东北地区保护地土壤拮抗放线菌的筛选

利用稀释分离法从东北蔬菜保护地37份土壤样品中共分离到各种放线菌菌株543株,采用活体对峙培养法共获得对植物病原菌具有拮抗作用的放线菌菌株19株,将其中抑菌圈直径超过15mm的菌株进行液体发酵试验,以叶霉病菌作为指示菌,采用杯碟法测定各菌株发酵液的抑菌活性,其中发酵液抑菌圈直径超过15mm的菌株有5株。对编号为MY02、MY04的菌株,进行了进一步的验证试验,测得其对叶霉病菌的抑菌圈直径分别为24 mm、23 mm。同时进行了这两个菌株的抑菌谱试验,结果表明无论其活体还是其发酵液对番茄叶霉病菌、番茄灰霉病菌等多种植物病原真菌均表现强烈的抑菌效果。菌株MY02除茄子黄萎病菌外,对试验的其他10种病原真菌抑制率均接近或超过90%。同时菌株MY02、MY04对植物病原细菌也具有很好的抑菌活性,试验结果表明,对番茄溃疡病菌、水稻白叶枯病菌、大白菜软腐病菌、果树根癌病菌、烟草野火病菌的抑菌圈直径分别可达20mm、16mm以上。     

番茄内生拮抗细菌的分离鉴定及培养条件研究

针对番茄生产上灰霉病和叶霉病两大瘸害,为寻找安全、高效无污染的生防菌株及其最佳培养条件,本试验采用组织分离法从健康的番茄植株中分离出642个内生细菌菌株,并采用平板对峙法筛选出对番茄灰霉病菌和叶霉病菌拮抗作用强且稳定的两个菌株Thyy1和Jcxy8。通过形态学观察及生理生化特征测定,初步鉴定Jcxy8属环状芽孢杆菌（Bacillus circulans）,菌株Thhy1属枯草芽孢杆菌（Bacillus subtilis）。内生拮抗细菌在以豆饼粉为原料的6号培养基中生长速度快,发酵滤液对两种病原菌的抑制作用强。培养基初始pH值、培养时间、温度、通气量等对菌株生长及其抗菌物质的分泌有明显影响。以豆饼粉培养基、初始pH6．7、培养时问48h、温度30qc、并尽量增大培养通气量为菌株的最佳培养条件。     

牛场沼液对几种蔬菜病原菌抑制作用的研究

实验室条件下,用不同贮存时间的牛场沼液原液和滤液对7种蔬菜病原菌（番茄叶霉病菌、番茄灰霉病菌、番茄早疫病菌、辣椒疫病病菌、辣椒绵腐病菌、黄瓜炭疽病菌、茄子灰霉病菌）的抑菌效果进行试验研究,为沼液应用于蔬菜病害农田防治的进一步研究提供基础数据。结果表明,新鲜沼液原液对番茄灰霉病菌、番茄早疫病菌、辣椒疫病病菌、辣椒绵腐病菌、黄瓜炭疽病菌和茄子灰霉病菌均有较强的抑制作用;随着贮存时间的增加,沼液原液对番茄灰霉病菌、番茄早疫病菌、黄瓜炭疽病菌和茄子灰霉病菌的抑菌率没有显著变化,对辣椒疫病病菌和辣椒绵腐病菌的抑菌率显著下降;新鲜沼液滤液对番茄灰霉病菌和茄子灰霉病菌有较好的抑制作用,但对其他5种病原菌的抑制效果不明显;随着贮存时间的增加,滤液对番茄灰霉病菌的抑菌率没有显著变化,对茄子灰霉病的抑制率表现出先显著下降而后升高的趋势。     

