Crop Science 40:713-716 (2000)
© 2000 Crop Science Society of America
CROP ECOLOGY, MANAGEMENT & QUALITY
Chitinase Activity in Tall Fescue Seedlings as Affected by Cultivar, Seedling Development, and Ethephon
S.M. Mareka,
C.A. Robertsb,
A.L. Karrc and
D.A. Sleperb
a Dep. of Plant Pathology, Univ. of California, Davis, CA 95616 USA
b Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211 USA
c Dep. of Plant Pathology, Univ. of Missouri, Columbia, MO 65211 USA
robertscr{at}missouri.edu
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ABSTRACT
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Recent reports indicate that tall fescue (Festuca arundinacea Schreb.) may be selected for increased disease resistance with the use of a marker such as chitinase, a defense protein associated with disease resistance in tall fescue. The objective of this study was to determine if chitinase activity in tall fescue cultivars differs consistently across seedling stage, and to determine if chitinase activity could be increased with ethephon ([2-chloroethyl]phosphonic acid), a growth regulator used as a chemical elicitor. Ten cultivars of tall fescue were planted in a greenhouse, and seedlings were harvested at 14, 28, and 42 d after germination. Seedlings were treated with and without ethephon 3 d prior to each harvest. Foliage was analyzed for total and specific chitinase activity. Both total and specific chitinase activity differed (P < 0.01) among cultivars and seedling stages. Highest ranking cultivars expressed at least 16% more total chitinase activity and 18% more specific activity than the lowest ranking cultivars. Though chitinase activity changed drastically over seedling development, there were no cultivar x seedling stage interactions (P < 0.01) for total or specific activity. Ethephon increased total and specific activity only at the 0.06 and 0.07 probability level and was far less effective than biological elicitors used to increase chitinase in previous studies. We concluded that chitinase could serve as a consistent marker among tall fescue cultivars across seedling stages, but a more effective chemical elicitor would be desirable to increase chitinase activity.
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INTRODUCTION
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MANY NEW TALL FESCUE FORAGE CULTIVARS lack the fungal endophyte, Neotyphodium coenophialum (Morgan-Jones and Gams) Glenn, Bacon, and Hanlin comb. nov., a mutualistic fungal endophyte that increases resistance to plant pathogens. These new Neotyphodium-free cultivars, initially released to prevent outbreaks of tall fescue toxicosis (Hoveland, 1997; Latch, 1993), also lack persistence (Latch, 1997). Their lack of persistence is related in part to susceptibility to diseases caused by nematodes (Kimmons et al., 1990; West et al., 1988), fungi (Latch, 1993; White and Cole, 1985), and viruses (West et al., 1990).
It should be possible to select for new Neotyphodium-free cultivars with improved resistance to these pathogens. In order to select efficiently, the use of marker-assisted techniques could be employed to allow initial screening of seedlings, thereby delaying the use of extensive field tests that are time consuming, environmentally influenced, and expensive.
Recent reports suggest that initial screening for disease resistance might be possible with a marker such as chitinase, an antifungal hydrolase present in a wide range of plant species (Neuhaus, 1999). In many crops, chitinase activity increases in response to pathogen infection (Neuhaus, 1999; Vogeli et al., 1988), indicating its importance in disease defense responses (Collinge et al., 1993). One example of this involves early research with transgenic tobacco (Nicotiana tabacum L.) and intraspecific tobacco hybrids (Nicotiana spp.), which showed that plants expressing elevated levels of chitinase activity exhibited increased resistance to fungal diseases (Broglie et al., 1991; Goy et al., 1992).
Chitinase appears to be a reliable marker for screening tall fescue for resistance to pathogenic disease as well. Recent literature indicates that it is expressed in tall fescue and that its specific activity increases in response to infection with the nematode Meloidogyne marylandi Jepson and Golden (Roberts et al., 1992). Also, chitinase in tall fescue is higher in `Neotyphodium-infected Kentucky 31', a cultivar known to be persistent, than in the less persistent `Johnstone'. Should screening be based on chitinase activity, a rapid procedure is already in place for screening large populations (Roberts et al., 1994).
Before chitinase can be used as a general marker for disease resistance, several questions need to be addressed, some of which are agronomic. The first question deals with possible effects of physiological development on chitinase activity. In a previous report (Roberts et al., 1992), chitinase activity in Neotyphodium-infected Kentucky 31 tall fescue fluctuated through seedling development as seedlings grew from 10 to 50 d. If this fluctuation occurs differently for each cultivar, a cultivar x seedling stage interaction would prevent the use of chitinase for a portion of the seedling stage.
