Creation of Stable Associations Between Perennial Ryegrass Or Tall Fescue and Fungal Endophytes W

Home , Acremonium, Lolium persicum

Creation of stable associations between perennial ryegrass or tall fescue and fungal endophytes W. Naffaa, C. Astier, C. Ravel, J.J. Guillaumin

To cite this version:

W. Naffaa, C. Astier, C. Ravel, J.J. Guillaumin. Creation of stable associations between perennial ryegrass or tall fescue and fungal endophytes. Agronomie, EDP Sciences, 1999, 19 (2), pp.133-144. hal-02690408

HAL Id: hal-02690408 https://hal.inrae.fr/hal-02690408 Submitted on 1 Jun 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original article

Creation of stable associations between perennial ryegrass or tall fescue and fungal endophytes

Walid Naffaaa Claude Astierb Catherine Ravelb Jean-Jacques Guillaumin

a Unité de pathologie végétale et mycologie, centre de Clermont-Ferrand, Inra, 234, avenue du Brézet, 63039 Clermont-Ferrand cedex 02, France b Unité d’amélioration des plantes, centre de Clermont-Ferrand, Inra, 234, avenue du Brézet, 63039 Clermont-Ferrand cedex 02, France

(Received 1 March 1998; accepted 15 January 1999)

Abstract - The ability of 15 isolates of endophytic fungi isolated from ten species of grasses to form compatible associations following artificial infection with perennial ryegrass (Lolium perenne, cv. Vigor) and tall fescue (Festuca arundinacea, cv. Clarine) was examined. Twenty-two associations were obtained, only eight failed. The species Neotyphodium lolii, N. coenophialum, LpTG-2, Epichloe festucae, E. bromicola and Gliocladium-like endophyte, could be associated with both L. perenne and F. arundinacea. Acremonium sp. from L. persicum could be associated with L. perenne only. The observation of infected plants after 6 months and the study of mycelial transmission to the seeds showed the stability of the following associations: tall fescue with N. coenophialum and N. lolii, perennial ryegrass with N. lolii, LpTG-2, Epichloe festucae and Gliocladium-like. Acremonium sp. from Lolium persicum was not able to survive in L. perenne for a long time. Various aspects of partial incompatibility were observed among the associations: tall fescue/LpTG-2, tall fescue / Gliocladium-like and perennial ryegrass/N. coenophialum. The study suggests that selection of suitable endophytic strains for fodder grasses can be enlarged to endophytes naturally harboured by other host species. (© Inra/Elsevier, Paris.)

Lolium perenne / Festuca arundinacea / endophytes / artificial infection / seed transmission

Résumé - Infection artificielle du ray-grass anglais et de la fétuque élevée par des champignons endophytes provenant de dix espèces différentes de graminées. On a examiné la capacité de 15 isolats d’endophytes, provenant de dix espèces de graminées différentes, à former des associations avec le ray-grass anglais (Lolium perenne, cv. Vigor) et la fétuque élevée (Festuca arundinacea, cv. Clarine). Vingt-deux associations sur trente ont pu être obtenues. Les taxa Neotyphodium lolii, N. coenophialum, LpTG-2, Epichloe festucae, E. bromicola et Gliocladium-like ont été capables de s’associer avec L. perenne et F. arundinacea. Acremonium sp. provenant de L. persicum a pu s’associer avec L. perenne, mais non avec F. arundinacea. L’observation après six mois des plantes inoculées et l’étude de la transmission du

Communicated by Yves Brygoo (Versailles, France)

* Correspondence and reprints [email protected] champignon aux graines ont montré la stabilité de six associations: la fétuque élevée avec N. coenophialum et N. lolii, le ray-grass anglais avec N. lolii, LpTG-2, Epichloe festucae et le Gliocladium-like. L’isolat d’Acremonium sp. provenant de Lolium persicum ne s’est pas maintenu au-delà de quelques mois sur L. perenne.Différentes manifestations d’incom- patibilité ont été observées sur les associations: fétuque élevée / LpTG-2, fétuque élevée / Gliocladium-like et ray-grass anglais / N. coenophialum. Cette étude montre que la sélection de souches d’endophytes pour introduction dans les gram- inées fourragères peut être étendue à des endophytes naturellement hébergés par d’autres espèces de graminées. (© Inra/Elsevier, Paris.)

