生物有机肥防控黄瓜土传立枯病效果及防控机理
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摘要
黄瓜(Cucumis sativus Linn)广泛分布于世界各地,并且为主要的温室产品之一。黄瓜土传立枯病是一种土传病害,主要由立枯丝核菌(Rhizoctonia solani Kuhn)引起,在世界各地均有不同程度的发生,每年造成了大量的经济损失。土传立枯病的防控是一个世界性的难题,传统的化学和农业防控措施通常会带来环境问题、食品安全问题、成本高和劳动强度大等问题,因此不能成功的应用于农业生产。而利用生物防控土传病害由于具有高效、环保等优点日益受到人们重视。
·本文筛选出了对土传立枯病病原菌立枯丝核菌具有高效拮抗作用的拮抗菌哈茨木霉SQR-T37(T37)和短小芽孢杆菌SQR-N43(N43).利用这两株高效拮抗菌及其生物有机肥进行黄瓜土传立枯病盆栽防控试验,并检测了施用拮抗菌后根际土壤中病原菌和拮抗菌的数量,以及根际土壤中微生物区系的变化。研究了两株高效拮抗菌在黄瓜根系的定殖作用,以及对病原菌拮抗的作用机理。继而研究了植物根系分泌物对两株高效拮抗菌的影响。
主要结果如下：
1.利用科赫法则验证立枯丝核菌Q1是黄瓜土传立枯病的致病菌株。采用平板对峙法从黄瓜根际土壤中分离出的400余株细菌菌株中筛选出16株对立枯丝核菌具有拮抗效果的菌株,抑菌带直径在0.8～1.9cm之间；并从中选出3株抑菌带直径在1.6cm以上的菌株N33、N35和N43,三株拮抗菌均能产生可溶性和挥发性的拮抗物质,结合形态、生理生化特性及16S rDNA序列比对分析,鉴定N33菌株(登录号HM439651)为荧光假单胞菌Pseudomonas fluorescens, N35和N43菌株(登录号GQ465936和GQ465935)为短小芽孢杆菌Bacillus pumilus。复筛确定短小芽孢杆菌SQR-N43和哈茨木霉Trichoderma harzianum SQR-T37菌株用于后续试验。
2.通过盆栽试验研究了拮抗菌及其生物有机肥对黄瓜的促生效果和土传立枯病的生物防控效果。结果表明短小芽孢杆菌SQR-N43和哈茨木霉SQR-T37能显著增加黄瓜地上部鲜重,与对照相比分别增加了38.6%和55.9%。拮抗菌N43和T37可以防控黄瓜土传立枯病,而且T37第二季的防效是第一季防效的1.25倍；此外,生物有机肥的防效(68.2%和81.8%)显著高于单独施用拮抗菌N43和T37的防效(22.7%和27.3%)。施用拮抗菌后,植物根际病原菌的数量由每克土106ITS拷贝数降低到每克土104ITS拷贝数。单独施用拮抗菌T37,接种后第0天到第20天哈茨木霉的数量从的每克土107ITS拷贝数降低到每克土105ITS拷贝数；生物有机肥的使用维持了拮抗菌T37的数量在每克土107ITS拷贝数；生物有机肥处理N43总数和N43芽孢数量所占百分比分别为1.67×108CFU g-1土和51.9%,与单独施用拮抗菌N43的处理(2.20×107CFUg-1土和78.6%)相比有显著差异。拮抗菌N43和T37及其生物有机肥降低了土壤中有害真菌的种类,如立枯丝核菌Rhizoctonia solani、玉米大斑病菌Setosphaeria sp和油瓶霉Lecythophora;增加了土壤细菌的种类和数量。综上所述,拮抗菌和生物有机肥可以促进黄瓜生长、防控黄瓜土传立枯病、降低根际土壤病原菌数量和改善根际土壤微生物区系,其中生物有机肥的处理效果最好。
3.为了阐述哈茨木霉SQR-T37和短小芽孢杆菌SQR-N43对立枯丝核菌的拮抗机理,显微观察了两株拮抗菌对病原菌菌丝的作用,并检测了T37菌株产生的几丁质酶和p-1,3-葡聚糖酶活性,以及N43菌株分泌的拮抗物质。结果表明：T37菌丝缠绕并穿透病原菌菌丝,造成病原菌菌丝细胞质渗漏和细胞皱缩。新鲜的和烘干的病原菌细胞壁都可以诱导T37菌株几丁质酶(9.49和10.80nkat ml-1粗酶液)和p-1,3-葡聚糖酶的产生(37.57和43.18nkat ml-1粗酶液)。N43菌株在对数生长期产生对立枯丝核菌Q1具有抑制作用的拮抗物质,这种物质造成了病原菌菌丝的顶端膨大和细胞质的渗漏。当培养基中NaCl浓度为1%(w/v)时,拮抗物质的活性达到最大值。70%饱和度的硫酸铵可以完全沉淀发酵液中的拮抗物质。拮抗物质经蛋白酶K和胰蛋白酶处理后活性显著下降,与对照相比分别降低了79%和53%。这种物质分子量在500-1000Da之间,对高温敏感,对碱性条件具有耐受性。拮抗物质与N43细胞悬液对土传立枯病的防控效果无显著差异。综上所述,T37菌株对立枯丝核菌Q1的拮抗机理为重寄生作用；N43菌株产生对立枯丝核菌Q1具有拮抗作用的短肽类活性物质。
