水稻恶苗病拮抗菌的筛选、鉴定及其抑菌活性

从水稻根际土壤中筛选出拮抗水稻恶苗病的菌株,初步研究其抑菌作用及生防效果。采用平板稀释法从水稻根际土壤中分离获得菌株,以水稻恶苗病菌为靶标菌采用平板对峙法筛选出拮抗菌;通过形态学特征、生理生化特征及16S r DNA序列分析对筛选出的拮抗菌进行鉴定;检测拮抗菌无菌发酵液对水稻恶苗病菌菌丝生长的影响,同时测定拮抗菌的抑菌谱及进行盆栽实验。分离得到6株拮抗菌,其中有一株对水稻恶苗病菌拮抗作用较强的菌株SH15,经鉴定菌株SH15为多粘类芽孢杆菌。菌株无菌发酵液对水稻恶苗病菌菌丝生长有显著抑制作用;菌株SH15抑菌谱广,对水稻恶苗病菌、层出镰孢菌、棉花枯萎病菌、辣椒疫病菌、棉花黄萎病、黄瓜黑斑病菌均有一定的抑菌活性。水稻盆栽实验表明,接种多粘类芽孢杆菌SH15可显著降水稻恶苗病的发病指数,平均防效高达65.68%。因此,多粘类芽孢杆菌SH15在水稻恶苗病的生物防治方面具有一定的应用价值。     

土壤放线菌P3-2的分类鉴定及抗菌活性研究

对从贵州土壤微生物中筛选到的放线菌菌株P3-2进行了分类学和抗菌活性的研究。采用多相分类法,对该菌株的形态特征、培养特征、生理生化特性以及16S rDNA基因序列进行了研究。结果表明,放线菌P3-2菌株属于链霉菌属;16SrDNA序列长度为1 456 bp,序列分析和系统进化树分析表明其序列与Streptomyces recifenis ST100的同源性最高,为99.4%。但与S.recifenis ST100相比较,P3-2菌株的培养特征和生理生化特性中多项指标都存在着不同,初步确定菌株P3-2为链霉菌属中S.recifenis ST100的一个亚种,暂定名为Streptomyces sp.P3-2。P3-2菌株的10倍稀释发酵液对油菜菌核病菌、黄瓜灰霉病菌、小麦赤霉病菌、水稻纹枯病菌及半夏立枯病菌的抑制率高达99%,对烟灰霉病菌、玉米小斑病菌等9种病原真菌均有不同程度的抑制作用,对金黄色葡萄球菌、蜡状芽孢杆菌和枯草芽孢杆菌有一定的抑制作用。     

多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究

对一株已经商业化的可防治植物枯萎病的生防菌-多粘类芽孢杆菌HY96-2发酵液中抗真菌活性物质进行了分离纯化,采用酸化、正丁醇萃取、乙酸乙酯沉淀、硅胶柱层析、Sephadex LH-20柱层析、高效液相色谱(HPLC)等方法分离得到了一个抗真菌活性化合物6B,经NMR、MS、MS/MS等光谱学方法鉴定其为Fusaricidin A。琼脂扩散法抑菌试验结果表明,6B对西瓜枯萎病菌、水稻纹枯病菌、灰霉病菌等15种植物病原真菌的最小抑菌浓度为12.5～50μg/mL。盆栽实验结果表明,6B对黄瓜灰霉病具有明显的防治效果,当6B的浓度达到250μg/mL时,防治效果高达95%。     

一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定

【目的】筛选对水稻纹枯病菌(Rhizoctonia solani)具有强拮抗作用的细菌菌株。【方法】用指示菌法筛选拮抗菌株;通过形态观察、生理生化实验、Biolog及16S rDNA序列分析鉴定目标菌株;利用平板双向培养法和滤纸片扩散法测定抑菌谱及拮抗性质。【结果】分离到一株高活力的水稻纹枯病菌拮抗菌株YB-3,该菌株属于解淀粉芽胞杆菌(Bacillus amyloliquefaciens);菌株YB-3对常见的14株病原真菌和7株细菌具有较强的拮抗作用,并发现其对亲缘关系较近的芽孢菌属有较强的拮抗作用;该菌株的抑制活性具有温度稳定、耐酸、但对蛋白酶敏感的特点。【结论】通过指示菌法筛选到一株对水稻纹枯病菌有强拮抗作用的解淀粉芽胞杆菌(B.amyloliquefaciens)YB-3,它具有广谱、高效的植物病原菌拮抗活性。     

