河南省小麦根、茎部内生真菌多样性及平板拮抗活性研究

To investigate the community composition and diversity of fungal endophytes in wheat roots and stems from different soil types in Henan Province, tissue isolation method was used to isolate and purify endophytic fungi from wheat plants collected from 22 counties/districts in five soil types, and the identification of the fungal endophytes fungi was based on morphology and internal transcribed spacer (ITS) sequence. The community composition and diversity of endophytic fungi were analyzed. The results showed that 426 endophytic fungi were affiliated with 23 genera, among which Chaetomium spp. had the highest number accounting for 23.2% of the total strain number, followed by Penicillium spp.. Five strains were tested for the antifungal activity against both Rhizoctonia Cerealis and Fusarium pseudograminearums on plate, and their inhibition rates against R. Cerealis ranged from 44.8% to 70.5% (zone widths 2.00~9.67 mm)while from 35.4% to 49.3% (zone widths 3.67~6.67 mm) against F. pseudograminearum. This paper clarifies the major taxa and their diversity of fungal endophytes in the roots and stems of wheat in different soil types in Henan Province, and their inhibitory effects on soil-borne fungi, providing a basis for the biological control of sharp eyespot and crown rot of wheat.     

进境苇状羊茅和多年生黑麦草种子携带内生真菌带菌率的研究

苇状羊茅内生真菌和多年生黑麦草内生真菌曾给一些国家的畜牧业造成巨大损失.为防止内生真菌随进境草籽传入我国并在中国扩散危害,我们对该2种进境草籽内生真菌的带菌率进行了研究.首先通过分离培养,得到了苇状羊茅内生真菌和黑麦草内生真菌,然后采用染色法对1998年以来收集到的从天津口岸进境的部分苇状羊茅和黑麦草种子进行了初步检验,经检验,37份苇状羊茅样品中有18个品种带菌,34份多年生黑麦草样品中14个品种带菌,带菌率最高可达86.7%.早熟禾、剪股颖等其它禾本科草籽目前尚未发现带有内生真菌.     

Endophytic fungi and ergot alkaloids in native Turkish grasses

Mature inflorescences of 76 samples of ten genera of native grasses were collected in the Ankara province of Turkey. Microscopic examination of individual seeds revealed the presence of endophytic fungi in four seed samples, The endophytes were isolated in pure culture and identified. Seeds were also ground and subjected to ELISA and HPLC analyses to confirm the levels of ergot alkaloids typically produced by endophytic fungi of grasses.     

Diversity of endophytes across the soil-plant continuum for Atriplex spp. in arid environments

Endophytes are hypothesized to be transferred across the soil-plant continuum, suggesting both the transfers of endophytes from environment to plant and from plant to soil. To verify this hypothesis and to assess the role of locality, we evaluated the similarity of microbial communities commonly found both in soils and endophytic communities in three arid regions, i.e. the Jornada LTER(Long Term Ecological Research) site in New Mexico, USA, and the research station of Jordan University of Science and Technology(JUST) and Khanasri research station in Badia region of Jordan. Rhizosphere and non-rhizosphere soils, leaves and seeds of Atriplex spp. were sampled. Diversity and distribution of bacteria and fungi across the soil-plant continuums were assessed by tag-encoded FLX amplicon pyrosequencing and sequence alignment. Of the total bacterial OTUs(operational taxonomic units), 0.17% in Khanasri research station, 0.16% in research station of JUST, and 0.42% in Jornada LTER site were commonly found across all the plant and soil compartments. The same was true for fungi in two regions, i.e. 1.56% in research station of JUST and 0.86% in Jornada LTER site. However, in Khanasri research station, 12.08% of total fungi OTUs were found in at least one soil compartment and one plant compartment. Putative Arthrobacter, Sporosarcina, Cladosporium and members of Proteobacteria and Actinobacteria were found across all the soil-plant continuums. Ascomycota, mainly including Didymellaceae, Pleosporaceae and Davidiellaceae were present across all the soil-plant continuums. Microbial communities in two regions of Jordan were similar to each other, but both of them were different from the Jornada LTER site of USA. SIMPER(similarity percentage) analysis of bacterial and fungal taxa for both soil and endophyte communities revealed that dissimilarities of two bacterial genera(Arthrobacter and Sporosarcina) and two fungal genera(Cladosporium and Alternaria) are very high, so they play key roles in the soil-plant continuums. A weighed Pearson correlation analysis for the specific bacterial OTUs in the soil-plant continuums only showed high similarity between the two regions of Jordan. However, fungal groups showed higher similarities among all regions. This research supports the hypothesis of continuity of certain bacterial and fungal communities across the soil-plant continuums, and also explores the influences of plant species and geographic specificity on diversity and distribution of bacteria and fungi.     

