作物杂志, 2024, 40(6): 194-204 doi: 10.16035/j.issn.1001-7283.2024.06.026

生理生化·植物营养·栽培耕作

燕麦地上部可培养内生真菌多样性分析及其功能研究

李晓婷,1, 张婷婷2, 张艳丽1, 李志伟1, 韩丽1, 赵鑫瑶1, 张永平1, 李立军,1

1内蒙古农业大学农学院,010019,内蒙古呼和浩特

2乌兰察布市农林科学研究所,012000,内蒙古乌兰察布

Diversity Analysis and Function Study of Culturable Endophytic Fungi in Oat Shoot

Li Xiaoting,1, Zhang Tingting2, Zhang Yanli1, Li Zhiwei1, Han Li1, Zhao Xinyao1, Zhang Yongping1, Li Lijun,1

1Agricultural College of Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia, China

2Ulanqab Institute of Agriculture and Forestry Sciences, Ulanqab 012000, Inner Mongolia, China

通讯作者: 李立军,研究方向为寒旱区农作制与农业生态,E-mail:imaullj@163.com

收稿日期: 2023-07-28   修回日期: 2023-10-31   网络出版日期: 2024-07-30

基金资助: 内蒙古自治区科技计划项目(2022YFXZ0031)
呼和浩特市科技计划项目(2020-农-1)

Received: 2023-07-28   Revised: 2023-10-31   Online: 2024-07-30

作者简介 About authors

李晓婷,研究方向为农牧交错区农作制与农业生态,E-mail:lixt1229@163.com

摘要

为探究燕麦叶和茎中可培养内生真菌分布情况、群落组成及其功能特性,通过组织培养分离法对阴山北麓区燕麦拔节期叶片和茎的内生真菌分离纯化,利用ITS鉴定并分析其多样性,同时测定其溶磷、解钾和分泌生长素(IAA)能力。结果发现,燕麦的叶和茎中分离得到74株内生真菌,总定殖率为61.94%,总分离率为20.56%,经过分子鉴定属于2门12属,其中优势菌门为子囊菌门(Ascomycota),优势菌属为链格孢菌属(Alternaria,分离率=5.56%,分离频率=27.03%)和镰刀菌属(Fusarium,分离率=8.06%,分离频率=39.19%)。多样性分析发现,茎中分离的内生真菌多样性高于叶,其多样性指数和辛普森指数分别为3.51和0.78。相似性分析发现,叶和茎中内生真菌的相似系数为0.15。对分离得到的内生真菌的功能分析发现,79.73%的菌株具有溶磷能力,72.97%的菌株具有解钾能力,40.54%的菌株能够分泌IAA。根据内生真菌的功能将74株内生真菌分为4类,分别有41、5、21和7株菌株,最优第4类菌株大部分均具有溶磷、解钾能力,且分泌IAA的能力最强。燕麦地上部组织中的内生真菌物种较为丰富,可为内生真菌的应用提供菌种资源,不同内生真菌的功能差异较大,功能性较强菌株在农业生产中可能会发挥其潜在促生长特性。

关键词: 燕麦; 内生真菌; 多样性; 溶磷; 解钾; 生长素

Abstract

In order to investigate the distribution, community composition, and functional characteristics of culturable endophytic fungi leaves and stems of oats (Avena sativa L.), the endophytic fungi in leaves and stems of oats at the jointing stage in the northern foothills of Yinshan were isolated and purified by the tissue culture isolation method. Their diversity was identified and analyzed by using ITS, and the abilities of phosphate- solubilizing and potassium-solubilizing, secreting auxin (IAA) were measured. The results showed that a total of 74 endophytic fungi were isolated from the leaves and stems of oats, with the total colonization rate and the total isolation rate of 61.94% and 20.56%, respectively, they were identified as belonging to two phyla and 12 genera by molecular identification, among which Ascomycota was the dominant phylum, and Alternaria (isolation rate=5.56%, isolation frequency=27.03%) and Fusarium (isolation rate=8.06%, isolation frequency=39.19%) were the dominant species. Diversity analysis showed that the diversity of endophytic fungi isolated from stems was higher than those from leaves, with the diversity index and Simpson index being 3.51 and 0.78, respectively. Similarity analysis showed that the similarity coefficient of endophytic fungi in leaves and stems was 0.15. The functional analysis of the isolated endophytic fungi showed that 79.73% of the strains had phosphate- solubilizing, 72.97% had potassium-solubilizing, and 40.54% could secrete IAA. According to the function of endophytic fungi, 74 strains of endophytic fungi were divided into four categories, including 41, 5, 21 and 7 strains, respectively. Most of the optimal strains of the 4th category had the ability to dissolve phosphorus and potassium, and the ability to secrete IAA was the highest. There are abundant endophytic fungi species in the leaves and stems of oat, which would provide strain resources for the application of endophytic fungi. The functions of different endophytic fungi are quite distinct, and the strains with stronger functions may exert their potential growth-promoting characteristics in agricultural production, which is beneficial to the sustainable development of agriculture.

