.生防放线菌菌株的分离、筛选及鉴定试验从茶树、红豆树、银杏树、白菜及油菜的根际土壤和根、茎、叶、枝条等材料中一共分离出放线菌368株。通过测定其对根肿病菌休眠孢子萌发的抑制作用初筛得到16株拮抗放线菌,复筛获得A316和A10两个具生防潜能的菌株。
采用传统分类、化学分类与分子分类相结合的方法,对根肿病菌生防放线菌A316和A10进行了分类鉴定。菌株A316与链霉菌中灰红链霉菌(Streptomyces griseoruber) NBRC 12873的16S rDNA同源性高达99.9%,形态与培养特征、生理生化特性等也与灰红链霉菌高度相似,因此,将菌株A316初步鉴定为灰红链霉菌(S. griseoruber);而菌株A10在构建发育树和16S rDNA同源性分析时与GenBank数据库中多个相似性序列的亲缘关系都较近,鉴于其形态与培养特征、生理生化特性等,菌株A10仅能归于链霉菌中的灰褐类群(Streptomyces sp.)。菌株A316和A10在GenBank的序列登录号分别为HQ335354和HQ335355。
2.菌株A316和A10对根肿病的防效及抗菌谱来自白菜根际土壤的A316和来自红豆树根部的A10对根肿病菌休眠孢子萌发的抑制作用最好,其培养液的抑制率分别高达77.58%和72.60%。菌株A316和A10的发酵液及菌悬液对根肿病菌休眠孢子的萌发均具有一定的抑制作用,因此2菌抑制根肿病菌的活性物质同时存在于发酵液和菌丝体中。通过传代实验表明,菌株A316和A10连续传代10次没有明显的活性退化现象,表明2种生防菌菌株的遗传稳定性较好。
经室内盆栽试验测定有效抑制休眠孢子萌发的16株拮抗菌株对根肿病的防治效果发现,菌株A316和A10的盆栽防效好于其他菌株,2菌株对白菜根肿病的室内盆栽防效分别为73.69%、70.37%,对油菜根肿病的室内盆栽防效分别达75.68%、71.65%。菌株A316和A10在大田对防治根肿病同样有效,2菌株对白菜根肿病的田间小区防效分别为65.91%、60.25%,对油菜根肿病的田间小区防效分别达67.92%、61.32%。生防菌株A316和A10还可在一定程度上提高白菜的产量,其对白菜的亩增产率依次达96.1%、64.9%。
抗菌谱实验表明,菌株A316和A10对玉米大斑病菌(Exserohilum turcicum)、玉米小斑病菌(Bipolaris maydis)、玉米弯孢病菌(Curvularia lunata)、西瓜枯萎病菌(Fusarium oxysporum)、枯草芽孢杆菌(Bacillus subtilis)等多种植物病原真菌及细菌都有不同程度的抑制作用,菌株A10较A316更具广谱性。但菌体本身与发酵液的抑菌活性之间没有必然联系。
3.菌株A316和A10的发酵液稳定性菌株A316和A10发酵液产生的活性物质在紫外线、中性或酸性条件下比较稳定,且对蛋白酶K有较强的稳定性。pH值调到11处理后,菌株A316和A10对根肿病菌休眠孢子萌发的抑制率分别下降达28.06%和32.73%。发酵液在100℃处理30 min和60 min时,A316对根肿病菌休眠孢子萌发的抑制率仅分别下降了9.71%和13.31%,而A10的抑制率分别下降了27.69%和32.73%,因此菌株A316的发酵产物对热的稳定性也较好,菌株A10在中低温条件下稳定。菌株A316的发酵液低温储藏稳定性较好,而菌株A10发酵液的储藏稳定性不及A316,在4℃和室温下放置22 d时,菌株A316发酵液的抑制率分别下降了14.08%和28.16%,菌株A10发酵液的抑制率分别下降了25.27%和36.82%。
