花椒流胶病拮抗菌的分离鉴定及其生防机制

doi:10.3969/j.issn.1004-1524.20230077

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (2): 373-382.DOI: 10.3969/j.issn.1004-1524.20230077

花椒流胶病拮抗菌的分离鉴定及其生防机制

赵吉桃(), 何静^*(), 丁德东, 李彦湘, 候彩霞, 赵倩

甘肃农业大学 a,林学院;b,甘肃省枸杞无害化栽培工程研究中心,甘肃兰州 730070

收稿日期:2023-01-18 出版日期:2024-02-25 发布日期:2024-03-05
作者简介:赵吉桃(1996—),男,甘肃临洮人,硕士研究生,主要从事经济林有害生物防治研究。E-mail:528451677@qq.com
通讯作者: *何静,E-mail:hejing268@aliyun.com
基金资助:
甘肃省自然科学基金重点项目(20JR10RA508);中央财政推广示范项目(2018ZYTG15)

Isolation, identification and biocontrol mechanism of antagonistic fungus against Chinese pepper gummosis

ZHAO Jitao(), HE Jing^*(), DING Dedong, LI Yanxiang, HOU Caixia, ZHAO Qian

College of Forestry; b. Wolfberry Harmless Cultivation Engineering Research Center of Gansu Province, Gansu Agricultural University, Lanzhou 730070, China

Received:2023-01-18 Online:2024-02-25 Published:2024-03-05

摘要/Abstract

摘要：

由三线镰刀菌(Fusarium tricinctum)引起的花椒流胶病是近年来危害严重的花椒病害之一。为获得F. tricinctum的优良拮抗菌株,采用平板对峙法,筛选分离得到一株具较强拮抗作用的花椒内生真菌HJ-18,其对F. tricinctum的抑制率达58.85%。基于形态学和ITS分子鉴定,初步确定HJ-18为裂褶菌(Schizophyllum commune,GenBank登录号为OP502072);经抑菌谱与稳定性测试发现,HJ-18对4种供试植物病原真菌均具抑制效果,培养滤液经不同光照、温度和pH处理后,抗真菌效果稳定,但对紫外线敏感;当菌株HJ-18培养滤液体积分数为50%时,对F. tricinctum菌丝生长抑制率为66.40%;扫描电镜结果显示,经菌株HJ-18培养滤液处理后,F. tricinctum菌丝出现褶皱,且表面有碎屑产生;盆栽试验表明,菌株HJ-18对花椒流胶病的相对防效为52.65%,与22.5%啶氧菌酯100倍液处理具有相同防效。以上结果表明,菌株HJ-18是一株具有生防潜力的内生真菌,可作为生防菌剂进行开发利用。

关键词: 花椒流胶病, 内生真菌, 拮抗菌株, 培养滤液, 生防机制

Abstract:

Chinese pepper gummosis caused by Fusarium tricinctum is a serious disease of Zanthoxylum bungeanum in recent years. In order to obtain the excellent antagonistic strain of F. tricinctum, and the endophytic fungi with strong antagonistic effect were screened and isolated by plate confrontation method. A strain with high antagonistic activity was obtained, and its inhibition rate reached 58.85%, which was named HJ-18. Based on morphological and ITS molecular identification, it was preliminarily identified as Schizophyllum commune (GenBank accession number: OP502072). The antifungal spectrum test showed that it had inhibitory effect on 4 plant pathogenic fungi. After treatment with different light, temperature and pH, the antifungal effect was stable, but sensitive to ultraviolet ray. When the volume fraction of culture filtrate was 50%, the inhibition rate of mycelium growth of F. tricinctum was the highest, reaching 66.40%. Scanning electron microscope observation showed that after treatment with culture filtrate, the mycelium of F. tricinctum was wrinkled, and debris was produced on the surface. The results of pot experiment showed that the relative prevention effect of strain HJ-18 was 52.65%, which was the same as 100-fold dilution of 22.5% picoxystrobin treatment. The above results show that strain HJ-18 is an endophytic fungus with biocontrol potential and can be used as a biocontrol agent.

Key words: Chinese pepper gummosis, endophytic, antagonistic strains, culture filtrate, biocontrol mechanism

中图分类号:

S435.73

赵吉桃, 何静, 丁德东, 李彦湘, 候彩霞, 赵倩. 花椒流胶病拮抗菌的分离鉴定及其生防机制[J]. 浙江农业学报, 2024, 36(2): 373-382.