Lolium perenne / Festuca arundinacea / endophytes / inoculation artificielle / transmission dans les graines

1. INTRODUCTION found in several wild species of Festuca [24]. An endophyte of Koeleria cristata was also referred to as E. festucae from its morphological characteris- Endophytic fungi belonging to the genus tics, isozyme pattern and ITS sequence [29]. Neotyphodium Glenn, Bacon & Hanlin The Neotyphodium are considered to be (= Acremonium sect. Albo-lanosa Morgan-Jones & species mutualistic [9] because they improve the resistance Gams) establish mutualistic associations with many of their hosts against abiotic stresses (drought, grasses of the subfamily Festucoideae [10, 14]. and also confer on their are related to nitrogen depletion, etc.) Neotyphodium species closely species the of resis- of Epichloe (family of Clavicipitaceae), with which hosts, through presence mycotoxins, tance to insects and herbivorous mammals [36, 38]. they share many characteristics. However, the sexual stage (perithecia produced in mycelial stromata Certain grass species also harbour non-clavicipi- surrounding host tillers) is lacking in Neotyphodium taceous fungal endophytes which are less frequent species, which are regarded as asexual descendants than e-endophytes and belong to two different of Epichloe [34]. The term ’e-endophytes’ is often types: i) the p-endophytes include Gliocladium-like used to regroup both Neotyphodium and Epichloe isolates in perennial ryegrass and Phialophora-like species. isolates in Festuca spp. [19, 37] (the letter ’p’ refer- to the of the conidio- Two species of fodder grasses have been more ring penicillate disposition phores, common to Gliocladium-like and thoroughly studied as regards their endophytes: tall Phialophora-like isolates); ii) the a-endophytes fescue (Festuca arundinacea) and perennial rye- to Acremonium sp. sensu stricto have grass (Lolium perenne). The former has belonging species been found in annual of Lolium been shown to harbour three different taxonomical only species [28, 29]. The are found in co- groups or TG: FaTG-1 or Neotyphodium p-endophytes frequently symbiosis with e-endophytes and they do not seem coenophialum, FaTG-2 and FaTG-3, which were to synthesize mycotoxins. According to Schmidt distinguished on the basis of their macro- and of Festuca characteristics, [35], Phialophora-like endophyte micromorphological mycotoxin affects its hosts. analysis and isozyme phenotypes [6, 27, 33]. pratensis negatively Recently, Malinowski et al. [26] contrasting results Although FaTG-2 and FaTG-3 are likely to be reported under controlled conditions which were explained species, they did not receive latin names. Using the by specific interactions between meadow fescue same criteria, two e-endophytes were described genotypes and Phialophora-like strains. The a- from Lolium perenne: LpTG-1 or Neotyphodium are still known as concerns their lolii and which did not receive a latin endophytes poorly LpTG-2, with their hosts. name [5, 6, 20]. Several species have also been relationships described within the genus Epichloe, among which The presence of e-endophytes has contrasting the more common are the parasitic species E. typhi- implications for agriculture: they improve the resis- na (Pers.:Fr.) Tul., which infects many grass species tance of their hosts to abiotic and biotic stresses, and the pleiomorphic species (generally symptom- and especially to insects and nematodes through less) E. festucae Leuchtmann, Schardl and Siegel, two groups of mycotoxins: peramine and lolines. However, through two other groups of toxins into Lolium perenne [4, 17] or the host; for instance, (ergotic compounds and lolitrems), they can cause Christensen [4], observed the death of host tissues severe toxicoses in livestock consuming infested of the shoot apices of tall fescue infected with N. forage. Cases of toxicosis are common in the USA uncinatum or LpTG-2, while Christensen et al. [7] and New Zealand [1, 12], and they have recently reported that certain isolates of E. festucae caused been observed in Europe [31]. However, it was increased mortality, stunted growth and chlorotic shown that a large variability (both at the intra- and leaf symptoms on infected seedlings of perennial interspecific levels) exists among e-endophytes as ryegrass, tall fescue and meadow fescue. These dif- concerns their capacity to synthesize mycotoxins ferent manifestations of incompatibility could [6, 32]. Therefore, plant breeders can select endo- explain that certain associations are not stable and phyte strains with a low rate of synthesis for ergot- can disappear over time (especially at the critical ic compounds and lolitrems and try to introduce level of seed transmission). Anyhow, in most of them into valuable cultivars of ryegrass and fescue these studies (with the exception of Koga et al. in order to use the beneficial effect of this mutualis- [18]), the transmission of the mycelium of the endo- tic symbiosis [13]. However, this strategy implies phyte to the seeds of the inoculated plant has not that these selected strains could easily be intro- been studied, so it is not really possible to assess the duced into hosts by artificial inoculation. stability of these artificial associations. Although natural infection of grasses by Our objective was to study the possibility of cre- Neotyphodium in nature seems to occur entirely ating associations as diverse as possible and to through seed transmission, artificial infection could assess the stability of these associations by observa- be obtained by inoculating seedlings [4, 7, 20], tion of transmission of the endophyte to the seeds of mature plants [30] and callus cultures [16]. This the plant. Infection of seedlings of ryegrass and tall artificial infection could have important practical fescue was tried with 15 isolates belonging to ten consequences, as it could be the first step towards different fungal species distributed between the the creation of new associations between commer- three groups: e-, p- and a-endophytes. cial grass cultivars and endophyte isolates selected for their harmlessness to mammals and beneficial effects on the host. As stated by Fletcher and Easton 2. MATERIALS AND METHODS [13], a high rate (close to 100 %) of transmission of the fungus to the seeds is a necessary (but not suffi- 2.1 Plant material cient) condition to hope to use in practice a new association. Recently, Koga et al. [18] showed that the resistance to some insects could be transmitted arundinacea cv. Clarine and Lolium by artificial inoculation of endophytes. In New Festuca perenne cv. Vigor were used in the study. For both species, endo- Zealand, a number of natural iso- by checking high phyte-free seeds were obtained from endophyte-free lates of the of it was endophytes perennial ryegrass, plants. Vigor is a cultivar that is currently endophyte- possible to select certain strains which were poten- free. As concerns Clarine, the endophyte-free plants tially of interest because they were free of the two from which the seeds were collected were obtained by main groups of mammalian mycotoxins [13]. Raynal (pers. comm.) from a selection within a partially infected population. In addition, 100 seeds of both culti- In inoculations are most successful general, vars were examined to verify that they were really free of when isolates are transferred between closely relat- endophytes. ed grasses [23]. However, some evidence of incom- can be observed when an isolate is intro- patibility 2.2 isolation duced into another host, even if it is not very distant Fungal from the original one; incompatibility can concern either the fungus which shows distorted, vacuolized The 15 isolates were freshly isolated from their hosts hyphae, as when N. coenophialum is introduced for the experiment. A majority of isolates were obtained from the leaf sheaths of growing plants either collected ney-like, 6-7 μm in length. The isolates of this group in nature (Frn-1, Fg-1a, Kc-1) or maintained in nurseries have typical isozyme patterns on MDH and PGM. Fa-3, accord- (Lp-1, Lp-12, Lp-13, Lp-14, Lp-80, Fa-6), - The N. coenophialum isolates are similar to those of ing to the method of Clark et al. [8]. Lpr-1 was obtained LpTG-2 with regards to their growth rate, the of from seeds [29] and Pn-1, Pd-1, Be-1 from aspect Dg-1, their thalli on PDA and their of conidia stromas shape (kidney- mycelial [8]. like). They differ from LpTG-2 by the large size of their conidia (8-12 μm in length) and their isozyme patterns.

