house et at., 1988). The RPV subgroup consists of viruses transmitted ... cifically by Sitobion avenae (MAV; see Lister &. Sward, 1988) or Sehizaphis graminum ...
Plant Pathology (1993) 42, 1-5
Barley yellow dvvarf viruses in Japanese pasture grasses and lack of correlation with the presence of fungal endophytes p. L. GUY* Division of Microbiology, National Institute of Agro-Environmental Seienees, Tsukuba. Ibaraki, Japan Pasture grasses from temperate Japan were tested for infection with barley yellow dwarf viruses (BYDVs) and fungal endophytes. BYDVs from both the MAV and RPV subgroups were detected, but no symptoms attributable to BYDV infection were observed. Not all isolates from the MAV subgroup could be clearly discriminated as MAV or PAV solely on ELISA results, and may have been intermediate serotyiJes or mixed infections. BYDVs were found to infect fescue (Festuea arundinaeea: 17%), ryegrass {Loliumperenne: 41%), timothy (Phleumpratense. 94%) and Poa spp. (20%). Fescue and ryegrass were predominantly infected with RPV and PAV, respectively. The small collections of Poa spp. were only infected with PAV, while timothy was only tested for MAV subgroup viruses. In fescue 26% of tillers were infected with Aeremonium eoenophlalum, and 60% of ryegrass tillers from an ecotype collection were infected with Aeremonium lolii. There was no correlation between BYDV infection and the presence of endophytes for the above species or for Epiehloe fy/jftma-infected (50%) timothy. An ELISA test for A. loliidid not detect /4. eoenophiatum in fescue or E. typhina in timothy but showed good agreement with epidermal staining of A. lolii in ryegrass leaf sheaths.
INTRODUCTION Barley yellow dwarf viruses (BYDVs) have been reported to infect Agropyron tsukttsiense (Toriyama & Yora, 1972) and cereals in Japan (Kojima et at., 1983) but there are no reports on BYDVs in Japanese pasture grasses. BYDVs can be divided into two groups depending on their genome organisation (Martin et al., 1990) and serological properties (Waterhouse et at., 1988). The RPV subgroup consists of viruses transmitted specifically by either Rhopalosiphum maidis (RMV) or by R. padi (RPV). The MAV subgroup contains viruses transmitted specifically by Sitobion avenae (MAV; see Lister & Sward, 1988) or Sehizaphis graminum (SGV), or non-specifically by a number of species including R. padi and S. avenae (PAV). Serological relationships have been found within but not between the two subgroups. Viruses from both the MAV and RPV subgroups infect C3 pasture species. In North America, fescue (^Festuea arundinaeea) is infected with MAV, PAV and RPV (Fargette et ai, 1982; Paliwal, 1982). In Australia, fescue is predominantly infected with PAV even when nearby cocks• Present address: Univeriity of Otago, Botany Department PO Box 56, Dunedin, New Zealand.
foot and Fhalaris plants are infected with RPV (Guy, 1988). Ryegrass (Lolium perenne) is predominantly infected with MAV subgroup viruses. Holmes (1985) reported that in Scotland MAV and PAV occurred as both single and mixed infections, but that PAV predominated in ryegrass. RPV occurs in Australian ryegrass pastures as single or mixed infections, but on the island of Tasmania, infection with PAV is more common (Guy et ai, 1986; Guy, 1988), while in Victoria PAV predominates over MAV at most sites (Eagling et ai, 1989). Fungal endophytes of grasses are grouped in the tribe Balansiae of the Clavicipitaceae. Within this tribe are the genera Balansia, Balansiopsis, Atkinsonella and Myrlogenospora which infect grasses with C4 photosynthetic pathways. Epiehloe typhina is found on a number of important C> pasture species. Aeremonium coenophialum and A. tola, the endophytes of fescue and ryegrass, may be anamorphic forms of Epiehloe (Siegel et ai, 1987). In Japan, E. typhina is confined to timothy (Phleumpratense) where it causes typical choke symptoms (Shimanuki & Sato, 1983). There are no reports of endophyte-infected fescue (Udagawa & Tsubaki, 1978). A. lolii was recently isolated from ryegrass and large-scale screenings of imported fescue and ryegrass seedlots delected
