Fungal endophytes of invasive Phagramites australis populations vary in species composition and fungicide susceptibility

Phragmites australis subsp. australis (Poaceae) is an aggressively invasive reed that threatens both freshwater and saltwater ecosystems in North America. We sampled P.a. australis plants for fungal endophytes at seven sites across a short geographic range near a freshwater lake in Michigan. Compared to previous studies, our data reveal novel variation in the diversity and abundance of fungal endophytes within P.a. australis. Within each sampling site we observed 4–10 morphologically unique, culturable fungi. Since fungal endophytes can confer significant benefits to their plant host, we hypothesized that fungal endophytes are important for mediating plant invasions. To test this hypothesis we first had to establish a protocol to experimentally control fungal endophytes within P.a. australis, which does not easily grow from sterile seed. We therefore investigated the effect of fungicides as a potential method for eliminating fungi from living plant tissue such as rhizomes. We selected the ten most abundant fungi isolated from P.a. australis and tested their susceptibility to three commonly available fungicides. Response to the fungicides varied across fungal isolates, demonstrating physiological variation and fungicide-resistant phenotypes.     

Fungal endophytes and phytochemistry of oak foliage: determinants of oviposition preference of leafminers?

Sedentary insect herbivores, such as gallformers and leafminers, are usually non-randomly distributed among and within host plants. Dispersion of these insects is largely a function of female oviposition choice. In field experiments and observations spanning two growing seasons, we tested the hypothesis that selective oviposition on individual leaves within trees by the dominant herbivore of Emory oak, the monophagous leaf-miner Cameraria sp. nov., is determined by the probability of colonization by endophytic fungi. These fungi are alleged to act as plant mutualists by deterring, killing, or inhibiting the growth of insect herbivores. We found that leaves selected by females for oviposition and paired, unmined leaves were equally likely to be colonized by fungal endophytes. Furthermore, condensed and hydrolyzable tannin levels, purported inhibitors of fungal infection, and protein content did not vary between leaves selected by females and unmined leaves, or between leaves with and without endophyte infections. We conclude that female Cameraria do not choose leaves within trees for oviposition on the basis of propensity for endophytic fungal infection or on phytochemical parameters that might indicate probability of future infections. At this spatial scale at least, fungal endophytes do not explain the highly aggregated distribution of Cameraria among leaves and associated costs in terms of increased larval mortality. Fungal endophytes may, nevertheless, affect leafminer dispersion and abundance at larger spatial scales, such as host plant populations or species. We did find, however, that the amount of mining activity on leaves is positively associated with increased colonization by fungal endophytes. We suggest that mining activity increases endophyte fungal infections by facilitating spore germination and hyphal penetration into the leaf or by altering leaf phytochemistry. The facilitation of endophyte colonization by leafmining activity coupled with the lack of predictability of endophyte infections based on leaf phytochemistry and almost 100% infectivity of all oak leaves during sporadic wet years may prevent female leafminers from discriminating leaves for oviposition on the basis of current or future levels of endophytes in leaves.     

Epichlo? endophytes grow by intercalary hyphal extension in elongating grass leaves.

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

Colonisation of barley roots by endophytic Fusarium equiseti and Pochonia chlamydosporia: Effects on plant growth and disease

Colonisation of plant roots by endophytic fungi may confer benefits to the host such as protection against abiotic or biotic stresses or plant growth promotion. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Fusarium equiseti is a naturally occurring endophyte in vegetation under stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode egg-parasitic fungus with a worldwide distribution. Both fungi have the capacity to colonise roots of non-host plants endophytically and to protect them against phytopathogenic fungi under laboratory conditions. The aim of this study was to evaluate the root population dynamics of these fungi under non-axenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence of disease caused by the pathogen Gaeumannomyces graminis var. tritici were monitored periodically. Both fungi colonised barley roots endophytically over the duration of the experiment and competed with other existing fungal root colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted plant growth. Although a clear suppressive effect on disease could not be detected, F. equiseti isolates reduced the mean root lesion length caused by the pathogen. Results of this work suggest that both F. equiseti and P. chlamydosporia are long-term root endophytes that confer beneficial effects to the host plant.     

Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

Colonization of Onions by Endophytic Fungi and Their Impacts on the Biology of Thrips tabaci

Endophytic fungi, which live within host plant tissues without causing any visible symptom of infection, are important mutualists that mediate plant–herbivore interactions. Thrips tabaci (Lindeman) is one of the key pests of onion, Allium cepa L., an economically important agricultural crop cultivated worldwide. However, information on endophyte colonization of onions, and their impacts on the biology of thrips feeding on them, is lacking. We tested the colonization of onion plants by selected fungal endophyte isolates using two inoculation methods. The effects of inoculated endophytes on T. tabaci infesting onion were also examined. Seven fungal endophytes used in our study were able to colonize onion plants either by the seed or seedling inoculation methods. Seed inoculation resulted in 1.47 times higher mean percentage post-inoculation recovery of all the endophytes tested as compared to seedling inoculation. Fewer thrips were observed on plants inoculated with Clonostachys rosea ICIPE 707, Trichoderma asperellum M2RT4, Trichoderma atroviride ICIPE 710, Trichoderma harzianum 709, Hypocrea lixii F3ST1 and Fusarium sp. ICIPE 712 isolates as compared to those inoculated with Fusarium sp. ICIPE 717 and the control treatments. Onion plants colonized by C. rosea ICIPE 707, T. asperellum M2RT4, T. atroviride ICIPE 710 and H. lixii F3ST1 had significantly lower feeding punctures as compared to the other treatments. Among the isolates tested, the lowest numbers of eggs were laid by T. tabaci on H. lixii F3ST1 and C. rosea ICIPE 707 inoculated plants. These results extend the knowledge on colonization of onions by fungal endophytes and their effects on Thrips tabaci.     

A fungal endophyte defensive symbiosis affects plant-nematode interactions in cotton

Background and aims

Most investigations of fungi as nematode antagonists have focused on their interactions with nematodes in the soil. This study tested a foliar-isolated endophytic Phialemonium inflatum for its effects against the root-knot nematode as an endophyte in cotton using a seed treatment inoculation.

Methods

Cotton seeds were inoculated with P. inflatum spore suspensions prior to planting. Nematode infection and reproduction were quantified at Day 12 and 6 weeks after nematode egg inoculation, respectively. To establish whether the observed negative effects on nematodes were due to P. inflatum in the soil or as an endophyte in the plant, we also applied a soil fungicide treatment at the seedling stage to kill the fungi outside the plant.

Results

Persistent suppression of nematode penetration and galling, as well as subsequent reproduction, were observed in endophyte-treated plants independent of fungicide treatment, consistent with an endophytic mode of nematode suppression; and these negative effects did not depend on the concentration of fungal inoculum used to treat to the seed.

Conclusions

Our study highlights a novel role for P. inflatum as part of a plant-fungal defensive symbiosis in cotton, as well as the need for a broader understanding of endophyte-plant-nematode ecological interactions.

     

Using direct amplification and next-generation sequencing technology to explore foliar endophyte communities in experimentally inoculated western white pines

Fungal endophytes can influence survivability and disease severity of trees. Here we characterized the endophyte community in Pinus monticola (western white pine), an important species in the northwest USA, largely decimated by pathogenic fungi. We also assessed the ability to successfully inoculate seedlings with desirable endophytes, with the long-term goal of providing a protective microbiome and added defense from pathogens. P. monticola seedlings were inoculated in the field with potential pathogen antagonists and fungi isolated from healthy mature trees. Following inoculations direct amplification and next generation sequencing were used to characterize fungal endophyte communities, and explore interspecific competition, diversity, and co-occurrence patterns in needle tissues. Negative co-occurrence patterns between inoculated fungi and potential pathogens, as well as many other species, suggest early competitive interactions. Our study explores early endophyte community assemblage and shows that fungal inoculations may influence tree growth.     

Culturable Endophytes Associated with Soybean Seeds and Their Potential for Suppressing Seed-Borne Pathogens

Seed-borne pathogens in crops reduce the seed germination rate and hamper seedling growth, leading to significant yield loss. Due to the growing concerns about environmental damage and the development of resistance to agrochemicals among pathogen populations, there is a strong demand for eco-friendly alternatives to synthetic chemicals in agriculture. It has been well established during the last few decades that plant seeds harbor diverse microbes, some of which are vertically transmitted and important for plant health and productivity. In this study, we isolated culturable endophytic bacteria and fungi from soybean seeds and evaluated their antagonistic activities against common bacterial and fungal seed-borne pathogens of soybean. A total of 87 bacterial isolates and 66 fungal isolates were obtained. Sequencing of 16S rDNA and internal transcribed spacer amplicon showed that these isolates correspond to 30 and 15 different species of bacteria and fungi, respectively. Our antibacterial and antifungal activity assay showed that four fungal species and nine bacterial species have the potential to suppress the growth of at least one seed-borne pathogen tested in the study. Among them, Pseudomonas koreensis appears to have strong antagonistic activities across all the pathogens. Our collection of soybean seed endophytes would be a valuable resource not only for studying biology and ecology of seed endophytes but also for practical deployment of seed endophytes toward crop protection.     