4.菌株A316和A10对白菜防御酶系的影响通过测定白菜植株体内防御酶系的变化发现,接种根肿病菌的同时使用A316或A10的发酵液灌根后,白菜叶片的PAL、POD、PPO活性均明显上升,且呈现“先升后降”的趋势。对于菌株A316,PAL、POD、PPO酶活峰值分别出现在第7d (36.87 U/g-h)、第5d (141.5 U/g-min)、第5d(28.97 U/g-min),比仅接种根肿病菌时的酶活峰值依次提高了42.63%、40.45%、53.69%；而对于菌株A10, PAL、POD、PPO酶活峰值分别出现在第9d(33.4U/g-h)、第7d (133.65 U/g-min)、第7d (26.14 U/g-min),比仅接种根肿病菌时的酶活峰值依次提高了29.21%、32.66%、38.67%。
5.菌株A316和A10的定殖采用传统的抗生素标记法,设置自然土与无菌土2个处理,分别测定了生防放线菌A316和A10的抗利福平突变菌株在土壤中及白菜植株内的定殖能力。2菌株在土壤中及植株体内均具有较强的定殖能力。自然土中,菌株A316在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在14d (6.79×106cfu/g土)、17d(4.33×104cfu/g根)、17 d (2.82×103cfu/g茎)、24 d (3.8×102cfu/g叶),菌株A10在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在21 d (9.23×106 cfu/g土)、17d(9.06x103cfu/g根)、17d (3.23×102cfu/g茎)、24 d (0.981×102cfu/g叶)；灭菌土中,菌株A316在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在14 d(2.36×106cfu/g土)、24d(1.77×104cfu/g根)、24d(7.53×102 cfu/g茎)、24d(2.71×102 cfu/g叶),菌株A10在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在21 d(5.4×106cfu/g土)、17d(3.79×103cfu/g根)、24 d(2.23×102 cfu/g茎)、24d(0.851×102 cfu/g叶)。土壤中A10的定殖能力好于A316,而在植株体内A316的定殖能力好于A10。灭菌土中2菌的定殖菌量均小于自然土中的定殖菌量,灭菌土中的菌量变化幅度大于自然土。2生防菌株在植株的根内定殖数量最大,茎中次之,叶中最少。
6.菌株A316和A10的发酵优化采用正交设计法改良生防放线菌的发酵培养基,并使用单因素分析法优化其培养条件。菌株A316的较佳培养基组成为：1%葡萄糖,1%大豆粉,0.5%酵母膏；其理想的发酵培养条件为：种子液种龄36 h,接种量8%,初始pH 7,装液量40%,温度28℃,摇床转速180r/min,发酵时间5 d。菌株A10的较佳培养基组成为：1.5%葡萄糖,1%大豆粉,0.3%蛋白胨；其理想的发酵培养条件为：种子液种龄60 h,接种量10%,初始pH 7.5,装液量30%,温度28℃,摇床转速180r/min,发酵时间6 d。
7.菌株A316抗根肿病菌活性物质的初步分离及鉴定灰红链霉菌(Streptomyces griseoruber) A316的发酵产物经溶剂萃取、薄层层析、硅胶柱层析等进行分离纯化,得到了其抑制根肿病菌的活性物质。活性组分经气相色谱-质谱(GC-MS)联用分析后发现主要含有2种物质。化合物Ⅰ与通用型酚类抗氧剂2,2’-亚甲基双-(4-甲基-6-叔丁基苯酚)的匹配度达97.84%；化合物Ⅱ极性较大,应该是含有活泼氢的物质,与邻苯二甲酸单乙基已基酯的匹配度为74.28%。
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 A10
A316 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.
