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© 2008 Plant Management Network. Susceptibility of Selected Cultivars of Forage Grasses to Magnaporthe oryzae Isolates from Annual Ryegrass and Relatedness of the Pathogen to Strains from Other Grasses P. Vincelli, E. Dixon, and M. Farman, Department of Plant Pathology, University of Kentucky, Lexington 40546-0312 Corresponding author: P. Vincelli. pvincell@uky.edu Vincelli, P., Dixon, E., and Farman, M. 2008. Susceptibility of selected cultivars of forage grasses to Magnaporthe oryzae isolates from annual ryegrass and relatedness of the pathogen to strains from other grasses. Online. Forage and Grazinglands doi:10.1094/FG-2008-0226-01-RS. Abstract Isolates of Magnaporthe oryzae obtained from annual ryegrass were inoculated onto two or three cultivars each of seedlings and adult plants of annual ryegrass, perennial ryegrass, tall fescue, meadow fescue, and festulolium. All plant species tested were very susceptible as seedlings, although modest differences in susceptibility among cultivars were sometimes observed. Adult plants of all species tested exhibited blighting as well, suggesting that all could allow inoculum buildup in pastures where they are sown. DNA fingerprinting tests indicate that the M. oryzae isolates causing disease on annual ryegrass appear to be from the same pathogen population responsible for the gray leaf spot epidemics on perennial ryegrass. This disease appears to pose a significant threat to the use of annual ryegrass sown in late summer on Kentucky dairy and beef farms. Introduction As a forage crop, annual ryegrass (Lolium multiflorum) is adapted throughout much of the southern United States (1). When seeded in late summer in Kentucky, it can produce high-quality forage suitable for grazing in late autumn and through most of the spring. However, recent efforts to use annual ryegrass on dairy- and beef-cattle farms in Kentucky have sometimes resulted in failure due to severe leaf blighting by Magnaporthe oryzae B. Couch (Fig. 1) ( P. Vincelli, unpublished), a member of the M. grisea species complex (2).
M. oryzae ([the asexual stage is known as Pyricularia oryzae Cavara [=Pyricularia grisea (Cooke) Sacc.]) is known to cause sporadically severe epidemics on annual ryegrass in several states along the Gulf of Mexico (9,10,13). Over the past 20 years, M. oryzae has caused sporadically severe epidemics on turf-type perennial ryegrass in the Mid-Atlantic and much of the Midwest and eastern Great Plains (7,14,15). Until now, however, it was not known whether strains of M. oryzae from annual ryegrass are related to those that cause epidemics on perennial ryegrass. The primary objective for this study was to evaluate the susceptibility of a selection of entries of annual ryegrass and other forage grass species to isolates of M. oryzae obtained from annual ryegrass. Seedlings were tested separately from adult plants because of their substantially greater susceptibility (13). A second objective was to use DNA fingerprinting to determine the genetic relationship between strains of M. oryzae from annual ryegrass and those from perennial ryegrass. Tests of Seedling Resistance We tested susceptibility of two or three cultivars of each of the following forage grasses: diploid and tetraploid cultivars of both perennial ryegrass (Lolium perenne L.), annual ryegrass (Lolium multiflorum Lam.), endophyte-infected and endophyte-free cultivars of tall fescue [Lolium arundinaceum (Schreb) Darbysh. = Festuca arundinacea Schreb.], meadow fescue [Lolium pratense (Huds.) Darbysh. = Festuca pratensis Huds.], and festulolium [Festulolium loliaceum (Huds.) P.V. Fournier]. With the exception of endophyte-infected and endophyte-free tall fescue, cultivars representing each species/ploidy class were arbitrarily selected from those available in the University of Kentucky Forage Variety Evaluation Program. Forty to 50 seeds of the cultivars tested were sown into clean 35-mm film canisters filled with soil-less potting mix. Pots were watered using a wick system. After seeding, pots