Keywords: Oat; Endophytic fungi; Diversity; Phosphate-solubilizing; Potassium-solubilizing; IAA

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本文引用格式

李晓婷, 张婷婷, 张艳丽, 李志伟, 韩丽, 赵鑫瑶, 张永平, 李立军. 燕麦地上部可培养内生真菌多样性分析及其功能研究. 作物杂志, 2024, 40(6): 194-204 doi:10.16035/j.issn.1001-7283.2024.06.026

Li Xiaoting, Zhang Tingting, Zhang Yanli, Li Zhiwei, Han Li, Zhao Xinyao, Zhang Yongping, Li Lijun. Diversity Analysis and Function Study of Culturable Endophytic Fungi in Oat Shoot. Crops, 2024, 40(6): 194-204 doi:10.16035/j.issn.1001-7283.2024.06.026

内生真菌是植物微生物的一部分,存在于健康的植物组织中,对宿主无不良影响[1]。植物体内有丰富的内生真菌,其多样性会受到寄主、地理位置、取样部位和生长环境等影响[2-4]。Ofek-Lalzar等[5]利用高通量测序技术从不同基因型的小麦中分离得到67个不同的分类单元,其核心菌落为链格孢属(Altemaria)。Yuan等[6]从野生水稻的根中分离获得58株内生真菌。辛赫文等[7]发现,小麦根、茎部有丰富的内生真菌,其中子囊菌是主要类群,根、茎部的优势类群分别是毛壳菌属(Chaetomium)和青霉菌属(Penicillium)。秦华伟等[8]从山东不同地区玉米叶片中分离获得857株内生真菌,其中链格孢属(Alternaria)和镰刀菌属(Fusarium)的定殖率较高。农业系统内生真菌丰富的多样性表明,农作物也是研究内生真菌的潜在资源。

内生真菌在陆地生态系统中发挥着关键作用,极大地影响植物进化、群落结构和生态适应性[9]。内生真菌与植物形成共生关系,参与植物体内的各种生物过程,增强宿主植物的环境适应性,提高生长速度,增强竞争力[10]。已有报道[11]表明,从植物中分离到的大多数内生真菌能够产生不同种类的植物生长激素,如吲哚乙酸(IAA,又称生长素)和赤霉素(GA)。内生真菌通过分泌有机酸溶解无机磷和有机磷[12],分解不溶性钾[13],还可固定空气中的氮[14]。植物―内生真菌共生体不仅会影响植物的生长和健康,还能有效改善土壤质量和养分循环[15]。总之,内生真菌作为天然的微生物资源,在提高农作物适应性、促进农业可持续发展等方面具有重要意义[16-17]

许多农作物地上组织中内生真菌群落的多样性、组成和功能性方面的研究仍有待挖掘,尤其是关于旱作区农作物内生真菌群落多样性的研究甚少。作为内蒙古特色作物,燕麦主要种植在阴山北麓农牧交错带,具有较强的耐旱、耐瘠薄特性。有关燕麦的研究主要集中于其抵抗非生物胁迫生理机制、高产栽培技术及种质资源方面,关于不同组织可培养内生真菌多样性及其功能的研究较少。因此,本研究探索农牧交错区燕麦地上部组织中存在的可培养内生真菌,并初步评价其多样性和分布情况。采用系统聚类分析和判别分析统计学方法综合评价燕麦内生真菌的功能,为燕麦内生真菌在农业生产中的开发利用提供理论基础。

1 材料与方法

1.1 样品采集

本研究于2019年6月10日在内蒙古武川县内蒙古农业大学试验基地的燕麦田中取样,取样时期为燕麦拔节期,选择长势均匀一致的燕麦,随机取10株健康无病症的植株,每间隔50 m取1次样,共取6次。所有样品去除表面杂物后立即置于塑料袋并及时带回实验室,置于4 ℃冰箱保存,于48 h内处理样品。

1.2 培养基配制

马铃薯葡萄糖琼脂培养基(PDA):去皮马铃薯200 g,葡萄糖20 g,琼脂15 g,水1000 mL。

有机磷液体培养基(每升):葡萄糖10 g,(NH4)2SO4 0.5 g,NaCl 0.3 g,KCl 0.3 g,MgSO4·7H2O 0.3 g,FeSO4·7H2O 0.03 g,MnSO4·4H2O 0.03 g,CaCO3 5 g,卵磷脂0.2 g,pH 7.0~7.5。

无机磷液体培养基(每升):葡萄糖10 g,(NH4)2SO4 0.5 g,NaCl 0.3 g,KCl 0.3 g,MgSO4·7H2O 0.3 g,FeSO4·7H2O 0.03 g,MnSO4·4H2O 0.03 g,Ca3(PO4)2 5.0 g,pH 7.0~7.5。不加琼脂即为无机磷液体培养基。

解钾液体发酵培养基(每升):蔗糖10 g,MgSO4·7H2O 0.5 g,CaCO3 1.0 g,(NH4)2SO4 1.0 g,NaCl 0.1 g,酵母膏0.5 g,Na2HPO4 2.0 g,钾长石粉10 g,pH 7.4。

含有L-色氨酸的液体R2A培养基(每升):酵母膏 0.5 g,胰蛋白胨0.5 g,酪蛋白氨基酸0.5 g,葡萄糖0.5 g,可溶性淀粉0.5 g,丙酮酸钠0.3 g,K2HPO4 0.3 g,MgSO4·7H2O 0.05 g,L-色氨酸0.5 g,pH 7.2±0.2。

1.3 燕麦内生真菌的分离与纯化

采用组织培养的方法进行内生真菌的分离[18]。将采集的燕麦地上部用流水冲洗30 min,去除表面沉积物,然后用无菌的剪刀将燕麦植株的茎和叶分开,并剪成3 cm长小段。燕麦各组织表面的消毒在超净工作台进行,具体操作步骤如下,将燕麦茎和叶片段分别于75%的酒精浸泡2 min,无菌水冲洗1次,3% NaClO溶液浸泡3~4 min(茎浸泡时间长,叶的浸泡时间短),再用75%的酒精浸泡1 min,最后用无菌水清洗5次,每次2 min,将最后一次洗涤的无菌水用涂布棒均匀涂抹在PDA培养基上,作为对照检测燕麦组织表面消毒情况。将表面消毒的茎和叶置于无菌滤纸上,吸干表面水分,用无菌剪刀剪去叶片和茎段被消毒剂浸染的边缘组织,剪成约1 cm的小段,用无菌镊子将叶和茎的片段置于含有硫酸链霉素的PDA培养基上培养,每个PDA平板上放置9个组织块,茎和叶片各制备20个平板。