A second agronomic question deals with possible chitinase responses to alternate elicitors. Chitinase in seedlings has increased six-fold in response to M. marylandi (Roberts et al., 1992). Although such an increase was beneficial in expediting detection and elevating pathogenesis-related isoforms of chitinase, it required the use of nematodes. This in turn required the tedious maintenance of populations in stock cultures as well as monitoring survival in hot greenhouses. If chitinase activity can be increased via a chemical elicitor, the screening process would be greatly simplified.
The objectives of our study, therefore, were to determine if the chitinase activity in tall fescue cultivars differs consistently across seedling stage and to determine if chitinase activity could be increased with the chemical elicitor, ethephon.
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Materials and methods
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Plant Propagation
Seed of 10 tall fescue cultivars were obtained from MFA, Inc. (Columbia, MO). Cultivars included Neotyphodium-infected and -free Kentucky 31, `Stargrazer', `Fawn', `Mozark', `Johnstone', `Martin', `Phyter', `AU-Triumph', and `Festorina'. Five grams seed of each cultivar were planted into small pots containing 50 g commercial peat-based growing medium (Promix, Premier Horticulture, Dorval, Quebec). Seedlings were grown under natural lighting and greenhouse temperatures between 20 and 28°C.
Seedlings were harvested at 14, 28, or 42 d after germination. There were no repeat harvests on seedlings, and therefore no repeated measurements on experimental units. Three days before each harvest, plants were sprayed with the control, deionized distilled water, or with 1.2 g L-1 ethephon in the formulation Ethrel (Rhône-Poulenc Ag Co., Research Triangle Park, NC). Harvested seedlings were trimmed to remove roots, freeze dried, and ground with a cyclone type grinder to pass a 1-mm sieve. Ground tissue was stored at -20°C until laboratory analysis.
Chitinase Analysis
Total and specific chitinase activity were determined by near infrared reflectance spectroscopy as reported by Roberts et al (1994); instrumentation included a Pacific Scientific 6250 scanning monochromator (Silver Spring, MD) with software developed by Infrasoft International (Port Matilda, PA). Reference values for calibration equations were obtained according to the procedure reported by Molano et al. (1977) with modifications specified below. Spectral calibrations were developed with forward stepwise multiple regression and validated with samples not used in calibration. Calibration and validation coefficients of determination ranged from 0.83 to 0.86 for total activity and specific activity. Means and standard errors of performance for total activity were 15.1 ± 1.4 disintegrations per minute per milligram dry matter (dpm mg-1 DM); for specific activity, they were 89.0 ± 8.1 disintegrations per minute per milligram protein (dpm mg-1 protein).
The modified procedure of Molano et al. (1977) included the following steps. One gram of tissue was extracted in 15 mL of extraction buffer: 0.1 M sodium acetate, pH 5.4, 0.001 M EDTA, 0.02 M sodium meta-bisulfite, 2% (w:v) polyvinyl polypyrrolidone, 2% (w:v) polyvinyl pyrrolidone. Extracts were filtered through one layer of miracloth (Calbiochem, Inc., La Jolla, CA ), and filtrates were centrifuged at 15 000 x g for 15 min; the supernatant was retained, and a 200-µL aliquot was incubated with 50 µL [3H]chitin (1 Ci kg-1 chitin) suspension for 30 min at 21°C. The reaction was stopped with 500 µL 10% (w:v) trichloroacetic acid. The solution was centrifuged at 11 300 x g for 5 min. Except for incubation at 21°C, all other steps were performed at 4°C.
To quantify activity, a 400-µL aliquot of the supernatant was mixed with 4.5 mL of scintillation cocktail, and solubilized label was quantified with a Beckman LS2000 scintillation counter (Beckman Instruments, Inc., Fullerton, CA). Total chitinase activity was expressed as disintegrations per minute per milligram dry matter (dpm mg-1 DM). Specific activity was expressed as disintegrations per minute per milligram protein (dpm mg-1 protein); crude protein was based on micro-Kjeldahl nitrogen x 6.25.
Experimental Design and Statistical Analysis
Treatments were arranged in a factorial combination of cultivar, seedling stage, and chemical elicitor. The experimental design was a randomized complete block with three replications. All treatments were considered as fixed effects. The experiment was repeated in another trial, and analyses of variance indicated no statistical difference (P < 0.05) between the two trials for either main effects or interactions. Data were pooled, and significance of treatment main effects and interactions was assessed by standard analysis of variance techniques (Steel and Torrie, 1980). Significant treatment means were separated by the least significant difference test (Steel and Torrie, 1980).