were placed at 28°C under continuous fluorescent light (40 to 60 µE/m²/sec) for 2.5 weeks. Plants were trimmed to a height of approximately 4.5 to 5 cm 8 days after seeding and at 3- to 4-day intervals subsequently. A mixture of three isolates (designations PL1-1, PL2-1, and PL3-1) collected from annual ryegrass in Pulaski County, KY were grown on dilute oatmeal agar for 7 to 10 days. Spores (conidia) were harvested with sterile distilled water, counted, and diluted with sterile water to achieve a concentration of 3 × 105 spores/ml. This spore suspension was sprayed over all leaves to runoff (four replicate pots for each cultivar). Inoculated plants were placed into a sealed humidity chamber and incubated for 96 h of leaf wetness (24 h in darkness at 25°C, then 72 h of continuous light at 28°C). Humidity chambers were then unsealed and plants were allowed to grow for 3 days while disease progress continued. Plants were harvested 7 days after inoculation by cutting at ground level, selecting 10 tillers arbitrarily, and scoring each visually for foliar blighting. Plants were scored as 0%, 2%, 5%, 10% foliar blighted, and thereafter at 10% increments up to 100%. Disease severity was averaged for all tillers in a pot. For this and all subsequent tests, all data were arcsine-transformed and analyzed by ANOVA as a completely randomized design, and statistical inference was based on the Waller-Duncan k-ratio t-test (k = 100, P = 0.05). The experiment was repeated once as described above (referred to below as "high disease pressure tests") and once with a reduced inoculum level (4 × 104 conidia/ml) and a shorter period of post-inoculation leaf wetness (24 h dark followed by 24 h light) in order to evaluate seedling resistance under reduced disease pressure. In the high disease pressure tests, all cultivars of all grasses tested exhibited moderately high to high levels of leaf blighting, with means ranging from 48 to 88% (Table 1). The correlation coefficient between treatment means in Experiment 1 vs Experiment 2 was 0.85, indicating good agreement in results between the two experiments. Cultivars of diploid annual ryegrass were among most severely affected; ‘Gulf’, a known susceptible cultivar (11), exhibited the highest level of leaf blighting in the tests. All cultivars of perennial ryegrass (diploid and tetraploid) and tall fescue, as well as two cultivars of festulolium, exhibited statistically less blighting than ‘Gulf’ annual ryegrass (P = 0.05). However, all of these exhibited levels of blighting exceeding 52%. Cultivars of meadow fescue were among the least affected across both high-pressure tests, with blighting levels around 50%. Table 1. Severity of leaf blighting caused by strains of M. oryzae collected from annual ryegrass on cultivars of several species of forage grassx.
x Means within the same column followed by the same letter are not significantly different, Waller-Duncan k-ratio t-test (k = 100, P = 0.05). ANOVA and analysis by the multiple comparison test were performed on arcsine-transformed data; however, means presented are untransformed, for ease of interpretation. y Common names of hosts tested are: tall fescue (Lolium arundinaceum), annual ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), festulolium, meadow fescue (Festuca pratensis). "d" and "t" = diploid and tetraploid, respectively. In the reduced disease pressure test, levels of leaf blighting were lower than in high disease pressure tests. As above, cultivars of diploid annual ryegrass were all among the most susceptible, with disease severities ranging from 49.5 to 53.3%. At least one cultivar each of tetraploid annual ryegrass, tetraploid perennial ryegrass and festulolium exhibited a disease severity level which was statistically equal to ‘Gulf’ annual ryegrass. Cultivars of tall fescue and diploid perennial ryegrass all exhibited lower severity (P = 0.05) than ‘Gulf’ annual ryegrass. Cultivars of meadow fescue exhibited the lowest disease severity observed in the test (P = 0.05). Tests of Adult Plant Resistance Six pots of each cultivar were