Published August 16, 2017

RESEARCH

Tolerance in St. Augustinegrass Germplasm against insularis Barber (: )

Susana R. Milla-Lewis,* Katharine M. Youngs, Consuelo Arrellano, and Yasmin J. Cardoza

S.R. Milla-Lewis, Dep. of Crop and Soil Sciences, North Carolina ABSTRACT State Univ., Raleigh, NC 27695-7620; K.M. Youngs and Y.J. Cardoza, St. Augustinegrass [Stenotaphrum secundatum Dep. of Entomology and Plant Pathology, North Carolina State Univ., (Walt.) Kuntze] is a widely used lawn grass Raleigh, NC 27695-7613; C. Arrellano, Dep. of Statistics, North in the southern United States due to its Carolina State Univ., Raleigh, NC 27695-8203. Received 20 May stoloniferous growth habit and shade tolerance. 2016. Accepted 1 Dec. 2016. *Corresponding author (susana_milla- However, St. Augustinegrass is prone to [email protected]). Assigned to Associate Editor Ambika Chandra. thatch accumulation, which is conducive to Abbreviations: FPLI, functional plant loss index; PI, plant introduction; pest problems, with the southern chinch bug SCB, southern chinch bug. ( Barber, SCB) being the most economically important one. Previous work to identify additional sources of SCB resistance t. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] reported genotypes with comparatively high Sis widely used as a lawn grass in warm, tropical, and sub- numbers of recovered but low damage tropical regions of the world (Sauer, 1972). It is a popular choice ratings. This study was conducted (i) to evaluate for lawns in the southern United States due to its aesthetically the performance of these materials in response pleasing appearance and shade tolerance (Busey and Davis, 1991; to varying SCB feeding densities, and (ii) Trenholm and Nagata, 2005). However, St. Augustinegrass is to determine feeding and oviposition under prone to thatch accumulation (Horn et al., 1973), which is con- no-choice scenarios. Genotypes exposed to 0, 10, or 30 adult SCBs were evaluated after 4 ducive to and disease problems (Haygood and Martin, wk for damage and insect survival. Significant 1990). The southern chinch bug (SCB, Blissus insularis Barber) is differences were observed among genotypes. the most economically important insect pest of St. Augustinegrass Across infestation levels, while recovered insect and is found throughout the Gulf States, from Texas to Florida numbers for susceptible check ‘Seville’ and and further north into Georgia and North Carolina (Henry and plant introductions 509038 and 509039 were Froeschner, 1988; Sweet, 2000). Southern chinch bugs have not significantly different, damage ratings were piercing-sucking mouthparts and feed on the phloem of grass significantly lower for the latter, indicating that plants within meristematic tissues (Painter, 1928). In doing so, these materials were tolerant to SCB feeding. SCB deposit their salivary sheaths in the plant tissue at the site In the no-choice experiments, survival levels of feeding (Backus, 1988). These insects normally reside in the of both males and females on week 4 were thatch area of the turfgrass stand and prefer to feed on the lower significantly lower for resistant check ‘FX10’, PI leaf sheaths and crown area of the plant (Anderson et al., 2006). 365031, and PI 289729. These genotypes, along with PIs 291594, 300129, and 647924, showed Affected areas turn yellow, then brown, and ultimately die. As significantly lower SCB oviposition and feeding the season progresses, these areas can coalesce into large areas or compared with Seville. Our study was able to entire lawns of dead grass (Reinert et al., 1995). confirm that two PIs display tolerance to SCB feeding, and five additional PIs have antibiosis Published in Crop Sci. 57:S-26–S-36 (2017). activity against adult SCB (likely antibiosis), doi: 10.2135/cropsci2016.05.0361 representing sources of SCB resistance for future St. Augustinegrass breeding efforts. © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