ZHAO Jitao, HE Jing, DING Dedong, LI Yanxiang, HOU Caixia, ZHAO Qian. Isolation, identification and biocontrol mechanism of antagonistic fungus against Chinese pepper gummosis[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 373-382.

图/表 9

图1 培养7 d菌株HJ-18对三线镰刀菌的抑制作用 A,对照(正面);B,对照(反面);C,对峙效果(正面);D,对峙效果(反面)。培养时间为7 d。

Fig.1 Inhibitory effect of strain HJ-18 on Fusarium tricinctum A, Control (front); B, Control (reverse); C, Confrontation effect (front); D, Confrontation effect (reverse). Culture time was 7 days.

图2 菌株HJ-18的形态特征 A,菌落形态(正面);B,菌落形态(反面),C,菌丝;D,孢子。

Fig.2 Morphological characteristics of strain HJ-18 A, Colony morphology (front); B, Colony morphology (reverse); C, Mycelium; D, Spore.

图3 菌株HJ-18基于ITS序列的系统发育树

Fig.3 The phylogenetic tree of strain HJ-18 based on ITS sequence

图4 菌株HJ-18对几种植物病原真菌的抑制效果柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.4 Inhibitory effect of strain HJ-18 on several plant pathogenic fungi The bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图5 不同条件下菌株HJ-18培养滤液的稳定性

Fig.5 Stability of the culture filtrate of strain HJ-18 under different conditions

图6 菌株HJ-18培养滤液对三线镰刀菌菌丝生长的影响 A,菌落直径;B,抑制率。

Fig.6 Effects of culture filtrate of strain HJ-18 against the mycelial growth of Fusarium tricinctum A, Colony diameter; B, Inhibition ratio.

图7 菌株HJ-18培养滤液对三线镰刀菌菌丝形态的影响 A,对照(×1 000);B,对照(×4 000);C,HJ-18培养滤液处理(×1 000);D,HJ-18培养滤液处理(×4 000)。

Fig.7 Effect of culture filtrate of strain HJ-18 on mycelial morphology of Fusarium tricinctum A, Control (×1 000); B, Control (×4 000); C, Treated by HJ-18 culture filtrate (×1 000); D, Treated by HJ-18 culture filtrate (×4 000).

图8 不同处理的花椒幼苗、茎部形态 A,接种三线镰刀菌;B,三线镰刀菌+菌株HJ-18处理;C,22.5%啶氧菌酯100倍液处理;D,无菌水处理;E,先接种菌株HJ-18再接种三线镰刀菌。

Fig.8 Morphology of Zanthoxylum bungeanum seedlings and stems under different treatments A, Inoculation with Fusarium tricinctum; B, Fusarium tricinctum+HJ-18 treatment; C, 100-fold dilution of 22.5% picoxystrobin treatment; D, Sterile water treatment; E, Inoculation with strain HJ-18 before the Fusarium tricinctum.

表1 菌株HJ-18对花椒流胶病的生防效果

Table 1 Control effect of strain HJ-18 on Chinese pepper gummosis

处理 Treatment	病情指数 Disease index	相对防效 Relative control effect/%
病原菌F. tricinctum	77.78±2.22 a
病原菌+菌株HJ-18	36.67±3.33 c	52.65±5.10 a
F. tricinctum+HJ-18
病原菌+啶氧菌酯	40.00±0.00 c	48.48±1.52 a
F. tricinctum+ picoxystrobin
病原菌+无菌水	63.33±3.33 b	18.56±3.61 b
F. tricinctum+sterile water
菌株HJ-18+病原菌	33.33±6.67 c	56.82±9.19 a
HJ-18+F. tricinctum

