Pathogen Identification and Botanical Fungicides for Soft Rot Disease on Guichang Kiwifruit
-
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.
下载: 导出CSV
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
下载: 导出CSV
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
下载: 导出CSV
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
下载: 导出CSV
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
下载: 导出CSV
-
[1] 马松涛, 宋晓斌, 张学武, 等.猕猴桃花果病害研究现状及趋势[J].西北林学院学报, 2000, 15(3):86-90. doi: 10.3969/j.issn.1001-7461.2000.03.019MA S T, SONG X B, ZHANG X W, et al. Present situation and trend of the researches on flower and fruit diseases of kiwifruit[J]. Journal of Northwest Forestry University, 2000, 15(3): 86-90.(in Chinese) doi: 10.3969/j.issn.1001-7461.2000.03.019 [2] 韩礼星, 黄贞光, 李明, 等.加入WTO后我国猕猴桃产业的发展策略[J].果树学报, 2003, 20(3):218-223. http://d.old.wanfangdata.com.cn/Periodical/gskx200303016HAN L X, HUANG Z G, LI M, et al. On the strategies for development of kiwifruit industry in China after accessed to WTO[J]. Journal of Fruit Science, 2003, 20(3): 218-223.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/gskx200303016 [3] HAWTHORNE B T, REES-GEORGE J, SAMUELS G J. Fungi associated with leaf spots and post-harvest fruit rots of kiwifruit (Actinidia chinensis) in New Zealand[J]. New Zealand Journal of Botany, 1982, 20(2):143-150. doi: 10.1080/0028825X.1982.10428835 [4] 丁爱冬, 于梁, 石蕴莲.猕猴桃采后病害鉴定和侵染规律研究[J].植物病理学报, 1995(2):149-153. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=QK199500674990DING A D, YU L, SHI Y L. Studies on the identification of pathogenic fungi and their ways of post-harvest rot disease infection on kiwifruit[J]. Acta Phytopathologica Sinica, 1995 (2): 149-153.(in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=QK199500674990 [5] 张承, 李明, 龙友华, 等.采前喷施壳聚糖复合膜对猕猴桃软腐病的防控及其保鲜作用[J].食品科学, 2016, 37(22):274-281. doi: 10.7506/spkx1002-6630-201622042ZHANG C, LI M, LONG Y H, et al. Control of soft rot in kiwifruit by pre-harvest application of chitosan composite coating and its effect on preserving and improving kiwifruit quality[J]. Food Science, 2016, 37(22): 274-281.(in Chinese) doi: 10.7506/spkx1002-6630-201622042 [6] 黎晓茜, 曾彬, 尹显慧, 等.修文县猕猴桃腐烂病病原鉴定及防治药剂筛选[J].中国南方果树, 2016, 45(5):101-104. http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201605026LI X X, ZENG B, YIN X H, et al. Identification of pathogenic fungi causing fruit rot of kiwifruit in Xiuwen county and the toxicity assay of fungicites[J]. South China Fruits, 2016, 45(5): 101-104.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201605026 [7] 吴文能, 张起, 雷霁卿, 等.贵长猕猴桃软腐病病原菌分离鉴定及抑菌药剂筛选[J].北方园艺, 2018(16):47-54. http://d.old.wanfangdata.com.cn/Periodical/bfyany201816008WU W N, ZHANG Q, LEI J Q, et al. Identification and pharmaceutical screening of kiwifruit soft rot disease on 'Guichang' gooseberry[J]. Northern Horticulture, 2018 (16): 47-54.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/bfyany201816008 [8] 潘慧, 胡秋舲, 张胜菊, 等.贵州六盘水市猕猴桃病害调查及病原鉴定[J].植物保护, 2018, 44(4):125-131. http://d.old.wanfangdata.com.cn/Periodical/zwbh201804023PAN H, HU Q L, ZHANG S J. et al. Kiwifruit disease investigation and pathogen identification in liupanshui city, Guizhou province[J]. Plant Protection, 2018, 44(4): 125-131.