解淀粉芽孢杆菌HMB33604的抑菌物质及对马铃薯黑痣病的防治效果

【目的】由立枯丝核菌（Rhizoctonia solani AG-3）引起的马铃薯黑痣病是马铃薯生产中重要的土传病害,严重影响马铃薯的产量和质量。利用微生物杀菌剂防治马铃薯黑痣病是环境友好、切实可行的措施之一。本研究旨在获得有效防治马铃薯黑痣病的生防细菌并明确其防病作用方式,为生防细菌发酵工艺的优化、微生物杀菌剂的合理施用提供科学依据。【方法】通过对峙培养法和温室盆栽试验筛选有效防治马铃薯黑痣病的生防细菌,通过Biolog微生物鉴定系统、16S rDNA序列以及gyrA、gyrB、rpoB和rpoC多基因序列分析对生防细菌进行分类鉴定。通过盆栽试验比较生防细菌菌株发酵液、无菌体上清液和菌体悬浮液对马铃薯黑痣病的防治效果,利用高效液相色谱（HPLC）对脂肽提取物进行分离,通过抑菌活性测定结合质谱（UPLC-Triple TOF-MS/MS）技术鉴定生防细菌产生的抑菌活性物质。采用实时荧光定量PCR（real-time PCR）技术测定马铃薯根际黑痣病菌的DNA拷贝数。【结果】通过对2 106株细菌进行筛选,获得3株有效防治马铃薯黑痣病的生防细菌,其中HMB33604菌株对马铃薯黑痣病的防治效果较高,达到52.9%。通过生理生化及多基因序列比对,将HMB33604菌株鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。该菌株发酵液和上清液对马铃薯黑痣病具有显著的防治效果,分别为52.2%和66.4%,而菌体悬浮液对马铃薯黑痣病的防治效果仅为16.9%,证明该菌株主要通过产生抑菌活性物质而起到防治马铃薯黑痣病的作用。HMB33604菌株可以产生泛革素（fengycin）、伊枯草菌素A（iturin A）和表面活性素（surfactin）,抑菌试验证明泛革素和伊枯草菌素是HMB33604菌株产生的主要抑菌活性物质,这两种脂肽类抗生素能够显著抑制黑痣病菌的生长,并且造成菌丝畸形。菌株发酵液和无菌体上清液均显著降低马铃薯根际黑痣病菌的DNA拷贝数,与对照相比分别降低了60.3%和64.0%;而菌体悬浮液处理后根际黑痣病菌的DNA拷贝数仅降低10.3%。【结论】获得一株有效防治马铃薯黑痣病的生防细菌——解淀粉芽孢杆菌HMB33604,该菌株主要通过产生抑菌活性物质而发挥生防作用。泛革素和伊枯草菌素是HMB33604菌株产生的主要抑菌活性物质。泛革素和伊枯草菌素不仅造成黑痣病菌菌丝畸形,还可显著降低马铃薯根际黑痣病菌的数量,从而有效防治马铃薯黑痣病。     

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.     

捺细菌性根癌病菌生物型鉴定及生物防治

从福建的古田、沙县和长汀等地采集到的捺细菌性根癌病标样上分离到１２株能致癌的菌株，经细菌学鉴定后认为属根癌土壤杆菌生物型Ⅱ，其中有１０株菌株对土壤杆菌素８４（ａｇｒｏｃｉｎ８４）敏感．用Ｋ８４菌液浸捺砧木的根部３０ｍｉｎ后种植在用分离到的致癌病菌接种过的土壤中，株发病率为２．９６％；用清水处理的砧木种植在用致癌病菌接种过的土壤中和不接种的土壤中，株发病率分别为１９．８９％和３．８２％，表明Ｋ８４菌对捺细菌性根癌病有一定的防治效果     

大樱桃根癌病原细菌分类地位鉴定

从烟台地区采集13份大樱桃根癌病样本,分离纯化出26个菌株,致病性测定表明23个菌株在指示寄主番茄上具有致病性;进一步经生理生化反应、碳源利用、选择性培养基等试验鉴定出16个根癌病菌菌株为Agrobacterium tumefaciens生物型2,占69.57%,为优势种群,其余7个菌株为生物型1.对大樱桃根癌病菌分类地位的确定为今后病原菌的系统研究和病害防治提供了理论基础.     

