Published online 2 December 2005
Published in Crop Sci 46:98-104 (2006)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
FORAGE & GRAZINGLANDS
Chemical Control of Neotyphodium spp. Endophytes in Perennial Ryegrass and Tall Fescue Seeds
C. Leyronasa,*,
B. Mériauxb and
G. Raynalc
a UMR Epidémiologie végétale et écologie des populations, INRA-INA P.G., BP 01, F-78840 Thiverval-Grignon, France
b FNAMS, Le Verger, F-49800 Brain sur l'Authion, France
c UMR Epidémiologie végétale et écologie des populations, INRA-INA P.G., BP 01, F-78840 Thiverval-Grignon, France
* Corresponding author (leyronas{at}grignon.inra.fr)
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ABSTRACT
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Seed transmitted Neotyphodium endophytes are widespread in grasses. These fungi may modify agronomic plant traits and induce synthesis of mycotoxins that lead to possible cattle toxicoses. For these reasons, the Technical Permanent Committee for Breeding recommends the grass varieties submitted to the French Official List to contain less than 20% of seeds with living Neotyphodium. Several fungicides were tested as seed treatments on perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreber). Phytotoxic effects were measured. Their efficacy was evaluated in greenhouse and in field conditions of seed production. Triticonazole [(RS)-(E)-5-(4-chlorobenzylidene)-2,2-dimethyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol] was effective but much too phytotoxic. The tested doses of bitertanol [1-(biphenyl-4-yloxy)-3,3-dimethyl-1-(1H-1,2,4-triazol- 1-yl)butan-2-ol] and fluquinconazole [3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2,4-triazol-1-yl)quinazolin-4(3H)-one] have few negative effects on germination but very few effects on endophytes. Prochloraz [N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-1H-imidazole-1-carboxamide] (0.75 g kg–1) represents the best balance between high efficacy and low phytotoxicity on perennial ryegrass and tall fescue seeds. No difference among varieties and species was detected in their response to prochloraz treatment. In greenhouse trials, less than 10% of living endophyte remained in perennial ryegrass and tall fescue seed lots, even in highly infected ones (66% of E+ seeds), when treated with prochloraz. This seed treatment was also efficient in field conditions where the level of endophyte was reduced by more than 60% in highly endophyte-infected ryegrass seed lots. This study shows that, even if seed germination is a little decreased, prochloraz could be used during grass breeding so that subsequent generations of seed will be free from endophyte with good germination rate.
Abbreviations: E+, endophyte-infected • E–, endophyte-free
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INTRODUCTION
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NEOTYPHODIUM spp. endophytes are widespread in numerous grass species and particularly in the genera Lolium and Festuca, in which they grow in aerial parts and are disseminated by seed (Ravel et al., 1994; Leyronas and Raynal, 2001; Rolston et al., 2002). These fungi are symbionts and can confer to plants a greater resistance to drought, improve their nitrogenous nutrition, and increase their resistance to herbivores (Ravel et al., 1997). These characteristics are due to alkaloids having effects on invertebrates (mainly peramine) and vertebrates (mainly lolitrem B and ergovaline) including cattle (Siegel et al., 1990; Stuedemann and Hoveland, 1988).
In European soil and climate conditions, beneficial effects of endophyte-infected (E+) versus endophyte-free (E–) grasses are not clear. Studies about abiotic factors such as limiting water avaibility, clipping, shading, low nitrogen fertilization, or low soil pH showed that the plant response varied depending on the genotype of the plant–endophyte combination (Lewis et al., 1996; Lewis, 2004; Hesse et al., 2004). Moreover, Argentine stem weevil [Listronotus bonariensis (Kuschel)], which attacks endophyte-free ryegrass in New Zealand, does not occur in Europe, and the presence of endophyte seems to have no deterring effect on the two main major insect pests of northern Europe grasses, frit fly (Oscinella spp.) and leatherjackets (Tipula spp.) (Lewis and Vaughan, 1997). However toxicosis cases are sporadically observed (Raynal, 1991; Bony et al., 1998; Benkhelil et al., 2004) and may be underestimated because symptoms can be confused with other cattle diseases.