Antioxidants in Festuca rubra L. seeds affected by the fungal symbiont Epichloë festucae

Vertically transmitted fungal endophytes can be beneficial for host grasses. While the alkaloid-mediated mechanism for herbivore resistance has been widely studied, underlying physiological mechanisms for increased tolerance to abiotic stress remain scarcely explored. In this study we used three maternal lines of perennial grass Festuca rubra to examine the role of antioxidants in endophyte-mediated effects on seed viability over long-term storage. Uncolonized plants (E?) were generated by removing the endophyte from ramets of naturally endophyte-colonized (E+) plants. The E?+ and E? ramets were planted in a common garden in Salamanca, Spain. Seeds produced in 2009, 2010 and 2011 were harvested at maturity, dried and stored at 10 °C until 2011 when we tested seed and endophyte viability, and measured antioxidants. Seed viability and α-tocopherol antioxidant were negatively affected by the endophyte in two maternal lines. In these same lines, the endophyte viability was lowest at the longest storage time. In the maternal line that showed the highest negative effect of endophyte on seed viability, the pattern of glutathione was opposite to that observed for tocopherols since it was higher for E?+ than for E? seeds. In all maternal lines, the glutathione half-cell reduction potential (E_GSSG/2GSH) and % glutathione disulphide (GSSG) increased with storage time but there was no clear pattern associated with endophyte symbiosis. Whether these parameters are good predictors of seed and endophyte longevity in storage and natural conditions should be further explored.     

The influence of host plant,nitrogen fertilization and fungicide application on the abundance and seasonal dynamics of vesicular-arbuscular mycorrhizal fungi in arable soils of northern Germany

The present investigation examines whether the crop plant, increased nitrogen (N) fertilization and fungicide application influence the pattern of vesicular-arbuscular mycorrhizal fungi (VAM) populations. For this purpose, two arable field locations in Lower Saxony (Hotteln and Langreder) were chosen and the formation of mycorrhiza, spore density, number of infectious propagules (MPN) and frequency of spore types within VAM populations were investigated. The influence of crop plants was examined over two cultivation periods (1986/1987 and 1987/1988) in Hotteln, comparing winter wheat, winter barley and sugar beet. The effects of increased N fertilization and fungicide application were investigated on winter wheat in Langreder in the cultivation period 1988 only. Both the frequency of mycorrhizal infection and the spore dynamics in soil differed with the crops grown. Spore density and MPN increased until harvesting when host plants (winter wheat, winter barley) were cultivated, whereas both diminished with a non-host plant (sugar beet). Different spore types increased or decreased, according to the plant species grown, but the predominating types of the location remained constant. Increased N fertilization caused marginal inhibition of mycorrhizal infection and sporulation on winter wheat, whereas both leaf and base application of fungicides resulted in minor increases in mycorrhizal colonization of roots and sporulation in soil. Both increased N fertilization and fungicide application distinctly decreased the sporulation of one type in May, but the characteristic compositions of the VAM populations remained unchanged.     

Two fungal symbioses collide: endophytic fungi are not welcome in leaf-cutting ant gardens

Interactions among the component members of different symbioses are not well studied. For example, leaf-cutting ants maintain an obligate symbiosis with their fungal garden, while the leaf material they provide to their garden is usually filled with endophytic fungi. The ants and their cultivar may interact with hundreds of endophytic fungal species, yet little is known about these interactions. Experimental manipulations showed that (i) ants spend more time cutting leaves from a tropical vine, Merremia umbellata, with high versus low endophyte densities, (ii) ants reduce the amount of endophytic fungi in leaves before planting them in their gardens, (iii) the ants'' fungal cultivar inhibits the growth of most endophytes tested. Moreover, the inhibition by the ants'' cultivar was relatively greater for more rapidly growing endophyte strains that could potentially out-compete or overtake the garden. Our results suggest that endophytes are not welcome in the garden, and that the ants and their cultivar combine ant hygiene behaviour with fungal inhibition to reduce endophyte activity in the nest.     

