Beaumont Site Visit: Management of Stem Borers and Aphids Attacking , Energycane, , and Rice

Project Investigators: Gene Reagan, LSU AgCenter M.O. Way, Texas A&M AgriLife Research Mike Stout, LSU AgCenter Julien Beuzelin, LSU AgCenter, Dean Lee Research Center

Graduate Assistants: Matt VanWeelden Blake Wilson

Cooperators: David Kerns, Associate Professor, LSU AgCenter, Macon Ridge Research Station Jeff Davis, Associate Professor, LSU AgCenter, Department of Entomology Ted Wilson, Professor and Center Director, Texas AgriLife Beaumont Yubin Yang, Texas AgriLife Beaumont Bill White, USDA ARS Sugarcane Research Scientist, Houma, LA Tony Prado, Rio Grande Valley Sugar Growers Inc. Allan Showler, USDA ARS, Kerrville, TX Andy Scott, Rio Farms, TX

and

Rebecca Pearson, Texas AgriLife Beaumont Suhas Vyavhare, Texas AgriLife Beaumont Randy Richard, USDA ARS Sugarcane Research Station

15 September, 2015

This work has been supported by grants from the USDA NIFA AFRI Sustainable Bioenergy, and Crop Protection and Pest Management Programs, USDA Crops at Risk, and U.S. EPA Strategic Agricultural Initiative programs. We also thank the Texas Rice Research Foundation, the American Sugar Cane League and Rio Grande Valley Sugar Growers Inc, participating Agricultural Chemical Companies, the Texas Department of Agriculture and the Louisiana Department of Agriculture and Forestry for their support. Stem Borers and Aphids Attacking Sugarcane, Rice, and Sorghum

(b) Sugarcane borer larva (a) Adult female sugarcane borer

(c) Adult female Mexican rice borer (d) Mexican rice borer larva

(e) Sugarcane aphids on sorghum (f) Honeydew on sugarcane leaves from aphid feeding

Photos: (a) B. Castro; (b) J. Saichuk; (c) F. Reay-Jones; (e) B. Wilson; (d)(f) A. Meszaros

Texas A&M AgriLife Research and Extension Center at Beaumont

LSU AgCenter

USDA, Houma, LA

Gene Reagan, Mo Way, Julien Beuzelin, Mike Stout, David Kerns, Jeff Davis, Matt VanWeelden, Blake Wilson, Bill White, Ted Wilson, and Yubin Yang

The Beaumont Center will host a “Site Visit” on September 15, 2015 to discuss recent research results regarding pests (particularly Mexican rice borer and sugarcane aphid) attacking sugarcane, sorghum and rice. The goal of this visit is to educate stakeholders about progress towards managing stalk borers and aphids. Attendees will meet in the auditorium to hear presentations from LSU and Texas A&M entomologists before going to the field to observe stalk borers and aphids in experiments before lunch. This will be an informal visit with time for questions and discussion. Following the field visit, attendees will again meet in the auditorium to complete CEUs. Below is a summary of the details of the site visit:

Where: Beaumont Center, 1509 Aggie Dr., Beaumont, TX

When: Tuesday September 15, 2013

Time: Starts at 8:30 AM and ends about 1:00 PM

Snack lunch at 12:00

Contact: Mo Way, [email protected], 409-658-2186 for more information, if needed.

Please RSVP to Mo by email if you plan to attend---this will help determine sandwich, snack and drink orders.

Hope to see you September 15---drive safely!

Beaumont Site Visit: Meeting Agenda September 15th, 2015 – 8:30 AM – Inside Presentations (10–15 minute presentations).

Introduction Gene Reagan Stem borer research in sugarcane, rice, and bioenergy feedstocks – Movement of MRB into Louisiana and sugarcane varietal resistance (Blake Wilson) – MRB in conventional and bioenergy and (Matt VanWeelden) – Rice IPM studies in Louisiana and Texas (Dr. Mike Stout) – The big perspective of insect and disease management in bioenergy crops along the U.S. Gulf Coast (Dr. L.T. Wilson and Dr. Yubin Yang)

The sugarcane aphid as a pest of sorghum and sugarcane – Sugarcane aphid in Louisiana sugarcane (Dr. Julien Beuzelin) – Host plant resistance to the sugarcane aphid in sorghum (Dr. David Kerns) – Perspectives on the new USDA-NIFA IPM grant work (Dr. Jeff Davis)

Wrap up with MRB and aphids in rice, sugarcane, and sorghum (Dr. L.T. Wilson)

Field observations of Mexican rice borer and sugarcane aphid infestations (Dr. Mo Way)

Snack lunch (12:00 PM)

Table of Contents Stem borers and aphids attacking sugarcane, rice, and sorghum……………………….…………i Site visit announcement…………………………………………..……………………………….ii Meeting agenda……………………………………………………..…………………………….iii Sugarcane Stem Borer Research Mexican rice borer movement and pest status in Louisiana………………………………………1 Mexican rice borer in conventional and bioenergy sugarcane and sorghums…………………….3 Insecticidal control of the sugarcane borer in Louisiana sugarcane…………………….………...6 The big perspective of insect and disease management in bioenergy crops along the U.S. Gulf Coast………………………………………..…………………………………………..7 Rice Insect Management Research Integrating management tactics for the invasive Mexican rice borer into existing management programs in rice……………………………………………………………………10 Trapping for Mexican rice borer in the Texas rice belt………………………………………….12 Evaluation of seed treatments on XL753 and Antonio varieties for control of rice water weevil and stem borers. Eagle Lake, TX, 2014..…….………………………….…………...…………..13 Sugarcane Aphid Research The sugarcane aphid: A pest of sugarcane in Louisiana…………………….…….………….….15 Evaluating grain sorghum for resistance to sugarcane aphid…………………………………….17 Insecticidal control of the sugarcane aphid in sorghum. Beaumont, TX, 2014.…………………19 Biology, ecology, and impact of Melanaphis sacchari in sorghum and sugarcane production systems – New USDA-NIFA national competitive grant………………..21 Published Journal Article Reprints Yield response to Mexican rice borer (: ) injury in bioenergy and conventional sugarcane and sorghum. J. Econ. Entomol. DOI: 10.1093/jee/tov190…….…..….23 Expansion of the Mexican rice borer (Lepidoptera: Crambidae) into rice and sugarcane in Louisiana. Environ. Entomol. 44: 757–766………………………………...…32 A relative resistance ratio for evaluation of Mexican rice borer (Lepidoptera: Crambidae) susceptibility among sugarcane cultivars J. Econ. Entomol. DOI:10.1093/jee/tov07…………...42 Feeding by sugarcane aphid, Melanaphis sacchari, on sugarcane cultivars with differential susceptibility and potential mechanism of resistance. Entomol. Experimentalis et Applicata 150: 32–44…………………………………………………………………………….50

Mexican Rice Borer Movement and Pest Status in Louisiana B.E. Wilson1, M.T. VanWeelden1, T.E. Reagan1, J.M. Beuzelin2, and J. Meaux3 1LSU AgCenter, Department of Entomology 2LSU AgCenter, Dean Lee Research Station 3LSU AgCenter, Calcasieu Parish Extension Office Cooperative studies on the Mexican rice borer (MRB), Eoreuma loftini, between the LSU AgCenter, Texas A&M University AgriLIFE, the Texas Department of Agriculture, and the Louisiana Department of Agriculture and Forestry (LDAF) have been on-going since 2001 to monitor the movement of this devastating pest of sugarcane into Louisiana. Since its initial detection in Louisiana in 2008, MRB has been moving eastward into rice and sugarcane in southwestern parishes. Extensive trapping of MRB is being been conducted in southwest Louisiana by LDAF and LSU AgCenter personnel with more than 100 monitored in 15 parishes. To date, pheromone traps have detected MRB in Calcasieu, Cameron, Jefferson Davis, Beauregard, Allen, Vermillion, Acadia, and Evangeline Parishes (Fig. 1). Average monthly trap captures were greatest in Calcasieu and Jefferson Davis Parishes in both 2014 (Table 1) and 2015 (Table 2). Sentinel traps near the eastern edge of the known range of MRB are monitored by LSU AgCenter county extension agents in St. Mary, St. Martin, St. Landry, Iberia, and Lafayette Parishes to provide early detection of MRB expansion. Additional LDAF sentinel trapping efforts are focused in Pointe Coupe and West Baton Rouge Parishes as well as trapping near sugarcane mills in St. Mary and Iberia Parishes during harvest. Larval infestations have been recorded in sugarcane fields in Calcasieu, Jefferson Davis, and Vermilion Parishes. While these infestations have remained at low levels in most areas, mixed infestations of sugarcane borer (SCB) and MRB were detected in Calcasieu and Jefferson Davis in 2015 which reached levels of >20% injured stalks in some sugarcane fields. Damaging MRB infestations in Calcasieu rice fields that did not receive insecticidal seed treatments have been observed each year since 2012. However, only minimal infestations were observed in fields planted with Dermacor X-100 treated seed. The eastern most trap capture to date in 2015 is south of Rayne, LA in Vermilion Parish, representing little eastward expansion since 2014. Based on its average rate of spread since 2008 of roughly 15 mi/yr, MRB may become established throughout Vermillion and Acadia and may be detected in Lafayette, Rapides, and St. Landry Parishes during the 2016 growing season.

Table 1. MRB pheromone trap captures in SW Louisiana in 2014. No. of Mean MRB/Trap/Day Parish trap sites Mar Apr May Jun July Aug Sept Oct Nov Calcasieu 11 0.25 0.76 0.84 0.88 0.97 0.82 0.54 1.12 1.08 Jeff. Davis 16 0.34 2.78 1.33 1.13 2.02 1.16 3.73 1.02 2.12 Cameron 5 0.08 0.90 1.36 1.44 1.42 1.11 0.72 1.06 0.70 Beauregard 5 0 0 0.01 0.03 0.05 0.03 0.01 0.01 0.01 Allen 5 0 0.06 0.08 0.15 0.23 0.38 0.16 0.10 0.05 Acadia 10 NA NA 0.03 0.01 0.03 0.06 0.01 0.04 0.04 Vermilion 4 NA NA 0.02 0.02 0.03 0.04 0.0 0.0 0.0 Evangeline 1 NA NA 0.03 0 0 0.03 0.0 0.0 0.0 Table 2. MRB pheromone trap captures in SW Louisiana in 2015. No. of Mean MRB/Trap/Day Parish trap sites March April May June July Calcasieu 10 0.230 1.466 0.826 0.625 1.117 Jeff. Davis 17 0.939 2.962 0.913 0.817 2.351 Cameron 5 0.743 2.897 0.782 0.397 0.733 Allen 5 0.000 0.256 0.412 0.204 0.289 Beauregard 5 0.011 0.052 0.118 0.026 0.053 Acadia 11 0.054 0.152 0.064 0.132 0.166 Vermilion 5 0.000 0.099 0.060 0.110 0.026 Evangeline 1 0.006 0.004 0.000 0.000 0.046