planted in 7.6-cm pots in a mixture of 50% Promix and steamed topsoil. The pots were grown under greenhouse conditions for 10 weeks, divided and transplanted, and grown for another 8 to 10 weeks in the greenhouse. Plants were fertilized every 10 to 14 days with 175 ppm of Peter’s 20-20-20 and trimmed every 3 to 4 weeks to a height of 10 cm. Twelve weeks after sowing, triadimefon (0.375 g/liter) was applied to runoff to control powdery mildew. Four-month-old plants were trimmed, allowed to grow for 3 to 5 days, then acclimated in a growth chamber for 5 days prior to inoculation. Chamber conditions were 29/24°C day/night temperature and 14 to 16 h of florescent light. Four pots of each cultivar were sprayed with 1.5 × 105 spores/ml (40-ml/12-pot tray). Inoculated plants were sealed in transparent plastic bags to maintain high humidity and returned to the growth chamber, in which lights were extinguished for 18 h before returning to a diurnal cycle. Plants remained within bags for 4 days, then were held in the growth chamber for 3 additional days. Seven days after inoculation, visual evaluations of leaf blighting were made as described above. For each cultivar tested, a set of two uninoculated control pots was incubated under identical conditions in order to monitor for foliar necrosis from causes other than M. oryzae. As in the seedling assay, cultivars of diploid annual ryegrass were among the most susceptible adult plants, with ‘Gulf’ appearing to be the most susceptible (Table 1). The tetraploid annual ryegrass ‘Aurelia’ was significantly less susceptible than all three annual ryegrass cultivars (P = 0.05). Cultivars of tall fescue, perennial ryegrass (both diploid and tetraploid), festulolium, and meadow fescue were all significantly less affected than ‘Gulf’, although all exhibited over 20% leaf blighting in at least of one of the tests. There was reasonably good agreement in results between the two experiments testing for adult-plant resistance (correlation coefficient = 0.72 between treatment means for Experiment 4 vs Experiment 5). In order to test the degree of correspondence between an entry’s seedling susceptibility vs adult-plant susceptibility, we calculated means for Experiment 2 and plus Experiment 3 and tested for correlation of these means to treatment means for Experiment 4 plus Experiment 5, yielding a correlation coefficient of 0.55. DNA Fingerprinting To determine if the M. oryzae isolates obtained from annual ryegrass are related to those causing disease in perennial ryegrass, we used the Pot2 transposon probe (6) to perform DNA fingerprinting. Approximately 200 ng of genomic DNA were digested separately with BamHI (New England Biolabs, Bethesda, MD) and PstI and electrophoresed through a 0.7% agarose gel. The gel was Southern blotted to a membrane (Biodyne B, Pall Industries, East Hills, NY) The membrane was then probed with a 0.9 kb BamHI fragment internal to the Pot2 transposon (4) that had been labeled with ³²P using the Prime-a-Gene labeling kit from Promega (Madison, WI). After washing to high stringency, the blot was exposed to a phospor screen for 24 h, which was then scanned in a Typhoon phosphorimager (Amersham Biosciences, Pittsburgh, PA). The resulting image revealed that the Pot2 fingerprints of the M. oryzae isolates from annual ryegrass were very similar to those obtained for the ones from perennial ryegrass (Fig. 2 shows the results obtained with BamHI). In fact, the fingerprint of isolate PL1-1 was almost identical to that observed for CHRF (Fig. 2, lanes marked with asterisks). PL3-1 was the most different, as it exhibited many more hybridizing fragments than the others. However, closer inspection revealed that most of the fragments that are shared among the annual ryegrass and perennial ryegrass isolates are also present in PL3-1. Therefore, it appears that this isolate has simply experienced amplification of Pot2. Overall, the molecular data support the hypothesis that M. oryzae isolates found on annual ryegrass belong to the same pathogen population as that which infects perennial ryegrass.