Identification of novel Trichoderma hamatum genes expressed during mycoparasitism

Trichoderma species are currently used as biocontrol agents for crop diseases caused by a number of fungal plant pathogens. However, their biocontrol performance in the field can be unreliable and it is likely that more consistent performance could be achieved through knowledge and manipulation of the genes involved. For example, induction of the genes could be optimised for variable environmental and physiological conditions, superior strains could be selected more effectively and novel st…     

小麦基因型对根癌农杆菌菌株敏感性研究

以栽培冬小麦运丰优、临优145、中优9507的成熟胚愈伤组织为外植体,用C58c1、EHA105和LBA4404 3个根癌农杆菌菌株(都含有pCAMBI1301质粒)研究小麦基因型对根癌农杆菌的敏感性,以及根癌农杆菌对小麦的侵染能力。结果表明,小麦基因型对根癌农杆菌的敏感性存在显著差异,以临优145最敏感。根癌农杆菌菌株对小麦成熟胚侵染能力不同,含有pCAMBI1301(p1301)质粒的C58c1侵染能力较强,但差异未达极显著水平。     

汉麻内生菌的分离及抑菌活性研究

本研究以汉麻为植物材料,对其内生菌进行较为系统的分离,并进行抑菌活性的筛选,以期得到能够产生较高杀菌活性物质的菌株。为筛选具有生防作用的汉麻内生菌提供理论依据和技术支持。筛选出的汉麻新菌种可以用作生物农药,与多种生物肥料复配,生产出新型复合微生物菌剂。     

三种生防细菌对新疆灰霉病菌(Botrytis cinerea)的拮抗作用

[目的]明确生防芽孢杆菌A57、A178、N1729在植物病害生物防治中的实用价值,并为后续的生防制剂的研发提供科学依据,对其在新疆各地蔬菜灰霉病菌的抑菌活性进行了研究.[方法]采用平板对峙法检测三种生防细菌抑菌活性,并检测其无细胞滤液的抑菌效果,无细胞滤液利用摇瓶发酵后经微孔滤膜过滤制备,用滤纸片法测定抑菌圈,确定抑菌效果.[结果]A178的活菌体拮抗效果最强,平均抑菌率达45.67;, N1729菌株分泌物的拮抗效果最强,平均抑菌率为44.61;,可作为灰霉病的有效生防菌;而A57的抑菌效果最差,不能作为后灰霉病菌有效的生防菌使用.[结论]A178、N1729这两个菌株具有很好的开发应用前景.     

农用植物酵素液中梨树腐烂病菌拮抗细菌的筛选和防病效果

从农用植物酵素液中筛选对梨黑腐皮壳菌（Valsa pyri）具有抑菌活性的细菌菌株,明确其对梨树腐烂病的防治潜力,以梨黑腐皮壳菌为靶标菌,通过平板对峙培养和牛津杯法从分离自农用植物酵素液的细菌中筛选拮抗菌株,并分析拮抗菌株对病原菌孢子萌发和菌丝生长的影响及防病效果。结果表明,从农用植物酵素液中分离的68株细菌中初筛出15株对梨黑腐皮壳菌具有抑菌作用的细菌,占分离菌株数的22.1%,抑菌率为41.25%～74.14%。经过复筛,4个菌株（gjfn2、gjfn4、PFN41、PGY-FX67）的发酵液和除菌发酵滤液抑菌率分别大于70%和60%,显著高于其他菌株。采用形态特征、生理生化特性及16S rRNA和gyrA基因序列分析,将gjfn2、gjfn4和PGY-FX67鉴定为贝莱斯芽孢杆菌（Bacillus velezensis）,PFN41鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。4个拮抗菌株发酵液和除菌发酵滤液对梨黑腐皮壳菌分生孢子的萌发有明显的抑制作用,gjfn4、PFN41菌株发酵液及除菌发酵滤液对梨黑腐皮壳菌分生孢子的抑制率36 h均可达到100%,并使梨黑腐皮壳菌菌丝形态畸形、弯曲、末端膨大。离体枝条法防效测定结果表明,4个菌株的发酵液防效均达到79%以上,除菌发酵滤液防效均达到73%以上,其中gjfn4菌株的防效最佳,其发酵液、除菌发酵滤液及除菌发酵滤液50倍稀释液对梨树腐烂病防效分别均达到84.79%、81.57%和67.27%。由此表明,从农用植物酵素液中筛选获得的拮抗细菌菌株具有防治梨树腐烂病的良好潜力。     