将PDA平板置于28 ℃培养箱中持续培养,每天观察培养基情况。当PDA培养基中长出真菌菌丝,及时转移到新鲜的培养基中培养,每个PDA培养基中接种一个内生真菌,在28 ℃培养箱中继续培养,进一步纯化。最后将纯化的内生真菌置于30%甘油中,于-80 ℃超低温冰箱保存。

1.4 燕麦内生真菌的鉴定

将各菌种于PDA平板上活化培养7 d,用无菌接种钩刮取一定样品置于2 mL离心管中,用于内生真菌DNA的提取,参照Edwards等[19]改进的CTAB法进行提取。

根据Schoch等[20]的方法进行内生真菌PCR扩增程序,扩增引物是ITS1(5’-TCCGTAGGTGAA CCTGCGG-3’)和ITS4(5’-TCCTCCGCTTATTGA TATGC-3’),扩增反应体系为基因组DNA(20 ng/μL)1.0 μL,10× Buffer(含2.5 mmol/L Mg2+)5.0 μL,Taq聚合酶(5 U/μL)1.0 μL,dNTP(10 mmol/L)1.0 μL,ITS1引物(10 μmol/L)1.5 μL,ITS4引物(10 μmol/L)1.5 μL,ddH2O39.0 μL,总体积50.0 μL。PCR扩增反应程序为95 ℃预变性5 min;95 ℃变性30 s,58 ℃退火30 s,72 ℃延伸1 min,35个循环数;72 ℃延伸7 min。

反应完成后,用1%琼脂糖凝胶电泳检测PCR产物,使用AxyPrep DNA凝胶回收试剂盒回收PCR产物。用测序仪ABI3730-XL进行PCR产物DNA测序,获得的序列用NCBI Blast程序将拼接后的序列文件与NCBI核酸数据库中的数据进行比对,并在Genbank中上传获得登录号。

1.5 燕麦内生真菌促生特性

1.5.1 溶磷能力

参照詹寿发等[13]的方法进行有效磷含量的测定。在150 mL三角瓶中分别加入50 mL有机磷液体培养基和无机磷液体培养基。接种3块直径5 mm菌饼(用灭菌打孔器在培养7 d的内生真菌边缘直接打孔获得),对照为3块5 mm PDA培养基,在28 ℃ 160转/min下分别振荡培养4 d,所有处理重复3次,将菌体悬浮液10 000转/min离心10 min,采用钼锑抗比色法[21]测定磷含量。

1.5.2 解钾能力

参照罗娜等[22]的方法进行有效钾含量的测定。在150 mL三角瓶中分别加入50 mL解钾液体培养基。接种3块5 mm菌饼,对照用3块5 mm PDA培养基代替,在28 ℃,160转/min下分别振荡培养4 d,所有处理重复3次,将菌体悬浮液10 000转/min离心10 min,采用火焰光度法[21]测定钾含量。

1.5.3 产IAA能力

参照刘丽辉等[23]的方法测IAA含量并做一定的修改。将5 mm菌饼3块分别接种于盛有50 mL R2A培养基的三角瓶中,每一菌株重复3次,将三角瓶置于28 ℃,160转/min摇床培养4 d,取菌悬液1 mL置于试管中,同时加1 mL Salkowski试剂,以不加菌株的作为阳性对照,在比色液中只加入50 μL 50 mg/L的IAA作为阴性对照。将试管置于室温下避光放置30 min后观察其颜色变化。颜色变红者为阳性,表示能够分泌IAA,颜色越深表示分泌IAA活性越强,不变色为阴性,表示不能分泌IAA。对初筛获得能够分泌IAA的内生真菌进行定量测定,培养条件同上。将菌体悬浮液10 000转/min离心10 min,取1 mL上清液,再加入等体积的Salkowski试剂,避光静置30 min,采用分光光度法测定其OD530值,然后计算IAA含量。

1.6 燕麦内生真菌多样性分析

燕麦内生真菌多样性的分析参照秦华伟等[8]和罗鑫等[24]的方法进行处理。

定殖率(colonization rate,CR,%)=N0/Nt×100,式中,N0为样品中内生真菌侵染组织块数,Nt为培养组织块数;

分离率(isolation rate,IR,%)=Ni/Nt×100,式中,Ni为分离总菌株数,Nt为培养组织块数;

卡玛戈指数(Camargo’s index)=1/S(S为物种数),Pi=n/N,式中,n为某种类的菌株数量,N为所有种类的总菌株数量,当某种菌株的Pi大于卡玛戈指数时为优势属;

分离频率(isolation frequency,IF,%)=n/N× 100,式中,n为某种类菌株的总数量,N为所有内生真菌的菌株数;

多样性指数H′(Shannon-Weiner diversity index)=∑ki=1·Pi·lnPi,式中,k为宿主体内生真菌种类总数,Pi为某种内生真菌的总株数占全部内生真菌菌株总数的百分比;

均匀度指数E(ACE)=H′/lnS,式中,H′为多样性指数,S为物种总数;

丰富度指数R(Chao1)=(S-1)/lnN,式中S为宿主体内生真菌种类总数,N为内生真菌菌株总数;

辛普森指数D(Simpson)=1-∑Pi2,式中,Pi为某种内生真菌相对分离频率;

