Abstract
Breeding and improvement of new bermudagrass (Cynodon spp.) cultivars with superior nematode tolerance are essential because sting nematode (Belonolaimus longicaudatus Rau) is a major limitation for use of bermudagrass in the sandy coastal soils of the southeastern United States. The screening of both African (Cynodon transvaalensis) and common (C. dactylon) bermudagrass is necessary to develop triploid hybrid cultivars. Five commercial cultivars and 46 germplasm accessions of bermudagrass were tested for nematode responses in two greenhouse trials in 2009. Turfgrass was grown in sand-filled plastic conetainers and inoculated with 50 sting nematodes per conetainer. Nematode and root samples were collected 90 d after nematode inoculation. Fifteen bermudagrass accessions did not have measurable root loss from inoculation with sting nematode. Seven bermudagrass accessions, including ‘Celebration’, produced longer roots in sting nematode-infested soil than the standard ‘Tifway’. Differences in final nematode numbers were identified among the genotypes, and different relative responses were identified in variable ploidy levels and origins. This could aid a turfgrass breeding program by elucidating the genetic diversity available for breeding future bermudagrass cultivars for golf course cultivation.
Bermudagrass (Cynodon spp.) is the predominant turfgrass used in the southern United States and other warm regions in the world. A limitation for the use of bermudagrass in the southeastern United States is the sting nematode (Belonolaimus longicaudatus), which is frequently found in sandy coastal soils. It has been considered the most damaging plant–parasitic nematode on bermudagrass in Florida (Crow, 2005; Luc et al., 2007) where it causes damage to greens, fairways, and rough areas on golf courses as well as athletic fields and lawns (Crow and Han, 2005). With the cancellation of fenamiphos (Nemacur; Bayer CropScience, Research Triangle Park, NC), turfgrass managers are in need of new nematode management strategies. Use of resistant or tolerant cultivars would be the most desirable, least costly nematode management practice with the minimum number of ecological effects on non-target species (Giblin-Davis et al., 1992b). Breeding and improvement of new bermudagrass cultivars with superior nematode responses are essential. Giblin-Davis et al. (1992b) tested the sting nematode tolerance and resistance of seven commercial bermudagrass cultivars and 30 experimental accessions. They found that 26 accessions showed a significant reduction in root dry weights compared with the uninoculated controls and that 25 supported reproduction of sting nematode. They also determined that ‘Tifway’ was among the most tolerant of the cultivars evaluated. Currently, there are few known sting nematode-resistant or -tolerant bermudagrass genotypes available. Except for ‘TifEagle’, all bermudagrass cultivars used on greens are related to each other; therefore, environmental pressure exists for the development of a significant pest problem on bermudagrass greens. This highlights the need to select new sources of genetically superior bermudagrass accessions for use in a breeding program. Most bermudagrass cultivars that have been widely used on golf courses are triploids [Cynodon dactylon (L.) Pers. var. dactylon × C. transvaalensis Burtt-Davy] derived from hybridizations of tetraploid common bermudagrass and diploid African bermudagrass. Previous development of sterile, triploid hybrids has focused primarily on the selection of a superior common bermudagrass parent. This was attributable, in part, to a lack of knowledge regarding the genetic diversity and potential of improvement for African bermudagrass. Information is now available that indicates that improvement of African bermudagrass is possible for several turfgrass performance traits (Kenworthy et al., 2006). This necessitates the screening of both African and common bermudagrass. The University of Florida bermudagrass breeding program through multiyear evaluations has identified superior, Florida-adapted, experimental accessions of common and African bermudagrass to use in crosses to develop new sterile bermudagrass hybrids for use on golf courses. Sting nematode responses to these bermudagrass accessions remain uncharacterized and could provide valuable information in the selection of parents to develop progeny and cultivars resistant to this serious turfgrass pest. The objectives of this study were to test the responses caused by sting nematodes among superior University of Florida accessions of common and African bermudagrass and to select accessions with superior responses for future cultivar breeding and development.
Materials and Methods
Plant materials.
Five commercial cultivars (Celebration, TifEagle, Tifway, TifSport, and TifGrand) and 46 germplasm accessions of bermudagrass were tested in two sequential experimental trials in 2009 in a greenhouse at the University of Florida Turfgrass Envirotron in Gainesville, FL. The bermudagrass accessions with origin information are listed in Table 1.
Mean root length of five cultivars and 46 germplasm accessions of bermudagrass at 90 d after inoculation with Belonolaimus longicaudatus in two experimental trials.
Inoculum preparation.
