黄瓜根结线虫病生防细菌的筛选与评价

[13]。121℃灭菌20minLB培养基胰蛋白10g，酵母提取物5g，NaCl10g，1.5%琼脂，加蒸馏水定容至1L。121℃灭菌20min。BPY培养基酵母膏5.0g，葡萄糖10.0g，氯化钠5.0g，蛋白胨10.0g，牛肉膏5.0g，加蒸馏水定容至1L。121℃灭菌20min。LB液体培养基胰蛋白10g,酵母提取物5g,NaCl10g,加蒸馏水定容至1L。121℃灭菌20min。其他氯霉素和四环素购于索莱宝生物科技有限公司（于-4℃下保存）。实验方法线虫饲养将根部放入蒸馏水中，在显微镜下剥取黄瓜根部的卵块，将卵块放在装有双层滤网的培养皿中，加入蒸馏水孵化，待其在28℃孵化至2龄幼虫备用REF_Ref19570\r\h[14]。细菌的分离、纯化与保存细菌的分离用计量天平准确称量有虫和健康土壤样品各10.0g，分别倒入200mL容量的锥形瓶中，用量筒称取90.0mL无菌水加入锥形瓶中，充分混匀土壤，并向其中加入3-5颗无菌玻璃珠。将锥形瓶置于37℃恒温摇床中，以180rpm的速度震荡40min，使土壤颗粒分散，并使土壤菌体复苏REF_Ref20103\r\h[15]。用移液器吸取1.0mL土壤悬液转移至装有9.0mL无菌水的试管中，震荡摇匀，此为10-1浓度梯度，从10-1浓度的土壤悬液中吸取1.0mL转移至装有9.0mL无菌水的试管中，震荡摇匀，此为10-2浓度，依次稀释至10-3、10-4、10-5、10-6浓度梯度，完成系列稀释。操作前，提前半小时打开操作台的紫外灯关紫外，开通风，稍抬高玻璃挡板，5min后开照明，用75%酒精喷洒手腕及手部轻晃酒精灯，以检测灯内酒精余量待手上酒精挥发完全后，方可在离酒精灯较远处用打火机打火，再点燃酒精灯用手感受酒精灯的无菌圈范围，此后无菌操作应严格在无菌圈范围内执行拆开培养皿，整齐堆码在酒精灯左侧在无菌圈内打开锥形瓶封口，并始终保持锥形瓶瓶口在酒精灯外焰处右手托住锥形瓶瓶底，左手取培养皿，沿板交界处在外焰上灼烧一圈后，在无菌圈范围内打开培养皿倒培养基时，锥形瓶瓶口不应与培养皿的大小板接触，培养基不能倒在小板外壁或酒精灯上当培养基没过一半的小板时，左手盖合培养皿大小板，并沿操作台边缘将培养皿平推入操作台中以3-4个培养皿为一摞，待培养基凝固后可将培养皿倒置，以减少皿中水汽。将10-2、10-3、10-4、10-5浓度梯度稀释土壤悬液于平板上涂布，每个梯度作3个平行，随后用封口膜密封平板周围，置于37℃培养箱中恒温培养24h，观察菌落的生长情况，最后选择梯度为10-3，进行涂布平板。右手持200μL移液枪，并装上200μL黄枪头左手取培养皿，沿大小板交界处在外焰上灼烧一圈后，在无菌圈范围内打开培养皿，用量程调为100μL的移液枪取100μL稀释液，并将其打入培养基正中央，通过玻璃珠在平板上来回轻轻滚动，使稀释液在平板上涂均匀，涂干。平板倒置，封口膜封口，倒置于37℃培养箱中恒温培养24h培养。细菌的纯化取出空培后的培养皿，整齐堆码在酒精灯左侧,将表面消毒过的试管架放在操作台的右后侧，将接种环浸泡于含95%酒精的大试管中,拆开含有需接种菌株平板的保鲜膜,左手持板，右手将浸有95%酒精的接种环，朝下，于外焰上灼烧,默念30s，待接种环稍凉后，左手将培养皿沿大小板交界处在外焰上灼烧一圈，稍稍打开培养皿，将接种环伸入平板中，可用拇指隔着大板感受接种环的温度,接种环挑取单菌落,左手放下板，再取一个空培后的培养皿，沿大小板交界处在外焰上灼烧一圈后，在无菌圈范围内打开培养皿,一区划线，培养皿转90°,将接种环浸泡在含95%酒精的大试管中，并在酒精灯外焰处灼烧灭菌,从一区引线，进行二区划线，培养皿转90°,将接种环浸泡在含95%酒精的大试管中，并在酒精灯外焰处灼烧灭菌,从二区引线，进行三区划线，培养皿转90°,平板倒置，封口膜封口，倒置于37℃培养箱中恒温培养24h培养。细菌的保存短期保存，采用斜面保藏法，根据菌株原先的培养皿选择相对应的斜面培养基，将待保存的菌株接种到准备好的斜面培养基上。放在37℃培养箱中恒温培养24h后，转移至4℃冷藏室中保藏。在使用期间，需要定期将细菌转移到新的斜面培养基上。菌株发酵及其杀虫效力的测定菌株的发酵菌株活化：将4℃下保藏的菌株于LB固体培养基上进行三区划线，置于恒温培养箱中37℃培养24h。发酵种子液的制备：挑取活化后菌株的单菌落，接种于装有3~5ml的BPY液体培养基的离心管中，置于摇床中在37℃下以180rpm恒温培养12h。发酵液的制备：将发酵种子液按照1%接种量的比例接种到装有3ml的BPY液体培养基的离心管中，置于摇床中在37℃下以180rpm恒温培养48h。发酵上清液的制备：移液器吸取发酵液至1800uL离心管中，在4℃下以12000rpm离心13min，注射器吸取上清液1400ul，过0.22μm滤膜过滤除菌，获得滤菌上清液后于4℃下保藏备用。