目前,国内外对根肿病尚无有效的防治方法。在抗病育种方面,虽已育成白菜高抗品种(如秋利皇、春福皇等),但对众多的十字花科蔬菜和油料作物尚无抗病品种；化学药剂防治虽有一定效果,但同时也带来环境污染、农药残留、破坏土壤微生态环境等诸多不利影响。由于根及根茎病害的病原物存在和危害根部,且土传病害的病原菌在土壤中越冬,故对此类病害进行生物防治有极大的潜力。因而,开展根肿病生物防治的应用基础研究和生防实践,对根肿病的生物防治具重要的理论及实践意义,从而为控制该病提供了一条有希望的途径。生防微生物的研究和开发是生物防治的重要内容,在生防微生物类群中,放线菌资源丰富,而且是微生物代谢产物中具有生物活性物质的主要产生菌,具开发应用前景。为获得能有效防治十字花科作物根肿病且性状优良的生防放线菌,本研究从根际土壤及植株体内分离并筛选放线菌入手,开展了生防放线菌的防效测定、菌株的鉴定、发酵液稳定性、抗菌谱、对防御酶系的影响、定殖状况、发酵条件的优化、活性物质的初步分离纯化及鉴定等方面的研究,取得了以下结果：
1.生防放线菌菌株的分离、筛选及鉴定试验从茶树、红豆树、银杏树、白菜及油菜的根际土壤和根、茎、叶、枝条等材料中一共分离出放线菌368株。通过测定其对根肿病菌休眠孢子萌发的抑制作用初筛得到16株拮抗放线菌,复筛获得A316和A10两个具生防潜能的菌株。
采用传统分类、化学分类与分子分类相结合的方法,对根肿病菌生防放线菌A316和A10进行了分类鉴定。菌株A316与链霉菌中灰红链霉菌(Streptomyces griseoruber) NBRC 12873的16S rDNA同源性高达99.9%,形态与培养特征、生理生化特性等也与灰红链霉菌高度相似,因此,将菌株A316初步鉴定为灰红链霉菌(S. griseoruber);而菌株A10在构建发育树和16S rDNA同源性分析时与GenBank数据库中多个相似性序列的亲缘关系都较近,鉴于其形态与培养特征、生理生化特性等,菌株A10仅能归于链霉菌中的灰褐类群(Streptomyces sp.)。菌株A316和A10在GenBank的序列登录号分别为HQ335354和HQ335355。
2.菌株A316和A10对根肿病的防效及抗菌谱来自白菜根际土壤的A316和来自红豆树根部的A10对根肿病菌休眠孢子萌发的抑制作用最好,其培养液的抑制率分别高达77.58%和72.60%。菌株A316和A10的发酵液及菌悬液对根肿病菌休眠孢子的萌发均具有一定的抑制作用,因此2菌抑制根肿病菌的活性物质同时存在于发酵液和菌丝体中。通过传代实验表明,菌株A316和A10连续传代10次没有明显的活性退化现象,表明2种生防菌菌株的遗传稳定性较好。
经室内盆栽试验测定有效抑制休眠孢子萌发的16株拮抗菌株对根肿病的防治效果发现,菌株A316和A10的盆栽防效好于其他菌株,2菌株对白菜根肿病的室内盆栽防效分别为73.69%、70.37%,对油菜根肿病的室内盆栽防效分别达75.68%、71.65%。菌株A316和A10在大田对防治根肿病同样有效,2菌株对白菜根肿病的田间小区防效分别为65.91%、60.25%,对油菜根肿病的田间小区防效分别达67.92%、61.32%。生防菌株A316和A10还可在一定程度上提高白菜的产量,其对白菜的亩增产率依次达96.1%、64.9%。
抗菌谱实验表明,菌株A316和A10对玉米大斑病菌(Exserohilum turcicum)、玉米小斑病菌(Bipolaris maydis)、玉米弯孢病菌(Curvularia lunata)、西瓜枯萎病菌(Fusarium oxysporum)、枯草芽孢杆菌(Bacillus subtilis)等多种植物病原真菌及细菌都有不同程度的抑制作用,菌株A10较A316更具广谱性。但菌体本身与发酵液的抑菌活性之间没有必然联系。
3.菌株A316和A10的发酵液稳定性菌株A316和A10发酵液产生的活性物质在紫外线、中性或酸性条件下比较稳定,且对蛋白酶K有较强的稳定性。pH值调到11处理后,菌株A316和A10对根肿病菌休眠孢子萌发的抑制率分别下降达28.06%和32.73%。发酵液在100℃处理30 min和60 min时,A316对根肿病菌休眠孢子萌发的抑制率仅分别下降了9.71%和13.31%,而A10的抑制率分别下降了27.69%和32.73%,因此菌株A316的发酵产物对热的稳定性也较好,菌株A10在中低温条件下稳定。菌株A316的发酵液低温储藏稳定性较好,而菌株A10发酵液的储藏稳定性不及A316,在4℃和室温下放置22 d时,菌株A316发酵液的抑制率分别下降了14.08%和28.16%,菌株A10发酵液的抑制率分别下降了25.27%和36.82%。