参考文献 39

[1]	袁海梅, 邱露, 谢贞建, 等. 花椒属植物生物碱类成分及其药理活性研究进展[J]. 中国中药杂志, 2015, 40(23): 4573-4584.
	YUAN H M, QIU L, XIE Z J, et al. Research progress on alkaloids constituents from Zanthoxylum and their pharmacological activities[J]. China Journal of Chinese Materia Medica, 2015, 40(23): 4573-4584. (in Chinese with English abstract)
[2]	于胜男. 花椒的研究概述[J]. 中国调味品, 2012, 37(12): 10-12.
	YU S N. Study on the Chinese prickly ash[J]. China Condiment, 2012, 37(12): 10-12. (in Chinese with English abstract)
[3]	张树衡, 丁德东, 何静, 等. 两种生物肥料配施对再植花椒生长及光合特性的影响[J]. 西北农业学报, 2021, 30(9): 1355-1364.
	ZHANG S H, DING D D, HE J, et al. Effect of two biofertilizer mixtures on growth and photosynthetic characteristics of replanted Zanthoxylum bungeanum[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2021, 30(9): 1355-1364. (in Chinese with English abstract)
[4]	韩富军, 彭海, 任静, 等. 花椒树流胶原因及防治措施探讨[J]. 农业与技术, 2019, 39(15): 12-13.
	HAN F J, PENG H, REN J, et al. Discussion on the causes and control measures of gum flow in Zanthoxylum bungeanum[J]. Agriculture and Technology, 2019, 39(15): 12-13. (in Chinese)
[5]	王秀娟, 何静, 王斌, 等. 花椒流胶病病原鉴定及其生物学特性[J]. 甘肃农业大学学报, 2019, 54(1): 123-128.
	WANG X J, HE J, WANG B, et al. Identification and biological characteristics of pathogens of Chinese pepper gummosis[J]. Journal of Gansu Agricultural University, 2019, 54(1): 123-128. (in Chinese with English abstract)
[6]	杜超. 花椒流胶病病原菌及化学防治研究[D]. 雅安: 四川农业大学, 2020.
	DU C. Study on pathogen and chemical control of gummosis of Zanthoxylum bungeanum[D]. Yaan: Sichuan Agricultural University, 2020. (in Chinese with English abstract)
[7]	冯江鹏, 邱莉萍, 梁秀燕, 等. 草莓胶孢炭疽菌拮抗细菌贝莱斯芽孢杆菌JK3的鉴定及其抗菌活性[J]. 浙江农业学报, 2020, 32(5): 831-839.
	FENG J P, QIU L P, LIANG X Y, et al. Identification of antagonistic bacteria Bacillus velezensis JK3 against anthracnose of strawberry and its antipathogenic activity[J]. Acta Agriculturae Zhejiangensis, 2020, 32(5): 831-839. (in Chinese with English abstract)
[8]	ADELEKE B S, AYILARA M S, AKINOLA S A, et al. Biocontrol mechanisms of endophytic fungi[J]. Egyptian Journal of Biological Pest Control, 2022, 32(1): 46.
[9]	YAN L, ZHU J, ZHAO X X, et al. Beneficial effects of endophytic fungi colonization on plants[J]. Applied Microbiology and Biotechnology, 2019, 103(8): 3327-3340.
[10]	ADELEKE B S, BABALOLA O O. The plant endosphere-hidden treasures: a review of fungal endophytes[J]. Biotechnology & Genetic Engineering Reviews, 2021, 37(2): 154-177.
[11]	ATALA C, ACUÑA-RODRÍGUEZ I S, TORRES-DÍAZ C, et al. Fungal endophytes improve the performance of host plants but do not eliminate the growth/defence trade-off[J]. The New Phytologist, 2022, 235(2): 384-387.
[12]	GUPTA S, KULKARNI M G, WHITE J F, et al. Epigenetic-based developments in the field of plant endophytic fungi[J]. South African Journal of Botany, 2020, 134: 394-400.
[13]	HU Z B, WU Z H, SU Q H, et al. Metabolites with phytopathogenic fungi inhibitory activities from the mangrove endophytic fungus Botryosphaeria ramose[J]. Bioorganic Chemistry, 2020, 104: 104300.
[14]	李梅, 田莹, 蒋细良. 植物内生木霉菌研究进展[J]. 中国生物防治学报, 2020, 36(2): 155-162.
	LI M, TIAN Y, JIANG X L. Advances in research on endophytic Trichoderma in plants[J]. Chinese Journal of Biological Control, 2020, 36(2): 155-162. (in Chinese with English abstract)