发酵液杀虫效力的测定杀虫体系构建：总体系100μL，其中发酵上清液30μL；线虫总数大于50条的虫悬液30μL；50IU/mL的氯霉素10μL；50IU/mL的四环素10μL；无菌水20μL。对照组为线虫总数大于50条的虫悬液30μL；50IU/mL的氯霉素10μL；50IU/mL的四环素10μL；无菌水50μL。（加入抗生素抑制虫悬液中细菌的生长）配置好杀虫体系，分成三部分虫液，菌液，抗生素液（氯霉素四环素无菌水）充分吸打混匀+，每株菌设立三组，对照组也一致，加入96孔板中，每株菌的杀虫体系之间最好间隔一格，所以每个板每次最多做18株菌，对照组要远离菌株的杀虫体系，避免交叉污染，将其置于28℃培养箱中恒温培养12h。将96孔板中的杀虫体系吸取至显微镜下镜检，进行线虫计数，记录总数与死亡数，据其计算死亡率与校正死亡率。平均死亡率超过60％的第二次新发酵验证，发酵液需要重新制备，其他不变。然后选取两次验证中平均死亡率超过90%的进行第三次梯度验证，梯度验证中发酵液用第二次制备的，改变发酵液体积，分别设置20ul和10ul，其他不变，无菌水体积对应变为30ul和40ul，对照组不变。设置两组。结果与分析细菌的分离与纯化细菌的分离结果基于菌落培养特征的不同（图1），本研究从发病和未发病的黄瓜株系根际土壤中初筛到600株可培养细菌。由于仅根据菌落培养特征作为分离和纯化的标准，很难排除重复菌株的出现。本分离结果仅作为细菌的初分离和纯化，菌株仅用于杀虫效果的测定，后续将对杀虫效果好的菌株，开展相关的菌种鉴定工作。图SEQ图\*ARABIC1菌落培养特征（部分）细菌的纯化结果在纯化后的细菌培养物中，我们可以清晰地观察到目标细菌的形态特征，如菌落的颜色、形状、大小等，这些特征都表明了细菌纯化成功。挑取从未发病的黄瓜株系根际土壤分离的600株菌进行进一步的纯化和保存（图2）。图SEQ图\*ARABIC2细菌纯化培养结果（部分）从图2可以看出，经过2次纯化后的菌落培养特征一致，且生长良好。分离菌株的杀虫效力的测定 在两次发酵验证中，编号为153、162、47、27、26、89、45、4、95等多个菌株都表现出较好的杀虫作用。尤其是153和162不仅两次验证中平均校正死亡率超过了90%，最高还达到了100%。虽然在梯度实验验证中表明，低浓度下很多菌株并没有表现出明显的杀虫作用，但是Hg-CK-153和Hg-CK-162在低浓度的20uL和10ul的添加量下的平均校正致死率分别达到97.06%、79.00%和94.00%、70.76%，从图3可以看出菌株Hg-CK-153菌落呈乳黄色，Hg-ck-162呈乳白色。两株菌的单菌落都呈圆形，表面凸起且光滑。图SEQ图\*ARABIC3黄瓜-CK-153(左）和黄瓜-ck-162（右）的单菌落纯化结果4.24.34.14.24.34.1图SEQ图\*ARABIC4发酵上清液中根结线虫的虫体形态注：图4-1：hg-ck-153，4-2：hg-ck-162，4-3对照组从图4中可以看出，4-1和4-2中能明显观察到线虫死亡，身体僵硬呈直线型，不再表现出活动能力。4-3中线虫充满活力，身体灵活呈弯曲。表3第一次验证（30ul发酵液）试验线虫死亡率编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-162100.00%100.00%100.00%100.00%Hg-ck-164100.00%97.62%100.00%99.21%Hg-ck-8996.67%100.00%100.00%98.89%Hg-ck-79100.00%96.92%96.43%97.78%Hg-ck-190100.00%97.87%95.24%97.70%Hg-ck-3792.31%100.00%100.00%97.44%Hg-ck-4596.30%100.00%93.75%96.68%Hg-ck-15790.00%100.00%100.00%96.67%Hg-ck-16595.70%95.95%97.65%96.43%Hg-ck-6695.35%100.00%93.75%96.37%Hg-ck-7497.75%96.30%95.00%96.35%Hg-ck-1396.97%93.33%96.23%95.51%Hg-ck-14892.45%95.00%96.74%94.73%Hg-ck-6593.10%90.00%100.00%94.37%Hg-ck-2696.00%91.67%95.24%94.30%Hg-ck-15396.15%86.67%100.00%94.27%Hg-ck-4893.75%95.00%92.31%93.69%Hg-ck-18495.24%94.74%90.91%93.63%Hg-ck-4790.38%93.18%96.15%93.24%Hg-ck-495.08%94.12%90.48%93.23%Hg-ck-12192.00%94.74%91.18%92.64%Hg-ck-4393