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a Department of Plant Pathology, University of Wisconsin, Madison, WI 53706
b USDA-ARS, U.S. Dairy Forage Research Center, Madison, WI 53706
* Corresponding author (jung{at}plantpath.wisc.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: VCG, vegetative compatibility group
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dollar spot is a major disease of turfgrass throughout the world. This disease is the most prevalent and economically important turf disease in North America, particularly on intensively managed golf course putting greens and closely mown fairways (Couch, 1995; Vargas, 1994). Symptoms occur from spring through fall, but are most prevalent during warm and humid days with cool nights in spring, early summer, and fall. The optimum temperature for disease development is 21 to 27°C, though the fungus will grow over a wider range of temperature, 21 to 32°C. On bentgrass turf maintained at low mowing heights such as golf greens or fairways, symptoms of dollar spot appear as round or irregularly shaped sunken, straw colored patches approximately 3 cm in diameter or about the same size as a silver dollar (Walsh et al., 1999). When disease pressure is high, isolated spots coalesce to form large, irregular patches. Since this fungus is not known to produce conidia or a sexual stage in North America, the organism most likely spreads via mycelia or transport of colonized leaf tissue by wind, water, machinery such as mowers, or by human traffic (Baldwin and Newell, 1992).
Dollar spot severity can be reduced by management practices such as blending seeds of resistant and susceptible cultivars (Abernathy et al., 2001). Cultural practices include removal of foliar dew by mowing or poling (Williams et al., 1996), and applications of late spring N fertilizer or multiple applications of composts (Boulter et al., 2002). Even though cultural practices reduce disease severity, dollar spot management is still highly dependent on fungicide applications. However, isolates of S. homoeocarpa have developed resistance to benzimidazole, dicarboximide, and demethylation inhibitor fungicides (Cole et al., 1968; Warren et al., 1974; Detweiler et al., 1983; Golembiewski et al., 1995). Moreover, some fungicides have not been reregistered due to environmental concerns. These factors have further stimulated research into alternative disease management strategies such as host resistance.
Development of creeping bentgrass cultivars that exhibit resistance to S. homoeocarpa would greatly reduce the costs and environmental impacts of fungicide applications. Although host resistance to S. homoeocarpa is reported (Bonos et al., 2003), the genetic mechanisms of dollar spot resistance are poorly understood. A thorough understanding of both pathogen and host biology is required to develop resistant cultivars for deployment. Knowledge of the genetic variability of the pathogen is needed to predict genetic shifts affecting pathogenicity, which affects the durability of host resistance.
Information about the genetic composition of S. homoeocarpa populations can be derived from the study of vegetative compatibility groups (VCGs). Sonoda (1988) identified 54 VCGs among 119 dollar spot isolates from Paspalum notatum Flueggé in Florida, while Bernick (2002) detected seven different VCGs using 1297 isolates collected from different locations in the midwestern U.S. and the Netherlands. A high degree of genetic similarity (
90%) was found among the isolates (Bernick, 2002), similar to previous studies by Raina et al. (1997) and Hsiang and Mahaku (1999). The authors hypothesized that due to a lack of sexual reproduction, isolates of S. homoeocarpa were genetically similar and did not form isolated populations representing particular geographical locations. Previous studies have shown that there is more variation among the VCGs compared to variation within a VCG (Bernick, 2002). But there has not been any study on the aggressiveness of isolates representing different VCGs and susceptibility of bentgrass cultivars and species.
Bentgrass cultivar field evaluations by the National Turfgrass Evaluation Program (NTEP) (www.ntep.org/data/bt03g/bt03g_05-2/bt03g05t19.txt; verified 19 Jan. 2005) have demonstrated significant differences among cultivars in their susceptibility to S. homoeocarpa. Moreover, field studies (Bonos et al., 2003) using multiple dollar spot isolates have shown that resistance to S. homoeocarpa is a quantitatively inherited trait. It is unknown if these results were due to major resistance genes specific to each isolate, or due to a number of genes indicating that resistance to dollar spot is a quantitative trait.
Therefore, studying host resistance and race specificity interactions using isolates representing different VCGs will provide valuable information about the nature of resistance. This information will help the breeders to make decisions about sources of dollar spot resistance through germplasm screening and developing breeding lines for improved dollar spot resistance.