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zwbh201804023 [9] 李诚, 蒋军喜, 冷建华, 等. 6种杀菌剂对猕猴桃主要腐烂病菌的室内毒力测定[J].中国南方果树, 2012, 41(1):27-29. http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201201007LI C, JIANG J X, LENG J H, et al. Indoor toxicity test of six fungicides to main pathogenic fungi causing fruit rot of kiwifruit[J]. South China Fruits, 2012, 41(1): 27-29.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201201007 [10] 刘达富, 王井田, 陆雪峰, 等.猕猴桃果实腐烂病田间药效试验[J].浙江林业科技, 2015, 35(2):70-73. doi: 10.3969/j.issn.1001-3776.2015.02.016LIU D F, WANG J T, LU X F, et al. Field experiment on fungicides on fruit rot of actinidia chinensis[J]. Journal of Zhejiang Forestry Science and Technology, 2015, 35(2): 70-73.(in Chinese) doi: 10.3969/j.issn.1001-3776.2015.02.016 [11] 王小洁, 李士谣, 李亚巍, 等.猕猴桃软腐病病原菌的分离鉴定及其防治药剂筛选[J].植物保护学报, 2017, 44(5):826-832. http://d.old.wanfangdata.com.cn/Periodical/zwbhxb201705016WANG X J, LI S Y, LI Y W, et al. Pathogen identification of kiwifruit soft rot and fungicide screening for control of the disease[J]. Journal of Plant Protection, 2017, 4(5): 826-832.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zwbhxb201705016 [12] 李诚, 蒋军喜, 冷建华, 等.猕猴桃枝枯病病原菌鉴定[J].北方园艺, 2013(24):130-133. http://d.old.wanfangdata.com.cn/Periodical/bfyany201324038LI C, JIANG J X, LENG J H, et al. Identification of the pathogen causing shoot blight of Kiwifruit[J]. Northern Horticulture, 2013(24): 130-133.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/bfyany201324038 [13] 凌丹燕.蓝莓主要真菌病害的分离鉴定与防治研究[D].金华: 浙江师范大学, 2016.LING D Y. The study of isolation and identification of major fungal diseases of blueberry and disease control[D]. Jinhua: Zhejiang Normal University, 2016.(in Chinese) [14] 何军, 马志卿, 张兴.植物源农药概述[J].西北农林科技大学学报(自然科学版), 2006, 34(9):79-85. doi: 10.3321/j.issn:1671-9387.2006.09.017HE J, MA Z Q, ZHANG X. Review of botanical pesticide[J]. Journal of Northwest A & F University(Natural Science Edition), 2006, 34(9): 79-85.(in Chinese) doi: 10.3321/j.issn:1671-9387.2006.09.017 [15] 陈昂.五种植物源农药的环境毒性评价[D].长沙: 中南大学, 2010.CHEN A. Environmental toxicity evaluation of five botanical pesticides[D]. Changsha: Central South University, 2010.(in Chinese) [16] 汤丽梅, 周清, 吴尚, 等.大蒜素对猕猴桃果实致病菌的抑制活性及贮藏防效研究[J].中国南方果树, 2014, 43(4):15-18. http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201404004TONG L M, ZHOU Q, WU S, et al. Study on inhibitory activity of allicin to pathogens of kiwifruit and its efficacy on fruit storage[J]. South China Fruits, 2014, 43(4): 15-18.(in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgnfgs201404004 [17] 王强, 冯岩, 何小静, 等.植物源杀菌剂的筛选[J].福建农业学报, 2012, 27(11):1246-1249. doi: 10.3969/j.issn.1008-0384.2012.11.019WANG Q, FENG Y, HE X J, et al. Screening of the botanical fungicides[J]. Fujian Journal of Agricultural Sciences, 2012, 27(11): 1246-1249.(in Chinese) doi: 10.3969/j.issn.1008-0384.2012.11.019 [18] 周军, 赵杰.植物性杀菌剂对桃炭疽病菌和桃果腐病菌的毒力测定[J].上海农业科技, 2018(5):113. doi: 10.3969/j.issn.1001-0106.2018.05.055ZHOU J, ZHAO J. Toxicity determination of botanical fungicides to anthracnose and fruit rot on Peach[J]. Shanghai Agricultural Science and Technology, 2018 (5): 113.(in Chinese) doi: 10.3969/j.issn.1001-0106.2018.05.055 -
点击查看大图
图(4) / 表(5)
计量
- 文章访问数: 1454
- HTML全文浏览量: 266
- PDF下载量: 34
- 被引次数: 0
