Identification, Biological characteristics, and Fungicides screening of the Pathogen causing Leaf spot disease on Pittosporum tobira
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摘要:
目的 明确引起重庆市万州区海桐叶斑病的病原及其生物学特性，并筛选出有效防治药剂。 方法 采用组织分离法从具有典型症状的海桐叶片分离病原菌，进行致病性测定，采用形态学和分子生物学方法对病原菌进行鉴定，用菌丝生长速率法进行病原菌生物学特性和室内药剂筛选。 结果 从病叶中分离获得菌株HT10，将其回接于健康叶片上，接种后病斑中央呈灰褐色，边缘棕褐色，外围有黄色晕圈，与田间症状一致，符合柯赫氏法则的致病性测定；PDA培养基上菌丝呈棕黑色，分生孢子倒棒状、卵形或近椭圆形，表面具横隔和纵隔；经多基因（ITS、GAPDH和RPB2）系统发育分析，其与长柄链格孢（Alternaria longipes）聚集在同一分支；A. longipes最适生长条件为：PDA培养基，全光照，28 ℃和pH 6.0，碳源和氮源分别是蔗糖和甘氨酸，致死温度41 ℃ / 15 min。室内毒力测定结果表明，25%抑霉·咯菌腈悬乳剂抑菌作用最强，EC50为0.799 μg·mL−1，400 g·L−1克菌·戊唑醇悬乳剂抑菌作用最弱，EC50为370.457 μg·mL−1。 结论 海桐叶斑病的致病菌为长柄链格孢（A. longipes），这是A. longipes侵染海桐引起叶斑病的首次报道；25%抑霉·咯菌腈悬乳剂对A. longipes有较强的抑制作用。 Abstract:Objective The present paper aimed to identify the pathogen causing leaf spot on Pittosporum tobira, to study the biological characteristics of the pathogen, and screen out effective fungicides to control this disease. Method The pathogen was isolated from the leaves with typical symptoms by tissue separation method and verified by pathogenic test. The morphological and molecular biology methods were used to identify species of the pathogen. The mycelium growth in plate was used to investigate the biological characteristics and fungicides toxicity of the pathogen. Result The HT10 strain was isolated from the disease leaves and inoculated onto healthy P. tobira. After inoculation, the center of the disease spot was grayish brown, the edge was brown, and the periphery was yellow halo, which was consistent with the field’s symptoms. On PDA media, the fungal colonies displayed brown and black. Conidia were inverted rod, ovoid or nearly elliptic, with transverse and longitudinal septa. The phylogenetic analysis used multiple genes of ITS, GAPDH and RPB2 clustered the isolate with Alternaria longipes clade. The optimum growth condition on PDA were full light, 28 ℃ temperature and, 6.0 pH. The optimal carbon source or nitrogen source is sucrose or glycine, respectively. The lethal temperature is 41 ℃/15 min. Laboratory toxicity test showed that the 25% SC of Imazalil·Fludioxonil has the best inhibitory effect to pathogenic fungus with EC50 of 0.799 μg·mL−1 and the 400 g·L−1 SC of Captan·Tebuconazole has the worst inhibitory effect with EC50 of 370.457 μg·mL−1. Conclusion The pathogen causing the leaf spot disease on P. tobira is A. longipes, which is the first report of leaf spot disease on P. tobira caused by A.longipes. 25% SC Imazalil·Fludioxonil has a strong inhibitory effect on the growth of A. longipes. -
图 1 海桐叶斑病田间发病症状及致病性测定
A.田间发病症状；B.发病初期症状；C.发病后期症状；D.接种发病症状。
Figure 1. Disease symptoms in the field and pathogenicity determination of Pittosporum tobira leaf spot
A. Symptoms of disease in the field; B. Symptoms of initial stage; C. Symptom of late stage; D. Symptoms of inoculation.
图 2 海桐叶斑病病原菌HT10形态特征
A-C. 病原菌在PDA培养基上培养3 d、5 d、7 d时菌落形态；D-E. 分生孢子；F. 分生孢子梗。
Figure 2. Morphological characteristics of HT10 pathogen of Pittosporum tobira leaf spot
A-C. Colony morphology of pathogenic fungi cultured on PDA medium for 3, 5 and 7 days; D-E. Conidia; F.Conidiophore.
图 3 基于ITS、GAPDH和RPB2序列构建的多基因系统发育树
分支上的数值为 1000 次重复后的 Bootstrap；B. oryzae（CBS 157.50）为外类群；HT10为本研究分离的菌株。
Figure 3. Multi-loci phylogenetic analysis based on ITS, GAPDH, and RPB2 sequences
The value on the branch is the bootstrap value after 1000 repetitions; B. Oryzae （CBS 157.50） is an outgroup; HT10 is the strain isolated in this study.
图 4 不同培养条件对海桐长柄链格孢菌丝生长的影响
图中数据为平均数 ± 标准差，不同字母表示经邓肯氏新复极差法检验在 P ＜ 0.05 水平差异显著。
Figure 4. Effects of different culture conditions on hyphal growth of A. longipes haitung
Data are mean±SD，different letters indicate significant difference at P ＜ 0.05 level by Duncan’s new multiple range test.