Figure 1. Mexican rice borer range expansion in SW Louisiana as of July 2015. Traps present in Rapides, St. Martin, St. Landry, Iberia, Lafayette, and St. Mary Parishes have not yet detected MRB. Mexican Rice Borer (Lepidoptera: Crambidae) in Conventional and Bioenergy Sugarcane and Sorghum M.T. VanWeelden1, B.E. Wilson1, J.M Beuzelin2, T.E. Reagan1, and M.O. Way3 1Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 2Louisiana State University Agricultural Center, Dean Lee Research Center, Alexandria, LA 3Texas A&M AgriLIFE Research and Extension Center, Beaumont, TX The Mexican rice borer (MRB), Eoreuma loftini, is a pest of sugarcane and sorghum; however, its impact on dedicated bioenergy crops has not been examined. Because of the increase in demand for production of renewable fuel sources, research on pest complexes affecting current conventional crops is needed to reduce potential yield losses in bioenergy crops. A field experiment was conducted to assess the susceptibility of sugarcane, energycane, high- biomass sorghum, and sweet sorghum cultivars to MRB infestations and to measure subsequent yield losses from MRB injury. Two energycane (L 79-1002, Ho 02-113), two sugarcane (HoCP 04-838, HoCP 85-845), two high-biomass sorghum (ES 5200, ES 5140), and one sweet sorghum (M81E) cultivar(s) were subjected to four natural and artificially established MRB infestations levels (Table 1). Injury at natural MRB infestations ranged from 5.1 to 14.1% bored internodes, with injury being lowest in energycane and MRB-resistant sugarcane HoCP 85-845; however, resistance in HoCP 85-845 was reduced under enhanced infestations. Negative impacts to sucrose concentration from MRB injury were greatest in energycane, high-biomass sorghum, and sweet sorghum cultivars. Even under heavy MRB infestations, energycane and high-biomass sorghum were estimated to produce more ethanol than all other cultivars under suppressed infestations. A second field study was conducted to examine the impact of nitrogen (N) fertilization on MRB infestations and subsequent yields in cultivars of high-biomass and sweet sorghum. Percentage of bored internodes increased with higher N rates (Fig. 1), ranging from 2.9% at 0 kg N/ha to 8.4% at 135 kg N/ha across all cultivars. In ES 5200, injury increased over 2-fold when N was increased from 45 to 135 kg/ha, and injury in ES 5140 increased over 2.5-fold when N was increased from 45 to 135 kg/ha. In addition, adult emergence per stalk increased up to 7.4- fold when N was increased from 45 kg/ha to 135 kg/ha. Yield data indicated that N rate was positively associated with increases in stalk weight, sucrose concentration, and ethanol productivity (Table 2). Because higher N rates were associated with increased yields despite having greater levels of E. loftini injury, our data suggests that increases in yield from additional N outweigh decreases from additional MRB injury. Fertilization rates maintained between the recommended 45 and 90 kg N/ha should be used to minimize the risk of negative area-wide impacts from increased production of MRB adults while still allowing for optimum yields. To further examine the susceptibility of these crops to MRB, an oviposition preference experiment was conducted in the greenhouse. Mature plants possessed greater availability of dry leaf material compared to immature plants, and all MRB eggs were observed exclusively on dry leaf material. Sweet sorghum (M81E) was preferred over sugarcane (HoCP 04-838, HoCP 85- 845) and high-biomass sorghum (ES 5200, ES 5140) when measuring eggs per plant. The number of eggs per oviposition event was also greater on sweet sorghum than on sugarcane. This research showed that bioenergy crops are more resistant than conventional crops, demonstrating their compatibility in the Gulf Coast region. Because bioenergy crops are less tolerant to MRB in some instances, proper management practices should be continued.

Table 1. Energycane, sugarcane, high-biomass sorghum, and sweet sorghum parameter estimates across varying MRB infestation levels. Jefferson Co, TX, 2012‒2013. Ethanol Infestation Percent Bored Sucrose Cultivar Productivity Level Internodes Concentration (10^3 L ha-1) Suppressed 0.4 f 6.3 e‒h 16.1 Energycane Natural 6.0 b‒f 5.7 h 13.4 L 79-1002 Enhanced 12.4 a‒c 5.8 h 10.1 Highly-Enhanced 12.2 a‒d 5.9 gh 11.3 Suppressed 0.5 f 7.9 a‒d 18.5 Energycane Natural 5.2 b‒f 7.8 b‒e 16.1 Ho 02-113 Enhanced 7.2 b‒f 7.6 b‒e 14.7 Highly-Enhanced 11.9 a‒e 7.6 b‒f 13.7 Suppressed 0.8 ef 9.4 a 9.3 Sugarcane Natural 11.8 a‒e 8.9 ab 7.3 HoCP 04-838 Enhanced 16.3 ab 9.1 ab 6.3 Highly-Enhanced 19.7 a 9.0 ab 5.3 Suppressed 1.0 d‒f 8.6 a‒c 6.7 Sugarcane Natural 5.1 c‒f 8.6 a‒c 5.3 HoCP 85-845 Enhanced 14.4 a‒c 8.7 a‒c 4.4 Highly-Enhanced 15.0 a‒c 8.8 a‒c 4.6 Suppressed 0.0 f 6.1 f‒h 22.2 High-biomass Natural 14.1 a‒c 5.8 h 16.5 Sorghum ES 5200 Enhanced 9.8 a‒f 5.8 h 15.4 Highly-Enhanced 16.1 a‒c 5.7 h 15.8 Suppressed 0.2 f 5.5 h 11.5 High-Biomass Natural 9.8 a‒f 5.7 h 7.9 Sorghum ES 5140 Enhanced 13.4 a‒c 5.1 h 7.3 Highly-Enhanced 19.5 a 5.0 h 6.6 Suppressed 0.2 f 7.3 c‒f 9.6 Sweet Natural 8.8 a‒f 6.4 d‒h 6.0 Sorghum M81E Enhanced 8.0 b‒f 6.3 e‒h 6.6 Highly-Enhanced 10.3 a‒f 6.3 e‒h 5.0 *Means sharing the same letter are not significantly different (α=0.05).

12.0 0 kg N/ha a

SE) 45 kg N/ha

± 10.0 a 90 kg N/ha ab 8.0 135 kg N/ha abc

6.0 bc bc bc bc bc bc bc 4.0 c

2.0 PercentageBoredof Internodes ( 0.0 ES 5200 ES 5140 M81E

Figure 1. Evaluation of MRB injury in high-biomass (ES 5200, ES 5140) and sweet sorghum (M81E) across varying nitrogen (N) rates, Beaumont, TX, 2013. Columns with the same letter are not significantly different (α=0.05).

Table 2. High-biomass (ES 5200, ES 5140) and sweet sorghum (M81E) injury attributed to MRB and yield parameters, Beaumont, TX, 2013. N Rate Fresh Weight Sucrose Ethanol Productivity Cultivar (kg/ha) (kg) per Stalk Concentration (L/ha) 0 0.42 5.4 23238.0 High-biomass 45 0.49 5.7 23578.0 Sorghum ES 5200 90 0.54 5.7 26986.0 135 0.63 6.1 35627.0 0 0.18 4.7 10235.0 High-biomass 45 0.16 4.8 8440.0 Sorghum ES 5140 90 0.17 4.7 8300.1 135 0.28 5.5 14585.0 0 0.06 7.0 3192.4 Sweet Sorghum 45 0.10 6.7 5547.1 M81E 90 0.12 6.8 6998.9 135 0.17 6.1 6382.4

Insecticidal Control of the Sugarcane Borer in Louisiana Sugarcane J.M. Beuzelin1, B.E. Wilson2, M.T. VanWeelden2, and T.E. Reagan2. 1LSU AgCenter, Dean Lee Research Station, Alexandria, LA 2LSU AgCenter, Department of Entomology, Baton Rouge, LA The efficacy of insecticides labeled for sugarcane borer (SCB), saccharalis, management in sugarcane was evaluated over two years at Enterprise Plantation in Jeanerette, LA. Insecticide treatments (10 in 2013, 8 in 2014) and a non-treated check, replicated four times, were assessed in 4-row wide and 24-ft long plots of variety HoCP 96-540. Experimental plots were separated by 6-ft alleys. Pretreatment infestation counts in 2013 indicated SCB populations were above the recommended threshold of 5% of plants with larvae feeding on plant surfaces and insecticide applications were made on 1 July. In 2014, inspection of 300 randomly selected stalks on 28 Jul revealed SCB infestations were extremely low (0% with live larvae on plant surfaces). However, inspection of 100 stalks on 15 Aug showed that infestations attained 3% of stalks with at least one treatable larva, nearing the action threshold. Insecticides were applied on 15 Aug, 2014. Insecticides were applied with a CO2-pressurized backpack sprayer calibrated to deliver 10 GPA at 40 PSI. Differences between treated and non-treated plots were detected for percent bored internodes in both 2013 and 2014 (Figure 1). SCB injury in untreated plots was greater in 2013 (17.1% bored internodes) than in 2014 (7.4%). In 2013, Diamond, Belt, Prevathon, and Besiege provided successful control of SCB. However, numerical trends in the data suggest that Confirm provided less than adequate control of SCB in this test. Application timing and reduced residual of Confirm relative to other chemistries tested may have influenced the efficacy observed in this trial. However, it is possible that reduced susceptibility of SCB to Confirm is present and insecticide resistance management and utilization of alternative control strategies may be needed. Confirm was the least effective again in 2014; however, all treatments reduced SCB injury to < 2.0% bored internodes in this test.

18

16 14 2013 12 10 2014 8 6 4

Percent bored internodes bored Percent 2 0

Figure 1. SCB injury, small plot insecticide trial. Enterprise Plantation, Jeanerette, LA. The Big Perspective of Insect and Disease Management in Bioenergy Crops along the U.S. Gulf Coast Y. Yang1, L.T. Wilson1, J.M. Beuzelin2, T.E. Reagan3, and J. Wang1 1Texas A&M AgriLife at Beaumont 2LSU AgCenter, Dean Lee Research Station 3LSU AgCenter, Department of Entomology Integrated Crop-Pest Forecast and Analysis system The Mexican rice borer (MRB) and sugarcane borer (SCB) have a wide host range, which allows rapid movement and establishment over relatively large areas. Their seasonal and regional population dynamics are greatly affected by weather, crop distribution, abundance of non-crop hosts, and management practices. An integrated crop-pest forecast and analysis system has been developed to identify optimal field-scale and area-wide stem borer management strategies in multi-use agricultural landscapes. Major components include: 1) Pest models that simulate the dynamics of both pests in major conventional and bioenergy host crops, and in major weed hosts, 2) Crop models that accurately predict host plant growth, development, and yield as impacted by agronomic management and insect injury, 3) Weather, soil, and cropland distribution databases for analysis at multiple temporal and spatial scales, and 4) An interface that allows users to identify which management options provide the greatest economic return for the selected growing region and crop configuration. The pest models incorporate host-specific development, fecundity, survival and dispersal. The crop models simulate seasonality and availability of the 4 most abundant host crops (rice, corn, sorghum, and cane - sugarcane and energycane: Wilson et al., 2010; Yang et al., 2011) (Figure 1) and 4 non-crop hosts (johnsongrass, vaseygrass, ryegrass and brome: Beuzelin, 2011) (Figure 2) in the agricultural landscapes of the Upper Golf Cost. Non-crop host buffers around the edges of each field mimic actual field conditions. The seasonal growth of each weed host is dictated by its development threshold and thermal requirements to reach maturity. Biomass is simulated as a function of cumulative degree-days above each base development threshold. Model calibration and validation includes pest population dynamics and pest dispersal. Pest population dynamics is based on field-scale observational data. Pest dispersal is based on historic MRB trap data of pest spread from south to southeast Texas and to western Louisiana. Landscape-Wide Pest Management Strategies Field- and regional-scale pest management strategies are being developed. Major factors considered in the analysis include 1) Level of host plant resistance for sugarcane and energycane varieties, 2) Planting dates, 3) Field- and regional crop rotation patterns, 4) Nitrogen fertilization and irrigation management, 5) Temperatures and precipitation, 6) Management of weeds surrounding crop fields, 7) Seasonal pest density, and 8) Insecticidal control. Each factor will be analyzed alone and in combination, and optimal strategies will be identified. Regional management will focus on how to mitigate pest pressure within both bioenergy and associated conventional crops. A critical component of area-wide management is SCB and MRB movement between non-crop, conventional, and bioenergy crop hosts as their availability changes throughout the seasons (Figure 3).

