一株乳突赤壳属蕨麻致病菌的鉴定及生物学特性

doi:10.13304/j.nykjdb.2022.0973

摘要/Abstract

摘要：

为了明确引起蕨麻根腐病的病原菌及其生物学特性，从青海省门源蕨麻人工种植基地患根腐病的蕨麻块根部分离得到菌株MY-BSX4-1，对其进行致病性检测、形态学观察、rDNA-ITS序列分析鉴定，同时分析不同温度、pH、光照、碳源、氮源、培养基对该病原菌生长及其产孢量的影响。结果表明，根据形态学观察和 rDNA-ITS 序列分析将MY-BSX4-1菌株鉴定为乳突赤壳属（Thelonectria olida）。致病性研究表明，该菌株符合柯赫氏法则，是导致蕨麻根腐病的病原真菌。生物学特性研究表明，菌株MY-BSX4-1菌丝生长和产孢的最适温度均为25 ℃；光照有利于菌丝生长，且对菌落产孢无明显影响；pH 4.0~12.0时菌丝均能较好生长，其中菌丝生长的最适pH为6.0~9.0，产孢最适pH为8.0；碳源中可溶性淀粉利于菌丝生长，乳糖利于产孢，果糖不利于菌丝生长；氮源中牛肉浸膏利于菌丝生长，硝酸铵不利于菌丝生长；蕨麻煎汁培养基和蕨麻煎汁培养基（硝酸钠）有利于菌丝生长和产孢；菌丝致死温度为47 ℃、10 min。

关键词: 蕨麻, 根腐病, 乳突赤壳属菌, 生物学特性

Abstract:

In order to identify the pathogen causing the root rot of Potentilla anserina L. and its biological characteristics， the strain MY-BSX4-1 was as material， which isolated from the tuberous root of Potentilla anserina in the artificial planting base of Menyuan， Qinghai province. The strain MY-BSX4-1 was analyzed by the pathogenic detection， morphological observation， rDNA-ITS sequence analysis. The growth and spore production of the pathogen were analyzed under different temperatures， pH， light， carbon sources， nitrogen sources and culture media. The results showed that MY-BSX4-1 was identified as Thelonectria olida according to its morphological characteristics and rDNA-ITS sequence analysis. It was identified to be the pathogenic fungus of Potentilla anserina L.according to Koch’s rule. The biological characteristics showed that the optimum temperature of strain MY-BSX4-1 was 25 ℃for mycelial growth and spore production. The light condition was beneficial to mycelium growth， but had no obvious effect on spore production. The mycelium could grow well at pH 4.0~12.0， which the optimum pH for mycelium growth was 6.0~9.0 and the optimum pH was 8.0 for spore production. In carbon sources， soluble starch was beneficial to mycelial growth， lactose was beneficial to spore production， while fructose was unfavorable to mycelial growth. In nitrogen sources， beef extract was beneficial to mycelial growth， while ammonium nitrate was unfavorable to mycelial growth. Potentilla anserina L juice medium （JM） and JM （sodium nitrate） were beneficial to mycelial growth and spore production. The lethal temperature of mycelium was 47 ℃ for 10 min.

Key words: Potentilla anserina L., root rot, Thelonectria olida, biological characteristics

中图分类号:

王鑫慈, 李军乔, 李晨芹, 田甜, 曲俊儒. 一株乳突赤壳属蕨麻致病菌的鉴定及生物学特性[J]. 中国农业科技导报, 2023, 25(5): 87-95.

Xinci WANG, Junqiao LI, Chenqin LI, Tian TIAN, Junru QU. Identification and Biological Characteristics of a Thelonectria Pathogenic Fungi of Potentilla anserina L.[J]. Journal of Agricultural Science and Technology, 2023, 25(5): 87-95.

图/表 10

图1 蕨麻根腐病感病田间症状A：田间发病症状；B：患病蕨麻全株；C：患病块根纵切面；D：患病块根横切面

Fig. 1 Field symptoms of Potentilla anserina L. root rot infectionA： Symptoms of disease in the field；B： Whole plant of diseased Potentilla anserina L.；C： Longitudinal section of diseased root tuber；D： Transverse section of diseased root tuber

图2 菌株MY-BSX4-1的形态学鉴定A：田间病部黑色子实体形态；B：正面菌落形态；C：反面菌落形态；D：子囊壳；E：有隔菌丝；F：大型分生孢子；G：小型分生孢子；H：产孢结构

Fig. 2 Morphological identification of MY-BSX4-1A： Morphology of black fruiting bodies of diseased parts in the field； B： Colony morphology （front）； C： Colony morphology （back）； D： Perithecium； E： Septum hyphae； F： Megaconidium； G： Microconidium； H： Sporogenous structure