薄皮甜瓜采后病害生防菌株的筛选及鉴定

[目的]从薄皮甜瓜体表分离筛选对引起薄皮甜瓜采后病害主要病原真菌具有拮抗作用的生防菌株,并对其进行鉴定确定分类地位,为薄皮甜瓜采后病害的防治提供高效菌株.[方法]采用活体生测的方法从甜瓜瓜表筛选生防菌株,利用非寄养非肠道革兰氏阴性杆菌鉴定试剂盒（比色法）API 20 NE自动鉴定系统测定菌株C3的生理生化指标.[结果]从甜瓜表面分离筛选出2株对粉红单端孢（Trichothecium roseum）防治效果达80％以上的生防菌株C3、B3;通过平板对峙试验表明,菌株C3对引起采后病害的2种病原菌粉红单端孢（Trichothecium roseum）和镰刀菌（Fusarium spp.）的抑菌能力均优于B3;菌株C3对粉红单端孢（Trichothecium roseum）的抑菌效果较好,达到67.7％;对镰刀菌（Fusarium spp.）的抑菌率次之,为27.9％;应用API 20 NE系统分析,通过形态学观察、革兰氏染色将菌株C3初步鉴定为浅黄假单胞菌Pseudomonas luteola.[结论]从甜瓜表面分离筛选获得1株生防菌株C3,该菌株对引起薄皮甜瓜采摘后病害的主要病原菌粉红单端孢（Trichothecium roseum）具有较强的拮抗作用,并初步鉴定为浅黄假单胞菌（Pseudomonas luteola）.     

防治小麦纹枯病有益内生细菌的筛选

从采自河南西部地区的38份健康小麦根系内分离到小麦内生细菌296株.通过活体植株筛选,从中获得的13株细菌,可显著降低小麦纹枯病的发病率和严重度,其中菌株Z8-15的生防效果最好,防效达到81.82％.在平板上测定了13个菌株对小麦纹枯病菌和小麦全蚀病菌的拮抗能力以及产生葡聚糖酶、几丁质酶和蛋白酶等真菌细胞壁降解酶的能...     

Screening of antagonistic Trichoderma strains and their application for controlling stalk rot in maize

Maize is one of the major crops in China, but maize stalk rot occurs nationwide and has become one of the major challenges in maize production in China. In order to find an environment-friendly and feasible technology to control this disease, a Trichoderma-based biocontrol agent was selected. Forty-eight strains with various inhibition activities to Fusarium graminearum, and Fusarium verticillioides were tested. A group of Trichoderma strains(DLY31, SG3403, DLY1303 and GDFS1009) were found to provide an inhibition rate to pathogen growth in vitro of over 70%. These strains also prevented pathogen infection over 65% and promoted the maize seedling growth for the main root in vivo by over 50%. Due to its advantage in antifungal activity against pathogens and promotion activity to maize, Trichoderma asperellum GDSF1009 was selected as the most promising strain of the biocontrol agent in the Trichoderma spectrum. Pot experiments showed that the Trichoderma agent at 2–3 g/pot could achieve the best control of seedling stalk rot and promotion of maize seedling growth. In the field experiments, 8–10 g/hole was able to achieve over 65% control to stalk rot, and yield increased by 2–11%. In the case of natural morbidity, the control efficiency ranged from 27.23 to 48.84%, and the rate of yield increase reached 11.70%, with a dosage of Trichoderma granules at 75 kg ha~(-1). Based on these results, we concluded that the Trichoderma agent is a promising biocontrol approach to stalk rot in maize.     