The objectives of this study were to define the genetic relatedness of 23 isolates of S. homoeocarpa representing 11 VCGs, evaluate susceptibility of 79 clones representing 10 bentgrass cultivars by greenhouse inoculations with one isolate of S. homoeocarpa, followed by greenhouse inoculations of 12 bentgrass cultivars with 18 isolates of S. homoeocarpa representing 10 VCGs, and examine the aggressiveness of the isolates and the presence of race specific interactions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirty Operon RAPD primers were arbitrarily chosen (B11, 17, C11, 19, D2, 3, 16, 20, E3, 6, 14, F13, 14, G7, 10, 11, H4, 13, 14, K2, 8, 9, 12, P8, T14, Y5, 9, 13, 14, 15; Operon Technologies Inc., Alameda, CA) and were utilized for the RAPD reactions. The reactions were performed in 96 well plates with a total volume of 10 µL using an MJ PTC-100 thermocycler (MJ Research, Watertown, MA) programmed to the same conditions as described by Scheef et al. (2003). Thermal cycling conditions of 91°C for denaturation, 42°C for annealing, and 72°C for elongation were used for the first cycle followed by 39 cycles with first cycling time of 60 s for denaturation, 15 s for annealing, and 70 s for elongation. Subsequent cycles were the same except for a 15-s denaturation period. PCR products were separated by a 1.5% (w/v) agarose gel for 2 h at 300 V, stained in ethidium bromide (1.5 µg mL–1) for 30 min and subsequently destained in distilled water for 20 to 30 min. Only bright and reproducible bands were scored manually for the presence or absence of bands as 1 and 0, respectively. One data matrix was generated by pooling categorical data from thirty primers and used for genetic relatedness analysis.
S. homoeocarpa Inoculum Preparation and Greenhouse Inoculations
Of the 23 isolates in Table 1, only 18 isolates representing 10 VCGs were selected by considering several factors such as VCGs, genetic relatedness, and the growth ability of mycelium. These isolates were grown on PDA for 1 wk at 21°C under constant fluorescent light. Inoculum was prepared by twice autoclaving 15 g of oat (Avena sativa L.) seeds with 20 mL Difco potato dextrose broth (PDB) in 125 mL Pyrex flasks. Four 4-mm-diameter culture plugs were excised from the growing edge of each fungal colony and transferred to the oat seed medium and allowed to grow for 3 wk at room temperature, with 12 h of light and daily shaking to prevent clumping of the seeds.
Seventy-nine clones of 10 cultivars of the creeping, colonial, velvet, and dryland bentgrass species were grown from individual seeds for the 2001 inoculation experiments in May and September. Three replications were used for each experiment, and different vegetative replicates of the same clones were used in each experiment, maintained in the greenhouse and established in 6.5-cm-diameter pots using identical protocols for each experiment. Two inoculation experiments were conducted in 2003 using 12 seeded bentgrass cultivars in factorial combinations with 18 isolates of S. homoeocarpa under similar conditions of temperature, humidity, and inoculum dose in September and December. The plants were started from 0.05 g of seeds evenly spread on soilless potting media (Metro Mix 366-P, the Scotts Company, Marysville, OH) on each 6.5-cm-diameter pot. Turf was trimmed twice every month to a height of 1 to 1.5 cm for 4 mo. There were four replications of each of the cultivars, and plants were inoculated when they completely filled the pots and mimicked a short-trimmed green or tee.
For all four experiments in 2001 and 2003, plants were inoculated by placing four infested oat seeds on the surface of the turf canopy in the center of each pot. This number of seeds was chosen based on previous inoculation data because the number gave most reliable disease under infection and colonization conditions used in the study. In these previous inoculations (N. Chakraborty, unpublished data, 2003), it was noted that changing the number of seeds by one, such as from two to three seeds or three to four seeds, did not lead to a significant difference in dollar spot severity. Therefore the amount of mycelia on each seed was estimated visually and seeds with equal amount of mycelia were used as inoculum.
