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- Materials and Methods
The presence of fungal endophytes in the seeds of Lolium perenne L. was first observed by Guérin (1898). McLennan (1920) studied the distribution and development of the mycelium in the different organs of the grass. Sampson (1937) was the first to isolate an endophytic fungus from L. perenne and to grow it in pure culture.
In New Zealand, a damaging tremorgenic disease of sheep and cattle, ‘ryegrass staggers’, had long been reported and attributed to the consumption of perennial ryegrass by grazing animals (Cunningham & Hartley, 1959). Later on, ryegrass staggers was shown to be caused by alkaloids (Aasen et al., 1969) and the implication of the fungal endophyte in the toxicosis was eventually established by Fletcher & Harvey (1981).
Sampson (1933) observed the presence of two different endophytic fungi in Lolium perenne and Latch et al. (1984) isolated them. The first type was thin, poorly stainable and highly branched. It was generally sterile in culture, but the production of a few conidiophores resembling Gliocladium led to this species being called ‘Gliocladium-like’ (Philipson, 1989).
The second, more frequent, type consisted of twisted, poorly branched hyphae. It was shown to belong to clavicipitaceae and described as a new species, Acremonium loliae Latch, Christensen and Samuels (later changed to Acremonium lolii). Within the genus Acremonium, this species was classified in a section Albo-lanosa created by Morgan-Jones & Gams (1982) to include the anamorphs of the clavicipitaceae. However, the fungi belonging to this section were reclassified by Glenn et al. (1996) into the new genus Neotyphodium, the clavicipitaceous endophytes of L. perenne being named N. lolii (Latch, Christensen & Samuels) Glenn, Bacon & Hanlin.
Christensen et al. (1991, 1993) introduced an additional distinction among the clavicipitaceous endophytes of perennial ryegrass on the basis of growth rate, isolation delay, sporulation at 23°C and isozyme pattern. The majority of isolates could be accommodated as Neotyphodium lolii. However, a few isolates showing higher growth were just named by the abbreviation LpTG-2.
The clavicipitaceous endophytes of perennial ryegrass are known to synthesize several mycotoxins, among which three are particularly important: lolitrem B, a tremorgenic molecule responsible for ‘ryegrass staggers’; ergovaline, a compound belonging to the ergopeptine group, which has vasoconstrictive effects and causes various diseases on grazing mammals (‘fescue-foot’ and ‘fescue toxicosis’); and peramine, a tripeptide which is repellent and toxic for insects but not for mammals. The strains of LpTG-2 which have been studied synthesized ergovaline and peramine, but not lolitrem B, while variation was observed among strains of N. lolii for the synthesis of lolitrem B, ergovaline and peramine (Christensen et al., 1993). Schardl et al. (1994) showed that LpTG-2 is a heteroploid species originating from interspecific hybridization between Neotyphodium lolii and the parasitic species Epichloë typhina.
The dual action of clavicipitaceous endophytes (beneficial effects on the host vs risk of toxicity for grazing animals) sets forage grass breeders special problems. Sometimes, these have been solved by removing the endophyte from the cultivars selected for pasture and by maintaining or introducing it into varieties selected for turf. Another strategy, developed mainly in New Zealand (Latch, 1989), proposed to select harmless clavicipitaceous endophytes (producing no or little ergovaline or lolitrem) and to inoculate these strains to commercial cultivars. This strategy involves an extensive study of the variability of the endophytes of Lolium perenne. Such research was conducted on a large scale in New Zealand (Latch, 1994; Fletcher & Easton, 1997).
Very few studies have been conducted on this subject in Europe, despite the fact that Lolium perenne probably originated from the Near East and has diversified in Europe (Balfourier et al., 2000). Maximum genetic variability would therefore be expected in Europe.
In France, 547 natural populations of Lolium perenne were collected from 1983 to 84 (Charmet et al., 1990, 1993; Balfourier & Charmet, 1991). About half of these populations were sampled at random and checked for the presence of endophytes (Lewis et al., 1997; Ravel, 1997). One, or several, endophytes were found in 188 populations out of 262 (72%). The present study was conducted on part of this material. The objectives were to identify the fungal species involved and to describe intraspecific variability, particularly as concerns the production of mycotoxins.