大豆灰霉病和黑斑病拮抗菌株的筛选和鉴定

生物防治病虫害是当前农业可持续发展的重要方向之一。大豆灰霉病(Botrytis cinerea)和黑斑病(Alternaria tenuissima)是大豆上的主要病害,为了控制灰霉病和黑斑病对大豆的危害,通过生物防治的方法,采用土壤稀释法从江西省东乡野生稻根际土壤中分离获得64株拮抗菌,经反复筛选及平板对峙法测定菌株A2、A6、A10、F7和G6对大豆灰霉病菌和黑斑病菌有稳定拮抗作用。通过菌株发酵滤液和化学农药抑菌效果比较,菌株发酵液的抑菌率远超化学农药,其中拮抗菌的发酵滤液对灰霉病最佳抑制率达91. 7%,对黑斑病最佳抑制率在60. 9%～87. 0%之间;而灰霉病特效素和苯醚甲环唑对灰霉病和黑斑病的最佳抑制率仅为68. 0%和47. 3%。根据菌株的生理生化特征及16S r DNA分子鉴定,5株菌分别为米修链霉菌(Streptomyces misionensis)、绛红产色小单孢菌(Micromonospora purpureochromogenes)、圈卷产色链霉菌(Streptomyces ansochromogenes)、链霉菌(Streptomyces sp.)和类芽孢杆菌(Panenibacillus sp.)。本研究为大豆灰霉病和黑斑病的生防菌开发和利用提供了一定的支撑。     

爵床提取物的抑菌杀虫活性研究

爵床(Justicia procumbens var.procumbens L.)是一种传统中药。为探明爵床提取物的抑菌杀虫活性,采用冷浸和超声波提取相结合的方法,分别获得了爵床甲醇、氯仿、丙酮、乙酸乙酯和正己烷等溶剂提取物;并应用抑菌圈法研究了5种提取物对柑橘炭疽病菌(Colletotrichum gloeosprioides)、芦笋茎枯病菌(Phomopsisasparagi)、稻瘟病菌(Pyricularia grisea)、棉花红腐病菌(Fusarium graminearum)和草莓灰霉病菌(Botrytiscinerea)等5种植物病原真菌的抑制效果,测定了甲醇提取物对柑橘炭疽病、芦笋茎枯病、草莓灰霉病和柑橘溃疡病(Xanthomonas campestris)等4种植物病菌及白蚊伊蚊(Aedes albopictus)、家蝇(Musca domestica)和菜青虫(Pieris rapae)等3种害虫的毒力。结果表明,不同极性溶剂提取物8 mg.mL 1对5种植物病原真菌均有一定的抑制作用,其中甲醇提取物对柑橘炭疽病菌、芦笋茎枯病菌、草莓灰霉病菌的抑制率均达50%以上。爵床甲醇提取物具有较强的抑菌杀虫活性,对柑橘炭疽病菌、芦笋茎枯病菌和草莓灰霉病菌菌丝生长的抑制中浓度(EC50)分别为5.94 mg.mL 1、4.61 mg.mL 1和5.27 mg.mL 1,EC90分别为63.69 mg.mL 1、58.01 mg.mL 1和54.57 mg.mL 1;0.25~1.00 mg.mL 1爵床甲醇提取物对柑橘溃疡病病菌显示强抑菌作用,0.125 mg.mL 1显示中度抑菌作用,最小抑制浓度(MIC)为0.062 5 mg.mL 1;爵床甲醇提取物对白蚊伊蚊、家蝇和菜青虫的致死中浓度(LC50)分别为0.195 8 mg.mL 1、0.351 4 mg.mL 1和0.287 7 mg.mL 1,LC95分别为0.988 4 mg.mL 1、3.053 2mg.mL 1和2.584 4 mg.mL 1。因此,爵床提取物作为生物农药,在农业生产中具有较好的应用前景。     

