应用TaqMan-MGB探针进行松材线虫的实时荧光定量检测技术研究

 松材线虫是国际公认的最重要的检疫性有害生物之一,也是我国2类检疫危险性有害生物,我国口岸多次从货物的木质包装中截获该线虫。由于松材线虫与拟松材线虫在形态上极其相似,难以区分,幼虫更无法用于鉴定。传统的形态学鉴定、生化以及其他分子技术等方法存在费时、准确度不高、灵敏度低等缺点,不易形成标准。我们设计筛选一对引物以及一条MGB探针,对松材线虫进行实时荧光PCR检测。建立了一条从1 pg到10⁴pg标准曲线,相关系数r=0.965。该检测方法省时、准确、快速、无污染。     

Penetration, Development and Emigration of Juveniles of the Nematode Meloidogyne arenaria in Myrobalan Plum (Prunus cerasifera) Clones Bearing the Ma Resistance Genes

Penetration, development and emigration of M. arenaria in the roots of three Myrobalan plum (Prunus cerasifera) clones genetically characterized for their resistance to root-knot nematodes (RKN) were studied during the 10 (penetration) and 15 (emigration) days following the date of inoculation (D) of 2500 juveniles (J2s) per plant into the soil. Miniaturized tests were conducted on the two resistant clones P.2175 (Ma1 gene) and P.1079 (Ma2 gene) and the susceptible clone P.2032 (recessive for both genes), obtained from micropropagated plantlets and grown in mini-containers under controlled conditions at 25°C in a growth chamber. For penetration and development studies, nematodes in the roots were recovered by the acid fuchsin-lactophenol staining technique. Equivalent numbers of J2s were recovered in all the clones at D+1 and D+2. Subsequently, the numbers increased rapidly in P.2032 and were significantly different from those in P.1079 and P.2175 that remained at a low level. No swollen larvae were observed in the resistant clones. In P.2032, the first swollen larvae were observed at D+4, the first females were observed at D+12, whereas the first females with attached egg sacs and the first new-generation J2s were obtained between D+21 and D+28. Our data suggest that the resistance phenomenon does not act on the very early nematode penetration but acts later by preventing feeding-site induction and development into the third-stage. For emigration studies, plants in which J2s had been allowed to penetrate for two days (from D to D+2) were washed free of soil, repotted and then, after various periods of growth, soil-free roots were placed under a mistifier to evaluate the numbers of emigrating individuals. Emigration of J2s from the roots occured mainly from D+2 to D+4 in all the genotypes and was very limited from D+4 to D+10. There was no significant differences in the number of emigrated juveniles between the resistant and susceptible clones, indicating that emigration cannot explain the difference in the numbers of nematodes recovered in the roots.     

莨菪烷碱防治蔬菜根结线虫药效研究

本文报道了莨菪烷碱（hyoscyamine)防治蔬菜根结线虫（Meloidogyne incognita)效果，该制剂以有效成分用量7.5-10.5g/667m^2对水稀释后穴施，药后150d对线虫防效达73.78%-82.84%，优于3％米乐尔（isazofos)颗粒剂有效成分用量150g/667m^2穴施药后150d的防效65．30％，并有一定刺激生长作用，有较高推广应用价值。     

侵染广西番茄的番茄黄化曲叶病毒的分子鉴定

番茄黄化曲叶病毒病是番茄生产上的毁灭性病害,严重影响番茄产量及品质。2019年从广西百色市采集疑似番茄黄化曲叶病叶片样品,采用滚环扩增(RCA)及基因克隆等方法,获得了4个烟粉虱传双生病毒的全基因序列,4个分离物的全长序列分别为2 776、2 781、2 752、2 781 bp,均编码6个开放阅读框;核苷酸相似性比较发现,4个分离物彼此间的相似性均在90%以上,与已报道的番茄黄化曲叶病毒Tomato yellow leaf curl virus (TYLCV)各分离物间的相似性也在91%以上;系统进化分析表明,TYLCV广西分离物BS-2和BS-4与TYLCV-Hunan、BS-1和BS-3与TYLCV-YN6553等分离物处于独立的小分支,说明TYLCV广西分离物与TYLCV-Hunan、TYLCV-YN6553具有较近的亲缘关系。本研究首次报道TYLCV在广西发生。     

4种杀虫剂对甘草胭珠蚧的防治效果

甘草胭珠蚧Porphyrophora sophorae(Arch.)是人工栽培甘草的主要害虫。采用灌根、土壤处理法研究了35%吡虫啉悬浮剂、70%噻虫嗪水分散粒剂、48%毒死蜱乳油和5%毒死蜱颗粒剂4种杀虫剂对甘草胭珠蚧的防治效果。结果表明,8月中旬施药各药剂的防治效果均低于50%,但可有效降低翌年春季田间的虫口密度;6月中旬采用35%吡虫啉悬浮剂和70%噻虫嗪水分散粒剂灌根的防效可达80%以上。建议生产中可在6月中旬或之前采用灌溉后进行35%吡虫啉悬浮剂1 500倍或70%噻虫嗪水分散粒剂7 500倍液300mL/株灌根的措施防治甘草胭珠蚧。     