图3 PCR扩增产物电泳结果

Fig. 3 Results of PCR amplification products

图4 菌株MY-BSX4-1的系统发育树

Fig. 4 Phylogenetic tree of MY-BSX4-1

图5 人工接种MY-BSX4-1后蕨麻植株的发病症状A：有伤口离体回接处理；B：有伤口离体回接处理横截面；C：无伤口离体回接处理；D：无伤口离体回接处理横截面；E：有伤口离体回接CK；F：有伤口离体回接CK横截面；G：无伤口离体回接CK；H：无伤口离体回接CK横截面；I：活体回接植株感病叶腹面部位症状；J：活体回接植株感病叶背面部位症状；K：活体回接植株感病叶部位症状；L：活体回接植株感病茎叶部位症状；M：活体回接植株整株症状；N：活体回接CK

Fig. 5 Symptoms of diseased plant after artificial inoculation of MY-BSX4-1A： In vitro tieback treatment of wound； B： Cross section of wound in vitro tie-in treatment； C： In vitro tieback without wound； D： Cross section of wound free in vitro tieback treatment； E： Connect CK in vitro with wound； F： Cross section of CK connected in vitro with wound； G： No wound in vitro connection CK； H： Cross section of CK without wound in vitro graft； I： Symptoms on the ventral part of infected leaves of living graft plants； J： Symptoms on the back of infected leaves of living grafted plants； K： Symptoms of infected leaves of living grafted plants； L： Symptoms of diseased stems and leaves of living grafted plants； M： Symptoms of whole plant grafted back in vivo； N： Live connection CK

图6 不同生物学特性下MY-BSX4-1菌丝的生长和产孢A：温度；B：光照；C：pH。不同小写字母表示不同处理间在P<0.05水平差异显著

Fig. 6 Mycelium growth and sporulation of MY-BSX4-1 under different biological characteristicsA：Temperature； B： Illumination； C： pH. Differenet lowercase letters indicate significant differences between different treatments at P<0.05 level

表1 不同碳源下MY-BSX4-1的菌丝生长及产孢

Table 1 Growth and sporulation of MY-BSX4-1 mycelium under different carbon source

碳源 Carbon source	菌落直径 Colony diameter/cm	产孢量 Sporulation/（×10⁵·mL^-1）
缺碳对照CK without carbon	4.83±0.03 f	13.33±1.67 cd
葡萄糖Glucose	5.92±0.02 b	23.33±1.67 b
蔗糖Sucrose	5.38±0.02 c	8.33±1.67 de
乳糖Lactose	5.27±0.04 d	40.00±2.89 a
可溶性淀粉Soluble starch	6.97±0.02 a	18.33±1.67 bc
D-果糖D-fructose	3.65±0.05 g	20.00±2.89 b
D-甘露醇D-mannitol	4.93±0.03 e	3.33±1.67 e

表2 不同氮源下MY-BSX4-1的菌丝生长及产孢

Table 2 Mycelium growth and sporulation of MY-BSX4-1 under different nitrogen source

氮源 Nitrogen source	菌落直径 Colony diameter/cm	产孢量 Sporulation/（×10⁵·mL^-1）
缺氮对照CK without nitrogen	5.52±0.02 c	0.00±0.00 d
甘氨酸Glycine	5.02±0.04 d	0.00±0.00 d
磷酸氢铵Ammonium phosphate	4.52±0.03 e	11.67±1.67 c
硝酸铵Ammonium nitrate	3.02±0.04 f	3.33±1.67 d
硫酸铵Ammonium sulphate	5.58±0.04 c	21.67±3.33 b
蛋白胨Peptone	6.00±0.00 b	33.33±3.33 a
牛肉浸膏Beef extract	7.12±0.03 a	16.67±4.41 bc

表3 不同培养基下MY-BSX4-1菌丝生长及产孢

Table 3 Mycelium growth and sporulation of MY-BSX4-1 under different mediums

培养基 Medium	菌落直径 Colony diameter/cm	产孢量 Sporulation/（×10⁵·mL^-1）
蕨麻煎汁培养基P. anserina juice medium	6.82±0.04 c	45.00±2.89 a
蕨麻煎汁培养基（硝酸钠） P. anserina juice medium （sodium nitrate）	7.00±0.00 b	43.33±1.67 a
蕨麻煎汁培养基（葡萄糖） P. anserina juice medium （glucose）	7.20±0.00 a	33.33±3.33 b
LB培养基LB medium	3.43±0.02 f	31.67±1.67 bc
马铃薯培养基PDA medium	4.55±0.06 d	25.00±2.89 c
查氏培养基Czapek medium	3.90±0.03 e	28.33±1.67 bc

图7 MY-BSX4-1的菌丝致死温度注：不同小写字母表示不同温度间在P<0.05水平差异显著。

Fig. 7 Fatal temperature of MY-BSX4-1 myceliumNote： Different lowercase letters indicate significant differences between different temperatures at P<0.05 level.

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