黑附球菌XF_1菌株对马铃薯晚疫病的生防作用
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摘要
黑附球菌(Epicoccum nigrum)XF1菌株是由本课题组筛选出的一株对致病疫霉(Phytophthora infestans)有较强拮抗作用的生防真菌,本论文旨在研究其对P. infestans的抑制作用方式和对马铃薯晚疫病的防治效果,并初步探索固体发酵条件,为其将来应用于马铃薯晚疫病的生物防治奠定基础。主要内容如下:
采用玻片对峙法研究了XF1菌丝与P. infestans菌丝的相互作用,显微观察显示,对峙区域内P. infestans菌丝内原生质发生割裂、凝集,并出现畸形菌丝,而XF1菌丝无明显变化。运用纤维素膜法,证明了E. nigrum分生孢子萌发生长48 h后所产生的通过纤维素膜(截留分子量8000-14000)渗透并积累到黑麦培养基中的胞外分泌物,对P. infestans孢子囊直接萌发的抑制率达到90%以上,对其休止孢萌发抑制率则达100%。通过麦粒固体发酵所得到的XF1无菌发酵滤液对P. infestans游动孢子的释放具有较强的抑制作用,释放率仅为0.36%;对其休止胞萌发和休止胞芽管的延伸抑制率分别为50%和75%。以上结果都说明,XF1生长过程中能分泌一些强烈抑制P. infestans的物质,即其抑制作用方式主要表现为拮抗。
离体叶片试验结果表明,室内条件下浓度为1.0×107个/mL和1.0×108个/mL的XF1分生孢子悬浮液对马铃薯晚疫病的预防作用,与0.2%可杀得无显著性差异。温室条件下,将XF1分生孢子水悬浮液(浓度为1.0×107个/mL)在不同时间喷施于马铃薯植株的叶片上,到达处理时间后,剪下叶片带回室内接种P. infestans。结果显示,接种前1 h喷施XF1对马铃薯晚疫病的防效最高,达到88.89%;接种前12 h、24 h、48 h和96 h喷施的防效逐渐降低,但均在65%以上;接种P. infestans前检查发现,不同时间喷施于马铃薯叶片上的XF1分生孢子几乎未萌发,萌发率均低于1%,然而接种P. infestans后保湿4 d再次检查XF1分生孢子的萌发情况表明,接种前1 h喷施的XF1分生孢子萌发率最高,为45.27%,显著高于接种前48 h(萌发率为23.56%)和96 h(萌发率为19.26%)喷施的处理。此外,XF1分生孢子灭活后(121℃,20 min)即失去对马铃薯晚疫病的防治效果。因此说明,只有活的XF1分生孢子才对马铃薯晚疫病具有防治作用,而且其喷施于马铃薯植株后的存活状况(萌发能力)是其对晚疫病防治效果的决定因素。室内试验结果显示,XF1分生孢子经紫外灯照射24 h后的萌发率显著降低,由此表明,XF1分生孢子喷施于马铃薯植株后,影响其存活能力和对晚疫病防治效果的重要因素之一可能为紫外线。
以黑麦和小麦粒为固体基质,进行了XF1的固体发酵,结果表明,在培养40 d时,以黑麦为发酵基质的产孢量较高,然而两种基质所得到的分生孢子活力均较高,萌发率均在90%以上。
在本研究的基础上,对XF1应用于马铃薯晚疫病生物防治的前景和存在问题进行了讨论。
Epicoccum nigrum, strain XF1 was collected as a potential biological control agent for the late blight of potatoes caused by Phytophthora infestans. This study focused on the inhibition and control effect of XF1 against P. infestans in vitro and in vivo, and preliminarily tested the solid-state fermentation conditions of XF1 as well.