表 1 杀菌剂对长柄链格孢菌丝生长的抑制效果
Table 1. Inhibition effect of fungicides on mycelium growth of Alternaria longipes
杀菌剂
Fungicide毒力回归方程
Virulent regression equation相关系数R2
Correlation coefficient抑制中浓度
EC50/（μg·mL−1）25%抑霉·咯菌晴悬乳剂
Imazalil·Fludioxonil 25% SC (Suspension Concentrate, SC)y = 0.2647x + 5.0583 0.9900 0.799 25%多菌灵可湿性粉剂
Carbendazim 25% WP (Wettable Powder, WP)y = 0.2761x + 3.8609 0.9964 61.908 45%咪鲜胺微乳剂
Prochloraz 45% ME (Micro Emulsion, ME)y = 0.3644x + 4.7093 0.9918 2.214 30%吡唑醚菌酯乳油
Pyraclostrobine 30% EC (Emulsifiable Concentrate, EC)y = 0.7618x + 0.8093 0.9783 246.434 400 g·L−1克菌·戊唑醇悬浮剂
Captan·Tebuconazole 400 g·L−1 SC (Suspension Concentrate, SC)y = 0.931x − 0.4898 0.9708 370.457 
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[1] 杨壹, 刘建军, 孙继斌. 5种屋顶绿化常绿植物的耐热性研究 [J]. 西北林学院学报, 2021, 36(5):233−237, 280.YANG Y, LIU J J, SUN J B. Heat tolerance of five evergreen plants for roof greening [J]. Journal of Northwest Forestry University, 2021, 36(5): 233−237, 280.(in Chinese) [2] RJEIBI I, NCIB S, BEN SAAD A, et al. Evaluation of nutritional values, phenolic profile, aroma compounds and biological properties of Pittosporum tobira seeds [J]. Lipids in Health and Disease, 2017, 16(1): 206. doi: 10.1186/s12944-017-0596-1 [3] 李玲玲, 周文明, 洪东风, 等. 海桐种子及果皮抑菌活性初步研究 [J]. 西北农业学报, 2007, 16(3):274−276.LI L L, ZHOU W M, HONG D F, et al. Preliminary study on antimicrobial activity of the seeds and pericarp of Pittosporum tobira ait [J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2007, 16(3): 274−276.(in Chinese) [4] 张虹, 向俊锋, 谭莉, 等. 海桐皮提取物的抗肿瘤活性及其机制研究 [J]. 药学学报, 2009, 44(12):1359−1363.ZHANG H, XIANG J F, TAN L, et al. Anti-tumor activity of Erythrina variegata L. extract and its mechanism of action [J]. Acta Pharmaceutica Sinica, 2009, 44(12): 1359−1363.(in Chinese) [5] 方中达. 植病研究方法 [M]. 3版. 北京: 中国农业出版社, 1998. [6] 孙广宇, 宗兆锋. 植物病理学实验技术 [M]. 北京: 中国农业出版社, 2002. [7] 卢东升, 王金平, 谢正萍, 等. 信阳市园林植物真菌性病害调查与鉴定 (Ⅱ) [J]. 安徽农业科学, 2009, 37(19):9030−9031. doi: 10.3969/j.issn.0517-6611.2009.19.093LU D S, WANG J P, XIE Z P, et al. Investigation and identification of the fungal diseases of garden plants in Xinyang city (Ⅱ) [J]. Journal of Anhui Agricultural Sciences, 2009, 37(19): 9030−9031.(in Chinese) doi: 10.3969/j.issn.0517-6611.2009.19.093 [8] 桑维钧, 徐芳玲, 杨汝, 等. 贵阳市园林植物真菌病害的初步调查 [J]. 中国森林病虫, 2006, 25(6):22−25.SANG W J, XU F L, YANG R, et al. Investigation on the fungal diseases in the landscape plants in Guiyang [J]. Forest Pest and Disease, 2006, 25(6): 22−25.(in Chinese) [9] LIU D, LI P, HU J L, et al. First report of leaf spot caused by Alternaria tenuissima on Pittosporum tobira in Anqing, China [J]. Plant Disease, 2018, 102(3): 676. [10] 谢莉华. 海桐煤污病的病原菌分离与杀菌剂室内毒力测定 [J]. 山地农业生物学报, 2007, 26(6):532−538. doi: 10.3969/j.issn.1008-0457.2007.06.014XIE L H. The pathogenic isolation of pittosporium sooty blotch and toxicity determination of germicide [J]. Journal of Mountain Agriculture and Biology, 2007, 26(6): 532−538.(in Chinese) doi: 10.3969/j.issn.1008-0457.2007.06.014 [11] YAMAOKA Y, KUROKI D, MATSUTANI K, et al. Pathogenicity of Fusarium solani associated with a bark beetle, Scolytogenes birosimensis, to Pittosporum tobira [J]. Journal of Forest Research, 2015, 20(6): 514−521. doi: 10.1007/s10310-015-0505-2 [12] 张书建, 何月秋. 介绍一种简单的真菌单孢子分离法 [J]. 云南农业大学学报, 2003, 18(3):315−316.ZHANG S J, HE Y Q. A simple method to isolate single fungal spore [J]. Journal of Yunnan Agricultural University, 2003, 18(3): 315−316.