S-26 www.crops.org crop science, vol. 57, july–august 2017 Historically, management of the SCB has been In a previous study looking at new sources of resistance achieved through insecticide applications (Watson and to SCB, complete antixenosis was not detected among a set Bratley, 1929; Kelsheimer, 1952; Wolfenbarger, 1953; of plant introductions (PIs) of St. Augustinegrass (Youngs Kerr, 1956). However, heavy reliance on chemical con- et al., 2014). Nonetheless, a number of PIs within the tested trol and lack of proper rotation of chemical formulations group exhibited damage ratings that were not significantly (Cherry and Nagata, 2005; Vázquez et al., 2011), com- different from those of the resistant checks. Further evalu- bined with more generations per year of SCB living in ation of these materials for neonate development identified warm climates, have led to development of resistance to nine PIs with low survival and slower development of SCB organophosphates and organochlorines (Kerr, 1958, 1961; compared with susceptible checks (Youngs et al., 2014). Reinert, 1982; Reinert and Niemczyk, 1982; Reinert and Four of the genotypes identified as having antibiosis- Portier, 1983) and to bifenthrin in Florida (Cherry and based resistance (PI 509038, PI 509039, PI 600734, and PI Nagata, 2005) and permethrin (Vázquez et al., 2011) in 647924) are diploid, which facilitates their use for transfer Florida. Currently, clothianidin (Arena, Valent Profes- of resistance into commercial cultivars. sional Products, Walnut Creek, CA) is the best insecticide In the research described here, we furthered our in the market for chemical control of resistant chinch bug efforts to assess potential tolerance in selected genotypes populations in Florida. and reference cultivars of St. Augustinegrass. Our objec- Host-plant resistance can provide a feasible alterna- tives were: (i) to evaluate the performance of selected PIs tive to chemical SCB management. Host-plant resistance in response to feeding by varying densities of SCB, and (ii) encompasses a range of adaptations evolved by plants, to assess potential adult feeding or oviposition deterrence. which reduce the impact of pests and diseases, improving plant survival and reproduction (Hayes, 1935; Fleschner, MATERIALS AND METHODS 1952; Price, 1986). Traditionally, host-plant resistance has St. Augustinegrass Maintenance been the most successful approach for controlling SCB in and Experimental Propagation St. Augustinegrass (Horn et al., 1973, Reinert and Dudeck, A total of five cultivars and 11 PIs of St. Augustinegrass were 1974; Crocker et al., 1982, 1989; Busey and Zaenker, 1992). used in this study (Table 1). Additionally, two accessions of There are three clearly delineated categories of host-plant pembagrass (Stenotaphrum dimidatum), a close relative of St. resistance: antixenosis, antibiosis, and tolerance. Anti- Augustinegrass, were included. The cultivars were chosen to xenosis includes any plant characteristic that leads to the serve as points of reference for ascribing tolerance–susceptibil- pest’s nonpreference for a resistant plant when compared ity scores to the uncharacterized accessions. The group included with a susceptible plant (Painter, 1951; Kogan and Ortman, SCB-resistant ‘FX-10’ (Busey, 1990), ‘Captiva’ (Nagata and 1978). Antibiosis is due to chemical plant properties that Cherry, 2003), and ‘Raleigh’ (Anderson et al., 2006; Chong can negatively affect the life history of the pest, leading to et al., 2009; Youngs et al., 2014) and SCB-susceptible Flora- increased mortality, decreased fecundity, or reduced lon- tam (Busey and Center, 1987; Nagata and Cherry, 2003) and ‘Seville’ (Crocker et al., 1989). Plant introductions were chosen gevity (Painter, 1951). A plant exhibiting tolerance, on according to the results of Youngs et al. (2014), who reported the other hand, is an acceptable and adequate host for the genotypes with comparatively high numbers of recovered pest, but it can withstand large infestations and can com- insects but low damage ratings. Cultivars and PIs were obtained pensate for any damage caused by the noxious organism from the North Carolina State University (Raleigh, NC) turf- (Painter, 1958). Southern chinch bugs have been able to grass breeding program’s germplasm collection. consistently overcome management strategies ranging from All plant materials were propagated and maintained accord- chemical to host-plant resistance. For example, ‘Floratam’ ing to Youngs et al. (2014) and grown at the North Carolina State St. Augustinegrass, which was released in 1973 (Horn et University greenhouse complex (Raleigh, NC) at 28 ± 5°C and al., 1973) and subsequently planted throughout the south- under natural light with an approximate 14:10-h light:dark cycle. ern United States (Reinert and Dudeck, 1974; Crocker et al., 1982, 1989), exhibited a high level of SCB antibiosis Blissus insularis Colonies (Reinert and Dudeck, 1974). However, resistance-breaking All SCB used in this study were offspring from adult SCB col- SCB populations in Floratam fields were reported in Flor- lected from infested residential lawns in Wilmington, NC. ida by 1985 (Busey and Center, 1987) and have continued Southern chinch bug colonies were maintained by transferring 40 to 70 SCB adults into new flats (15.25 ´ 20.32 cm) of SCB- to spread since then (Cherry and Nagata, 1997; Reinert, susceptible cultivar Seville every 4 wk. All SCB colonies were 2008; Vázquez et al., 2011). The identification of resistance- maintained under greenhouse conditions, as described above. breaking populations, in combination with environmental To ensure that levels of genetic diversity were maintained, 300 concerns over pesticide use and the need for more eco- to 400 field-collected late instar and adult SCB were added to nomically feasible management strategies, has renewed the greenhouse colonies three to four times each summer. interest in identifying additional sources of SCB resistant St. Augustinegrass germplasm.

crop science, vol. 57, july–august 2017 www.crops.org S-27 Table 1. St. Augustinegrass (Stenotaphrum spp.) genotypes used to evaluate response to southern chinch bug (Blissus insularis) feeding and to identify deterrence to southern chinch bug (SCB) feeding and oviposition. Genotype Species Type Ploidy† FR‡ OFP‡ Captiva secundatum Resistant check diploid (2n = 18) X Floratam secundatum Susceptible check aneuploid (2n = 32) X FX-10 secundatum Resistant check aneuploid (2n = 30) X Raleigh secundatum Resistant check diploid (2n = 18) X X Seville secundatum Susceptible check diploid (2n = 18) X X PI 290888 secundatum Plant introduction aneuploid (2n = 30) X PI 291594 secundatum Plant introduction triploid (2n = 27) X PI 300129 secundatum Plant introduction triploid (2n = 27) X PI 300130 secundatum Plant introduction aneuploid (2n = 30) X PI 410360 secundatum Plant introduction diploid (2n = 18) X PI 410361 secundatum Plant introduction diploid (2n = 18) X X PI 509038 secundatum Plant introduction diploid (2n = 18) X X PI 509039 secundatum Plant introduction diploid (2n = 18) X X PI 600734 secundatum Plant introduction diploid (2n = 18) X PI 647924 secundatum Plant introduction diploid (2n = 18) X PI 647925 secundatum Plant introduction diploid (2n = 18) X PI 289729 dimidatum Plant introduction tetraploid (2n = 36) X PI 365031 dimidatum Plant introduction hexaploid (2n = 54) X † According to Milla-Lewis et al. (2013).