After inoculation, the pots were arranged in a randomized complete block design and then placed in a mist chamber for 3 d with humidity >90% and temperature ranging from 22 to 27°C which is optimal for maximum pathogenicity (Walsh et al., 1999). The duration of daylight varied according to the season during the four experiments due to lack of artificial lights in the mist chamber. For the inoculation experiment in May, the average daylength was 14 h, for the September experiment it was 12 h, whereas for the December inoculation, it was approximately 9 to 10 h. After 3 d, the pots were moved from the mist chamber to the greenhouse. Two days later, the infested seeds were removed and disease symptom severity was scored as a percentage of area of symptomatic, whitish colored turf in each pot. To increase accuracy and precision of estimating disease severity, transparencies with grids of 1 cm2 (Apollo, Ronkonkoma, NY) were used to trace the area of symptomatic bleached tissue area in each pot and then converted into percentage.
Statistical Analysis
RAPD data pooled from 30 primers were analyzed using the NTSYS software package (Rohlf, 1993). The genetic similarity matrix was constructed based on the simple matching coefficient method (Sneath and Sokal, 1973). The unweighted pair group method with arithmetic averages (UPGMA) was then used to analyze the matrix for hierarchical clustering. Analysis of Molecular Variance (AMOVA, Arlequin version 2.000) was used to estimate the variance among the isolates within a VCG and the variance between VCGs (Schneider et al., 2000).
Each pair of pathogenicity experiments (2001 and 2003) was analyzed by ANOVA, combined over experiments and analyzed separately for each experiment. In both cases, the combined ANOVA detected significant clone or cultivar x experiment interaction, so only the separate analyses were used in reporting results. For the 2001 experiments, the source of variation for bentgrass species was partitioned into three orthogonal contrasts: velvet bentgrass vs. others (diploid genome A1A1 vs. tetraploid genomes A2A2 A3A3 and A1A1 A2A2), creeping bentgrass vs. colonial and dryland bentgrasses (different tetraploid genomes A2A2 A3A3 vs. A1A1 A2A2), and colonial vs. dryland bentgrass (similar tetraploid genomes A1A1 A2A2 vs. A1A1 A2A2). Cultivars within species, and clones within cultivars comprised the remainder of the variability among the 79 clones.
For the 2003 experiments, the source of variation for bentgrass species was partitioned into two orthogonal contrasts: velvet bentgrass vs. creeping and colonial bentgrasses, and creeping vs. colonial bentgrass. The method of contrasts was also used to partition the interactions of the two bentgrass species contrasts with S. homoeocarpa VCG and isolates within VCG. All effects were assumed to be fixed, except for replicates, clones (in the 2001 experiments), and experiments (for the combined analyses), which were assumed to be random (SAS Institute, 1999). Contrasts were computed according to the procedures of Steel et al. (1996). Mixed models analysis was used to compute standard errors of least squares treatment means.
Normality of the residuals was tested by the Shapiro–Wilk test and heterogeneity of residual variances was determined by visually inspecting plots of residuals vs. predicted values (SAS Institute, 1999). For both the 2001 and 2003 experiments, there was some evidence for non-normality, largely by a slightly elevated frequency of extreme observations. However, the linearity of normal probability plots and the general absence of variance heterogeneity suggested that data transformation was not necessary.
Field data on dollar spot disease ratings of seven bentgrass cultivars were obtained from the NTEP web site (NTEP, 2001, 2003). The data consist of means over 10 locations, representing a wide range of climatic regions, and were based on 1 to 3 yr of data at each location under natural inoculum loads. Ratings were made on a 1-to-9 scale, where 1 = completely diseased and 9 = no disease. Rank correlation coefficients and Kendall's coefficient of concordance were used to compare average disease rankings for the greenhouse studies with the NTEP field data (Conover, 1971; Steel et al., 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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84% similar to each other but they digressed along with isolate A7 from the rest and shared only 48% similarity with all the other isolates. The other 19 isolates shared 62% similarity between them.
