Pathogen Identification and Botanical Fungicides for Soft Rot Disease on Guichang Kiwifruit
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摘要:
目的 明确贵长猕猴桃软腐病致病病原菌，筛选具有防控作用的绿色植物源杀菌剂，为猕猴桃软腐病的绿色防控提供科学依据。 方法 采用组织分离法分离、纯化并结合回接试验确定病原菌。通过形态学和分子生物学对病原进行鉴定，并采用菌丝生长速率法测定6种植物源杀菌剂对病原菌的毒力。 结果 分离获得的8株有效菌株中RF2和RF2-4可引发软腐病，将该菌株的rDNA-ITS序列在NCBI上进行BLAST比对，菌株RF2和RF2-4分别与葡萄座腔菌Botryosphaeria dothidea、拟茎点霉菌Phomopsis sp.同源性达100%和99%，结合RF2和RF2-4病原菌形态特征分析，明确两株致病菌为葡萄座腔菌B.dothidea、拟茎点霉菌Phomopsis sp.。0.5%苦参碱AS对葡萄座腔菌B.dothidea和拟茎点霉菌Phomopsis sp.的EC50分别为0.442 mg·L-1和0.322 mg·L-1，0.3%丁子香酚SL的EC50则分别为0.680 mg·L-1和0.301 mg·L-1，两者毒力均高于其他植物源杀菌剂。 结论 引起贵长猕猴桃软腐病的病原菌为葡萄座腔菌B.dothidea和拟茎点霉菌Phomopsis sp.；0.5%苦参碱AS和0.3%丁子香酚SL对葡萄座腔菌B.dothidea和拟茎点霉菌Phomopsis sp.菌丝生长具有较强的抑制作用，该研究结果可为贵长猕猴桃软腐病的田间药剂防治提供筛选依据。 Abstract:Objective Pathogens that cause the soft rot disease on Guichang kiwifruit and potential botanical fungicides for the disease control were investigated. Method Suspected microbes were isolated using tissue culture and verified as the causation pathogens by means of artificial infection. The pathogens were identified morphologically and molecular biologically. In an indoor toxicity test using the mycelium growth method, 6 selected botanical fungicides were applied on the identified pathogenic isolates to determine their potencies for the disease control. Result Among the 8 isolated strains, RF2 and RF2-4 induced the soft rot disease on kiwifruits. The BLAST comparison on NCBI of the rDNA-ITS sequences of these strains suggested that RF2 was Botryosphaeria dothidea with a perfect match, and RF2-4 Phomopsis sp. with a 99% homology. The morphological analysis further confirmed the identifications. The EC50 of the organic fungicides were found for 0.5% matrine AS to be 0.442 mg·L-1 against B. dothidea and 0.322 mg·L-1 against Phomopsis sp.; and for 0.3% eugenol SL, 0.680 mg·L-1 against B. dothidea and 0.301 mg·L-1 against Phomopsis sp. The potencies were greater than other botanical fungicides tested. Conclusion B. dothidea and phomopsis sp.were positively identified as the pathogens that caused the soft rot disease on kiwifruits; and as botanical fungicides, 0.5% matrine AS and 0.3% eugenol SL seemed most effective for the disease control. -
图 1 猕猴桃软腐病病原菌
注：A为RF2菌落正面；B为RF2菌落反面；C为RF2菌丝形态；D为RF2-4菌落正面；E为RF2-4菌落反面；F为RF2-4菌丝形态。
Figure 1. Photos of kiwifruit soft rot pathogens
Note: A:front of RF2 colony; B:back of RF2 colony; C:mycelial morphology of RF2; D:front of RF2-4 colony; E:back of RF2-4 colony; F:mycelial morphology of RF2-4.
图 2 猕猴桃软腐病病原菌回接发病情况
注：CK为对照组；A为RF2未刺伤接种；B为RF2刺伤接种；C为RF2-4未刺伤接种；D为RF2-4刺伤接种。
Figure 2. Incidence of soft rot infection after inoculating isolates on kiwifruits
Note: CK:control group; A:unpunctured inoculation of RF2; B:puncture inoculation of RF2; C:unpunctured inoculation of RF2-4; D:puncture inoculation of RF2-4.
