© 2006 Plant Management Network.
Salinity and Salinity Tolerance Alter Rapid Blight in Kentucky Bluegrass, Perennial Ryegrass, and Slender Creeping Red Fescue
James J. Camberato, Agronomy Department, Purdue University, West Lafayette, IN 47907; Paul D. Peterson, and S. Bruce Martin, Entomology, Soils, and Plant Sciences Department, Pee Dee Research and Education Center, Clemson University, Florence, SC 29506
Camberato, J. J, Peterson, P. D., and Martin, S. B. 2006. Salinity and salinity tolerance alter rapid blight in Kentucky bluegrass, perennial ryegrass, and slender creeping red fescue. Online. Applied Turfgrass Science doi:10.1094/ATS-2006-0213-01-RS.
Rapid blight, caused by Labyrinthula terrestris (D. W. Bigelow, M. W. Olsen, and Gilb.), occurs on cool-season grasses in arid and coastal regions of the USA with elevated irrigation salinity. The impacts of irrigation salinity and cultivar salinity tolerance on rapid blight were examined in greenhouse experiments. Four cultivars each of Kentucky bluegrass (Poa pratensis L.), perennial ryegrass (Lolium perenne L.), and slender creeping red fescue (Festuca rubra spp. littoralis (G. F. W. Meyer) Auquier), known to differ in rapid blight susceptibility, were irrigated daily with solutions of 0.2, 1.3, 2.5, 3.6, and 4.8 dS/m, with or without inoculation with L. terrestris. Treatment effects were assessed by rating chlorotic leaf tissue and calculating area under the leaf chlorosis curve (AULCC). Rapid blight had little effect on AULCC of any grass at ≤ 1.3 dS/m. Slender creeping red fescues showed little AULCC due to rapid blight even at ≥ 2.5 dS/m. Perennial ryegrasses and Kentucky bluegrasses had substantial rapid blight-induced AULCC at ≥ 2.5 dS/m. Leaf chlorosis due to rapid blight was less and slower to develop in ‘North Star’ Kentucky bluegrass and ‘Hawkeye’ and ‘Peregrine’ perennial ryegrasses than in other cultivars of these species. Leaf chlorosis due to salinity of 4.8 dS/m without inoculation correlated well with leaf chlorosis due to rapid blight at 2.5 dS/m, suggesting mechanisms imparting salinity and rapid blight tolerance may be similar. Irrigation water management practices reducing rootzone salinity and selection of tolerant species and cultivars are recommended to lessen rapid blight.
Rapid blight of cool-season grasses has been recognized since 1995 and documented on over 100 golf courses in at least 11 states in the USA (17,18) and in the United Kingdom (6). Areas of occurrence in the USA are the west, southwest, and coastal south and southeast. Irrigation water electric conductivities from 8 golf courses in Arizona, California, Texas, and Utah with rapid blight on annual bluegrass (Poa annua L.) from which L. terrestris isolates were obtained ranged from 0.5 to 3.5 dS/m, averaging 1.4 dS/m (Peterson, Martin, and Camberato, unpublished data). Rapid blight occurrences in coastal South Carolina and North Carolina occurred during successive years of below normal rainfall. Irrigation water salinities on 8 golf courses in this region with confirmed rapid blight on rough bluegrass (Poa trivialis L.) ranged from 1.0 to 6.0 dS/m with a mean of 3.1 dS/m (Camberato and Martin, unpublished data). Annual and rough bluegrass are considered very sensitive to salinity with decreases in top growth at irrigation water salinities >1.0 dS/m (3).
The causal agent of rapid blight is Labyrinthula terrestris, a newly recognized species in the genus Labyrinthula (2,4). Other species of Labyrinthula have been associated only with marine environments (20). High salinity levels may provide a better environment for the pathogen and may predispose the host plant to infection so that disease develops. In one study increased salinity from 0.5 to 8.0 dS/m increased rapid blight in ‘Brightstar SLT’ perennial ryegrass (13).