p. L. Guy a low incidence of endophyte infection (T. Shimanuki, 12 June 1990, HAES, personal communication). Elsewhere, high incidences of endophytes occur in pastures and wild populations. Siegel et al. (1984) sampled 37 fescue pastures in Kentucky and found the range of ^4. eoenophialum incidence was 10-100% (average: 64%). Shelby & Dalrymple (1987) found the mean infection rate for fescue in 26 states was 58%, while for ryegrass pastures. Latch & Christensen (1982) and Guy (1992) reported 0-96% and 4-94% (average for both: 66%) incidence in nine New Zealand and 27 Tasmanian pastures, respectively. Clay & Leuchtmann (1989) found the incidence in Indiana woodland grasses was generally very high. For example, more than 50% of Festuea obtusa plants collected from each offivesites were infected with an Aeremonium species endophyte. Most previous studies have concentrated on either endophytes or BYDVs in pastures. This report centres on the incidences of both BYDVs and endophytes in wild populations of tall fescue (F. arundinaeea), a native of Japan and Eurasia (Hubbard, 1968) and on ryegrass, an introduced species. In view ofthe report (Siegel et ai, 1987) that Acremonium-\n[tclt(i grasses can resist insect attack, it was of particular interest to see if there was a (negative) correlation between incidence of BYDVs, which are aphid transmitted, and endophytes. MATERIALS A N D METHODS Sample collection Single tillers were collected from fescue plants growing at one site in each of Akita, Chiba, Hokkaido and Saitama prefectures, and two sites in Ibaraki prefecture (8-10 tillers per site). Adaxial epidermal leaf-sheath strips (from each tiller) were mounted in 0 05% lactophenol cotton blue and examined under a microscope for the presence of endophyte mycelia. Pieces of three leaves from each tiller were ground together in buffer (1/ 10 w/v) and stored frozen before ELISA testing for BYDVs. Tillers from ryegrass plants in the ecotype collection of the National Grasslands Research Institute (NGRI) at Nishi-Nasuno, and a pasture at the Hokkaido Agricultural Experimental Station (HAES) at Hitsujigaoka, were ground and stored in the same way. Epidermal strips were prepared from 40% of the NGRI samples and compared with the A. tolii ELISA results. Tillers from nine choke-afTectcd and nine choke-free 2-
year-old timothy plants were collected from a trial at HAES and processed. Tillers of Poa spp. were collected from two sites in Fukushima prefecture and ground in bufTer as above. ELISA procedures Antisera to four barley yellow dwarf viruses and freeze-dried oat and/or ryegrass tissue infected with the homologous viruses were used in ELISA tests: an RPV virus isolated from Hordeum murinum (Guy, 1993), a PAV-bke virus (T-OA6) from oat (Guy et ai, 1986), and type PAV and MAV (Rochow, 1984). The ^. /o//i anti-mycelium and anti-culture protein antisera (Guy, 1992) were used in conjunction with locally acquired endophyte-infected and endophyte-free ryegrass and fescue. The ELISA methods were essentially those described by Clark & Adams (1977) with the following changes. The working volume for each step was 100 /il per well in Nunc Maxisorb plates. All steps except plate washing and substrate development for the A. lolii tests were done at 4°C. A. lolii plates were coated with 1 /ig/ml IgG and conjugate was added at 1/600 dilution. RPV, PAV and MAV plates were coated with 3,1 and I /ig/ml IgG, respectively, while all BYDV conjugates were used at 1 /2000 dilution. Samples being tested for BYDV were incubated in plates at 30 C for 4 h. After washing, the conjugates were added and incubated at 4 C for 16 h. Absorbance values (AMU) which were more than two and three times the maximum negative control value for the A. lolii and BYDV assays, respectively, were regarded as positive. RESULTS Virus incidence BYDVs were detected at all 11 sites. All four species were infected with BYDVs (Table 1) but none ofthe tillers showed symptoms of infection. Fescue tillers (17"o) were predominantly infected with RPV. Ryegrass (83%) from the ecotype collection was predominantly infected with PAV although MAV, RPV and mixed infections were also found. At least 26° o ofthe ryegrass pasture samples (which were not tested for RPV) wete infected. Many of the ryegrass and timothy sap extracts reacted strongly in both the MAV and PAV ELISA tests and could not be clearly discriminated as MAV or PAV types. These results may indicate mixed infections or serotypes intermediate between MAV and PAV. Most (17
Barley yellow dwarf viruses and endophytes in Japanese grasses Table 1. Barley yellow dwarf viruses in Japanese grasses Barley yellow dwarf viruses
Festuea arundinaeea Lolium perenne Ecotypes, NGRIJ Pasture, HAES§ Phleum pratense Poa spp.
iNumDer ot tillers
MAV
MAV sub*
PAV
P-t-Rt
RPV
53
0
0
2
0
7
37 110 18 10
0 3
1 12 14 0
20 14 3 2
7
3 — — 0
0 0
—f
— 0
• MAV-subgroup, isolates could not be serotyped reliably beyond this level. t P + R, dual PAV and RPV infections. X National Grasslands Research Institute, Nishi-Nasuno. § Hokkaido Agricultural Experimental Station, Hitsujigaoka. f Not tested.