Figure 1. Crop hosts: (1) Biomass as a function of Figure 2. Grassy weed hosts (1) Biomass as a function thermal time. A. Rice; B. Corn; C. Biomass of thermal time: A. Johnsongrass; B. Vaseygrass; C. sorghum; D. grain Sorghum; E. Cane; (2) Plant Ryegrass; D. Brome; (2) Plant height as a function of height as a function of thermal time. F. Rice; G. thermal time: E. Johnsongrass; F. Vaseygrass; G. Corn; H. Biomass sorghum; I. grain Sorghum; J. Ryegrass; H. Brome Cane

References Beuzelin, J.M., 2011. Agroecological factors impacting stem borer (Lepidoptera: Crambidae) dynamics in Gulf Coast sugarcane and rice. Deparement of Entomology. Louisiana State University, Baton Rouge, p. 213. Wilson, L.T., Yang, Y., Wang, J., 2010. Integrated Agricultural Information and Management System (iAIMS): Cropland Data. March 2010 (http://beaumont.tamu.edu/CroplandData) (accessed 8/6/2013). Yang, Y., Wilson, L.T., Wang, J., Li, X., 2011. Development of an integrated cropland and soil data management system for cropping system applications. Computers and Electronics in Agriculture 76, 105-118.

Figure 3. Simulated spread of Mexican rice borer over a multi-use landscape in southeast Texas from a hypothetical initial single-field infestation in 2003: Top figure (Cropland distribution); Middle figure (Spread after 3 years); bottom figure (Spread after 6 years) Integrating Management Tactics for the Invasive Mexican Rice Borer into Existing Management Programs in Rice M.J. Stout1, J.M. Beuzelin2, M.O. Way3, D. Groth4, B. Tubana5, and D. Ring1 1LSU AgCenter, Department of Entomology 2 LSU AgCenter, Dean Lee Research Station 3Texas A&M AgriLife at Beaumont 4LSU AgCenter, Rice Research Station 5LSU AgCenter, School of Plant, Environmental, and Soil Sciences The Mexican rice borer (MRB) is a destructive pest of graminaceous crops such as rice, , and sugarcane. The larval stage of this insect damages crop plants by boring into stems and feeding internally. This insect invaded south Texas from Mexico in the early 1980s, rapidly becoming the major pest of Texas sugarcane. Within 10 years, its range expanded into the rice- producing areas of Texas, and over the past two decades the MRB has become an important pest of Texas rice. Recently, the MRB has invaded and begun to establish in the major rice-producing region of Louisiana, and continued range expansion of the MRB threatens other important rice-, sugarcane-, sorghum-, and maize-producing regions in the southern U.S. Although other stem- boring species such as the sugarcane borer have long been present at low levels in Louisiana rice, Louisiana rice producers have historically not suffered large losses from borers. They have, however, suffered large losses from other insect and disease pests such as the rice water weevil and sheath blight, and rice producers regularly employ management tactics such as resistant varieties and pesticide applications against these long-established pests. As the MRB continues to spread and establish in Louisiana, there is an urgent need to develop and implement a comprehensive pest management program that reduces the impact of MRB on rice in a sustainable manner, retards the spread of the insect into other important production areas, and is compatible with existing management practices for other pests of rice. In the fall of 2014, researchers from the LSU AgCenter and Texas A&M received funding from the USDA (Crop Protection and Pest Management Program) to address the emerging threat of the MRB in the context of the existing pest complex in rice. The goal of the project is to develop and implement a cost-effective, sustainable management program in rice for the invasive MRB that is compatible with management programs for the existing complex of rice pests, including the rice water weevil, the sugarcane borer, and sheath blight. The Objectives of this project include evaluating commercial rice varieties for resistance to multiple disease and insect pests, characterizing the interactive effects of nitrogen (N) and silicon (Si) fertilization on multiple insect and disease pests, and optimizing rates of chlorantraniliprole (Dermacor X-100) seed treatments for simultaneous rice water weevil and stem borer management. Additional experiments are proposed to quantify yield impacts of MRB infestation in rice and to investigate the influence of early-season infestations by the rice water weevil on later-season attacks by stem borers. A diverse set of extension activities are proposed to educate stakeholders about the MRB management program, increase grower involvement in monitoring, and evaluate adoption of IPM tactics. Experiments initiated in the first year of the funding period are still ongoing. Several obstacles were encountered in meeting grant objectives for 2014, including wet weather that interfered with planting, difficulties in establishing laboratory colonies of stem borers, and low borer populations in Crowley, Louisiana, one of the two primary field sites. Several field and greenhouse experiments were conducted to evaluate the independent and combined effects of N and Si fertility and chlorantraniliprole seed treatments on rice water weevils and stem borers. Two large experiments were conducted to evaluate commonly grown varieties for multiple pest resistance. The influence of early season rice water weevil infestations on late season stem borer infestations is also being evaluated. Many of these experiments have not yet been harvested and most data have not yet been analyzed. The expected outcome of the project is a coordinated set of multi-tactic management programs for the pest complex in rice that will be adopted by growers in areas where the MRB has invaded or will invade. From a broader perspective, the proposed research explores the fundamental issue of integration in integrated pest management (IPM). In theory, integration is an important aspect of IPM programs at all levels; in practice, however, management tactics for a pest are often developed or studied in isolation from other tactics, and in isolation from management programs for other pests or from prevailing agronomic practices. Resolution of conflicts in recommendations are often left to producers, who typically make decisions based on ease of implementation, short-term economic considerations, and perceived rather than actual importance of one pest vis-à-vis other pests. The invasion and ongoing establishment of a new pest in rice, a crop with a well-studied complex of existing pests, will allow us to explore how establishment of a new pest influences other pests, and how management tactics for the new pest interact with one another and with management tactics for other pests. Trapping for Mexican Rice Borer in the Texas Rice Belt M.O. Way, R.A. Pearson, J. Vawter, and N. Jordan. Texas A&M AgriLife at Beaumont Mexican rice borer (MRB) pheromone traps were set up in selected counties of the Texas Rice Belt. MRB was detected for the first time in Louisiana in November 2008. MRB was collected for the first time in Orange Co. in September 2010. Data are being used to follow the progress of MRB population densities over time in the TRB. In December 2012, an MRB was found in a light trap in Florida. Trap captures were greatest in Colorado and Chambers Counties.

Table 1. Monthly totals of Mexican rice borer adults from pheromone traps (2 traps/county) located next to rice on the Texas Upper Gulf Coast in 2014. Month Chambers Co. Colorado Co. Jefferson Co. Orange Co. January 0 1 0 0 February 0 2 0 0 March 53 108 2 1 April 251 886 2 2 May 55 204 4 2 June 105 500 25 0 July 39 240 31 0 August 59 109 25 0 September 1 39 3 0 October 51 64 13 1 November 47 2 15 0 December 58 3 1 0

Evaluation of Seed Treatments on XL753 and Antonio Varieties for Control of Rice Water Weevil and Stem Borers. Eagle Lake, TX, 2014. M.O. Way and R.A. Pearson Texas A&M AgriLife at Beaumont Two separate replicated field studies at Eagle Lake, TX evaluated seed treatments and foliar applied insecticides in drill seeded XL753 and Antonio rice. Treatments included Dermacor X- 100, CruiserMaxx, and NipsIt INSIDE applied to seed at labeled rates. The Cruiser Maxx and NipsIt INSIDE treatments also received a foliar application of Karate Z applied on July 3, 2014. Rice water weevil (RWW) cores (5 cores per plot, each core 4 inches diameter, 4 inches deep, containing at least one rice plant) were collected on Jun 12 and Jun 20. Core samples were stored in a cold room, later washed through 40 mesh screen buckets and immature RWW counted. Panicle counts (3, 1 ft counts/plot) and whitehead (WH) counts (4 rows) were taken on Jul 25 (main) and Oct 30 (ratoon). White heads are a measure of stem borer injury. Plots were harvested on Aug 20 (main crop) and on Nov 3 and 19 (ratoon crop). In both experiments, rice water weevil (RWW) populations were above treatment threshold (about 15 larvae/pupae per 5 cores) in the untreated on the 1st sample date (Table 1). None of the seed treatments performed as well as expected on either variety, but the Dermacor X-100 treatment gave the best control of RWW (Tables 1 and 2). I am uncertain as to the reason(s) for this lack of control. RWW populations were similar between the two varieties. Panicle density was about the same, regardless of treatment. Whiteheads (WHs) are a measure of stem borer activity. Based on sample dissections, the vast majority of stalk borers in both experiments were Mexican rice borer. WH density was high in the untreated and significantly lower in all insecticidal treatments for the main crop (Tables 1 and 2). This suggests the Karate Z applications at late boot/early heading and the Dermacor X-100 seed treatment effectively controlled stem borer damage in the main crop. However, for the ratoon crop only Dermacor X- 100 seed treatment significantly reduced WH density in both experiments. This strongly suggests Dermacor X-100 controlled stem borer damage both in the main and ratoon crops. WH density was also reduced in the ratoon crop of NipsIt treated plots in the XL753 experiment. Also, applications of Karate Z on the main crop did not control stalk borer damage on the ratoon crop. Furthermore, data from both experiments indicate Dermacor X-100 seed treatment actually controls stem borers infesting the ratoon crop. The number of WHs was approximately 2-fold greater in the Antonio experiment than in the XL753 experiment. Main crop yields were numerically higher in all insecticide treatments compared to the untreated in both experiments (Table 3 and 4). Ratoon crop yields were also numerically higher in all insecticide treatments compared to the untreated with the exception of Cruiser Maxx in the XL753 test. Ratoon yields in the Dermacor X-100 treatment in the Antonio experiment were significantly higher than in the untreated. In addition, total yields (main + ratoon crop yields) were significantly higher than the untreated for Dermacor X-100 and NipsIt INSIDE + Karate Z treatments in the Antonio experiment. The Dermacor X-100 treatment outyielded the untreated, in terms of main + ratoon crops, by 1,417 lb/A in the Antonio experiment. No differences in total crop yields were detected between treated and untreated plots in the XL753 experiment. Dermacor X-100 provided a degree of control (about 50% control) of RWW and effective control of stem borers on both main and ratoon crops in both the Xl753 and Antonio experiments.