‡ FR, feeding response experiments; OFP, feeding and oviposition preference experiments. Evaluation of Tolerance to SCB Feeding added to paper bags over a 2-wk period, after which time no more in Selected Germplasm insects were recovered. The experiment was a complete random- Six genotypes were screened for tolerance in the spring of 2013: ized block design with seven replicates, occurring over three trials Seville, Raleigh, PI 410360, PI 410361, PI 5090338, and PI 509039 of two replicates each and one with a single replicate obtained (Table 1). The four PIs were selected based on the comparatively from February to April of 2013. high number of insects recovered from them and low damage rat- ings observed in Youngs et al. (2014). Four rooted stolons were Adult Oviposition and Fecal Output planted in a 16-cm-diameter clay pot, with each stolon containing on Selected Plant Lines precisely three nodes and trimmed to approximately 8 cm every This experiment was designed to determine feeding and ovi- 2 wk to standardize the aerial plant tissue available to the insects position by adult SCB under no-choice scenarios. Based on the and to mimic standard growing conditions. Growing conditions neonate performance results of Youngs et al. (2014), 13 PIs were and maintenance throughout the experiment were as described used for this experiment along resistant and susceptible con- above. Three pots of each genotype were planted per replicate trols (Table 1). Eight stolons of each genotype were planted to be exposed to 0, 10, or 30 adult SCB for 4 wk. At the end into 16-cm pots and maintained as described above. Meth- of the 4 wk, damage ratings, functional plant loss index (FPLI), ods described by Rangasamy et al. (2006) were used to obtain and survival of male and female insects were recorded to com- adults of similar age. Briefly, fifth instars were selected from pare across grass genotypes. Damage was rated on a scale of 1 to the experimental colony and then provided with fresh Seville 5 (1, 0–10%; 2, 11–30%; 3, 31–50%; 4, 51–70%; and 5, 71–100% grass clippings. Insects that molted to the adult stage within a damage) according to Heng-Moss et al. (2002) and assessed twice, 5-d period were selected for the experiment. One male and one with a 3-d period in between. Ratings for each plant were aver- female were confined to a single stolon for 1 wk at a time using a aged to obtain a mean damage rating per genotype, which was Petri dish cage and then transferred to a new stolon for a period subsequently used for data analysis. The FPLI was calculated for of four consecutive weeks (Fig. 1). After each weekly interval, each genotype according to Panda and Heinrichs (1983) using the insect survival was recorded and live insects were transferred formula: 1− [dry wt. of infested plant/dry wt. of control plant] ´ gently using a mouth aspirator to a new stolon on the same [1 − (damage rating/5)]. Plants were unearthed, collected, thor- plant. Excretory spots and eggs were counted weekly and aver- oughly washed to remove soil from roots, air dried for 3 h, and aged over the duration of the experiment. Aluminum foil was then placed within paper bags into an oven at 75°C for 60 h to used in the arena to facilitate recording of fecal spots and eggs. obtain dry weights. For insect counts, a surface layer (including This procedure was repeated for four consecutive weeks. The foliage) of 3.81 cm of soil was removed from each pot and placed experiment was set up in singlet or duplicates and repeated over in a paper bag (14 ´ 21 ´ 45 cm [width ´ length ´ height]). time to obtain a total of six replicates from July 2012 until May Fresh, thoroughly washed corn (Zea mays L.) ears were cut into 2013. Male and female survival, number of eggs, and number of 8- to 10-cm pieces and placed into each bag to lure insects away fecal spots were recorded weekly to compare across genotypes. from the dying St. Augustinegrass material. Insects were collected from corn pieces and counted every 2 d. Fresh corn pieces were