Table 2. Barley yellow dwarf viruses and endophytes in three pasture species Endophyte-infected Speeies
Number of tillers
BYDV +
53 37 18
4 20 9
1 * Festuea arundinaeea 2 Lolium perenne 3 Phleum pratense
BYDV10 2 0
Endophyte-free BYDV+
BYDV-
5 11 8
34 4 1
* Endophytes (I) Acremonium eoenophiatum, (2) Acremonium lolii, (3) Epiehloe typhina.
18:94%) ofthe timothy plants were infected with MAV-subgroup viruses. The small number of Poa spp. collected were infected (2/10) with PAV. Endophytes and BYDV infection A. coenophialum, as determined by microscopy, infected 26% of tillers from native fescue plants at most (4/6) sites, and A. lolii infected 60% of ryegrass tillers from the ecotype collection (Table 2). There was no difference (x^ = 1891, NS) between the incidence of BYDV infection (or any specific serotype) of endophyte-infected/free fescue and ryegrass nor for the E. typhina-Mected timothy. None of the endophytes protected its host against BYDV infection (Table 2). ELISA tests for A. tola mycelium and culture proteins failed to detect intercellular A. eoenophiatum in fescue or E. typhina in timothy. The mycelium ELISA test did give a positive reaction against high concentrations (0-5 mg/ml; Guy, 1991) of A. eoenophialum mycelium from broth cultures and E. typhina stroma taken from fertile
tillers. However, this was of no use in field studies of these endophytes. There was good agreement between the mycelium ELISA results and epidermal staining of A. lolii in ryegrass leaf sheaths. All (15/15) samples gave identical results with both tests (see also Guy, 1992). DISCUSSION R. maidis, R. padi, S. avenae and S. graminum. the definitive vectors of BYDV, occur in Japan but only R. padi and 5. avenae are reported as vectors of Japanese isolates (Toriyama & Yora, 1972; Kojima et ai, 1983). The latter two species together can transmit all the serotypes and mixtures found during the present study, which has shown that BYDVs are widespread throughout temperate Japan. The wide spacing (c. 0 5 x I m) of plants in the ryegrass ecotype collection and the timothy plots probably contributed to BYDV incidences above those reported for most pastures (e.g. Lindstcn & Gerhardson, 1969; Holmes, 1985; Guy et ai.
p. L. Guy 1986). Plant spacing affects risk of infection by a number of aphid-borne viruses (A'Brook, 1968; Johnstone et al., 1982). Many aphid spwcies are attracted to host plants surrounded by bare earth or contrasting foliage. A'Brook (1973) trapped larger numbers of BYDV vectors over widely spaced drill lines of cocksfoot than over a closely drilled crop. BYDV incidence in grasses can also be related to pasture age (Latch, 1977; Guy et al, 1986; Guy, 1988) but unfortunately, with the exception of the timothy plots, information on the age structure of the plant communities was not available. The same limitation was encountered by Guy et ai (1987) when sampling Tasmanian native grass species; 13% of native Pooideae, of undetermined ages, were infected. In Japan 17% of fescue and 20% of Poa spp. (both Pooideae) were infected. The incidence of A. lolii'm ryegrass was similar to that reported from Australia and New Zealand (Latch & Christensen, 1982; Guy, 1992) while the incidence of A. coenophialum in fescue was lower than that reported from North American pastures (Siegel et al., 1984; Shelby & Dalrymple, 1987) and woodlands {Acremonium spp.. Clay & Leuchtmann, 1989). A striking difference between Acremoniuminfected and uninfected fescue and ryegrass is the infected plant's ability to resist insect attack (Siegel et al., 1987). However, none ofthe endophytes protected its host against aphid-borne BYDV infection in the present study. It has already been suggested that endophyte infection of fescue and ryegrass does not affect the behaviour of at least three BYDV vectors: Metopolophium dirhodum, S. avenae and 5. fragariae (Johnsonetal., 1985; Latchetal., 1985). However Latch et at. (1985) reported that R. padi avoided fescue plants infected with A. coenophialum, but noted that after 1-3 weeks an average of 1-4 R. padi were still present on each .4 coenophialuminfectcd fescue plant in their glasshouse. Johnson et al. (1985) also found that A. coenophialuminfected fescue had a repellent effect on R. padi, and on .S'. graminum, but found that no aphids survived when confined to these plants for 3-6 days. Preliminary reports suggest that loline alkaloids, produced in .A. coenophiaium-infecled plants, are responsible for the antifeedant and insecticidal effects on R. padi and S. graminum (Dahlman et al.. 1991). However, the aphid studies used fescue plants grown under temperature-controlled conditions with no intcr-plant comrwtition for regularly supplied fertilizer. In
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