From a legal point of view, in France, the new varieties submitted for the Official List registration must undergo tests evaluating their agronomic values. In these tests, the real effect of the variety only must be evaluated. In the case of an endophyte-infected variety, the effect of the variety is confounded by the effect of the fungus genotype. For this reason, CTPS (Technical Permanent Committee for Breeding) determined that grass varieties proposed to the Official List must have endophyte infection levels less than 20%. Therefore, because of this legal point and owing to existing toxicosis risks and low benefits of endophyte-infected grasses in European conditions, a method to reduce Neotyphodium spp. in grass seeds was needed.
Previous studies dealt with fungicide treatments of E+ plants or seeds of tall fescue and ryegrass. Many fungicides were ineffective in controlling the endophytes when applied on plants (Dernoeden et al., 1990; Latch and Christensen, 1988). The best way to control Neotyphodium appeared to be seed treatment because seeds are the only way for endophyte transmission and dissemination. It appears that, on tall fescue seeds, triadimenol [(1RS,2RS;1RS,2SR)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol], triadimefon [1-(4-chlorophenoxy)-3,3-diemthyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one], bitertanol, and propiconazole [cis-trans-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole] were the most effective fungicides, but they could reduce germination of tall fescue seeds (Williams et al. 1984; Bilotti et al., 1989; Maddaloni et al. 1989). On ryegrass seeds, propiconazole and prochloraz were the most effective chemicals against endophyte, but phytotoxic effects could be observed (Harvey et al., 1982; Latch and Christensen, 1982).
The aim of our study conducted from 2001 to 2004 was to determine fungicide treatment effectiveness to eliminate Neotyphodium spp. in ryegrass and tall fescue seeds. Trials were run in laboratory and in the field. Phytotoxicity and efficacy of fungicides were assessed to find the best balance between endophyte eradication and seed viability.
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MATERIALS AND METHODS
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Seed Material
Several ryegrass and tall fescue seed lots were used in this study. In 2001, trials were conducted with 2 perennial ryegrass seed lots (one forage variety) harboring an LpTG1 endophyte (Neotyphodium lolii Latch, Christensen & Samuels). One seed lot (L1) showed a low infection (14%). The other one (L2) was heavily infected (98%). In 2002 and 2003, two seed lots of ryegrass turf varieties were used in each year that showed contrasting endophyte contamination levels. The seed lots with low contamination (L3 in 2002 and L5 in 2003) had an average contamination of 20%. The highly contaminated lots (L4 in 2002 and L6 in 2003) averaged 70%. The Neotyphodium strain was not identified in any of the lots. All seed lots had a high germination rate before treatments (>91%). Seed lots used in 2003 originated from the 2002 harvest of untreated plots. In 2004, the seed treatments were performed on four tall fescue and six ryegrass seed lots of different varieties, showing various contamination levels.
Seed Treatments
Five fungicides were tested at various doses (Table 1). Triticonazole, bitertanol, and fluquinconazole are systemic triazoles and inhibit ergosterol synthesis. Prochloraz is an imidazole and is known for its translaminar properties. In 2004, another seed treatment was tested, containing the active ingredient fludioxonil [4-(2,2-difluoro-1,3-benzodioxol-4-yl)pyrrole-3-carbonitrile], a phenylpyrrole that is slightly penetrating. Doses were modified from 1 yr to another depending on the results. Fungicides were applied with a batch seed treatment machine HEGE 11 (HEGE, Germany). Seed batches of 500 g were treated by the slurry method. Slurry was applied at 20 L/100 kg of seeds. After chemical application, seeds were dried during several days in ambient conditions and stored at 5°C until efficacy and phytotoxicity analyses.