4.本章构建了绿色荧光蛋白(Green Fluorescent Protein, GFP)标记的短小芽孢杆菌SQR-N43菌株(GFP-N43),并研究了GFP-N43菌株和哈茨木霉SQR-T37在黄瓜根系的定殖能力。结果表明：构建的GFP-N43菌株能发出绿色荧光；生长到89代时,还有43.33%的菌株含有pHAPⅡ质粒；GFP-N43菌株对立枯丝核菌Q1的抑制效果较N43菌株相比无显著差异；在每克土107和108GFP-N43CFU处理中,GFP-N43在植物根尖和伸长区定殖并形成了稳健的生物膜,保护植物根系不受病原菌侵害；定量结果表明在每克土107和108GFP-N43CFU处理中,GFP-N43菌体浓度有所下降,接种后14天的菌体浓度分别是每克土4.6×106CFU和1.1×107CFU；0到14天,105和106浓度的处理中GFP-N43的数量呈上升趋势,第14天菌体浓度分别是第0天的3.55和2.09倍；T37菌丝可以缠绕黄瓜根系,在黄瓜根表形成保护层。
5.利用高效液相色谱鉴定了黄瓜根系分泌的有机酸,并检测了拮抗菌短小芽孢杆菌SQR-N43对有机酸的趋化作用、哈茨木霉SQR-T37对有机酸的利用和有机酸对T37孢子萌发和菌丝生长的影响。结果表明：黄瓜根系分泌物中有机酸主要有草酸、苹果酸、乙酸、柠檬酸、琥珀酸和延胡索酸。N43菌株对苹果酸和柠檬酸具有浓度依赖性的正向趋化作用,趋化系数分别为3.1和2.3。除琥珀酸外,另4种有机酸都对T37孢子萌发具有不同程度的促进作用；30μgm1-1的草酸具有最大促进作用,T37孢子萌发数较对照处理提高了39%。5种有机酸对T37菌丝生长都具有不同程度的促进作用；80μgm1-1的苹果酸具有最大促进作用,T37菌落直径是对照处理的1.1倍。在5种有机酸都存在的情况下,T37优先利用乙酸、草酸和苹果酸。
Cucumber (Cucumis sativus Linn) is one of the main greenhouse vegetables which widely distributes around the world. The soil-born disease of cucumber Rhizoctonia damping-off is mainly caused by Rhizoctonia solani, which occurs around the world with different degree and causes a lot of economic loss every year. The control of the disease is a world wide problem. Traditional chemical and farming control methods result in serious environmental, foot safety problems and time-and labor-consuming, thus can not be successfully applied in agricultural production. Biological control of soil-born disease is an efficient and environmentally friendly way and is receiving increased attention.
In the present study, we isolated and screened Trichoderma harzianum SQR-T37(T37) and Bacillus pumilus SQR-N43(N43) which showed highly antagonistic effect against Rhizoctonia solani, the pathogen of damping-off disease. The two antagonistic strains and their bio-organic fertilizers were used in pot experiments to assess their control efficiencies of cucumber damping-off disease. The populations of pathogen and antagonistic strains and microbial communities in the rhizosphere soils after applied with antagonistic strains and their bio-organic fertilizers were also determined. Besides, the abilities of colonization in cucumber rhizosphere and the mechanisms of antagonism against the pathogen of the antagonists were studied. The influences of plant root exudates on the antagonistic strains were also investigated.