Promotion of growth, health and stress tolerance of Styrian oil pumpkins by bacterial endophytes

Substantial yield losses of Styrian oil pumpkin caused by the fungus Didymella bryoniae and bacterial pathogens were recently reported. Here we applied bacterial endophytes with a broad antagonistic activity to pumpkin plants by seed priming. Effects of the bacterial inoculants with and without chemical seed treatments on plant growth and health were evaluated during three different field trials in two consecutive years (2010 and 2011). Biological seed treatments strongly supported the germination of pumpkin seeds. In 2010, the germination of the biologically treated seeds was comparable to the rate following a chemical treatment; whilst in 2011 effects of biological seed treatments were more obvious, including an increased emergence rate up to 109?% by Serratia plymuthica S13. Furthermore, tolerance against desiccation stress was observed for Serratia as well as for Lysobacter gummosus L101 treatment. The biological treatment showed different effects against fungal diseases: no effect on fruit rot was observed, whereas powdery mildew could be significantly suppressed by Paenibacillus polymyxa PB71 and L. gummosus L101 in 2010. In addition, both strains led to reproducible increases in harvest yields. In this study, we found bacterial endophytes suitable as inoculants for plant growth promotion, biocontrol, as well as for enhancing stress tolerance in Styrian oil pumpkins.     

Diversity,plant growth-promoting traits,and biocontrol potential of fungal endophytes of Sorghum bicolor

The diversity of fungal endophytes in Sorghum bicolor was investigated in samples collected from 10 different geographical regions of Karnataka state, India. A total of 360 endophytes were isolated from leaf, stem, and root tissues and were assigned to 26 fungal species based on morphology and molecular characterization using ITS sequences. All the endophytes belonged to the phylum Ascomycota. The diversity (Shannon H, 2.57; Simpson_1-D, 0.92) and species richness (Margalef's, 4.68; Menhinick, 3.61) were found to be higher for the endophytes isolated from leaf tissues. The species evenness of the endophytic assemblage was strongly influenced by tissue type, followed by geographical location. The biocontrol potential of isolated endophytes was evaluated against economically destructive sorghum grain mould pathogens such as Fusarium thapsinum, Epicoccum sorghinum, Alternaria alternata, and Curvularia lunata using the dual culture method. Biocontrol potential was exhibited by 26 endophytic isolates, of which Trichoderma asperellum recorded broad-spectrum activity against target pathogens, followed by E. nigrum and A. longipes. Most (82%) endophytes showed plant growth-promoting traits. Biosynthesis of indole-3-acetic acid (IAA) was observed in 84% of isolates, and phosphate solubilization, siderophore production, and cellulase activity was observed in 69%, 23%, and 27% of isolates, respectively. Seeds treated with T. asperellum exhibited a significantly higher seed vigour index (2096), germination percentage (94%), and yield under greenhouse and field conditions. The results were substantiated by the confocal microscopy analysis, which clearly demonstrated the colonization of treated endophyte in root tissues. The present study reveals an ecofriendly approach to explore T. asperellum in sorghum disease management.     

