Identification of the pathogens causing root rot on Polygonatum cyrtonema Hua in Fujian
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摘要:
目的 明确福建地区多花黄精根腐病的发病率和病原菌种类,并为该病害的防治提供理论依据。 方法 调查福建省3个多花黄精种植基地的根腐病发病率,并采集具有典型根腐病症状的植株和块根病样,分离纯化获得病原菌,利用形态学特征、分子生物学特征及致病性测定对其进行鉴定。 结果 多花黄精种苗的根腐病平均发病率约为10.50%,采收时根茎的根腐病平均发病率为17.65%。分离纯化获得到98株菌株,结合菌株的形态学特征、特异性引物FOF1/FOR1和F8/R8、tef-1α 基因序列分析,分别鉴定为尖孢镰刀菌Fusarium oxysporum、腐皮镰刀菌F. solani和藤仓镰刀菌F. fujikuroi,三者的分离频率依次为75.51%、20.41%和4.08%。代表性菌株经回接根茎进行致病性检测发现其发病症状与田间表现一致,且符合柯赫氏法则。 结论 尖孢镰刀菌、腐皮镰刀菌和藤仓镰刀菌是福建地区多花黄精根腐病的主要病原菌,其中藤仓镰刀菌可引起我国多花黄精根腐病为首次报道。 Abstract: :Objective The purpose of this paper is to clarify the root rot incidence rate of Polygonatum cyrtonema Hua in Fujian area and identify the causing pathogens, which will provide a theoretical basis for the control of root rot disease. Methods The incidence of root rot were investigate in the three planting areas of P. cyrtonema in Fujian province. The roots and rhizomes with typical root rot symptoms were collected from the fields. The pathogens were isolated and identified with morphology, molecular biology and pathogenicity test. Results The average incidence of root rot was about 10.50% in the seedlings phase and 17.65% in the rhizomes during harvesting phase. A total of 98 isolates were obtained from the diseased samples. The pathogens were finally identified as Fusarium oxysporum, F. solani and F. fujikuroi based on morphological characteristics, FOF1/FOR1 and F8/R8 PCR amplification of specific primer, and sequence analysis of translation elongation factor-1α (tef-1α) gene. The separation frequencies of the tree pathogens were 75.51%, 20.41% and 4.08%, respectively. Inoculating the pathogens on the healthy rhizomes reproduced the same symptoms previously observed in the field. Pathogenicities of the tree pathogens were confirmed by fulfilling the Koch’s postulates. Conclusion It was confirmed that F. oxysporum, F. solani and F. fujikuroi were the pathogens of root rot of P. cyrtonema in Fujian province. This is the first report of F. fujikuroi causing root rot of P. cyrtonema in China. -
Key words:
- Polygonatum cyrtonema Hua /
- Root rot /
- Fusarium oxysporum /
- Fusarium solani /
- Fusarium fujikuroi
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图 2 多花黄精根腐病病原菌的第I类菌株形态特征
A-B:菌落在PDA培养基上的形态;C:大型和小型分生孢子;D:厚垣孢子;E:产孢细胞。
Figure 2. Morphological characteristics for the class I of the pathogen causing root rot of Polygonatum cyrtonema
A-B: Colony grown on potato dextrose agar; C: macroconidia and microconidia; D: chlamydospore; E: sporulation cells.
图 3 多花黄精根腐病病原菌的第II类菌株形态特征
A-B:菌落在PDA培养基上的形态;C:大型和小型分生孢子;D:厚垣孢子;E:产孢细胞。
Figure 3. Morphological characteristics for the class II of the pathogen causing root rot of Polygonatum cyrtonema
A-B: Colony grown on potato dextrose agar; C: macroconidia and microconidia; D: chlamydospore; E: sporulation cells.
图 5 基于尖孢镰刀菌特异性引物FOF1/FOR1的代表性供试菌株的PCR扩增结果
M:DL2 000;1-20:分离菌株;21-23:分别为尖孢镰刀菌、腐皮镰刀菌和藤仓镰刀菌的阳性对照;24:无菌水阴性对照。
Figure 5. PCR amplification of DNA for the representative tested pathogen strains using specific primer set FOF1/FOR1 of Fusarium oxysporum
M: DL2 000; Lane 1-20: the tested strains; Lane 21-23: positive control for F. oxysporum,F. solani and F. fujikuroi; Lane 24: negative control for sterile water.
图 6 基于腐皮镰刀菌特异性引物F8/R8的代表性供试菌株的PCR扩增结果
M:DL2 000;1-20:分离菌株;21-23:分别为尖孢镰刀菌、腐皮镰刀菌和藤仓镰刀菌的阳性对照;24:无菌水阴性对照。
Figure 6. PCR amplification of DNA for the representative tested pathogen strains using specific primer set F8/R8 of Fusarium solani
M: DL2 000; Lane 1-20:the tested strains; Lane 21-23: positive control for F. oxysporum,F. solani and F. fujikuroi; Lane 24: negative control for sterile water.