Table 1. Mean insect and panicle count data for XL753 seed treatments. a a Rate No. RWW /5 cores Panicles/ft No. WHs /4 rows Treatment (fl oz/cwt) Jun 12 Jun 20 of row Main Ratoon Untreated --- 45.3 a 9.8 a 22.8 16.3 a 15.5 a CruiserMaxx Rice + 7 + 36.5 ab 9.0 a 21.3 1.5 b 14.0 a Karate Zb 0.03 lb ai/A Dermacor X-100 5.0 21.8 c 2.8 b 21.0 5.0 b 5.0 b NipsIt INSIDE + 1.92 + 30.0 bc 6.3 ab 21.4 1.5 b 6.0 b Karate Zb 0.03 lb ai/A aRWW = rice water weevil; WH = whitehead. bApplied at late boot/heading. Means in a column followed by the same letter are not significantly different (P > 0.05). Table 2. Mean insect and panicle count data for Antonio seed treatments. a a Rate No. RWW /5 cores Panicles/ft No. WHs /4 rows Treatment (fl oz/cwt) Jun 12 Jun 20 of row Main Ratoon Untreated --- 34.8 a 12.8 24.0 32.0 a 32.5 a CruiserMaxx Rice + 7 + 26.8 ab 6.8 25.5 6.8 b 35.8 a Karate Zb 0.03 lb ai/A Dermacor X-100 1.75 15.8 c 2.5 25.5 4.8 b 8.5 b NipsIt INSIDE + 1.92 + 22.8 bc 7.3 23.4 4.8 b 32.0 a Karate Zb 0.03 lb ai/A aRWW = rice water weevil; WH = whitehead. bApplied at late boot/heading. Means in a column followed by the same letter are not significantly different (P > 0.05). Table 3. Mean yield data for XL753 seed treatments. Rate Yield (lb/A) Treatment (fl oz/cwt) Main Ratoon Total Untreated --- 10740 3911 14651 CruiserMaxx Rice + Karate Za 7 + 0.03 lb ai/A 10994 3787 14781 Dermacor X-100 5.0 10823 4420 15242 NipsIt INSIDE + Karate Za 1.92 + 0.03 lb ai/A 11213 4120 15333 aApplied at late boot/heading. Means in a column are not significantly different (P > 0.05). Table 4. Mean yield data for Antonio seed treatments. Rate Yield (lb/A) Treatment (fl oz/cwt) Main Ratoon Total Untreated --- 7811 1327 b 9137 c CruiserMaxx Rice + Karate Za 7 + 0.03 lb ai/A 8092 1520 b 9613 bc Dermacor X-100 1.75 8317 2237 a 10554 a NipsIt INSIDE + Karate Za 1.92 + 0.03 lb ai/A 8266 1534 b 9800 b aApplied at late boot/heading. Means in a column followed by the same letter are not significantly different (P > 0.05).

The Sugarcane Aphid: A Pest of Sugarcane in Louisiana J.M. Beuzelin1, M.T. VanWeelden2, B.E. Wilson2, T.E. Reagan2, and W.H. White3 1LSU AgCenter, Dean Lee Research Station, Alexandria, LA 2LSU AgCenter, Department of Entomology, Baton Rouge, LA 3USDA-ARS, Sugarcane Research Unit, Houma, LA

The sugarcane aphid (SCA), Melanaphis sacchari (Zehntner), was first detected in the continental U.S. in Florida in 1977 and was first observed in Louisiana near Houma in September 1999. A survey conducted immediately following the first detection of the SCA in Louisiana found it occurring in 8 of the 21 sugarcane producing parishes. At the time, the potential pest status of the insect for Louisiana sugarcane was unknown. LSU AgCenter and USDA-ARS scientists have studied aspects of SCA biology and management in sugarcane, including population dynamics, pest status, varietal resistance, the role of natural enemies, and insecticide efficacy. The SCA has been the most abundant aphid in Louisiana sugarcane during the past decade. Typically, infestations start low in the spring, increase in May and June, peak in July, and then decline. The SCA is a virus vector, transmitting Sugarcane yellow leaf virus (ScYLV), which causes yellow leaf. Field experiments over two growing seasons showed that whenever there was an increase in SCA populations, there was an increase in yellow leaf incidence. Losses in sugar yield associated with ScYLV infections have attained 11-14% in Louisiana sugarcane varieties. However, absence of yield loss is also observed. ScYLV-free certification is now required for micro-propagated seed cane. The SCA can also transmit mosaic caused by strains of Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV). In addition to viruses, SCA population build up is associated with honeydew accumulation on leaves and subsequent sooty mold development, which can reduce photosynthesis and increase plant stress. Grid sampling of four fields recording aphid abundance in the summer and end-of-season yield could not provide conclusive evidence of the negative impact of increasing numbers of SCA on sugarcane yield, TRS, and sugar yield. Thus, the SCA is currently considered a sporadic sugarcane pest that can cause substantial yield losses on occasion. Sugarcane variety can have a substantial effect on SCA population size. Greenhouse and field experiments studied five commercial varieties: LCP 85-384, HoCP 91-555, Ho 95-988, HoCP 96-540, and L 97-128. In the field, population build up was lowest on HoCP 96-540 and HoCP 91-555. In the greenhouse, SCA did not prefer feeding on a particular variety whereas feeding on HoCP 91-555 decreased fecundity and longevity. In addition, feeding on this resistant cultivar negatively impacted intrinsic rate of natural increase (rm), rate of increase (λ), and doubling time (DT). The absence of certain free amino acids (nitrogen-containing nutrients) in the sap of HoCP 91-555 is thought to confer resistance to the SA. Natural enemies of the SA include fungi like Verticillium lecanii, parasitoids like the parasitic wasp Lysiphlebus testaceipes, and predators like the lady beetle Diomus terminatus. Fungi are believed to be the most effective natural enemies. Natural enemies, as a group, may mitigate SCA outbreaks but are not used as a SCA management tool. Only pyrethroids are currently labeled for SCA control in sugarcane. These insecticides applied in a high volume of water as directed sprays can control the SCA. However, in the most recent insecticide efficacy evaluation, which used a CO2-pressurized backpack sprayer to apply insecticides approximately 2 ft above the canopy at 10 gpa, the pyrethroid Baythroid XL did not suppress or control SCA populations. In the same evaluation, Transform WG at 1.5 oz/A provided the greatest level of control, but products containing imidacloprid provided satisfactory control. Thus, labeled pyrethroids are generally not recommended for SCA control because of lack of efficacy and potential non-target effects. In addition, thresholds assisting in timing insecticide applications have not been developed. In conclusion, the SCA has become a pest of sugarcane in Louisiana since its introduction into the state over 15 years ago. However, the status of the SCA as a pest of sugarcane remains unclear. There is now national interest in managing the SCA because the insect has become a severe problem in sorghum nationwide. Although there are numerous differences between the situation in sugarcane and in sorghum, there is an urgent need to (1) quantify life table parameters for SCA populations on resistant and susceptible varieties of sugarcane and sorghum, (2) develop an efficient field sampling protocol, (3) determine the susceptibility and injury-yield response of sugarcane and sorghum varieties, and (4) develop an integrated decision-making tool. Evaluating Grain Sorghum for Resistance to Sugarcane Aphid David L. Kerns LSU AgCenter, Macon Ridge Research Station Since its introduction into U.S. grain sorghum in 2013, the sugarcane aphid has been a difficult and costly pest to manage for sorghum producers from New Mexico to the Carolinas. Managing sugarcane aphid populations requires the use of an integrated approach that includes hybrid selection, planting date, insecticide seed treatments, foliar insecticide selection, targeted application techniques, foliar insecticide timing, other pest considerations, crop desiccation and harvest conditions. Early or timely planting will help avoid sugarcane aphid infestations and hybrid selection is fundamental in reducing aphid populations while maximizing yield throughout the growing season. Hybrids with taller stalks – more space between panicle and leaf canopy – may facilitate easier harvest by reducing the amount of honeydew coated chaff moving through the combine. Additionally, there is strong evidence that some sorghum hybrids offer resistance to sugarcane aphid. Currently all major sorghum seed companies and university breeding programs are actively breeding sorghum for resistance to sugarcane aphid. There have been a number of new inbred lines identified that appear to offer excellent resistance to sugarcane aphid. However, it could be several years before these traits are available in highly productive commercial hybrids. In the meantime we are primarily forced to rely on insecticides and currently available hybrids. Fortunately some commercially available sorghum hybrids appear to offer some resistance to sugarcane aphid. However, this resistance may not be expressed completely throughout the season or may be insufficient under high aphid numbers.

Figure 1. Sorghum Association seedling screening for resistance to sugarcane aphid, LSU AgCenter.

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Figure 3. Aphids per lower and flag leaf at harvest. Insecticidal Control of the Sugarcane Aphid in Sorghum. Beaumont, TX 2014. M.O. Way and R.A. Pearson, Texas A&M AgriLife at Beaumont An experiment was conducted to evaluated selected insecticides for control of the sugarcane aphid (SCA) in sorghum at the Beaumont research station in 2012. The experiment was a randomized complete block with 9 treatments and 4 replications. Plot size was four 20 long rows with a 30 in row spacing. Sorghum (variety Dekalb S53-67) was drill planted on 21May, 2014 and urea was applied at planting and again on 28 July. Plots were irrigated as needed. Natural SCA infestations were enhanced by clipping infested sweet sorghum leaves to leaves in all plots on Aug 22. Insecticide treatments were applied on Sep 24 with a 3-nozzle spray boom (800067 tips, 50 mesh screens, 25 gal/acre final spray volume). Pre-treatment infestations were recorded by counting the number of SCA on one leaf from 10 plants per plot on Sept 23. The number of SCA on one leaf from 20 plants per plot was recorded on Sept 26, Sept 29, and Oct 22. The experiment was planted late in hopes of encouraging SCA populations. The experiment was planted in 2 blocks normally reserved for rice experiments; thus, we were able to irrigate our grain sorghum plots when needed (blocks were flush irrigated---temporary flood immediately followed by draining). We selected the hybrid variety Dekalb S53-67 based on observations by local farmers who grew this variety in 2013 and experienced problems with SCA. We did not observe any SCA in plots until grain sorghum was well past heading (late August). Unfortunately, blackbird pressure was severe, so our yield data are suspect and unreliable. However, in mid-August, we applied AV-1011 to all plots. AV-1011 is a bird repellent manufactured by Arkion Life Sciences. Although AV-1011 is not registered on grain sorghum, the application was made to our research plots which are exempt from registration requirements. Following the experiment, grain sorghum heads as well as vegetation were destroyed and did not enter the food chain. We did not observe many birds in plots after application of AV-1011, so we believe the repellent was effective. We plan on applying AV-1011 in the future to our grain sorghum research plots. In addition, we did not observe any AV-1011 effects on SA populations. To complicate yield losses further, we did not specifically control any other insect pests, such as stink bugs, head worms and sorghum midge which were not monitored, but probably were present in the plots. Counting SCAs in the field is not exact. With experience, we were able to estimate SA populations by counting aphids in groups of 10. Pretreatment counts were not significantly different among treatments (Table 1). Counts 2, 5 and 9 days after treatment (DAT) showed significantly lower populations (compared to the untreated) in the following treatments: Transform WG (both rates), Sivanto, Endigo ZCX, and Centric 40WG. At 16 DAT, populations in the untreated were low, so results are not reliable. Data suggest the low rate of Transform WG performed as well as the high rate. Although Lorsban Advanced, Dimethoate 4EC and Fulfill treatments did not perform as well as the other treatments, they did reduce SCA populations.

