Construction and Identification of Yeast Surface Display Libraries of Ovis aries DRA Gene Exon 2

The specific primers were designed according to Ovis aries DRA gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1,which was a vector used for protein surface display on Saccharomyces cerevisiae.The gene encoding DRA was amplified by PCR using the genomic RNA of Ovis aries.The 762 bp fragment was cloned and released in GenBank and registration number was KR422362.The PCR product was inserted into the yeast surface display plasmid vector pYD1 by double enzyme digestion.It was indicated that DRA gene was successfully integrated into the genome.Dot mutation was made at both ends of exon 2 in DRA gene for making restriction enzyme cutting site and design the exon 2 specific primers according to mutated Ovis aries DRA gene sequence.Sequenced exon 2 amplification products based on DNA pooling of sheep large sample template was analyzed the polymorphic loci.The polymorphic exon 2 246 bp fragment was obtained by double enzyme digestion and connected to surface display restructuring mutation carriers pYD1-DRA by the same double enzyme digestion,and then we successfully constructed yeast surface display libraries.We transformed it into Saccharomyces cerevisiae EBY100 cell.Yeast monoclone was identified by PCR amplification and sequencing,and we confirmed that DRA gene had been integrated into Saccharomyces cerevisiae genome.After galactose induced,it was detected that DRA gene library had been successfully demonstrated on the yeast cell surface under the fluorescence microscope by immunofluorescence method.     

葡萄VvSUC12基因启动子的克隆及上游调控因子鉴定

枝干病害葡萄溃疡病近年来严重限制了葡萄产业发展。研究表明葡萄蔗糖转运蛋白参与寄主植物和病原菌的互作过程。为解析蔗糖转运蛋白VvSUC12在葡萄免疫反应过程中的功能,本研究克隆了VvSUC12基因翻译起始位点上游1 500 bp的启动子区,通过生物信息学分析发现该区域包含4个Dof转录因子结合序列(A/TAAAG)及多种激素调节与防御相关的顺式元件。实时荧光定量分析显示,接种可可毛色二孢菌显著诱导VvSUC12和VvDof19基因的表达。通过酵母单杂交实验筛选得到与VvSUC12启动子互作的转录因子VvDof19。酵母双杂交实验证实VvDof19具有自激活活性;进一步通过烟草瞬时表达发现Dof19-GFP的相对GUS活性约为对照GFP的9倍,表明转录因子VvDof19能够激活VvSUC12基因的表达。研究结果为深入研究蔗糖转运蛋白在葡萄免疫反应中的功能奠定基础。     

卡利比克迈耶氏酵母对水果采后病害的抑制及对展青霉素的降解机制

为了明确拮抗酵母卡利比克迈耶氏酵母Meyerozyma caribbica对果实采后病害的抑制效果及其对毒素的降解机制, 本文研究了卡利比克迈耶氏酵母结合辅助因子褐藻寡糖(alginate oligosaccharide, AOS), 对草莓、梨、苹果、番茄的青霉病、灰霉病和黑斑病的抑制效果, 以及拮抗酵母不同处理液对展青霉素(patulin, PAT)体外降解的机制。结果表明, 浓度为1×108 cfu/mL的M. caribbica可有效抑制草莓、梨、苹果青霉病, 草莓、番茄、葡萄灰霉病以及番茄、梨、葡萄黑斑病的发生, 添加5 g/L的AOS能显著增强M. caribbica的抑制效果; M. caribbica发酵液可以有效降解苹果伤口处的PAT, 处理后8 d其降解率为30.35%, 经AOS诱导后, 其对PAT的降解效率达到39.15%。体外条件下, 接种M. caribbica后27 h可将10 μg/mL的PAT完全降解; M. caribbica主要是通过活细胞代谢降解PAT, 同时AOS可显著增加M. caribbica 的降解能力, 说明卡利比克迈耶氏酵母是一株生防潜力较好的菌株, 本研究为该生防菌株的进一步应用奠定了基础。     

拮抗菌JKJ-23菌株诱导香蕉抵抗枯萎病机理的初探

摘要：以巴西蕉盆栽苗为试材,通过接种病原菌和灌根拮抗菌菌悬液等4个处理,探索不同的处理下香蕉苗防御酶活性、叶绿素含量及根系活力的差异,从而探究生防菌抗病机理。结果表明：JKJ-23菌悬液处理香蕉苗CAT、PPO、SOD、POD活性明显提高,各处理最大峰值分别是对应时间对照的3.72、2.03、1.72、2.61倍;JKJ-23菌悬液能够很好的抑制病原菌对香蕉苗叶绿素的破坏;无论有无病原菌的存在,JKJ-23拮抗菌菌悬液能够明显地提高香蕉苗根系活力。     

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.     

