辽宁药用植物土壤微生物多样性及生防菌株研究
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摘要
辽宁省位于长白山、华北和蒙古3个植物区系交汇地带,种植中草药具有得天独厚的地理环境和生物资源,大力发展中药材生产已成为辽东山区农民走多种经营致富道路的重要途径。
在本研究中,首次以辽宁省主栽药用植物:五味子、细辛、穿山龙、移山参、龙胆、黄花乌头、平贝母、百合、黄芪、防风、苍术、玉竹、威灵鲜和白鲜皮等近20种药用植物土壤为研究对象,应用平板稀释法和磷脂脂肪酸测定法对土壤微生物多样性进行研究,通过研究明确了影响土壤微生物多样性的因素;土壤微生物与药用植物关系;以药用植物土壤微生物为供试菌株,药用植物近年来主要病害为靶标菌,进行生防菌株筛选。本论文主要研究结果如下。
1.分析辽宁省药用植物土壤可培养微生物数量,生长年限是影响药用植物土壤微生物数量的主要原因,其次是药用植物入药部位,和土壤肥力、种植方式和季节变化也有一定关系;供试的14种4年生药用植物土壤微生物数量从高到低变化趋势依次为黄花乌头、穿山龙、五味子、平贝母、百合、移山参、黄芪、防风、苍术、龙胆草、细辛、玉竹、威灵鲜和白鲜皮。
2.首次采用磷脂脂肪酸(PLFA)测定法对药用植物土壤微生物多样性构成和群落进行了识别和定量描述。五味子土壤微生物PLFA含量随种植年限的加长而增加;健康五味子土壤微生物PLFA总量略高于罹病五味子土壤微生物PLFA总量;沈阳农业大学药材园4种药用植物土壤微生物PLFA含量高低顺序依次为:黄花乌头、穿山龙、黄芪、五味子;五年生人参土壤微生物PLFA含量明显高于两年生的含量。4年生和5年生五味子土壤微生物的优势种群为14:0、i15:0、a15:0、16:0、16:1w7t、17:0、18:0、a18:0、18:1w7、cy19:0和20:0。
3.从14种4年生药用植物土壤中共分离到真菌菌株185株,分属于30个属,其中以镰孢菌属Fusarium、木霉菌属Trichoderma和青霉菌属Penicillium出现频率较高。药用植物土壤真菌多样性与药用植物入药部位和药用植物种类有一定关系,和地理环境关系不明显。在真菌属的水平上研究14种药用植物之间关系,把14种药用植物分成5组,百合、平贝母、人参、防风和黄芪为一组;龙胆草、苍术和玉竹为一组;五味子、威灵仙、穿山龙和细辛为一组;白鲜皮和黄花乌头自成一组。关系最近的两种药用植物为百合和平贝母,最远的为白鲜皮和穿山龙。
4.通过形态学方法,对从药用植物土壤中分离到真菌优势种群进行鉴定,共鉴定出镰孢菌属10种,尖孢镰孢菌F.oxysporum为优势种群,其中大部分为药用植物根腐病的病原菌;木霉菌属9种,哈茨木霉T.harzianum为优势种群;青霉菌属6种,微紫青霉P.janthinellum为优势种群。
5.从14种4年生药用植物土壤中共分离到细菌109株,分属于17个属,其中芽孢杆菌属Bacillus为优势种群:分离到放线菌213株,分属于链霉菌属Streptomyces、诺卡氏菌Nocardia、链孢囊菌Streptosporangium、小多孢菌Micropolyspora和小单孢菌Micromonospora。其中优势种群为链霉菌属。从土壤细菌种群水平上对辽宁14种4年生药用植物进行分析,把14种药用植物分成2组,苍术、白鲜皮、五味子、百合、和细辛为一组:人参、威灵仙、黄花乌头、龙胆草、平贝母、玉竹、黄芪、穿山龙、防风为一组。为一组。
6.首次从辽宁药用植物土壤中筛选出对靶标病原菌(人参锈腐病菌Cylindrocarponpanicicola、穿山龙黑斑病菌Alternania dioscoreae、五味子茎基腐病菌Fusarium.oxysporum、人参黑斑病菌Rhizoctonia sp.和草莓灰霉病菌Botrytis cinerea)抑菌效果较好的菌株,其中细菌3株,放线菌4株,木霉菌1株。筛选出的细菌和放线菌可以在不同程度上使病原菌菌丝出现变形,肿大:顶端出现囊泡;菌丝变透明:菌丝体溶解;原生质体外渗等现象。无菌发酵液在一定程度上抑制菌丝生长和孢子萌发。木霉菌株T1对供试的五种病原菌抑制率均在75%以上,通过缠绕、穿透和侵入病原菌菌丝,与病原菌菌丝平行生长等现象分解病原菌菌丝物质,促使病原菌菌丝逐渐解体。说明药用植物土壤中广泛存在拮抗效果较好的生防菌株,为药用植物病害生物防治提供了理论基础。
本论文创新点为,首次以辽宁药用植物土壤为研究对象,将磷脂脂肪酸(PLFA)测定法应用到药用植物土壤微生物多样性研究上,并首次进行了辽宁药用植物土壤微生物中生防菌株筛选研究。
Liaoning Province is located in the joint zone of three flora of changbai mountain, north China and Mongolia,which enjoys nature-endowed geographic environments and biological resources in planting Chinese herbal medicine.The development of Chinese herbal medicine is one of main methods for peasants to increase income in the eastern mountain area of Liaoning.
