生态学报  2014, Vol. 34 Issue (23): 6789-6796

文章信息

王欣禹, 周勇, 任安芝, 高玉葆
WANG Xinyu, ZHOU Yong, REN Anzhi, GAO Yubao
内生真菌感染对宿主羊草抗病性的影响
Effect of endophyte infection on fungal disease resistance of Leymus chinensis
生态学报, 2014, 34(23): 6789-6796
Acta Ecologica Sinica, 2014, 34(23): 6789-6796
http://dx.doi.org/10.5846/stxb201303080377

文章历史

收稿日期:2013-03-08
网络出版日期:2014-03-19
内生真菌感染对宿主羊草抗病性的影响
王欣禹, 周勇, 任安芝, 高玉葆     
南开大学生命科学学院, 天津 300071
摘要:以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichloë bromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C. lunataC. sp.侵染后的病斑数或病斑长度,但对B. sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E. bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。
关键词内生真菌    病原菌    羊草    抗病性    
Effect of endophyte infection on fungal disease resistance of Leymus chinensis
WANG Xinyu, ZHOU Yong, REN Anzhi, GAO Yubao     
College of Life Science, Nankai University, Tianjin 300071, China
Abstract:Endophytes are commonly defined as fungi that live asymptomatically within healthy plant tissue, such as leaves, stems or roots, for a part or all of their life cycle. The largest plant family hosting these endophytes is Poaceae. Studies on the grass-endophyte symbiosis indicate that endophytes can not only protect the host grass from abiotic stresses but also enhance its resistance to biotic disturbance, including herbivores, nematodes, bacteria and pathogens. However, the response of grass-endophyte symbiosis to fungal pathogens is less studied. Most previous reports on resistance of endophytes to pathogens have focused on inhibition of fungal pathogens by endophytes in vitro or by inoculation of fungal pathogens on detached leaves of the symbiont, and these studies have demonstrated that endophytes can inhibit growth of certain species of fungal pathogens to some degree. Up to now, only a few studies have centered on influence of endophyte infection on disease resistance of live plants. In this paper we used the endophyte Epichloë bromicola, which was isolated from Leymus chinensis. L. chinensis is a natural grass, widely distributed in the Inner Mongolian steppe. Three species of fungal pathogens were chosen, i.e. Curvularia lunata, Bipolaris sorokiniana and Cladosporium sp.. The experiment comprised three parts, i.e. fungal pathogens inhibition experiment by endophyte, infected detached leaves and infected intact plants experiments. The questions were: (1) whether E. bromicola could inhibit the growth of the three fungal pathogens in vitro, and (2) whether endophyte-infected (E+) and endophyte-free (E-) plants differed in resistance to the pathogenic fungi in detached leaves and live plants of L. chinensis. The results suggested that E. bromicola significantly inhibited the growth of C. lunata, B. sorokiniana and C. sp. in vitro, but anti-fungal activities of the endophyte to different pathogenic fungi were different. The inhibition rates were 56.22, 46.93 and 45.15%, respectively. Culture filtrate of endophyte also effectively reduced pathogenic spore germination and the average germination rate were 30.4, 15.7 and 16.4%, respectively. The main antagonism mechanisms involved in competition and producing anti-fungal chemical compounds. As for our research, the main strategy was the latter. Leaf inoculation trial showed that all fungal pathogens were able to cause lesions on detached leaves regardless of endophyte status. Either the number or length of disease lesions on E+ L. chinensis leaves caused by C. lunata or C. sp. decreased compared with those on E- leaves. But lesion number and length of B. sorokiniana leaf spots were dramatically higher on E+ leaves compared with E- leaves. Detached leaves extraction inhibited three pathogens to varying degrees. The reason might be that some inhibitory substances differed in vitro and in the whole plant. The intact E+ leaves had greater resistance to all of three species of pathogens than E- leaves. The explanation may be that the defense of plant associated with endophyte increased through resistance enhancement and secondary metabolites production. In a word, the endophyte E. bromicola had a positive effect on disease resistance of the host plant.
Key words: endophyte    fungal pathogens    Leymus chinensis    disease resistance    