Table 1. Mean data for sorghum insecticide screening study. Beaumont, TX. 2014. Wet No. sugarcane aphids/leafa Rate wt./seed Treatment (fl oz/A) Pret. 2 DAT 5 DAT 9 DAT 16 DAT head (g) Transform WGb 0.75 oz/A 20.7 4.8 d 1.8 d 1.1 cd 0.6 bc 21.3 Transform WGb 1.5 oz/A 57.8 7.9 cd 2.8 d 0.2 d 0 c 22.1 Lorsban Advancedb 24 20.6 21.2 abc 25.9 ab 15.1 a-d 8.6 bc 18.5 Sivantoc 5 39.9 2.5 d 10.7 cd 0.4 d 0.7 bc 18.1 Dimethoate 4ECb 16 40.1 16.9 a-d 16.4 abc 16.8 abc 24.8 a 20.5 Endigo ZCXc 5 65.1 10.3 bcd 14.4 bcd 1.4 bcd 2.4 bc 21.2 Fulfilld 5 oz/A 56.4 31.2 ab 24.4 abc 24.0 ab 2.2 bc 20.9 Centric 40WGc 2.5 oz/A 43.9 8.0 cd 6.9 bcd 0.6 cd 0.2 bc 20.2 Untreated --- 67.6 43.5 a 46.2 a 36.7 a 10.0 b 19.6 a Pret. = pretreatment; DAT = days after treatment b Also includes COC @ 1% v/v c Also includes NIS @ 0.25% v/v d Also includes MSO @ 1% v/v Means in a column followed by the same or no letter are not significantly (NS) different (P = 0.05, ANOVA and LSD)

Biology, Ecology, and Impact of Melanaphis sacchari in Sorghum and Sugarcane Production Systems – New USDA-NIFA National Competitive Grant Jeff Davis1, T.E. Reagan1, J.M. Beuzelin2, F.P.F. Reay-Jones3, L.T. Wilson4, and Y. Yang4 1LSU AgCenter, Department of Entomology 2LSU AgCenter, Dean Lee Research Station 3Clemson University, Pee Dee Research Station 4Texas A&M AgriLife at Beaumont Populations of the sugarcane aphid, Melanaphis sacchari, reached unprecedented levels in 2013 and 2014, devastating grain sorghum throughout the mid-south and southeastern United States. Melanaphis sacchari is also an important pest of sugarcane throughout the world, including Louisiana, Florida, and Texas as a vector of sugarcane yellow leaf virus. Current management tactics available for M. sacchari in sugarcane and sorghum rely solely on the use of broad spectrum insecticides without economic thresholds, cultural practices, or resistant varieties. Development of biology- and ecology-based management strategies which reduce reliance on insecticides is needed. Our project aims to quantify the effects of host crop, variety, plant phenology, and nitrogen fertilization level on M. sacchari life table parameters. Additionally, the project will develop an efficient rapid aphid density assessment rating (RADAR) sampling protocol easily implemented in the field. Aphid density-yield response studies will be used in development of economics to improve sustainability of M. sacchari control. Greenhouse assays and replicated field studies will identify factors which influence the biology, ecology, and impact of M. sacchari infestations in sugarcane and sorghum. Results from these studies will be incorporated into an integrated forecasting model. A web-based predictive tool will be developed and delivered to stakeholders to aid in making IPM decisions based on forecasted infestations and resulting impacts on yield. Coordinated outreach efforts involving LSU AgCenter, Texas AgriLife and Clemson University research and extension services will deliver key knowledge and technologies to stakeholders through multiple media channels. Such information will be promoted nationally and internationally. Life system parameters on plants and host crops other than sugarcane are presented in Table 1, and the differences among several varieties of sugarcane for the sugarcane aphid are given in Table 2. Table 1. Life Table parameters for M. sacchari on various host grasses (J. Davis unpub.). Host Plant rm TG R0 DT Grain Sorghum (Sorghum bicolor) 0.57 9.4 67.1 1.21 Johnsongrass (S. halepense) 0.36 8.6 14.6 1.91 Rice (Oryza sativa) -0.02 7.5 0.9 69.09 Wheat (Triticum aestivum) 0.23 6.1 8.3 3.64 rm = intrinsic rate of increase, TG = generation time (days), R0 = net reproductive rate (progeny/female), and DT = doubling time (days).

Table 2. Life history parameters of M. sacchari reared on sugarcane.* Prereproductive Reproductive Variety Fecundity Fecundity/d Longevity (d) period (d) period (d) LCP 85-385 8.0 a 14.4 b 15.8 a 1.12 a 25.0 ab HoCP 91-555 10.2 a 10.6 b 3.4 b 0.32 b 24.0 b Ho 95-988 9.8 a 15.6 ab 11.8 ab 0.72 ab 28.6 ab HoCP 96-540 10.4 a 14.6 ab 11.8 ab 0.82 ab 28.4 ab L 97-128 7.6 a 21.2 a 19.6 a 0.92 a 31.6 a Means within a column which share a letter are not different (P > 0.05 Tukey’s HSD test). *Data from Akbar et al. 2010. The long term goal of our project is to identify factors which influence the biology, ecology and impact of M. sacchari for use in development and implementation of a sustainable IPM program for this pest in sugarcane and sorghum. Specific objectives are: 1. Quantify the life table parameters for M. sacchari populations on resistant and susceptible varieties of sugarcane and sorghum at different phenological stages and nitrogen fertilization levels (Research Objective) 2. Develop a Rapid Aphid Density Assessment Rating (RADAR) for efficient field sampling (Research Objective) 3. Determine the susceptibility and injury-yield response of sorghum and sugarcane varieties (Research Objective) 4. Develop an integrated and predictive RADAR-based decision tool. (Extension Objective) 5. Implement a multi-channel extension outreach program for M. sacchari management. (Extension Objective) This program is a compilation of efforts by the LSU AgCenter, Texas A&M AgriLife at Beaumont, and Clemson University Research and Extension Center in Florence, South Carolina.

Journal of Economic Entomology Advance Access published July 6, 2015

FIELD AND FORAGE CROPS Yield Response to Mexican Rice Borer (Lepidoptera: Crambidae) Injury in Bioenergy and Conventional Sugarcane and Sorghum

1,2 1 3 2 4 M. T. VANWEELDEN, B. E. WILSON, J. M. BEUZELIN, T. E. REAGAN, AND M. O. WAY

J. Econ. Entomol. 1–9 (2015); DOI: 10.1093/jee/tov190 ABSTRACT The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae) is an invasive stem borer of sugarcane, Saccharum spp., and sorghum, Sorghum bicolor (L.), and poses a threat against the production of dedicated bioenergy feedstocks in the U.S. Gulf Coast region. A 2-yr field study was conducted in Jefferson County, TX, to evaluate yield losses associated with E. loftini feeding on bioenergy and conventional cultivars of sugarcane and sorghum under natural and artificially established E. loftini infestations. Bioenergy sugarcane (energycane) ‘L 79-1002’ and ‘Ho 02-113’ and sweet sorghum ‘M81E’ exhibited reduced E. loftini injury; however, these cultivars, along with high-biomass sorghum cultivar ‘ES 5140’, sustained greater losses in fresh stalk weight. Negative impacts to sucrose concentration from E. loftini injury were greatest in energycane, high-biomass sorghum, and sweet sorghum cultivars. Even under heavy E. loftini infestations, L 79-1002, Ho 02-113, and ‘ES 5200’ were estimated to produce more ethanol than all other cultivars under suppressed infestations. ES 5200, Ho 02-113, and L 79-1002 hold the greatest potential as dedicated bioenergy crops for production of ethanol in the Gulf Coast region; however, E. loftini management practices will need to be continued to mitigate yield losses.

KEY WORDS Eoreuma loftini, Saccharum spp., Sorghum bicolor, invasive species, yield loss

With the onset of the Energy Independence and Secur- the greatest potential for production because of exten- ity Act of 2007, the U.S. government has mandated an sive arable land, abundant rainfall, and mild winters. In increase in the minimum levels of renewable fuel used addition, Gulf Coast states (e.g., Texas, Louisiana, and in transportation fuel to 136.3 billion liters by 2022 Florida) already possess infrastructure required for (U.S. Environmental Protection Agency 2007), which growing related conventional graminaceaous crops will require an increase in acreage of feedstocks for such as sugarcane (Saccharum spp.) and grain sorghum production of biofuels, specifically ethanol. Most etha- [Sorghum bicolor (L.) Moench], and will require fewer nol produced in the United States is derived from costs to establish large-scale production of bioenergy starch in corn (Zea mays L.) and other grain crops crops. grown in the Midwest that have a high input to output Before large-scale production of dedicated bioenergy ratio relative to other graminaceous feedstocks (Solo- crops can be implemented in the Gulf Coast region, re- man et al. 2007). Dedicated bioenergy feedstocks pro- lationships between pests and bioenergy duced for lignocellulosic biomass, such as energycane crops should be understood. One insect pest antici- (Saccharum spp.), high-biomass sorghum [Sorghum bi- pated to threaten bioenergy crop production is the color (L.) Moench], and switchgrass ( virgatum Mexican rice borer, Eoreuma loftini (Dyar), which L.) have the potential to produce ethanol more effi- feeds on closely related graminaceous crops including ciently because of higher fiber content, which can be sugarcane, sorghum, corn, and rice (Oryza sativa L.; hydrolyzed into additional sugars for ethanol produc- Van Zwaluwenburg 1926, Johnson 1984, Showleretal. tion (Soloman et al. 2007). English et al. (2006) com- 2012). E. loftini has been the most destructive pest of pared the economic competitiveness of bioenergy crop sugarcane in the Lower Rio Grande Valley since enter- production across various regions of the United States ing Texas in 1980 (Johnson and van Leerdam 1981). and concluded that the southern United States holds This species has expanded its geographical range north- east through the southeast Texas rice production area (Reay-Jones et al. 2007b) and eastward into rice and sugarcane in Louisiana as of 2013 (Wilson et al. 2015). 1 Department of Entomology, Louisiana State University Agricul- tural Center, 404 Life Sciences Bldg., Baton Rouge, LA 70803. Economic losses associated with E. loftini in Louisiana 2 Corresponding author, email: [email protected]. sugarcane may reach as high as U.S.$220 million when 3 Dean Lee Research Station, Louisiana State University Agricul- the insect becomes fully established in the state (Reay- tural Center, 8105 Tom Bowman Dr., Alexandria, LA 71302. Jones et al. 2008); however, the impact of this pest on 4 Texas A&M AgriLife Research and Extension Center at Beau- mont, Texas A&M University System, 1509 Aggie Dr., Beaumont, TX the production of dedicated bioenergy crops has not 77713. been examined; hence, the objectives of this study