4.菌株A316和A10对白菜防御酶系的影响通过测定白菜植株体内防御酶系的变化发现,接种根肿病菌的同时使用A316或A10的发酵液灌根后,白菜叶片的PAL、POD、PPO活性均明显上升,且呈现“先升后降”的趋势。对于菌株A316,PAL、POD、PPO酶活峰值分别出现在第7d (36.87 U/g-h)、第5d (141.5 U/g-min)、第5d(28.97 U/g-min),比仅接种根肿病菌时的酶活峰值依次提高了42.63%、40.45%、53.69%；而对于菌株A10, PAL、POD、PPO酶活峰值分别出现在第9d(33.4U/g-h)、第7d (133.65 U/g-min)、第7d (26.14 U/g-min),比仅接种根肿病菌时的酶活峰值依次提高了29.21%、32.66%、38.67%。
5.菌株A316和A10的定殖采用传统的抗生素标记法,设置自然土与无菌土2个处理,分别测定了生防放线菌A316和A10的抗利福平突变菌株在土壤中及白菜植株内的定殖能力。2菌株在土壤中及植株体内均具有较强的定殖能力。自然土中,菌株A316在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在14d (6.79×106cfu/g土)、17d(4.33×104cfu/g根)、17 d (2.82×103cfu/g茎)、24 d (3.8×102cfu/g叶),菌株A10在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在21 d (9.23×106 cfu/g土)、17d(9.06x103cfu/g根)、17d (3.23×102cfu/g茎)、24 d (0.981×102cfu/g叶)；灭菌土中,菌株A316在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在14 d(2.36×106cfu/g土)、24d(1.77×104cfu/g根)、24d(7.53×102 cfu/g茎)、24d(2.71×102 cfu/g叶),菌株A10在土壤及白菜根、茎、叶内的最大定殖菌量分别出现在21 d(5.4×106cfu/g土)、17d(3.79×103cfu/g根)、24 d(2.23×102 cfu/g茎)、24d(0.851×102 cfu/g叶)。土壤中A10的定殖能力好于A316,而在植株体内A316的定殖能力好于A10。灭菌土中2菌的定殖菌量均小于自然土中的定殖菌量,灭菌土中的菌量变化幅度大于自然土。2生防菌株在植株的根内定殖数量最大,茎中次之,叶中最少。
6.菌株A316和A10的发酵优化采用正交设计法改良生防放线菌的发酵培养基,并使用单因素分析法优化其培养条件。菌株A316的较佳培养基组成为：1%葡萄糖,1%大豆粉,0.5%酵母膏；其理想的发酵培养条件为：种子液种龄36 h,接种量8%,初始pH 7,装液量40%,温度28℃,摇床转速180r/min,发酵时间5 d。菌株A10的较佳培养基组成为：1.5%葡萄糖,1%大豆粉,0.3%蛋白胨；其理想的发酵培养条件为：种子液种龄60 h,接种量10%,初始pH 7.5,装液量30%,温度28℃,摇床转速180r/min,发酵时间6 d。
7.菌株A316抗根肿病菌活性物质的初步分离及鉴定灰红链霉菌(Streptomyces griseoruber) A316的发酵产物经溶剂萃取、薄层层析、硅胶柱层析等进行分离纯化,得到了其抑制根肿病菌的活性物质。活性组分经气相色谱-质谱(GC-MS)联用分析后发现主要含有2种物质。化合物Ⅰ与通用型酚类抗氧剂2,2’-亚甲基双-(4-甲基-6-叔丁基苯酚)的匹配度达97.84%；化合物Ⅱ极性较大,应该是含有活泼氢的物质,与邻苯二甲酸单乙基已基酯的匹配度为74.28%。
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 A10
A316 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.
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