两株铁皮石斛内生真菌对组培苗和原球茎生长的影响

在铁皮石斛内生真菌的研究中,获得了对组培苗的生长呈现不同影响的两株内生真菌DCN-1和DCN-3,为探究其对组培苗和原球茎生长的影响,首先对已分离纯化的菌株DCN-1和菌株DCN-3进行菌株形态和ITS1-5.8S-ITS2区段序列鉴定;采用孢子悬浮液注射和菌丝体附着伤口的方法对铁皮石斛组培苗和原球茎进行接种,观察组培苗的生长情况;参照柯赫氏法则对菌株进行重新分离,并结合切片观察结果验证侵染情况。结果显示:菌株DCN-1和菌株DCN-3分别与Myceliophthora verrucosa、G ibberella moniliformis的相似度最高,其中菌株DCN-1对组培阶段和移栽后的组培苗和原球茎的生长无明显影响,菌株DCN-3会导致组培阶段的植株苗枯萎甚至死亡而对移栽后的组培苗影响不大,但抑制原球茎的生长。徒手组织切片观察及二次分离结果分析显示:两株内生真菌均成功侵染植株体内,并确定相应植株的生长状况与该两株内生真菌的侵染相关。两株内生真菌在在营养较丰富的组织培养条件下对铁皮石斛组培苗和原球茎造成不同的影响,为探究其与铁皮石斛的相互作用和促进铁皮石斛植株生长及有效活性物质产生的复合型菌制剂提供了依据。     

花生内生菌的种群及动态分析

１９９４－１９９７年,先后从山东省主要花生产区监沂、烟台等地采集约１０００株不同类型的花生无病 ,进行了植株内主要微生物类群分析。结果表明,不同品种花生的内生菌都表细菌和真菌,未分离到放线菌和酵母菌,细菌以芽孢杆菌（Ｂａｃｉｌｌｕｓｓｐｐ．）黄单孢菌（Ｘａｎｔｈｏｍｏｎａｓｓｐｐ．）和欧文氏菌（Ｅｒｗｉｎｉａｓｐｐ．）为优势种群。其中芽孢杆菌属以巨大芽孢杆菌（Ｂ．ｍｅｇａｔｅｒｉｕｍ）、蜡样芽孢杆     

植物内生真菌及其展望

内生真菌在自然界广泛存在,有些内生真菌可以促进植物营养吸收、提高植物抗生物逆境和非生物逆境的能力、刺激植物产生活性物质等。因此,内生真菌是植物病虫害生物防治领域的重要研究内容之一。本文就内生真菌与寄主共生的分子基础、内生真菌对植物影响机制、内生真菌与寄主特异性相互作用、特殊的内生真菌--植物病原真菌、内生真菌形成的分子机制和内生真菌研究展望等6个方面对内生真菌进行了叙述,期望为科学地利用内生真菌资源提供一些参考。     

Review: Endophytic microbes and their potential applications in crop management

Endophytes are microbes (mostly bacteria and fungi) present asymptomatically in plants. Endophytic microbes are often functional in that they may carry nutrients from the soil into plants, modulate plant development, increase stress tolerance of plants, suppress virulence in pathogens, increase disease resistance in plants, and suppress development of competitor plant species. Endophytic microbes have been shown to: (i) obtain nutrients in soils and transfer nutrients to plants in the rhizophagy cycle and other nutrient‐transfer symbioses; (ii) increase plant growth and development; (iii) reduce oxidative stress of hosts; (iv) protect plants from disease; (v) deter feeding by herbivores; and (vi) suppress growth of competitor plant species. Because of the effective functions of endophytic microbes, we suggest that endophytic microbes may significantly reduce use of agrochemicals (fertilizers, fungicides, insecticides, and herbicides) in the cultivation of crop plants. The loss of endophytic microbes from crop plants during domestication and long‐term cultivation could be remedied by transfer of endophytes from wild relatives of crops to crop species. Increasing atmospheric carbon dioxide levels could reduce the efficiency of the rhizophagy cycle due to repression of reactive oxygen used to extract nutrients from microbes in roots. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