Sting nematode was maintained on ‘FX-313’ st. augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] grown in clay pots filled with pure sand under greenhouse conditions (Giblin-Davis et al., 1992a). Juveniles and adults of sting nematode were extracted and collected from soil by using Cobb's decanting and sieving technique (Cobb, 1918; Flegg, 1967). The average number of nematodes was counted from five 1-mL aliquots and extrapolated to the total volume of the suspension.
Nematode responses of germplasm accessions.
Nematode-free aerial stolons of each cultivar/accession were vegetatively propagated into (3.8 cm diameter × 21 cm deep) ultraviolet-stabilized Ray Leach “Cone-tainers”™ (SC10; Stuewe & Sons, Inc., Tangent, OR) filled with 100% USGA specification greens sand (USGA, 1993). The bottom of conetainers was filled with Poly-fil (Fairfield Processing Corporation, Danbury, CT) to prevent sand from escaping from the drainage holes. Two pieces of terminal aerial stolons with one node each were planted into each conetainer. Two minutes of overhead mist irrigation was applied six times daily for 2 weeks to allow the sprigs to establish. From the third week onward, the irrigation was reduced to once a day in the morning for 6 min and 3 min a day from the beginning of the fifth week. Six weeks after establishment, grass was inoculated with no nematodes or 50 sting nematodes per conetainer. Before inoculation, suspensions of sting nematodes were concentrated to 10 nematodes/mL and set at room temperature for 3 h. None or a total of 5 mL of the suspensions were divided and inoculated into two 3-cm-deep holes made 1 cm from the surface center of the conetainer for the uninoculated and inoculated treatments, respectively. The holes were covered with a light layer of sand and moistened with a light mist. Turfgrasses were maintained in a randomized complete block design with six replications for each genotype with a total of 51 bermudagrass genotypes. To provide insulation from temperature fluctuation, conetainers were placed in (60 × 35 × 15 cm) Beaver Plastics Styroblock™ (Stuewe & Sons, Inc., Tangent, OR). The experiments were maintained under a temperature range of 24 to 34 °C with natural daylight in a greenhouse. Turfgrass was mowed once a week at a height of 2.5 cm and fertilized once a week using 24–8–16 (N-P2O5-K2O) at a rate of 0.5 kg N/100 m2 per growing month.
Experiments were harvested 90 d after the inoculation of nematodes. Root and soil samples were collected from each conetainer. Roots were collected by removing the shoots and Poly-fil. Roots were washed free of soil on an 853-μm (20-mesh) sieve and put into a 50-mL plastic tube submerged with water. Roots were digitally scanned using WinRHIZO root scanning equipment and software (Regent Instruments, Ottawa, Ontario, Canada). Root length was measured from the scanned images. Nematodes were extracted from the entire soil volume from the conetainer by using the modified centrifugal flotation technique (Jenkins, 1964). Nematode solution extracted from each conetainer was poured into a counting dish, and the final nematode population densities were counted under an inverted microscope at ×40.
Data analysis.
To determine if a particular cultivar or accession was damaged by sting nematode, we compared total root length of the inoculated treatment with the non-inoculated control using linear contrasts at P ≤ 0.1. Use of this P value level provided a more conservative threshold for identifying accessions that were not different in total root length between treatments. To determine if an accession performed better than a standard cultivar, the root lengths of the inoculated treatment from each accession were compared with ‘Tifway’ using the Fisher's protected least significant difference (lsd) test. Final nematode population densities were subjected to analysis of variance, and genotypes were compared by the Fisher's protected lsd test at P ≤ 0.1. Statistical analysis was conducted by using the SAS program (SAS Institute, Cary, NC).
Results
Statistical analysis showed that there was a significant difference among the genotypes (P < 0.0001), treatment (P < 0.0001), and no interaction between them (P = 0.8926). A significant genotype × trial interaction (P = 0.0139) occurred, so results are presented separately for the two trials. For all genotypes, root lengths varied from 650 to 1599 cm (Trial 1) and 920 to 2358 cm (Trial 2) for the uninoculated controls and 490 to 1268 cm (Trial 1) and 426 to 1817 cm (Trial 2) for the inoculated treatments. Results of the linear contrast showed that genotypes ‘Tifway’, ‘Celebration’, 304, 445, 528, PI291590, AB42, UFC12, and UFC29 had significant reductions in root length from sting nematodes in both trials (P ≤ 0.1), whereas ‘TifEagle’, 132, 171, 173, 295, 296, 301, 343, PI290868, AB1, AB3, AB33, AB37, UFC11, and UFC26 did not show root reduction in either trial (Table 1). Several genotypes were found to have greater root length in the inoculated treatment than ‘Tifway’ in both trials (P ≤ 0.1). These were ‘Celebration’, 132, 157, 481, PI291590, AB3, and AB33 (Table 1).