S-28 www.crops.org crop science, vol. 57, july–august 2017 were not significantly different from this check (Table 2). At infestation level 30, Seville was again the most sus- ceptible genotype. Only PI 410361 had a damage rating equivalent to that of Seville. Meanwhile, PI 509038 and PI 509039 yielded damage ratings not significantly differ- ent than that of resistant Raleigh (Table 2). The number of insects recovered from plants at the end of the experiments was significantly impacted by infestation level (F = 9220.24; df = 2, 84; p < 0.0001), plant genotype (F = 143.60; df = 6, 84; p < 0.0001), and their interaction (F = 82.31; df = 12, 84; p < 0.0001). At infestation level 10, the number of insects recovered from all plant lines was equivalent to those from Seville, except Fig. 1. Setup for experimentation on feeding and oviposition of southern chinch bugs on St. Augustinegrass. One male and one for Raleigh, from which the fewest insects were recovered female were restricted to a single stolon for 1 wk at a time for (Table 2). The same was observed when the infestation four consecutive weeks. Aluminum foil was used in the arena to level was increased to 30 insects per plant. (Fig. 2B) facilitate recording of fecal spots. Excretory spots and eggs were Dry weight was significantly impacted by infestation counted weekly and averaged over the duration of the experiment. level (F = 52.86; df = 2, 84; p < 0.0001), plant genotype Statistical Analysis (F = 14.79; df = 6, 84; p < 0.0001), and by the interac- Data for the evaluation of tolerance was analyzed with ANOVA tion between infestation level and plant genotype (F = using PROC GLM (SAS Institute, 2014) to test for the effects 4.05; df = 12, 84; p < 0.0001). Under non-infested con- of plant genotype, infestation level, and their interaction on ditions (infestation level 0), dry weights for Raleigh and damage rating, number of recovered insects, dry weight of Seville were equivalent to each other and were signifi- plant material, and FPLI. Analysis for the adult feeding and cantly higher than those of all other genotypes (Table 2). oviposition experiment was conducted using a multivariate However, once infested, dry weight mass was negatively ANOVA using PROC GLM (SAS Institute, 2014) to test for impacted at infestation level 10, markedly for Seville and the multivariate effect of plant genotype on number of eggs slightly for PI 410360 and PI 410361. On the other hand, and number of fecal spots, followed by a univariate effect of dry weight for PI 509038 and PI 509039 were not as nega- plant genotype on each dependent variable (response) at each tively affected, even at infestation levels of 30 chinch bugs week. Surviving males and females over the 4-wk period were plant−1. Raleigh’s dry weight was not as reduced by insect subjected to a nonparametric life table survival analysis (Stokes infestation level, as was that of Seville, and it remained the et al., 2012) using the procedure LIFETEST of SAS software (SAS Institute, 2014). Wilcoxon homogeneity test of survival top performing genotype at both infestation levels. It is curves among genotypes was used, followed by pairwise com- important to note that dry weights for PI 509038 and PI parisons between genotypes. The Sidak correction for multiple 509039 were not significantly different among infestation comparisons was applied using a significance level of 0.05 to levels (Fig. 2C). protect against falsely rejecting the null hypothesis of no differ- Functional plant loss index was significantly impacted ence between genotypes. by infestation level (F = 128.84; df = 1, 84; p < 0.0001) and plant