水稻启动子OsN1p的克隆与功能分析

将OsN1基因上游881 bp启动子(OsN1p)序列取代pBI121中gus基因上游的35S启动子,构建植物表达载体pBIN1p,经农杆菌介导转化水稻品种‘日本晴’,获得转基因植株。GUS组织化学染色结果表明,由该启动子驱动的gus基因能在愈伤组织中较低水平地表达;稻瘟病菌接种转基因植株24 h后,GUS活性为未接种前的4.2倍;5 mmol.L-1水杨酸(SA)和0.5 mmol.L-1茉莉酸甲酯(MeJA)分别喷施转基因植株叶面6 h后,GUS活性分别为处理前的5.9和2.4倍。表明,OsN1p启动子具有启动活性,同时明显具有受稻瘟病菌、SA和MeJA诱导表达的特性。     

Carbendazim sensitivity in populations of Colletotrichum gloeosporioides complex infecting strawberry and yams in Hubei Province of China

The ascomycete fungus Colletotrichum gloeosporioides is a devastating plant pathogen with a wide host range and worldwide distribution. Carbendazim has been widely used to control anthracnose caused by the C. gloeosporioides complex in China for more than 30 years and resistance to carbendazim has been reported in China. A total of 125 Colletotrichum isolates of strawberry and yam were collected from different geographical regions in Hubei Province, China. Approximately 52.8% of Colletotrichum spp. isolates showed resistance to carbendazim. The isolates tested in this study belong to four species, and the frequencies of resistant isolates differed across Colletotrichum species. Resistant isolates were found in C. siamense and C. fructicola. In contrast, all isolates of C. gloeosporioides and C. aenigma were sensitive to carbendazim. Highly carbendazim-resistant isolates harbored the E198 A mutation in the β-tubulin 2(TUB2) gene, whereas moderately carbendazim-resistant isolates harbored the F200 Y mutation in the TUB2 gene. Carbendazim-sensitive Colletotrichum isolates in this study were not genetically similar enough to form a separate cluster from resistant isolates. The result of this study emphasizes the importance of knowing which Colletotrichum sp. is present, when strategies for disease control are made.     

水稻OsN1基因5’端启动子不同区域序列分析

为了研究水稻OsN1基因启动子(OsN1p)的表达调控机理,用OsN1基因ATG(ATG中的A为0)上游-1 089 bp、-881 bp、-489 bp和-245 bp的启动子序列分别取代pBI121中的35S启动子,构建植物表达载体pBIN1.1p、pBIN0.9p、pBIN0.5p和pBIN0.2p。将这些表达载体经农杆菌介导转化水稻日本晴,获得转基因植株。对启动子的表达特点和启动活性进行了分析,结果表明,由-1 089 bp启动子、-881 bp启动子和-489 bp启动子驱动的gus基因能在愈伤组织中表达,-245 bp启动子不能驱动gus基因在愈伤组织中表达,说明启动子OsN1p具有启动活性的最小启动序列为ATG上游的-489 bp;-489 bp启动子驱动的gus基因在转基因植株根中表达,在根冠不表达;用5 mmol/L水杨酸(SA)喷施转基因植株叶片后,GUS定量分析结果表明转基因植株的GUS活性增高,转-881bp启动子植株的GUS活性比转-1 089 bp启动子植株的活性高。可见,启动子OsN1p具有组织特异性表达和受SA诱导表达的特性。     

西瓜噬酸菌pilN基因功能分析

为了分析菌毛组装蛋白家族基因对瓜类细菌性果斑病菌致病性的影响,以xjl12菌株为背景构建转座子(Tn5)插入的突变体文库,通过浸种处理和子叶注射接种方法筛选致病性相关突变体,通过两亲交配获得突变株,并对所得的突变体、野生型及互补等多个菌株进行致病性、过敏性反应、游动性等相关表型进行测定,用反转录PCR(qRT-PCR)方法验证xjl12和突变体中hrpG、hrcV、celA表达量的差异。结果表明:突变体文库中筛选得到1株致病力明显下降的突变体Δxj-7,经亚克隆鉴定为pilN基因,其功能主要与菌毛(pili)生物合成相关。两亲交配后所得突变菌株ΔpilNac相较于野生型,其致病性、游动性、胞外纤维素酶活性降低,同时丧失烟草过敏性反应。qRT-PCR试验结果显示,Ⅲ型分泌系统的主要调控基因以及纤维素酶生物合成基因在ΔpilNac中的转录水平明显下调。证明菌毛生物合成相关基因pilN对致病性有重要的调控作用。     

Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79

Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 10⁸ CFU/g) was much larger than in stems (6.78 × 10⁷ CFU/g) or roots (3.56 × 10⁷ CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m²) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast.     