- Three isolates (Frn-1, and Kc-1) were consid- 2.3 Fungal isolates Fg-1a ered as belonging to Epichloe festucae. They grow fairly rapidly on PDA, the thalli are white and fluffy on the upper side, brown on the lower side. They sporulate in Fifteen isolates from ten of Poaceae were used species culture and their kidney-like conidia are similar to those in this These isolates were distributed study (table I). of LpTG-2, although slightly smaller (4-6 μm in length). between the main of in groups endophytes reported Although Kc-1 has been isolated from a species Poaceae (Neotyphodium spp., Epichloe spp., p-endo- (Koeleria cristata) not belonging to the genus Festuca and In the were phytes a-endophytes). experiment, they and the tribe Poaeae, it has been identified as E. festucae identified several characteristics: their macromor- by by Naffaa et al. [29] from all its characteristics, includ- in culture on their asexual in phology PDA, sporulation ing isozyme patterns. vitro and the morphology and size of their conidia, and their isozyme pattern for three systems: MDH (malate - The isolates belonging to Epichloe typhina (Dg-1 deshydrogenase), PGI (phosphoglucose isomerase) and and Pn-1, isolated from Dactylis glomerata and Poa PGM (phosphoglucomutase). nemoralis, respectively) cannot be distinguished from those of Epichloe festucae by the shape and size of their The characteristics of the isolates were as follows. conidia. However, their isozyme patterns are different.