烟草赤星病生防菌的分离与鉴定

从连作障碍较严重的烟草根际土壤中分离出49株细菌活体纯培养物,依次编号为HHHB1～HHHB49,并筛选出对烟草赤星病病原菌（Alternaria alternate）具有拮抗活性的菌株HHHB7和HHHB19,二者抑菌圈直径分别为5、23 mm。二者初步定性为具有生防活性。多相鉴定结果表明,活性最强的菌株HHHB19隶属于假单胞菌属（Pseudomonas）,暂定名Pseudomonas sp. HHHB19。盆栽试验证实,HHHB19菌株悬液叶面喷雾混合处理对烟草赤星病不同发病阶段均表现出一定的防治效果,其中喷施14 d后防治效果达61.83%。菌株HHHB19可作为良好的生防菌备用资源。     

Construction of engineering Trichoderma strains and their characteristics against tomato gray mold

The transformed Trichoderma strains Ttrm31, Ttrm34 and Ttrm55 were obtained from Trichoderma wild strain T21 mutated by REMI technique for more effective biocontrol of tomato gray mold (Botrytis cinerea) with Trichoderma agent. Those transformants appeared much better in biocontrol activity in vitro or in vivo against tomato gray mold were better than that of wild strain T21. The main results were as follow: The transformed Trichoderma strains were detected in their genetic …     

木霉菌·芽孢杆菌混剂对水稻纹枯病的田间防治效果

为评价生防制剂木霉菌·芽孢杆菌混剂防治水稻纹枯病的田间药效,本试验于2017—2018连续2年采用大田对比试验对木霉菌·芽孢杆菌混剂的防效和增产率进行研究。2017年结果表明在水稻纹枯病发生前或初期施用木霉菌·芽孢杆菌混剂2次(100 g/667 m~2),防效和增产率可达53.61%和28.9%,超过木霉菌芽孢杆菌单一菌剂的防效和增产率(47.8%和12.5%;32.2%和26.2%)且持效期性较好。2018年结果表明,在水稻纹枯病发生前或发生初期施用木霉菌·芽孢杆菌混剂2次(200 g/667 m~2;300 g/667 m~2),防效和增产率分别达到68.52%和8.08%;79.23%和12.8%,增产显著,且对水稻安全无药害,值得进一步研究和推广应用。该研究结果将为减少化学农药的使用量,提高绿色防控水稻纹枯病的水平提供技术支撑。     

淡紫拟青霉与放线菌代谢物复配对南方根结线虫的防治

对淡紫拟青霉和放线菌的拮抗特性及其复配菌剂对南方根结线虫的防治进行了研究.采用杯碟法和平板对峙法测定了淡紫拟青霉菌株PL1和PL2与放线菌菌株LH117和LH121的拮抗性,结果表明两种生防菌4个菌株之间没有明显的拮抗作用;效价法测定结果显示,LH121的发酵上清液对PL2的菌丝生长有轻微抑制作用,抑制率为14.5%.室内生物测定表明,按等体积比例配成的淡紫拟青霉孢子液和放线菌发酵上清液组合的4种复配菌剂处理对根结线虫的卵孵化抑制率和二龄幼虫杀死率分别可达到66.6%和78.0%,明显高于各单菌株处理;温室盆钵试验结果表明,PL1孢子液和LH117发酵上清液的复配菌剂对番茄的根结形成和根结线虫卵块形成的抑制率分别为68.7%和58.4%.上述结果表明:淡紫拟青霉与放线菌的复配菌剂对根结线虫的防治效果比单菌株的防治效果显著,复配菌剂的防治效果与生防菌株间的相瓦拮抗性可能密切相关.     