The interaction between XF1 and P. infestans mycelia were studied by means of the dual culture on slides. Results showed that the mycelia growth of P. infestans was inhibited by XF1 evidently, microscopic vision indicated the hyphae of P. infestans became stunted and distorted adjacent to the boundary of XF1 colony, and the protoplasm of P. infestans mycelia was degenerated in the inhibition zone. However, the hyphae of XF1 had no significant difference compared with normal ones. After 48 h incubation of conidia of XF1 on the membrane (molecular weight cut off 8000-14000) with RA (Rye agar) underneath, the extra cellular substances secreted by XF1 and penetrated into the RA through the membrane can significantly inhibit the direct germination of sporangia and cystspores of P. infestans, with inhibitory rate of 90% and 100%, respectively. Releasing of zoospores from sporangia of P. infestans was inhibited intensively by the conidia free filtrates of solid fermentation products of XF1 with wheat grains, the release rate was only 0.36%, so did on cystospore germination and germ tube elongation, with inhibitory ratio was 50% and 75%, respectively. Those results showed that the substances that intensely inhibited P. infestans can be produced by XF1 during growth, the action mode for inhibition, in other words, mainly relyed on antagonism.
Results of detached leaves test showed that conidia suspension of XF1 with concentration of 1.0×107 conidia/mL and 1.0×108 conidia/mL could provide the same control effect on potato late blight with 0.2% Kocide DF under indoor conditions. The surface of selected leaves of potato cultivar in greenhouse were sprayed with conidia suspension of XF1 at different time and then detached and brought into lab inoculated with P. infestans. And it was showed that control effect decreased one by one of 1, 12, 24, 48 and 96 hours sprayed before inoculation. The conidia of XF1 did not germinate before inoculation, with germination ratio were less than 1%. However, after inoculation and incubated under wetness for 4 hours, germination of conidia of XF1 were promoted, the highest germination rate, showed by the treatment of 1 hour spray before inoculation, was 45.27%, which significantly higher than the treatments of 48 and 96 hours spray before inoculation (with germination rate were 23.56% and 19.26, respectively). In addition, conidia of XF1 loosed control effect against potato late blight after sterilize (121℃, 20 min). Therefore, it was indicated that only viable conidia of XF1 can have control effect against potato late blight, and the survival status (germination ability) play the critical role on its control effect against potato late blight. Lab test showed that the germination rate of conidia of XF1 would significantly decrease after irradiated by ultraviolet light (UV) for 24 h, which suggests that after spraying of conidia of XF1 on potato plants, UV may be one of the important factors on their viability and control effect on potato late blight.
Production of conidia of XF1 was tested in solid-state fermentation with rye and wheat grains. It was showed that the highest conidia production obtained after 40 days fermentation with rye. While the conidia of XF1 have high viability from both of the two grains, germination ratio were higher than 90%.
Based on this study, the prospects and problems of applying the XF1 into biocontrol of potato late blight on field were discussed.
采用玻片对峙法研究了XF1菌丝与P. infestans菌丝的相互作用,显微观察显示,对峙区域内P. infestans菌丝内原生质发生割裂、凝集,并出现畸形菌丝,而XF1菌丝无明显变化。运用纤维素膜法,证明了E. nigrum分生孢子萌发生长48 h后所产生的通过纤维素膜(截留分子量8000-14000)渗透并积累到黑麦培养基中的胞外分泌物,对P. infestans孢子囊直接萌发的抑制率达到90%以上,对其休止孢萌发抑制率则达100%。通过麦粒固体发酵所得到的XF1无菌发酵滤液对P. infestans游动孢子的释放具有较强的抑制作用,释放率仅为0.36%;对其休止胞萌发和休止胞芽管的延伸抑制率分别为50%和75%。以上结果都说明,XF1生长过程中能分泌一些强烈抑制P. infestans的物质,即其抑制作用方式主要表现为拮抗。
离体叶片试验结果表明,室内条件下浓度为1.0×107个/mL和1.0×108个/mL的XF1分生孢子悬浮液对马铃薯晚疫病的预防作用,与0.2%可杀得无显著性差异。温室条件下,将XF1分生孢子水悬浮液(浓度为1.0×107个/mL)在不同时间喷施于马铃薯植株的叶片上,到达处理时间后,剪下叶片带回室内接种P. infestans。结果显示,接种前1 h喷施XF1对马铃薯晚疫病的防效最高,达到88.89%;接种前12 h、24 h、48 h和96 h喷施的防效逐渐降低,但均在65%以上;接种P. infestans前检查发现,不同时间喷施于马铃薯叶片上的XF1分生孢子几乎未萌发,萌发率均低于1%,然而接种P. infestans后保湿4 d再次检查XF1分生孢子的萌发情况表明,接种前1 h喷施的XF1分生孢子萌发率最高,为45.27%,显著高于接种前48 h(萌发率为23.56%)和96 h(萌发率为19.26%)喷施的处理。此外,XF1分生孢子灭活后(121℃,20 min)即失去对马铃薯晚疫病的防治效果。因此说明,只有活的XF1分生孢子才对马铃薯晚疫病具有防治作用,而且其喷施于马铃薯植株后的存活状况(萌发能力)是其对晚疫病防治效果的决定因素。室内试验结果显示,XF1分生孢子经紫外灯照射24 h后的萌发率显著降低,由此表明,XF1分生孢子喷施于马铃薯植株后,影响其存活能力和对晚疫病防治效果的重要因素之一可能为紫外线。