- The two lolii isolates and Neotyphodium (Lp-13 Lp- - The isolate Be-1 was obtained from a stroma on 14) are characterized by their very slow growth on PDA, Bromus erectus, it also shows the same macroscopical brain-like, cream-coloured colonies, absence of sporula- aspect in culture and the same shape and size of conidia tion in culture and of MDH, PGI and specific patterns as E. This strain to the PGM. typhina. probably belongs species Epichloe bromicola, recently described by Leuchtmann - The isolates belonging to LpTG-2 (Lp-1 and Lp-12) and Schardl [25]. Its MDH and PGI patterns are the same grow slowly and form white, fluffy colonies on PDA. as those of E. festucae, its PGM pattern is the same as They sporulate in culture, their conidia are scarce, kid- that of E. typhina. - The isolate Pd-1 was isolated from a stroma on phytes. Epidermal strips removed from leaf sheaths were Puccinellia distans. It is also similar to E. typhina by its mounted in aniline blue stain, as described by Clark et al. aspect in culture, growth rate and conidia, its isozyme [8] and examined with a compound microscope. patterns have not been determined. Confirmation of successful inoculation was obtained by from surface-sterilized leaf sheaths on - isolating fungi The isolate Lp-80 belonging to p-endophytes does PDA, and by comparing conidial dimensions and colony not sporulate in vitro, it grows slowly on PDA at 23 °C, characteristics with those of the original isolates. but more rapidly at 18 °C. The colonies are white, cot- of these isolates were also and convex. The are of Isozyme profiles compared tony isoenzyme patterns typical with those of the isolates the methods p-endophytes. original using described by Leuchtmann and Clay [22]. - The isolate Lpr-1 belonging to a-endophytes is well Thirteen out of the 22 successful associations were characterized by its rapid growth on PDA, the pinkish, selected in order to the transmission of mycelium fluffy aspect of its mycelium on this medium (with yel- study to the seeds (tables II and III). The selection was made low pigment on the lower side), the abundant sporulation so as to in the field one association in culture, rod-shaped conidia and isozyme patterns [29]. plant representative per host plant/endophyte species combination. The isolates are conserved at Inra Clermont-Ferrand. The E+ plants of these 13 associations were conserved for about 4 months in the greenhouse before planting in the field. During this period, the plants of the two asso- 2.4 Inoculation of plants ciations with Epichloe bromicola (Clarine/Be-1 and Vigor/Be-1) died, so that only 11 associations were planted, 6 months after inoculation (in the autumn of Endophyte-free seeds were surface sterilized by soak- 1996). Just before planting, these plants were submitted ing in 6 % sodium hypochlorite for 4 h and rinsing in to a second examination for the presence of endophyte sterile water. Seeds were then placed on water agar in and new isolations were carried out. Petri dishes (ten per dish), and incubated in darkness at Transmission to the seeds was studied for these 11 23 °C ± 1 for 7 days. During this time, the seeds germi- associations in 1997 (table IV). Several plants nated and those that appeared contaminated with fungi or spring (2-8) were randomly selected from each association. bacteria were removed. A longitudinal slit was cut Three ears or panicles (fescue) were random- through the tissue of the young seedlings at the junction (ryegrass) selected from each of these plants. About 20 seeds of the mesocotyl and coleoptile and a drop of solution of ly from each ear or panicle were examined, using the ground mycelium in sterile water was placed in the slit. method of Clark et al. [8], for the presence of mycelium The mycelium used for inoculation was taken at the The percentage of seeds showing mycelium periphery of the colonies. For those isolates which sporu- endophyte. and the confidence interval (α = 5 %) were calculated for late in vitro (all isolates except Lp-13, Lp-14 and Lp-80), each association. Generally, seeds from different plants the inoculum probably consisted of a mixture of myceli- and different ears or of a association were um and conidia. panicles given mixed and observed in bulks. However, for two associa- Control seedlings received similar treatments except tions which showed symptoms of partial incompatibility no was on the slit of the that mycelium placed seedlings. (N. coenophialum on perennial ryegrass and Gliocladium-like on tall the of the After inoculation the seedlings were incubated for 4 fescue), presence days in darkness, then 3 days in light prior to planting in endophyte was checked separately on the seeds of the different and ears Seeds from non- pots. Then, the plants were, after adaptation, grown in plants (or panicles). the greenhouse, in summertime conditions (temperatures infected plants (controls) were also examined. between 22 and 28 °C), and watered twice a week.

3. RESULTS 2.5 Detection of endophytes and transmission to the seeds 3.1 Success of artificial inoculation

The seedlings were submitted to a first examination 023 of Clarine + approximately 8 weeks after infection for the presence of In total, 3 855 seedlings (2 intercellular hyphae characteristic of the used endo- 1 832 of Vigor) were inoculated. Of these, 1 450