Efficacy of Chemical Seed Treatments
The efficacy of chemical treatments was measured in the greenhouse on young plants obtained from treated seeds and in field trials on seeds harvested on plants obtained from treated seeds. From 2001 to 2003, only non phytotoxic doses were sown in field trials (Table 1).
Greenhouse Trials
Experiments were performed in greenhouses at INRA (National Institute of Agronomical Research) in Grignon (France). Seeds were sown in a mixed substrate (2/5 topsoil + 2/5 clay soil + 1/5 sand) and placed in greenhouse at about 20°C, with 12-h photoperiod. Fifty seeds were sown in each 30- x 30-cm tray. No foliar fungicide treatment was applied. After 2 to 3 mo, 50 young plants were analyzed to identify the presence of living endophyte.
Field Trials
To evaluate seed treatments efficacy in natural conditions, field trials were conducted in two experimental sites of FNAMS (French National Federation of Seed Growers) in 2001, 2002, and 2003: Brain sur l'Authion located in the west of France and Troyes located in the east. The soil type of Brain sur l'Authion was clayey sand limestone and the soil type of Troyes was clayey silky sand limestone. Four replicates were sown for each treatment in each site at the end of September or beginning of October. Sowing was performed with a hand-sower EBRA (EBRA, France). Plots consisted of three rows 25 to 35 cm apart and 10 m long. Harvests were performed at seed maturity in July. Efficacy was evaluated by analyzing 50 seeds per plot.
Endophyte Detection
The Neotyphodium detection in young plants was made by removing a piece of internal tissue of leaf sheath, placing it on a microscope slide and staining with aniline blue. Slides were warmed a few seconds to accelerate and improve the staining. Neotyphodium spp. mycelium appeared straight and unbranched, running between plant cells, sometimes with a few convolutions. We chose to analyze only one tiller per seedling because preliminary results showed that when a seedling is endophyte-infected, fungus can be detected in any tiller. Tissue pieces were observed from all the successive leaf sheaths (about 3–5) of a chosen tiller to ensure validity of analysis. The endophyte detection in harvested seeds was made by soaking them for 48 h in 3% (v/v) sodium hydroxide solution, rinsing with tap water, and then staining with anilin blue. Before placing on a slide for microscopy, each seed was dissected to remove starch which could hide the presence of mycelium. Neotyphodium mycelium is unbranched and much more convoluted than in leaf sheath, located between aleurone cells (Siegel et al., 1987).
Phytotoxicity
Seed treatment phytotoxicity was evaluated by standard test of germination following ISTA (International Seed Testing Association) method (ISTA, 2003). Three repetitions of 100 seeds were put on moist blotting paper to germinate at 20°C during 14 d. A first count of normal seedlings was made 7 d after sowing. Seven days later, normal and abnormal seedlings and nongerminated seeds were counted.
Statistical Analyses
Statistical analyses of endophyte incidence in field trials were done with ANOVA performed by StatBox (version 2.5, Grimmer Software). For laboratory trials (efficacy and phytotoxicity), results were statistically analyzed with mean comparisons performed with ISTA tolerance tables (ISTA, 2003). Statistical inferences were made at the 5% level of significance, unless otherwise stated.
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RESULTS
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In 2001, the germination measured in the laboratory was slightly reduced in the two lots of ryegrass seeds at the triticonazole doses of 2.5 and 5 g a.i. kg–1 seeds. Triticonazole had little effect on endophytes of the young plants grown in the greenhouse in 2001. For each seed lot there was no significant difference in effectiveness of different triticonazole doses (Table 2). Field trials gave the same results: at both sites, the doses of 2.5 and 5 g a.i. kg–1 had no significant effect on the endophyte for the same seed lot. Regarding these unsatisfactory results, it was decided to try other active ingredients and higher doses of triticonazole in the following years.
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Table 2. Triticonazole phytotoxicity and endophyte incidence evaluated in 2001 on ryegrass plants in greenhouse and in field trials.