genotype (F = 32.44; df = 6, 84; p < 0.0001), but not by their interaction. Functional plant loss index RESULTS values increased in accordance with infestation level for all Evaluation of Tolerance to SCB Feeding genotypes tested (Table 2). At infestation level 10, Seville in Selected Germplasm yielded the highest FPLI values compared with all other Damage rating was significantly impacted by infestation plant genotypes, followed by PI 410360 and PI 410361. level (F = 149.41; df = 2, 84; p < 0.0001), plant genotype The lowest FPLI values were obtained for PI 509039 and (F = 32.52; df = 6, 84; p < 0.0001), and by the interaction PI 509038. Results for FPLI values at infestation level 30 between infestation level and plant genotype (F = 6.82; df followed a similar trend, with values for PI 410361 and = 12, 84; p < 0.0001). Damage ratings for the susceptible Seville significantly higher than those for all other geno- control Seville significantly increased with increasing SCB types. The lowest FPLI values were again obtained for PI infestation levels (Fig. 2A). On the other hand, damage 509039 and PI 509038 (Fig. 2D). ratings for Raleigh were not significantly increased across SCB infestation levels. At infestation level 10, damage rat- Adult Oviposition and Fecal Output ings for all tested germplasm were significantly lower than on Selected Plant Lines that of Seville (Table 2). The lowest damage rating at this Percent male survival was more impacted by plant gen- level was that of Raleigh, but PI 509038 and PI 509039 otype as weeks of evaluation went by, with p-values

crop science, vol. 57, july–august 2017 www.crops.org S-29 Fig. 2. Means for (A) damage ratings, (B) number of insects recovered, (C) plant dry weight, and (D) functional plant loss index (FPLI) for St. Augustinegrass germplasm subjected to three levels of southern chinch bug infestation. Within a genotype, mean separation for infestation levels followed by the same letter are not statistically different (Tukey’s mean separation test P ³ 0.05).

Table 2. Damage rating, number of recovered insects, dry weight, and functional plant loss index (FPLI) by infestation level for St. Augustinegrass germplasm subjected to three levels on southern chinch bugs infestation. Values represent mean ± SE for five replicates per plant genotype. Means for genotypes within infestation levels followed by the same letter are not statistically different (Tukey’s mean separation test P ³ 0.5). Infestation level† Genotype Damage rating‡ Recovered insects Dry weight FPLI 0 PI 410360 1.00 ± 0.00 a – 2.92 ± 0.30 b – PI 410361 1.00 ± 0.00 a – 2.82 ± 0.35 b – PI 509038 1.00± 0.00 a – 3.40 ± 0.36 b – PI 509039 1.00 ± 0.00 a – 3.00 ± 0.28 b – Raleigh 1.00 ± 0.00 a – 5.08 ± 0.20 a – Seville 1.40 ± 0.24 a – 5.04 ± 0.23 a – 10 PI 410360 2.00 ± 0.00 b 9.80 ± 0.20 a 2.34 ± 0.28 b 51.20 ± 3.48 b PI 410361 2.00 ± 0.00 b 10.00 ± 0.00 a 2.32 ± 0.44 b 52.34 ± 4.84 b PI 509038 1.00 ± 0.00 c 9.00 ± 0.00 ab 3.04 ± 0.35 ab 28.62 ± 1.58 c PI 509039 1.40 ± 0.24 bc 9.20 ± 0.20 ab 2.88 ± 0.34 ab 30.34 ± 5.80 c Raleigh 1.20 ± 0.20 c 6.00 ± 0.45 c 4.20 ± 0.36 a 36.44 ± 7.13 b c Seville 3.20 ± 0.20 a 9.40 ± 0.24 ab 2.90 ± 0.34 ab 79.74 ± 2.42 a 30 PI 410360 2.80 ± 0.37 bc 29.20 ± 0.20 a 1.22 ± 0.14 bc 80.82 ± 3.71 ab PI 410361 3.80 ± 0.20 ab 29.40 ± 0.60 a 0.70 ± 0.14 c 92.88 ± 2.79 a PI 509038 2.20 ± 0.20 c 28.00 ± 0.55 a 2.66 ± 0.33 a 56.20 ± 4.28 c PI 509039 2.20 ± 0.20 c 27.6 0 ± 0.68 a 2.50 ± 0.27 ab 52.52 ± 4.65 c Raleigh 1.60 ± 0.40 c 12.00 ± 1.10 b 2.78 ± 0.33 a 61.38 ± 8.13 bc Seville 4.40 ± 0.24 a 29.20 ± 0.20 a 0.78 ± 0.19 c 97.52 ± 1.08 a † Number of insects added.