Multitrophic interactions among fungal endophytes,bees, and <Emphasis Type="Italic">Baccharis dracunculifolia</Emphasis>: resin tapering for propolis production leads to endophyte infection

The tropics are known for their high diversity of plants, animals, and biotic interactions, but the role of the speciose endophytic fungi in these interactions has been mostly neglected. We report a unique interaction among plant sex, bees, and endophytes on the dioecious shrub, Baccharis dracunculifolia (Asteraceae). We assessed whether there was an association between resin collection by bees and fungal endophytes considering the host plant sex. We hypothesized that resin collection by the Africanized honey bee, Apis mellifera L. (Apidae) could favor the entry of endophytes in B. dracunculifolia. Specifically, we tested the hypotheses that (1) bees damage the leaf buds of female and male plant at different proportions; (2) damage on leaf buds increases the richness of endophytic fungi; (3) endophyte richness differs between female and male plants; and (4) in vitro growth of endophytes depends on the sex of the plant individual from which the resin was extracted. Endophyte richness and proportion of leaf bud damage did not vary between the plant sexes. However, species similarity of endophytes between female and male plants was 0.33. Undamaged leaf buds did not show culturable endophytes, with all fungi exclusively found in damaged leaf buds. Endophyte composition changed with the plant sex. The endophytes exclusively found in female plants did not develop in the presence of male resin extract. These findings highlight that resin collection by A. mellifera for propolis production favors the entry of endophytic fungi in B. dracunculifolia. Additionally, endophyte composition and growth are influenced by plant sex.     

内生真菌感染对宿主羊草抗病性的影响

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Epichlo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.lunata和C.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。     

Correction to: Neighbourhood stories: role of neighbour identity,spatial location and order of arrival in legume and non-legume initial interactions

Background and aims

We characterized fungal endophytes of seeds of invasive, non-native Phragmites from three sites in the Great Lakes region to determine if fungal symbiosis could contribute to invasiveness through their effects on seed germination and seedling growth.

Methods

Field-collected seeds were surface sterilized and plated on agar to culture endophytes for ITS sequencing. Prevalence of specific endophytes from germinated and non-germinated seeds, and from seedlings, was compared.

Results

One-third of 740 seeds yielded endophyte isolates. Fifteen taxa were identified with Alternaria sp. representing 54% of all isolates followed by Phoma sp. (21%) and Penicillium corylophilum (12%). Overall germination of seeds producing an isolate (36%) was significantly higher than seeds not producing an isolate (20%). Penicillium in particular was strongly associated with increased germination of seeds from one site. Sixty-three isolates and 11 taxa were also obtained from 30 seedlings where Phoma, Penicillium and Alternaria respectively were most prevalent. There was a significant effect of isolating an endophyte from the seed on seedling growth.

Conclusions

These results suggest that many endophyte taxa are transmitted in seeds and can increase seed germination and seedling growth of invasive Phragmites. The role of fungal endophytes in host establishment, growth and invasiveness in nature requires further research.

     

Fungal endophytes which invade insect galls: insect pathogens,benign saprophytes,or fungal inquilines?

Fungi are frequently found within insect galls. However, the origin of these fungi, whether they are acting as pathogens, saprophytes invading already dead galls, or fungal inquilines which invade the gall but kill the gall maker by indirect means, is rarely investigated. A pathogenic role for these fungi is usually inferred but never tested. I chose the following leaf-galling-insect/host-plant pairs (1) a cynipid which forms two-chambered galls on the veins of Oregon white oak, (2) a cynipid which forms single-chambered galls on California coast live oak, and (3) an aphid which forms galls on narrowleaf cottonwood leaves. All pairs were reported to have fungi associated with dead insects inside the gall. These fungi were cultured and identified. For the two cynipids, all fungi found inside the galls were also present in the leaves as fungal endophytes. The cottonwood leaves examined did not harbor fungal endophytes. For the cynipid on Oregon white oak, the fungal endophyte grows from the leaf into the gall and infects all gall tissue but does not directly kill the gall maker. The insect dies as a result of the gall tissue dying from fungal infection. Therefore, the fungus acts as an inquiline. Approximately 12.5% of these galls die as a result of invasion by the fungal endophyte.     