外源油菜素甾酮处理水稻对褐飞虱取食和产卵选择性等行为的影响

为了解经油菜素甾酮处理后水稻对褐飞虱Nilaparvata lugens的抗性,通过在水稻培养液中添加不同浓度的油菜素甾酮,观察水稻对褐飞虱取食和产卵、若虫生长发育、羽化等行为的影响,评估油菜素甾酮在水稻抗虫防御反应中的作用。结果表明,水稻培养液中的油菜素甾酮可被水稻有效吸收并产生生理活性。经0.01～10μmol/L油菜素甾酮处理后水稻叶片弯曲程度均显著增加,其叶角分别为对照的3.3～11.0倍。褐飞虱取食和产卵嗜好性与油菜素甾酮处理浓度密切相关,经较低浓度0.005～0.05μmol/L油菜素甾酮处理的水稻能显著降低褐飞虱雌成虫的取食和产卵嗜好性,其产卵量为对照的35.0%～73.9%;而较高浓度0.1～5μmol/L油菜素甾酮处理则增加褐飞虱雌成虫的取食和产卵嗜好性,其产卵量为对照的1.3～1.8倍。0.01、1、5、10μmol/L油菜素甾酮处理的水稻显著降低褐飞虱若虫的羽化率,为对照的32.5%～53.3%。0.1～10μmol/L油菜素甾酮处理显著缩短褐飞虱若虫发育历期,仅为对照的94.1%～96.9%。褐飞虱若虫存活数和初羽化雌成虫体重分别仅在0.1、10μmol/L油菜素甾酮处理下显著降低。表明油菜素甾酮处理水稻可对水稻抗虫防御反应产生影响,其具有作为生态功能分子用于褐飞虱田间防控的潜力。     

玉米细菌性枯萎病菌的环介导恒温扩增(LAMP)检测方法

为了提高口岸和基层实验室检疫和监测玉米细菌性枯萎病菌的准确性和工作效率,利用环介导恒温扩增技术(LAMP),根据内切葡聚糖酶(EGase)基因前导序列,设计2个内引物和2个外引物,对玉米细菌性枯萎病菌进行快速检测。结果表明,使用玉米细菌性枯萎病菌的近缘种或引致相似症状的病原菌菊欧文氏菌玉米致病变种Erwinia chrysanthemi pv.zeae、玉米内州萎蔫病菌Clavibacter michiganensis subsp.nebraskensis、燕麦假单胞菌Pseudomonas avenae、杓兰欧文氏菌Erwinia cypripedii检测其特异性,仅玉米细菌性枯萎病菌有扩增。LAMP检测灵敏度达到2 pg DNA,为普通PCR的100倍;与其它检测方法相比,LAMP方法检测时间短,效率高,不仅降低了设备投入,易于操作,而且具有较高的灵敏度和特异性,适合玉米细菌性枯萎病菌的现场检疫和大规模检测。     

薰衣草油与肉桂油对朱砂叶螨的生物活性和行为分析

为明确在室内条件下薰衣草油、肉桂油对朱砂叶螨Tetranychus cinnabarinus(Boisduval)的生物活性,采用喷雾、熏蒸法测定了薰衣草油和肉桂油对朱砂叶螨的触杀活性、产卵抑制活性和熏蒸活性,并分析了2种精油的亚致死剂量对雌成螨运动行为的影响。结果表明:与肉桂油相比,薰衣草油对朱砂叶螨的触杀活性较强,经浓度为1%的薰衣草油喷雾处理后,雌成螨、若螨24 h后的死亡率分别为64.07%和52.22%,LC_(50)分别为0.41%和0.89%;浓度为2%的薰衣草油对雌成螨产卵的抑制作用最强;与肉桂油相比,薰衣草油的熏蒸活性也较强,熏蒸雌成螨6、12和24 h后的死亡率分别为70.37%、85.19%和95.19%,LD_(50)分别为0.94、0.59和0.39μL;随精油剂量增加雌成螨运动轨迹紊乱,触壁反射活动减少,移动范围明显减小,而移动频率先升高后降低。研究表明,与肉桂油相比,在朱砂叶螨的生物防治中薰衣草油具有更高的应用价值。     

不同温度下的豌豆蚜实验种群生命表研究

在6种温度(12、15、18、21、24℃和27℃)和相对湿度为80%的条件下,研究了温度对豌豆蚜发育和增殖的影响。组建了生命表,并统计出种群内禀增长率、净增殖率、世代平均周期、种群加倍时间和周限增长率等种群动态参数。结果表明,在试验温度范围内,豌豆蚜的发育历期随温度升高而缩短。种群的内禀增长率在24℃条件下最大(rm=0.281 0),相应的种群净增殖率(R0)、周限增长率(λ)、平均世代周期(T)和种群加倍时间(t)分别为17.030 9、1.324 5、10.089 3 d和2.466 7 d。     

利用贴牌水培法快速鉴定稗草对二氯喹啉酸的抗药性水平

本文通过室内贴牌水培法及整株法,快速鉴定并验证了2013年采自湖南省不同地区稻田的50个稗草生物型对二氯喹啉酸的抗药性,试验结果表明:贴牌水培法所用的二氯喹啉酸甄别剂量为80mg/L;稗草死亡率越低,其抗性倍数(resistance factor,RF)越高,其中死亡率为0时,整株法测定的RF值高达896.07~1 209.38;贴牌水培法测定的稗草样本对二氯喹啉酸的ED50远低于整株法,但两种方法得到的不同稗草生物型对二氯喹啉酸的抗性水平趋势高度吻合。笔者认为以贴牌水培法测定的死亡率小于40%的稗草样本对二氯喹啉酸的抗性风险较高,值得深入研究。