内生真菌(endophyte)是在植物体内完成全部或部分生活史,暂时不形成明显侵染的一大类真菌[1],其宿主植物涉及多个类群,但在禾本科植物中尤为常见。自1977年Bacon等[2]首次从毒性高羊茅(Festuca arundinacea Shreb.)中分离出内生真菌以后,内生真菌与植物之间关系的研究正式开展起来。如内生真菌可以增强宿主植物对许多有害昆虫的抗性[3, 4, 5],有些染菌植物对大型食草动物,特别是家畜产生毒性[6]。此外,内生真菌对线虫、细菌以及病原菌等其它生物也存在抗性[4, 7]。然而,相对于对昆虫及食草动物的研究,对共生体抗真菌活性的研究尚涉及不多[8, 9]

在过去的一个世纪,尽管在病原真菌致病性以及控制策略方面进行了大量的工作,但植物真菌病害仍然是一个巨大的农业问题[10, 11]。关于内生真菌和病原真菌关系的研究,早期实验多集中在体外纯培养的内生真菌或是共生体离体叶片对病原菌侵染的抗性方面。许多在离体条件下的工作都证实内生真菌对某些植物病原真菌有抑制作用,Siegel和Latch[12]发现:高羊茅中分离的内生真菌Phialophora-like sp.可以抑制多种禾草病原菌;Nan和Li[13]的研究表明:接种Alternaria alternataFusarium avenaceumFusarium culmorum 3d后,感染内生真菌的圆柱披碱草(Elymus cylindricus)离体叶片有更少、更小的病斑。然而,内生真菌-禾草共生体对病原菌的抵抗能力不仅与共生体双方的独立作用有关,而且与双方的相互作用有关,如Welty等[14]发现离体条件下获得的结果与在体实验并不一致,因而植物在体条件下对病原菌抗性的研究在近年来逐渐展开。但就仅有的研究来看,结果是令人鼓舞的,如在对非禾草内生真菌的研究中,Arnold等[15]就发现:感染内生真菌的可可树(Theobroma cacao)可以显著减少Phytophthora sp.对叶片的伤害;Herre等[16]也发现:内生真菌感染能够增强宿主植物抵御P. palmivora的侵染。内生真菌对宿主禾草抗病能力的增益效应一旦被证实,就可以将带有内生真菌的草种纳入抗病育种的范围,这必将对植物生长和农业生产有重要的理论意义与应用前景[17]

羊草(Leymus chinensis)是禾本科赖草属多年生草本植物,在内蒙古阿巴嘎旗,内生真菌感染率在60%以上[18]。本文以羊草-内生真菌共生体为研究对象,首先通过体外纯培养,进而比较染菌(E+)和不染菌(E-)羊草在离体和在体条件下对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)3种病原菌的抗病性,通过一系列病害相关指标的测定,以探究:(1)体外培养条件下,内生真菌是否能够抑制3种病原菌的生长;(2)羊草离体和在体条件下,染菌和不染菌植物对3种病原菌的抗性是否有差异?进而为在农业生态系统中全面了解和利用禾草内生真菌提供科学依据和基础资料。

1 材料和方法 1.1 实验材料

(1)内生真菌 分离自羊草(L. chinensis),菌落白色,绵状疏松,气生菌丝较丰富,产孢多,生长较慢。通过形态学和分子系统学相结合的方法,本课题组将它命名为Epichlo bromicola [19]

(2)病原菌 新月弯孢(C. lunata)和根腐离蠕孢(B. sorokiniana)由兰州大学草地农业科技学院提供;枝孢霉(C. sp.)由华南农业大学资源环境学院植物病理系提供。