died after transfer to the greenhouse while 2 405 association Vigor/Lpr-1 and the re-isolations were could be observed. Six hundred and thirty seedlings negative for this association. Most of the other ini- appeared to be infected (346 of Clarine + 284 of tially successful infections of seedlings had persist- Vigor), representing 26.2 % of the seedlings which ed. However, Gliocladium-like endophyte had dis- could be observed. appeared in 30 % of infected plants of Clarine. Fifteen associations were obtained in this study. LpTG-2, N. lolii, Gliocladium-like endophyte, N. coenophialum, Epichloe bromicola from B. erectus, 3.2 Incompatibility reactions E. festucae from F. rubra nigrescens, F. gigantea and Koeleria cristata, were able to form associations with L. perenne and F. arundinacea. We could Two types of incompatibility reactions were not infect rye-grass or tall fescue with Epichloe observed. i) Hyphae of N. coenophialum were high- typhina from Dactylis glomerata or Poa nemoralis, ly vacuolated in many plants of L. perenne with nor from Epichloe sp. from Puccinellia distans. which this endophyte formed associations. Perennial ryegrass, but not tall fescue could be Vacuolated hyphae were not observed for the other infected with Acremonium sp. from L. persicum endophytes. ii) Stunted tillers were present in all F. (Lpr-1). arundinacea plants successfully inoculated with Gliocladium-like or LpTG-2, and of L. perenne The percentage of inoculated seedlings in estab- plants inoculated with Gliocladium-like or N. lished associations varied (tables II and III). greatly coenophialum. Many stunted plants of F. arundi- Whatever the host, it was particularly high for the nacea infected by Gliocladium-like occasionally Gliocladium-like and for one of endophyte (Lp-80) produced healthy tillers. No intercellular the isolates of E. (Frn-1). apparently festucae hyphae could be observed in these tillers and In the particular case of Acremonium sp. (isolat- attempts to re-isolate from these tillers were unsuc- ed from Lolium persicum), only 13 plants could be cessful. Of the F. arundinacea plants infected by observed and the endophyte was found in five LpTG-2, 62.5 % died, this phenomenon not being cases. The mycelial fragments were rare and observed with the other associations. Staining and restricted to the very base of the foliar sheaths. observation of the mycelium of Gliocladium-like in F. arundinacea were difficult. In con- After these first microscopic observations, re- plants very the of this in L. isolations were attempted from all observed plants. trast, mycelium endophyte perenne The results showed that 0-5 % of apparently endo- plants was easily stained and observed. phyte-free plants, according to microscopic observations, gave rise to mycelium after isolation. This phenomenon was frequent for the association 3.3. Transmission of the endophytes to seeds Vigor/Lpr-1 (Acremonium sp. from L. persicum). of artificially infected plants On the other hand, colonies of endophyte were obtained from all plants in which mycelium had been observed. Morphological and isozyme study The seeds from the different plants and ears were of five isolates from each association showed that observed in bulk in nine cases out of 11 (table IV). all endophytes re-isolated from artificially infected For two particular associations (the Gliocladium- plants were identical to the original isolates accord- like endophyte in seeds of F. arundinacea and ing to their conidial shape and dimensions, colony N. coenophialum in seeds of L. perenne) which characteristics and isozyme patterns for MDH, showed symptoms of incompatibility, the different PGM and PGI. plants and the different ears or panicles of one plant were checked and not in bulk (table V). Eleven associations were re-examined after separately about 6 months for the presence of intercellular No mycelium was observed in the seeds for the hyphae. At this time, no mycelium was found in the association Vigor/Lpr-1 (Acremonium sp.). The other endophytes were generally transmitted seedlings inoculated with endophytes from another at a high rate (> 67.3 %) in L. perenne and F. arun- host have failed to persist for more than 6-8 dinacea seeds. This rate reached 100 % for some months. In the present study, in the association associations. Vigor/Acremonium sp. (from L. persicum), the could be detected after 2 but not In the association F. arundinacea/Gliocladium- mycelium months, after 6 whatever the method like, all the seeds (from three different panicles) months, (microscopic observation or re-isolation). Moreover, from plant number 4 were endophyte-free. Gliocladium-like in F. arundinacea did not However, on the other four persist- plants, endophyte- ed in about 30 % of infected Of the other infected seeds were found in all the plants. panicles nine the second observa- observed, the frequency of endophyte-infected associations, microscopic tion (after 6 months) showed that the mycelium was seeds from a given panicle was fairly homogeneous still present. These include four associations that and never