The main medicinal plants in Liaoning Province are schisandra,asarum,dioscorea, panax,gentiana,aconitum,fritillaria,lilium,astragalus,saposhnikovia,atractylodes,polygonatum, clematis,dictamnus,etc.The soils of nearly 20 species of the medicinal plant were studied in this paper.The differences among different medicinal plants were analyzed.The species and quantity of fungi,bacteria and antinomycete in soil of medicinal plants were clear.With the pathogen of main diseases of medicinal plants as target,the strains in the control of pathogen were screened from soil of medicinal plants.The main results are as follows.
1.The quantity of culturable microorganisms in medicinal plants' soil was analyzed. The results showed that the main reason affecting the number of microorganisms in soil was root exudates of medicinal plants,and next came soil fertility and planting pattern.The effect of changes of seasons was subtle.The soil microorganisms of perennial medicinal plants were more than of annual and biennial ones.The results showed that the soil microorganisms number of the 14 4-year-old medicinal plants decreased in the order of aconitum,dioscorea, schisandra,fritillaria,lilium,panax,astragalus,saposhnikovia,atractylodes,gentiana,asarum, polygonatum,clematis,dictamnus.
2.The composition of phospholipid fatty acid(PLFA) can be used as 'signatures' to analyze changes in microbial communities structure and microbial biomass.PLFA content of soil microorganisms of schisandra increased with the increase of planting age;PLFA quantity of healthy schisandra were slightly higher than those of infected ones.PLFA content of 4 medicinal plants decreased in the order of aconitum,dioscorea,astragalus,schisandra.PLFA content of 5-year-old panax were obviously higher than those of 2-year-old ones.The dominant species of al and a2 were 14:0,i15:0,a15:0,16:0,16:1w7t,17:0,18:0,a18:0, 18:1w7,cy19:0 and 20:0;
3.185 fungi strains were isolated from soil of 14 4-year-old medicinal plants,which belonged to 30 genera.Among them the frequency of Fusarium,Trichoderma and Penicillium were higher and could be isolated from all 14 medicinal plants.The frequency of Fusarium, Cylindrocarpon and Rhizoctonia inducing root disease were higher,too.The results indicated that the diversity of soil fungi of medicinal plants related with species and using parts of medicinal plants,and had no obvious relationship with geographic environment.Lilium and fritillaria might be the most closely related species,and dictamnus and dioscorea might be farthest.
4.The dominant fungi species from 63 medicinal plants soil samples were identified through morphological methods.The results showed that 10 species of Fusarium were identified,among them Fusarium oxysporum was the dominant one;9 species of Trichoderma were identified,among them Trichoderma harzianum was the dominant one;7 species of Penicillium were identified,among them Penicillium janthinellum was the dominant one.
5.109 bacteria strains were isolated from soil of 14 4-year-old medicinal plants,which belonged to 17 genera.Most of them were gram-negative bacteria.4 genera of gram-positive bacteria were identified,among them Bacillus was the dominant one.213 antinomycete were isolated,of which Streptomyces,Nocardia,Streptosporangium,Micropolyspora and Micromonospora were identified.The dominant genera was Streptomyces.The results indicated that the diversity of soil bacteria of medicinal plants might be related with species, using parts of medicinal plants and geographic environment.Clematis and panax might be the most closely related species,and astragalus,schisamdra and atractylodes might be farthest.
6.3 bacteria,4 antinomycete and 1 Trichoderma against 5 target pathogen were screened from soil of medicinal plants.The screened bacteria and antinomycete could cause the deformation of pathogen hyphal growth in different degree.The phenomena of Vesicles, swelling and protoplasm leaking out were observed.The germfree ferment filtrate could inhibit hyphal growth and spore germination of pathogen.The inhibition rate of Trichoderma T1 against 5 pathogen were all over 75%.T1 could decompose the pathogen hypha through interweaving,penetrating,invading and paralleling.Good biocontrol strains are widely distributed in medicinal plant soil.The results provides theoretical base for biocontrol of medicinal plant disease.