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 2JOURNAL OF ECONOMIC ENTOMOLOGY were to evaluate susceptibility of energycane, sugar- Company, Yuma, AZ) applied once every other week at cane, high-biomass sorghum, and sweet sorghum culti- a rate of 140 g ai/ha with a CO2-pressurized backpack vars to E. loftini injury, and to determine E. loftini- sprayer calibrated to deliver 96 liter/ha. Insecticide induced yield reduction. applications were initiated on 22 June 2012 and 18 June 2013 and were discontinued 2 wk before harvest. Natural E. loftini infestations were neither suppressed Materials and Methods nor enhanced. Enhanced and highly enhanced E. lof- Field Experiment. A 2-yr study was conducted in tini infestation levels were established in the field by 2012 and 2013 at the Texas A&M AgriLife Research clipping wax paper strips (5 cm in length by 1 cm in and Extension Center at Beaumont in Jefferson width) containing viable E. loftini egg masses (30–50 County, TX, to examine the impact of E. loftini injury eggs) onto the basal leaf sheaths of plants, the site on yields of energycane, sugarcane, high-biomass- where natural oviposition often occurs (Van Leerdam sorghum, and sweet sorghum. The study included two 1986). Eggs were obtained from a laboratory colony at energycane cultivars, ‘L 79-1002’ and ‘Ho 02-113;’ two the Texas A&M AgriLife Research and Extension Cen- sugarcane cultivars, ‘HoCP 04-838’ (E. loftini suscepti- ter in Weslaco, TX, which was supplemented by ble standard) and ‘HoCP 85-845’ (E. loftini-resistant monthly collections of larvae and pupae from sugar- standard; Reagan et al. 2012); two high-biomass sor- cane fields located in Hidalgo County, TX. Larvae were ghum cultivars, ‘ES 5200’ and ‘ES 5140’ (Blade Energy reared on artificial diet (Martinez et al. 1988)at25C, Crops, Thousand Oaks, CA); and one sweet sorghum 65% relative humidity, and at a photoperiod of 14:10 cultivar, ‘M81E’ (MAFES Foundation Seed Stocks, (L:D) h (Showler and Castro 2010b). Each subplot Mississippi State, MS). Energycane cultivars L 79-1002 (main plot by strip) within a strip subjected to and Ho 02-113 are hybrids of Saccharum officinarum enhanced or highly enhanced infestations received one L., Saccharum spontaneum L., Saccharum barberi Jes- or three egg masses per row every 2 wk, respectively. wiet, and Saccharum sinense Roxburgh Amend. Jeswiet In 2012, enhancement of E. loftini infestations was ini- released by the Louisiana State University Agricultural tiated on 6 July. In 2013, enhancement of E. loftini Center (LSU AgCenter) and U.S. Department of Agri- infestations was initiated on 18 June in energycane and culture–Agricultural Research Services (USDA-ARS) sugarcane and 21 August in high-biomass and sweet Sugarcane Research Unit, respectively (Bischoff et al. sorghum, with delayed enhancement on sorghum 2008, Hale et al. 2012). These energycane cultivars are because of slowed plant development early in the sea- “Type I” sugarcane feedstocks because of their low son from sugarcane aphid, Melanaphis sacchari (Zehnt- sucrose (10–14%) and high-fiber contents (14–20%; ner) infestations. Enhancement of E. loftini infestations Tew and Cobill 2008, White et al. 2011). Seedcane was was continued for 9–12 wk and discontinued 2 wk obtained from the LSU AgCenter Sugar Research Sta- before harvest. tion, St. Gabriel, LA. High-biomass sorghum ES 5200 Within-Season E. loftini Injury Determination. and ES 5140 are Sorghum spp. and Sorghum spp. Periodic sampling was conducted to monitor E. loftini drummondii (sudangrass) hybrids, respectively, bred to injury throughout each growing season. Each subplot produce thick-stemmed, late-heading stalks suitable for was sampled six times in 2012 (21 May, 22 June, 6 July, dedicated bioenergy production (Blade Energy Crops 17 July, 9 August, and 24 August) and four times in 2012). Sweet sorghum M81E is a conventional Sor- 2013 (2 July, 7 August, 21 August, and 18 September) ghum spp. inbred released by the USDA-ARS for pro- by examining 10 randomly selected stalks and duction of fermentable sugar (Broadhead et al. 1981). recording the number of injured internodes. A randomized strip-plot block design with four blocks Internodes were classified as injured if feeding signs (one replication per block) was used in this experiment. characteristic of stem borer injury including window- Each block was 21.9 m in length and 33.6 m in width paning and frass in the leaf sheaths or entry holes in (21 rows), and was divided into seven vertical main the stalk were present. Injured internodes were plots and four horizontal strips. Main plots were 21.9 m expressed as the proportion of injured to total in length and 4.8 m in width (three rows), and strips internodes. were 5.5 m in length and 33.6 m in width (21 rows). End-of-Season E. loftini Injury and Yield Cultivars were randomized to main plots. Sugarcane Determination. Experiments were harvested on 15 and energycane cultivars were planted as whole stalks October in 2012 and 27 November in 2013 to collect on 2 November 2011 at a density of two stalks per 2 m end-of-season E. loftini injury and yields. Four ran- row; plant and ratoon crops were used in 2012 and domly selected stalks were collected from each row of 2013, respectively. High-biomass and sweet sorghum each subplot (12 stalks collected per subplot), stripped cultivars were planted on 20 April 2012 and 13 May of leaf material, and the numbers of bored internodes 2013 using a hand planter (Precision Garden Seeder, and numbers of total internodes were recorded. Earthway, Bristol, IN) calibrated to deliver 210,039 Sampled stalks were weighed to determine fresh stalk seeds per hectare. Cultivars were not re-randomized in weight (kg) and then crushed using a sugarcane stalk 2013. Four E. loftini infestation levels, suppressed, nat- crusher (Skyfood Equipment, Miami, FL) to separate ural, enhanced, and highly enhanced, were randomized juice from bagasse (stalk fiber). The volume of each to strips each year of the study. The suppressed infesta- juice sample was recorded and 1 ml from each of the tion level was established to provide an uninjured con- samples was analyzed using a handheld refractometer trol using tebufenozide (ConfirmVR 2F, Gowan (Reichert Technologies, Depew, NY) to determine Brix 2015 VANWEELDEN ET AL.: YIELD RESPONSE TO Eoreuma loftini INJURY 3

(% w/w soluble solids). From the Brix reading, sucrose fixed effects. Random effects included year, block concentration in stalks (% w/w sucrose) from each sam- (year), cultivar block(year), E. loftini infestation ple was calculated using the equation: level block(year), and E. loftini infestation level cultivar block(year). Least square means 6 SEs Sucrose concentration ¼ Brix=1 0:85=1:71975; from the LSMEANS statement output (Proc GLIM- MIX, SAS Institute 2008) are reported. Mean pairwise where 1 is a factor converting Brix to soluble solid con- comparisons were made using the LSMEANS state- centration in juice (% w/v) assuming a juice relative ment and the Tukey–Kramer adjustment (a ¼ 0.05). density of 1, 0.85 is the purity factor for converting The relationship between E. loftini end-of-season juice sucrose to normal juice sucrose (Reay-Jonesetal. injury and each yield parameter (fresh stalk weight, 2005), and 1.71975 is a constant estimated from calcu- sucrose concentration, and ethanol productivity) was lating the relationship between fresh stalk weight and determined using a multiple linear regression (PROC juice volume from data collected in the experiment. REG, SAS Institute 2008). The explanatory variables Ethanol productivity from sucrose in liter/ha was were a continuous variable for percentage of bored estimated using the equation from Vasilakoglou et al. internodes and binomial indicator variables for each (2011): cultivar and year:

d ^ ^ ^ Ethanol productivity from sucrose Yield ¼ b0 þ b1 PercentBoredþb2ES 5140 ¼ sucrose concentration fresh biomass 6:5 ^ ^ ^ þ b3M81E þ b4Ho 02-113 þ b5L79-1002 0:85 ½1:00=0:79 ; ^ ^ þ b6HoCP 04-838 þ b7HoCP 85-845 þ b^ where sucrose concentration is the sucrose concentra- 8Year tion in stalks (% w/w) from each sample calculated ^ from the previous equation, fresh biomass is the total Where b0 is predicted yield for ES 5200 at 0% ^ fresh stalk weight in metric ton/ha, 6.5 is the conversion bored internodes (intercept); b1 is the coefficient for factor of ethanol from sucrose, 0.85 is the efficiency the continuous variable PercentBored (i.e., slope); and ^ ^ ^ ^ ^ ^ ^ constant from converting sucrose into ethanol, and b2, b3, b4,b5, b6, b7,andb8 are coefficients of the 1.00/0.79 is the specific gravity of ethanol in g/ml. indicator variables used to adjust the predicted yield at Because experimental stalk populations were lower 0% bored internodes for cultivars ES 5140, M81E, Ho than expected in density but spatially homogenous, 02-113, L 79-1002, HoCP 04-838, and HoCP 85-845, stalk populations were standardized using published and year, respectively. Regression parameter estimates population estimates released by LSU AgCenter, were also used to determine the percent yield loss per ^ USDA-ARS, and Ceres Inc., and multiplied by fresh percent bored internode for each cultivar. jb1j (magni- stalk weights to determine biomass per hectare for tude change in yield per percent bored internode) was each sample. Bagasse from each sample was weighed divided by the yield at 0% bored internodes (intercept) and subsamples were collected, weighed, and allowed and multiplied by 100. to dry for 4 wk. Dry bagasse samples were weighed to determine percentage moisture loss. Total bagasse Results weight of each sample was multiplied by 100 % moisture loss to estimate dry biomass for each sample. E. loftini Within-Season Injury. Although the sug- Dry biomass weights were multiplied by a factor of arcane borer, Diatraea saccharalis (F.), is present in 465.3, the theoretical ethanol yield in liter per metric Jefferson County, TX, all larvae collected in both years ton from bagasse (U.S. Department of Energy 2013), were E. loftini and all injury characteristic of stem bor- to estimate lignocellulosic ethanol productivity. Total ers resulted from E. loftini feeding. In 2012, differen- ethanol productivity was calculated as the sum of esti- ces were detected in the percentage of injured mated ethanol yields from sucrose and lignocellulosic internodes among cultivars (F ¼ 8.9; df ¼ 6, 23.1; material. P < 0.0001), E. loftini infestation levels (F ¼ 26.6; Statistical Analyses. Analyses of variance (ANOVAs) df ¼ 3, 74.2; P < 0.0001), and sampling dates (F ¼ 7.2; were conducted using PROC GLIMMIX (SAS Insti- df ¼ 5, 3252; P < 0.0001). A cultivar by sampling date tute 2008, Cary, NC). The Kenward–Roger method interaction was also detected (F ¼ 3.3; df ¼ 27, 3235; was used for calculation of error degrees of freedom. P < 0.0001). Although low levels of infestations were Season-long injury data were analyzed using three- detected in suppressed subplots on the third sampling way ANOVAs with cultivar, E. loftini infestation level, date (6 July), injury was <2.0% injured internodes for sampling date, and all associated two- and three-way all cultivars. Injury in subplots with natural infestation interactions as fixed effects. Random effects included levels fluctuated from 0.0 to 8.8 6 1.2% injured intern- block, block cultivar, block E. loftini infestation odes, with levels of injury on the first sampling date (21 level, and block cultivar E. loftini infestation May) not exceeding that of the final sampling date (18 level. Data were analyzed separately for each year. September) across all cultivars excluding L 79-1002, Injury and yield data collected at harvest were ana- where the percentage of injured internodes decreased lyzed using two-way ANOVAs with cultivar, E. loftini from 3.3 6 1.1to1.66 1.2% between the first and last infestation level, and the two-way interaction as sampling dates, respectively. The highest percentage of 4JOURNAL OF ECONOMIC ENTOMOLOGY

