Z-3-Hexenylacetate emissions induced by the endophyte Epichloë occultans at different levels of defoliation during the host plant's life cycle

Vertically transmitted fungal endophytes are common defensive symbionts of cool-season grasses. Protection against herbivores has been generally associated with alkaloids produced in the grass-endophyte symbiosis. However, many other changes occur in host metabolism like the release of VOCs. We aimed at characterizing the profile of volatile organic compounds (VOCs) induced by simultaneous fungal endophyte symbiosis and defoliation during the entire life cycle of the annual host grass and the asexual symbiont. We designed an outdoor factorial experiment with plots dominated by intact and damaged Lolium multiflorum plants with high and low infection levels with Epichloë occultans. After exploring the entire VOC profile, the green leaf volatile (Z)-3-hexenyl acetate (Z3-HAC) was found as the main compound emitted by the plants under field conditions. While in low-infected patches there were no differences in volatile emission during the plant life cycle, highly infected patches emitted more Z3-HAC in seedling and vegetative phases than in the reproductive phase. The role of Z3-HAC provided by symbiotic to neighbouring non-symbiotic plants in the associational protection against herbivores and fungal pathogens is discussed.     

Contrasting diurnal variations in soil organic carbon decomposition and root respiration due to a hysteresis effect with soil temperature in a Gossypium s. (cotton) plantation

Cool-season grasses commonly harbor fungal endophytes in their aerial tissues. However the effects of these symbionts on soil microbial communities have rarely been investigated. Our objective was to explore microbial community responses in soils conditioned by plants of the annual grass Lolium multiflorum with contrasting levels of infection with the endophyte Neotyphodium occultans. At the end of the host growing season, we estimated the functional capacity of soil microbial communities (via catabolic response profiles), the contribution of fungi and bacteria to soil activity (via selective inhibition with antibiotics), and the structure of both microbial communities by molecular analyses. Soil conditioning by highly infected plants affected soil catabolic profiles and tended to increase soil fungal activity. We detected a shift in bacterial community structure while no changes were observed for fungi. Soil responses became evident even without changes in host plant biomass or soil organic carbon or total nitrogen content, suggesting that the endophyte modified host rhizodepositions during the conditioning phase. Our results have implications for the understanding of the reciprocal interactions between above and belowground communities, suggesting that plant-soil feedbacks can be mediated by this symbiosis.     

A fungal endophyte of an annual weed reduces host competitive ability and confers associational protection to wheat

Epichloid fungal endophytes (Epichloë spp., Ascomycota: Clavicipitaceae) inhabit aerial tissues of several cool-season grasses, and enhance host growth and defence against herbivores. The presence of these symbionts can also affect interactions between the host and other non-epichloid plants. The role of an epichloid endophyte on interspecific competition has been tested using perennial grasses with contrasting results, but it has been scarcely tested using annual species in agroecosystems. We evaluated the impact of Epichloë-grass symbiosis on the competitive interaction between a non-host cereal crop (Triticum aestivum, wheat) and a host weed (Lolium multiflorum, ryegrass), growing in the presence of invertebrate herbivores (aphids) under no resource limitation. We conducted an outdoor mesocosm experiment with wheat plants growing in monoculture or in mixture with low or high proportions of ryegrass plants. Ryegrass plants presented either low (E-) or high (E+) incidence of Epichloë occultans (i.e. frequency of epichloid endophytic plants). We measured wheat vegetative and reproductive yield and its natural aphid infestation. Although epichloid endophyte incidence did not affect ryegrass biomass, wheat reproductive yield in mixtures (relative to wheat monocultures) was 45% higher when grown with E+ ryegrass plants than E- conspecific plants. Aphids preferred wheat plants grown with E- plants rather than wheat plants grown with E+ plants, but only in mixtures with high proportion of ryegrass. Our results demonstrate that epichloid endophyte incidence can decrease host competitive ability and confers associational protection to the non-endophytic neighbouring plants. Thus, ryegrass-endophyte symbiosis can increase crop yield by positive neighbourhood effects through different mechanisms probably related to the density of the weed. The benefits of this endosymbiont cannot be considered host-exclusive since they can be disseminated to non-endophytic plants. Furthermore, our results suggest that the epichloid endophyte incidence on annual weeds can contribute to agroecosystem sustainability by influencing pest management and increasing crop yield.     