Accessions also showed differences in host status to sting nematode (Table 2). The final nematode population densities were significantly different among genotypes (P ≤ 0.001). The final nematode numbers were compared with the initial inoculum level of 50 sting nematodes/conetainer. Nematode numbers increased (more than 50 sting nematodes/conetainer) in ‘Celebration’, ‘TifGrand’, ‘157’, ‘445’, ‘AB21’, and ‘AB39’ in Trial 1 with the maximum increase of 2.3-fold in AB39. In Trial 2, ‘TifGrand’, ‘Tifway’, 157, ‘347’, 355, 445, ‘481’, AB21, AB42, and ‘UFC03’ increased the numbers of sting nematode, and the highest increase was 2.4-fold in 355. However, to select better nematode response, we considered accessions on which the nematode numbers decreased (less than 50 sting nematodes/conetainer) in both trials as potential good candidates for breeding. Based on this, nine African and 28 common bermudagrass accessions could move forward in the selection process (Table 2).
Final mean population density of Belonolaimus longicaudatus on five cultivars and 46 germplasm accessions of bermudagrass 90 d after inoculation with 50 B. longicaudatus/conetainer in two experimental trials.
Discussion
Genotypes with better sting nematode tolerance, which either had no reduction in root length or had greater root length than ‘Tifway’, were found in various sources, and there was no relationship between nematode response and the origin of genotypes. According to Giblin-Davis et al. (1992b), ‘Tifway’ was relatively tolerant to sting nematode, whereas at the same time it was a host. Because ‘Tifway’ is the most commonly used bermudagrass cultivar in the southeastern United States (Burton, 1966), it was used as the standard cultivar for comparison in our trials. Nematode tolerance in bermudagrass has been defined as a genotype either suffering minimal root reduction from the nematodes or as being able to produce more roots than a standard genotype in the presence of the nematodes (Pang et al., 2011). The inoculated to non-inoculated comparisons allow us to determine the degree to which the genotype is damaged by nematodes. Comparisons to ‘Tifway’ allow us to determine the relative tolerance compared with a standard cultivar. Both types of comparisons are vital for genotype selection and exclusion of only one type of comparison can lead to improper conclusions. For example, UFC11 did not have root length differences between inoculated and non-inoculated. However, when we compare the root lengths of UFC11 with ‘Tifway’, we see that it had poor root production overall and would be a poor choice to move forward in the selection process. Conversely, PI291590 is a vigorously rooting accession compared with ‘Tifway’, but it was severely damaged by sting nematode. Genotypes whose roots were not significantly impacted by sting nematode and also were more vigorously rooting than ‘Tifway’ in both trials were 132, AB3, and AB33. Furthermore, the final nematode population densities could provide an additional reference for germplasm selection and cultivar breeding for nematode response. Accessions that maintained a final nematode density of less than 50 sting nematodes/conetainer in these studies would have a potential to suppress nematode reproduction and lower the number of nematodes in the field or golf courses, which can be used as a strategy for nematode management. Genotypes 132 and AB33 not only maintained vigorous rooting under sting nematode-infested soil, but also were good candidates to reduce nematode population densities based on the fact that the final nematode numbers were 50% lower than the initial density in this study (Table 2).
Overall, differential responses to sting nematode were identified among bermudagrass accessions with diverse genetic backgrounds (diploid, triploid, tetraploid, pentaploid, and hexaploid) and sources. Accessions were identified with increased and decreased rooting under sting nematode pressure when compared with ‘Tifway’. No obvious relationship was found to exist between rooting response and ploidy level or origin of the accessions. This information should be of value to select parents for future breeding and cultivar improvement. The results of this study should not be considered definitive because multiple nematode species or pathogens exist in natural fields, and the nematode or pest responses of these genotypes need to be assessed with future field studies.
Conclusions
This study indicated that bermudagrass germplasm accessions respond differently in host suitability and tolerance to sting nematodes and that selection of genotypes with improved responses to nematodes is possible. Accessions 132, AB3, and AB33 were not measurably affected by sting nematode and were more vigorously rooting than the standard cultivar, Tifway. Accessions that are relatively tolerant to sting nematode were identified with variable ploidy levels and origins. Accessions with larger root systems tend to be more tolerant, which might be potentially useful for turfgrass breeding and development for host tolerance against nematodes. This is the first report of sting nematode responses on African bermudagrass and variable responses were observed. The use of diploid and tetraploid genotypes identified in this research may prove to be highly valuable genetic resources for future improvement of bermudagrass and serve to reduce dependence on nematicides.
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