小麦幼苗内生菌多样性的宏基因组分析

小麦是世界各地广泛种植的重要禾本科作物,在生长过程中与内生菌形成和谐共生的微生态体系,内生菌次生代谢产物具有抗病、防虫与促生等重要作用。宏基因组技术是研究环境微生物及内生菌多样性的重要手段。本研究首先富集纯化幼苗期小麦内生菌的宏基因组DNA,PCR扩增内生细菌的16S rDNA及内生真菌rDNA-ITS序列,构建了大肠杆菌克隆文库,选取阳性克隆测序。根据16S rDNA信息及系统进化树对内生细菌群初步分析,划分为9个分类操作单元(operational taxonomic unites,OTUs),主要包括γ-变形菌纲肠杆菌科(95.59%)、梭菌纲(1.47%)及不可培养细菌(2.94%),其中肠杆菌科菌群为优势菌。内生真菌rDNA-ITS克隆序列根据序列相似性和系统发育分析划归为13个OTUs,主要为不可培养真菌(37.50%)与子囊菌(62.50%),后者分属于半子囊菌纲(30.00%)、座囊菌纲(5.00%)与粪壳菌纲(27.50%)3个菌纲,其中不可培养真菌为优势菌。上述研究结果表明小麦幼苗内生菌遗传多样性分布较广,本研究为进一步挖掘利用小麦内生菌资源提供了基础信息。     

Fungi Associated with Sorghum vulgare under Different Storage Conditions in India

Abstract

Seeds of Sorghum vulgare stored under different conditions were collected to isolate the fungi associated with them. Species of Helminthosporium, Gloeocercospora, Curvularia, Aspergillus and Rhizopus were isolated. It was observed that incidence of fungi was greater in the case of the seeds stored in gunny bags* than with those stored in panicles. Species of the first three fungi were found to cause seed rot as well as seeding blight and the latter two were found to have a detrimental effect on germination of seeds. Seed treatment with Agrosan G.N.** and Ceresan dry at the rate of 2.5 g/kg of seed were found to be most effective in controlling these fungi.

An attendant problem of Jowar (Sorghum vulgare) growing is a number of diseases, many of them due to fungi associated with seeds. Investigations were undertaken to discover the fungi associated with seed during storage causing rot of seedlings and methods of their control. Pathogenicity tests and seed treatments with different fungicides were also conducted.     

Arbuscular mycorrhizal fungal community structure and diversity are affected by host plant species and soil depth in the Mu Us Desert,northwest China

The vertical diversity and distribution of arbuscular mycorrhizal (AM) fungi were investigated in the Mu Us Desert, northwest China. Soils were sampled to 50?cm in depth in the rhizospheres of Hedysarum laeve, Artemisia ordosica, and Psammochloa villosa and 44 AM fungal species belonging to 10 genera were isolated. Several of these species have peculiar morphological features, which are distinct from other habitats. AM fungal diversity and distribution differed significantly among the three host plants and the five soil layers. Spore density, species richness, and the Shannon-Wiener index of AM fungi were 0.55–4.3 spores g^?1 soil, 7–36 and 1.78–2.89, respectively. Spore density and species richness had a significant positive correlation with soil total phosphorus content (0.0377–0.1129?mg?g^?1), and a negative correlation with soil pH (7.19–7.64). Nonmetric multidimensional scaling, PerMANOVA, and structural equation model analysis demonstrated that host plant species and soil depth significantly and directly influenced the structure of AM fungal communities. We concluded that diversity and distribution of AM fungi might be influenced by plant species, soil depth patterns, and soil nutrient availability in desert ecosystems. This research into AM fungal communities may lead to the development of AM fungi treatment for the mitigation of soil erosion and desertification using mycorrhizal plants, such as H. laeve, A. ordosica, and P. villosa.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

Endophytic fungi in the invasive weed Impatiens glandulifera: a barrier to classical biological control?