The main results obtained were listed as follows:
1. In this study, Rhizoctonia solani Q1was verified as the pathogen of cucumber damping-off disease using Koch's postulates. More than400bacterial strains were isolated from rhizosphere soil of cucumber plants, and the dual culture method was used to screen bacterial antagonists against R. solani. Sixteen strains of bacterial antagonists were isolated and their inhabiting zone against R. solani ranged from0.8cm to1.9cm. Three strains (N33, N34and N43) with inhabiting zone more than1.6cm could produce volatile antibiotics and soluble antibiotics. Based on the results of morphologic characteristics, physiological and biochemical properties and phylogenetic analysis of16S rDNA, the three strains (Genbank accession number HM439651, GQ465936and GQ465935, respectively) were identified as Pseudomonas fluorescens, Bacillus pumilus and Bacillus pumilus, respectively. Antagonists of B. pumilus SQR-N43and Trichoderma harzianum SQR-T37were further chosen for following experiments.
2. Pot Experiments were performed to assess the promotional efficiency on the growth of cucumber and in vivo disease-control efficiency of the antagonistic microbes and bio-organic fertilizers (BIOs). Results indicated that compared with control, Bacillus pumilus SQR-N43and Trichoderma harzianum SQR-T37increased the dry weight of cucumber shoot by38.6%and55.9%, respectively. The antagonists N43and T37could control damping-off disease of cucumber; further more, the control efficiency of T37increased during the second growth cycle by25.0%as compared with the first growth cycle. Besides, the control efficiencies of BIO products (68.2%and81.8%, respectively) were higher than those applied with antagonist N43or T37solely (22.7%and27.3%, respectively). The population of R. solani in the rhizosphere soil of cucumber was decreased from106ITS copies g-1soil to104ITS copies g-1soil in the presence of the antagonists. Twenty days after incubation, the population of T. harzianum was107ITS copies g-1soil in the BIO treatment, which was much higher than that in the treatment only T37was applied (105ITS copies g-1soil). Significant differences in the number of CFUs and the percentage of spores of N43were recorded between the N treatment (2.20×107CFU g'1of soil and79%, respectively) and the BIO treatment (1.67×108CFU g-1of soil and52%, respectively). The antagonists N43and T37and BIOs decreased the number of harmful fungal species, such as Rhizoctonia solani, Setosphaeria sp. and Lecythophora, and increased the population of bacteria and the number of bacterial species. As a conclusion, the antagonistic microbes and BIOs could promote the growth of cucumber, prevent the damping-off disease of cucumber, decrease the population of the pathogen in the rhizosphere soil, and improve the rhizosphere microbial community. Among of them, the BIOs had the best efficiency.
3. For clarification of the mechanisms of the antagonism of Trichoderma harzianum SQR-T37and Bacillus pumilus SQR-N43against Rhizoctonia solani Q1, the effect of two antagonistic strains on the pathogen mycelium was microscopically observed and the activities of chitinase and β-1,3-glucanase produced by T37and production of antibiotic by N43were determined. Result showed that the mycelia of T37coiled themselves round and penetrated into the mycelia of pathogen, causing leakage of cytoplasm and crimple of pathogen cell walls. Both fresh and dried cell walls of the pathogen could induce the production of chitinase (9.49and10.80nkat ml-1crude enzyme, respectively) and β-1,3-glucanase (37.57and43.18nkat ml-1crude enzyme, respectively). N43produced antibiotic substances towards R. solani Ql at logarithmic growth phase. The antibiotics caused enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1mycelia. The activities of the antibiotics had the highest value when the concentration of NaCl in the medium was1%(w/v).70%saturation of ammonium sulfate made complete precipitation of the antibiotics in culture broth. When treated with protease K and trypsase, the activities of antibiotics were decreased by79%and53%, respectively, compared with control. The antibiotics were sensitive to high temperature and were alkaline stable. The molecular weights of the substances were between500-1000Da. The control efficiencies of the antibiotics and cell suspension of N43on damping-off disease had no significant difference. As a conclusion, the mechanism of antagonism of T37against R. solani Q1was mycoparasitism. N43produced oligopeptides which had inhibitory effect on R. solani Q1.