(3)植物材料 2010年8月自内蒙古阿巴嘎旗(43°5′01′′N,115°20′36′′E)采回,种于网室。

1.2 实验方法 1.2.1 内生真菌的抑菌实验

内生真菌活菌对病原菌的抑制:参考刘晓光等的方法[20]进行,以计算抑菌率。

抑菌率(%)= (对照半径R0-对峙培养病原菌半径R)/对照半径R0×100%

内生真菌培养滤液对病原菌孢子萌发的抑制:参考张广民等的方法[21]制备内生真菌的培养滤液,之后在无菌条件下将病原菌接到平板PDA培养基上培养1周,制备成孢子悬液,并用血球计数板测量孢子浓度。参考谢凤行等[22]的方法,在清洁无菌的双凹载玻片的凹陷穴中,滴加200 μL的内生真菌培养滤液,晾干后加入150 μL病原菌孢子悬液,设加无菌水的为对照,培养10 h后检查孢子萌发率。

1.2.2 离体叶片的抗菌侵染实验

离体叶片的接种和病害的测定:参考Nan和Li的方法[13]进行,从接种24 h后每天观察病斑数,测量病斑长度。连续观测7 d后,测量离体叶片侵染后的孢子浓度。

生长速率(cm/d) = 菌落直径cm-0.5 cm 培养天数

叶片提取液对病原菌生长的抑制:参考田沛的方法[17]进行,24 h后开始连续7 d测量菌落直径。

1.2.3 在体植株的抗菌侵染实验

活体植株的接种和病害的测定:参考Nan和Li的方法[13]进行,喷完后立即用黑色塑料袋将盆罩住,使之保持湿润36 h后把塑料袋取下。7 d后,依次到每个花盆里剪取叶片,在每个花盆中随机剪取5片叶片,进行病斑数和病斑长度的测定。测定完成后,进行孢子浓度的测定。

1.3 数据处理

采用Microsoft Excel和SPSS13.0中的独立样本T检验(Independent-samples T Test)进行处理和分析。

2 结果与分析 2.1 内生真菌的抑菌实验 2.1.1 内生真菌活菌对病原菌的抑制

对峙培养的结果表明,3种病原菌的生长速度均快于内生真菌,但内生真菌对3种病原菌都有抑制作用,只是不同病原菌受抑制的表现形式不同。在与C. lunata的对峙培养中,接种的第5天两菌相交。相交时C. lunata的菌落半径远小于其对照的半径,菌丝密度较对照也明显稀疏,菌落边缘颜色加深,并产生清晰的拮抗带,抑菌率达56.22%(图 1A,B和表 1)。在与B. sorokiniana的对峙培养中,接种第5天两菌相交。相交时B. sorokiniana的菌丝密度较对照变化不明显,但在两菌相邻的边缘菌丝密度很低,且菌落半径明显小于对照,抑菌率达46.93%(图 1C,D和表 1)。在与C. sp.的对峙培养中,两菌在接种第6天相交。相交时C. sp.的菌丝密度较对照明显稀疏,菌落半径也小于对照,并产生清晰的拮抗带,抑菌率为45.15%(图 1E,F和表 1)。

图 1 内生真菌对病原菌抑制作用 Fig. 1 Inhibition of fungal pathogens by endophyte A:新月弯孢对照Curvularia lunata control; B:新月弯孢与Epichlo bromicola对峙培养C. lunata antagonistically cultured with E. bromicola; C:根腐离蠕孢对照Bipolaris sorokiniana control; D:根腐离蠕孢与E. bromicola对峙培养B. sorokiniana antagonistically cultured with E. bromicola; E:枝孢霉对照Cladosporium sp. control; F:枝孢霉与E. bromicola对峙培养; C. sp. antagonistically cultured with E. bromicola
表1 内生真菌的抑菌率以及培养滤液对病原菌孢子萌发率的影响 Table 1 Inhibition rate of endophyte and Effect of culture filtrate on germination rate of the pathogenic spores
病原菌 Pathogens抑菌率 Inhibition rate/%平均萌发率Average germination rate/%
对照Control内生真菌E. bromicola
新月弯孢 Curvularia lunata56.2291.6b30.4a
根腐离蠕孢 Bipolaris sorokiniana46.9384.0b15.7a
枝孢霉 Cladosporium sp.45.1589.6b16.4a
2.1.2 内生真菌培养滤液对病原菌孢子萌发的抑制