The rust fungus, Puccinia komarovii var. glanduliferae, has been introduced into the UK for biological control of the invasive weed, Impatiens glandulifera (Himalayan balsam). However, establishment of the pathogen has differed across the country, which may be partly explained by variation in plant genotype. The aim of this study was to examine whether there is a further layer of phenotypic resistance, provided by indigenous foliar endophytic fungi. Culturable endophytes were isolated from a number of different balsam populations, and the commonest species were inoculated into ‘clean’ balsam plants, to test their interactions with the rust. We found that endophyte communities within balsam are low in diversity and become more dissimilar with increasing distance between populations. Three endophytes (Colletotrichum acutatum, Alternaria alternata and Cladosporium oxysporum) were common and appeared to be antagonistic to the rust, reducing pustule number and mitigating the effect of the pathogen on plant biomass. I. glandulifera thus partially conforms to the endophyte-enemy release hypothesis, in that as an introduced species, it has an impoverished endophyte complement, acquired from the local environment. However, these endophytes represent a potential barrier to effective biological control and future weed control strategies need to find strains of rust that can overcome plant genetic resistance and the overlaying phenotypic resistance, conferred by endophytes. Future classical biological control programmes of weeds must therefore take into account the fungal bodyguards that invasive species may acquire in their introduced ranges.     

Significant in vitro antagonism of the laurel wilt pathogen by endophytic fungi from the xylem of avocado does not predict their ability to control the disease

The ascomycete Raffaelea lauricola causes laurel wilt, a lethal vascular disease of avocado, Persea americana, and other members of the Lauraceae plant family. Few effective control measures for laurel wilt exist and new measures are needed. In this study, biological control of the disease with endophytic fungi from avocado was examined. Thirty‐two endophytes (24 operational taxonomic units or OTUs) isolated from the xylem of healthy trees (the infection court of R. lauricola) were evaluated against R. lauricola with in vitro dual‐culture assays. Nine OTUs that showed strong in vitro antagonism of the pathogen were tested in planta against laurel wilt. In three greenhouse experiments, grafted avocado plants of Simmonds or Russell cultivars, which are both susceptible to laurel wilt, were inoculated with endophytes and, after 10–16 days, inoculated with the same isolate of R. lauricola that was used in the in vitro assays. Within 14 days of inoculation with R. lauricola, laurel wilt developed in plants that were not treated with endophytes (positive controls) but also developed in endophyte‐treated plants to the extent observed in the positive controls (P = 0.05). The pathogen colonized plants rapidly and systemically, but endophytes generally did not colonize xylem more than 2 cm above the point at which plants were inoculated. Although the tested endophytes strongly antagonized the pathogen in vitro, this did not translate to an ability to reduce development of laurel wilt. The management of laurel wilt and other plant diseases with fungal endophytes is discussed.     

Implications of bacterial contaminated seed lots and endophytic colonization by Pseudomonas fuscovaginae on rice establishment

The economic impact of seedborne bacterial diseases on rice production provides a major motivation for research on seed health. This paper reports on the endophytic growth of a rifampicin‐marked strain of the seedborne rice pathogen Pseudomonas fuscovaginae. The bacterium was found in most tested seeds indicating that, even without visible discolouration, seed transmission is possible. Crushed discoloured seeds contained more bacterial cells than did non‐crushed discoloured seeds. These bacteria were released during seed soaking, contaminating clean seed and lowering seed germination. Cells of a rifampicin‐resistant strain of P. fuscovaginae, which had been inoculated onto rice seeds, were subsequently recovered from different growth stages and from different rice tissues, thereby indicating endophytic colonization. These results have implications for seedling establishment, as symptomless seeds do not assure disease‐free seeds, and the presence of seedborne bacteria results in poor germination and poor seedling establishment. Elimination of seedborne bacteria by soaking in sodium hypochlorite can increase seed germination. This could be used in developing control strategies, and, if practised regularly, reduce entry of seedborne disease‐causing organisms into crops, resulting in lower disease pressure.     