‡ Damage was rated on a scale of 1 to 5 (1, 0–10%; 2, 11–30%; 3, 31–50%; 4, 51–70%; and 5, 71–100% damage), according to Heng-Moss et al. (2002).

S-30 www.crops.org crop science, vol. 57, july–august 2017 Table 3. Significance of genotype effects over weeks of With the exception of FX-10 on week 1, male percent evaluation revealed by ANOVA from the adult oviposition and feeding preference experiments in St. Augustinegrass. survival was not significantly different across genotypes during the first 2 wk of evaluation (Table 4). However, Trait Week 1 Week 2 Week 3 Week 4 while not significant, marked differences were observed in Male survival 0.0355 0.0266 0.0003 <0.0001 Female survival 0.4660 0.5958 0.0293 <0.0001 percent survival on week 2, with PI 365031 showing the No. of eggs <0.0001 <0.0001 <0.0001 <0.0001 lowest survival numbers. On week 3, this PI and FX-10 No. of fecal spots <0.0001 <0.0001 <0.0001 <0.0001 had significantly lower levels of survival compared with susceptible control Seville. While numerically higher, a decreasing from 0.0355 in week 1 to <0.0001 in week 4 few genotypes had levels of survival not significantly dif- (Table 3). This was also the case for percent female sur- ferent from PI 365031 and FX-10. On week 4, Seville vival, but for this trait, differences were significant only still maintained 100% survival, while PI 365031, FX-10, in weeks 3 and 4. Wilcoxon survival curve homogeneity and PI 289729 were the best-performing entries with 0% test among genotypes was significant c( 2 = 89.46, df = survival. For female survival, Wilcoxon homogeneity 17, p < 0.001), indicating different patterns for genotype survival curve test showed significant differences among survival curves along these 4 wk. Meanwhile, number of genotypes (c2 = 62.60, df = 17, p < 0.001). Surviving eggs and fecal spots observed were significantly different female percent did not differ significantly among geno- among plant genotypes across weeks of evaluation. types for weeks 1 and 2, but on week 3, FX-10 and PI 291594 showed significantly lower female percent survival

Table 4. Weekly percent surviving male and female southern chinch bugs during oviposition and feeding preference experiments in St. Augustinegrass. Values represent mean ± SE for six replicates per plant genotype. Values within above columns followed by the same letter are not statistically different (Tukey’s mean separation test P ³ 0.5). Survivors Genotype Week1 Week 2 Week 3 Week 4 Males PI 289729 100.0 ± 0.00 a 83.3 ± 16.67 a 50.0 ± 22.36 ab 00.0 ± 0.00 c PI 290888 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 291594 100.0 ± 0.00 a 83.3 ± 16.67 a 66.6 ± 21.08 ab 50.0 ± 22.36 abc PI 300129 100.0 ± 0.00 a 100.0 ± 0.00 a 83.3 ± 16.67 ab 16.6 ± 16.66 bc PI 300130 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 33.3 ± 21.08 abc PI 365031 100.0 ± 0.00 a 50.0 ± 22.36 a 16.6 ± 16.66 b 00.0 ± 0.00 c PI 410361 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 509038 100.0 ±0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 509039 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 83.3 ± 16.67 ab PI 600734 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 abc PI 647924 100.0 ± 0.00 a 100.0 ± 0.00 a 50.0 ± 22.36 ab 50.0 ± 22.36 abc PI 647925 83.3 ± 16.67 ab 66.6 ± 21.08 a 66.6 ± 21.08 ab 66.6 ± 21.08 abc Floratam 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 50.0 ± 22.36 abc FX-10 66.6 ± 21.08 b 66.6 ± 21.08 a 16.6 ± 16.66 b 00.0 ± 0.00 c Captiva 100.0 ± 0.00 a 83.3 ± 16.67 a 50.0 ± 22.36 ab 33.3 ± 21.08 abc Raleigh 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 50.0 ± 22.36 abc Seville 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a Females PI 289729 100.0 ± 0.00 a 100.0 ± 0.00 a 83.3 ± 16.67 ab 16.6 ± 16.66 b PI 290888 100.0± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 291594 83.3 ± 16.67 b 83.3 ± 16.67 a 33.3 ± 21.08 b 16.6 ± 16.67 b PI 300129 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 33.3 ± 21.08 b PI 300130 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 66.6 ± 21.08 ab PI 365031 100.0 ± 0.00 a 100.0 ± 0.00 a 50.0 ± 22.36 ab 16.6 ± 16.66 b PI 410361 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 509038 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 509039 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a PI 600734 100.0 ± 0.00 a 100.0 ± 0.00 a 83.3 ± 16.67 ab 66.6 ± 21.08 ab PI 647924 100.0 ± 0.00 a 83.3 ± 16.67 a 83.3 ± 16.67 ab 50.0 ± 22.36 ab PI 647925 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 66.6 ± 21.08 ab Floratam 100.0 ± 0.00 a 100.0 ± 0.00 a 83.3 ± 16.67 ab 50.0 ± 22.36 ab FX-10 100.0 ± 0.00 a 83.3 ± 16.67 a 33.3 ± 21.08 b 16.6 ± 16.66 b Captiva 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 16.6 ± 16.66 b Raleigh 100.0 ± 0.00 a 100.0 ± 0.00 a 66.6 ± 21.08 ab 50.0 ± 22.36 ab Seville 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a 100.0 ± 0.00 a crop science, vol. 57, july–august 2017 www.crops.org S-31 compared with Seville, PI 290888, PI 410361, PI 509038, genotypes during week 2 tended to drop for most geno- and PI 509039 (Table 4). On week 4, FX-10, Captiva, PI types compared with week 1, except for Seville, PI 410361, 365031, PI 289729, PI 291594, and PI 300129 had sig- PI 509038, PI 509039, and Raleigh. A similar pattern nificantly lower percent female survival compared with was observed for week 3. On the final week, FX-10, PI Seville, PI 290888, PI 410361, PI 509038, and PI 509039. 289729, PI 291594, PI 300129, PI 365031, and PI 647924 Overall, multivariate test for number of eggs indicated experienced no oviposition at all. Meanwhile, only Seville, significant differences among genotypes (Pillai’s trace test, PI 410361, PI 509038, and PI 509039 continued to yield