在室温下培养10 h后,显微镜下观察孢子萌发可知(表 1),与对照相比,经内生真菌培养滤液处理过的病原菌孢子萌发率均有不同程度的降低,其中以C. lunata受抑制程度最小,而其它两种病原菌受抑制程度较大;同时还发现,内生真菌对病原菌的抑制作用不仅表现在孢子的萌发上,而且也表现在萌发孢子的生长上,经内生真菌培养滤液处理过的病原菌孢子即使萌发,其上长出的菌丝长度也显著低于对照(图 2)。

图 2 内生真菌培养滤液对C. lunata孢子萌发的影响 Fig. 2 Influence of culture filtrate of endophyte on germination of C. lunata spores 黑色箭头所指的是病原菌孢子上长出的菌丝
2.2 离体叶片的抗菌侵染实验 2.2.1 羊草离体叶片上病害及侵染后病原菌孢子浓度的测定

不论是否感染内生真菌,所接种的3种病原菌均能在48 h内在离体叶片上形成明显可见的病斑。如表 2所示,在接种C. lunata后,E-离体叶片上的病斑数一直有高于E+叶片的趋势,这种差异从第5天开始达到显著,但病斑长度二者未见显著差异,经显微镜检测,离体叶片侵染后的孢子浓度E-显著高于E+。但是接种B. sorokiniana后,从第3天开始,E+离体叶片上的病斑数就显著高于E-,到第5天,病斑长度也显著高于E-,侵染后E+和E-离体叶片上的孢子浓度具有显著差异。与前两种病原菌相比,接种C. sp.后,E+和E-离体叶片上的病斑数在测定期内没有显著差异,但病斑长度从第6天开始,E-叶片上的病斑长度显著高于E+,而E+和E-离体叶片侵染后的孢子浓度没有显著差异。

表2 染菌 (E+) 和不染菌 (E-) 羊草离体叶片受病原菌侵染后的病斑数和病斑长度以及侵染前-后病原菌的孢子浓度 Table 2 Mean number and length of lesions developing on detached leaves of E+ and E- L. chinensis after inoculation with pathogens and Spore concentration of pathogens(×105/ mL)before and after inoculation
病原菌 Pathogens接种后 的天数/d Days after inoculation叶片病斑数 Mean number of lesions 病斑长度/mm Mean length of lesions 病原菌的孢子浓度/(×105/ mL) Spore concentration of pathogens
接种前 Before inoculation 接种后 After inoculation
E+E- E+E- E+E-
E+:感染内生真菌的羊草叶片,Endophyte-infected leaves;E-:不感染内生真菌的羊草叶片,Endophyte-free leaves
新月弯孢 20.55a 1.20a6.50a 7.60a19.21.83a3.17b
C. lunata31.50a 2.90a6.92a 7.89a
42.40a 3.65a7.65a 8.54a
52.50a 5.90b7.82a8.98a
64.25a 8.05b7.87a 9.22a
75.50a10.45b8.49a 9.49a
根腐离蠕孢 21.00a0.65a9.81a 7.07a7.832.17b1.00a
B. sorokiniana33.50b1.45a9.94a 7.27a
45.35b 2.80a10.06a 7.51a
57.75b 4.10a10.49b 7.64a
69.30b6.45a10.99b 7.97a
712.75b8.70a11.77b 9.71a
枝孢霉 C. sp.21.10a 0.95a6.44a 7.31a6.831.67a 1.83a
32.05a 2.50a6.88a 7.73a
42.80a 3.30a7.10a 8.06a
54.19a 4.70a7.49a 8.63a
64.81a 5.55a7.64a 10.25b
79.31a 9.95a8.64a10.66b
2.2.2 离体叶片提取液对病原菌生长的抑制