在本研究中,首次以辽宁省主栽药用植物:五味子、细辛、穿山龙、移山参、龙胆、黄花乌头、平贝母、百合、黄芪、防风、苍术、玉竹、威灵鲜和白鲜皮等近20种药用植物土壤为研究对象,应用平板稀释法和磷脂脂肪酸测定法对土壤微生物多样性进行研究,通过研究明确了影响土壤微生物多样性的因素;土壤微生物与药用植物关系;以药用植物土壤微生物为供试菌株,药用植物近年来主要病害为靶标菌,进行生防菌株筛选。本论文主要研究结果如下。
1.分析辽宁省药用植物土壤可培养微生物数量,生长年限是影响药用植物土壤微生物数量的主要原因,其次是药用植物入药部位,和土壤肥力、种植方式和季节变化也有一定关系;供试的14种4年生药用植物土壤微生物数量从高到低变化趋势依次为黄花乌头、穿山龙、五味子、平贝母、百合、移山参、黄芪、防风、苍术、龙胆草、细辛、玉竹、威灵鲜和白鲜皮。
2.首次采用磷脂脂肪酸(PLFA)测定法对药用植物土壤微生物多样性构成和群落进行了识别和定量描述。五味子土壤微生物PLFA含量随种植年限的加长而增加;健康五味子土壤微生物PLFA总量略高于罹病五味子土壤微生物PLFA总量;沈阳农业大学药材园4种药用植物土壤微生物PLFA含量高低顺序依次为:黄花乌头、穿山龙、黄芪、五味子;五年生人参土壤微生物PLFA含量明显高于两年生的含量。4年生和5年生五味子土壤微生物的优势种群为14:0、i15:0、a15:0、16:0、16:1w7t、17:0、18:0、a18:0、18:1w7、cy19:0和20:0。
3.从14种4年生药用植物土壤中共分离到真菌菌株185株,分属于30个属,其中以镰孢菌属Fusarium、木霉菌属Trichoderma和青霉菌属Penicillium出现频率较高。药用植物土壤真菌多样性与药用植物入药部位和药用植物种类有一定关系,和地理环境关系不明显。在真菌属的水平上研究14种药用植物之间关系,把14种药用植物分成5组,百合、平贝母、人参、防风和黄芪为一组;龙胆草、苍术和玉竹为一组;五味子、威灵仙、穿山龙和细辛为一组;白鲜皮和黄花乌头自成一组。关系最近的两种药用植物为百合和平贝母,最远的为白鲜皮和穿山龙。
4.通过形态学方法,对从药用植物土壤中分离到真菌优势种群进行鉴定,共鉴定出镰孢菌属10种,尖孢镰孢菌F.oxysporum为优势种群,其中大部分为药用植物根腐病的病原菌;木霉菌属9种,哈茨木霉T.harzianum为优势种群;青霉菌属6种,微紫青霉P.janthinellum为优势种群。
5.从14种4年生药用植物土壤中共分离到细菌109株,分属于17个属,其中芽孢杆菌属Bacillus为优势种群:分离到放线菌213株,分属于链霉菌属Streptomyces、诺卡氏菌Nocardia、链孢囊菌Streptosporangium、小多孢菌Micropolyspora和小单孢菌Micromonospora。其中优势种群为链霉菌属。从土壤细菌种群水平上对辽宁14种4年生药用植物进行分析,把14种药用植物分成2组,苍术、白鲜皮、五味子、百合、和细辛为一组:人参、威灵仙、黄花乌头、龙胆草、平贝母、玉竹、黄芪、穿山龙、防风为一组。为一组。
6.首次从辽宁药用植物土壤中筛选出对靶标病原菌(人参锈腐病菌Cylindrocarponpanicicola、穿山龙黑斑病菌Alternania dioscoreae、五味子茎基腐病菌Fusarium.oxysporum、人参黑斑病菌Rhizoctonia sp.和草莓灰霉病菌Botrytis cinerea)抑菌效果较好的菌株,其中细菌3株,放线菌4株,木霉菌1株。筛选出的细菌和放线菌可以在不同程度上使病原菌菌丝出现变形,肿大:顶端出现囊泡;菌丝变透明:菌丝体溶解;原生质体外渗等现象。无菌发酵液在一定程度上抑制菌丝生长和孢子萌发。木霉菌株T1对供试的五种病原菌抑制率均在75%以上,通过缠绕、穿透和侵入病原菌菌丝,与病原菌菌丝平行生长等现象分解病原菌菌丝物质,促使病原菌菌丝逐渐解体。说明药用植物土壤中广泛存在拮抗效果较好的生防菌株,为药用植物病害生物防治提供了理论基础。
本论文创新点为,首次以辽宁药用植物土壤为研究对象,将磷脂脂肪酸(PLFA)测定法应用到药用植物土壤微生物多样性研究上,并首次进行了辽宁药用植物土壤微生物中生防菌株筛选研究。
Liaoning Province is located in the joint zone of three flora of changbai mountain, north China and Mongolia,which enjoys nature-endowed geographic environments and biological resources in planting Chinese herbal medicine.The development of Chinese herbal medicine is one of main methods for peasants to increase income in the eastern mountain area of Liaoning.