拮抗菌株Kc-t99的鉴定及其抑菌活性研究

通过传统分类与分子生物学技术相结合的方法对从京郊菜园土壤中分离筛选的拮抗菌株Kc-t99进行鉴定,并采用生物测定的方法评价其抑菌活性.结果表明,菌株Kc-t99的形态学特征和生理生化特性与枯草芽孢杆菌基本一致,其16S rDNA序列与GenBank中已鉴定的枯草芽孢杆菌的16S rDNA序列同源性高达98.06%,据此初步确定其为枯草芽孢杆菌.抑菌试验表明该菌株对供试的5种病原真菌和4种细菌均具抑菌活性,其中对甘蓝枯萎病菌、黄瓜角斑病菌和桃褐腐病菌抑制作用显著.     

羊耳菊抗菌物质及其抑菌特性分析

通过硅胶柱层析法,结合活性追踪,从羊耳菊中分离到1种对水稻纹枯病菌具有较强抑菌活性的化合物,经菌丝生长速率法测定,其抑菌中浓度为47 mg/L。通过分析氢谱和碳谱数据,该化合物被鉴定为百里香酚,具有耐热、耐酸碱、抗紫外光和荧光的特性。百里香酚对15种植物病原真菌和4种植物病原细菌具有抑制作用,其中对水稻纹枯病菌、茉莉枝枯病菌、烟草黑胫病菌、香蕉弯孢霉叶斑病菌、白菜软腐病菌和木薯细菌性枯萎病菌的抑菌活性较强。     

饲用微生物的分离鉴定及其对杏鲍菇菌糠发酵的效果

【目的】菌糠的营养素含量齐全,但纤维素含量过高是阻碍其饲料化利用的主要因素。故本研究筛选适合于发酵杏鲍菇菌糠的微生物菌株,以改善其饲用品质。【方法】首先,本研究采用纤维素-刚果红、苯胺蓝和MRS-Ca (De Man, Rogosa, Sharpe-Ca)筛选培养基,结合纤维素、木质素酶活力及抑菌活性的测定,从EM (effective microorganisms)原液发酵的杏鲍菇菌糠中分离筛选具有较强纤维素、木质素降解能力及抑菌能力的细菌/真菌。通过细菌16S rRNA和真菌18S rDNA基因序列分析确定菌株所属种属。其次,将筛选出的菌株菌液等体积混合制成复合菌剂用于固态发酵杏鲍菇菌糠。测定不同发酵时长菌糠营养成分含量以确定最佳发酵时间,并与相同工艺条件下EM原液发酵的杏鲍菇菌糠进行饲用品质比较。【结果】筛选并鉴定得到纤维素酶活性较高的特基拉芽孢杆菌(Bacillus tequilensis)菌株P11、发酵毕赤酵母(Pichia fermentans)菌株R8和马克斯克鲁维应变酵母(Kluyveromyces marxianus)菌株MU5;木质素酶活性较高的解淀粉芽孢杆菌(Bacillus amyloliquefaciens subsp.plantarum)菌株MU7;抑菌活性较高的类肠膜魏斯氏菌(Weissella paramesenteroides)菌株R4和乳酸片球菌(Pediococcus acidilactici)菌株R9。使用以上菌株复合发酵杏鲍菇菌糠7 d后,各项指标达到稳定。与EM原液发酵的杏鲍菇菌糠相比,复合菌剂发酵杏鲍菇菌糠的NDF和ADF分别显著降低了19.6%和21.44%(P0.05);CP (crude protein)、CA (crude ash)和EE (ether extract)含量分别显著提高了10.44%、5.26%和123.53%(P0.05)。【结论】本研究筛选得到的芽孢杆菌、酵母菌和乳酸菌优势菌株复合后用于发酵杏鲍菇菌糠可以很好地改善其饲用品质,效果优于生产中常用市售EM原液。     