Flowers for <Emphasis Type="Italic">Neotyphodium</Emphasis> endophytes detection: a new observation method using flowers of host grasses

Neotyphodium endophytes are vertically transmitted fungal symbionts of grasses. Being pest-repelling and growth-promoting agents for their hosts, and also potential mycotoxin producers, their detection in plants is important. Observation of chemically cleared flowers of infected grasses (Festuca arundinacea, F. pratensis, Lolium perenne, and L. multiflorum) using differential interference contrast microscopy revealed the existence of endophytes within immature ovaries of host plants. This observation method provides an accurate and easy way to detect and distinguish Neotyphodium endophytes in flowering host grasses and to investigate the seed transmission process, which is critical to their life cycle, and the practical use of infected plants.     

Family issues: fungal endophyte protects host grass from the closely related pathogen Claviceps purpurea

Certain cool season grasses establish systemic and asymptomatic symbioses with clavicipitaceous fungi of the genus Neotyphodium, which affect multiple biotic interactions within host neighborhood. The presumed symbiont-mediated plant resistance to pathogens is mostly based on studies performed under laboratory and greenhouse conditions. Here we investigated, in two outdoor experiments, the relation between two fungi of the same family with opposite effects on Lolium multiflorum plants: the mutualist endophyte Neotyphodium occultans, and the pathogen Claviceps purpurea. Natural infection and its consequences on symbiotic and non-symbiotic plants were studied under varying conditions of stress by herbicide. In both experiments, N. occultans reduced significantly the infection by C. purpurea at population levels (70 % less). The percentage of spikes infected by C. purpurea was almost three times lower in endophyte-symbiotic plants than in non-symbiotic ones. However, the protective effect was not maintained under stress condition. Our results show that constitutive symbionts such as the systemic fungal endophytes mediate the interaction between host grasses and pathogens, although the effect may depend on the level of stress in the environment.     

Hybridization of Neotyphodium endophytes enhances competitive ability of the host grass

? Associations with microbial symbionts may lead to niche differentiation of their host. Vertically transmitted Neotyphodium endophytes of grasses often hybridize in nature. Infection by these hybrid symbionts may result in different host-plant phenotypes from those caused as a result of infection by nonhybrid symbionts. Observations of wild Arizona fescue (Festuca arizonica) populations show that hybrid Neotyphodium-infected (H+) grasses dominate in resource-poor environments, whereas nonhybrid endophyte-infected (NH+) grasses dominate in environments with more resources. We studied the hypothesis that hybridization of endophytes increases stress tolerance of the host. ? To test whether hybridization of Neotyphodium affects performance and competitive abilities of the host depending on resources, we conducted a glasshouse experiment where competition, nutrients and watering were manipulated. ? H+ plants had greater wet biomass than NH+ and endophyte-free plants, when grown in competition, but only in low-water and low-nutrient treatments. By contrast, NH+ plants did not perform better than H+ or endophyte-free plants regardless of the treatment combination. ? Our results suggest that hybridization of symbiotic Neotyphodium endophytes may increase competitive potential of the host in stressful environments and that this hybridization may be underlying niche expansion of Arizona fescue in the environments with low resources.     

禾草内生真菌的遗传多样性及其共生关系

 近百年的禾草内生真菌研究历经了由浅入深的过程,从最初的家畜中毒事件认识到是一种共生内生真菌存在的缘故,到如今利用分子生物学技术揭示其共生机制,人类发现这类植物内生真菌并非想象中的对生态系统无足轻重。Epichloë及其无性型Neotyphodium与禾本科植物是系统发生的互利共生关系,尤其是Neotyphodium可提高宿主抵抗环境胁迫的能力和抵御动物的取食,增强植物的竞争力。禾草内生真菌有3种生活史：有性生活史、无性生活史和兼性生活史,后者表明真菌在不同的宿主及环境下既能营有性生殖也可营无性繁殖,是一种更灵活而有效的生活史对策。对内生真菌分子系统学、生活史以及与宿主禾草协同进化的研究发现,Neotyphodium起源于禾草致病真菌Epichloë的某些种,或是Epichloë与Neotyphodium的种间杂交后代。植物和内生真菌各异的生活史策略,真菌的种间杂交,两者的协同进化亦或种群间基因流的差异,都促成了共生体多样化的基因组合（Genetic combination ）,也是其共生关系多样化的根源。内生真菌对宿主的有益作用只在特定基因型真菌、宿主和一定环境条件下才起作用,自然生态系统的共生关系要比农业系统复杂得多,是一个从互利共生至寄生关系的连续系统。未来对于更多共生体的遗传背景和基因与环境相互作用的阐明将有助于对禾草内生真菌共生关系本质更加深入的认识。     

