利用线虫相关真菌防治番茄根结线虫病害的研究
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摘要
根结线虫是一种严重危害农业生产的植物寄生虫,严重威胁着农业生产,特别是随着保护地蔬菜种植面积的扩大,已成为大棚蔬菜生产中的最大障碍,其生物防治也越来越受到人们的广泛重视。
本研究从根结线虫发生严重的蔬菜(黄瓜和番茄)地块采集40份样品。在根和根围土两个不同生境的根结线虫雌虫和卵中分离到537株真菌,测定菌株的产蛋白酶和几丁质酶的能力。结果表明,共有269株真菌能够产生蛋白酶,透明圈直径与菌落直径的比值为1.05-2.52；148株具有几丁质酶活,酶活为0.13-0.55 U/min ml,其中20株菌株蛋白酶活高于2.00和几丁质酶活性高于0.2 U/min ml.选取这20株高产蛋白酶和几丁质酶菌株进行温室试验,对主要由南方根结线虫(Meloidogyne incognita Kofoid & White)引起的番茄根结线虫病的相对防效为29.0%-58.4%,促生作用为0.4-25.6%。同时测定它们对南方根结线虫的二龄幼虫(J2)死亡率、卵寄生率和孵化率。我们通过相关性分析,研究发现,20株寄生菌株的温室防效与蛋白酶产量、几丁质酶活具有明显的相关性(r=0.82,0.80)；与J2死亡率也有较高的相关性(r=0.76),但是与寄生率和卵孵化率相关性较低(r=0.51,0.52)。由此推测真菌胞外蛋白酶或几丁质酶可作为筛选根结线虫根结线虫颉颃真菌的重要指标。
选择淡紫拟青霉XT18、XCS46和厚垣孢轮枝菌XT124,研究它们在田间(江苏省宿迁)条件下对番茄根结线虫病的防病增产效果及其对根围生态的影响。2007年,它们的相对防效分别为60.7%、51.5%和48.1%；番茄相对增产率分别为62.0%、43.1%和40.4%。2008年的相对防效分别为67.8%、57.3%和53.2%；番茄相对增产率分别为72.1%、54.5%和51.9%。两年的试验结果均表明,不论在防效还是在增产方面,三株菌均优于阿维菌素。同时,两年的试验结果还显示,番茄移栽40 d后(第二次生防菌灌根处理10d后),采用平板检测法检测微生物的菌落数,三株生防菌施入土壤后能够显著地影响根围土壤中微生物的生存,成为优势种群,三个菌株的数量均能达到≥105 CFU/g根围样品,与对照相比,明显的降低根围其他真菌、细菌和放线菌存在的数量。
为了解防御酶系是否与淡紫拟青霉XT18、XCS46和厚垣孢轮枝菌XT124的生防作用相关,本文将它们的孢子悬浮液处理番茄叶片,对照用灭菌水处理；移栽3d后,接种M. incognita,检测接种0、1、2、3、4、5、6、和7d后,植株体内苯丙氨酸解氨酶PAL (Phenylalanine Ammonia-Lyase)、多酚氧化酶PPO (polyphenoloxidase)、过氧化物酶POD (Peroxidase)、超氧化物歧化酶SOD (Superoxide Dismutase)的活性变化。结果表明,与单独用M. incognita处理的对照2和用无菌水处理的对照1相比,XT18、XCS46和XT124处理提高了番茄体内PAL、PPO、POD与SOD酶的活性。四种酶的活性高峰分别出现在生防菌处理后4、5、2、3d。该研究结果显示相关防御酶酶活性的增加与生防菌XT18、XCS46和XT124诱导植物抗病性有关。
研究了淡紫拟青霉菌XT18在不同培养液、温度、离心率条件下的的生长特性(菌丝干重、孢子量)及其上清液对M. incognita的J2的相对致死率。结果表明,不同培养条件对该菌株的生长及其上清液对J2的致死作用都有明显影响。在玉米和马铃薯培养液中培养8d后,菌丝量分别达到672.5mg/100ml和635.4mg/100ml;该菌株在马铃薯和查氏培养液中产孢量较高,分别能够达到34.8×107spores/ml和34.6×107spores/ml;且在察氏培养液中的上清液对J2致死率较高,达到67.2%；25℃为菌体生长和孢子产生的最合适温度,而在29℃C培养时,上清液对J2的致死率最高；菌丝生长与孢子生成的最佳摇床离心率为13g,该条件下培养10 d的上清液对J2的相对致死率达到51.4%。
Root-knot nematodes(Meloidogyne spp.) are a group of important pathogens on various crops, causing the severe damage to the crop production. Especially with the increasing of cultivated area of greenhouse vegetables, root-knot nematodes have become the biggest obstacle to the production of greenhouse vegetables. In this background, the biological control for this disease has drawn more and more attention recently.