图 3 基于rDNA-ITS序列构建的菌株RF2系统发育分析
Figure 3. Phylogenetic analysis of strain RF2 based on rDNA-ITS sequence
图 4 基于rDNA-ITS序列构建的菌株RF2-4系统发育分析
Figure 4. Phylogenetic analysis of strain RF2-4 based on rDNA-ITS sequence
表 1 供试植物源杀菌剂
Table 1. Botanical fungicides tested
药剂名称
Fungicides剂型
Dosage form生产厂家
Manufacturer0.5%苦参碱0.5% matrine 水剂(AS) 山东兴禾作物科学技术有限公司 80%乙蒜素80% ethylicin 乳油(EC) 河南科邦化工有限公司 0.5%小檗碱0.5% berberine 水剂(AS) 河北万特生物化学有限公司 1%蛇床子素1% osthole 水乳剂(EW) 江苏省苏科农化有限责任公司 0.3%丁子香酚0.3% eugenol 可溶性液剂(SL) 保定市亚达化工有限公司 0.5%大黄素甲醚0.5% physcion 水剂(AS) 内蒙古清源保生物科技有限公司 23%嘧菌·噻霉酮23% azoxystrobin·benziothiazolinone 悬浮剂(SC) 陕西西大华特科技实业有限公司 注：23%嘧菌·噻霉酮为化学对照药剂。
Note: Chemical agent，23% azoxystrobin·benziothiazolinone，was used as control for comparison.
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表 2 植物源杀菌剂对RF2的质量浓度梯度
Table 2. Concentration gradient of botanical fungicides used on RF2
编号
Numbering药剂名称
Fungicides质量浓度梯度
Concentration gradient/(mg·L-1)T1 T2 T3 T4 T5 1 0.5%苦参碱0.5% matrine 0.063 0.125 0.250 0.500 1.000 2 80%乙蒜素80% ethylicin 10.000 20.000 40.000 80.000 160.000 3 0.5%小檗碱0.5% berberine 3.125 6.250 12.500 25.000 50.000 4 1%蛇床子素1% osthole 6.250 12.500 25.000 50.000 100.000 5 0.3%丁子香酚0.3% eugenol 0.188 0.375 0.750 1.500 3.000 6 0.5%大黄素甲醚0.5% physcion 3.125 6.250 12.500 25.000 50.000 7 23%嘧菌·噻霉酮23% azoxystrobin·benziothiazolinone 0.898 3.594 14.374 57.498 229.990
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表 3 植物源杀菌剂对RF2-4的质量浓度梯度
Table 3. Concentration gradient of botanical fungicides used on RF2-4
编号
Numbering药剂名称
Fungicides质量浓度梯度Concentration gradient/(mg·L-1) T1 T2 T3 T4 T5 1 0.5%苦参碱0.5% matrine 0.078 0.156 0.312 0.625 1.250 2 80%乙蒜素80% ethylicin 12.500 25.000 50.000 100.000 200.000 3 0.5%小檗碱0.5% berberine 3.125 6.250 12.500 25.000 50.000 4 1%蛇床子素1% osthole 6.250 12.500 25.000 50.000 100.000 5 0.3%丁子香酚0.3% eugenol 0.038 0.075 0.150 0.300 0.600 6 0.5%大黄素甲醚0.5% physcion 3.125 6.250 12.500 25.000 50.000 7 23%嘧菌·噻霉酮23% zoxystrobin·benziothiazolinone 0.180 0.719 2.875 11.501 46.003
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表 4 植物源杀菌剂对葡萄座腔菌的室内毒力
Table 4. Toxicities of botanical fungicides against B. dothidea
药剂名称
Fungicides回归方程
Regression equationEC50
/(mg·L-1)相关系数(R)
Correlation coefficient0.5%苦参碱AS 0.5% matrine AS Y=5.422+1.191X 0.442 0.978 0.3%丁子香酚SL 0.3% eugenol Y=5.365+2.180X 0.680 0.991 1%蛇床子素EW 1% osthole EW Y=4.201+0.628X 18.752 0.981 80%乙蒜素EC 80% ethylicin EC Y=2.065+1.522X 84.745 0.993 0.5%大黄素甲醚AS 0.5% physcion AS Y=2.858+1.086X 93.919 0.971 0.5%小檗碱AS 0.5% berberine AS Y=4.100+0.287X 1362.110 0.943 23%嘧菌·噻霉酮SC 23% zoxystrobin·benziothiazolinone SC Y=4.275+0.835X 7.384 0.946
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表 5 植物源杀菌剂对拟茎点霉菌的室内毒力
Table 5. Toxicities of botanical fungicides against Phomopsis sp.
药剂名称
Fungicides回归方程
Regression equationEC50 /(mg·L-1) 相关系数(R)
Correlation coefficient0.3%丁子香酚SL 0.3% eugenol Y=5.710+1.360X 0.301 0.997 0.5%苦参碱AS 0.5% matrine AS Y=5.925+1.882X 0.322 0.923 1%蛇床子素EW 1% osthole EW Y=3.178+1.290X 25.847 0.996 80%乙蒜素EC 80% ethylicin EC Y=3.139+1.174X 38.521 0.953 0.5%大黄素甲醚AS 0.5% physcion AS Y=4.117+0.502X 57.205 0.991 0.5%小檗碱AS 0.5% berberine AS Y=3.533+0.614X 244.928 0.991 23%嘧菌·噻霉酮SC 23% zoxystrobin·benziothiazolinone SC Y=4.377+1.002X 4.184 0.977
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