Table 1. Statistical comparisons for selected injury and yield infestations, injury to M81E and ES 5200 decreased parameters, Jefferson Co, TX, 2012–2013 1.1- and 1.4-fold, respectively, under enhanced infesta- tions (Table 2). > Parameter F df P F Fresh stalk weight (Table 1) ranged from 0.55 6 0.03 Percentage of bored internodes kg per stalk in HoCP 04-838 to 0.19 6 0.03 kg per stalk Cultivar 3.00 6,42.3 0.0156 in M81E. Stalk weights of HoCP 04-838 were 2.0 and E. loftini infestation level 36.66 3,20.9 <0.0001 Cultivar E. loftini infestation level 2.11 18,125.1 0.0090 1.9 times greater than in L 79-1002 (0.27 6 0.03 kg per Fresh stalk weight stalk) and Ho 02-113 (0.29 6 0.03 kg per stalk), respec- Cultivar 64.63 6,42.4 <0.0001 tively. Enhancement of E. loftini infestations decreased E. loftini infestation level 52.35 3,20.4 <0.0001 stalk weights across all cultivars (Table 1). Maximum Cultivar E. loftini infestation level 2.52 18,122.7 0.0015 stalk weight was achieved under suppressed infestation Sucrose concentration levels (0.48 6 0.03 kg per stalk) and decreased 1.3-fold Cultivar 34.86 6,48.1 <0.0001 E. loftini infestation level 10.74 3,146.2 <0.0001 under natural infestations. Stalk weight in plants sub- Cultivar E. loftini infestation level 2.14 18,146.2 0.0071 jected to highly enhanced infestations decreased 1.5- Ethanol productivity fold compared with plants under suppressed infesta- Cultivar 67.12 6,45.6 <0.0001 tions. Differences in stalk weight were not observed E. loftini infestation level 31.13 3,22.7 <0.0001 between enhanced and highly enhanced infestation lev- Cultivar E. loftini infestation level 1.48 18,123.3 0.1067 els. Cultivars responded differently to changes in infes- tation level, as noted by the cultivar by infestation interaction for stalk weight (Table 1). Changes in stalk injured internodes, 10.7 6 1.4%, was recorded in L 79- weights between suppressed and natural infestations 1002 with enhanced infestations on the fourth sampling ranged from 1.2-fold in Ho 02-113 and HoCP 04-838 date (17 July). to 1.6-fold in M81E (Table 2). In addition, stalks of In 2013, differences were detected in the percentage HoCP 85-845, ES 5200, and ES 5140 sustained no fur- of injured internodes across infestation levels (F ¼ 6.7; ther decreases in weight under highly enhanced df ¼ 3, 24.7; P ¼ 0.0018) and sampling dates (F ¼ 8.2; infestations. df ¼ 3, 3055; P < 0.0001). There was limited evidence A negative relationship between the percentage of (P < 0.1) for differences in injury among cultivars, but bored internodes and stalk weight was detected across a cultivar by infestation level interaction was detected cultivars and years (F ¼ 146.0; df ¼ 645; P < 0.0001; (F ¼ 2.3; df ¼ 18, 24.6; P ¼ 0.0281). Injury in subplots R2 ¼ 0.6398). Each unit increase in the percentage of with suppressed infestations remained<1.0% injured bored internodes resulted in a 0.005 6 0 kg reduction internodes across all cultivars and sampling dates. The in stalk weight. Reductions in stalk weight in 2012 percentage of injured internodes ranged from ranged from 0.74 to 1.80% per percent bored inter- 0.0 6 1.6% in sorghum cultivars to 7.6 6 1.6% in L 79- node, with expression of damage being highest in 1002, with minimum and maximum injury levels occur- M81E and lowest in HoCP 04-838 and HoCP 85-845 ring during the first (2 July) and final (18 September) (Fig. 1A). In 2013, reductions in stalk weight per per- sampling dates, respectively. By the final sampling date cent bored internode ranged from 0.85% in HoCP 04- (18 September), enhancement of infestations increased 838 to 2.70% in M81E, consistent with results from levels of injury in Ho 02-113, HoCP 04-838, ES 5200, 2012. In addition, the reduction of stalk weight per per- and ES 5140, reaching as high as 12.8 6 1.6% injured cent bored internode in HoCP 85-845 was comparable internodes in ES 5200. with that observed in HoCP 04-838 in 2012 and 2013. End-of-Season E. loftini Injury and Impact on Sucrose concentration in stalks was highest in HoCP Yield. A difference in the percentage of bored interno- 04-838 (9.1 6 0.3% w/w), and was 1.5 and 1.2 times des was detected among cultivars (Table 1). Percen- greater than in L 79-1002 and Ho 02-113, respectively. tages of bored internodes ranged from 6.2 6 3.1% in Sucrose concentration in M81E (6.6 6 0.3% w/w) was Ho 02-113 to 12.1 6 3.1% in HoCP 04-838. Injury in greaterthaninES5140(5.36 0.3% w/w; t ¼ 3.62; HoCP 04-838 was 1.4 times greater than in HoCP 85- df ¼ 48.3; P ¼ 0.0118), but not different than in ES 845, whereas injury in ES 5140 was 1.6 times greater 5200 (5.8 6 0.3% w/w; t ¼ 2.12; df ¼ 48.3; P ¼ 0.3562). than in M81E. E. loftini infestation levels impacted Differences in sucrose concentration were detected bored internodes in all cultivars in both years. Applica- among E. loftini infestation levels (Table 1), despite no tions of tebufenozide were successful in reducing injury reductions in sucrose concentration occurring between to 1.0% bored internodes under suppressed infesta- natural (7.0 6 0.2% w/w), enhanced (6.9 6 0.2% w/w), tions. Enhanced and highly enhanced infestations and highly enhanced infestations (6.9 6 0.2% w/w). increased the percentage of bored internodes 1.3- and Across both years, minimum and maximum sucrose 1.7-fold, respectively, relative to natural infestations. An yields were achieved under highly enhanced and sup- interaction was detected between cultivar and infesta- pressed infestation levels, respectively. Sucrose concen- tion level (Table 1), as a result of inconsistent fluctua- trations among cultivar by infestation level ranged from tions in injury to ES 5200 and M81E in response to 5.0 6 0.3% w/w in ES 5140 at highly enhanced infesta- enhancement of E. loftini infestation levels. Although L tions to 9.4 6 0.3% w/w in HoCP 04-838 at suppressed 79-1002, Ho 02-113, HoCP 85-845, HoCP 04-838, and infestations (Table 2). The greatest reduction in sucrose ES 5140 had 1.4-fold greater percentage of bored concentration occurred in M81E between suppressed internodes when increasing from natural to enhanced and natural infestations, decreasing 1.0 6 0.3% w/w. 2015 VANWEELDEN ET AL.: YIELD RESPONSE TO Eoreuma loftini INJURY 5

Table 2. Energycane, sugarcane, high-biomass sorghum, and sweet sorghum parameter estimates (LS means) across varying E. loftini infestation levels. Jefferson Co, TX, 2012–2013

Cultivar E. loftini Percent bored Fresh weight Sucrose Ethanol productivity infestation level internodes (kg)/stalk concentration (w/w) (103 liter ha1) (6 3.5 [SE]) (6 0.04 [SE]) (6 0.3 [SE]) (6 1.2 [SE]) Energycane L 79-1002 Suppressed 0.4f 0.34e–g 6.3e–h 16.1 Natural 6.0b–f 0.25f–h 5.7h 13.4 Enhanced 12.4a–c 0.23f–h 5.8h 10.1 Highly enhanced 12.2a–d 0.24f–h 5.9gh 11.3 Energycane Ho 02-113 Suppressed 0.5f 0.34e–g 7.9a–d 18.5 Natural 5.2b–f 0.29f–h 7.8b–e 16.1 Enhanced 7.2b–f 0.27f–h 7.6b–e 14.7 Highly enhanced 11.9a–e 0.25f–h 7.6b–f 13.7 Sugarcane HoCP 04-838 Suppressed 0.8ef 0.70a 9.4a 9.3 Natural 11.8a–e 0.57b–d 8.9ab 7.3 Enhanced 16.3ab 0.48c–e 9.1ab 6.3 Highly enhanced 19.7a 0.46de 9.0ab 5.3 Sugarcane HoCP 85-845 Suppressed 1.0d–f 0.70ab 8.6a–c 6.7 Natural 5.1c–f 0.54cd 8.6a–c 5.3 Enhanced 14.4a–c 0.44de 8.7a–c 4.4 Highly enhanced 15.0a–c 0.45de 8.8a–c 4.6 High-biomass sorghum ES 5200 Suppressed 0.0f 0.62a–c 6.1f–h 22.2 Natural 14.1a–c 0.45de 5.8h 16.5 Enhanced 9.8a–f 0.44de 5.8h 15.4 Highly enhanced 16.1a–c 0.44de 5.7h 15.8 High-biomass sorghum ES 5140 Suppressed 0.2f 0.34ef 5.5h 11.5 Natural 9.8a–f 0.24f–h 5.7h 7.9 Enhanced 13.4a–c 0.21gh 5.1h 7.3 Highly enhanced 19.5a 0.21gh 5.0h 6.6 Sweet sorghum M81E Suppressed 0.2f 0.27f–h 7.3c–f 9.6 Natural 8.8a–f 0.17h 6.4d–h 6.0 Enhanced 8.0b–f 0.16h 6.3e–h 6.6 Highly enhanced 10.3a–f 0.15h 6.3e–h 5.0 a Means within the same column followed by different letters are significantly different (P < 0.05). b Means within columns for % bored internodes, fresh weight / stalk, sucrose concentration, and ethanol productivity have the same SE.

Sucrose concentrations remained comparable between P < 0.0001; R2 ¼ 0.6821). Each unit increase in the per- enhanced and highly enhanced infestations among all centage of bored internodes resulted in a loss of cultivars. 0.1 6 0.02 103 liters of ethanol per hectare. Reduc- A negative relationship between the percentage of tions in ethanol productivity per percent bored inter- bored internodes and sucrose concentration in stalks node in 2012 ranged from 0.67% in ES 5200 to 1.78% was detected across cultivars and years (F ¼ 197.4; in HoCP 85-845 (Fig. 1C). In 2013, reductions in etha- df ¼ 647; P < 0.0001; R2 ¼ 0.7057). For each unit nol production per percent bored internode ranged increase in the percentage of bored internodes, sucrose from 0.76% in ES 5200 to 2.56% in HoCP 85-845. concentration decreased by 0.01% 6 0.00 w/w. In 2012, Ethanol productivity reduction per percent bored inter- reductions in sucrose ranged from 0.13 to 0.22% per node was greater in sugarcane cultivars than in energy- percent bored internode in HoCP 04-838 and ES cane and high-biomass cultivars. 5140, respectively (Fig. 1B). In 2013, reductions in sucrose concentration per percent bored internode Discussion ranged from 0.14 to 0.25% in HoCP 04-838 and ES 5140, respectively. Overall, sucrose concentration This study is the first to show increasing levels of E. reductions per percent bored internode were greater in loftini injury and associated yield loss in energycane energycane and sorghum cultivars than in sugarcane and high-biomass sorghum cultivars bred as dedicated cultivars. bioenergy feedstocks anticipated to be highly resistant Ethanol productivity ranged from 17.5 6 1.0 103 to stem borers. As predicted, energycane cultivars were liter/ha in ES 5200 to 5.2 6 1.0 103 liter/ha in HoCP less susceptible to injury than sugarcane and high-bio- 85-845, and ethanol productivity in ES 5200 was 1.1- mass sorghum cultivars, including E. loftini resistant and 2.4-fold greater than the most productive energy- sugarcane cultivar HoCP 85-845. Plants in this study cane (Ho 02-113) and sugarcane (HoCP 04-838) culti- were under natural and enhanced E. loftini infestations. vars, respectively. Increases in E. loftini infestations Thus, differences in injury between energycane and reduced ethanol productivity 1.3-, 1.4-, and 1.5-fold sugarcane cultivars were most likely associated with under natural, enhanced, and highly enhanced infesta- resistant traits affecting E. loftini immature performance tions levels, respectively. (e.g., larval survival). Thin stalks, which average 1.40 A negative relationship between the percentage of and 1.48 cm in diameter in L 79-1002 (Bischoff et al. bored internodes and ethanol productivity was detected 2008) and Ho 02-113 (Hale et al. 2012), respectively, across cultivars and years (F ¼ 171.0; df ¼ 626; might convey resistance to E. loftini injury. However, 6JOURNAL OF ECONOMIC ENTOMOLOGY

3.00 A 2.50

2.00

1.50

1.00

0.50 Percent Bored Internode Percent Bored

0.00 Loss in Fresh Stalk Weight (%) per (%) Weight Fresh Stalk Loss in L 79-1002 Ho 02-113 HoCP 04-838 HoCP 85-845 ES 5200 ES 5140 M81E

2012 2013

0.30 B 0.25

0.20

0.15

0.10

0.05 per Percent Bored Internode per Percent Bored

Loss in Sucrose Concentration (%) 0.00 L 79-1002 Ho 02-113 HoCP 04-838 HoCP 85-845 ES 5200 ES 5140 M81E

2012 2013

3.00 C 2.50

2.00

1.50

1.00

0.50 per Percent Bored Internodeper Percent Bored

Loss in Ethanol Productivity (%) (%) Ethanol Productivity Loss in 0.00 L 79-1002 Ho 02-113 HoCP 04-838 HoCP 85-845 ES 5200 ES 5140 M81E

2012 2013

Fig. 1. Percent yield loss in (A) fresh stalk weight, (B) sucrose concentration, and (C) ethanol productivity per percent bored internode across seven cultivars of energycane, sugarcane, high-biomass sorghum, and sweet sorghum, estimated using multiple linear regression, Jefferson Co, TX, 2012 and 2013.