玉米内生芽胞杆菌的抗菌活性物质及其拮抗玉米大斑病菌机理的初步研究

为了研究生防菌株对玉米大斑病菌的抑菌作用,深化对生防菌抗菌机制的认识,本研究从玉米(Zea mays)植株体内分离拮抗玉米大斑病菌(Setosphaeria turcica)的内生细菌,对其抗菌物质及其抑菌机理进行初步研究。结果表明,所分离的内生菌株YY1经形态学观察、生理生化测定及16SrDNA序列分析,鉴定为枯草芽胞杆菌(Bacillus subtilis)。菌株YY1发酵液的硫酸铵沉淀物具有抑菌活性,且在硫酸铵50%饱和度时抑菌活性最强,说明YY1菌株产生的抗菌活性物质可能是蛋白类物质。该菌株及其蛋白粗提液均对禾谷镰刀菌(Fusarium graminearum)、苹果轮纹病菌(Botryosphaeria dothidea)、灰霉病菌(Botrytis cinerea)、玉米弯孢霉叶斑病菌(Curvularia lunata)等7种植物病原真菌有较强的拮抗作用。用蛋白粗提液处理菌丝、分生孢子、原生质体后经显微观察发现,大斑病菌的基内菌丝由丝状畸变为串珠状,当蛋白粗提液浓度为0.78μg/μL时,可完全抑制分生孢子萌发,并导致原生质体裂解。通过抑制孢子萌发过程中信号途径相关基因的半定量RT-PCR分析和玉米大斑病菌不同信号途径相关基因突变体的抑制率统计,初步判定该抑菌过程主要通过cAMP信号转导途径发挥作用。本研究为寻找玉米大斑病菌新的防治方法和途径提供基础资料。     

不同杀菌剂对番茄灰霉病的防治效果分析

近年来,在番茄的栽培过程中,番茄灰霉病一直是对番茄产量最有影响的病害之一。为了解决番茄灰霉病影响产量这一实际问题,本文就采用离体叶片法测定了2种新杀菌剂,42.8%氟吡菌酰胺·肟菌酯悬浮剂(SC)和22.5%啶氧菌酯SC对番茄灰霉病的作用,经田间试验结果表明,两种新药剂对番茄灰霉病的防治效果显著。     

一株百合内生细菌 Burkholderia sp. FJb-2的分离鉴定及其体外抑菌促生效应

从北京市房山区百合资源圃采集红芯百合(Fangio)植株样品,通过组织块分离法分离内生细菌;进一步以百合灰霉病菌—灰葡萄孢菌( Botrytis cinerea)ACCC36423、百合叶尖干枯病菌—葡萄座腔菌( Botryosphaeria dothidea)ACCC37263和百合枯萎病菌—尖孢镰刀菌( Fusarium oxysporum)F01139等几种主要百合病原真菌为指示菌,采用平板对峙法筛选具有拮抗活性的拮抗菌株;并对拮抗菌株进行产 1-氨基环丙烷 -1-羧酸(ACC)脱氨酶活性、产生长素(IAA)、溶磷、产铁载体等多种植物促生特性测定;最后通过形态观察及 16S rRNA基因序列分析,初步鉴定其种属。结果从百合茎部分离获得一株内生细菌菌株 FJb-2;该菌株对灰葡萄孢菌、葡萄座腔菌和尖孢镰刀菌均有较高拮抗活性,对病原真菌生长的抑制率范围为 60%～ 81%;该菌株产 ACC脱氨酶活性为     

低温寡照影响番茄幼苗根系有机酸代谢和养分吸收

低温寡照气象灾害严重制约设施番茄产量和品质,本研究拟从番茄根系有机酸代谢和养分吸收的角度,探究低温寡照影响番茄生长的潜在机制。通过人工控制试验,设置不同低温（最高温/最低温：12℃/2℃、14℃/4℃、16℃/6℃、18℃/8℃）和弱光（200、400μmol·m-2·s-1）的交互处理,研究低温寡照处理2、4、6、8、10d后苗期番茄根系活力、氮磷钾含量、植株干重以及根系分泌低分子量有机酸（LMWOAs）的动态变化。结果表明：低温寡照显著抑制番茄根系活力和氮、磷、钾吸收,抑制根、茎叶干重增加,温度越低抑制作用越强;最低温（12℃/2℃）和最弱光（200μmol·m-2·s-1）处理下,番茄根系活力仅为对照的7.70%～22.1%,根系氮、磷、钾的净吸收量分别为对照的3.75%～18.1%、1.28%～27.1%和19.1%～35.5%,根系、茎叶干重分别为对照的23.4%～55.9%和42.6%～66.5%。低温寡照胁迫下番茄根系分泌低分子量有机酸的总量显著降低,土壤pH值升高,其中草酸的分泌量最大,下降幅度最明显。表明低温寡照对番茄生长的抑制作用可能与根系活力下降,草酸分泌减少和养分吸收降低有关,推测在低温寡照胁迫初期施加适量草酸或氮、磷、钾复合肥料或许可提高番茄的抗灾能力。     