烟草orf25基因植物表达载体的构建及遗传转化

为更好地研究烟草雄性不育的机理,构建orf25基因的植物表达载体。orf25基因是ATP合成酶F(0)部分的4个亚基因之一。以雄性不育烟草MS革新3号为受体材料,利用组成型启动子Ca MV35S构建orf25基因植物表达载体,将表达载体p BI121-orf25导入根癌农杆菌LBA4404。采用根癌农杆菌介导orf25基因遗传转化MS革新3号,结果证明载体上的目的基因orf25已整合到烟草基因组中,并获得了52株转p BI121-Ca MV35Sorf25基因烟草植株,经PCR鉴定,转基因阳性植株有14株,转化率为26.9%。为下一步研究该基因的功能及其与烟草雄性不育性的关系提供依据。     

真菌病害拮抗菌的筛选及其对多种植物病原真菌的拮抗活性测定

针对广西境内常见多发的12种植物真菌病害,对柑橘、香蕉、甘蔗、辣椒、药用植物等作物根际土壤进行拮抗菌筛选试验。结果从不同作物根际土壤中共分离得到386株菌株,其中有拮抗作用的34株,平皿拮抗效果较好的16株;再经复筛,得到一株对柑橘炭疽菌有较好抑制效果的拮抗菌Bs55;对峙培养试验表明,Bs55菌株对12种常见病原真菌具有较强的抗菌活性,抑菌率最高达87.7%。拮抗菌Bs55具有拮抗作用强、能大量繁殖、抑菌谱广等特点,是具有较好利用前景的生物防治材料。     

PRODUCTION OF NEW WAP-8294A CYCLODEPSIPEPTIDES BY THE BIOLOGICAL CONTROL AGENT LYSOBACTER ENZYMOGENES OH11

● Lysobacter enzymogenes mutants were generated for WAP-8294A biosynthesis. ● Essential and non-essential accessory genes for WAP-8294A biosynthesis were determined. ● Six new WAP-8294A analogs were identified using UHPLC-HR-MS/MS. ● Three deoxy analogs were detected supporting the function of ORF4 in asparagine hydroxylation. Naturally-occurring environmental microorganisms may provide ‘green’ and effective biocontrol tools for disease management in agricultural crops. Due to the constant threat of resistant pathogens there is a pressing and continual need to search for new biocontrol tools. This study investigated the production of new analogs of WAP-8294A compounds by the biocontrol agent Lysobacter enzymogenes OH11 through biosynthetic engineering. WAP-8294As are a family of natural cyclic lipodepsipeptides with potent activity against Gram-positive bacteria. A series of genetic manipulations was therefore conducted on the accessory genes in the WAP biosynthetic gene cluster. The resulting strains containing a single-point mutation in ORF4, which was predicted to encode a 2-ketoglutarate dependent dioxygenase, produced deoxy-WAP-8294As. This result provides evidence for the function of ORF4 in catalyzing β-hydroxylation of the D-asparagine residue in WAP-8294As. In addition, six new analogs of WAP-8294As were identified by UHPLC-HR-MS/MS. This is the first attempt to produce new WAP-8294As in Lysobacter and shows that the spectrum of the biocontrol compounds may be expanded through the manipulation of biosynthetic genes.     

产黄原胶降解酶放线菌筛选及发酵工艺研究

[目的]黄原胶是植物致病菌野油菜黄单胞菌所分泌的胞外多糖,其主链类似纤维素很难降解,可作为生物胶用于增稠剂、悬浮剂、乳化剂和稳定剂,还能引起十字花科植物黑腐病.筛选分离对黄原胶有显著降解作用的放线菌,以生物方法降解黄原胶,降解产物黄原胶寡糖可有效防治黑腐病,具有开发为生物农药的潜力.[方法]从自然土壤样品中进行分离筛选、纯化、16S rRNA鉴定及诱变选育,获得高效降解黄原胶放线菌菌株,通过发酵工艺研究,确定最适产酶条件.[结果]优化产酶培养基配方为:蔗糖3;,( NH4 )2SO4 0.5;,KNO31;,酵母膏0.1;.菌株发酵产酶培养条件为:发酵温度28℃,pH7.5,500mL瓶装量为150 mL,底物浓度0.5;,接种量为5;.[结论]获得一株高效降解黄原胶的链霉菌(Streptomyces sp.),优化其产黄原胶降解酶的摇瓶培养基及培养条件,发酵液黄原胶降解酶的酶活达到200 IU/L.