相似性指数Cs(sorenson’s similarity coefficients)=2j/(a+b),式中,j为2个种群共有的种类数量,a为第1个种群内所有种类数量,b为第2个种群内所有种类的数量。

1.7 数据处理

采用Microsoft Office Excel 2019进行多样性指数的整理分析,采用Origin 2021绘制饼图,采用SPSS 25.0进行系统聚类分析。

2 结果与分析

2.1 燕麦内生真菌多样性

2.1.1 分离鉴定

在PDA平板中培养的各内生真菌经ITS测序后,获得对应的序列信息,将各菌株的DNA序列在NCBI中比对,选择相似度最大的序列作为物种鉴定结果(表1)。从燕麦中共分离得到74株内生真菌,其中从茎中分离到28株,叶中46株,分属于2门、3纲、6目、8科、12属、19种,其中有70株属于子囊菌门(Ascomycota),2株属于担子菌门(Basidiomycota),2株未分类的真菌,子囊菌门内生真菌数量最多,占比94.59%。

表1   燕麦内生真菌序列鉴定

Table 1  Sequence identification of endophytic fungi in oat

菌株
Strain
登录号
GenBank ID
来源
Source
DNA鉴定结果
DNA identification result
相似性
Identity (%)
菌株
Strain
登录号
GenBank ID
来源
Source
DNA鉴定结果
DNA identification result
相似性
Identity (%)
OL-1ON495971链格孢菌99.81OL-38ON500580木贼镰孢菌100.00
OL-2ON495943木贼镰孢菌99.61OL-39ON500581链格孢菌100.00
OL-3ON495944链格孢菌100.00OL-40ON500582真菌99.40
OL-4ON495945互隔链格孢99.44OL-41ON500583子囊菌99.60
OL-5ON495946木贼镰孢菌99.41OL-42ON500584木贼镰孢菌99.80
OL-6ON495947木贼镰孢菌100.00OL-43ON500585木贼镰孢菌99.61
OL-7ON495948内生真菌99.63OL-44ON500586互隔链格孢99.81
OL-8ON495949木贼镰孢菌99.61OL-45ON500587木贼镰孢菌99.61
OS-9ON495950木贼镰孢菌99.81OS-46ON500588紧密帚枝霉99.45
OL-10ON495951木贼镰孢菌99.22OS-47ON500589裂褶菌99.50
OL-11ON500553木贼镰孢菌99.22OL-48ON500590细极链格孢99.44
OS-12ON500554互隔链格孢99.26OL-49ON500591互隔链格孢99.63
OL-13ON500555木贼镰孢菌100.00OS-50ON500592芸苔链格孢99.26
OL-14ON500556木贼镰孢菌99.41OL-51ON500593木贼镰孢菌99.80
OS-15ON500557木贼镰孢菌100.00OL-52ON500594子囊菌99.40
OS-16ON500558互隔链格孢99.63OL-53ON500595互隔链格孢100.00
OS-17ON500559芸苔链格孢99.81OL-54ON500596木贼镰孢菌99.41
OS-18ON500560派伦霉99.21OL-55ON500597木贼镰孢菌99.61
OS-19ON500561茎点霉99.60OL-56ON500598木贼镰孢菌99.41
OL-20ON500562派伦霉99.21OL-57ON500599木贼镰孢菌99.41
OS-21ON500563高粱附球菌99.40OL-58ON500600木贼镰孢菌99.80
OS-22ON500564互隔链格孢99.44OL-59ON500601木贼镰孢菌99.03
OS-23ON500565高粱附球菌99.60OL-60ON500602互隔链格孢99.63
OL-24ON500566子囊菌99.40OS-61ON500603廯囊腔菌99.61
OS-25ON500567雪球微座孢99.42OL-62ON500604木贼镰孢菌99.61
OS-26ON500568派伦霉99.40OL-63ON500605木贼镰孢菌99.41
OS-27ON500569小双孢腔菌99.40OL-64ON500606木贼镰孢菌99.61
OS-28ON500570镰刀菌属99.61OS-65ON500607互隔链格孢99.45
OS-29ON500571派伦霉99.21OS-66ON500608链格孢菌100.00
OS-30ON500572春孔菌99.62OL-67ON500609木贼镰孢菌99.61
OS-31ON500573链格孢菌99.44OL-68ON500610子囊菌99.40
OS-32ON500574木贼镰孢菌99.61OS-69ON500611廯囊腔菌99.61
OL-33ON500575小双孢腔菌99.60OS-70ON500612雪球微座孢99.23
OL-34ON500576木贼镰孢菌99.61OS-71ON500613紧密帚枝霉99.45
OL-35ON500577木贼镰孢菌99.42OL-72ON500614链格孢菌100.00
OL-36ON500578子囊菌99.40OL-73ON500615芸苔链格孢99.81
OL-37ON500579澳大利亚小光壳99.20OS-74ON500616互隔链格孢99.44

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2.1.2 多样性分析

表2可知,从燕麦地上组织中分离出74株内生真菌,分别隶属于12个属。子囊菌门内生真菌在属水平下分离频率最高的菌为镰刀菌属(Fusarium),分离到29株菌株,占总菌株数的39.19%,其次为链格孢属(Alternaria),分离到20株菌株,占总菌株数的27.03%,均为优势菌属;分离到的亚隔孢壳属(Didymella)、附球菌属(Epicoccum)、茎点霉属(Phoma)、小光壳属(Leptosphaerulina)、派伦霉属(Peyronellaea)、帚枝霉属(Sarocladium)、微座孢属(Microdochium)、小不整球壳属(Plectosphaerella),占比较小,在子囊菌门内生真菌中还有5株为未识别的子囊菌。担子菌门内生真菌只分离到2株,分别属于春孔菌属(Earliella)和裂褶菌属(Schizophyllum),各占菌株总数的1.35%。