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In 2002, the percentage of E+ plants decreased significantly (P = 0.05) for both ryegrass seeds lots treated with prochloraz at 0.5 and 1 g kg–1 (Fig. 1). Endophyte percentage was reduced to zero in the least endophyted-infected lot (L3) treated with prochloraz at 1 g kg–1. With the same treatment, only 4% of the plants of the highly endophyte-infected lot were still infected (72% in the untreated control). The endophyte incidence in lot four plants was also significantly reduced after seed treatment with triticonazole at 5 and 10 g kg–1. It was not significant for lot 3. The percentage of E+ plants was not significantly reduced by bitertanol and fluquinconazole treatments. Germination was affected by prochloraz and triticonazole treatments, the most severe effect being due to prochloraz at 1 g kg–1. Indeed, seed germination fell down to 64% in seed lot 3 and 58% in seed lot 4. The germination decrease is due to 30% of abnormal seedlings and 12% of ungerminated seeds in lot 4 (Table 3). In lot 3, there were 31% of abnormal seedlings and 5% of ungerminated seeds. Abnormal seedlings showed short roots and/or short thicker coleoptiles. Bitertanol at 1 and 2 g kg–1 and fluquinconazole at 2.5 and 5 g kg–1 did not affect germination. In the field, only prochloraz (0.5 g kg–1), triticonazole (10 g kg–1), bitertanol (2 g kg–1) and fluquinconazole (5 g kg–1) were tested. The treatment efficacies observed in greenhouse tests were confirmed by field trials. Indeed, the most effective treatments were prochloraz 0.5 g kg–1 and triticonazole 10 g kg–1 for the two seed lots at the two locations (Fig. 2). Fluquinconazole at 5 g kg–1 was not effective and the 2 g kg–1 dose of bitertanol seemed to be too low to have a complete effect on endophyte eradication. Percentages of E+ harvested seeds were significantly greater in Troyes compared to Brain (Fig. 2): there was a site effect (P < 0.0001), but no interaction between treatments and sites. These efficacy differences may be due to differences in climate and soil.
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Fig. 1. Endophyte incidence after seed treatment measured on fifty, 3-mo-old ryegrass plants for each lot in 2002. Germination rate evaluated at 14 d. Different letters indicate significant differences at P = 0.05.
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Table 3. Evaluation of treatments phytotoxicity on ryegrass by measuring abnormal seedlings and ungerminated seeds 14 d after treatment in 2002.
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Fig. 2. Endophyte incidence in seeds harvested from two perennial ryegrass lots grown in fields in 2002 at two locations, whose parental seeds were treated by fungicides. Different letters indicate significant differences at P = 0.05. ANOVA were performed on each lot at each location (lowercase letters for Troyes and uppercase letters for Brain).
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As a consequence of the 2002 results, triticonazole was not tested in 2003 because the effective doses had affected germination rates, and 2001 results showed that lower doses were not effective enough. Thus it appeared difficult to find a balance between endophyte elimination and seed viability with triticonazole seed treatment. Doses of prochloraz between 0.5 and 1 g kg–1 were tested to lower effects on germination and maintain an effect on the endophyte. Bitertanol and fluquinconazole doses were increased because of low germination incidence and low efficacy observed in 2002. In 2003, significant differences (P = 0.05) were found in greenhouse tests (Fig. 3). Indeed, prochloraz at 0.5, 0.75, and 0.9 g kg–1 reduced considerably the percentage of E+ ryegrass plants. The percentage of plants remaining endophyte-infected was contained between 0 to 2% in both lots, which is extremely low compared with the untreated plants (18% for the least infected lot and 68% for the highly infected lot). Moreover, the germination was not, or only slightly, affected by prochloraz treatments, and few abnormal seedlings were observed (between 0 and 4%) (Table 4). In comparison, bitertanol and fluquinconazole were much less effective. Indeed, the 10 g kg–1 dose was slightly effective, but at this dose, the germination was significantly reduced and the number of abnormal seedlings was increased (Table 4). The results of plant analyses were confirmed by field trials. Analyses have been made on 3 replicates. Prochloraz significantly reduced endophyte incidence in both lots at both locations (Fig. 4). The 0.75 g kg–1 dose totally controlled the endophyte in the highly infected lot (L6) in Brain, and at the most, 3% of the seeds in Troyes remained E+. For L5, the level of endophyte was 1% in the two locations. When prochloraz was applied at 0.9 g/kg, at most 5% of the seeds in L6 in Troyes remained E+. Fluquinconazole and bitertanol reduced the percentage of E+ seeds but were less effective than prochloraz: on average 30% of seeds remained endophyte-infected.