(in Chinese) [13] 谢宝贵. 倒置显微镜单孢分离法 [J]. 中国食用菌, 1997, 16(1):35.XIE B G. Single spore separation by inverted microscope [J]. Edible Fungi of China, 1997, 16(1): 35.(in Chinese) [14] WHITE T J, BRUNS T, LEE S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics [M]//PCR Protocols. Amsterdam: Elsevier, 1990: 315－322. [15] BERBEE M L, PIRSEYEDI M, HUBBARD S. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences [J]. Mycologia, 1999, 91(6): 964−977. doi: 10.1080/00275514.1999.12061106 [16] GRUYTER J, NOORDELOOS M. Contributions towards a monograph of Phoma (Coelomycetes) - I. 1. Section Phoma: Taxa with very small conidia in vitro [J]. Persoonia - Molecular Phylogeny and Evolution of Fungi, 1992, 15(1): 71−92. [17] 宁平, 黄艳花, 黄微, 等. 喜树叶斑病病原菌鉴定及其防治药剂筛选 [J]. 西南农业学报, 2022, 35(6):1348−1354.NING P, HUANG Y H, HUANG W, et al. Pathogen identification and screening fungicide of leaf spot of Camptotheca acuminata [J]. Southwest China Journal of Agricultural Sciences, 2022, 35(6): 1348−1354.(in Chinese) [18] YIN Q X, AN X L, WU X, et al. First report of Alternaria longipes causing leaf spot on tea in China [J]. Plant Disease, 2021, 105(12): 4167. [19] SIRNMONS E G. Alternaria themes and variations [J]. Myeotaxon, 1981, 13(1): 16−34. [20] 杨志英, 卢佳欣, 李涛, 等. 贵州开阳地区茶叶斑病相关病原菌长柄链格孢菌(Alternaria longipes)的研究 [J]. 中国植保导刊, 2020, 40(11):5−12, 34.YANG Z Y, LU J X, LI T, et al. Study of tea leaf spot related Alternaria longipes in Kaiyang region, Guizhou Province [J]. China Plant Protection, 2020, 40(11): 5−12, 34.(in Chinese) [21] 彭希文, 刘光珍, 杨永柱, 等. 云南省烟草赤星病(Tobacco brown spot)病原研究及其防治药剂的筛选 [J]. 西南农业大学学报, 2000, 22(2):153−156.PENG X W, LIU G Z, YANG Y Z, et al. Identification of pathogens for brown spots on tobacco plants in Yunnan Province and selection for effective fungicides for its control [J]. Journal of Southwest Agricultural University, 2000, 22(2): 153−156.(in Chinese) [22] SUWANNARACH N, KUMLA J, LUMYONG S. First report of Alternaria leaf blight disease on oil palm caused by Alternaria longipes in Thailand [J]. Phytoparasitica, 2015, 43(1): 57−59. doi: 10.1007/s12600-014-0429-y [23] 龙亚琴. 金刚藤叶斑病病原学、发生规律及防治技术研究 [D]. 武汉: 华中农业大学, 2009.LONG Y Q. Studies on etiology, occurrence and control of leaf spot of Smilax China [D]. Wuhan: Huazhong Agricultural University, 2009. (in Chinese) [24] 黎妍妍, 杨涛, 贾欣欣, 等. 湖北省烟草赤星病菌生物学特性研究 [J]. 湖北农业科学, 2018, 57(22):27−31.LI Y Y, YANG T, JIA X X, et al. Study on biological characteristics of pathogenic bacteria causing tobacco brown spot disease in Hubei Province [J]. Hubei Agricultural Sciences, 2018, 57(22): 27−31.(in Chinese) [25] 王义, 胡美姣, 李敏, 等. 草海桐链格孢叶斑病菌生物学特性及杀菌剂的室内筛选 [J]. 热带作物学报, 2020, 41(8):1634−1641. doi: 10.3969/j.issn.1000-2561.2020.08.018WANG Y, HU M J, LI M, et al. Biological characteristics of Alternaria leaf blight of Scaevola taccada and screening of fungicides in vitro [J]. Chinese Journal of Tropical Crops, 2020, 41(8): 1634−1641.(in Chinese) doi: 10.3969/j.issn.1000-2561.2020.08.018 [26] 于秀敏, 彭飞, 于淑玉, 等. 6种杀菌剂对胶孢炭疽菌和长柄链格孢菌的室内毒力测定 [J]. 河北科技师范学院学报, 2021, 35(2):78−80.YU X M, PENG F, YU S Y, et al. Toxicity tests of six fungicides against Glomerella cingulata spauld and Alternaria longipes [J]. Journal of Hebei Normal University of Science & Technology, 2021, 35(2): 78−80.(in Chinese) -
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