内生真菌对羽茅生长及光合特性的影响

通过温室栽培实验,以感染两种内生真菌(Neotyphodium sibiricum和Neotyphodium gansuence)和未感染内生真菌的羽茅(Achnatherum sibiricum)为实验材料,分析感染不同种内生真菌对宿主植物的生长及光合特性的影响。结果表明,感染两种内生真菌羽茅的株高和CO₂补偿点显著低于未染菌的羽茅,而染菌羽茅的蒸腾速率和气孔导度显著高于未染菌羽茅,但对于感染不同种内生真菌的羽茅,无论是分蘖数与生物量的积累还是光合生理值之间均无显著差异。     

Can seed-borne endophytes promote grass invasion by reducing host dependence on mycorrhizas?

Symbiotic interactions between plants and microorganisms have recently become the focus of research on biological invasions. However, the interaction between different symbionts and their consequences in host-plant invasion have been seldom explored. Here, we propose that vertically transmitted fungal endophytes could reduce the dependency of invasive grasses on mycorrhizal fungi allowing host establishment in those environments where the specific mutualist may be not present. Through analyzing published studies on nine grass species, we evaluated the effect of seed-borne Epichloë endophytes on the relationship of invasive and non-invasive grasses with arbuscular mycorrhizal fungi (AMF), a symbiosis known to be fundamental for plant fitness and invasion success. The endophyte effect on AMF colonization differed between invasive and non-invasive grasses, reducing mycorrhization only on invasive species but with no impact on their biomass. These results allowed us to propose that Epichloë endophytes could reduce the dependency of host plants on the mutualism with AMF, promoting host grass establishment and subsequent invasion. Simultaneous interactions with different types of mutualists may have profound effects on the host-plant fitness facilitating its range expansion. Our findings suggest that some specific mutualistic fungi such as epichloid endophytes facilitate host invasion by reducing the requirements of the benefits derived from other mutualisms.     

Three symbionts involved in interspecific plant-soil feedback: epichloid endophytes and mycorrhizal fungi affect the performance of rhizobia-legume symbiosis

Aims

Plants interact by modifying soil conditions in plant-soil feedback processes. Foliar endophytes of grasses exert multiple effects on host rhizosphere with potential consequences on plant-soil feedback. Here, we hypothesize that the grass-endophyte symbiosis impairs soil symbiotic potential, and in turn influences legume performance and nitrogen acquisition.

Methods

Soil was conditioned in pots, growing Lolium multiflorum with or without the fungal endophyte Epichloë and with or without arbuscular mycorrhizal fungi (AMF). Then, Trifolium repens grew in all types of conditioned soils with high or low rhizobia availability.

Results

Endophyte soil conditioning reduced AMF spores number and rhizobial nodules (?27 % and ?38 %, respectively). Seedling survival was lower in endophyte-conditioned soil and higher in mycorrhizal soils (?27 % and +24 %, respectively). High rhizobia-availability allowed greater growth and nitrogen acquisition, independent of soil conditioning. Low rhizobia-availability allowed both effects only in endophyte-conditioned soil.

Conclusion

Endophyte-induced changes in soil (i) hindered symbiotic potential by reducing AMF spore availability or rhizobia nodulation, (ii) impaired legume survival irrespective of belowground symbionts presence, but (iii) mimicked rhizobia effects, enhancing growth and nitrogen fixation in poorly nodulated plants. Our results show that shoot and root symbionts can be interactively involved in interspecific plant-soil feedback.