In this study, we isolated 537 fungal strains from females and eggs of Meloidogyne spp. collected from plant roots and infested soil in Jiangsu, China, and examined their in vitro protease productions and chitinase activities. Then we selected 20 strains, each with a protease production higher than 2.00 (protease production was measured as the ratio of diameters of the zone of clearing to that of the colony) and a chitinase activity higher than 0.2 U/min ml. For these strains, we examined their egg parasitic rates, mortality rates of stage 2 juvenile (J2), egg hatching rates and evaluated their biocontrol efficacies under greenhouse conditions. The biocontrol efficacies of these 20 potential biocontrol fungal strains ranged from 29.0%to 58.4%.The actual biocontrol efficacy against Meloidogyne root-knot was highly correlated with the in vitro protease production (r= 0.82), chitinase activity (r= 0.80), and the mortality of J2 (r= 0.76), but poorly correlated with the egg parasitic rate (r= 0.51) and the egg hatch rate (r= 0.52).Based on these results, we suggested that in vitro protease productions or chitinase activities of the fungal strains could be an applicable indicator for assessing biocontrol efficacy against nematodes.
We screened out three biocontrol strains of Paecilomyces lilacinus XT18, XCS46 and Pochonia chlamydosporia XT124 and determined their biocontrol efficacy against root-knot disease, the yield increasement and the effect on diversity of rizosphere in the field trial in Suqian of Jiangsu Province. In 2007, the results indicated that biocontrol efficacies of XT18, XCS46 and XT12 were 60.7%,51.5%, and 48.1% respectively. And the yield increasements of these three strains were 62.0%,43.1%, and 40.4%, respectively. In 2008, the results showed that biocontrol efficacies of XT18, XCS46 and XT124 were 72.1%,54.5% and 51.9%, respectively. Either the biocontrol efficiency or the yield increasement of them was better than that of abamectin in the two years. The results of the two years also showed that after 40 d of transplanting the tomatoes (Secend treated with antagonist spore suspensions of XT18、XCS46 and XT124)), they significantly affected the micro-ecological of rizosphere after treatment and became the dominant species in all the habitats, and the number of each strain was above 105 CFU/g sampled soils by detecting the number of isolates on plates, and they could reduce the quantity of other fungi, bacteria and actinomycetes compared with the control.
In order to investigate whether the antioxidant enzymes were associated with the biocontrol mechanisms in Paecilomyces lilacinus XT18, XCS46 and Pochonia chlamydosporia XT124, the roots of the tomatoes were treated with the spore suspensions of XT18, XCS46 and XT124, and 3 d after transplanting, the tomatoes were inoculated with M. incognita, the activities of Phenylalanine Ammonia-Lyase (PAL) polyphenoloxidase (PPO), Peroxidase (POD) and Superoxide Dismutase (SOD) were measured 0,1,2,3,4,5,6,7 d after inoculation. The results showed that XT18, XCS46 and XT124 significantly increased the activities of PAL, PPO, POD and SOD, when compared to the control 1 (the tomatoes were only inoculated with M. incognita) and control 2 (the roots of the tomatoes were only treated with sterile water). The highest activites of these four enzymes appeared 4,5,2, and 3 d after inoculation, respectively. These results indicated that the increased activities of antioxidant enzymes might be correlated with the induced resistance in the tomatoes to M. incognita.
The growth characteristics of Paecilomyces lilacinus XT18(mycelial growth and spore production) under different media, temperatures, shaker's eccentricity were studied. The results showed that different cultured conditions would affect the growth of XT 18 and the activity of its supernatant which could cause death to the J2 of M. incognita. The dry weights of the mycelia were 672.5 mg/100 ml and 635.4 mg/100 ml, when cultured in the maize and potato liquid media for 8 d, respectively. The spore productions were higher in potato and Czapek liquid media and were 34.8×107spores/ml and 34.6x107spores/ml, respectively. And the activity of the supernatant in Czapek liquid media was highest against the J2 of M. incognita.The results also showed that 25℃was the most suitable temperature for the mycelial growth and spore production of XT 18 and when cultured under 29℃, the supernatant exhibited greatest activity against the M.incognita isolate J2. The best shaker's eccentricity for the mycelial growth and spore production of XT 18 was 110 rpm, and the mortility was 51.4% when the J2 of M. incognita was treated with the 10 d-cultured supernatant under this eccentricity.