4. In this study, green fluorescent protein tagged Bacillus pumilus SQR-N43strain (GFP-N43) was constructed and abilities of both GFP-N43and Trichoderma harzianum SQR-T37to colonize roots and the rhizosphere soil of cucumber were determined. Results showed that constructed GFP-N43strain could emit green fluorescent. When grew up to89generations,43.3%of population of the strain still contained plasmid pHAPⅡ. The inhibitory effect of GFP-N43against Rhizoctonia solani Q1was identical with that of N43strain. In the treatments inoculated with107and108GFP-N43CFU g-1of soil, the presence of GFP-N43through colonization and formation of biofilm on tips and elongation zones of plant roots could ensure the protection of the hosts from the pathogen. Quantitative measurement showed that in the treatments of10and10GFP-N43CFU g-1soil, the numbers of GFP-N43strain decreased to4.6×106CFU g-1soil and1.1×107CFU g-1soil, respectively, at14days after inoculation (DAI). However, in the treatments inoculated with105and106GFP-N43CFU g-1soil, the populations of GFP-N43strain had upward trends in the period from0to14DAI, and GFP-N43strain at14DAI were3.55and2.09times higher than those at0DAI, respectively. The hyphae of T37strain coiled round cucumber roots and formed protection layer on cucumber root surfaces.
5. The organic acids in cucumber root exudates were identified by high performance liquid chromatography (HPLC). Chemotaxis of B. pumilus SQR-N43towards organic acids, utilization of organic acids by T. harzianum SQR-T37strain and the influences of organic acids on T37conidial germination and mycelial growth were also determined. Results indicated that oxalic acid, malic acid, acetic acid, citric acid and succinic acid were the mainly organic acids in cucumber root exudates. N43exhibited positive, concentration-dependent chemotactic behavior towards malic acid and citric acid with a relative chemotaxis index3.1and2.3, respectively. Except for succinic acid, the organic acids had different degrees of promotional effects on T37conidial germination. The largest number of germinated conidia which increased by39%, compared with control, was obtained with30μg ml-1oxalic acid. The five organic acids had various degree of promotional effect on T37mycelial growth. The largest diameter of T37colony, which was1.1times wider than that in control, was obtained with80μg ml-1malic acid. When supplied with the five organic acids, T37strain had a preference for acetic acid, oxalic acid and malic acid.
·本文筛选出了对土传立枯病病原菌立枯丝核菌具有高效拮抗作用的拮抗菌哈茨木霉SQR-T37(T37)和短小芽孢杆菌SQR-N43(N43).利用这两株高效拮抗菌及其生物有机肥进行黄瓜土传立枯病盆栽防控试验,并检测了施用拮抗菌后根际土壤中病原菌和拮抗菌的数量,以及根际土壤中微生物区系的变化。研究了两株高效拮抗菌在黄瓜根系的定殖作用,以及对病原菌拮抗的作用机理。继而研究了植物根系分泌物对两株高效拮抗菌的影响。
主要结果如下：
1.利用科赫法则验证立枯丝核菌Q1是黄瓜土传立枯病的致病菌株。采用平板对峙法从黄瓜根际土壤中分离出的400余株细菌菌株中筛选出16株对立枯丝核菌具有拮抗效果的菌株,抑菌带直径在0.8～1.9cm之间；并从中选出3株抑菌带直径在1.6cm以上的菌株N33、N35和N43,三株拮抗菌均能产生可溶性和挥发性的拮抗物质,结合形态、生理生化特性及16S rDNA序列比对分析,鉴定N33菌株(登录号HM439651)为荧光假单胞菌Pseudomonas fluorescens, N35和N43菌株(登录号GQ465936和GQ465935)为短小芽孢杆菌Bacillus pumilus。复筛确定短小芽孢杆菌SQR-N43和哈茨木霉Trichoderma harzianum SQR-T37菌株用于后续试验。