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Fig. 3. Endophyte incidence after seed treatment measured in fifty, 3-mo-old ryegrass plants for each lot in 2003. Germination evaluated at 14 d. Different letters indicate significant differences at P = 0.05.
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Table 4. Evaluation of treatments phytotoxicity on ryegrass by measuring abnormal seedlings and ungerminated seeds 14 d after treatment in 2003.
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Fig. 4. Endophyte incidence in seeds harvested from two perennial ryegrass lots grown in fields in 2003 at two locations, whose parental seeds were treated by fungicides. Different letters indicate significant differences at P = 0.05. ANOVA were performed on each lot at each location (lowercase letters for Troyes and uppercase letters for Brain).
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In 2004, tests were conducted with prochloraz on ryegrass and tall fescue seeds. Fludioxonil was also tested. Fluquinconazole and bitertanol were abandoned. Fludioxonil at 0.2 g kg–1 had very little effect on tall fescue and ryegrass germination (Table 5). Only one lot (rg3) showed a significant decrease in germination after treatment, but it remained high (92%). Prochloraz was more phytotoxic on tall fescue and ryegrass seeds. Germination was significantly decreased in seven lots (tf1, tf4, rg1, rg2, rg3, rg4, rg6) compared with the untreated lots. The germination rates of tf1 and rg2 fall under the legal limit fixed for seed lots commercialized in France (80%). It must be noticed that the germination decrease was due partly (50%) to abnormal seedlings, and partly to ungerminated seeds. The malformations due to prochloraz seed treatment were stubby roots, very short coleoptiles, and twisted leaves. Fludioxonil was effective on seed lots of ryegrass and tall fescue with a low percentage of infection, but on highly infected ones (rg4 and rg5), the endophyte was not completely controlled (Table 6). Prochloraz at 0.75 g kg–1 was quite effective on ryegrass and tall fescue seeds. The plants from lots with little or no infection were not analyzed (tf3, tf4, and rg6). Prochloraz seed treatments killed endophytes in the seven analyzed seed lots of ryegrass and tall fescue. The most highly infected lots, rg4 and rg5, respectively, still contained 6 and 2% of seeds with living endophytes.
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Table 5. Germination of ryegrass and tall fescue lots in 2004 after seed treatments with prochloraz and fludioxonil.
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DISCUSSION
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In 2003, prochloraz was the most effective and least phytotoxic in greenhouse and field trials on ryegrass seeds. However, it must be noted that 2002 and 2003 results are not quite the same concerning prochloraz. Indeed, the 0.5 g kg–1 dose was more effective in 2003 than in 2002 in greenhouse tests. It may be explained by the lots differences in 2002 and 2003. They were of the same varieties but not from the same harvest year. The differences in plant growth, harvest, or seed storage conditions may have induced a difference in endophyte vigor in seeds and modified the response to fungicide treatments. This could also explain the difference in germination between prochloraz at doses of 0.9 g kg–1 in 2003 and 1 g kg–1 in 2002. Indeed, the 0.9 g kg–1 dose induced no or very slight germination decrease, while the 1 g kg–1 dose caused a 30% decrease in germination. In 2004, the same phenomenon was encountered when germination was noticeably affected for certain lots but not for others.
