Isolation and identification of endophytic fungi from <i>Cymbidium faberi</i> ‘Hongxiangfei’ and their bacteriostatic effect in vitro

JIANG Chuchu; XIN Jingjing; XIA Shuquan; LUO Ping; SHAO Guoyuan; CUI Yongyi

doi:10.11833/j.issn.2095-0756.20220578

Volume 40 Issue 4

Aug. 2023

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Article Navigation > Journal of Zhejiang A&F University > 2023 > 40(4): 783-791

JIANG Chuchu, XIN Jingjing, XIA Shuquan, LUO Ping, SHAO Guoyuan, CUI Yongyi. Isolation and identification of endophytic fungi from Cymbidium faberi ‘Hongxiangfei’ and their bacteriostatic effect in vitro[J]. Journal of Zhejiang A&F University, 2023, 40(4): 783-791. doi: 10.11833/j.issn.2095-0756.20220578

Citation:

JIANG Chuchu, XIN Jingjing, XIA Shuquan, LUO Ping, SHAO Guoyuan, CUI Yongyi. Isolation and identification of endophytic fungi from Cymbidium faberi ‘Hongxiangfei’ and their bacteriostatic effect in vitro[J]. Journal of Zhejiang A&F University, 2023, 40(4): 783-791. doi: 10.11833/j.issn.2095-0756.20220578

Isolation and identification of endophytic fungi from Cymbidium faberi ‘Hongxiangfei’ and their bacteriostatic effect in vitro

doi: 10.11833/j.issn.2095-0756.20220578

1.
College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
2.
Tongxing Planting Professional Cooperative of Jiuzhaigou County in Sichuan, Jiuzhaigou 623499, Sichuan, China

Received Date: 2022-09-12
Accepted Date: 2023-01-01
Rev Recd Date: 2022-12-21
Publish Date: 2023-08-20

Abstract

Objective The objective is to screen and obtain the biocontrol fungi against orchid plant diseases from Cymbidium faberi ‘Hongxiangfei’ , so as to provide scientific basis for mycorrhizal cultivation and green biological control of C. faberi. Method The endophytic fungi were isolated, purified and identified from the healthy root segments of C. faberi ‘Hongxiangfei’ . With 5 common plant pathogens as indicator fungi, the endophytic fungi with the best biocontrol effect were screened by plate confrontation culture method, and their bacteriostatic effect was verified by in vivo inoculation. Result 6 endophytic fungi were isolated from the root segment culture of healthy plants of C. faberi ‘Hongxiangfei’ , and 4 strains belonged to Chaetomium spp., 1 strain belonged to Fusarium spp. and 1 belonged to Scedosporium spp. It was found in the plate confrontation culture that all 6 endophytic fungi had certain inhibitory effect on 5 pathogenic strains, among which Z2 strain (Fusarium solani) had an obvious inhibitory effect on F. oxysporum (the causative agent of orchid stem rot), with an inhibition rate of 67.51%. The inhibition rate of Z3 strain (Dichotomopilus funicola) against Colletotrichum gloeosporioides (the pathogen of orchid anthracnose) was 68.56%. The in vivo inoculation test of Cymbidium goeringii ‘Green Cloud’ showed that the rate of diseased leaves and disease index of the experimental group treated with Z3 strain were significantly lower than those of the control group (P＜0.01). Conclusion Z3 is selected as the dominant antagonistic biocontrol strains against orchid diseases. [Ch, 4 fig. 2 tab. 35 ref.]
- Cymbidium faberi ‘Hongxiangfei’,
- endophytic fungi,
- Chaetomium spp.,
- Fusarium spp.,
- bacteriostatic