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- Materials and Methods
Isolation of endophytes from germinating seeds was very successful. The 94 isolates obtained belonged to the three endophyte species which had already been described in Lolium perenne: Neotyphodium lolii, LpTG-2 and the ‘Gliocladium-like’ fungus. N. lolii was the most common, representing 90% of the isolates. No other species were detected. The specific identification of these three species was based on their isozyme patterns for the two enzymes MDH and/or PGM, as described by Christensen et al. (1993) and Naffaa et al. (1998). The presence of conidia in culture for LpTG-2 at 20–24°C (N. lolii is always sterile at this temperature), is another criterion advocated to distinguish between these two clavicipitaceous species (Christensen et al., 1993). The nine isolates of LpTG-2 isolated in our study were checked for the presence of conidia on PDA at 23°C. Conidia were observed for four isolates only. On the other hand, they were not observed on the cultures of five isolates of N. lolii which had been drawn by lot. The presence of conidia in cultures of LpTG-2 at room temperature seems to be a sufficient, but not necessary, condition for identification.
Pure culture macromorphology may be useful for species identification, since LpTG-2 and Gliocladium-like show a specific and very homogenous morphology (MG VII and MG VIII, respectively). Neotyphodium lolii varies greatly in morphology and in growth rate. Linear growth was preferred to biomass production as being potentially more discriminant. The strong correlation observed between macromorphology and linear growth is not surprising: the very slow apical growth of the mycelial hyphae of some strains is concomitant with a high rate of branching, the result being a three-dimensional growth of the colony, which is stroma-like, being as high as wide. This morphology, which has sometimes been named ‘brain-like’, characterizes MGs I and II; in MG II, the central prosenchyma is surrounded by a band of flat mycelium while in MG I, it falls abruptly into the culture medium. These two groups are clearly distinct from each other and from all the other morphological types which can be described within N. lolii. The morphologies I and II appear stable with subculturing and are retained after a storage at low temperatures (−80°C). The flat aspect of the young colony characterized all the other morphological groups described; the MGs IV, V and VI show typical morphological aspects, and contain only a few isolates. MG III is less homogeneous; in this group, the centre of the colony can undergo different development after 20 d of growth: for example appearance of cottony warts or of radial and concentric furrows. This type is the one that contained the highest number of isolates. The typical appearance of the colonies of LpTG-2 on PDA has already been described by Christensen et al. (1991). These authors had also reported the high variability of the isolates of N. lolii and published photos resembling our groups II and III.
The variability within N. lolii was also considerable with respect to the synthesis of the three main mycotoxins: lolitrem B, ergovaline and peramine. Four isolates, all of French origin (6% of the isolates) did not synthetize lolitrem B. 23 isolates (37%) did not synthetize ergovaline. Of these 23, 20 originated from France, one from Poland, one from Yugoslavia and one from Greece. Six isolates (10%) (excluding those with traces of peramine lower than the quantification threshold) did not synthetize peramine. Among these six, all of French origin, one was also lolitrem B-deficient, two were ergovaline-deficient, and three synthetized both ergovaline and lolitrem B.
The most striking observation was the close link between the deficiency for ergovaline or lolitrem B in N. lolii and the morphology of the isolates: all four isolates which did not produce lolitrem B were from MG I and no isolate of MG I produced lolitrem B. Among the 23 isolates that did not produce ergovaline, 22 belonged to MG II. In MG II, no isolate produced ergovaline. By contrast, the peramine-deficient strains distributed among several morphological groups, including MG I and MG II.
As the MG I and MG II morphologies coincide with a slow linear growth rate, it seems that the ‘mutation’ (in the broad sense) which led to the absence of synthesis of lolitrem or ergovaline is regularly accompanied by disturbances of the mycelial growth. The loss of the ability to synthetize ergovaline (or lolitrem) could be the result of a chromosome deletion which could also result in the loss of genes playing a role in mycelial growth and morphogenesis. As the deficiencies for ergovaline and lolitrem are independent and accompanied by different morphological disturbances, the two deletions probably concern different chromosomes or chromosome fragments. The loss of a minor chromosome (a ‘B chromosome’, according to Kisler & Mia (1992)) could also be an explanation: Kuldau et al. (1999) found eight chromosomes in N. lolii of which three were smaller than 3 Mb.