Role of Diverse Non-Systemic Fungal Endophytes in Plant Performance and Response to Stress: Progress and Approaches

Plant–fungal symbiotic associations are ubiquitously distributed in natural plant communities. Besides the well-studied mycorrhizal symbiosis and grass systemic clavicipitaceous endophytes, recently, nonsystemic and horizontally transmitted fungal endophytes serving as plant symbionts have been increasingly recognized. Pure culture isolation and culture-independent molecular methods indicate that all parts of healthy plant tissues potentially harbor diverse and previously unknown fungal lineages. Limited evidence also supports a hypothesis that endophytic mycobiota dynamics may have a role in evolution of plants. High variability or “balanced antagonism” can be generally characterized with host–endophyte interactions, which implies that the outcome of symbiotic interactions can fall within a continuum ranging from mutualism to commensalism, and ultimately pathogenicity. Despite this complicated system, admittedly, fungal endophytes really endow the host with an extended phenotype. Accumulating facts illustrate that plant nutrition acquisition, metabolism, and stress tolerance may be strengthened or modulated via fungal symbionts. Piriformospora indica, a member of the order Sebacinales, simultaneously confers host resistance to biotic and abiotic stress. The ecological relevance of other fungal groups, including foliar endophytes, root dark septate endophytes (DSEs), some opportunistic and avirulent microsymbionts (for example, Trichoderma and Fusarium), and even uncultured fungi structurally and physiologically integrated with host tissues, are also being deeply exploited. Production of bioactive metabolites by fungi, overexpression of stress-related enzymes, and induced resistance in hosts upon fungal colonization are responsible for direct or indirect beneficial effects to hosts. More knowledge of endophyte-mediated enhancement of host performance and fitness will offer alternatively valuable strategies for plant cultivation and breeding. Meanwhile, with unprecedented loss of biodiversity, discovery of indigenously novel symbiotic endophytes from natural habitats is urgently needed. In addition, we present some approaches and suggestions for studying host–endophyte interactions.     

醉马草内生真菌共生体研究进展

有关醉马草(Achnatherum inebrians)内生真菌(Epichloë gansuensis, E. inebrians)共生体的研究, 代表了我国禾草内生真菌研究领域的重要方向, 使中国的醉马草-内生真菌与美国的苇状羊茅(Festuca arundinacea)-内生真菌(E. coenophiana)和新西兰的多年生黑麦草(Lolium perenne)-内生真菌(E. festucae var. lolii)成为禾草内生真菌国际三大研究分支。该文综述了近30年来对醉马草内生真菌共生体的系统研究, 包括: 内生真菌的分布、带菌率、检测方法、多样性, 内生真菌提高宿主的抗旱、耐寒、耐盐碱、耐重金属、抗虫、抗病等抗逆性及其机理, 共生体产生的生物碱等次生代谢物, 对草食动物的毒性, 及其在草地生态系统中的作用等。研究者实验证实了醉马草本身无毒, 只有当内生真菌与醉马草共生并产生麦角新碱和麦角酰胺等麦角类生物碱后才能导致采食醉马草家畜中毒。文章展望了醉马草内生真菌基因组学和功能分析, 利用杀菌剂杀死内生真菌进行醉马草脱毒, 利用无毒内生真菌菌株进行饲用醉马草新品种选育, 利用有毒醉马草内生真菌共生体进行抗虫防鸟的机场绿化新品种选育及生物源农药与医药开发等。     

Consequences of simultaneous interactions of fungal endophytes and arbuscular mycorrhizal fungi with a shared host grass

Plants simultaneously associate with multiple microbial symbionts throughout their lifetimes. To address the question of whether the effects of simultaneous symbionts are contingent on the specific identities, we conducted a greenhouse experiment manipulating the presence and identities of arbuscular mycorrhizal fungi (AMF) and fungal endophytes on the shared host grass Elymus hystrix. Each plant host was inoculated with one of two AMF species having varying effects on host growth, or a sterile soil control. Further, we used naturally occurring endophyte‐infected (E+) and uninfected (E–) individuals from two populations of the endophyte Epichloë elymi that varied in their interaction with E. hystrix. We then measured responses of plants, AMF, and fungal endophytes. Overall, we found that the combined effects of AMF and fungal endophytes on plant growth were additive, reflecting the mutualistic quality of each symbiont independently interacting with host plants. However, fungal endophyte infection differentially altered hyphal colonization of the two AMF species and the identity of the coinfecting AMF species affected fungal endophyte fitness traits. The results of this study demonstrate that the outcome of interspecific symbiotic interactions varies with partner identity such that the effects of simultaneous symbioses can not be generalized.