枸杞炭疽病生防菌株筛选、生物功能测定及防治效果

为研发对枸杞炭疽病有良好防治效果的生防制剂,利用枸杞炭疽病菌Colletotrichum acutatum对实验室已分离保存的芽胞杆菌菌株进行室内筛选,并对拮抗效果较好的菌株进行形态学和分子生物学鉴定、病原菌孢子萌发抑制试验、生物学功能测定、稳定性测定、抑菌谱测定以及室内离体防治效果试验和田间防治效果试验。结果表明,菌株F3A对枸杞炭疽病菌有较好的拮抗作用,对枸杞炭疽病菌菌丝的抑制率为62.13%;结合形态学特征、16S rDNA以及gyrA基因序列分析,将菌株F3A鉴定为贝莱斯芽胞杆菌Bacillus velezensis;菌株F3A具有溶解有机磷和无机磷的能力,在含色氨酸和不含色氨酸的金氏培养液中产吲哚乙酸量分别为5.76 mg/L和5.74 mg/L;菌株F3A产蛋白酶和葡聚糖酶的活性较高;菌株F3A连续培养20代后,对枸杞炭疽病菌的抑制率为61.21%,该菌对棉花枯萎病菌Fusarium oxysporum f.sp.vesinfectum、番茄早疫病菌Alternaria solani、黄瓜枯萎病菌F.oxysporum f.sp.cucumerinum、番茄灰霉病菌Botrytis cinerea、番茄叶霉病菌Cladosporium fulvum、茄子菌核病菌Sclerotinia sclerotiorum和黄芪根腐病菌F.solani的抑制率分别为40.71%、53.58%、32.00%、53.00%、79.27%、71.13%和66.08%;菌株F3A发酵液的保护作用明显优于治疗作用,菌株F3A发酵液1倍稀释液室内预防效果为90.32%;喷施菌株F3A发酵液1倍稀释液3 d和7 d后的田间防治效果分别为78.26%和63.19%。表明菌株F3A有作为开发枸杞炭疽病生物农药的潜力。     

拟康宁木霉Tk1的分离鉴定、拮抗作用及其生物学特性

为了分离利用高效植物病害生物防治菌株,从广东药用植物金丝皇菊根部分离获得一株木霉菌株Tk1。经过形态观察、rDNA-ITS和EF-1α序列分析明确其为拟康宁木霉Trichoderma koningiopsis。用平板对峙培养法测定,该菌株对金丝皇菊枯萎病菌（尖孢镰刀菌Fusarium oxysporum）和柑橘炭疽病菌（胶胞炭疽菌Colletotrichum gloeosporioides）的抑制率分别为68.5%和77.1%,且在后期能完全覆盖或部分覆盖病原真菌。其无菌发酵液对金丝皇菊枯萎病菌的抑制率为45.55%。该菌株营养生长和产孢的最适碳源分别为乳糖和葡萄糖,最适氮源是酵母膏;在pH 4~11的培养基上均可生长、产孢,最适pH为7;12 h交替光照条件下菌丝的生长速度最快,全光照条件下产孢数量最大;该菌株的致死温度为73℃处理10 min。     

草地贪夜蛾寄生蜂资源及其调控寄主免疫反应的研究

草地贪夜蛾Spodoptera frugiperda（J.E.Smith）原产于美洲,自2019年初入侵我国以来迅速向北蔓延,对农业造成了巨大损失。其寄生蜂作为重要的天敌资源之一,在害虫防治中具有不可替代的作用。本文对目前已鉴定到的9个科170种草地贪夜蛾寄生蜂天敌等进行了分类总结,而后讨论了其在害虫生物防治方面的应用前景,并围绕草地贪夜蛾寄生蜂寄生因子调控寄主免疫反应的分子机理展开详细论述。该研究旨在丰富草地贪夜蛾的生物防治资源素材,并为该害虫的绿色防控提供重要的理论与实践指导意义。     

松毛虫赤眼蜂携带球孢白僵菌防治亚洲玉米螟技术研究与应用

高效的载菌技术是提高载菌赤眼蜂防治效果的重要基础。本研究对比了不同吸附助剂对松毛虫赤眼蜂羽化时间、羽化量、存活时间及载菌量影响,证明了不同助剂及吸附球孢白僵菌分生孢子后对赤眼蜂无不利影响。试验结果表明,0.1%（w/v）淀粉溶液作为吸附助剂可显著提高赤眼蜂的载菌量,达3.6×10⁴孢子/蜂以上,并应用该助剂建立了赤眼蜂高效携带白僵菌方法。从被害株数、蛀孔数、活虫数和僵虫数4个方面测定了载菌赤眼蜂田间防治玉米螟效果。结果表明,载菌赤眼蜂较单一使用赤眼蜂防治效果显著提高,被害株数、蛀孔数和活虫数均显著降低,分别降低了28.1%、22.8%和24.5%,并且载菌赤眼蜂处理组中僵虫数量显著高于单独使用赤眼蜂处理组,说明载菌赤眼蜂对玉米螟具有协同防控作用。利用该方法进行玉米螟田间防治,能够降低防治成本,提高防治效率,节约劳动力,具有巨大的应用和推广价值。