[15]	文才艺, 吴元华, 田秀玲. 植物内生菌研究进展及其存在的问题[J]. 生态学杂志, 2004, 23(2): 86-91.
	WEN C Y, WU Y H, TIAN X L. Recent advances and issues on the endophyte[J]. Chinese Journal of Ecology, 2004, 23(2): 86-91. (in Chinese with English abstract)
[16]	STROBEL G A. Fungal endophytes in plants[M]. Switzerland: MDPI, 2018.
[17]	陈金阳, 陆儒涵, 王玲, 等. 药用植物内生菌抗氧化活性研究进展[J]. 中草药, 2016, 47(20): 3720-3727.
	CHEN J Y, LU R H, WANG L, et al. Recent progress in study on anti-oxidant activity of endophytes in medicinal plants[J]. Chinese Traditional and Herbal Drugs, 2016, 47(20): 3720-3727. (in Chinese with English abstract)
[18]	李磊, 许敏, 王美琴. 生防菌解淀粉芽孢杆菌Ht-q6可湿性粉剂的研制及对番茄病害的田间防效[J]. 中国生物防治学报, 2018, 34(5): 738-745.
	LI L, XU M, WANG M Q. Preparation of biocontrol agent Bacillus amyloliquefaciens Ht-q6 wettable powder and the field control against tomato diseases[J]. Chinese Journal of Biological Control, 2018, 34(5): 738-745. (in Chinese with English abstract)
[19]	韩忠明, 孙卓, 王妍, 等. 防风根腐病拮抗真菌的筛选鉴定及生防作用研究[J]. 中国生物防治学报, 2022, 38(5): 1288-1295.
	HAN Z M, SUN Z, WANG Y, et al. Screening, identification and biological control of antagonistic fungus against root rot of Saposhnikovia divaricata[J]. Chinese Journal of Biological Control, 2022, 38(5): 1288-1295. (in Chinese with English abstract)
[20]	方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社, 1998.
[21]	张小彦, 何静, 侯彩霞, 等. 枸杞根腐病菌拮抗菌株的筛选与鉴定[J]. 浙江农业学报, 2020, 32(5): 858-865.
	ZHANG X Y, HE J, HOU C X, et al. Screening and identification of antagonistic strains of wolfberry root rot[J]. Acta Agriculturae Zhejiangensis, 2020, 32(5): 858-865. (in Chinese with English abstract)
[22]	魏景超. 真菌鉴定手册[M]. 上海: 上海科学技术出版社, 1982.
[23]	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.
[24]	许乐, 王子强, 张爽, 等. 丹参根腐病拮抗细菌筛选、鉴定及生防机理研究[J]. 中国生物防治学报, 2021, 37(4): 846-854.
	XU L, WANG Z Q, ZHANG S, et al. Screening, identification and biocontrol mechanism of antagonistic bacteria against root rot of Salvia miltiorrhiza[J]. Chinese Journal of Biological Control, 2021, 37(4): 846-854. (in Chinese with English abstract)
[25]	罗江会, 漆巨容, 眭顺照, 等. 重庆地区桃树流胶病发生规律研究[J]. 西南园艺, 2004(5): 1-3.
	LUO J H, QI J R, SUI S Z, et al. Study on the occurrence regularity of peach gum disease in Chongqing[J]. Southwest Horticulture, 2004(5): 1-3. (in Chinese)
[26]	石晶盈, 陈维信, 刘爱媛. 植物内生菌及其防治植物病害的研究进展[J]. 生态学报, 2006, 26(7): 2395-2401.
	SHI J Y, CHEN W X, LIU A Y. Advances in the study of endophytes and their effects on control of plant diseases[J]. Acta Ecologica Sinica, 2006, 26(7): 2395-2401. (in Chinese with English abstract)
[27]	HARMAN G E, HOWELL C R, VITERBO A, et al. Trichoderma species: opportunistic, avirulent plant symbionts[J]. Nature Reviews Microbiology, 2004, 2: 43-56.
[28]	HERMOSA R, VITERBO A, CHET I, et al. Plant-beneficial effects of Trichoderma and of its genes[J]. Microbiology, 2012, 158(Pt 1): 17-25.
[29]	DESHMUKH S K, GUPTA M K, PRAKASH V, et al. Endophytic fungi: a source of potential antifungal compounds[J]. Journal of Fungi, 2018, 4(3): 77.
[30]	ADAMS P B. The potential of mycoparasites for biological control of plant diseases[J]. Annual Review of Phytopathology, 1990, 28: 59-72.