Fval = 3.54, dfnum = 68, dfden = 176, p < 0.0001). Our higher numbers of eggs compared with other germplasm. susceptible check, Seville, had significantly higher number The relationship between female survival and number of of eggs compared with all other genotypes over the 4 wk eggs is presented in Fig. 3. For genotypes with 100% female of evaluation (Table 5). Insects oviposited on all plant lines survival, numbers of eggs were high and did not follow and cultivars tested, albeit to varying degrees, starting on the same trend as number of surviving females, with the week 1, but levels of oviposition drastically decreased and exception of PI 290888 (Fig. 3A). On the other hand, for reached zero for resistant control FX-10 and several of the genotypes with <100% survival, a concomitant reduction evaluated PIs as weeks of the experiment went by. On week in number of eggs was observed as female survival went one, insects oviposited on all genotypes, with the highest down, with the exception of PI 647925 (Fig. 3B). number of eggs on Seville and the lowest number of eggs Fecal output, a proxy for insect feeding, was observed on PI 365031. The number of eggs oviposited across all on all tested genotypes. Overall, there were significant

Table 5. Mean number of eggs by genotype per week obtained from adult oviposition and feeding preference experiments in St. Augustinegrass. Values represent mean ± SE for six replicates per plant genotype. Values within columns followed by the same letter are not statistically different (Tukey’s mean separation test P ³ 0.5). Variable Genotype Week1 Week 2 Week 3 Week 4 No. eggs PI 289729 4.0 ± 0.58 efg 0.3 ± 0.21 f 1.0 ± 0.45 f 0.0 ± 0.00 e PI 290888 3.8 ± 0.87 efg 2.2 ± 0.83 ef 5.0 ± 0.58 def 2.7 ± 0.88 cde PI 291594 1.3 ± 0.61 fg 0.0 ± 0.00 f 0.2 ± 0.17 f 0.0 ± 0.00 e PI 300129 1.0 ± 0.52 fg 0.0 ± 0.00 f 0.3 ± 0.33 f 0.0 ± 0.00 e PI 300130 3.3 ± 0.49 efg 0.7 ± 0.21 ef 1.7 ± 0.67 ef 0.7 ± 0.33 de PI 365031 0.7 ± 0.49 g 0.2 ± 0.17 f 0.0 ± 0.00 f 0.0 ± 0.00 e PI 410361 16.2 ± 2.02 b 17.7 ± 3.18 b 17.7 ± 2.82 b 14.2 ± 1.92 b PI 509038 7.0 ± 1.29 cdef 7.5 ± 1.15 cde 9.8 ± 1.05 cd 12.2 ± 1.49 b PI 509039 11.2 ± 1.58 bc 13.2 ± 2.06 bc 15.7 ± 1.31 bc 14.0 ± 1.95 b PI 600734 6.0 ± 0.77 cdefg 2.5 ± 0.56 ef 2.5 ± 0.67 ef 0.8 ± 0.40 de PI 647924 1.3 ± 0.56 fg 0.0 ± 0.00 f 0.3 ± 0.33 f 0.0 ± 0.00 e PI 647925 6.7 ± 1.12 cdefg 3.0 ± 0.26 ef 3.7 ± 0.76 def 8.0 ± 2.00 bc Floratam 4.8 ± 0.65 defg 2.3 ± 0.42 ef 3.5 ± 0.76 def 0.8 ± 0.54 de FX-10 2.0 ± 0.58 fg 0.3 ± 0.21 f 0.0 ± 0.00 f 0.0 ± 0.00 e Captiva 5.2 ± 0.95 cdefg 2.8 ± 0.48 ef 1.7 ± 0.76 ef 0.2 ± 0.17 e Raleigh 10.7 ± 1.26 bcd 11.0 ± 2.16 bcd 9.8 ± 2.06 cd 7.3 ± 2.68 bcd Seville 22.5 ± 2.91 a 26.3 ± 3.56 a 29.2 ± 3.98 a 23.2 ± 2.96 a No. fecal spots PI 289729 10.5 ± 0.76 fgh 8.2 ± 0.48 fgh 7.8 ± 0.91 fg 5.8 ± 2.43 def 290888 16.1 ± 0.54 de 15.8 ± 1.33 cde 13.5 ± 0.85 def 17.2 ± 0.70 bc PI 291594 9.2 ± 0.40 h 12.0 ± 1.03 defg 10.0 ± 0.63 ef 4.0 ± 1.69 def PI 300129 11.0 ± 0.45 fgh 13.3 ± 1.09 def 10.7 ± 0.49 def 3.8 ± 1.01 def PI 300130 13.8 ± 0.48 efg 10.7 ± 0.67 efgh 13.7 ± 0.76 def 5.5 ± 1.86 def PI 365031 9.0 ± 0.58 h 6.0 ± 0.45 h 2.8 ± 0.48 g 1.3 ± 0.80 f PI 410361 26.2 ± 0.87 ab 24.0 ± 1.29 ab 27.5 ± 0.85 ab 28.3 ± 0.71 a PI 509038 24.7 ± 1.48 ab 19.8 ± 1.11 bc 26.3 ± 1.31 ab 25.3 ± 1.17 ab PI 509039 22.8 ± 0.79 bc 21.2 ± 1.30 bc 22.0 ± 0.73 bc 25.2 ± 0.91 ab PI 600734 14.7 ± 1.20 def 11.7 ± 0.56 defg 16.2 ± 1.05 cd 10.2 ± 2.15 cde PI 647924 8.0 ± 0.73 h 8.3 ± 0.61 fgh 10.5 ± 1.59 def 9.2 ± 1.89 cdef PI 647925 11.0 ± 0.82 fgh 14.2 ± 0.95 de 11.0 ± 2.05 def 11.5 ± 2.39 cd Floratam 18.8 ± 0.70 cd 17.2 ± 1.70 cd 13.7 ± 2.32 def 11.0 ± 3.51 cde FX-10 9.8 ± 0.60 gh 7.0 ± 0.73 gh 2.2 ± 0.54 g 0.7 ± 0.49 f Captiva 10.8 ± 0.79 fgh 13.7 ± 1.05 def 7.8 ± 1.01 fg 2.7 ± 0.61 ef Raleigh 18.3 ± 0.84 d 20.3 ± 1.67 bc 15.7 ± 1.20 de 11.5 ± 2.75 cd Seville 28.5 ± 1.18 a 28.2 ± 1.62 a 30.7 ± 1.26 a 30.3 ± 0.95 a

S-32 www.crops.org crop science, vol. 57, july–august 2017 Fig. 3. Total number of surviving females across days (black solid line) and total number of eggs across days (dotted blue line) for (A) plant genotypes with 100% insect survival and (B) plant genotypes with lower than 100% survival obtained from adult oviposition and feeding preference experiments in St. Augustinegrass. crop science, vol. 57, july–august 2017 www.crops.org S-33 differences among genotypes (Pillai’s trace test, Fval = 2009; Reinert et al., 2011). Those assays, however, were