Our results can be compared with those of Latch and Christensen (1982) concerning ryegrass plants analyses showing that prochloraz provides a good control on endophytes when applied as a seed treatment at 0.5 and 1 g per kg of seeds. Counting numbers of emerged seedlings at 7 and 14 d showed that prochloraz had phytotoxic effects. In our analyses of young plants, the 1 g kg–1 dose also reduced seedling emergence but the 0.5 g kg–1 dose showed a low phytotoxicity and a good endophyte control, which was confirmed by field results in standard cultural conditions.
The data obtained with bitertanol treatment can be compared with those obtained by Siegel et al. (1984) on F. arundinacea seeds where fescue plants were analyzed at 10 to 12 wk after seed treatment with bitertanol at 5.2 g kg–1. They observed a 100% reduction of E+ plants (untreated seed lot had an endophyte incidence of more than 80%). All the tested triazoles induced a control of endophyte in seeds but induced also germination decrease, except bitertanol. On the contrary, we showed that on ryegrass seeds neither bitertanol nor fluquinconazole, which is a triazole, induced proper control of the endophyte. Moreover, both affected germination when doses applied were 10 g kg–1. Then we can wonder whether a fungicide able to control a ryegrass endophyte is able to control a fescue endophyte. Indeed, efficacy of fungicides may be linked with the endophyte species. Saiga et al. (2003) showed that soaking trials of ryegrass seedlings harboring N. lolii and tall fescue harboring N. coenophialum Morgan-Jones & Gams in benomyl ([3-(butylcarbamoyl)-3H-benzoimidazol-2-yl]aminoformic acid methyl ester), triforine [N,N'-{piperazine-1,4-diylbis[(trichloromethyl)methylene]}diformamide] or thiophanate methyl ([2-(methoxycarbonylthiocarbamoylamino)phenyl]thiocarbamoylaminoformic acid methyl ester) proved that benomyl is effective in eliminating both N. lolii and N. coenophialum. Triforine was effective against N. coenophialum but not really against N. lolii. The contrary was observed for thiophanate methyl. If prochloraz treatments have to be used on other grass species (F. pratensis L. or F. rubra L., for example), it would be useful to check its efficacy on these species.
In summary, prochloraz seems to be able to control ryegrass and tall fescue endophytes. Some seed lots maintained a good germination while others fell below 80% of germination. However, if prochloraz is to be used during grass breeding, or in the very early generations of multiplication, one can accept a rather low germination of parental seeds. The seed treatment could be used once during the selection. Then, subsequent generations of seeds will be free from endophytes, and the germination rate will be conserved.
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CONCLUSIONS
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Prochloraz was found efficient in eliminating Neotyphodium endophytes from perennial ryegrass and tall fescue seeds. It makes it possible to decrease levels of seeds with living endophytes under the limit of 20% fixed by the French regulations. It would be now very interesting to test its efficacy on other endophyte-infected grasses used as forage or turf. When the transmission mode of other Neotyphodium spp. having teleomorphs belonging to the Epichloë genus will be more precisely described, it may be also useful to test prochloraz to reduce choke disease in orchard grass seed-producing fields.
Since the late 1990s, strategies have been developed to find endophyte– associations which have positive agronomic characteristics and little or no lolitrem and ergovaline production. These strategies are mainly conducted in countries where biotic and abiotic stresses are severe and where obvious benefits in cultivating E+ grasses exist, generally for ryegrass and tall fescue (Hill et al., 2002). One strategy consists of finding endophyte–grass associations occurring in nature and using phenotypic selection to enhance desirable traits and reduce undesirable traits. Another strategy is to free a plant from its endophyte and to reinfect it with a low toxin-producing isolate (Bouton and Hopkins, 2003). An endophyte-free host is therefore needed. Our endophyte control technique with prochloraz seed treatment could fit into this strategy.
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ACKNOWLEDGMENTS
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We thank Dr C. Huyghe and F. Deneufbourg for their reviewing.
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NOTES
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This study was supported by the French Ministry of Agriculture, CTPS and French forage seed companies.
Received for publication February 11, 2005.
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ABSTRACT
INTRODUCTION