[31]	姜钰, 董怀玉, 徐秀德, 等. 放线菌在植病生防中的研究进展[J]. 杂粮作物, 2005, 25(5): 329-331.
	JIANG Y, DONG H Y, XU X D, et al. Progress on the research of actinomycetes in biocontrol of plant disease[J]. Rain Fed Crops, 2005, 25(5): 329-331. (in Chinese with English abstract)
[32]	张富美, 尚晓静, 李思, 等. 蓝莓根腐病生防真菌的筛选鉴定及其生防机制分析[J]. 植物保护学报, 2022, 49(6): 1673-1684.
	ZHANG F M, SHANG X J, LI S, et al. Screening and identification of biocontrol fungi against blueberry root rot and studies on its biocontrol mechanisms[J]. Journal of Plant Protection, 2022, 49(6): 1673-1684. (in Chinese with English abstract)
[33]	宋晓宇. 内生真菌裂褶菌和间座壳菌活性挥发物抑菌作用及其机制初探[D]. 北京: 中国林业科学研究院, 2020.
	SONG X Y. Antifungal activity and preliminary mechanism of active volatiles from endophytic fungal Schizophyllum commune and Diaporthe spp.[D]. Beijing: Chinese Academy of Forestry, 2020. (in Chinese with English abstract)
[34]	郝苑汝, 庞俊倩, 赵鑫丹, 等. 核桃内生真菌SYS-5-2的鉴定及其抑菌活性[J]. 西北林学院学报, 2020, 35(1): 158-164.
	HAO Y R, PANG J Q, ZHAO X D, et al. Identification and antimicrobial activity of endophytic fungus SYS-5-2 from walnut[J]. Journal of Northwest Forestry University, 2020, 35(1): 158-164. (in Chinese with English abstract)
[35]	张美红, 王萌, 杨书珍, 等. β-蒎烯抑制柑橘意大利青霉作用机制初步研究[J]. 华中农业大学学报, 2018, 37(6): 91-97.
	ZHANG M H, WANG M, YANG S Z, et al. Possible action mode of beta-pinene against Penicillium italicum[J]. Journal of Huazhong Agricultural University, 2018, 37(6): 91-97. (in Chinese with English abstract)
[36]	钱秀萍, 杨庆尧. 几种营养条件对裂褶菌生长及产L-苹果酸的影响[J]. 食用菌学报, 1996, 3(3): 18-24.
	QIAN X P, YANG Q Y. Effects of some nutrient conditions on Mycelium growth and L-malic acid production of Schizophyllum commune[J]. Acta Edulis Fungi, 1996, 3(3): 18-24. (in Chinese with English abstract)
[37]	张伟, 任怡莲, 葛谦, 等. 不同基质配方对裂褶菌菌丝生长影响研究[J]. 食品研究与开发, 2022, 43(5): 130-133.
	ZHANG W, REN Y L, GE Q, et al. Effect of different matrix formulae on the mycelial growth of Schizophyllum[J]. Food Research and Development, 2022, 43(5): 130-133. (in Chinese with English abstract)
[38]	王恒煦, 徐伟慧, 杨友财, 等. 一株Fusarium oxysporum f. sp. niveum拮抗菌的筛选、鉴定及其抑菌特性[J]. 浙江农业学报, 2019, 31(10): 1671-1680.
	WANG H X, XU W H, YANG Y C, et al. Screening, identification, and characteristics of an antagonistic strain against Fusarium oxysporum f.sp. niveum[J]. Acta Agriculturae Zhejiangensis, 2019, 31(10): 1671-1680. (in Chinese with English abstract)
[39]	陈思杰, 张涛, 贾宝森, 等. 深色有隔内生真菌对枸杞根腐病菌抑菌活性[J]. 园艺学报, 2022, 49(7): 1519-1531.
	CHEN S J, ZHANG T, JIA B S, et al. Study on antibacterial activity of dark septate endophytes against Fusarium oxysporum in Chinese wolfberry[J]. Acta Horticulturae Sinica, 2022, 49(7): 1519-1531. (in Chinese with English abstract)

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花椒流胶病拮抗菌的分离鉴定及其生防机制

Isolation, identification and biocontrol mechanism of antagonistic fungus against Chinese pepper gummosis

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[5]	黄华毅, 王佳琳, 马荣, 梁英梅, 田呈明. 枯草芽孢杆菌STO-12抑菌活性及其抑菌物质分析[J]. 浙江农业学报, 2017, 29(1): 81-88.
[6]	王国平;鲁书玲;郑必强;章初龙 . 紫杉木霉ZJUF0986代谢产物及其对番茄灰霉病的抑制作用[J]. , 2008, 20(2): 0-108.
[7]	申屠旭萍;俞晓平. 银杏中抗病原真菌的内生真菌的分离筛选[J]. , 2006, 18(5): 0-320.
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