The main medicinal plants in Liaoning Province are schisandra,asarum,dioscorea, panax,gentiana,aconitum,fritillaria,lilium,astragalus,saposhnikovia,atractylodes,polygonatum, clematis,dictamnus,etc.The soils of nearly 20 species of the medicinal plant were studied in this paper.The differences among different medicinal plants were analyzed.The species and quantity of fungi,bacteria and antinomycete in soil of medicinal plants were clear.With the pathogen of main diseases of medicinal plants as target,the strains in the control of pathogen were screened from soil of medicinal plants.The main results are as follows.
1.The quantity of culturable microorganisms in medicinal plants' soil was analyzed. The results showed that the main reason affecting the number of microorganisms in soil was root exudates of medicinal plants,and next came soil fertility and planting pattern.The effect of changes of seasons was subtle.The soil microorganisms of perennial medicinal plants were more than of annual and biennial ones.The results showed that the soil microorganisms number of the 14 4-year-old medicinal plants decreased in the order of aconitum,dioscorea, schisandra,fritillaria,lilium,panax,astragalus,saposhnikovia,atractylodes,gentiana,asarum, polygonatum,clematis,dictamnus.
2.The composition of phospholipid fatty acid(PLFA) can be used as 'signatures' to analyze changes in microbial communities structure and microbial biomass.PLFA content of soil microorganisms of schisandra increased with the increase of planting age;PLFA quantity of healthy schisandra were slightly higher than those of infected ones.PLFA content of 4 medicinal plants decreased in the order of aconitum,dioscorea,astragalus,schisandra.PLFA content of 5-year-old panax were obviously higher than those of 2-year-old ones.The dominant species of al and a2 were 14:0,i15:0,a15:0,16:0,16:1w7t,17:0,18:0,a18:0, 18:1w7,cy19:0 and 20:0;
3.185 fungi strains were isolated from soil of 14 4-year-old medicinal plants,which belonged to 30 genera.Among them the frequency of Fusarium,Trichoderma and Penicillium were higher and could be isolated from all 14 medicinal plants.The frequency of Fusarium, Cylindrocarpon and Rhizoctonia inducing root disease were higher,too.The results indicated that the diversity of soil fungi of medicinal plants related with species and using parts of medicinal plants,and had no obvious relationship with geographic environment.Lilium and fritillaria might be the most closely related species,and dictamnus and dioscorea might be farthest.
4.The dominant fungi species from 63 medicinal plants soil samples were identified through morphological methods.The results showed that 10 species of Fusarium were identified,among them Fusarium oxysporum was the dominant one;9 species of Trichoderma were identified,among them Trichoderma harzianum was the dominant one;7 species of Penicillium were identified,among them Penicillium janthinellum was the dominant one.
5.109 bacteria strains were isolated from soil of 14 4-year-old medicinal plants,which belonged to 17 genera.Most of them were gram-negative bacteria.4 genera of gram-positive bacteria were identified,among them Bacillus was the dominant one.213 antinomycete were isolated,of which Streptomyces,Nocardia,Streptosporangium,Micropolyspora and Micromonospora were identified.The dominant genera was Streptomyces.The results indicated that the diversity of soil bacteria of medicinal plants might be related with species, using parts of medicinal plants and geographic environment.Clematis and panax might be the most closely related species,and astragalus,schisamdra and atractylodes might be farthest.
6.3 bacteria,4 antinomycete and 1 Trichoderma against 5 target pathogen were screened from soil of medicinal plants.The screened bacteria and antinomycete could cause the deformation of pathogen hyphal growth in different degree.The phenomena of Vesicles, swelling and protoplasm leaking out were observed.The germfree ferment filtrate could inhibit hyphal growth and spore germination of pathogen.The inhibition rate of Trichoderma T1 against 5 pathogen were all over 75%.T1 could decompose the pathogen hypha through interweaving,penetrating,invading and paralleling.Good biocontrol strains are widely distributed in medicinal plant soil.The results provides theoretical base for biocontrol of medicinal plant disease.
引文
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