表2   燕麦内生真菌群落组成

Table 2  Community composition of endophytic fungi in oat

门Phylum纲Class目Order科Family属Genus种SpecieNIR (%)IF (%)
Ascomycota子囊菌门座囊菌纲格孢腔菌目格孢菌科链格孢属互隔链格孢102.7813.51
芸苔链格孢30.834.05
链格孢菌51.396.76
链格孢菌10.281.35
细极链格孢10.281.35
亚隔孢壳科亚隔孢壳属小双胞腔菌20.562.70
附球菌属高粱附球菌20.562.70
茎点霉属茎点霉10.281.35
小光壳属澳大利亚小光壳10.281.35
派伦霉属派伦霉20.562.70
Peyronellaea sp.派伦霉20.562.70
子囊菌纲肉座菌目丛赤壳科镰刀菌属木贼镰孢菌287.7837.84
镰刀菌10.281.35
Sarocladiaceae帚枝霉属紧密帚枝霉20.562.70
炭角菌目微座囊菌科微座孢属Microdochium bolleyi20.562.70
小丛壳目小不整球囊菌科小不整球壳属癣囊腔菌20.562.70
未识别的子囊菌子囊菌51.396.76
Basidiomycota担子菌门伞菌纲多孔菌目多孔菌科春孔菌属春孔菌10.281.35
伞菌目裂褶菌科裂褶菌属裂褶菌10.281.35
Unclassified_fungi未识别的真菌20.562.70
总计Total7420.56100.00

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对燕麦不同部位内生真菌的定殖率和分离率分析(表3)发现,叶片和茎中内生真菌的定殖率分别为90.56%和88.89%,分离率分别为28.22%和17.50%,叶片内生真菌的定殖率和分离率均大于茎。

表3   燕麦内生真菌定殖率和分离率

Table 3  Rates of colonization and isolation of endophytic fungi in oat

项目Index叶Leaf茎Stem
培养组织块数(Nt)Number of tissue blocks180180
有菌组织块数(N0)Number of bacterial tissue blocks163160
分离菌株数(Ni)Number of isolated strains4628
定殖率(CR)Colonization rate (%)90.5688.89
分离率(IR)Isolation rate (%)28.2217.50

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对燕麦茎和叶中内生真菌分离频率分析(图1)可知,茎和叶中共有内生真菌为链格孢属(Alternaria)、亚隔孢壳属(Didymella)、镰刀菌属(Fusarium)和派伦霉属(Peyronellaea)。叶片中特有的内生真菌为小光壳属(Leptosphaerulina),还有5株未识别的子囊菌和2株未识别的真菌,茎中特有的内生真菌为春孔菌属(Earliella)、微座孢属(Microdochium)、附球菌属(Epicoccum)、茎点霉属(Phoma)、小不整球壳属(Plectosphaerella)、帚枝霉属(Sarocladium)、裂褶菌属(Schizophyllum)。叶片中优势菌属为镰刀菌属(Fusarium),分离频率为54.348%;而茎中的优势菌属为链格孢属(Alternaria),分离频率为32.143%。

图1

图1   燕麦不同部位内生真菌分离频率

Fig.1   Isolation frequency of endophytic fungi from different organs of oat


比较燕麦叶片和茎内生真菌多样性指数结果(表4)表明,叶和茎中的多样性指数分别为2.00和3.51,均匀度指数为1.03和1.46,丰富度指数为1.57和1.80,辛普森指数为0.62和0.78。综合来看,茎中内生真菌的多样性指数高于叶片,表明茎中的内生真菌物种较为丰富。叶和茎中内生真菌相似性指数为0.15,表明2种组织中的内生真菌组成有差异。

表4   燕麦不同部位内生真菌多样性指数和相似性指数

Table 4  Diversity of endophytic fungi in different organs of oat

项目Index
Leaf

Stem
内生真菌
Endophytic
fungi
物种数(S)Number of species5912
卡玛戈指数Camargo’s index0.200.110.083
多样性指数H
Shannon-Weiner diversity index
2.003.511.88
均匀度指数E(ACE)1.031.460.71
丰富度指数R(Chao1)1.571.803.02
辛普森指数D(Simpson)0.620.780.76
相似性指数Cs
Sorenson’s similarity coefficient
0.15

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2.2 燕麦内生真菌不同功能研究
2.2.1 功能聚类分析结果

对内生真菌功能特性分析发现,具有解有机磷能力的66株,解无机磷能力的67株,解钾能力的54株,分泌IAA能力的30株。对燕麦内生真菌功能测定的试验结果进行系统聚类分析,将74株内生真菌分为4类群(表5),类群Ⅰ中有41株内生真菌,大部分具有溶磷能力,其中能够溶解钾的仅有25株,解钾能力偏低,大部分菌株不能够分泌IAA;类群Ⅱ中有5株内生真菌,均具有溶解磷、钾的能力,且解钾的能力比较大,但分泌IAA能力较低;类群Ⅲ中有21株内生真菌,该组合中内生真菌均能够分泌IAA,IAA含量在37.68~75.26 mg/L,大部分内生真菌均具有解无机磷、有机磷和钾的能力;类群Ⅳ中有7株内生真菌,具有溶磷和解钾能力的内生真菌均为6株,各菌株分泌的IAA含量均较高,IAA含量在79.48~99.90 mg/L。

表5   燕麦内生真菌功能的分类

Table 5  Classification of growth promoting functions of oat endophytic fungi mg/L