Previous research (19) found rapid blight tolerance among cool season grass species to be generally related to their reported salinity tolerance; however no direct comparisons of rapid blight and salinity tolerance were made in this research. Species considered moderately tolerant to tolerant of salinity, including alkaligrass (Puccinellia distans (Jacq.) Parl.) (1,8,9,11,14), creeping bentgrass (Agrostis stolonifera L.) (14,15,24), and the fescues (Festuca spp.) (12,14) were moderately to highly tolerant of rapid blight. Species very sensitive to salinity, such as colonial (Agrostis capillaris L.) (7,14,15) and velvet (Agrostis canina L.) (15) bentgrasses and annual and rough bluegrasses (3) were also susceptible to rapid blight. However, rapid blight tolerance was not always related to reported salt tolerance. For five creeping bentgrass cultivars there was only a weak correlation between our assessment of rapid blight tolerance (19) and an independent assessment of salinity tolerance (15). Several perennial ryegrasses were susceptible to rapid blight, although as a group they are rated as moderate to highly tolerant of salinity (3). Substantial variation in rapid blight tolerance also occurred among cultivars of Kentucky bluegrass (19). Kentucky bluegrass has shown a wide range in salinity tolerance as well (7,10,22,23).
The objectives of this study were to (i) define the relationship between salinity and occurrence of rapid blight in susceptible cool-season grasses and (ii) examine the relationship between rapid blight tolerance and salinity tolerance among and within three grass species.
Evaluating Salinity and Rapid Blight Tolerance
Duplicate runs (4 replicates per run) of an experiment were conducted in a greenhouse environment to examine the effects of salinity on rapid blight disease of perennial ryegrass (PRG), slender creeping red fescue (SCRF), and Kentucky bluegrass (KB). Treatments consisted of 4 cultivars of each grass species at 5 levels of salinity with or without inoculation with L. terrestris.
Cultivars (Table 1) of PRG, SCRF, and KB were selected based on a wide range in susceptibility to rapid blight from previous research (ref. 19; all cultivars were previously examined except the SCRF cultivar Barcrown II). Grasses were grown in plastic pots containing 275 g of a 80:20 (v:v) sand:peat mix amended with ground triple super phosphate at 0.1 g/kg soil and dolomitic limestone at 0.25 g/kg soil. Seeding rates were 178,000, 200,000, and 306,000 seeds per m2 for PRG, SCRF, and KB, respectively. The grasses were germinated and established with tap water applied 1 to 4 times daily (more frequent at seeding and decreasing with increased establishment). Plants were clipped weekly prior to inoculation (not thereafter) to maintain heights of 5, 7.5, and 2.5 cm for PRG, SCRF, and KB, respectively. Greenhouse average daily minimum, maximum, and mean temperatures were 13.9, 29.2, and 18.4°C for run 1 and 12.3, 27.2, and 17.1°C for run 2. Nutrients were applied weekly in 15 ml solution to each pot from 14 to 70 days after seeding (DAS) in run 1 and from 11 to 71 DAS in run 2. Nutrients applied to both runs were N, P, and K at 13.6, 5.9, and 11.2 mg per kg of soil, respectively, and S, Mg, B, Cu, Fe, Mn, Zn, and Mo at 476, 340, 14, 34, 68, 34, 34, and 0.34 µg per kg of soil. Although no symptoms of S deficiency were evident, the low S content of the fertilizer and that of the lowest salinity treatment prompted the addition of more S to the weekly fertilization in run 2. Additional S was added as MgSO4, providing S at 1.2 mg/kg and Mg at 1.1 mg/kg of soil.
Table 1. Cultivars of perennial ryegrass, Kentucky bluegrass, and slender creeping red fescue examined for salinity tolerance and susceptibility to rapid blight disease.
Fungicides were applied to protect against infection of the grasses with Pythium spp.; azoxystrobin (Heritage) at 30 mg a.i./m2 at 15, 44, and 57 DAS in run 1 and 9, 17, 31, and 52 DAS in run 2. Mefanoxam (Subdue Maxx) was also applied in run 2 at 0.8 mg a.i./m2 at 14 DAS for additional control. Neither fungicide controls rapid blight (16).