高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力

【目的】从水稻根际筛选能高效抑制水稻病原菌的细菌,分析和鉴定其形态和生化特征,为开发新型绿色农药奠定基础。【方法】从水稻根际分离能以1-氨基环丙烷-1-羧酸(ACC)为唯一碳源的菌株,根据菌株形态和生化特性、16S r DNA序列比对和磷脂脂肪酸图谱,对该菌株进行鉴定。通过氯仿萃取抽提、高效液相色谱分析,确定菌株PA1201在PPM培养基和黄豆粉培养基中申嗪霉素和吩嗪-1-酰胺的产量。【结果】菌株PA1201能有效抑制水稻纹枯病菌和水稻白叶枯病菌的生长,属于铜绿假单胞菌(Pseudomonas aeruginosa sp.PA1201);PA1201产生两种抑菌代谢产物申嗪霉素和吩嗪-1-酰胺,在PPM和黄豆粉培养基中申嗪霉素的产量可达81.7 mg/L和926.9 mg/L,吩嗪-1-酰胺的产量亦可达18.1 mg/L和489.5 mg/L;PA1201产生大量胞外蛋白酶,对人肺腺癌细胞系A549和黑腹果蝇具有一定毒性。【结论】PA1201的申嗪霉素产量比现有生产菌株的出发菌株M18高3-4倍,还能产生另一种抑菌活性更高的衍生物吩嗪-1-酰胺,具有进一步开发的潜力。     

α-淀粉酶高产菌株选育方法的研究

在含有氨苄青霉素1—10μg/ml的LB液体培养基中,接入枯芽孢杆菌BF7658菌悬液,在37℃下振荡培养3—5天,经平板分离单菌落以及摇瓶发酵试验,初筛获得2213、2104和2120等α-淀粉酶高产菌株。将2213菌株的菌悬液涂布于含有4-6μg/ml氨苄青霉素的2%淀粉培养基上,复筛得到4213、4218、4237等α-淀粉酶高产菌株。4213菌株再经4次亚硝基胍诱变,未能得到蛋白酶活性低、α-淀粉酶活性高的突变株。     

α-淀粉酶高产菌株选育方法的研究

在含有氨苄青霉素1—10μg/ml的LB液体培养基中,接入枯芽孢杆菌BF7658菌悬液,在37℃下振荡培养3—5天,经平板分离单菌落以及摇瓶发酵试验,初筛获得2213、2104和2120等α-淀粉酶高产菌株。将2213菌株的菌悬液涂布于含有4-6μg/ml氨苄青霉素的2%淀粉培养基上,复筛得到4213、4218、4237等α-淀粉酶高产菌株。4213菌株再经4次亚硝基胍诱变,未能得到蛋白酶活性低、α-淀粉酶活性高的突变株。     

腐烂病菌侵染对苹果愈伤组织防御酶活性及丙二醛含量的影响

以苹果树腐烂病菌LXS080601、感病苹果品种‘富士’和抗病砧木‘平邑甜茶’愈伤组织为材料,测定腐烂病菌侵染后,愈伤组织内过氧化物酶（POD）、多酚氧化酶（PPO）、超氧化物歧化酶（SOD）和苯丙氨酸解氨酶（PAL）活性及丙二醛（MDA）含量的动态变化。结果显示,接种LXS080601后,‘富士’愈伤组织的发病严重度和病情指数均明显大于‘平邑甜茶’;感病品种MDA含量上升速度快,于接种后3 d增幅为28.02%,且变幅较大,为–0.32%~36.39%,而抗病砧木MDA含量变化较小,仅为–2.17%~7.46%。同时,腐烂病菌侵染提高了愈伤组织内4种防御酶活性,接种后1~2 d,PPO和POD酶活性达到高峰,接种后3~4 d,PAL和SOD酶到达活性高峰;除PPO外,‘平邑甜茶’PAL、SOD和POD酶活性变化均明显高于‘富士’,且整个侵染过程酶活性维持在较高水平,而‘富士’体内3种酶活性快速下降至对照水平,表明‘平邑甜茶’通过提高抗氧化酶活性减少体内活性氧的积累,降低膜脂过氧化产物MDA的形成,增强了对腐烂病菌侵染的抗性。     