     

禾草内生真菌对其它微生物的影响研究进展

禾草内生真菌在宿主植物的茎叶等地上组织中普遍存在,不仅能够提高禾草对生物与非生物逆境的抗性,而且能够对周围环境中的不同微生物类群产生影响。主要总结了禾草Neotyphodium/Epichlo内生真菌对病原真菌、丛枝菌根真菌和土壤微生物的影响及其作用机理。发现禾草内生真菌普遍存在对病原真菌的抑制作用,而对丛枝菌根真菌存在不对称的竞争作用,且因种类而异。禾草内生真菌对土壤微生物群落的作用则会随着土壤类型和时间等外界因素发生变化。禾草内生真菌对不同类群微生物的影响机制主要包括:通过生态位竞争、抑菌物质分泌、诱导抗病性等对病原真菌造成影响;通过根系化学物质释放、营养元素调节、侵染条件差异等对丛枝菌根真菌造成影响;通过根际沉积物和凋落物等对土壤微生物群落造成影响。禾草内生真菌产生的生物碱能提高宿主植物对包括昆虫在内草食动物采食的抗性,影响病原菌的侵入、定殖和扩展;根组织分泌物中包含次生代谢产物能够抑制菌根真菌、土传病原真菌及其它土壤微生物的侵染与群落组成;也可能通过次生代谢物影响禾草的其它抗性。因此,禾草内生真菌在植物-微生物系统中的作用应该给予更多的关注和深入研究。     

Plant nitrogen acquisition and interactions under elevated carbon dioxide: impact of endophytes and mycorrhizae

Both endophytic and mycorrhizal fungi interact with plants to form symbiosis in which the fungal partners rely on, and sometimes compete for, carbon (C) sources from their hosts. Changes in photosynthesis in host plants caused by atmospheric carbon dioxide (CO₂) enrichment may, therefore, influence those mutualistic interactions, potentially modifying plant nutrient acquisition and interactions with other coexisting plant species. However, few studies have so far examined the interactive controls of endophytes and mycorrhizae over plant responses to atmospheric CO₂ enrichment. Using Festuca arundinacea Schreb and Plantago lanceolata L. as model plants, we examined the effects of elevated CO₂ on mycorrhizae and endophyte (Neotyphodium coenophialum) and plant nitrogen (N) acquisition in two microcosm experiments, and determined whether and how mycorrhizae and endophytes mediate interactions between their host plant species. Endophyte‐free and endophyte‐infected F. arundinacea varieties, P. lanceolata L., and their combination with or without mycorrhizal inocula were grown under ambient (400 μmol mol⁻¹) and elevated CO₂ (ambient + 330 μmol mol⁻¹). A ¹⁵N isotope tracer was used to quantify the mycorrhiza‐mediated plant acquisition of N from soil. Elevated CO₂ stimulated the growth of P. lanceolata greater than F. arundinacea, increasing the shoot biomass ratio of P. lanceolata to F. arundinacea in all the mixtures. Elevated CO₂ also increased mycorrhizal root colonization of P. lanceolata, but had no impact on that of F. arundinacea. Mycorrhizae increased the shoot biomass ratio of P. lanceolata to F. arundinacea under elevated CO₂. In the absence of endophytes, both elevated CO₂ and mycorrhizae enhanced ¹⁵N and total N uptake of P. lanceolata but had either no or even negative effects on N acquisition of F. arundinacea, altering N distribution between these two species in the mixture. The presence of endophytes in F. arundinacea, however, reduced the CO₂ effect on N acquisition in P. lanceolata, although it did not affect growth responses of their host plants to elevated CO₂. These results suggest that mycorrhizal fungi and endophytes might interactively affect the responses of their host plants and their coexisting species to elevated CO₂.     

Callus induction and plant regeneration of <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Cattle breeding is an important economical activity in Argentina, highly dependent on grass production. In the last decades, grasslands zones were reduced and confined to less productive lands due to the advance of agronomical cultures. Therefore, it is important to develop new strategies to improve forage production. New eco-friendly trends in plant growth promotion include the use of microbial endophytes, but the in vitro studies of plant-bioinoculant interactions is limited by the scarce current technological development. In this work, we use a micropropagation protocol for Lolium multiflorum, developed in a previous work, to study the effect of bacterization with actinobacterial endophytes, isolated from Argentine native grasses, on the growth of L. multiflorum in vitro plantlets. To achieve this objective, L. multiflorum plantlets were inoculated with three Micromonospora strains (SB3, TW2.1 and TW2.2). The results obtained showed that the effect of actinobacterial inoculation depends on the Micromonospora strain used. The inoculation with SB3 promoted plant growth, increasing plant biomass, root length and the rate of plantlets ready to be acclimatized after 4 weeks of in vitro culture. Strain TW2.1 did not show, statistically, differences compared to control treatments, while TW2.2 inhibited plant growth, decreasing plant biomass, root length and the rate of plants ready to acclimatize. Our results showed that Micromonospora strain SB3 could be a good candidate to use in breeding programs for L. multiflorum and other grasses to increase their yield.     