.75%95.24%88.89%92.63%Hg-ck-390.91%94.74%92.00%92.55%Hg-ck-14993.33%87.50%94.12%91.65%Hg-ck-14588.73%90.24%91.53%90.17%Hg-ck-1488.89%93.75%87.50%90.05%Hg-ck-16885.54%91.84%88.68%88.69%Hg-ck-14786.27%89.61%89.87%88.59%Hg-ck-15075.61%90.00%100.00%88.54%续表3编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-90100.00%78.57%86.96%88.51%Hg-ck-18678.87%90.74%95.83%88.48%Hg-ck-10994.44%81.25%88.89%88.19%Hg-ck-11285.71%88.89%89.47%88.03%Hg-ck-11085.71%88.89%89.47%88.03%Hg-ck-18088.89%88.73%86.00%87.87%Hg-ck-4182.14%91.43%88.89%87.49%Hg-ck-7192.31%76.92%90.91%86.71%Hg-ck-7388.57%85.00%85.96%86.51%Hg-ck-17382.61%87.14%89.06%86.27%Hg-ck-191.67%95.45%71.43%86.18%Hg-ck-17785.71%90.12%79.75%85.19%Hg-ck-8085.71%92.00%76.47%84.73%Hg-ck-14371.15%88.52%92.77%84.15%Hg-ck-9576.67%89.47%85.71%83.95%Hg-ck-6375.86%76.67%95.83%82.79%Hg-ck-10582.50%81.82%80.00%81.44%Hg-ck-16690.91%83.33%69.23%81.16%Hg-ck-2777.27%97.22%66.67%80.39%Hg-ck-9282.35%78.26%80.00%80.20%Hg-ck-5288.89%65.85%81.82%78.85%Hg-ck-7781.97%77.61%76.47%78.68%Hg-ck-15677.78%84.04%70.37%77.40%Hg-ck-17271.67%77.11%79.73%76.17%Hg-ck-1669.57%83.33%75.00%75.97%Hg-ck-2569.84%70.15%82.81%74.27%Hg-ck-16369.84%79.10%73.13%74.03%Hg-ck-12385.14%62.71%73.33%73.73%Hg-ck-15168.52%80.33%70.42%73.09%Hg-ck-4271.43%75.00%70.21%72.21%Hg-ck-1560.67%70.91%46.15%59.90%Hg-ck-3650.00%61.29%68.43%59.68%Hg-ck-5751.52%60.87%66.67%59.24%Hg-ck-12100.00%62.50%15.00%59.17%Hg-ck-14346.15%53.52%67.77%59.15%Hg-ck-1955.56%100.00%20.00%58.52%Hg-ck-12064.29%46.51%64.29%58.36%Hg-ck-4955.97%57.10%62.24%58.43%Hg-ck-2258.82%83.33%31.25%57.80%Hg-ck-669.23%61.00%40.00%56.74%续表3编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-5037.50%65.00%60.71%54.40%Hg-ck-554.55%46.67%60.00%53.74%Hg-ck-956.52%61.29%42.31%53.37%Hg-ck-2472.73%66.67%20.69%53.36%Hg-ck-4028.57%70.00%61.00%53.19%Hg-ck-5841.67%55.56%60.61%52.61%Hg-ck-2358.82%77.78%17.86%51.49%Hg-ck-748.57%55.56%50.00%51.38%Hg-ck-11673.91%43.48%33.33%50.24%Hg-ck-840.00%52.00%51.28%47.76%Hg-ck-17449.25%46.67%46.48%47.47%Hg-ck-17538.89%42.25%53.01%44.72%Hg-ck-5340.63%45.83%47.37%44.61%Hg-ck-263.64%42.86