温度、补充营养和体型大小对白蛾周氏啮小蜂成虫寿命的影响

寄生蜂成虫能否找到并寄生足够的寄主,是实现其最大生殖力的重要限制性因素之一[1],该因素与寿命直接相关,故寿命在进化中受到较大的自然选择压力,从而成为寄生蜂重要的适应性特征之一[2];在害虫生物防治实践中,寿命是决定寄生蜂控制寄主害虫能力的重要参数,是常用的评价天敌     

短稳杆菌对草地贪夜蛾的杀虫活性及其体内解毒酶的影响

【目的】明确生物农药短稳杆菌对草地贪夜蛾的杀虫活性及其体内解毒酶活性的影响,为草地贪夜蛾的高效绿色防控提供理论依据。【方法】采用叶片浸渍法测定短稳杆菌对草地贪夜蛾1、2和3龄幼虫的室内杀虫活性;通过喷雾法测定短稳杆菌对草地贪夜蛾的田间控制效果;以短稳杆菌亚致死剂量处理草地贪夜蛾幼虫,利用酶标仪检测幼虫体内解毒酶（谷胱甘肽S-转移酶、羧酸酯酶）和乙酰胆碱酯酶活性的变化。【结果】室内杀虫活性测定结果显示,短稳杆菌对草地贪夜蛾幼虫具有较强的毒杀作用,取食经1.00×107孢子/mL短稳杆菌处理的玉米叶片72 h后,1、2和3龄幼虫的校正死亡率分别达86.21%、83.05%和58.33%。玉米田施用100亿孢子/mL短稳杆菌悬浮剂800和1000倍液7 d后,对草地贪夜蛾的防治效果分别为95.84%和93.78%,显著高于0.5%苦参碱水剂800倍液处理（P<0.05）,高于200 g/L氯虫苯甲酰胺悬浮剂2000倍液处理但差异不显著（P>0.05）。亚致死剂量短稳杆菌（LC₂₀）处理后,草地贪夜蛾幼虫体内谷胱甘肽S-转移酶、羧酸酯酶和乙酰胆碱酯酶活性均有不同程度下降。【结论】短稳杆菌对草地贪夜蛾低龄幼虫具有较好的室内杀虫活性和田间防治效,可作为防治草地贪夜蛾有效的生物农药使用。在玉米田草地贪夜蛾低龄幼虫盛发期,使用100亿孢子/mL短稳杆菌悬浮剂进行防治时,建议使用800~1000倍液喷施。     

Field evolution of insecticide resistance in the brown planthopper (Nilaparvata lugens Stål) in China

The brown planthopper (Nilaparvata lugens Stål) is one of the most destructive pests of rice crops in Asian countries including China, Vietnam, Thailand, etc. Evolution of resistance in this pest insect to isoprocarb, buprofezin, pymetrozine, imidacloprid and other neonicotinoid insecticides has been reported. In order to investigate the current status of resistance to commonly used insecticides, nine field populations of N. lugens were collected from Central China, East China and South China, and resistance to insecticides was monitored from 2009 to 2012. All the 9 field populations collected in 2012 had developed extremely high resistance to imidacloprid, with resistance ratios (RR) ranging from 209.3 to 616.6. Resistance to imidacloprid was much higher in 2012 than in 2009. The RR of thiamethoxam varied from 17.4 to 47.1, and the RR of nitenpyram varied from 1.4 to 3.7 in 2012. Of the 9 field populations, six populations showed higher resistance to nitenpyram in 2012 than in 2011. RR for buprofezin varied from 110.1 to 221.6 in 2012 whereas resistance was at a medium level (RR 20.4 ∼ 39.4-fold) in 2009. RR for pymetrozine ranged from 34.9 to 46.8 in 2012. As for isoprocarb, RR ranged from 21.7 to 38.1 in 2012. The obvious increase in resistance to widely applied insecticides indicates that insecticide resistance management strategies are urgently needed to prevent or delay further increase of insecticide resistance in N. lugens.     

早熟禾对精噁唑禾草灵耐药性的靶标酶机制研究

早熟禾对精噁唑禾草灵、精喹禾灵、高效氟吡甲禾灵、炔草酸、精吡氟禾草灵、氰氟草酯、烯禾啶和苯草酮具有耐药性．而对烯草酮和吡喃草酮敏感。早熟禾生物型的乙酰辅酶A羧化酶（ACCase）对精嗯唑禾草灵的活性显著高于敏感杂草日本看麦娘．5个早熟禾生物型的,IC50值为敏感杂草日本看麦娘,IC50值的10．46～11．98倍：早熟禾ACCase氨基酸序列1781位点存在亮氨酸／异亮氨酸的多态性现象,而亮氨酸的存在是其它禾本科杂草对ACCase抑制剂除草剂产生抗性的主要原因之一：5个早熟禾生物型的ACCase基因表达量为日本看麦娘的4．67～7.37倍。早熟禾与日本看麦娘的ACCase活性、基因序列及基因表达量差异可能是导致早熟禾对精嗯唑禾草灵具有耐药性的靶标酶机理。     

杀菌剂种子处理对镰孢菌侵染玉米的影响

采用室内培养皿法和室外盆栽两种方法,分析杀菌剂种子处理(嘧菌脂、咯菌腈、精甲霜灵、克菌丹)单独和复合使用对特定镰孢菌种(禾谷镰孢菌F. graminearum、轮枝镰孢菌F. verticillioides和层出镰孢菌F. proliferatum)的影响。盆栽试验方差分析表明,多个性状其菌株效应表现显著,大多性状的互作等变异效应不显著;室内试验的方差分析表明,除了品种×种子处理间的互作效应不显著以外,其余变异来源均表现显著或极显著。两种方法均表明, F. graminearum具有更强的致病力。盆栽试验表明,多数性状的种子处理间没有显著差别,就根长和干重来说,使用咯菌腈处理的效果相对较好。室内结果表明,含有咯菌腈和克菌丹处理的种子均具有较好效果,其中以单独使用咯菌腈效果最好。杀菌剂处理对不同镰孢菌菌株具有不同的影响,所有杀菌剂处理对F. graminearum的效果均差于对F. proliferatum和F. verticillioides的效果。嘧菌脂种子处理的效果最差,单独使用咯菌腈的效果最好。