不同传播方式的内生真菌感染对羽茅的生理生态影响北大核心CSCD

通过田间试验,研究了垂直传播的Neotyphodium属内生真菌和水平传播的Epichloё属内生真菌对羽茅(Achnatherum sibiricum)生理生态的影响。结果表明:Neotyphodium属内生真菌感染对羽茅的株高和叶长有显著的正效应,而Epichloё属内生真菌感染对羽茅株高和叶长的增益作用较少。感染Neotyphodium属内生真菌的羽茅最大净光合速率显著高于感染Epichloё属内生真菌的羽茅,Neotyphodium属内生真菌显著提升了羽茅的气孔限制值和水分利用效率,而实验测定的第一年结果显示:Epichloё属内生真菌对羽茅的各个光合指标具有明显的负效应(胞间CO2浓度除外)。Neotyphodium属内生真菌感染使得宿主植物积累的可溶性糖含量显著高于感染Epichloё属内生真菌的羽茅和不染菌的羽茅植株。感染Epichloё属内生真菌的羽茅中氮含量显著高于感染Neotyphodium属内生真菌的羽茅。总之,内生真菌的传播方式是影响羽茅生理生态学特性的一个因素,并且垂直传播的内生真菌对羽茅生长和生理特性的益处高于水平传播的内生真菌。     

内生真菌对草坪植物病原真菌抑制作用的比较

分别从野生牧草羽茅(Achnatherum sibiricum (L.) Keng)、栽培种高羊茅(Festuca arundinacea Schreb.)(品种Millennium)、栽培种黑麦草(Lolium perenne L.) (品种Justus)中分离出内生真菌Neotyphodium sp.、N. coenophialum和N. lolli,通过体外培养法比较了这3种内生真菌对草坪植物病原真菌的抑制作用.结果表明,从羽茅中分离的内生真菌Neotyphodium sp.在两菌相交前对所有供试的病原真菌都有一定的抑制作用,其中对枝孢霉属(Cladosporium sp.)、弯孢霉属(Curvularia sp.)和拟茎点属(Phomopsis sp.)病原真菌的抑制效果尤为显著,对峙培养3d后的抑菌率分别达70.1%、52.3%和30.9%,营养竞争作用、重寄生作用是其主要的拮抗机制;从高羊茅中分离的内生真菌N. coenophialum对枝孢霉属病原真菌存在一定的抑制作用;而从黑麦草中分离的内生真菌N. lolli与病原真菌对峙培养时,病原真菌菌落慢慢侵占整个营养空间,内生真菌停止生长并逐渐褐变死亡.体外培养结果说明Neotyphodium sp.对供试病原真菌的拮抗效果优于N. coenophialum和N. lolli,由此推测Neotyphodium sp.与宿主植物羽茅的共生可能有利于宿主植物抵抗病原真菌的侵扰.     

Symbiosis with systemic fungal endophytes promotes host escape from vector-borne disease

Plants interact with a myriad of microorganisms that modulate their interactions within the community. A well-described example is the symbiosis between grasses and Epichloë fungal endophytes that protects host plants from herbivores. It is suggested that these symbionts could play a protective role for plants against pathogens through the regulation of their growth and development and/or the induction of host defences. However, other endophyte-mediated ecological mechanisms involved in disease avoidance have been scarcely explored. Here we studied the endophyte impact on plant disease caused by the biotrophic fungus, Claviceps purpurea, under field conditions through (1) changes in the survival of the pathogen´s resistance structure (sclerotia) during overwintering on the soil surface, and (2) effects on insects responsible for the transportation of pathogen spores. This latter mechanism is tested through a visitor exclusion treatment and the measurement of plant volatile cues. We found no significant effects of the endophyte on the survival of sclerotia and thus on disease inocula. However, both pathogen incidence and severity were twofold lower in endophyte-symbiotic plants than in non-symbiotic ones, though when insect visits were prevented this difference disappeared. Endophyte-symbiotic and non-symbiotic plots presented different emission patterns of volatiles suggesting that they can play a role in this protection. We show a novel indirect ecological mechanism by which endophytes can defend host grasses against diseases through negatively interacting with intermediary vectors of the epidemic process.     