%23.81%43.43%Hg-ck-17842.03%43.04%44.19%43.08%Hg-ck-6168.42%15.38%43.33%42.38%Hg-ck-15438.98%49.28%38.10%42.12%Hg-ck-7052.38%47.37%26.32%42.02%Hg-ck-9944.19%42.86%38.64%41.89%Hg-ck-1050.00%37.50%37.93%41.81%Hg-ck-17639.58%42.11%41.46%41.05%Hg-ck-2138.46%40.00%44.19%40.88%Hg-ck-16743.94%37.97%38.10%40.00%Hg-ck-10043.48%38.89%33.33%38.57%Hg-ck-18539.06%42.59%30.16%37.27%Hg-ck-1730.30%40.91%33.33%34.85%Hg-ck-8840.00%41.67%18.75%33.47%Hg-ck-10260.00%23.53%16.67%33.40%Hg-ck-18832.20%31.58%33.33%32.37%Hg-ck-9837.50%37.93%21.43%32.29%Hg-ck-8312.20%55.56%28.30%32.02%Hg-ck-17124.56%34.25%36.73%31.85%Hg-ck-12636.36%35.00%23.53%31.63%Hg-ck-7846.43%28.00%19.35%31.26%Hg-ck-6220.00%26.09%47.62%31.24%Hg-ck-15833.33%29.69%29.11%30.71%Hg-ck-5644.44%14.29%33.33%30.69%Hg-ck-15928.77%33.33%29.58%30.56%续表3编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-18732.86%28.92%28.13%29.97%Hg-ck-11826.67%41.38%21.43%29.82%Hg-ck-2828.57%30.00%28.00%28.86%Hg-ck-14426.32%33.33%25.00%28.22%Hg-ck-8543.48%26.32%14.81%28.20%Hg-ck-18232.20%28.77%23.33%28.10%Hg-ck-1559.09%28.57%44.44%27.37%Hg-ck-3127.27%31.25%23.53%27.35%Hg-ck-9631.58%25.00%25.00%27.19%Hg-ck-1824.14%28.57%28.57%27.09%Hg-ck-3328.00%28.00%25.00%27.00%Hg-ck-3242.86%19.23%18.42%26.84%Hg-ck-11529.41%23.53%23.08%25.34%Hg-ck-10820.00%33.33%22.22%25.19%Hg-ck-9128.57%36.96%9.52%25.02%Hg-ck-18132.81%30.51%10.81%24.71%Hg-ck-5414.63%36.84%22.45%24.64%Hg-ck-17020.31%35.82%16.33%24.15%Hg-ck-18326.98%26.76%18.52%24.09%Hg-ck-16033.90%23.21%14.75%23.96%Hg-ck-12930.43%20.00%21.43%23.95%Hg-ck-10316.67%26.67%27.27%23.54%Hg-ck-12414.81%29.41%26.32%23.51%Hg-ck-15223.26%26.92%20.00%23.39%Hg-ck-11129.41%33.33%5.26%22.67%Hg-ck-7625.00%22.92%19.57%22.49%Hg-ck-16924.73%17.78%20.90%21.13%Hg-ck-17919.05%22.73%20.34%20.70%Hg-ck-18915.79%25.00%20.00%20.26%Hg-ck-3536.36%15.00%8.70%20.02%Hg-ck-6845.83%9.52%4.55%19.97%Hg-ck-2925.64%6.67%27.27%19.86%Hg-ck-1117.95%16.67%24.39%19.67%Hg-ck-14613.51%23.60%19.70%18.94%Hg-ck-16114.29%20.00%20.75%18.35%Hg-ck-8726.67%14.29%12.50%17.82%Hg-ck-11320.00%13.04%19.35%17.47%Hg-ck-3820.00%20.00%11.76%17.25%Hg-ck-4613.