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Results and discussion
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Cultivars differed significantly (P < 0.01) in both total chitinase and specific chitinase activity (Tables 1 and 2)
. Both Kentucky 31 cultivars ranked among the highest cultivars in chitinase activity, and there was no difference (P > 0.05) due to the presence or absence of the endophyte, N. coenophialum. The Kentucky 31 cultivars contained at least 16% more total chitinase and 18% more specific chitinase than the lowest ranking cultivars. The high chitinase content of Kentucky 31 reported in this study is verified by a previous study (Roberts et al., 1992).
In addition to Kentucky 31, one other cultivar that ranked consistently high was Stargrazer. The technical report issued with the release of Stargrazer stated that it demonstrated a highly significant resistance to stem rust (Puccinia spp.) (FFR Cooperative, 1987). Such a response may be expected from tall fescue populations with high concentrations of defense proteins. Our own observations during previous greenhouse experiments noted a lack of Rhizoctonia solani Kühn growing on Stargrazer as it contaminated other potted plants (unpublished data). Future experiments need to be conducted before a relationship between chitinase activity and Rhizoctonia or Puccinia resistance can be confirmed.
Stage of seedling growth also affected (P < 0.01) chitinase activity in tall fescue (Table 3)
, and there were no cultivar x seedling stage interactions for either total or specific activity. Total activity increased almost 10% from 14 to 28 d, then decreased 28% by the time seedlings had grown 42 d. Specific activity decreased steadily as seedlings grew from 14 to 42 d. For both parameters, all stages differed (P < 0.01) from one another.
The fluctuation in chitinase activity can be explained in light of the multiple developmentally regulated isoforms comprising total activity. Multiple isoforms were first reported in Kentucky 31 by Marek et al. (1993). Their presence, as well as their fluctuating concentrations, are important to recognize if chitinase activity is to be used as a marker for broad disease resistance. In most plants, and probably in tall fescue, most of these isoforms are related to physiological development, not disease resistance (Marek et al., 1993). Because these isoforms fluctuate, selection with a chitinase marker would require knowing when developmentally regulated isoforms were in low concentrations and the true pathogenesis-related proteins (Datta and Muthukrishnan, 1999) were in high concentrations.
The lack of a cultivar x seedling stage interaction for chitinase activity indicates that developmental fluctuation of chitinase is not unique to Kentucky 31 tall fescue. It also appears that the fluctuation occurs consistently among commercial cultivars for both parameters. This consistency among cultivars permits screening at a single stage of seedling development.
Regarding the use of a chemical elicitor, ethephon did not affect total or specific chitinase activity when tested at the 0.05 level of probability. However, the ethephon effect approached significance on total activity (P < 0.06) and specific activity (P < 0.07). In addition, it only increased chitinase activity 4% (Table 4)
. Ethepon's effect on tall fescue chitinase was far less than the six-fold increase that resulted when M. marylandi was used as an elicitor. The marginal effectiveness of ethephon indicates that it is an ineffective and unreliable elicitor of chitinase in tall fescue.
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Conclusions
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We concluded that chitinase could be a consistent marker to screening tall fescue seedlings for chitinase activity, and that it could be used through the first 42 d of seedling development without cultivar x seedling stage interactions. Further studies may verify the reliable use of chitinase beyond 42 d after germination. Because total chitinase activity of early seedlings is comprised of both developmentally regulated as well as pathogenesis-related isoforms, a chitinase marker would better serve as a marker for disease resistance toward the end of the 42-d period. We also concluded that ethephon did not increase chitinase activity at the effectiveness needed to serve as an dependable elicitor.Cooperative 1987
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ACKNOWLEDGMENTS
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We are grateful to Mr. Pat McCartney of MFA, Inc. for providing tall fescue seed used in this study. We are also grateful to Ms. Sena McGinn for her technical assistance in the greenhouse and laboratory.
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NOTES
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Mention of a specific product or trade name does not imply preferential endorsement. Contribution from the Univ. of Missouri Agric. Exp. Stn., Columbia, MO 65211. This research was a joint contribution from the Univ. of Missouri Agric. Exp. Stn., paper no. 12,950 and the USDA-ARS Dale Bumpers Family Farm Research Center under specific cooperative agreement no. 6227-31230-004-I5S.
Received for publication July 19, 1999.
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ABSTRACT
INTRODUCTION