equal to 0 or 100 %. The same situation exist in nature (N. lolii, LpTG-2 and Gliocladium- was observed with the association L. perenne/N. like on ryegrass, N. coenophialum on tall coenophialum: on plants numbers 3 and 4, all the perennial fescue), and five artificial associations already ears observed had 100 % endophyte-free seeds obtained by other authors (N. lolii and LpTG-2 on while on plants numbers 1 and 2, all the ears tall fescue, N. coenophialum on perennial ryegrass observed had at least a weak proportion of endo- and on both phyte-infected seeds (table V). Epichloe festucae hosts). Clay [11] suggested that for a Neotyphodium-grass association to be considered 4. DISCUSSION compatible, a viable endophyte must be seed transmitted at high frequency, preferably for more than one generation. In the present study, the fungus was This work demonstrates (through the observation transmitted to the seeds in ten cases out of the 11 of seed transmission) the stability of certain associ- studied. As was expected from the results of micro- ations. Among the 20 associations attempted, 15 scopical observations and re-isolations from the appeared to succeed after 2 months; however, the plants, the mycelium of Acremonium sp. (Lpr-1) plants of the two associations with E. bromicola was not found in the seeds. In addition, the trans- died later in the greenhouse. Leuchtmann [21] mission rate was low in two associations: showed that initially successful infections of Gliocladium-like endophyte on tall fescue and particularly N. coenophialum on perennial ryegrass plant. In contrast with Wilson and Easton’s results (37.4 and 19.5 %, respectively). For these two asso- [39], we did not observe endophyte-free ears on ciations, the counting of seeds with and without endophyte-infected plants; the three ears observed endophyte was effected separately on the different on a given plant appeared either all endophyte-free plants and the different ears or panicles of the same or all infected. However, only a few plants were observed. Transmission of the endophytes through presence of endophyte-free tillers that did not the following generations will be studied. However, appear to be associated with incompatibility. It most of the endophytes were seed transmitted at should be noted that these endophyte-free tillers high frequency; the transmission rate to the seeds of also develop in natural associations [15]. perennial ryegrass was 100 % for three isolates Endophyte-free tillers presumably develop from belonging to the species: N. lolii, LpTG-2 and E. axillary buds which have not been colonized by the festucae. If the first two taxa are natural endophytes endophyte. The mechanism for this failure is of perennial ryegrass, it is not the case for E. festu- unclear [4]. ii) The endophyte did not persist for cae. more than a few months in some unstunted plants of tall fescue initially infected. Several reactions of incompatibility were observed. The main criterion for compatibility was Such a deleterious effect of p-endophytes on the presence or absence of modifications affecting their hosts has been reported even in the case of nat- the hyphae. Hyphae of N. coenophialum were high- ural associations [35]. vacuolated in many of the L. perenne with ly plants The different used in this which this endophyte formed associations. This e-endophytes study as host Such a result confirms the endo- appear only partially specific. study incompatibility affecting confirms Christensen’s results which showed that phytic hyphae, previously observed in certain asso- the taxa identified in L. perenne, F. arundinacea ciations of F. arundinacea endophytes in L. perenne and F. pratensis were not specific to their natural [4, 17]. The that an accumulation of possibility hosts This author that the lack of host host-produced antifungal compounds in the hyphae [4]. suggested specificity probably reflected the very close genetic of the L. perenne/N. coenophialum association may relationships between perennial ryegrass, tall fes- contribute to the degeneration of the cytoplasm and cue and meadow fescue. Koga et al. [17] also plasma membrane cannot be discarded. However, if showed that N. lolii, N. coenophialum and LpTG-2 antifungal compounds were present, according to are not specific to their natural hosts. However, in Koga et al. [17], they were not the product of a the present study, associations with endophytes hypersensitive reaction as often occurs in plant/fun- from more distant grass species such as Bromus gus incompatibility, since surrounding host cells erectus and Koeleria crastata could also be showed no ultrastructural changes. This fungal obtained. However, the plants with Epichloe bromi- reaction of incompatibility was not linked to the cola died in the a few months after of stunted tillers or to the death of infect- greenhouse presence infection. The with E. from K. ed plants. plants festucae cristata (Kc-1) had not been conserved after the No change affecting the hyphae was observed for first observation, the isolate Frn-1 (from Festuca the tall fescue/LpTG-2 