04%19.05%18.75%16.95%Hg-ck-5921.88%13.79%14.63%16.77%续表3编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-11714.71%8.70%26.09%16.50%Hg-ck-4414.81%20.51%13.89%16.41%Hg-ck-1220.00%16.67%32.26%16.31%Hg-ck-9730.00%9.09%9.52%16.20%Hg-ck-8615.79%18.52%14.29%16.20%Hg-ck-3414.81%16.67%16.67%16.05%Hg-ck-11921.05%15.00%11.76%15.94%Hg-ck-12827.59%5.88%13.33%15.60%Hg-ck-816.25%20.00%19.05%15.10%Hg-ck-607.69%21.28%15.79%14.92%Hg-ck-12511.11%12.50%20.83%14.81%Hg-ck-12712.90%14.71%15.38%14.33%Hg-ck-11414.29%11.11%17.39%14.26%Hg-ck-6416.22%9.09%13.89%13.07%Hg-ck-19120.83%10.00%5.88%12.24%Hg-ck-10114.29%6.25%15.79%12.11%Hg-ck-208.82%15.38%11.11%11.77%Hg-ck-677.32%16.67%10.87%11.62%Hg-ck-5510.34%7.14%16.67%11.38%Hg-ck-19212.90%9.52%11.11%11.18%Hg-ck-14214.93%6.78%11.48%11.06%Hg-ck-1048.33%14.29%10.53%11.05%Hg-ck-519.09%7.84%10.00%8.98%Hg-ck-395.88%11.11%9.30%8.77%Hg-ck-1074.76%10.71%8.51%8.00%Hg-ck-307.14%8.89%7.69%7.91%Hg-ck-758.51%7.94%7.27%7.91%Hg-ck-826.25%11.11%6.06%7.81%Hg-ck-1066.45%8.82%6.90%7.39%Hg-ck-692.63%10.00%6.06%6.23%Hg-ck-945.88%0.00%8.70%4.86%Hg-ck-840.00%5.56%5.88%3.81%Hg-ck-934.76%0.00%5.26%3.34%Hg-ck-720.00%0.00%5.88%1.96%从表3可以看出，180株菌发酵液（30uL）对线虫的触杀试验导致线虫的平均校正死亡率在1.96%—100%之间，其中致线虫平均校正死亡率达到60%以上的菌株有58株，达到90%的菌株有26株，其编号为：Hg-ck-164,Hg-ck-162,Hg-ck-89,Hg-ck-79,Hg-ck-190,Hg-ck-37,Hg-ck-45,Hg-ck-157,Hg-ck-165,Hg-ck-66,Hg-ck-74,Hg-ck-13,Hg-ck-148,Hg-ck-65,Hg-ck-26,Hg-ck-153,Hg-ck-42,Hg-ck-184,Hg-ck-47,Hg-ck-4,Hg-ck-121,Hg-ck-43,Hg-ck-3,Hg-ck-149,Hg-ck-145,Hg-ck-14。表4第二次新发酵验证（30ul发酵液）试验线虫死亡率编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-153100.00%100.00%100.00%100.00%Hg-ck-47100.00%100.00%100.00%100.00%Hg-ck-27100.00%100.00%100.00%100.00%Hg-ck-2698.31%100.00%100.00%99.44%Hg-ck-8997.78%100.00%100.00%99.26%Hg-ck-4598.18%100.00%96.55%98.24%Hg-ck-496.43%97.67%100.00%98.03%Hg-ck-9596.08%100.00%97.92%98.00%Hg-ck-1397.62%95.24%100.00%97.62%Hg-ck-4394.23%100.00%97.92%97.38%Hg-ck-16296.00%94.74%100.00%96.91%Hg-ck-7495.74%94.64%100.00%96.80%Hg-ck-17297.26%98.44%94.20%96.63%Hg-ck-9298.15%93.88%97.37%96.46%Hg-ck-1494.74%92.59%100.00%95.78%Hg-ck-14998.18