类群
Group
菌株
Strain
解有机磷能力
Ability of organic phosphate-solubilizing
解无机磷能力
Ability of inorganic phosphate-solubilizing
解钾能力
Ability of potassium-solubilizing
IAA含量
IAA content
OL-121.61±2.2123.49±1.6517.50±1.320.00±0.00
OL-64.34±0.4933.05±1.3510.73±1.310.00±0.00
OS-92.01±0.2748.97±1.210.00±0.000.00±0.00
OL-110.00±0.0041.29±0.666.00±0.260.00±0.00
OL-131.38±0.2426.18±1.550.00±0.000.00±0.00
OL-141.67±0.9318.30±1.861.60±0.600.00±0.00
OS-151.51±0.8445.07±4.8611.05±0.930.00±0.00
OS-162.66±0.1227.28±1.3728.67±1.290.00±0.00
OS-217.37±0.9019.65±0.600.00±0.000.00±0.00
OS-2214.75±3.9328.13±1.580.00±0.000.00±0.00
OS-2313.57±1.0520.39±1.680.00±0.000.00±0.00
OL-247.18±0.520.00±0.000.00±0.000.00±0.00
OS-265.56±0.2723.54±2.7819.13±0.350.00±0.00
OS-276.23±0.5416.41±0.5816.55±1.270.00±0.00
OS-299.37±0.3420.89±1.5933.77±0.900.00±0.00
OS-3112.21±2.7534.29±2.7211.20±0.420.00±0.00
OS-321.93±0.1641.59±3.934.10±0.660.00±0.00
OL-335.23±0.3512.38±0.560.00±0.000.00±0.00
OL-344.20±0.2821.46±0.9420.10±0.570.00±0.00
OL-357.17±0.3437.22±2.723.47±0.570.00±0.00
OL-3610.70±0.460.00±0.000.00±0.000.00±0.00
OL-3712.95±2.1118.12±0.9910.27±0.360.00±0.00
OL-384.97±0.2328.50±2.470.00±0.000.00±0.00
OL-401.66±0.2218.76±1.620.00±0.000.00±0.00
OL-415.31±0.120.00±0.006.23±0.210.00±0.00
OL-430.00±0.0019.38±1.511.63±0.290.00±0.00
OL-449.67±0.799.46±0.370.00±0.000.00±0.00
OL-451.13±0.2620.08±2.9522.40±2.930.00±0.00
OS-471.47±0.1322.93±1.946.15±0.210.00±0.00
OL-485.14±0.9318.85±1.0224.37±5.400.00±0.00
OL-496.69±0.8124.13±1.8433.40±2.550.00±0.00
OL-526.87±0.1532.84±3.120.00±0.000.00±0.00
OL-584.79±0.240.00±0.000.00±0.000.00±0.00
OL-591.57±0.5328.86±1.830.00±0.000.00±0.00
OS-610.00±0.0047.13±3.580.00±0.0013.51±1.26
OL-640.00±0.0024.07±1.228.85±0.460.00±0.00
OL-689.71±0.2517.39±0.737.77±0.110.00±0.00
OS-7015.67±0.4124.62±1.310.00±0.000.00±0.00
OS-719.85±0.4030.84±3.744.57±0.380.00±0.00
OL-739.61±0.9424.57±1.0912.90±1.840.00±0.00
OS-740.00±0.0028.50±2.1214.90±1.980.00±0.00
OL-410.95±1.3327.37±2.0653.33±1.2521.12±0.26
OL-53.39±0.4329.58±1.0943.00±2.130.00±0.00
OS-254.57±0.2613.75±0.4251.77±1.890.00±0.00
OS-282.42±0.6147.13±1.9325.90±0.710.00±0.00
OL-621.32±0.1840.01±2.8740.67±1.760.00±0.00
OL-23.41±0.6927.97±0.9731.27±2.6253.33±1.22
OL-313.17±2.8120.38±1.1213.33±2.3237.68±1.88
OS-126.24±0.5424.93±1.1826.37±1.7849.36±0.46
OS-178.06±0.2116.09±0.1112.80±1.4753.98±1.85
OS-185.05±0.1118.10±0.7446.27±2.8757.22±0.62
OS-1914.81±0.2821.47±1.9927.37±2.9561.18±1.89
OL-2013.82±4.570.00±0.000.00±0.0058.75±0.88
OS-300.00±0.0041.28±2.840.00±0.0047.97±1.29
OL-424.84±0.2832.87±0.6434.25±2.3360.75±2.19
OL-518.97±0.2522.42±1.6922.14±3.3738.32±0.10
OL-5310.40±0.6123.62±2.4318.27±2.8360.05±2.06
OL-545.88±0.1728.72±1.2212.62±0.2165.99±3.35
OL-550.00±0.0024.34±1.5910.45±1.0647.46±1.38
OL-562.05±0.2519.13±1.138.70±0.1450.61±0.44
OL-571.09±0.090.00±0.0031.73±2.7575.26±2.24
OL-605.73±0.2722.60±1.4659.37±3.1653.99±0.24
OL-637.25±0.1326.34±0.8920.30±3.5649.52±0.89
OS-659.69±0.3522.23±0.460.00±0.0057.01±0.53
OL-675.87±0.1833.27±2.0711.90±1.6254.09±1.58
OS-691.56±0.1728.59±1.9533.63±2.6141.19±0.53
OL-726.63±0.7824.77±1.7317.00±1.7540.48±1.18
OL-78.49±0.4320.28±0.1918.05±0.4389.45±5.13
OL-83.61±0.1528.53±0.9711.65±0.3581.75±1.88
OL-100.00±0.0042.07±0.6233.30±1.6196.61±2.15
OL-397.51±0.320.00±0.007.63±0.3899.90±3.58
OS-462.55±0.1628.87±2.300.00±0.0085.08±2.16
OS-5015.14±1.8522.37±1.3312.20±1.4479.48±0.20
OS-6616.53±0.5224.02±0.4439.13±0.7593.71±3.55