水杨酸对香荚兰抗逆相关酶的活性和丙二醛含量的影响

对大田栽培的香荚兰植株进行不同浓度的水杨酸(SA)处理,结果表明：较低浓度(25～100mg/L)的SA,使其叶片的SOD、POD、PAL和PPO活性增加,MDA含量降低,抗逆性增强;而较高浓度的SA(150～200mg/L)则抑制了这些酶的活性、使MDA含量升高,抗逆性减弱。     

施硅增强水稻对纹枯病的抗性

采用水培的方法,从细胞学和生理生化方面研究了硅增强水稻对纹枯病的抗性作用。结果表明：加硅处理的水稻叶片硅化细胞和叶片表面的硅元素含量均显著高于缺硅处理（对照）：接种纹枯病菌后,加硅处理的MDA含量总体上低于缺硅处理,峰值尤为显著;加硅处理的SOD活性始终高于缺硅处理,接种后第4天加硅处理SOD活性较低时,其POD活性较高,而缺硅处理的POD活性较低,表明硅增强了SOD和POD之间的协调性;接种后硅对CAT和PAL活性没有产生明显影响,但降低了PP0活性;加硅能显著降低水稻植株的纹枯病病情指数。     

苗龄、光照强度和施氮量对抗褐飞虱水稻品种主要防御酶活性的影响

为探明苗龄、光照强度和施氮量对水稻主要防御酶活性的影响及其与抗褐飞虱的关系,分别就苗龄、光照强度和施氮量等因子对不同抗褐飞虱水稻品种苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)和过氧化物酶(POD)活性的影响进行研究。结果表明:IR56、Ptb33和570011对褐飞虱生物型II的抗性与PAL和PPO活性密切相关,RHT对II型的抗性与PAL、PPO和POD活性密切相关,RP1976-18-6-4-2对II型的抗性与PAL活性密切相关,570011对II型的抗性与PAL和PPO活性密切相关;IR56对褐飞虱孟加拉型的抗性与PAL、PPO和POD活性密切相关,Rathu Heenati(RHT)和RP1976-18-6-4-2对孟加拉型的抗性与与PPO密切相关,570011对孟加拉型的抗性与POD活性密切相关。苗龄、光照强度和施氮量的单独作用或交互作用对不同水稻中防御酶活性有明显影响,但品种不同,其影响程度也不同。苗龄、光照强度和施氮量单独或是交互作用显著影响IR56中PAL和PPO、RHT和RP1976-18-6-4-2中PPO、Ptb33中POD及570011中PAL和POD的活性,苗龄显著影响IR56和RP1976-18-6-4-2中POD的活性,光照强度和施氮量单独或交互作用显著影响RHT和RP1976-18-6-4-2中PAL的活性,施氮量显著影响RHT中POD的活性,光照强度显著影响Ptb33中PAL的活性;苗龄和光照强度单独或交互作用显著影响570011中PPO的活性。     

苹果树腐烂病菌不同致病力菌株对苹果的诱导效应

以‘富士’苹果叶片为材料, 采用刺伤接种法, 比较了苹果树腐烂病菌强致病菌LXS080601和弱致病菌LXS081501侵染对寄主体内丙二醛（MDA）含量、渗透调节物质及防御酶活性的影响。结果表明, 两菌株侵染后, 叶片MDA、蛋白质和可溶性糖含量均增加, 接种LXS080601叶片MDA含量快速上升, 最大增幅为141.15%, 而接种LXS081501叶片的MDA含量变化较小, 增幅仅为1.16%-16.24%, 但后者可溶性糖和蛋白质含量增幅分别高达158.12%和113.57%, 显著高于接种LXS080601的处理。同时, 两菌株均能诱导叶片内4种防御酶活性的升高, 但LXS081501诱导的苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）、过氧化氢酶（CAT）和多酚氧化酶（PPO）活性均显著高于LXS080601, 说明不同致病力腐烂病菌对寄主防御酶活性的影响存在显著差异。     

Changes in defence-related enzymes in rice responding to challenges by Rhizoctonia solani