本研究从根结线虫发生严重的蔬菜(黄瓜和番茄)地块采集40份样品。在根和根围土两个不同生境的根结线虫雌虫和卵中分离到537株真菌,测定菌株的产蛋白酶和几丁质酶的能力。结果表明,共有269株真菌能够产生蛋白酶,透明圈直径与菌落直径的比值为1.05-2.52；148株具有几丁质酶活,酶活为0.13-0.55 U/min ml,其中20株菌株蛋白酶活高于2.00和几丁质酶活性高于0.2 U/min ml.选取这20株高产蛋白酶和几丁质酶菌株进行温室试验,对主要由南方根结线虫(Meloidogyne incognita Kofoid & White)引起的番茄根结线虫病的相对防效为29.0%-58.4%,促生作用为0.4-25.6%。同时测定它们对南方根结线虫的二龄幼虫(J2)死亡率、卵寄生率和孵化率。我们通过相关性分析,研究发现,20株寄生菌株的温室防效与蛋白酶产量、几丁质酶活具有明显的相关性(r=0.82,0.80)；与J2死亡率也有较高的相关性(r=0.76),但是与寄生率和卵孵化率相关性较低(r=0.51,0.52)。由此推测真菌胞外蛋白酶或几丁质酶可作为筛选根结线虫根结线虫颉颃真菌的重要指标。
选择淡紫拟青霉XT18、XCS46和厚垣孢轮枝菌XT124,研究它们在田间(江苏省宿迁)条件下对番茄根结线虫病的防病增产效果及其对根围生态的影响。2007年,它们的相对防效分别为60.7%、51.5%和48.1%；番茄相对增产率分别为62.0%、43.1%和40.4%。2008年的相对防效分别为67.8%、57.3%和53.2%；番茄相对增产率分别为72.1%、54.5%和51.9%。两年的试验结果均表明,不论在防效还是在增产方面,三株菌均优于阿维菌素。同时,两年的试验结果还显示,番茄移栽40 d后(第二次生防菌灌根处理10d后),采用平板检测法检测微生物的菌落数,三株生防菌施入土壤后能够显著地影响根围土壤中微生物的生存,成为优势种群,三个菌株的数量均能达到≥105 CFU/g根围样品,与对照相比,明显的降低根围其他真菌、细菌和放线菌存在的数量。
为了解防御酶系是否与淡紫拟青霉XT18、XCS46和厚垣孢轮枝菌XT124的生防作用相关,本文将它们的孢子悬浮液处理番茄叶片,对照用灭菌水处理；移栽3d后,接种M. incognita,检测接种0、1、2、3、4、5、6、和7d后,植株体内苯丙氨酸解氨酶PAL (Phenylalanine Ammonia-Lyase)、多酚氧化酶PPO (polyphenoloxidase)、过氧化物酶POD (Peroxidase)、超氧化物歧化酶SOD (Superoxide Dismutase)的活性变化。结果表明,与单独用M. incognita处理的对照2和用无菌水处理的对照1相比,XT18、XCS46和XT124处理提高了番茄体内PAL、PPO、POD与SOD酶的活性。四种酶的活性高峰分别出现在生防菌处理后4、5、2、3d。该研究结果显示相关防御酶酶活性的增加与生防菌XT18、XCS46和XT124诱导植物抗病性有关。
研究了淡紫拟青霉菌XT18在不同培养液、温度、离心率条件下的的生长特性(菌丝干重、孢子量)及其上清液对M. incognita的J2的相对致死率。结果表明,不同培养条件对该菌株的生长及其上清液对J2的致死作用都有明显影响。在玉米和马铃薯培养液中培养8d后,菌丝量分别达到672.5mg/100ml和635.4mg/100ml;该菌株在马铃薯和查氏培养液中产孢量较高,分别能够达到34.8×107spores/ml和34.6×107spores/ml;且在察氏培养液中的上清液对J2致死率较高,达到67.2%；25℃为菌体生长和孢子产生的最合适温度,而在29℃C培养时,上清液对J2的致死率最高；菌丝生长与孢子生成的最佳摇床离心率为13g,该条件下培养10 d的上清液对J2的相对致死率达到51.4%。
Root-knot nematodes(Meloidogyne spp.) are a group of important pathogens on various crops, causing the severe damage to the crop production. Especially with the increasing of cultivated area of greenhouse vegetables, root-knot nematodes have become the biggest obstacle to the production of greenhouse vegetables. In this background, the biological control for this disease has drawn more and more attention recently.