2.通过盆栽试验研究了拮抗菌及其生物有机肥对黄瓜的促生效果和土传立枯病的生物防控效果。结果表明短小芽孢杆菌SQR-N43和哈茨木霉SQR-T37能显著增加黄瓜地上部鲜重,与对照相比分别增加了38.6%和55.9%。拮抗菌N43和T37可以防控黄瓜土传立枯病,而且T37第二季的防效是第一季防效的1.25倍；此外,生物有机肥的防效(68.2%和81.8%)显著高于单独施用拮抗菌N43和T37的防效(22.7%和27.3%)。施用拮抗菌后,植物根际病原菌的数量由每克土106ITS拷贝数降低到每克土104ITS拷贝数。单独施用拮抗菌T37,接种后第0天到第20天哈茨木霉的数量从的每克土107ITS拷贝数降低到每克土105ITS拷贝数；生物有机肥的使用维持了拮抗菌T37的数量在每克土107ITS拷贝数；生物有机肥处理N43总数和N43芽孢数量所占百分比分别为1.67×108CFU g-1土和51.9%,与单独施用拮抗菌N43的处理(2.20×107CFUg-1土和78.6%)相比有显著差异。拮抗菌N43和T37及其生物有机肥降低了土壤中有害真菌的种类,如立枯丝核菌Rhizoctonia solani、玉米大斑病菌Setosphaeria sp和油瓶霉Lecythophora;增加了土壤细菌的种类和数量。综上所述,拮抗菌和生物有机肥可以促进黄瓜生长、防控黄瓜土传立枯病、降低根际土壤病原菌数量和改善根际土壤微生物区系,其中生物有机肥的处理效果最好。
3.为了阐述哈茨木霉SQR-T37和短小芽孢杆菌SQR-N43对立枯丝核菌的拮抗机理,显微观察了两株拮抗菌对病原菌菌丝的作用,并检测了T37菌株产生的几丁质酶和p-1,3-葡聚糖酶活性,以及N43菌株分泌的拮抗物质。结果表明：T37菌丝缠绕并穿透病原菌菌丝,造成病原菌菌丝细胞质渗漏和细胞皱缩。新鲜的和烘干的病原菌细胞壁都可以诱导T37菌株几丁质酶(9.49和10.80nkat ml-1粗酶液)和p-1,3-葡聚糖酶的产生(37.57和43.18nkat ml-1粗酶液)。N43菌株在对数生长期产生对立枯丝核菌Q1具有抑制作用的拮抗物质,这种物质造成了病原菌菌丝的顶端膨大和细胞质的渗漏。当培养基中NaCl浓度为1%(w/v)时,拮抗物质的活性达到最大值。70%饱和度的硫酸铵可以完全沉淀发酵液中的拮抗物质。拮抗物质经蛋白酶K和胰蛋白酶处理后活性显著下降,与对照相比分别降低了79%和53%。这种物质分子量在500-1000Da之间,对高温敏感,对碱性条件具有耐受性。拮抗物质与N43细胞悬液对土传立枯病的防控效果无显著差异。综上所述,T37菌株对立枯丝核菌Q1的拮抗机理为重寄生作用；N43菌株产生对立枯丝核菌Q1具有拮抗作用的短肽类活性物质。
4.本章构建了绿色荧光蛋白(Green Fluorescent Protein, GFP)标记的短小芽孢杆菌SQR-N43菌株(GFP-N43),并研究了GFP-N43菌株和哈茨木霉SQR-T37在黄瓜根系的定殖能力。结果表明：构建的GFP-N43菌株能发出绿色荧光；生长到89代时,还有43.33%的菌株含有pHAPⅡ质粒；GFP-N43菌株对立枯丝核菌Q1的抑制效果较N43菌株相比无显著差异；在每克土107和108GFP-N43CFU处理中,GFP-N43在植物根尖和伸长区定殖并形成了稳健的生物膜,保护植物根系不受病原菌侵害；定量结果表明在每克土107和108GFP-N43CFU处理中,GFP-N43菌体浓度有所下降,接种后14天的菌体浓度分别是每克土4.6×106CFU和1.1×107CFU；0到14天,105和106浓度的处理中GFP-N43的数量呈上升趋势,第14天菌体浓度分别是第0天的3.55和2.09倍；T37菌丝可以缠绕黄瓜根系,在黄瓜根表形成保护层。
5.利用高效液相色谱鉴定了黄瓜根系分泌的有机酸,并检测了拮抗菌短小芽孢杆菌SQR-N43对有机酸的趋化作用、哈茨木霉SQR-T37对有机酸的利用和有机酸对T37孢子萌发和菌丝生长的影响。结果表明：黄瓜根系分泌物中有机酸主要有草酸、苹果酸、乙酸、柠檬酸、琥珀酸和延胡索酸。N43菌株对苹果酸和柠檬酸具有浓度依赖性的正向趋化作用,趋化系数分别为3.1和2.3。除琥珀酸外,另4种有机酸都对T37孢子萌发具有不同程度的促进作用；30μgm1-1的草酸具有最大促进作用,T37孢子萌发数较对照处理提高了39%。5种有机酸对T37菌丝生长都具有不同程度的促进作用；80μgm1-1的苹果酸具有最大促进作用,T37菌落直径是对照处理的1.1倍。在5种有机酸都存在的情况下,T37优先利用乙酸、草酸和苹果酸。
Cucumber (Cucumis sativus Linn) is one of the main greenhouse vegetables which widely distributes around the world. The soil-born disease of cucumber Rhizoctonia damping-off is mainly caused by Rhizoctonia solani, which occurs around the world with different degree and causes a lot of economic loss every year. The control of the disease is a world wide problem. Traditional chemical and farming control methods result in serious environmental, foot safety problems and time-and labor-consuming, thus can not be successfully applied in agricultural production. Biological control of soil-born disease is an efficient and environmentally friendly way and is receiving increased attention.
In the present study, we isolated and screened Trichoderma harzianum SQR-T37(T37) and Bacillus pumilus SQR-N43(N43) which showed highly antagonistic effect against Rhizoctonia solani, the pathogen of damping-off disease. The two antagonistic strains and their bio-organic fertilizers were used in pot experiments to assess their control efficiencies of cucumber damping-off disease. The populations of pathogen and antagonistic strains and microbial communities in the rhizosphere soils after applied with antagonistic strains and their bio-organic fertilizers were also determined. Besides, the abilities of colonization in cucumber rhizosphere and the mechanisms of antagonism against the pathogen of the antagonists were studied. The influences of plant root exudates on the antagonistic strains were also investigated.