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Susceptibility of Bentgrass Cultivars, Aggressiveness, and Race Specificity of S. homoeocarpa Isolates
In the first pathogenicity experiment of 2001, a significant cultivar effect (P < 0.0062) was detected (Table 2). In addition, a significant clone effect (P < 0.0002) was found within cultivars. These clone and cultivar effects were noted in the first experiment, but both sources of variation were of marginal significance (P = 0.06–0.07) in the second experiment. Nevertheless, the rank order of cultivars remained similar with a rank correlation coefficient of r = 0.73 (P < 0.05). More importantly, differences among species were also consistent between the two experiments. In both 2001 experiments, the colonial bentgrass cultivars (mean percentage of disease 11.4 ± 1.0 in Experiment 1; 26.3 ± 1.7 in Experiment 2) were significantly less susceptible to S. homoeocarpa isolate MN1 than the creeping bentgrass cultivars (23.0 ± 1.3 in Experiment 1; 42.0 ± 2.3 in Experiment 2) (Table 3).
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98% of the variance among species in both 2001 experiments (Table 2). The velvet bentgrass cultivar, which is diploid with the A1 genome, was not significantly different in susceptibility from the other tetraploid bentgrass cultivars with the A1 and A2 genomes. In both experiments of 2003 involving susceptibility of 12 commercial cultivars from three species of bentgrass to 18 isolates of S. homoeocarpa from 10 different VCGs (Table 4), cultivar and isolate main effects explained most of the variability in disease reaction. A significant cultivar effect (P < 0.0001) was detected (Table 4) and it explained 49 and 76% of the variance among bentgrass cultivars in the first and second experiments, respectively. The remainder of the variance among cultivars was explained by the difference between the creeping and colonial bentgrass cultivars (52 and 18% in the first and second experiments, respectively, Table 4). Disease severity did not differ between creeping and colonial bentgrass cultivars compared to the velvet bentgrass cultivars (Table 4). Colonial and velvet bentgrass cultivars were generally less susceptible than creeping bentgrass cultivars (Table 5). Cultivar x isolate interactions were generally not significant (Table 4).
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= 0.62 (P = 0.19) across all four experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found a similarity of 48% between the 23 isolates collected from golf courses in Arkansas, Illinois, Florida, Michigan, and Minnesota. This result is similar to Hsiang and Mahaku (1999) in which they found 66% similarity among isolates from Canada, but unlike those of previous studies (Raina et al., 1997) which have shown 80% genetic similarity among isolates from the USA. The lower level of genetic similarity in our study might be due to the small sample size of 23 isolates in comparison to the other studies. The genetic relatedness study was the first step to understand the similarity among the different isolates but additional isolates representing each VCG are required to further clarify our findings. The outcome of this relatedness study was sufficient to show the genetic dissimilarity among the isolates, so that we can choose different isolates representing multiple VCGs and significant genetic diversity for studying host resistance in bentgrass cultivars.
Previous studies (Hsiang and Cook, 1992), including NTEP, have performed field disease evaluation experiments with different bentgrass species relying on natural inoculum. Results from NTEP have shown significant difference among the commercial bentgrass cultivars in their response to dollar spot, but so far there have been no published data on the performance of the commercial cultivars based on individual isolates of the pathogen under greenhouse conditions.
An observed cultivar effect in the first pathogenicity experiment of 2001 indicated that the bentgrass cultivars were significantly different from each other in susceptibility. A significant cultivar effect in the pathogenicity experiments of 2003 using 18 isolates of S. homoeocarpa was similar to the findings of the 2001 experiments (Table 2). The significant clone effect within each cultivar in these experiments (Table 2) indicated that clones within individual cultivars showed significantly different disease responses. This was a reasonable outcome since commercial cultivars are all synthetic varieties and individual clones are genetically different from each other.
A possible explanation for finding significant differences in susceptibility among clones within cultivar as well as cultivars within species in only the first experiment and not in the second of 2001 was due to a higher MS error despite the similar inoculation procedures used (Table 2). Possible causes are differences in the age of the clones inoculated and in temperature during an incubation period between the two experiments. The second experiment included older plants which were maintained for a longer period of time (approximately 8 mo) and in general, had more disease compared to the first one (4 mo). This might be due to more senescent tissue, thatch built up and nutrient stress in the plants used in the second experiment. The temperature inside the growth chamber was influenced by outside temperature. Nonetheless, both the 2001 experiments were still similar in respect to the rank order (r = 0.73) of the cultivars and the species. These results also support the idea that dollar spot resistance is affected by different environmental conditions as reported by other researchers (Vincelli et al., 1997).