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兰科Orchidaceae植物是单子叶植物中的第一大科^[1]，兰花作为中国“十大名花”之一，誉有“花中四君子”的称号，其中许多种类可作为观赏植物或药用植物加以利用，经济价值较高。在兰花栽培中，常发生多种病害导致其生长及开花品质受到较大的影响，主要有胶胞炭疽菌Colletotrichum glososporioides引起的兰花炭疽病，尖孢镰刀菌Fusarium oxysporum引起的兰花茎腐病，灰葡萄孢菌Botrytis cinerea引起的兰花灰霉病等^[2]。目前，兰花病虫害的防控主要采用化学农药防治，即对病害植物发病部位进行喷施、灌根或涂抹处理，但长期使用化学农药，不仅会造成环境污染，破坏生态平衡，还会导致植物病原菌对化学农药产生抗性，影响防治效果^[3]。绿色生物防治是指利用拮抗微生物对病原菌进行防治，有防病抑病和保护环境的双重功效^[4]。目前，从植物中分离有益内生真菌用作生物防治已成为一大热点。研究表明：内生真菌不仅能促进植物生长发育，还能提高宿主植物抗逆性^[5−6]。兰科植物是典型的内生菌根植物，其根内分布着能促进植株生长发育的菌丝体，两者互利共生是兰科植物能在生态系统中存活并发挥作用的重要因素^[7]。有研究已从建兰Cymbidium ensifolium、蕙兰C. faberi、寒兰C. kanran^[8−9]、带叶兜兰Paphiopedilum hirsutissimum^[10]等多种野生兰科植物中分离获得的内生真菌均对植株表现出促生效应；从野生青天葵Nervilia fordii^[11]、野生春兰C. goeringii^[12]、铁皮石斛Dendrobium officinale^[4]中分离的内生真菌对叶斑病、根腐病、炭疽病等常见植物病害具有良好的拮抗作用。

蕙兰‘红香妃’C. faberi‘Hongxiangfei’原产于四川九寨沟高海拔地区，因其叶片基部呈紫红色且花开红色而得名，拥有典雅的花色和淡雅的花香，深受大众喜爱。但由于人为过度采挖，导致蕙兰‘红香妃’野生资源处于濒危灭绝状态。本研究对蕙兰‘红香妃’内生真菌进行分离鉴定，旨在筛选出能有效控制病害的生防真菌资源，为开发兰花生物肥及绿色生物防治研究提供科学依据。

1. 材料与方法

1.1. 材料

1.1.1. 供试植物和菌株

蕙兰‘红香妃’取自四川省九寨沟县，春兰‘绿云’C. goeringii‘Green Cloud’来自浙江农林大学花卉栽培与遗传改良实验室。抑菌试验中的指示菌菌株为该实验室保存的兰花茎腐病病原菌(尖孢镰刀菌)、兰花炭疽病病原菌(胶孢炭疽菌)、番茄早疫病病原菌(链格孢菌Alternaria alternata)、草莓根腐病病原菌(三线镰刀菌F. tricinctum)、水稻恶苗病病原菌(藤仓镰孢菌F. fujikuroi)等5种病原菌。

1.1.2. 培养基

马铃薯葡萄糖琼脂培养基(PDA)、马铃薯葡萄糖肉汤培养基(PDB)^[6]。

1.2. 菌株的分离与纯化

1.2.1. 内生真菌分离

采用组织分离法分离内生真菌^[13]。取蕙兰‘红香妃’新鲜健康根段，先用洗洁精洗去根表面的污垢，并用流水冲洗30 min。将洗净后的根段置于超净工作台，于体积分数为75%乙醇溶液中浸泡30 s，用无菌水漂洗2次，体积分数为2%次氯酸钠溶液表面灭菌5 min，再用无菌水漂洗3~5次；将已表面灭菌的根切成0.5 cm的根段，切面朝向PDA培养基，在28 ℃下恒温培养，每天观察菌丝生长情况。为验证表面灭菌是否彻底，取适量最后一次漂洗组织的无菌水，涂布于PDA培养基上，同等条件培养，观察有无菌落产生。