Figure 4 shows the frequency distribution of the concentrations of the three mycotoxins in the plants. The concentrations of lolitrem B and ergovaline are similar to those in the literature (Di Menna et al., 1992; Ball et al., 1997b; Lane et al., 1997a, 1997b). By contrast, the concentrations of peramine (median value: 6.3 µg g−1 for 53 isolates) are lower than those in the literature, which are often in the range of 10–30 µg g−1 (Ball et al., 1995a, 1995b, 1997a). However, for two particular populations, the concentrations were much higher (> 40 µg g−1). The genotype of the fungus, which controls the synthesis of a given mycotoxin, can also play a role in fixing the quantitative level of this mycotoxin within the plant. However, many other factors probably play a role, such as the host genotype, the mycelium density in the host tissues and various environmental factors; so it is difficult to assert that differences recorded in the three mycotoxins concentrations faithfully reflect genetic quantitative differences between the fungal isolates.
The isolates of MG II (which do not produce ergovaline) produced significantly more lolitrem B than the isolates of MG III. The isolates of MG III produce significantly more peramine than those of MG II. However, there was no correlation between the concentration of the three mycotoxins if all isolates are considered together.
Isolates deficient in both ergovaline and lolitrem B were not found. The predicted frequency of such strains would be the product of the individual frequencies: 6% × 37% = c. 2%. This low predicted frequency could explain why no such isolate was found. On the other hand, if it is true that both the ergovaline-free and the lolitrem B-free isolates of N. lolii are the result of genetic accidents (for instance of chromosome deletions), one would expect that the isolates carrying a double deletion would be particularly deficient and find it difficult to survive in the plant. However, screening of more than 1000 populations in New Zealand, has succeeded in finding such isolates (Fletcher & Easton, 1997).
The close link between the absence of ergovaline or lolitrem B and a specific morphology of the colonies in pure culture could, if it is confirmed on a higher number of strains from more various origins, considerably improve the process of selection of harmless strains. Such a selection could be carried out by laboratories equipped only for routine mycology and lacking the complex equipment and technical competence necessary for the analysis of mycotoxins by HPLC.
This study confirmed that the species LpTG-2, of hybrid origin (Schardl et al., 1994), is in a minority among the endophytes of L. perenne in France. Only three French isolates of this taxon were found in our study: two from the region of Marseille and one from Corsica. Four other isolates were isolated from populations from Italy and Spain. The two reference isolates obtained from AgResearch also have a Western Mediterranean origin. Thus, LpTG-2 appears to be a West-Mediterranean species.
The seven LpTG-2 isolates were homogeneous in their morphology, growth rate, isozyme pattern and mycotoxin synthesis (lolitrem B is not synthetized). The isozyme patterns MDH and PGM allow a certain identification of the species, but the morphology of the colonies on PDA may appear to be a safe method of identification.
The taxon ‘Gliocladium-like’ was also a minority among our isolates. The four isolates obtained appeared identical as concerned their isozyme pattern, macromorphology (MG VIII type), high growth rate, low isolation delay, and preference for cool temperatures (18°C). The populations concerned came from Northern France, Central France and Germany. As for LpTG-2, the identification can be easily carried out from the macromorphology alone. Neither conidiophores nor conidia were observed in planta or in culture.
In 12 cases (14%) two different endophytes were isolated from the same seed lot. As each lot had been harvested from only one plant, two different fungal isolates must have coexisted within the same plant. In one case, a Gliocladium-like fungus was found associated with an isolate of N. lolii. In three cases, the association was between LpTG-2 and N. lolii and in eight cases, between two different isolates of N. lolii. Indeed, we could only detect the situations in which the two associated isolates of N. lolii belonged to two different MGs. It is probable that other situations exist in which the two (or more) isolates are morphologically similar and could be detected only with molecular markers.
Coexistence between two endophytes in the same plant has already been reported for the associations between a clavicipitaceous and a nonclavicipitaceous endophyte (Schmidt, 1994; Siegel et al., 1995). The association between two different isolates of the same species of endophyte has already been reported and analysed by Meijer & Leuchtmann (1999) on the pair Brachypodium sylvaticum/Epichloë sylvatica. However, to our knowledge, the present work is the first mention of the presence of two different strains of Neotyphodium lolii in the same plant of Lolium perenne. Frequent coexistence between two clavicipitaveous endophytes in the same host may support parasexual hybridization in the continuum existing from parasitic Epichloë to mutualistic Neotyphodium, leading to new species.