水稻白叶枯病菌蛋白质激发子HarpinXoo诱导植物的防卫反应

摘要：含质粒pHRF4的表达菌株BLHR4在培养基中经IPTG诱导，16 h Harpin表达接近最高量。用不同浓度的HarpinXoo喷雾处理烟草(Nicotiana tabacum L.)、油菜(Brassica campestris L.)和番茄(Lycopersicon esculentum Mill.)，对烟草花叶病毒(TMV)、油菜菌核病(Sclerotinia sclerotiorum (lib.) de Bary)和番茄灰霉病(Botrytis cinerea Pers.)均表现较好的诱导抗病效果。以浓度为100μg/mL的HarpinXoo处理烟草后，分别测定叶片中与防卫反应相关的一些生理指标的变化，结果表明，处理叶片中苯丙氨酸解氨酶（PAL）、过氧化物酶（PO）和多酚氧化酶（PPO）活性都有不同程度的增强。三种酶活性高峰分别在处理后24、12和3 h出现，分别比清水对照增加110.1%、211.2%和273.0%；同时，两个病程相关蛋白（pathogenesis-related protein，PR）基因PR-1b和PR-1a在HarpinXoo处理24 h后被诱导显著表达，36 h时达到最高表达水平。这表明HarpinXoo与HarpinEa一样能够诱导烟草不同抗病信号通路的启动，从而使植株获得抗病性。     

Antifungal activity and biotransformation of diisophorone by Botrytis cinerea

Diisophorone (1) was tested against two strains of the necrotrophic plant pathogen Botrytis cinerea.Fungal sensitivity varied according to the strain. B. cinera 2100 was more sensitive than B. cinereaUCA992: its mycelial growth was significantly inhibited at concentrations of 50 ppm and above. Although diisophorone (1) showed an effective control of B. cinerea, a detoxification mechanism was present. The detoxification of racemic diisophorone (1) by B. cinerea was investigated. Incubation with two strains of B. cinerea gave one and four biotransformation products (2-5), respectively. Their structures were established as the known 8beta-hydroxydiisophorone (2), 6alpha-hydroxydiisophorone (3), 6beta-hydroxydiisophorone (4) and 8beta,14beta-dihydroxydiisophorone (5) on the basis of their spectroscopic data, including two-dimensional NMR analysis [heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY)] and an X-ray crystallographic study.     

胨冻样芽孢杆菌(Bacillus mucilaginosus)对烟草灰霉病菌(Botrytis cinerea)的抑制作用

本文首次报道胨冻样芽孢杆菌XDB1和D4B1对烟草灰霉病菌(Botrytis cinerea)的抑制作用。实验表明,XDB1和D4B1的发酵原液和无细胞提取物具有相同的抑菌效果,而经121℃处理30分钟后,抑菌活性丧失。D481菌株的抑菌活性高于XDB1,钾长石作为基质时的活性高于土壤矿物。但是,XDB1和D481对立枯丝核病菌(R..solani)、西瓜尖孢镰刀菌(F.oxysporum)、腐霉(Pythium sp)、小麦全蚀病菌(G.graminis)、烟炭疽病菌(C.gloeosporides)、烟草赤星病菌(A.alternata)、烟草黑胫病菌(P.nicotianae、串珠镰刀病菌(F.moniliforme)和水稻稻瘟病菌(P.grisea)无抑制作用。     

水稻根际耐镉细菌碳源代谢功能分析

为探明前期分离得到的3株水稻根际耐镉细菌碳源代谢特性,在进行生理生化鉴定的基础上,采用BIOLOG ECO微平板法,分析了3株细菌(菌株A、菌株B和菌株C)的碳源代谢功能。生理生化试验结果表明,3株菌均属于革兰氏阴性细菌,且均具有极生鞭毛,菌株C的生理生化鉴定为铜绿假单胞菌,菌株A与菌株B为假单胞菌属的其他种,结果与之前的分子生物学鉴定结果一致。BIOLOG分析结果表明,3株菌株在培养72 h的AWCD(平均颜色变化率)均接近最大值,在培养24 h时的AWCD存在显著差异,这种差异主要体现在对氨基酸的利用上。在对提供的31种单一碳源利用上,3株菌对N-乙酰-D-葡萄糖胺、L-精氨酸、L-天冬酰胺、甲叉丁二酸、腐胺、4-羟基苯甲酸、丙酮酸甲酯、吐温-40和吐温-80的利用率均较高,且对L-精氨酸、甲叉丁二酸、丙酮酸甲酯的利用率存在显著差异。本研究结果对深入了解耐镉菌株增殖所需要的碳源以及将来优化耐镉细菌最适培养基提供了理论依据,也为构建相应的基因工程菌提供了微生物资源。     