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2.2.2 判别分析

根据表5结果进行判别分析,结果见表6。内生真菌的解钾能力和IAA含量在3个分类上具有显著性,而解有机磷能力和解无机磷能力无显著性差异。典型判别分析发现,4个类别的正确判别率分别为92.68%、100.00%、95.24%和100.00%。不同分类的内生真菌点性判别分析结果如图2,各组别均以组质心为中心分布集中。

表6   不同分类的各指标平均值的同等检验

Table 6  Equivalent tests of the means of each indicator for different categories

指标IndexFF-valuedf1df2PP-value
解有机磷能力Ability of organic phosphorus-solubilizing0.4323700.730
解无机磷能力Ability of inorganic phosphate-solubilizing0.8053700.495
解钾能力Ability of potassium-solubilizing14.9613700.000
IAA含量IAA content642.8873700.000

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图2

图2   燕麦内生真菌分类散点图

Fig.2   Scatter plot of the classification of endophytic fungi in oat


表7可知,判别函数1的特征值为28.149,其方差贡献率最大,为97.94%,与组别间的典型相关系数为0.983;判别函数2的方差贡献率为2.03%,特征值和典型相关系数分别为0.584和0.607;判别函数3的方差贡献率、特征值和典型相关系数最小,分别为0.03%、0.009和0.092。依据前2个判别函数,74株内生真菌的分布如图2。由于函数判别函数1的方差贡献率最大,依据判别函数1将74株内生真菌分类,发现第1类为促生效果较差的内生真菌,总共有41株,该类菌株多数均具有解有机磷和无机磷的能力,而解钾能力较低,不能产生IAA;第2类为促生效果一般的菌株,共有4株,此类菌株均具有解有机磷、无机磷和钾的能力,但不能产生IAA;第3类为促生效果良好的菌株,共有21株,这类内生真菌大部分均可以解有机磷、无机磷和钾,且能够分泌IAA,IAA的含量较高;第4类为促生效果最好的菌株,此类菌株大部分具有磷、钾的增溶能力,且分泌IAA的含量最高。

表7   判别分析函数特征值

Table 7  Eigenvalues of discriminant analysis function

判别函数
Discriminant function
特征值
Eigenvalue
方差贡献率
Variance contribution rate (%)
累计贡献率
Accumulated contribution rate (%)
典型相关系数
Canonical correlation coefficient
128.14997.9497.940.983
20.5842.0399.970.607
30.0090.03100.000.092

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3 讨论

3.1 燕麦内生真菌多样性

内生真菌在不同植物中的定殖情况不同,已有研究[25]发现,野燕麦的内生真菌染菌率在80%以上,表明燕麦中存在一定数量的内生真菌。本研究利用植物组织培养技术从健康的燕麦茎和叶中分离到74株内生真菌,其中大部分的内生真菌(94.59%)属于子囊菌门(Ascomycota),这和玉米[8]、杜鹃[26]、桫椤[27]和龙葵[28]等植物的内生真菌的表现类似。子囊菌门内生真菌是植物体内可被培养的微生物类群,且占据优势,这表明子囊菌门内生真菌与寄主植物的关系较为密切[29]。本研究中内生真菌主要分属座囊菌纲(Dothideomycetes)和粪壳菌纲(Sordariomycetes),这与小麦中内生真菌的分布类似[5],这些内生真菌的优势可能是共同进化造成的。分离获得的燕麦内生真菌分为12个属,其中链格孢属和镰刀菌属内生真菌的分离频率较高,分别为27.03%和39.19%,为燕麦内生真菌的优势属。链格孢属和镰刀菌属内生菌已被证明是常见的内生真菌,从小麦[30]、玉米[31]和卷柏[32]中均可分离到,表明这2类菌落是各作物的常见菌群,能够通过生态位的分配抵御胁迫。同时,有益的镰刀菌和链格孢菌在植物体内的存在也可通过中增加次级代谢物的产生,促进寄主植物的生长[33-34]

本研究发现燕麦叶和茎中分离到的内生真菌共有菌属为链格孢属、亚隔孢壳属、镰刀菌属和派伦霉属,且叶中镰刀菌属占比较高,茎中以链格孢属的占比较高,这些菌属有广泛的寄主范围,寄主类型的特异性低[35]。小光壳属只在叶片中发现,茎中发现少部分的春孔菌属、微座孢属、附球菌属、茎点霉属、小不整球壳属、帚枝霉属、裂褶菌属,这些菌在燕麦中的分离频率较低,表明内生真菌具有一定的组织特异性和专一性,也表现除了组织偏好性。产生这一现象的原因可能是由于内生真菌群落的组成和寄主组织产生的化学物质有关[36],也可能是由于内生微生物在植物组织中的移动能力受限制。叶中内生真菌的定殖率和分离率高于茎,可能是由于叶片暴露在空气中的面积较大,为内生真菌与该器官的关联提供更多的机会[37]。本研究中燕麦茎内生真菌的多样性和丰富度均高于叶片,这与Sun等[38]对红树林内生真菌多样性的研究结果有差异,可能是由于不同组织的结构、生理和代谢物有一定的差异,内生真菌的定殖对生存空间和营养需求不同[39],从而造成内生真菌类群的差异。燕麦茎和叶中内生真菌的类型和数量并不完全相同,表明内生真菌群落结构会因宿主组织的不同而不同[40]