Sheath blight disease caused by Rhizoctonia solani Kuhn is becoming a major constraint to rice production, especially in the intensified cultivation system. To know the in rice, it is important to get the knowledge of the activity of defence-related enzymes due to the fungal infection. The pathogen induced superoxide dismutase (SOD) and chitinase activities in rice plants, while suppressing peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activities at 36 and 24 h after inoculation, respectively. Induction of two POD isozymes, POD-3 and -4, up to 48 h after inoculation and disappearance of the said isomers at 72 h onwards in rice–Rhizoctonia interaction implicated the role of these isomers in susceptible host–pathogen interaction. Apart from POD and SOD, the activities of other stress-related enzymes, viz. PAL, polyphenol oxidase (PPO) and β-1,3-glucanase were also studied. From this study, it was found that these defence-related enzymes are most significantly related to host–pathogenic interaction.     

壳寡糖诱导黄瓜抗黄瓜黑星病的初步研究

本文研究了壳寡糖诱导黄瓜对黑星病的抗性作用。利用6 mg/mL壳寡糖溶液对苗期黄瓜诱导,进行病情调查统计及测定处理前后黄瓜叶片的主要防御酶系———苯丙氨酸解氨酶,过氧化物酶,多酚氧化酶,超氧化物歧化酶,过氧化氢酶的活性变化。结果显示,壳寡糖对黄瓜黑星病在10 d和17 d的诱抗效果分别为60.25%和47.59%,且作为诱导因子可显著提高黄瓜叶片内苯丙氨酸解氨酶(PAL)活性,过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)活性也有所提高,但叶片内过氧化氢酶(CAT)活性无较明显变化。研究结果表明壳寡糖对黄瓜抗黑星病产生诱导作用,为研究壳寡糖作为新型生物农药提供了依据。     

Effects of herbivore stress by Aphis medicaginis Koch on the Malondialdehyde contents and the activities of protective enzymes in different alfalfa varieties

The study focused on the dynamics of Malondialdehyde (MDA) contents and the activities of protective enzymes in the leaves of alfalfa varieties with various resistances to Aphis medicaginis Koch. The results showed that susceptible varieties always had higher MDA contents than resistant varieties, and the MDA contents tended to rise in both susceptible and resistant varieties in period of the varieties were pierced and sucked by aphids. Superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activities in susceptible varieties were lower than those in resistant varieties, and in both susceptible and resistant varieties the SOD and POD activities tended to rise at first and then decline, and the PAL activities rose to their peaks and then tended to remain stable. In the susceptible and resistant varieties the catalase (CAT) activities appeared to rise and decline alternatively; the PPO activities in resistant varieties were lower than those in susceptible varieties in early growth, but higher than those in susceptible varieties in later growth. It follows that infested by aphids, susceptible and resistant varieties had the MDA contents, variations of SOD, POD, PAL and PPO activities were closely correlated with their aphid resistances, hence these indexes could be used as physiological indexes for testing aphid resistance of alfalfa, whereas the relations of their CAT activities to their resistances needed to be further studied.     

Effects of herbivore stress by Aphis medicaginis Koch on the Malondialdehyde contents and the activities of protective enzymes in different alfalfa varieties

The study focused on the dynamics of Malondialdehyde (MDA) contents and the activities of protective enzymes in the leaves of alfalfa varieties with various resistances to Aphis medicaginis Koch. The results showed that susceptible varieties always had higher MDA contents than resistant varieties, and the MDA contents tended to rise in both susceptible and resistant varieties in period of the varieties were pierced and sucked by aphids. Superoxide dismutase (SOD), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) activities in susceptible varieties were lower than those in resistant varieties, and in both susceptible and resistant varieties the SOD and POD activities tended to rise at first and then decline, and the PAL activities rose to their peaks and then tended to remain stable. In the susceptible and resistant varieties the catalase (CAT) activities appeared to rise and decline alternatively; the PPO activities in resistant varieties were lower than those in susceptible varieties in early growth, but higher than those in susceptible varieties in later growth. It follows that infested by aphids, susceptible and resistant varieties had the MDA contents, variations of SOD, POD, PAL and PPO activities were closely correlated with their aphid resistances, hence these indexes could be used as physiological indexes for testing aphid resistance of alfalfa, whereas the relations of their CAT activities to their resistances needed to be further studied.     

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.