In this study, we isolated 537 fungal strains from females and eggs of Meloidogyne spp. collected from plant roots and infested soil in Jiangsu, China, and examined their in vitro protease productions and chitinase activities. Then we selected 20 strains, each with a protease production higher than 2.00 (protease production was measured as the ratio of diameters of the zone of clearing to that of the colony) and a chitinase activity higher than 0.2 U/min ml. For these strains, we examined their egg parasitic rates, mortality rates of stage 2 juvenile (J2), egg hatching rates and evaluated their biocontrol efficacies under greenhouse conditions. The biocontrol efficacies of these 20 potential biocontrol fungal strains ranged from 29.0%to 58.4%.The actual biocontrol efficacy against Meloidogyne root-knot was highly correlated with the in vitro protease production (r= 0.82), chitinase activity (r= 0.80), and the mortality of J2 (r= 0.76), but poorly correlated with the egg parasitic rate (r= 0.51) and the egg hatch rate (r= 0.52).Based on these results, we suggested that in vitro protease productions or chitinase activities of the fungal strains could be an applicable indicator for assessing biocontrol efficacy against nematodes.
We screened out three biocontrol strains of Paecilomyces lilacinus XT18, XCS46 and Pochonia chlamydosporia XT124 and determined their biocontrol efficacy against root-knot disease, the yield increasement and the effect on diversity of rizosphere in the field trial in Suqian of Jiangsu Province. In 2007, the results indicated that biocontrol efficacies of XT18, XCS46 and XT12 were 60.7%,51.5%, and 48.1% respectively. And the yield increasements of these three strains were 62.0%,43.1%, and 40.4%, respectively. In 2008, the results showed that biocontrol efficacies of XT18, XCS46 and XT124 were 72.1%,54.5% and 51.9%, respectively. Either the biocontrol efficiency or the yield increasement of them was better than that of abamectin in the two years. The results of the two years also showed that after 40 d of transplanting the tomatoes (Secend treated with antagonist spore suspensions of XT18、XCS46 and XT124)), they significantly affected the micro-ecological of rizosphere after treatment and became the dominant species in all the habitats, and the number of each strain was above 105 CFU/g sampled soils by detecting the number of isolates on plates, and they could reduce the quantity of other fungi, bacteria and actinomycetes compared with the control.
In order to investigate whether the antioxidant enzymes were associated with the biocontrol mechanisms in Paecilomyces lilacinus XT18, XCS46 and Pochonia chlamydosporia XT124, the roots of the tomatoes were treated with the spore suspensions of XT18, XCS46 and XT124, and 3 d after transplanting, the tomatoes were inoculated with M. incognita, the activities of Phenylalanine Ammonia-Lyase (PAL) polyphenoloxidase (PPO), Peroxidase (POD) and Superoxide Dismutase (SOD) were measured 0,1,2,3,4,5,6,7 d after inoculation. The results showed that XT18, XCS46 and XT124 significantly increased the activities of PAL, PPO, POD and SOD, when compared to the control 1 (the tomatoes were only inoculated with M. incognita) and control 2 (the roots of the tomatoes were only treated with sterile water). The highest activites of these four enzymes appeared 4,5,2, and 3 d after inoculation, respectively. These results indicated that the increased activities of antioxidant enzymes might be correlated with the induced resistance in the tomatoes to M. incognita.
The growth characteristics of Paecilomyces lilacinus XT18(mycelial growth and spore production) under different media, temperatures, shaker's eccentricity were studied. The results showed that different cultured conditions would affect the growth of XT 18 and the activity of its supernatant which could cause death to the J2 of M. incognita. The dry weights of the mycelia were 672.5 mg/100 ml and 635.4 mg/100 ml, when cultured in the maize and potato liquid media for 8 d, respectively. The spore productions were higher in potato and Czapek liquid media and were 34.8×107spores/ml and 34.6x107spores/ml, respectively. And the activity of the supernatant in Czapek liquid media was highest against the J2 of M. incognita.The results also showed that 25℃was the most suitable temperature for the mycelial growth and spore production of XT 18 and when cultured under 29℃, the supernatant exhibited greatest activity against the M.incognita isolate J2. The best shaker's eccentricity for the mycelial growth and spore production of XT 18 was 110 rpm, and the mortility was 51.4% when the J2 of M. incognita was treated with the 10 d-cultured supernatant under this eccentricity.
引文
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