3.2 燕麦内生真菌功能特性研究

植物内生真菌通过解无机磷、有机磷和难溶性钾[41-42],促进养分转运和宿主对养分的吸收利用。本研究对燕麦内生真菌的促生功能研究发现,具有解有机磷、无机磷和钾能力的菌株分别占燕麦内生真菌的89.19%、90.54%和72.97%,表明燕麦中具有丰富的溶磷和解钾菌群。目前,关于内生真菌溶磷解钾功能的菌群研究报道较多的是根内生真菌,而本研究发现燕麦叶和茎的部分内生真菌也能提高磷、钾的增溶能力。唐嘉城等[43]和Nath等[44]均发现宿主作物不同组织(根、茎和叶)的内生菌能分解难溶性磷、钾元素,表明内生微生物对难溶性磷、钾的增溶活性与其来源和分类物质无关。刘军等[18]和Hassan[45]也发现部分叶面内生真菌具有不同的增磷能力。内生微生物能够分泌IAA,促进宿主作物的生长。本研究中40.54%的内生真菌能够分泌IAA,这一发现表明燕麦中的内生真菌可能是促进燕麦种子萌发和生长的因素之一。前人[46]研究发现,少数的内生真菌能够产生IAA,本研究结果证实了这一观点。具有分泌生长素的内生真菌在寄主植物的生长发育中起着重要的作用[23]。对74株内生真菌的促生功能进行综合分析发现,解钾能力和分泌IAA含量是决定内生真菌分类的主要因素,并将74株内生真菌分成4组,其中促生效果最好的是第4组,该部分内生真菌均具有溶磷、解钾和分泌IAA的能力。此类菌株的应用能够调节植物体内激素水平的变化,促进对养分的吸收利用,有助于提高作物的抗逆性,间接促进植物生长。

4 结论

本研究从燕麦的茎和叶片中分离获得74株内生真菌,分为12个属,其中从叶中分离到46株,茎中分离到28株。通过各促生特性试验发现,燕麦不同部位中分离获得内生真菌具有种群和功能多样性,燕麦内生真菌能够分泌生长素、溶解有机磷、无机磷和解钾的特性,其中第4组中菌株OL-7、OL-8、OS-50和OS-66在各方面的促生长特性均较强,为微生物制剂在生态农业和可持续农业的应用提供基础。

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In hot deserts, plants cope with aridity, high temperatures, and nutrient-poor soils with morphological and biochemical adaptations that encompass intimate microbial symbioses. Whereas the root microbiomes of arid-land plants have received increasing attention, factors influencing assemblages of symbionts in aboveground tissues have not been evaluated for many woody plants that flourish in desert environments. We evaluated the diversity, host affiliations, and distributions of endophytic fungi associated with photosynthetic tissues of desert trees and shrubs, focusing on nonsucculent woody plants in the species-rich Sonoran Desert. To inform our strength of inference, we evaluated the effects of two different nutrient media, incubation temperatures, and collection seasons on the apparent structure of endophyte assemblages. Analysis of >22,000 tissue segments revealed that endophytes were isolated four times more frequently from photosynthetic stems than leaves. Isolation frequency was lower than expected given the latitude of the study region and varied among species a function of sampling site and abiotic factors. However, endophytes were very species-rich and phylogenetically diverse, consistent with less arid sites of a similar latitudinal position. Community composition differed among host species, but not as a function of tissue type, sampling site, sampling month, or exposure. Estimates of abundance, diversity, and composition were not influenced by isolation medium or incubation temperature. Phylogenetic analyses of the most commonly isolated genus (Preussia) revealed multiple evolutionary origins of desert-plant endophytism and little phylogenetic structure with regard to seasonality, tissue preference, or optimal temperatures and nutrients for growth in vitro. Together, these results provide insight into endophytic symbioses in desert-plant communities and can be used to optimize strategies for capturing endophyte biodiversity at regional scales.

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Frontiers in Microbiology, 2021, 12:646302.

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The diversity and distribution of endophytes across biomes, plant phylogeny and host tissues: how far have we come and where do we go from here?

Environmental Microbiology, 2020, 22(6):2107-2123.

DOI:10.1111/1462-2920.14968      PMID:32115818      [本文引用: 1]

The interiors of plants are colonized by diverse microorganisms that are referred to as endophytes. Endophytes have received much attention over the past few decades, yet many questions remain unanswered regarding patterns in their biodiversity at local to global scales. To characterize research effort to date, we synthesized results from ~600 published studies. Our survey revealed a global research interest and highlighted several gaps in knowledge. For instance, of the 17 biomes encompassed by our survey, 7 were understudied and together composed only 7% of the studies that we considered. We found that fungal endophyte diversity has been characterized in at least one host from 30% of embryophyte families, while bacterial endophytes have been surveyed in hosts from only 10.5% of families. We complimented our survey with a vote counting procedure to determine endophyte richness patterns among plant tissue types. We found that variation in endophyte assemblages in above-ground tissues varied with host growth habit. Stems were the richest tissue in woody plants, whereas roots were the richest tissue in graminoids. For forbs, we found no consistent differences in relative tissue richness among studies. We propose future directions to fill the gaps in knowledge we uncovered and inspire further research.© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.

Yuan Z S, Liu F, Zhang G F.

Characteristics and biodiversity of endophytic phosphorus- and potassium-solubilizing bacteria in Moso Bamboo (Phyllostachys edulis)

Acta Biologica Hungarica, 2015, 66(4):449-459.

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Applied Ecology and Environmental Research, 2015, 13(3):877-891.

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Journal of Advanced Research, 2017, 8(6):687-695.

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Metarhizium robertsii produces indole-3-acetic acid, which promotes root growth in Arabidopsis and enhances virulence to insects

Microbiology, 2017, 163(7):980-991.

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