图 8 接种不同类型病原菌14d时多花黄精根茎的发病症状
A-B:尖孢镰刀菌FJAT-33155;C-D:腐皮镰刀菌FJAT-33165;E-F:藤仓镰刀菌FJAT-33320;G-H:尖孢镰刀菌西瓜专化型菌株FJAT-31362;I-J:无菌水对照。
Figure 8. The disease symptoms on the rhizome of Polygonatum cyrtonema inoculated with the pathogens for 14d
A-B: F. oxysporum FJAT-33155; C-D: F. solani FJAT-33165; E-F: F. fujikuroi FJAT-33320; G-H: F. oxysporum f. sp. niveum FJAT-31362; I-J: sterile water (CK).
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[1] 国家药典委员会. 中华人民共和国药典-一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020. [2] 陈晔, 孙晓生. 黄精的药理研究进展 [J]. 中药新药与临床药理, 2010, 21(3):328−330.CHEN Y, SUN X S. Advances in pharmacological research of Polygonatum sibiricum [J]. Traditional Chinese Drug Research and Clinical Pharmacology, 2010, 21(3): 328−330. (in Chinese) [3] 周先治, 苏海兰, 陈阳, 等. 多花黄精主要病害发生规律调查 [J]. 福建农业科技, 2017, (10):25−27.ZHOU X Z, SU H L, CHEN Y, et al. Occurrence regularity of major diseases of Polygonatum sibiricum [J]. Fujian Agricultural Science and Technology, 2017(10): 25−27. (in Chinese) [4] 韩凤, 林茂祥, 章文伟, 等. 多花黄精根腐病对根际土壤酶活性及真菌群落变化的影响 [J]. 西南大学学报(自然科学版), 2021, 43(4):53−61.HAN F, LIN M X, ZHANG W W, et al. Effects of Polygonatum cyrtonema root rot on rhizosphere soil enzyme activity and fungal community [J]. Journal of Southwest University (Natural Science Edition), 2021, 43(4): 53−61. (in Chinese) [5] 梁忠厚, 李静纳. 湖南多花黄精根腐病病原菌的分离与鉴定 [J]. 南方农业学报, 2021, 52(7):1923−1930. doi: 10.3969/j.issn.2095-1191.2021.07.022LIANG Z H, LI J N. Isolation and identification of the pathogenic fungi of Polygonatum cyrtonema Hua root rot in Hunan Province [J]. Journal of Southern Agriculture, 2021, 52(7): 1923−1930. (in Chinese) doi: 10.3969/j.issn.2095-1191.2021.07.022 [6] 吴依婷, 姚传威, 邓波侠, 等. 黄精根腐病分离菌及其拮抗内生细菌的鉴定 [J]. 浙江农业学报, 2018, 30(12):2087−2093. doi: 10.3969/j.issn.1004-1524.2018.12.14WU Y T, YAO C W, DENG B X, et al. Identification of isolated fungus from root rot of Polygonatum sibiricum and its antagonistic endophytic bacteria [J]. Acta Agriculturae Zhejiangensis, 2018, 30(12): 2087−2093. (in Chinese) doi: 10.3969/j.issn.1004-1524.2018.12.14 [7] LI D L, GUO M. First report of Fusarium oxysporum causing root rot on Polygonatum sibiricum in Chizhou, China [J]. Plant Disease, 2019, 103(11): 2956. [8] 王锋, 卫国羽, 赵微, 等. 贵州多花黄精根腐病病原菌鉴定及生防菌筛选 [J]. 广东农业科学, 2023, 50(8):143−153.WANG F, WEI G Y, ZHAO W, et al. Identification of pathogen causing root rot in Polygonatum cyrtonema Hua and screening of biocontrol bacterias [J]. Guangdong Agricultural Sciences, 2023, 50(8): 143−153. (in Chinese) [9] 杨林毅, 陈泽历, 赖清玉, 等. 滇黄精腐皮镰刀菌的分离鉴定 [J]. 湖北农业科学, 2019, 58(3):65−67,101.YANG L Y, CHEN Z L, LAI Q Y, et al. Isolation and identification of Fusarium solani from Polygonatum kingianum [J]. Hubei Agricultural Sciences, 2019, 58(3): 65−67,101. (in Chinese) [10] 张磊, 李辉山, 杨枝中, 等. 云南滇黄精根茎腐病病原鉴定 [J]. 植物病理学报, 2021, 51(6):1000−1004.ZHANG L, LI H S, YANG Z Z, et al. Identification of the pathogen causing rhizome rot on Polygonatum kingianum in Yunnan [J]. Acta Phytopathologica Sinica, 2021, 51(6): 1000−1004. (in Chinese) [11] 韩凤, 李巧玲, 韩如刚, 等. 渝产多花黄精根腐病病原菌的分离与鉴定 [J]. 分子植物育种, 2020, 18(11):3693−3698.HAN F, LI Q L, HAN R G, et al. Isolation and identification of pathogen of Polygonatum cyrtonema Hua root rot disease in Chongqing [J]. Molecular Plant Breeding, 2020, 18(11): 3693−3698. (in Chinese) [12] NEL B, STEINBERG C, LABUSCHAGNE N, et al. Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants [J]. Plant Pathology, 2006, 55(2): 207−216. doi: 10.1111/j.1365-3059.2006.01343.x [13] 周洁, 张玲玲, 袁继荣, 等. 生姜腐皮镰刀菌的分离鉴定及PCR快速检测方法构建 [J]. 植物病理学报, 2022, 52(4):681−690.ZHOU J, ZHANG L L, YUAN J R, et al. Isolation and identification of Fusarium solani from ginger rhizomes and establishment of a rapid PCR detection method [J]. Acta Phytopathologica Sinica, 2022, 52(4): 681−690. (in Chinese) [14] GEISER D M, DEL MAR JIMÉNEZ-GASCO M, KANG S, et al. FUSARIUM-ID v. 1.0: A DNA sequence database for identifying Fusarium [J]. European Journal of Plant Pathology, 2004, 110(5): 473−479. [15] O’DONNELL K, SUTTON D A, RINALDI M G, et al. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections [J]. Journal of Clinical Microbiology, 2010, 48(10): 3708−3718. doi: 10.1128/JCM.00989-10 [16] LESLIE J F, SUMMERELL B A. The Fusarium laboratory manual[C]. Ames, USA: Blackwell publishing, 2006. [17] 高芬, 任小霞, 王梦亮, 等. 中草药根腐病及其微生物防治研究进展 [J]. 中国中药杂志, 2015, 40(21):4122−4126.GAO F, REN X X, WANG M L, et al. Research progress in root rot diseases of Chinese herbal medicine and control strategy by antagonistic microorganisms [J]. China Journal of Chinese Materia Medica, 2015, 40(21): 4122−4126. (in Chinese) [18] 廖长宏, 陈军文, 吕婉婉, 等. 根和根茎类药用植物根腐病研究进展 [J]. 中药材, 2017, 40(2):492−497.LIAO C H, CHEN J W, LV W W, et al. Research progress on root rot of medicinal plants of roots and rhizomes [J]. Journal of Chinese Medicinal Materials, 2017, 40(2): 492−497. (in Chinese) [19] 肖荣凤, 陈燕萍, 陈梅春, 等. 太子参根腐病病原菌的鉴定及防治药剂筛选 [J]. 植物保护学报, 2020, 47(6):1333−1342.XIAO R F, CHEN Y P, CHEN M C, et al. Pathogen identification of root rot of Pseudostellaria heterophylla plant and fungicide screening for its efficient control [J]. Journal of Plant Protection, 2020, 47(6): 1333−1342. (in Chinese) [20] 高芬, 赵晓霞, 秦雪梅, 等. 山西省蒙古黄芪根腐病优势致病菌群分析 [J]. 植物保护学报, 2018, 45(4):878−885.GAO F, ZHAO X X, QIN X M, et al. Analysis of dominant pathogen community causing Astragalus membranaceus var. mongholicus root rot in Shanxi Province [J]. Journal of Plant Protection, 2018, 45(4): 878−885. (in Chinese) [21] 曹瑱艳, 杨怡华, 申屠旭萍, 等. 浙江省铁皮石斛根腐病病原真菌的鉴定 [J]. 植物保护学报, 2020, 47(1):178−186.CAO Z Y, YANG Y H, SHENTU X P, et al. Identification of the pathogenic fungi of root rot of traditional medicinal Dendrobium officinale in Zhejiang Province [J]. Journal of Plant Protection, 2020, 47(1): 178−186. (in Chinese) [22] 陈宏州, 杨红福, 姚克兵, 等. 水稻恶苗病病原菌鉴定及室内药剂毒力测定 [J]. 植物保护学报, 2018, 45(6):1356−1366.CHEN H Z, YANG H F, YAO K B, et al. Identification of rice bakanae pathogens and indoor toxicity test of fungicides [J]. Journal of Plant Protection, 2018, 45(6): 1356−1366. (in Chinese) [23] 王倩, 童睿, 檀根甲, 等. 栝楼果实腐烂病病原菌鉴定及防治药剂的筛选 [J]. 华南农业大学学报, 2023, 44(2):270−279. doi: 10.7671/j.issn.1001-411X.202202006WANG Q, TONG R, TAN G J, et al. Identification of the pathogen causing Trichosanthes kirilowii fruit rot and screening of fungicides [J]. Journal of South China Agricultural University, 2023, 44(2): 270−279. (in Chinese) doi: 10.7671/j.issn.1001-411X.202202006 [24] SUN W, LEI T Y, YUAN H Z, et al. Occurrence of root rot caused by Fusarium fujikuroi and Fusarium proliferatum on peanut in China[J]. Plant Disease, 2022. DOI: 10.1094/PDIS-02-22-0438-PDN. [25] ZHENG L, MENG H, LIU Y T, et al. Root rot disease of Torreya grandis caused by Fusarium fujikuroi in China[J]. Plant Disease, 2022. DOI: 10.1094/PDIS-03-22-0710-PDN.