内生真菌感染对C. sp.的抑制作用最为显著,表现在从生长的第2天开始,E+的抑制作用就显著高于E-;内生真菌感染对C. lunata的抑制作用次之,且从第2天到第5天,E+和E-提取液对病原菌的影响无显著差异,从第6天开始,E+的抑制作用才显著高于E-;内生真菌感染对B. sorokiniana的抑制作用不明显,只在生长的第3天,E+的抑制作用显著高于E-,从第4天开始,E+和E-之间再未出现显著差异(图 3)。

图 3 叶片提取液对3种病原菌生长的抑制作用 Fig. 3 The inhibition of leaves extraction to growth of the three pathogens 不同的字母表示同一天的生长有显著差异(P<0.05),相同字母表示差异不显著(P>0.05)
2.3 在体植株的抗菌侵染实验 2.3.1 羊草在体植株上病害及侵染后病原菌孢子浓度的测定

表 3所示,接种3种病原菌7 d后,在所侵染的E+和E-植株中,E-植株的发病率均显著高于E+植株,即E-植株叶片上的病斑数显著高于E+。但对于病斑长度,除了受C. lunata侵染后E-植株叶片的病斑长度显著高于E+外,其余两种病原菌侵染后,E+和E-植株叶片病斑长度未见显著差异。经显微镜检测,接种3种病原菌后,E-植株叶片上的孢子浓度均高于E+,且二者都具有显著差异。

表3 染菌 (E+) 和不染菌 (E-) 羊草在体植株受病原菌侵染7 d后的病斑数和病斑长度以及侵染前-后病原菌的孢子浓度 Table 3 Mean number and length of lesions developing on intact plants of E+ and E- L. chinensis after inoculation with pathogens 7 d and Spore concentration of pathogens before and after inoculation
病原菌 Pathogens叶片病斑数 Mean number of lesions 病斑长度/mm Mean length of lesions 病原菌的孢子浓度/(×105/ mL) Spore concentration of pathogens
接种前 Before inoculation 接种后 After inoculation
E+E- E+E- E+E-
新月弯孢 C. lunata3.13a4.87b6.39a10.27b4.170.39a0.78b
根腐离蠕孢 B. sorokiniana2.67a4.47b7.42a6.86a1.830.33a0.67b
枝孢霉C. sp.3.33a5.53b6.78a7.13a5.831.11a1.94b
3 讨论

已有研究表明,内生真菌不同菌系的纯培养都可以抑制植物病原菌的生长。例如,Neotyphodium coenophialum可以有效的抑制A. alternataC. cladosporioidesRhizoctonia cerealis的生长[23];Li等[24]通过分离自甘肃醉马草(Achnatherum inebrians)的14种内生真菌菌系对几种病原菌的体外培养发现:绝大多数内生真菌的菌系对A. alternataC. lunataB. sorokiniana的菌落生长有不同程度的抑制作用,出现明显可见的抑菌圈。我们的研究显示:E. bromicola可以显著抑制C. lunataB. sorokinianaC. sp.的菌落生长,但对不同病原菌的抑制作用存在差异,抑菌率分别为56.22%、46.93%、45.15%。内生真菌的培养滤液对不同病原菌孢子萌发率的抑制作用也不同,三者的平均萌发率分别为30.4%、15.7%和16.4%。关于内生真菌的抑菌机制,主要包括竞争作用和产生抑菌活性物质[22]。竞争作用主要是对养分和生存空间的争夺,这在生长速度较快的真菌中体现较为明显,而实验中所使用的内生真菌生长速度较慢,因此竞争可能不是起主要作用的机制,结合菌滤液对孢子萌发的抑制结果推测,抑菌机制主要为抗菌物质的产生。Yue等[25]已经从EpichloNeotyphodium物种中鉴定出一系列能够抵抗病原菌活性的化合物,其中最重要的抗菌物质是吲哚类衍生物,其余还有倍半萜烯类、醌类、酚类、多肽等,且不同的抑菌物质抑菌活性有所不同。