以黑麦和小麦粒为固体基质,进行了XF1的固体发酵,结果表明,在培养40 d时,以黑麦为发酵基质的产孢量较高,然而两种基质所得到的分生孢子活力均较高,萌发率均在90%以上。
在本研究的基础上,对XF1应用于马铃薯晚疫病生物防治的前景和存在问题进行了讨论。
Epicoccum nigrum, strain XF1 was collected as a potential biological control agent for the late blight of potatoes caused by Phytophthora infestans. This study focused on the inhibition and control effect of XF1 against P. infestans in vitro and in vivo, and preliminarily tested the solid-state fermentation conditions of XF1 as well.
The interaction between XF1 and P. infestans mycelia were studied by means of the dual culture on slides. Results showed that the mycelia growth of P. infestans was inhibited by XF1 evidently, microscopic vision indicated the hyphae of P. infestans became stunted and distorted adjacent to the boundary of XF1 colony, and the protoplasm of P. infestans mycelia was degenerated in the inhibition zone. However, the hyphae of XF1 had no significant difference compared with normal ones. After 48 h incubation of conidia of XF1 on the membrane (molecular weight cut off 8000-14000) with RA (Rye agar) underneath, the extra cellular substances secreted by XF1 and penetrated into the RA through the membrane can significantly inhibit the direct germination of sporangia and cystspores of P. infestans, with inhibitory rate of 90% and 100%, respectively. Releasing of zoospores from sporangia of P. infestans was inhibited intensively by the conidia free filtrates of solid fermentation products of XF1 with wheat grains, the release rate was only 0.36%, so did on cystospore germination and germ tube elongation, with inhibitory ratio was 50% and 75%, respectively. Those results showed that the substances that intensely inhibited P. infestans can be produced by XF1 during growth, the action mode for inhibition, in other words, mainly relyed on antagonism.
Results of detached leaves test showed that conidia suspension of XF1 with concentration of 1.0×107 conidia/mL and 1.0×108 conidia/mL could provide the same control effect on potato late blight with 0.2% Kocide DF under indoor conditions. The surface of selected leaves of potato cultivar in greenhouse were sprayed with conidia suspension of XF1 at different time and then detached and brought into lab inoculated with P. infestans. And it was showed that control effect decreased one by one of 1, 12, 24, 48 and 96 hours sprayed before inoculation. The conidia of XF1 did not germinate before inoculation, with germination ratio were less than 1%. However, after inoculation and incubated under wetness for 4 hours, germination of conidia of XF1 were promoted, the highest germination rate, showed by the treatment of 1 hour spray before inoculation, was 45.27%, which significantly higher than the treatments of 48 and 96 hours spray before inoculation (with germination rate were 23.56% and 19.26, respectively). In addition, conidia of XF1 loosed control effect against potato late blight after sterilize (121℃, 20 min). Therefore, it was indicated that only viable conidia of XF1 can have control effect against potato late blight, and the survival status (germination ability) play the critical role on its control effect against potato late blight. Lab test showed that the germination rate of conidia of XF1 would significantly decrease after irradiated by ultraviolet light (UV) for 24 h, which suggests that after spraying of conidia of XF1 on potato plants, UV may be one of the important factors on their viability and control effect on potato late blight.
Production of conidia of XF1 was tested in solid-state fermentation with rye and wheat grains. It was showed that the highest conidia production obtained after 40 days fermentation with rye. While the conidia of XF1 have high viability from both of the two grains, germination ratio were higher than 90%.
Based on this study, the prospects and problems of applying the XF1 into biocontrol of potato late blight on field were discussed.
引文
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