Epichloë endophyte affects the root colonization pattern of belowground symbionts in a wild grass

Plants host multiple symbionts that interact with each other affecting plant performance and regulating their establishment. Here, we analyzed how the association with Epichloë endophytes affects belowground colonization by Dark Septate Endophytes (DSE) and arbuscular mycorrhizal fungi (AMF) in the grass Bromus auleticus. Epichloë-symbiotic (E+) and Epichloë-non symbiotic (E−) plants were sampled from a long-term experimental plot and colonization structures were analyzed in the roots. We also examined the influence of Epichloë exudates on the in vitro growth of DSE Microdochium bolleyi isolated from roots. Epichloë symbiosis increased AMF colonization, although differences were not significant. Despite the lack of differences in total DSE colonization, in concordance with in vitro findings, a higher significant abundance of microsclerotia was observed in E+ plants. A negative correlation between total mycorrhizal and DSE was found. Our findings show a more uniform root colonization pattern in E+ plants, suggesting a root symbiosis modulating role.     

Endophytes of native grasses from South America: Biodiversity and ecology

We review and present preliminary results of studies on cool-season grass endophytes native to South America. These fungi have been studied only in Argentina, where they have been detected in 36 native grass species. The hybrid Neotyphodium tembladerae is present in an extremely wide host range found in diverse environmental conditions, but some other endophytes seem to be strictly associated with one host species in a particular environment. In host species that inhabit different environments, the incidence of endophytes is highly variable among populations and in most of the cases is clearly associated with environmental conditions. In these native grasses, Neotyphodium presents a mutualistic behaviour, conferring enhanced growth, promoting the symbiosis with arbuscular mycorrhizal fungi, and inhibiting growth of pathogenic fungi. In native forage grasses, preliminary analyses indicate that some Argentinian endophytes can produce lolines but are unlikely to produce lolitrem B or ergot alkaloids.     

Trade-off between seed number and weight: Influence of a grass–endophyte symbiosis

Plant fitness is enhanced by resource allocation to seed number (offspring number) or weight (offspring survival). Besides, there is a well known trade-off in resource allocation between both traits. Symbiotic interactions can influence plant resource allocation to reproduction, yet little research has been performed in this direction. We studied the consequences of a grass–fungus symbiosis on the trade-off between seed number and weight, using Lolium multiflorum and the endophyte Neotyphodium occultans as our study system. In ecological terms, we experimentally removed N. occultans from L. multiflorum plants, and compared reproductive allocation to seed number and weight in endophyte-symbiotic vs. non-symbiotic plants at different levels of nutrient availability (small pots vs. large pots). In evolutionary terms, we compared reproductive allocation between symbiotic vs. non-symbiotic plants for different host genotypes. All plants showed a negative association between seed number and weight, once standardized for total reproductive biomass. Under high nutrient availability, endophyte-symbiotic plants showed higher seed weight than non-symbiotic plants for any seed number. However, no differences were observed under low nutrient availability. Endophyte influence also varied according to L. multiflorum genotype; specifically, endophyte-symbiotic plants showed a lower slope in the relationship between seed number and weight than non-symbiotic plants for the ‘Marshall’ genotype but no endophyte influence was found for the “Pampean” genotype. The results implied a higher plasticity in seed weight and lower plasticity in seed number for symbiotic plants. Indeed, endophyte-symbiotic plants showed an overall lower slope in the association between seed number and total reproductive biomass than non-symbiotic plants. Our results suggest that N. occultans induces heavier seeds in L. multiflorum plants under environmental conditions favorable to plant growth or for certain plant genotypes. We propose that symbiotic interactions may influence the evolution of seed number and weight trade-off.