associations. However, rubra ssp. nigrescens) having been taken as repre- stunted tillers were often observed in this associa- sentative of the species E. festucae. On the other tion, which must be put together with the high fre- hand, it was not possible to obtain associations of quency of plants that did not survive: 62 % of fes- tall fescue and perennial ryegrass with Epichloe cue plants infected by LpTG-2 failed to survive for spp. from Dactylis glomerata, Poa nemoralis and more than 4 months. Christensen [4] reported that Puccinellia distans. in the association of tall fescue with LpTG-2, the We confirm in the present study that it is possible presence of stunted tillers was linked to necroses at to transfer certain to which are the base of the tillers, in meristematic tissues. We endophytes grasses not their natural hosts. The possibility of introduc- did not investigate the associations at this level. ing selected strains of endophytes into commercial A high frequency of tall fescue plants infected varieties of perennial ryegrass has been investigated with Gliocladium-like were able to survive, for several years in New Zealand, this strategy although these plants were often stunted. Two phe- being extensively analysed in a recent review by nomena were observed for this association. i) The Fletcher and Easton [13]. These authors showed that the selection of suitable strains (possibly with- [3] Bush L.P., Wilkinson H.H., Schardl C.L., alkaloids of out lolitrem or ergovaline) and their introduction Bioprotective grass-fungal endophyte sym- bioses, Plant 114 (1997) 1-7. into new hosts are only preliminary steps on the Physiol. way to obtaining commercial associations, which [4] Christensen M.J., Variation in the ability of requires the study of the interactions between the Acremonium endophytes of Lolium perenne, Festuca arundinacea and F. to form selected strain and commercial cultivars, the influ- pratensis compatible associations in the three grasses, Mycol. Res. 99 (1995) ence of the environment (including extreme climat- 466-470. ic on the of and sequences) synthesis mycotoxins, [5] Christensen M.J., Latch G.C., B.A., the behaviour of the associations in the field. The Tapper Variation within isolates of Acremonium endophytes in New are to strains selected Zealand likely belong from perennial rye-grasses, Mycol. Res. 95 (1991) to N. lolii (and LpTG-2?); however, our results, 918-923. after those of Latch and Christensen [20], Koga et [6] Christensen M.J., Leuchtmann A., Rowan D.D., al. [17], Christensen [4], Christensen et al. [7], Tapper B.A., Taxonomy of Acremonium endophytes of show that it could be possible to enlarge the selec- tall fescue (Festuca arundinacea), meadow fescue (F. tion of strains to endophytes of other grass species. pratensis) and perennial rye-grass (Lolium perenne), Res. 97 (1993) 1083-1092. As regards Lolium perenne, in addition to the two Mycol. natural e-endophytes of this host, the selection [7] Christensen M.J., Ball O.J.P., Bennett R.J., Schardl C.L., and host effects on com- could also concern Epichloe festucae, a species Fungal genotype patibility and vascular colonization by Epichloe festu- which, in our experiment, was 100 % transmitted to cae, Mycol. Res. 101 (1997) 493-501. the seeds of perennial ryegrass and did not show Clark E.M., White Patterson harmful effect on this host. Recent [8] J.F., R.M., Improved any investiga- histochemical techniques for the detection of tions found in Festuca rubra not in F. ergovalin (but Acremonium coenophialum in tall fescue and methods of gigantea), infected by E. festucae. [2, 3]. However, in vitro culture of the fungus, J. Microbiol. Methods 1 E. festucae, as a sexed fungus, is likely to be high- (1983) 149-155. variable was shown Christensen et al. ly (as by [7]) [9] Clay K., Fungal endophytes of grasses: a defen- and it will be interesting to study its variability as sive mutualism between plants and fungi, Ecology 69 concerns mycotoxin synthesis. In contrast, (1988) 10-16. Neotyphodium coenophialum, appeared poorly [10] Clay K., Recent advances in the ecology of adapted to Lolium perenne. endophytes, Hume D.H. (Ed.), Proceedings of the Second International Symposium on Acremonium / Tall fescue generally appears less accessible than Grass Interactions, Palmerston North, New Zealand, perennial ryegrass for endophytes which are not its 1993, pp. 11-30. natural symbionts. However, the selection of endo- [11] Clay K., The ecology and evolution of endophytes suitable for this host could concern (in addi- phytes, Agric. Ecosys. Environ. 44 (1993) 39-64. tion to its natural endophyte Neotyphodium [12] Fletcher L.R., Harvey I.C., An association of a and N. lolii. coenophialum), Epichloe festucae Lolium endophyte with ryegrass staggers, N. Z Vet. J. 29 (1981) 185-186. [13] Fletcher L.R., Easton H.S., The evaluation and REFERENCES use of endophytes for pasture improvement, in: Bacon C.W., Hill N.S. (Eds.), Third International Symposium on Neotyphodium/Grass Interactions, Athens, Georgia. [1] Bacon C.W., Porter J.K., Robbins J.D., Luttrell Proceedings Plenum Press, New York, 1997, pp. 209-225. E.S., Epichloe typhina from toxic tall fescue grasses, Appl. Environ. Microbiol. 