We detected a significant difference in percentage of disease between creeping vs. colonial and dryland bentgrass cultivars in both 2001 experiments, and this difference explained 98% of the variance among species. Creeping bentgrass is allotetraploid with A2 and A3 genomes whereas colonial and dryland bentgrass are also allotetraploid with common genomes A1 and A2 (Jones, 1956). Thus, colonial and dryland bentgrasses are more closely related to each other than to creeping bentgrass. The fact that differences in disease response were not significant between colonial vs. dryland bentgrass cultivars provides further support of this. Velvet bentgrass with A1 genome, which is diploid, was not significantly different in susceptibility from all the other tetraploid bentgrass species. This might be because velvet bentgrass has a genomic affinity to the colonial bentgrass in being less susceptible to dollar spot than the creeping bentgrass. It is possible that the A1 genome common in the velvet and colonial bentgrass species is actually conferring the dollar spot resistance but further experiments are needed to validate this hypothesis. Similarly, in the two 2003 experiments, the colonial and velvet bentgrass cultivars were more resistant to all the isolates of S. homoeocarpa compared to the creeping bentgrass cultivars.
One of the criteria used for selecting the 18 fungal isolates (Table 6) for the pathogenicity tests in the 2003 experiments was their genetic difference compared to the other isolates based on RAPD derived genetic similarity (Fig. 1). Isolates with significant genetic diversity will help to mimic the array of natural inocula, thereby providing a better understanding of the pathogenicity and host resistance which will facilitate bentgrass breeding for dollar spot resistance.
There was a significant isolate effect in both 2003 experiments, which shows that the isolates differ in their aggressiveness irrespective of cultivars and species. But the interactions between bentgrass cultivars and species with isolates were not significant; therefore differences in susceptibility of bentgrass cultivars to S. homoeocarpa appear to be race nonspecific although significant difference in aggressiveness was observed among isolates. This is very important information for turfgrass breeders, because it suggests that turfgrass breeders should be able to select for resistance to one or a few virulent isolates of the pathogen, and obtain resistance to a wide array of isolates irrespective of different VCGs and genetic diversity.
Moreover, the only exception in the ranking of dollar spot resistance of bentgrass cultivars from greenhouse tests was in the second experiment in 2003 (Table 7). This was due to compression of cultivar differences in the second experiment, which had higher disease pressure. Excluding Experiment 2 of 2003, increased the estimate of Kendall's coefficient of concordance to = 0.81 (P = 0.10). These results suggest that there is, generally, a positive rank correlation between field and greenhouse rankings for resistance to this pathogen.
Greenhouse assays used in our current study were different than field assays in several respects, particularly in the nature and amount of inoculum, plant age, and environmental factors. In our greenhouse study, we only noted the disease severity, whereas field ratings were based on both disease severity and the number of lesions. This limitation to greenhouse studies reduces the potential range of disease severity that can be observed among cultivars. Modification of certain variables, including plant age, temperature, and light duration will improve the reproducibility and the sensitivity of our greenhouse experiments. As with field studies, greenhouse evaluations must be repeated in time, allowing assessment of host symptoms across potentially different environmental conditions and disease severity levels.
Even with these limitations, data from pathogenicity tests have demonstrated significant differences among bentgrass clones, cultivars, and species in their response to the dollar spot pathogen, significant differences in levels of aggressiveness among S. homoeocarpa isolates and finally, a lack of race-specific interactions. Race nonspecific resistance is much more likely to be durable across a wide array of environments in which bentgrass is grown. Therefore, our findings assure turfgrass breeders that new bentgrass cultivars with improved dollar spot resistance will have broad utility for the golf course industry, irrespective of geographic variation in S. homoeocarpa isolates. The genetic transfer of resistance from colonial bentgrass to creeping bentgrass should be a relatively simple process, because these species readily hybridize with each other (Belanger et al., 2004). However, because hybrids between velvet bentgrass and creeping bentgrass are sterile, it will be more difficult to transfer resistance from the diploid velvet bentgrass to the tetraploid creeping bentgrass (Brilman, 2003).
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ACKNOWLEDGMENTS |
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Received for publication April 25, 2005.
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