Conclusions and Recommendations • In pastures with significant gray leaf spot activity, M. oryzae could severely limit successful establishment of annual ryegrass. Because M. oryzae survives in diseased leaf residue (5,15), fields with significant residue are at greatest risk. Untilled pastures where annual ryegrass is sown every year probably represent the greatest risk. • Rotation to nonhost crops should help reduce disease pressure. However, crop rotation has limited value against this disease for two reasons. — (i) Suitable choices of non-hosts are limited primarily to dicotyledonous forages, since our results and the known host range of M. oryzae (3) indicate that seedlings of most suitable forage grasses will be damaged under high disease pressure. — (ii) Damaging spore clouds could be blown in from neighboring fields in regions like southern Kentucky, with its mosaic of small fields and pastures. • Delaying seeding until mid-September may help reduce disease pressure. This would reduce — but not eliminate — the risk of warm, humid conditions that favor infection. • Choosing resistant varieties of annual ryegrass would be desirable, but options are severely limited. We are aware of only one cultivar with resistance to gray leaf spot (8), and neither its level of resistance nor suitability for Kentucky conditions have been reported. Several plant introductions of annual ryegrass with moderately high resistance to M. oryzae exist (12). These could be useful in breeding for resistance, but commercially useful varieties could take 7 to 10 years to develop. • Our adult-plant inoculations indicate that perennial ryegrass, tall fescue, meadow fescue, and festulolium all are susceptible to isolates of M. oryzae obtained from annual ryegrass. Therefore, these grasses could allow for buildup of inoculum, setting the stage for epidemics where highly susceptible cultivars of annual ryegrass or other species are sown subsequently. • Our DNA fingerprinting tests indicate that the M. oryzae isolates causing disease on annual ryegrass appear to be from the same pathogen population responsible for the gray leaf spot epidemics on perennial ryegrass. Acknowledgment Thanks are expressed to Robert Spitaleri for providing seed of all entries used in these experiments. Literature Cited 1. Ball, D. M., Hoveland, C. S., and Lacefield, G. D. 2002. Southern Forages, 3rd Edn. Potash and Phosphate Inst., Norcross, GA. 2. Couch, B. C., and Kohn, L.M. 2002. A multilocus gene geneology concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94:683-693. 3. Farr, D. F., Bills, G. F., Chamuris, G. P., Rossman, A. Y. 1989. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN. 4. Farman, M. L. 2002. Pyricularia grisea isolates causing gray leaf spot of perennial ryegrass: relationship to P. grisea from other host plants. Phytopathology 92:245-254. 5. Harmon, P. F., and Latin, R. 2005. Winter survival of the perennial ryegrass pathogen Magnaporthe oryzae in north central Indiana. Plant Dis. 89:412-418. 6. Kachroo, P. K., Chattoo, B. B., and Leong, S. A. 1994. Pot2, an inverted repeat transposon from Magnaporthe grisea. Mol. Gen. Genet. 245:339-348. 7. Landschoot, P. J., and Hoyland, B. F. 1992. Gray leaf spot of perennial ryegrass turf in Pennsylvania. Plant Dis. 76:1280-1282. 8. Prine, G. M., Blount, A. R., Dunavin, L. S., Mislevy, P., Stanley, R. L., Jr. 2002. Registration of 'Jumbo' annual ryegrass. Crop-Science. 42:1749. 9. Reith, P. E., Prine, G. M., Blount, A. R. 2001. Gray leaf spot: New-old peril to annual ryegrass. Proc. Soil Crop Sci. Soc. Fla. 60:143-146. 10. Rush, M. C., Lindberg, G. D., Carver, R. B. 1972. Blast: a serious new disease of forage grasses in Louisiana. (Abstr.) Phytopathology 62:806. 11. Trevathan, L. E. 1982. Pathogenicity on ryegrass and cultural variability of Mississippi isolates of Pyricularia grisea. Plant Dis. 66:592-594 12. Trevathan, L. E. 1982. Response of ryegrass plant introductions to artificial inoculation with Pyricularia grisea under greenhouse conditions. Plant Dis. 66:696-697. 13. Trevathan, L. E., Moss, M. A., and Blasingame, D. 1994. Ryegrass blast. Plant Dis. 78:113-117. 14. Uddin, W., Viji, G., and Vincelli, P. 2003. Gray leaf spot (blast) of perennial ryegrass: An emerging problem for the turfgrass industry. Plant Dis. 87:880-889. 15. Vincelli, P. 1999. Gray leaf spot, an emerging disease of perennial ryegrass. Turfgrass Trends 7:1-8. |
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