34 (1990) 576-581. [14] Glenn A.E., Bacon C.W., Price R., Hanlin R.T., Molecular of Acremonium and its taxonomic [2] D.R., Vicari M., Emmerich S., L., Lin phylogeny Bazely Filip 88 369-383. D., Inman A., Interactions between herbivores and endo- implications, Mycologia (1996) phyte-infected Festuca rubra from the Scottish islands [15] Hinton D. M., Bacon C.W., The distribution and of St. Kilda, Benbecula and Rum, J. Appl. Ecol. 34 ultrastructure of the endophyte of toxic tall fescue, Can. (1997) 847-860. J. Bot. 63 (1985) 36-42. [16] Johnson M.C., Bush L.P., Siegel M.R., Infection [29] Naffaa W., Ravel C., Guillaumin J.-J., A new of tall fescue with Acremonium coenophialum by means group of endophytes in European grasses, Ann. Appl. of callus cultures, Plant Dis. 70 (1986) 380-382. Biol. 132 (1998) 211-226. Christensen Bennett [17] Koga H., M.J., R.J., [30] Ravel C., Wartelle D., Charmet G., Artificial of some / Acremonium Incompatibility grass endophyte infection of tillers from mature Res. 97 1237-1244. perennial ryegrass plants associations, Mycol. (1993) with Acremonium endophytes, in: Krohn K., Paul V.H., [18] Koga H., Hirai Y., Kanda K., Tsukiboshi T., Thomas J. (Eds.), Proceedings of the First International Uematsu T., Successive transmission of resistance to Conference on Harmful and Beneficial Microorganisms Bluegrass Webworm to perennial ryegrass and tall fes- in Grassland, Pastures and Turf, Paderborn, Germany, cue plants by artificial inoculation with Acremonium 1994, pp. 123-125. endophytes, J. A. R. Q. 31 (1997) 109-115. [31] Raynal G., Observations françaises sur les [19] Latch G.C.M., Christensen M.J., Samuels G.J., Acremonium, champignons endophytes des graminées Five endophytes of Lolium and Festuca in New Zealand, fourragères, Fourrages 126 (1991) 225-237. Mycotaxon 20 (1984) 535-550. [20] Latch G.C.M., Christensen M.J., Artificial infec- [32] Rowan D., Lolitrems, peramine and paxilline: of the / tion of grasses with endophytes, Ann. Appl. Biol. 107 mycotoxins ryegrass endophyte interaction, (1985) 17-24. Agric. Ecosyst. Environ. 44 (1993) 103-122. [21] Leuchtmann A., Systematics, distribution and [33] Schardl C.L., Epichloe species: Fungal host specificity of grass endophytes, Nat. Toxins 1 Symbionts of Grasses, Annu. Rev. Phytopathol. 34 (1992) 150-162. (1996) 109-130. Leuchtmann A., variation in [22] Clay K., Isozyme Schardl Liu White Finkel the [34] C.L., J.S., J.F., R.A., Acremonium/Epichloe fungal endophyte complex, M.R., Molecular of Phytopathology 80 (1990) 1133-1139. Siegel phylogenetic relationships nonpathogenic grass mycosymbionts and clavicipita- [23] Leuchtmann A., Clay K., Nonreciprocal compat- ceous plant pathogens, Plant Syst. Evol. 178 (1991) ibility between Epichloe typhina and four host grasses, 27-41. Mycologia 85 (1993) 157-163. [35] Schmidt D., Effects of Acremonium uncinatum [24] Leuchtmann A., Schardl C.L., M.R., Siegel and a Phialophora-like endophyte on vigour, insect and Sexual compatibility and taxonomy of a new species of disease resistance of meadow fescue, in: Hume D. H. Epichloe symbiotic with fine fescue grasses, Mycologia (Ed.), Proceedings of the Second International 96 (1994) 802-812. Symposium on Acremonium / Grass Interactions, [25] Leuchtmann A., Schardl C.L., Mating compati- Palmerston North, New Zealand, 1993, pp. 185-187. bility and phylogenetic relationships among two new species of Epichloe and other congeneric European [36] Siegel M.R., Latch C.G.M., Johnson M.C., species, Mycol. Res. 102 (1998) 1169-1182. Fungal endophytes of grasses, Annu. Rev. Phytopathol. 25 (1987) 293-315. [26] Malinowski D., Leuchtmann A., Schmidt D., Nosberger J., Growth and water status in meadow fescue [37] Siegel M.R., Schardl C.L., Phillips T.D., is affected by Neotyphodium and Phialophora species Incidence and compatibility of nonclavicipitaceous fun- endophytes, Agron. J. 89 (1997) 673-679. gal endophytes in Festuca and Lolium grass species, 87 196-202. [27] Morgan-Jones G., Gams W., Notes on Mycologia (1995) An of Festuca arundinacea Hyphomycetes. endophyte MacDonald Acremonium and the of new taxa in one [38] VanHeeswijch R., G., anamorph Epichloe typhina, in and other of the two new sections of Acremonium, Mycotaxon 15 endophytes perennial ryegrass pasture grass- (1982) 311-318. es in Australia and New Zealand, Aust. J. Agric. Res. 43 (1992) 1683-1709. [28] Naffaa W., Ravel C., Guillaumin J.-J., Morphological and molecular variability among [39] Wilson S.M., Easton, H.S., Seed transmission of Acremonium isolates from 22 species of Poaceae in an exotic endophyte in tall fescue, in: Bacon C.W., Hill France, in: Krohn K., Paul V.H. (Eds.), Proceedings of N.S. (Eds.), Third International Symposium on the Second International Conference on Harmful and Neotyphodium/Grass Interactions, Athens, Georgia. Beneficial Microorganisms in Grassland, Pastures and Proceedings Plenum Press, New York, 1997, pp. Turf, Paderborn, Germany, 1995, pp. 185-200. 281-283.