%95.52%93.48%95.73%Hg-ck-15091.89%97.37%97.62%95.63%Hg-ck-16897.01%96.67%93.15%95.61%Hg-ck-7397.96%96.15%92.45%95.52%Hg-ck-193.75%95.24%96.88%95.29%Hg-ck-4297.62%88.89%97.30%94.60%Hg-ck-18495.52%93.59%94.20%94.44%Hg-ck-1688.89%97.06%95.24%93.73%Hg-ck-14895.52%89.87%93.24%92.88%Hg-ck-16591.43%93.06%88.89%91.12%Hg-ck-17789.61%91.78%88.71%90.03%Hg-ck-11283.05%95.92%90.91%89.96%Hg-ck-15777.14%90.91%100.00%89.35%Hg-ck-14590.48%87.14%88.52%88.71%Hg-ck-6586.27%84.09%93.33%87.90%Hg-ck-15684.51%87.30%91.80%87.87%Hg-ck-7781.97%77.61%90.57%83.38%Hg-ck-12169.39%81.03%90.91%80.44%Hg-ck-366.67%83.33%87.50%79.17%Hg-ck-12377.05%76.56%82.86%78.82%Hg-ck-16493.33%72.73%70.00%78.69%Hg-ck-7185.45%69.39%78.43%77.76%续表4编号死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-19083.33%75.00%68.42%75.58%Hg-ck-3778.33%71.70%66.67%72.23%Hg-ck-4864.71%67.65%75.93%69.43%Hg-ck-4167.31%66.67%56.86%63.61%Hg-ck-18654.24%61.19%55.56%57.00%Hg-ck-5251.35%56.25%57.63%55.08%Hg-ck-18057.81%51.90%53.45%54.39%Hg-ck-7945.61%49.25%64.15%53.01%Hg-ck-17349.06%56.92%52.54%52.84%Hg-ck-14753.70%49.21%53.23%52.05%Hg-ck-16350.00%56.16%49.06%51.74%Hg-ck-8052.38%53.70%48.48%51.52%Hg-ck-15151.52%50.63%52.17%51.44%Hg-ck-9048.21%51.67%52.27%50.72%Hg-ck-10550.00%48.44%42.00%46.81%Hg-ck-16644.12%55.56%37.50%45.72%Hg-ck-2535.82%37.29%37.25%36.79%Hg-ck-11034.55%34.04%29.55%32.71%Hg-ck-6627.27%30.77%37.29%31.78%Hg-ck-10910.26%30.36%22.45%21.02%Hg-ck-6315.87%16.67%14.29%15.61%在第一次杀虫验证的基础上，我们又对平均校正死亡率超过60%的58株菌株进行了重复验证。从表4可以看出，其中41株仍表现较高的杀虫效果，平均校正死亡率达到90%以上的菌株有26株，分别是Hg-ck-153,Hg-ck-47,Hg-ck-27,Hg-ck-26,Hg-ck-89,Hg-ck-45,Hg-ck-4,Hg-ck-95,Hg-ck-13,Hg-ck-43,Hg-ck-162,Hg-ck-74,Hg-ck-172,Hg-ck-92,Hg-ck-14,Hg-ck-149,Hg-ck-150,Hg-ck-168,Hg-ck-73,Hg-ck-1,Hg-ck-42,Hg-ck-184,Hg-ck-16,Hg-ck-148,Hg-ck-165,Hg-ck-177，与第一次的杀虫验证具有较好的重叠性，进一步证明了以上这些菌株具有高效杀虫作用。