6.76, dfnum = 68, dfden = 272, p < 0.0001). Throughout the conducted using insect populations from Texas. Texas has experiment, fecal output of SCB on PI 289729, PI 291594, a history of SCB populations with counter-adaptations PI 300129, PI 300130, PI 365031, and PI 647924 were not to host-plant resistance, and this may explain these dis- significantly different from that of SCB on the resistant check crepancies. Chong et al. (2009) inoculated different St. FX-10 (Table 5). Seville, on the other hand, had a signifi- Augustinegrass cultivars with 10 SCB nymphs and found cantly higher numbers of fecal spots compared with all other Raleigh to allow moderate levels of SCB development, genotypes. Plant introductions 410361, 509038, and 509039 yet it yielded lower levels of plant tissue damage. yielded fecal outputs equivalent to those of susceptible Seville St. Augustinegrass germplasm inhibiting or limiting (Table 5), indicating no negative effect on SCB feeding. adult feeding and oviposition can be helpful by slowing infestations from growing exponentially over time, effec- DISCUSSION tively reducing pest population size. In this study, adult Tolerant St. Augustinegrass germplasm offers the poten- preference based on oviposition and fecal output was also tial for maintaining green turfgrass throughout the year in significantly affected by plant genotype. Interestingly, spite of SCB presence, which could greatly reduce pesti- while Rangasamy et al. (2006) found that SCB produced 11 cide applications. Previous research by our group (Youngs and 5 times more eggs on Floratam compared with FX-10 et al., 2014) identified St. Augustinegrass PIs that exhib- and Captiva, respectively, in our study, SCB oviposition ited antibiosis and antixenosis when infested with SCB. and feeding preference on Floratam were significantly In the latter study, genotypes with comparatively high reduced compared with Seville. This agrees with findings numbers of recovered insects but low damage ratings were by Chong et al. (2009), who (using chinch bug populations observed; however, potential tolerance was not assessed. from South Carolina) reported that Floratam significantly The goal of the present study was to further that inves- suppressed SCB oviposition when compared with Raleigh. tigation by assessing plant response to different levels of Based on our results and the latter report, we conclude infestation. Additionally, we aimed at looking further into that Floratam has moderate resistance against more north- antibiosis by evaluating potential feeding and oviposition ern SCB populations, such as those in North Carolina and rates by adult pairs under no-choice scenarios. South Carolina. Unlike Florida and Texas, Floratam is In the feeding density experiments, significant dif- not widely planted in North Carolina; therefore, the SCB ferences were observed for damage ratings, number of used in this study have not been under the same selection insects recovered, dry weights, and FPLI among geno- pressure and have not yet developed counter-adaptations types. Across infestation levels, while the number of to this cultivar. This performance disparity may suggest insects recovered from susceptible check Seville and from genetic differences between Floridian and North Carolina PIs 509038 and 509039 were not significantly different, SCB populations. However, this is something that needs the damage ratings and FPLI were significantly lower for to be determined in future research. Oviposition on five these two genotypes. Therefore, we were able to deter- plant introductions—PI 289729, PI 291594, PI 300129, mine that these two PIs display tolerance to SCB feeding PI 365031, and PI 647924—was similar to that obtained even at the highest infestation level tested in this study. on the resistant reference cultivars FX-10 and Captiva. Both of these are diploid genotypes and also showed mod- Busey (1990) also reported significantly suppressed ovi- erate levels of antibiosis in a previous study (Youngs et al., position by both standard and resistance-breaking SCB 2014). Although Raleigh has previously been shown to on PI 365031. Moreover, feeding on the same five PIs, as exhibit antibiosis (Anderson et al., 2006), it was included assessed by fecal output, was equivalent or lower than that in this study because it allowed moderate levels of imma- observed on the resistant reference cultivars, indicating ture development during the antibiosis experiment yet markedly low consumption rates on these grass geno- had low damage ratings during the choice experiment types. It is interesting to note that, although adult SCB (Youngs et al., 2014). During the tolerance experiment, readily fed on most PIs, some even to levels equivalent to Raleigh’s damage ratings, dry weight, and FPLIs were not those of Seville, oviposition selection was more astringent. as affected by insect infestation levels (Fig. 2); however, Moreover, mortality of females was higher on some of these can be explained by the relatively high mortality our tested germplasm, indicating gender-biased lethality observed at both infestation levels. This confirms that on some of the lines tested herein. Raleigh’s mechanism of resistance is antibiosis rather than The SCB resistant St. Augustinegrass germplasm tolerance, which appears to be effective against adult SCB. identified in our study provide the basis for future research It is worth noting that, despite of our results and those of to develop more sustainable management alternatives for others (Anderson et al., 2006; Chong et al., 2009; Youngs SCB, while at the same time addressing concerns over et al., 2014), previous studies have found Raleigh to be pesticide use. Our study was able to confirm that two PIs susceptible to SCB (Crocker et al., 1989; Chong et al., display tolerance to SCB feeding and that five additional

S-34 www.crops.org crop science, vol. 57, july–august 2017 PIs have antibiosis activity against adult SCB. These mate- Crocker, R.L., R.W. Tolar, and C.L. Simpson. 1982. Bioassay of St. Augustinegrass lines for resistance to southern chinch rials represent valuable sources of SCB resistance for future bug (Hemiptera: ) and to St. Augustinegrass St. Augustinegrass breeding efforts. decline virus. J. Econ. Entomol. 75:515–516. doi:10.1093/ jee/75.3.515 Conflict of Interest Fleschner, C.A. 1952. Host-plant resistance as a factor influenc- The authors declare there to be no conflict of interest. ing population density of citrus red mites on orchard trees. J. Econ. Entomol. 45:687–695. doi:10.1093/jee/45.4.687 Acknowledgments Hayes, W.M. 1935. Biological races of insects and their bearing on We acknowledge George Kennedy, Rick Brandenburg, and host plant resistance. Entomol. News 46(1):20. Peter Hertl (NCSU, Entomology) for providing the field insect Haygood, R.A., and S.B. Martin. 1990. Characterization and populations used in this study. We also thank Matt Sidebottom, pathogenicity of species of Rhizoctonia associated with cen- Dina Espinoza, Paul Adams, and Stephanie Gorski (NCSU, tipedegrass and St. Augustinegrass in South Carolina. Plant Entomology) for their help with plant maintenance and insect Dis. 74:510–514. doi:10.1094/PD-74-0510 Heng-Moss, T.M., F.P. Baxendale, T.P. Riordan, and J.E. Fos- sorting. This work was funded, in part, by the Center for ter. 2002. Evaluation of buffalograss germplasm for resistance Turfgrass Environmental Research and Education at North to (Hemiptera: Lygaeidae). J. Econ. Entomol. Carolina State University and the North Carolina Crop 95:1054–1058. doi:10.1603/0022-0493-95.5.1054 Improvement Association. Henry, T.J., and R.C. Froeschner. 1988. Catalog of the Heterop- tera or true bugs of Canada and the continental United States. References CRC Press, Boca Raton, FL. Anderson, W.G., T.M. Heng-moss, F.P. Baxendale, L.M. 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