Irrigation Salinity Treatments
Salinity treatments were initiated 24 DAS (run 1) and 22 DAS (run 2). Irrigation volume was 50 ml per pot per day through 59 DAS (run 1) and 51 DAS (run 2). Seventy ml per pot was applied thereafter as the older and larger plants used more water. Irrigation water salt content reflected the composition of coastal South Carolina water supplies; 46.6% Na, 34.8% Cl, 14.9% HCO3, 1.3% Ca, 1.1% K, 0.8% Mg, 0.2% SO4-S, and 0.2% B (expressed as the percentage of ions in meq/liter). Total soluble salts were 78, 784, 1,569, 2,353, and 3,137 mg/liter with measured electrical conductivity (ECW) of 0.2, 1.3, 2.5, 3.6, and 4.8 dS/m. Reagent- and food-grade chemicals and deionized water were utilized to make the salt solutions.
Labyrinthula Production and Inoculum
Bulk inoculum of 5 isolates of L. terrestris was prepared by growing isolates on petri plates containing modified serum seawater agar for approximately 4 days. Growth media contained 1% horse serum (20) with artificial seawater adjusted to 3.5 dS/m (5). Equal numbers of agar plugs from plates of each isolate were transferred into modified serum seawater broth (SSB) and vortexed for 2 min. Inoculum was quantified to 140,000 cells per ml using a hemacytometer and Tween (0.25% v/v) was added to the solution.
All plants were wounded prior to inoculation by trimming with scissors. Inoculum was applied 36 DAS (run 1) and 37 DAS (run 2) arbitrarily to leaves at the rate of 1 ml per pot using a 3-cm3 needle syringe that resulted in the formation of droplets on leaf tissue. A solution containing SSB and Tween (0.25% v/v) was applied at the same rate as the inoculum to an equivalent set of cultivars (non-inoculated controls). Trays with all plants (inoculated and non-inoculated) were placed in a sealed container for 48 h post-inoculation, after which time the lids were removed and the plants continued to be watered daily with saline water treatments as before.
Measurements, Experimental Design, and Statistics
The percentage of chlorotic leaf tissue on a per pot basis was rated visually
3 times per week from 42 to 77 DAS (run 1) and 44 to 78 DAS (run 2). Leaf tissue
that became necrotic was retained in the percent chlorosis rating. Area under
the leaf chlorosis curve (AULCC) was calculated as:
The entire sand:peat root media was frozen after shoot removal for later determination of EC of the saturation paste extract (ECe). After thawing, water was added to the media to form the saturated paste. The paste was stirred again 1 h after formation, and water added if necessary. After another 1 h the solution was separated from the soil by vacuum through Whatman 42 filter paper. The conductivity of the solution was determined with an Orion 160 Conductivity Meter (Orion Research Incorporated, West Germany) and 4-electrode cell (Orion 016010).
The experimental design was a fully randomized split-plot with 4 levels of sub-plot and 4 replications. Salinity was the main-plot factor, inoculation was the first split, turfgrass species was the second split, cultivar within grass species was the third split, and sampling date was the fourth split (when applicable). The entire test was repeated. Analysis of variance, standard error of the mean, and regression were used to assess treatment effects.
Saturated paste EC (ECe) reflected irrigation water ECW indicating minimal salinity accumulation in the sand:peat root media (Table 2). Although grass species and inoculation with L. terrestris influenced root media ECe, effects were generally small compared to differences among salinity treatments. Turf species and inoculation most likely altered EC through effects on plant growth and therefore water use. Treatments producing the largest plants used more water, resulting in the incomplete leaching of salts from the rootzone. We monitored leachate volume weekly and adjusted irrigation volume to compensate for growth effects on water usage, but we may not have compensated fully when water usage was greatest.
Table 2. Saturated paste electrical conductivity of the sand:peat root media as affected by irrigation water of 0.2, 1.3, 2.5, 3.6, and 4.8 dS/m, inoculation with Labyrinthula terrestris, and one cultivar of each turfgrass species -- perennial ryegrass, Kentucky bluegrass, and slender creeping red fescue.
* Main effects of salinity, inoculation, and cultivar were significant at P > F of 0.0001, 0.002, and 0.002, respectively.
† Salinity by inoculation and inoculation by cultivar interactions were significant at P > F of 0.0006 and 0.007, respectively.