The antifungal activity of widdrol and its biotransformation by Colletotrichum gloeosporioides (penz.) Penz. & Sacc. and Botrytis cinerea Pers.: Fr

Widdrol (1) was tested against the necrotrophic plant pathogens Botrytis cinerea and Colletotrichum gloeosporioides. While 1 was found to be inactive against C. gloeosporioides, it showed a selective and effective control of B. cinerea, significantly inhibiting the mycelial growth of the fungus at concentrations of 100 ppm and above. In addition, the biotransformation of 1 by both fungi was studied. Incubation with C. gloeosporioides and B. cinerea afforded four and one biotransformation products (2-6), respectively. Biotransformation with C. gloeosporioides was highly regioselective, yielding for the most part oxidation products at C-10: 10-oxowiddrol (2), 10beta-hydroxywiddrol (3), 10alpha-hydroxywiddrol (4), and 14alpha-hydroxywiddrol (5). The structures of all products were determined on the basis of their spectroscopic data, including coupling constants, two-dimensional NMR analysis (heteronuclear multiple quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser enhancement spectroscopy), and nuclear Overhauser effect. The biotransformation products were then tested against B. cinerea and found to be inactive. These results shed further light on the structural modifications, which may be necessary to develop selective fungal control agents against B. cinerea.     

基于生物化学和基因组学方法的Bacillus velezensis19573-3生防潜力评价

  【目的】  筛选具有防病促生功能的芽孢杆菌优良菌株,为植物病原菌生物防治提供生态绿色、环境友好的菌种资源。  【方法】  采用菌丝生长速率抑制法和离体叶片防效筛选拮抗多种病原菌的高效菌株,通过HPLC法检测抑菌活性物质,借助基础生物学及UPLC-MS法,测定其产生植物激素等代谢物能力水平,并初步解析其促生防病相关特性。通过Biolog系统鉴定、多序列系统进化和全基因组序列分析,明确菌株19573-3的分类地位,并对其可能产生的相关次级代谢产物基因簇进行注释分析。  【结果】  菌株19573-3对于灰霉菌、腐霉菌、疫霉菌、镰刀菌等多种病原真菌具有良好的拮抗能力,具有广谱抑菌活性,且该菌株对番茄灰霉病有显著的防效,经HPLC检测发现该菌株能够产生抑菌活性物质丰原素 (fengycin)。此外,该菌株兼具产生纤维素酶、蛋白酶和铁载体的能力,还可产生水杨酸、细胞分裂素和生长激素等与植物促生密切相关的物质。通过生理生化指标、16S rRNA与gyrA基因序列系统进化分析以及基于基因组水平的ANI分析鉴定,菌株19573-3属于贝莱斯芽孢杆菌 (Bacillus velezensis)。B. velezensis 19573-3基因组全长3990203 bp,G+C含量为46.56%,共编码基因4164个,编码区总长度占全基因组的比例90.03%,染色体上存在86个tRNA,27个rRNA和8个sRNA。利用antiSMASH预测菌株B. velezensis 19573-3基因组中可能存在合成丰原素 (fengycin)、表面活性素 (surfactin)、铁载体 (bacillibactin) 等与拮抗和促生相关的13个次级代谢产物合成基因簇。  【结论】  细菌19573-3属于Bacillus velezensis,对引起植物病害的灰霉菌、腐霉菌和疫霉菌等多种病原真菌均具有良好的拮抗能力,菌株代谢物显示了根际促生和生物防治功能。19573-3菌株由一个环状染色体构成,不含质粒,基因组全长3990203 bp,G+C含量为46.56%。在B. velezensis 19573-3基因组序列之中,注释到了一个长度为37669 bp的基因簇,包含fenA、fenB、fenC、fenD、fenE、fenF等基因,负责产生丰原素 (Fengycin),注释到了合成铁载体 (bacillibactin) 的基因簇以及与生长激素合成相关的基因ysnS和yhcX,从基因角度证实了B. velezensis 19573-3具备产生激素的能力。     

Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3beta-hydroxy-kaurenoic acid

The antifungal activity on Botrytis cinerea of the diterpenoids 3beta-hydroxy-kaurenoic acid and kaurenoic acid, obtained from the resinous exudates of Pseudognaphalium vira vira, was determined. 3beta-Hydroxy-kaurenoic acid reduced the mycelial growth of B. cinerea in solid and liquid media. Additionally, the damage produced by the fungus on the surface of tomato leaves in the presence of the diterpenoids was evaluated. A higher protective effect was observed in the presence of the hydroxylated diterpene. On the other hand, the effect of the diterpenoids on the production of enzymes that participate in the plant infection by B. cinerea was analyzed. p-Nitrophenylbutyrate esterase production was induced by both diterpenoids, whereas laccase production was only induced by the hydroxylated diterpene. In the study of the mechanism of action of these compounds, it was determined that 3beta-hydroxy-kaurenoic acid would produce permeabilization of the cell membrane of B. cinerea.     

Fungal biotransformation of (+/-)-linalool

The biotransformation of (+/-)-linalool was investigated by screening 19 fungi. Product accumulation was enhanced by substrate feeding and, for the first time, lilac aldehydes and lilac alcohols were identified as fungal biotransformation byproduct using SPME-GC-MS headspace analysis. Aspergillus niger DSM 821, Botrytis cinerea 5901/02, and B. cinerea 02/FBII/2.1 produced different isomers of lilac aldehyde and lilac alcohol from linalool via 8-hydroxylinalool as postulated intermediate. Linalool oxides and 8-hydroxylinalool were the major products of fungal (+/-)-linalool biotransformations. Furanoid trans-(2 R,5 R)- and cis-(2 S,5 R)-linalool oxide as well as pyranoid trans-(2 R,5 S)- and cis-(2 S, 5 S)-linalool oxide were identified as the main stereoisomers with (3 S,6 S)-6,7-epoxylinalool and (3 R,6 S)-6,7-epoxylinalool as postulated key intermediates of fungal (+/-)-linalool oxyfunctionalization, respectively. With a conversion yield close to 100% and a productivity of 120 mg/L.day linalool oxides, Corynespora cassiicola DSM 62485 was identified as a novel highly stereoselective linalool transforming biocatalyst showing the highest productivity reported so far.     

Evidence for protein degradation by Botrytis cinerea and relationships with alteration of synthetic wine foaming properties

Botrytis cinerea is an important fungal pathogen particularly dreaded in the cool climate vineyard. It is responsible for important damage, especially the decrease in foamability of sparkling wines, such as Champagne. Different studies have shown that proteins are largely involved in the stabilization of Champagne foam despite their low concentration. Other works demonstrated changes in the electrophoretic characteristics of must proteins originating from botrytized grapes, although the cause of such alterations was never explained. In the first part of this study, results showed the release by B. cinerea of 3.5 mg/L total proteins in a synthetic liquid medium. Among these proteins, the presence of a protease activity on bovine serum albumin (BSA) and must proteins was demonstrated by using a colorimetric method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the model wine, the Bradford method showed a BSA loss of 66% after 24 h and a loss of 96% after 120 h. In the same model wine, the soluble must protein concentration decreased by 35% after 1 week and by 53% after 2 weeks while the control showed no protein loss. B. cinerea proteases were then able to degrade BSA and must proteins and were above all active at must and wine pH and in the presence of ethanol and SO(2). The second part of this work was dedicated to the relationship between the presence of B. cinerea proteases and its effects on the synthetic wine foaming properties. The addition of a B. cinerea culture medium (1/33 v/v) to the synthetic wine containing 21 mg/L soluble grape proteins induced a decrease in foamability by 60% after 1 week. For BSA in the model wine, the foamability decreased by 32% after 24 h and by 95% after 120 h, as shown by the colorimetric method. These experiments demonstrate for the first time the relationship between B. cinerea protease activity and the decrease in wine foaming properties.