1.2.2. 菌株纯化

对已分离的不同形态的菌落进行梯度稀释纯化^[14]。取5个2 mL无菌离心管，依次加入1 000、900、900、900、900 μL无菌水；用无菌牙签刮取适量菌落于1号离心管中，充分摇匀后吸取100 μL至2号离心管中，摇匀后再吸取100 μL至3号离心管中，以此类推，将5管稀释成10、100、1 000、10 000、100 000倍等；每管取50 μL菌落悬浮液均匀涂在PDA培养基上，每个倍数重复3次，在28 ℃恒温培养箱中暗培养5~7 d后，取最边缘的菌落接到新的PDA培养基上培养，多次转接直至菌落呈单一，将纯化的真菌于4 ℃冰箱中保存备用。

1.2.3. 发酵液制备

使用5 mm打孔器在已纯化菌饼边缘打孔，将菌饼投入到事先制备好的PDB培养基中。PDB培养基使用500 mL锥形瓶盛装，装液量为200 mL，在180 r·min⁻¹、28 ℃摇床中培养7 d，后用无菌水稀释至1×10⁹ CFU·L⁻¹以备用^[15]。

1.3. 内生真菌的鉴定

1.3.1. 真菌的形态观察

菌落宏观形态观察：取已纯化的真菌接种于新的PDA培养基上，28 ℃恒温培养7 d后观察菌落颜色、质地、形状等特征。菌落微观形态观察：用无菌牙签挑取微量菌丝于载玻片上染色制成临时装片，于光学显微镜下，观察菌丝体、孢子的大小及形状等显微特征^[16]。

1.3.2. 真菌的分子鉴定

采用CTAB法提取真菌DNA^[17]，采用通用引物ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)进行PCR扩增真菌ITS序列。PCR反应体系(20 μL)：ddH₂O 7 μL，ITS1 1 μL，ITS4 1 μL，Easy Taq Mix酶10 μL，DNA模板1 μL。PCR反应程序：94 ℃预变性4 min，94 ℃变性30 s，55 ℃退火30 s，72 ℃延伸45 s，72 ℃延伸10 min，共35个循环。切胶回收目的片段，送擎科生物科技有限公司测序。

1.3.3. 构建进化树

目的序列于美国国家生物技术信息中心(NCBI)数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行Nucleotide BLAST比对，选取并下载同源性较高的序列。先用Clustal X软件进行序列比对，再用MEGA 6.0软件中的邻接法(neighbor-joining method)构建系统进化树，其中Bootstrap检验重复次数为1 000次。

1.4. 体外抑菌试验

采用平板对峙法初步筛选出具有拮抗作用的内生真菌。在PDA平板中间位置放置直径为5 mm的指示菌菌饼，离指示菌菌饼上下左右各2 cm处放置相同大小的内生真菌，每个处理3次重复。以5 mm中间只接指示菌为对照，在28 ℃恒温培养箱中培养7 d后，用十字交叉法测量病原菌的菌落直径。计算内生真菌对病原菌的生长抑制率：抑制率=[(对照菌落直径−处理菌落直径)/对照菌落直径]×100%^[18]。

1.5. 活体植株病菌接种

选择胶孢炭疽菌初筛抑菌效果较好的内生真菌，针刺接种病菌于春兰‘绿云’进行活体试验^[19]。设定2组：对照组为清水+病原菌(胶孢炭疽菌)；处理组为内生真菌+病原菌(胶孢炭疽菌)。5株为1组，重复3组，用清水和内生真菌发酵液进行灌根和喷施处理30 d，再用病原菌胶孢炭疽菌发酵液进行灌根和喷施处理30 d。病情指数设定为6级，具体分级标准见表1。统计计算病叶率、病情指数、防治效果。病叶率= 病叶数/调查总叶数×100%；病情指数=∑(各级病株数×相对级数值)/(调查总株数×最高级值)×100%；防治效果=(对照病情指数−处理病情指数)/对照病情指数×100%。