‡ Within each main effects treatment in run 1, means followed by the same letter do not differ according to LSD (P < 0.05).
§ Salinity treatment means at the same or different level of inoculation followed by the same letter do not differ according to LSD (P < 0.05),
# Species means within an inoculation treatment followed by the same letter do not differ according to LSD P < 0.05).
** Kentucky bluegrass cultivars SR2284 and Arcadia were examined in run 1 and 2, respectively.
Salinity-induced Leaf Chlorosis
The effects of salinity and inoculation on leaf chlorosis varied dependent on turfgrass species and cultivar, changed with time within each experimental run, and differed in magnitude between experimental runs (interaction terms ‘run by inoculation by cultivar within species by rating date’ and ‘salinity by inoculation by cultivar within species by rating date’ were both significant at P > F of 0.0001). Data are presented for only 2 cultivars of each species, representing the least and most affected by salinity and rapid blight (Figs. 1 to 3). For clarity, time during the experiment is referred to as days after inoculation for inoculated treatments and days after non-inoculation for non-inoculated treatments.
One obvious difference between run 1 and run 2 is leaf chlorosis due to salinity in non-inoculated plants was much more severe in run 1 (Figs. 1 to 3, parts a and e versus c and g). Greater salinity stress in run 1 may have occurred because greenhouse temperatures from the initiation of salinity treatments through 3 week after non-inoculation were greater in run 1 than run 2. Daily maximum and minimum temperatures averaged 30.6 and 14.3°C in run 1 versus 26.7 and 12.4°C in run 2. Supraoptimal temperatures have been shown to increase the effects of salinity on germination and leaf firing of KB cultivars (21,22). Greater water use with higher temperatures may also have allowed greater concentration of salts in the rooting media prior to each irrigation, thus greater osmotic stress and increased leaf chlorosis.
Leaf Chlorosis due to Inoculation with Labyrinthula terrestris
Plants inoculated with L. terrestris exhibited leaf chlorosis symptoms consistent with those in non-inoculated plants, although to a greater magnitude, as well as other symptoms that were absent from non-inoculated plants. Infected tissues unique to inoculated plants had a grey cast, appeared to loose turgidity and collapsed forming a compressed mat of tissue. This initially occurred in green leaf tissue that would eventually become chlorotic, then necrotic.
Tissue chlorosis due to inoculation was increased by increased salinity for all species and cultivars (Figs. 1 to 3; parts b, d, f, and h). Area under the leaf chlorosis curve (AULCC) due to rapid blight (the cumulative difference in leaf tissue chlorosis between inoculated and non-inoculated plants) was calculated for the entire rating period (Fig. 4). Salinity, species, and cultivar interacted to influence rapid blight-induced AULCC (P > F of 0.0008). Little rapid blight-induced AULCC occurred at ECW ≤ 1.3 dS/m for any cultivar of any species (Fig. 4) even though Labyrinthula was cultured from the leaf tissue of inoculated plants at both salinity levels (data not shown). Little AULCC occurred in SCRF even at ECW of 3.6 and 4.8 dS/m, and variation in leaf chlorosis among the 4 SCRF cultivars examined was minimal. All cultivars of PRG and KB (except KBG ‘North Star’ in run 2 at ECW = 2.5 dS/m) had detectable AULCC values due to rapid blight at ECW ≥ 2.5 dS/m. Irrigation water ECW >2.5 dS/m increased AULCC due to rapid blight of some cultivars more than others. The KB ‘North Star’ and PRG ‘Hawkeye’ and ‘Peregrine’ had lower AULCC than other cultivars of the same species. Species and cultivar differences in these experiments were similar to those found in another study (19). The simple correlation between AULCC due to rapid blight at 2.5 dS/m reported in this paper and % disease in an earlier study (19) was 0.74 (excluding Barcrown II which was not examined previously).
The time to reach a value of 800 AULCC due to rapid blight was calculated as an indicator of the rapidity of disease progression (Table 3). Increased salinity decreased the time needed to attain 800 AULCC. Disease progression was slower in ‘North Star’ than the other KB cultivars and in PRG cultivars ‘Hawkeye’ and ‘Peregrine’ in comparison to Brightstar SLT or Penguin. The SCRF cultivars rarely attained 800 AULCC (data not shown).