The main results obtained were listed as follows:
1. In this study, Rhizoctonia solani Q1was verified as the pathogen of cucumber damping-off disease using Koch's postulates. More than400bacterial strains were isolated from rhizosphere soil of cucumber plants, and the dual culture method was used to screen bacterial antagonists against R. solani. Sixteen strains of bacterial antagonists were isolated and their inhabiting zone against R. solani ranged from0.8cm to1.9cm. Three strains (N33, N34and N43) with inhabiting zone more than1.6cm could produce volatile antibiotics and soluble antibiotics. Based on the results of morphologic characteristics, physiological and biochemical properties and phylogenetic analysis of16S rDNA, the three strains (Genbank accession number HM439651, GQ465936and GQ465935, respectively) were identified as Pseudomonas fluorescens, Bacillus pumilus and Bacillus pumilus, respectively. Antagonists of B. pumilus SQR-N43and Trichoderma harzianum SQR-T37were further chosen for following experiments.
2. Pot Experiments were performed to assess the promotional efficiency on the growth of cucumber and in vivo disease-control efficiency of the antagonistic microbes and bio-organic fertilizers (BIOs). Results indicated that compared with control, Bacillus pumilus SQR-N43and Trichoderma harzianum SQR-T37increased the dry weight of cucumber shoot by38.6%and55.9%, respectively. The antagonists N43and T37could control damping-off disease of cucumber; further more, the control efficiency of T37increased during the second growth cycle by25.0%as compared with the first growth cycle. Besides, the control efficiencies of BIO products (68.2%and81.8%, respectively) were higher than those applied with antagonist N43or T37solely (22.7%and27.3%, respectively). The population of R. solani in the rhizosphere soil of cucumber was decreased from106ITS copies g-1soil to104ITS copies g-1soil in the presence of the antagonists. Twenty days after incubation, the population of T. harzianum was107ITS copies g-1soil in the BIO treatment, which was much higher than that in the treatment only T37was applied (105ITS copies g-1soil). Significant differences in the number of CFUs and the percentage of spores of N43were recorded between the N treatment (2.20×107CFU g'1of soil and79%, respectively) and the BIO treatment (1.67×108CFU g-1of soil and52%, respectively). The antagonists N43and T37and BIOs decreased the number of harmful fungal species, such as Rhizoctonia solani, Setosphaeria sp. and Lecythophora, and increased the population of bacteria and the number of bacterial species. As a conclusion, the antagonistic microbes and BIOs could promote the growth of cucumber, prevent the damping-off disease of cucumber, decrease the population of the pathogen in the rhizosphere soil, and improve the rhizosphere microbial community. Among of them, the BIOs had the best efficiency.