表5第三次梯度验证（10ul和20ul发酵液）试验线虫死亡率10ul发酵液20ul发酵液编号死亡率/%死亡率/%平均死亡率/%死亡率/%死亡率/%平均死亡率/%Hg-ck-15380.50%77.50%79.00%94.12%100.00%97.06%Hg-ck-2711.11%16.67%13.89%97.78%95.83%96.81%Hg-ck-15025.64%35.71%30.68%96.00%97.30%96.65%Hg-ck-16277.65%63.86%70.76%94.12%93.88%94.00%Hg-ck-4211.76%20.00%15.88%83.33%95.24%89.29%Hg-ck-16862.46%32.26%47.36%88.52%83.02%85.77%Hg-ck-15723.68%66.67%45.18%82.86%85.71%84.29%Hg-ck-37.32%0.00%3.66%76.92%87.50%82.21%Hg-ck-4322.50%23.53%23.01%77.55%79.31%78.43%Hg-ck-410.71%8.00%9.36%73.33%68.75%71.04%Hg-ck-2626.83%28.85%27.84%70.59%68.52%69.55%Hg-ck-8912.12%11.43%11.77%66.67%68.75%67.71%Hg-ck-17721.31%23.44%22.37%69.84%63.64%66.74%Hg-ck-18428.17%29.69%28.93%61.11%66.13%63.62%Hg-ck-4522.64%13.04%17.84%56.82%65.45%61.14%Hg-ck-9513.89%16.67%15.28%63.64%57.14%60.39%Hg-ck-12133.33%35.85%34.59%48.00%61.97%54.99%Hg-ck-4734.88%30.43%32.66%53.33%53.85%53.59%Hg-ck-7436.84%26.83%31.84%49.12%55.74%52.43%Hg-ck-6621.82%23.73%22.77%50.75%53.70%52.22%Hg-ck-1310.26%9.38%9.82%54.05%48.00%51.03%Hg-ck-6514.47%9.26%11.87%51.16%50.88%51.02%Hg-ck-14962.32%59.68%61.00%51.85%49.21%50.53%Hg-ck-16415.38%5.17%10.28%53.85%35.71%44.78%Hg-ck-3787.04%84.13%85.58%42.86%42.25%42.56%Hg-ck-9212.20%14.29%13.24%42.25%38.33%40.29%Hg-ck-14574.60%73.24%73.92%38.33%36.21%37.27%Hg-ck-146.25%16.67%11.46%31.11%37.50%34.31%Hg-ck-15.88%12.20%9.04%28.57%38.10%33.33%Hg-ck-14866.67%68.12%67.39%25.00%33.33%29.17%Hg-ck-1614.29%14.29%14.29%42.31%15.15%28.73%Hg-ck-16533.33%35.19%34.26%23.40%32.81%28.11%Hg-ck-17240.74%30.00%35.37%15.49%21.88%18.68%Hg-ck-7947.62%47.54%47.58%15.63%16.90%16.26%Hg-ck-19026.42%18.37%22.39%15.28%6.45%10.86%Hg-ck-7322.73%14.29%18.51%6.67%7.58%7.12%通过两次的杀虫效果验证，我们选取了两次验证中平均校正死亡率超过90%的菌株进行了低浓度下的杀虫效果实验，从表5可以看出，36株菌中，10uL发酵液对线虫的触杀试验导致线虫的死亡率在3.66%—85.58%之间，20uL发酵液对线虫的触杀试验导致线虫的死亡率在7.12%—97.06%之间，其中Hg-CK-153菌株发酵液在20uL和10ul的添加量下的平均校正致死率分别达到97.06%和79.00%，Hg-CK-162菌株发酵液在20uL和10ul的添加量下的平均校正致死率分别达到94.00%和70.76%，是两株潜在的根结线虫的高效生防菌。总结与展望本研究从黄瓜根际土壤中共筛选到600株可培养细菌，其中利用LB培养基分离到439株，利用PDA培养基分离到161株。由于现阶段菌株的分离挑取标准是基于菌落培养特征进行的，难免出现菌株的重复挑取的情况，后续将开展相关菌株的菌种鉴定工作。本研究保存的600株可培养细菌，不仅可以作为黄瓜根结线虫病生防菌的初筛菌株，也可以作为黄瓜等其他农作物常见病虫害的初筛菌株，是一份潜在的微生物菌种资源库。初次发酵液杀虫效力验证试验发现，从黄瓜根际土壤中筛选到的600株可培养细菌，有58株细菌的发酵液（30uL）致象耳豆根结线虫的死亡率超过60%，说明这58株细菌可能存在较好的杀虫作用。第二次发酵液杀虫效力验证试验结合第一次验证结果发现，58株细菌中，有36株细菌发酵液（30uL）致象耳豆根结线虫的死亡率超过90%，说明这36株细菌具有较强的杀虫作用。进一步的发酵液梯度杀虫效力验证试验表明，低浓度发酵液（10uL和20uL）的杀虫体系，杀虫效果整体有较大的