北京地区主栽西瓜品种种子携带真菌检测及鉴定

用离体平皿法对北京西瓜产区主栽的11个西瓜品种进行种子带菌检测、分离纯化和形态学、ITS序列比对,确定其属或种地位。结果表明:不同品种所带真菌种类有差异,种子表面携带的优势菌群主要为曲霉属Aspergillus spp.、青霉属Penicilliumspp.、链格孢属Alternariaspp.和镰刀菌属Fusariumspp.;种子内部寄藏真菌主要为青霉属、根霉属Rhizopus spp.和曲霉属。种子外部检出的主要病原菌有镰刀菌属的F.verticillioides,F.proliferatum及茎点霉Phomasp.;种子内部检出的病原菌有F.verticillioides和F.oxysporum。不同品种间种子表面携带真菌量与种子内部携带真菌率差异显著,但种子外部带菌量和内部种仁带菌率之间无显著相关性。本研究对开展西瓜种子处理研究有借鉴意义。     

Use of ITS nuclear sequences from Phelipanche aegyptiaca as a direct tool to detect single seeds of broomrape species in the soil

Broomrape (Phelipanche and Orobanche spp.) are obligate holoparasites that attack roots of almost all economically-important crops in semiarid regions of the world. Broomrape seeds are extremely small (dust-like seeds), averaging 200 to 300?μm in size and because of the miniscule seed size it is difficult to detect and confirm via conventional methods. In this study our aim was to develop a PCR-based assay specific for broomrape soil-borne seeds and sensitive enough to detect a single or few broomrape seeds in a soil sample. For this purpose, we used complementary polymerase chain reaction (PCR) primers based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of Phelipanche aegyptiaca Pers., Orobanche cumana Wallr. and Phelipanche crenata Forsk. and subjected to PCR analysis. Using ITS-350 primers, a specific PCR product (350?bp) was amplified and detected in all samples containing broomrape species, but was not detected in soil sample free of broomrape seeds or tissues. Additionally, the PCR-based assay was sensitive enough to detect even a single broomrape seed in the soil. As expected the universal internal control primers amplified a PCR product (555?bp) of genomic DNA extracted from soil samples with or without broomrape tissues or seeds. This diagnostic method is simple, reliable and rapid and could help for assessment of broomrape seed contamination in a crop field.     

Control of Verticillium Yellows in Chinese Cabbage by the Dark Septate Endophytic Fungus LtVB3

ABSTRACT Three hundred forty-nine fungal endophytes were obtained from a total of 1,214 root segments of eggplant, melon, barley, and Chinese cabbage grown as bait plants in a mixed soil made up of samples from different forest soils in Alberta and British Columbia, Canada. Three of the 349 isolates, when inoculated in axenically reared Chinese cabbage seedlings grown in petri dishes, almost completely suppressed the effects of a postinoculated and virulent strain of Verticillium longisporum. Two isolates effective against the pathogen were Phialocephala fortinii, which had been obtained from the roots of eggplant and Chinese cabbage. The third isolate was a dark septate endophytic (DSE) fungus obtained from barley roots. Hyphae of P. fortinii grew along the surface of the root and formed microsclerotia on or in the epidermal layer. Hyphae of the DSE fungus heavily colonized root cells of the cortex. Seedlings grown for 1 week in the presence of the endophytes were then challenged with the Verticillium pathogen. In DSE-treated roots, some of cell walls in the epidermal and cortical layers showed cell wall appositions and thickenings, which appeared to limit the ingress of the pathogen into adjacent cells. Such marked host reactions were not observed in the root cells colonized by P. fortinii. Chinese cabbage preinoculated with the above endophytes and, for comparison, a previously reported disease-suppressive fungal endophyte, Heteroconium chaetospira, were transplanted into the field and disease symptoms were assessed. The DSE could most effectively inhibit the development of Verticillium yellows, with reductions in the percentages of external and internal disease symptoms of 84 and 88%, respectively. The protective values against the disease are extremely high compared with those of other isolates. Most of the DSE-treated plants in the plots achieved marketable quality.