3. For clarification of the mechanisms of the antagonism of Trichoderma harzianum SQR-T37and Bacillus pumilus SQR-N43against Rhizoctonia solani Q1, the effect of two antagonistic strains on the pathogen mycelium was microscopically observed and the activities of chitinase and β-1,3-glucanase produced by T37and production of antibiotic by N43were determined. Result showed that the mycelia of T37coiled themselves round and penetrated into the mycelia of pathogen, causing leakage of cytoplasm and crimple of pathogen cell walls. Both fresh and dried cell walls of the pathogen could induce the production of chitinase (9.49and10.80nkat ml-1crude enzyme, respectively) and β-1,3-glucanase (37.57and43.18nkat ml-1crude enzyme, respectively). N43produced antibiotic substances towards R. solani Ql at logarithmic growth phase. The antibiotics caused enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1mycelia. The activities of the antibiotics had the highest value when the concentration of NaCl in the medium was1%(w/v).70%saturation of ammonium sulfate made complete precipitation of the antibiotics in culture broth. When treated with protease K and trypsase, the activities of antibiotics were decreased by79%and53%, respectively, compared with control. The antibiotics were sensitive to high temperature and were alkaline stable. The molecular weights of the substances were between500-1000Da. The control efficiencies of the antibiotics and cell suspension of N43on damping-off disease had no significant difference. As a conclusion, the mechanism of antagonism of T37against R. solani Q1was mycoparasitism. N43produced oligopeptides which had inhibitory effect on R. solani Q1.
4. In this study, green fluorescent protein tagged Bacillus pumilus SQR-N43strain (GFP-N43) was constructed and abilities of both GFP-N43and Trichoderma harzianum SQR-T37to colonize roots and the rhizosphere soil of cucumber were determined. Results showed that constructed GFP-N43strain could emit green fluorescent. When grew up to89generations,43.3%of population of the strain still contained plasmid pHAPⅡ. The inhibitory effect of GFP-N43against Rhizoctonia solani Q1was identical with that of N43strain. In the treatments inoculated with107and108GFP-N43CFU g-1of soil, the presence of GFP-N43through colonization and formation of biofilm on tips and elongation zones of plant roots could ensure the protection of the hosts from the pathogen. Quantitative measurement showed that in the treatments of10and10GFP-N43CFU g-1soil, the numbers of GFP-N43strain decreased to4.6×106CFU g-1soil and1.1×107CFU g-1soil, respectively, at14days after inoculation (DAI). However, in the treatments inoculated with105and106GFP-N43CFU g-1soil, the populations of GFP-N43strain had upward trends in the period from0to14DAI, and GFP-N43strain at14DAI were3.55and2.09times higher than those at0DAI, respectively. The hyphae of T37strain coiled round cucumber roots and formed protection layer on cucumber root surfaces.
5. The organic acids in cucumber root exudates were identified by high performance liquid chromatography (HPLC). Chemotaxis of B. pumilus SQR-N43towards organic acids, utilization of organic acids by T. harzianum SQR-T37strain and the influences of organic acids on T37conidial germination and mycelial growth were also determined. Results indicated that oxalic acid, malic acid, acetic acid, citric acid and succinic acid were the mainly organic acids in cucumber root exudates. N43exhibited positive, concentration-dependent chemotactic behavior towards malic acid and citric acid with a relative chemotaxis index3.1and2.3, respectively. Except for succinic acid, the organic acids had different degrees of promotional effects on T37conidial germination. The largest number of germinated conidia which increased by39%, compared with control, was obtained with30μg ml-1oxalic acid. The five organic acids had various degree of promotional effect on T37mycelial growth. The largest diameter of T37colony, which was1.1times wider than that in control, was obtained with80μg ml-1malic acid. When supplied with the five organic acids, T37strain had a preference for acetic acid, oxalic acid and malic acid.
引文
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