在以离体叶片为材料的研究中,Li 等[24]研究N. gansuense、醉马草(A. inebrians)和植物病原菌之间的相互作用发现:内生真菌的感染可以有效降低A. alternataF. chlamydosporumF. oxysporumF. solani侵染后叶片的病斑数和病斑长度;Tian等[26]针对10种病原菌对黑麦草(Lolium perenne)E+和E-离体叶片的研究表明:感染内生真菌的E+叶片对多数病原菌具有抗病优势。然而,苏珍珠等[27]在马铃薯晚疫病菌(P. infestans)侵染离体叶片的实验中却发现:有些内生真菌菌株的存在反而扩大了病斑的面积,其叶片上存在更多的病菌孢子囊。在本研究的体外纯培养实验中,内生真菌对供试的3种病原菌均有显著的抑制作用,但离体叶片侵染实验中,内生真菌却对B. sorokiniana不起抑制作用,反而加重了E+叶片病情的发展。这种不一致性也许是由于对峙培养和离体叶片接种的生长条件不同所引起的[24]。Christensen[7]把这种不一致性归结为在植物体外和体内内生真菌产生的抗菌物质数量不同,当抵抗某一种菌起作用的有效成分浓度降低到一定值时,具有促进生长的成分将对这种病原菌的菌丝生长发挥促进作用[27]。田沛[17]认为,内生真菌对病原菌抗性的产生可能是由于真菌与真菌之间本身就有拮抗性,而离体纯培养直接使内生真菌产生的抗真菌活性物质最大限度的发挥作用,没有在活体中被稀释,而活体中抗菌物质产生的数量不足以保护植物免受病原菌的伤害。

在体植株的抗菌侵染实验结果显示,内生真菌对3种病原菌均有抗性,在受到这3种病原菌侵染7 d后,E-植株叶片上的病斑数均显著高于E+植株,且侵染后病原菌的孢子浓度E-也显著高于E+。与离体实验相比,对于C. lunataC. sp.侵染的表现基本具有一致性,而对B. sorokiniana的侵染则出现了不一致的情况。这可能是由于内生真菌长期生存于植物细胞内或细胞间隙这一特殊环境,在协同进化的过程中,它们与宿主植物建立了和谐的亲密关系,在体植株可以调节内生真菌的生物学特性,使之产生十分丰富的次生代谢产物,其中有的成分具有杀菌活性[28]。反过来,内生真菌也可能诱导宿主发生防御反应,产生一系列抗菌物质。Pirttil等[29]提出,当病原菌入侵时,内生真菌可以诱导宿主快速发生一系列防御反应,诱导植物产生更多的抗病相关物质。或者,内生真菌可以通过强化宿主的能力,如促进生长、提高抗逆性等间接增加植物对病害的抗性。Kuldau和Bacon[30]指出,植物生长的增加可以加强植物对一系列生物和非生物胁迫的抗性,其中包括病原菌的侵染。因此,我们的研究证明内生真菌E. bromicola对宿主植物羊草在抗某些病原菌侵染方面确实存在一定的增益作用。

目前国内外关于禾草内生真菌抗性的研究已经越来越多,研究者已经发现内生真菌的培养滤液对植物寄生线虫Meloidogyne incognita具有很高的致死率,并且内生真菌还可以增强与之共生的羽茅、高羊茅等植物抵御这种线虫的抗性[31],说明内生真菌可能成为一个存在巨大潜力的新型生物防治剂。这表明内生真菌不仅具有理论研究的深度,同时具有多方面的实际应用价值,阐明内生真菌与宿主禾草之间的相互关系及其在抗病及抗虫方面的机理定将对农牧业的发展带来十分积极的影响。

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