Table 3. Number of days needed to attain 800 units of area under the leaf chlorosis curve (AULCC) due to rapid blight for each cultivar in each run. Minimal leaf chlorosis occurred for ryegrasses and bluegrasses at ECW < 2.5 dS/m and for fescues at any salinity, therefore data are not shown for these treatments.
* NA = 800 AULCC not attained.
For all but one cultivar, AULCC of non-inoculated plants at 4.8 dS/m minus that at 0.13 dS/m a measure of salinity tolerance, was correlated with AULCC due to rapid blight at 2.5 dS/m, a measure of rapid blight tolerance (Fig. 5). The exception was KB ‘Arcadia,’ which had a greater AULCC due to rapid blight than expected based on AULCC due to salinity. The relationship between salinity and rapid blight tolerance differed in magnitude between run 1 and 2 because salinity was less in run 2 than run 1, however both had high R2. The correlation between salinity tolerance and rapid blight tolerance observed in this study among 12 cultivars of 3 species and earlier research that found rapid blight tolerance among 49 cultivars representing 24 cool-season grass species to be generally related to their reported salinity tolerance (19), suggests that mechanisms of grass tolerance to salinity and to rapid blight may be similar.
Increased irrigation water salinity increased the amount of leaf chlorosis resulting from inoculation with L. terrestris in all cultivars of SCRF, KB, and PRG examined. Perhaps this is not surprising as L. terrestris, the organism causing rapid blight, is a land-based species of a marine slime mold and we have not found the disease in turf irrigated with non-saline water. Thus, management practices that reduce salinity accumulation in the rootzone, such as periodic leaching and blending multiple irrigation sources to reduce salinity, should be helpful in reducing rapid blight.
In our greenhouse studies rapid blight was not evident with irrigation water ≤ 1.3 dS/m, however we have diagnosed rapid blight on golf course greens irrigated with slightly lower salinity. The level of salinity that promotes rapid blight in golf course greens may be lower than that found in our greenhouse studies for a number of reasons. First, golf course greens with rapid blight are typically annual or rough bluegrass. These turfgrass species are more susceptible to rapid blight than KB and PRG, and disease likely occurs at lower salinity levels. Second, rootzone salinity on golf course greens is likely higher than that of the irrigation water, whereas rootzone and irrigation water salinity were nearly identical in our greenhouse pots. Complete leaching of previously applied salts is rarely accomplished on a golf course green, but was relatively easy in greenhouse pots. Third, salinity stress, and perhaps rapid blight, is enhanced by low soil moisture. Most golf course greens have only 10 to 20% moisture at field capacity and are frequently allowed to dry considerably between irrigations. Our greenhouse pots had 31% moisture and were watered daily. Fourth, the additional stress of low mowing height on a golf course green, in contrast to the higher heights used in the greenhouse, may allow disease to develop at lower salinity levels due to enhanced overall plant stress.
Slender creeping red fescues were more tolerant of rapid blight and salinity than the KB and PRG cultivars. ‘North Star’ KB and ‘Hawkeye’ and ‘Peregrine’ PRG are more tolerant than other cultivar of these species. In situations where irrigation water salinity is elevated, selection of species and cultivars tolerant to salinity and rapid blight may be helpful in minimizing turf damage due to these factors. The correlation between salinity and rapid blight tolerance found in this study, in conjunction with earlier research examining 21 additional cool-season grass species, suggest species and cultivars with high levels of salinity tolerance will also have high levels of rapid blight tolerance.
Technical Contribution No. 5121 of the Clemson University Experiment Station. This material is based upon work supported by the CSREES/USDA, under project number SC-1700211.
The authors thank Debbie Cottingham, Sheila Godwin, Cristal Robbins, Amy Turner, and Jessica Winburn for technical assistance. We also acknowledge the United States Golf Association for partial financial support of this project. We also thank Seed Research of Oregon, Turf-Seed, Inc. and Barenbrug for provision of seed for these experiments.
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