病情等级代表值病叶数量/片

0 0 0
1 1 1~3
2 3 4~5
3 5 6~7
4 7 8~9
5 9 ≥10

Table 1. Classification standard of disease

1.6. 数据分析

数据采用Excel 2010和SPSS 22.0软件的Duncan检验法对各处理组数据进行0.05或0.01水平下的差异显著性分析。

3. 讨论

近年来，在园艺植物栽培中，生物防治作为一种环境友好型防治手段，越来越受到人们的关注。生防菌主要通过产生活性物质、重寄生或竞争营养来抑制病原微生物的生长及危害^[2]。据研究报道：引起兰花茎腐病的病原菌为镰刀菌，镰刀属真菌多为土传类病原菌，主要侵染植物根茎部，导致根腐、茎腐、枯萎等^[22]。镰刀属真菌虽被广泛认为是引起病害的主要致病菌^[23−24]，但会以致病菌或非致病菌的形式与兰花共生^[25]，而非致病性镰刀属真菌通常会以分解者或共生者的身份与兰花共生^[26]，有益于兰花的生长发育^[27]。本研究从蕙兰‘红香妃’根部分离、纯化、鉴定出6株内生真菌，其中4株属于毛壳属，1株属于镰刀属，另1株属于赛多孢属；同时其与5株植物病原指示菌进行对峙培养发现：6株内生真菌对5株病原菌均有抑制效果，其中腐皮镰刀菌对尖孢镰刀菌抑制效果最佳，说明腐皮镰刀菌可能与蕙兰‘红香妃’建立共生关系，并有益于其生长和抗病性。李梅蓉等^[4]发现：镰刀菌对铁皮石斛圆斑病具有良好的抑制作用。陈大为等^[28]在对黄瓜Cucumis sativus白粉病的研究中也验证了腐皮镰刀菌可增强黄瓜植株抗性，抑制白粉病发病率。

炭疽病是园艺作物中寄主十分广泛的一种病害，引起兰花炭疽病的病原菌是炭疽菌属Colletotrichum spp.真菌，主要危害植物叶片，导致叶枯，其中胶孢炭疽菌是最为常见的病原菌^[29−30]。本研究从蕙兰‘红香妃’中分离得到的蝇生二叉毛壳菌，对胶孢炭疽菌抑制效果显著。而毛壳属是极具生防潜力的真菌，在防治植物病害方面尤为突出^[31−32]。PHONG等^[33]探究了毛壳属真菌对茶树Camellia sinensis枯萎病的防治效果，发现毛壳属真菌均能显著抑制枯萎病菌菌丝的生长和产孢。SONG等^[34]发现：毛壳属真菌对水稻Oryza sativa稻瘟病病菌具有拮抗作用。徐姣等^[35]将5种人参Panax ginseng内生真菌与6种人参病原菌进行拮抗试验，发现毛壳属菌株对人参病害的抗菌谱较广，说明毛壳属真菌对植物不同病原菌均存在一定的拮抗作用，且同属不同种真菌发挥作用因寄主而异。为进一步验证抑菌效果，本研究选择菌株Z3蝇生二叉毛壳菌对春兰‘绿云’开展活体植株试验。结果表明：处理组病叶率、病情指数均极显著低于对照组，说明菌株Z3蝇生二叉毛壳菌具有较优的抑菌效果，有望开发为兰科植物生防菌，为兰科植物栽培及绿色生防提供科学依据。

4. 结论

本研究通过对四川省九寨沟县健康的蕙兰‘红香妃’根部内生真菌进行分离纯化、形态学鉴定及构建系统发育树等，确定内生真菌共6株。对峙试验明确6株内生真菌的抑菌谱较广，其中菌株Z2 (F. solani)和菌株Z3 (D. funicola)对5株病原菌均具有显著的抑制效果，具有生防潜力；通过菌株Z3对春兰‘绿云’活体植株进行浇灌喷施试验，证实菌株Z3具有一定的生防功效，但其抑菌相关机制有待进一步研究。

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处理	病叶率/%	病情指数	防治效果/%
对照组	26.43	17.53 ± 2.61 a
处理组	14.81	10.27 ± 2.17 b	41.41
说明：病情指数中“±”后为标准差；同列不同小写字母表示差异极显著(P＜0.01)。