Showler et al. (2011) and Beuzelin et al. (2013) showed et al. 2008, Tew and Cobill 2008). High stem fiber and that plant hosts with stalk diameter of <0.7 cm, includ- lignin contents, which are preferable in bioenergy feed- ing sudangrass (S. bicolor ssp. drummondii), johnson- stocks, have been associated with negative effects on grass (Sorghum halepense (L.) Persoon), and larval feeding and development on sugarcane in an Afri- barnyardgrass (Echinochloa spp. (L.) Beauvois), are can stem borer, Eldana saccharina Walker (Lepidoptera: highly suitable hosts. Resistance in energycane might Pyralidae) (Rutherford et al. 1993). Rind hardness is involve higher fiber content in L 79-1002 and Ho 02- another characteristic that might negatively affect larval 113 than in susceptible sugarcane cultivars (Bischoff entry into the stalk (Martin et al. 1975, Ring et al. 1991). 2015 VANWEELDEN ET AL.: YIELD RESPONSE TO Eoreuma loftini INJURY 7

Sugarcane cultivar HoCP 85-845, classified as E. lof- narrow stalk diameter where feeding would compro- tini resistant (Reay-Jones et al. 2003, Wilson et al. mise stalk integrity, as documented in other sorghum 2012), expressed resistance to E. loftini feeding in the cultivars (Youm et al. 1988). natural infestation treatment, but some of the cultivar’s Applications of tebufenozide reduced E. loftini resistance was lost under heavy natural (Reay-Jones injury; however, differences in yield parameters et al. 2003) and, in our study, artificial infestations. The between plants in suppressed and natural infestations weakening of resistance suggests an antixenotic effect were not always detected. Sugarcane yield (tonnage) because insertion of egg masses into the leaf sheaths in differences associated with insecticidal control have not our study bypassed resistance mechanisms that affect been observed despite reductions in E. loftini injury oviposition preference. One resistance characteristic in (Meagher et al.1994, Legaspi et al. 1999b, Reay-Jones HoCP 85-845 may arise from the lack of available E. et al. 2005), resulting in the discontinuation of insecti- loftini oviposition sites. Plant phenotypic characteristics cide applications against E. loftini infesting sugarcane or drought stress influences the availability of dry and in south Texas (Legaspi et al. 1997). More recently, an folded leaf tissue for E. loftini oviposition (Reay-Jones increase in sugar content was observed in fields treated et al. 2005, 2007a; Showler and Castro 2010a, 2010b). with novaluron timed according to recommended Analyses of free amino acid levels in HoCP 85-845 action thresholds (Wilson et al. 2012). High levels of showed higher levels of free proline, which is associ- variability may inherently exist in determining yield loss ated with high water potential in the plant, rendering it relationships, specifically with sugarcane, allowing for more drought tolerant (Reay-Jones et al. 2005). In inconsistencies among studies (Reay-Jones et al. 2005). addition, nutritional or biochemical factors, including Alternatively, insecticides cannot be used as a single fructose and free essential amino acids, may influence tactic to prevent yield losses from E. loftini (Legaspi oviposition preference in host plant species (Showler et al. 1999a). While small sample sizes in this study and Castro 2010a, Showler et al. 2011, Showler and may have been inadequate to determine precise yield Moran 2014). estimates, each stalk sampled for injury was processed Injury to M81E did not differ with that in HoCP 04- for yield determination to accurately assess the relation- 838 or HoCP 85-845, indicating comparable levels of ship between injury and yield. susceptibility among conventional crop cultivars In 2013, enhancement of E. loftini infestations was assessed in this study. Showler et al. (2012) compared delayed until 21 August because of early season injury E. loftini injury among corn, grain sorghum, and sugar- from severe infestations of M. sacchari on all high- cane under small-plot and commercial field conditions biomass and sweet sorghum cultivars. Although M. sac- and detected no difference between injury in sorghum chari has been described as a pest of sugarcane in Lou- and sugarcane prior to harvest. Moderate levels of isiana (White et al. 2001)andFlorida(Mead 1978), injury to sorghum/sudangrass hybrid ES 5140 in our severe infestations on sorghum had not been reported study might be attributed to higher levels of fructose, in the United States prior to 2013 (Villanueva et al. which has been associated with E. loftini preference in 2014). Lower levels of E. loftini injury in sorghum sub- sorghum/sudangrass hybrids (Showler and Moran plots with natural infestations could have resulted from 2014). a decrease in host preference or suitability caused by This is the first study to show yield parameter reduc- plant stress and development of black sooty mold on tions, including fresh stalk weight, sucrose concentra- leaves with aphid infestations (Narayana 1975). In addi- tion, and ethanol productivity from E. loftini injury in tion, aphid populations may have attracted the red energycane and high-biomass sorghum cultivars. imported fire ant, Solenopsis invicta Buren, which Although energycane cultivars were resistant to injury, tends M. sacchari in sugarcane (Akbar et al. 2011). losses to stalk weight, and sucrose concentration corre- Increased S. invicta populations could result in sponding with increases in injury were more evident increased E. loftini predation. The influence of aphid than in conventional sugarcane cultivars. Legaspi et al. populations on other ecological guilds has been previ- (1999b) showed that E. loftini injury negatively affected ously examined, revealing positive relationships yield parameters in sugarcane. E. loftini injury causing between aphid populations and predation of herbivores an average of 11 and 19% bored internodes in sugar- and predators by S. invicta (Vinson and Scarborough cane cultivars CP 70-321 and NCo 310, respectively, 1989, Tedders et al. 1990, Styrsky and Eubanks 2010). were associated with decreases of 1.3% in sugar pro- Yield reductions from E. loftini injury reported in duction per hectare and 0.6% in stalk weight for each this study note the potential threat it poses to bioenergy percent bored internodes (Legaspi et al 1999b), feedstock production in the Gulf Coast region. Because expressing similar yield reductions to those reported in ethanol can be derived from fiber and sucrose, and this study. When left uncontrolled in southeast Texas, higher levels of fiber confer resistance against some E. loftini was associated with a loss in sugar production , bioenergy feedstock production was planned of 502 kg per ha, or 0.5% yield loss for each percent with limited insect pest management inputs. We bored internodes in cultivars LCP 85-384 and HoCP observed greater ethanol yield tolerance to E. loftini 85-845 (Reay-Jones et al. 2005). Despite moderate lev- injury in bioenergy cultivars than sugarcane and sweet els of resistance to E. loftini injury, sweet sorghum sorghum cultivars, although ethanol yields can be M81E expressed the greatest losses to stalk weight per reduced when E. loftini infestations are not controlled. percent bored internode. Reasons for low varietal toler- To maximize ethanol yields, effective pest management ance to E. loftini injury in M81E might result from practices will likely need to be implemented. 8JOURNAL OF ECONOMIC ENTOMOLOGY

Acknowledgments Martinez,A.J.,J.Baird,andT.Holler.1988.Mass rearing sugarcane borer and Mexican rice borer for production of We thank Dr. David Blouin for statistical consultation. We parasites Allorhogas pyralophagus and Rhacanotus roslinen- thank Dr. Don Henne and Manuel Campos (Texas A&M sis. USDA-ARS-PPQ, APHIS 83-1. AgriLife Research and Extension Center at Weslaco) for Mead,F.W.1978.Sugarcane aphid, Melanaphis sacchari maintenance of the E. loftini colony. We thank Will Thomp- (Zehntner)—Florida-New continental United States record. son, Casey Herraghty, Tyler Torina, Najib Mahmud, Rachel Coop. Plant Pest Rep. 34: 475. Anderson (LSU AgCenter), Becky Pearson, and Caleb Verret Meagher,R.L.,J.W.Smith,Jr,andK.J.R.Johnson.1994. (Texas A&M AgriLife Research and Extension at Beaumont) Insecticidal management of Eoreuma loftini (Lepidoptera: for technical assistance in the field. We thank Randy Eason Pyralidae) on Texas sugarcane: A critical review. J. Econ. (Texas A&M AgriLife Research and Extension at Beaumont) Entomol. 87: 1332–1344. for preparation of fields for planting. This research was Narayana,D.1975.Screening for aphids and sooty molds in funded by the USDA-NIFA Agricultural and Food Research sorghum. Sorghum Newsl. 18: 21–22. Initiative (AFRI) Sustainable Bioenergy program grant 2011- Reagan,T.E.,B.E.Wilson,M.T.VanWeelden,J.M.Beu- 67009-30132. zelin, W. H. White, R. Richard, and M. O. Way. 2012. pp. 130–131, in Sugarcane Research Annual Report 2012. References Cited Louisiana State University Agricultural Center, Baton Rouge, LA. Akbar,W.,A.T.Showler,J.M.Beuzelin,T.E.Reagan,and Reay-Jones, F.P.F., M. O. Way, M. Se´tamou,B.L.Legen- K. A. Gravois. 2011. Evaluation of aphid resistance among dre,andT.E.Reagan.2003.Resistance to the Mexican sugarcane cultivars in Louisiana. Ann. Entomol. Soc. Am. rice borer (Lepidoptera: Crambidae) among Louisiana and 104: 699–704. Texas sugarcane cultivars. J. Econ. Entomol. 96: 1929–1934. 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B. E. WILSON,1,2 T. N. HARDY,3 J. M. BEUZELIN,1 M. T. VANWEELDEN,1 T. E. REAGAN,1 3 4 1 1 R. MILLER, J. MEAUX, M. J. STOUT, AND C. E. CARLTON

Environ. Entomol. 44(3): 757–766 (2015); DOI: 10.1093/ee/nvv016 ABSTRACT The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae), is an invasive pest of sugarcane, Saccharum spp., rice, Oryza sativa L., and other graminaceous crops in the Gulf Coast region of the United States. Traps baited with E. loftini female sex pheromones were used to document establishment and distribution of E. loftini near sugarcane, rice, and noncrop hosts in seven southwest Louisiana parishes from 2009 to 2013. Additional field surveys documented larval infestations in com- mercial sugarcane and rice. After its initial detection in 2008, no E. loftini were detected in Louisiana in 2009 and only two adults were captured in 2010. Trapping documented range expansion into Cameron, Beauregard, and Jefferson Davis parishes in 2011 and Allen, Acadia, and Vermilion parishes in 2013. During the course of this study, E. loftini expanded its range eastward into Louisiana 120 km from the Texas border (22 km/yr). Surveys of larval infestations provided the first record of E. loftini attacking rice and sugarcane in Louisiana. Infestations of E. loftini in rice planted without insecticidal seed treat- ments in Calcasieu Parish reached damaging levels.

KEY WORDS monitoring, pheromone trap, Eoreuma loftini, invasive species

The Mexican rice borer, Eoreuma loftini (Dyar) (Lepi- uses a broad range of host plants, eradication of this doptera: Crambidae), is an invasive insect originating pest is not feasible (Johnson and Van Leerdam 1981; from Mexico, which has become established as a major Showler et al. 2011, 2012; Beuzelin et al. 2011, 2013). pest of graminaceous crops in Texas. E. loftini attacks Development of population management strategies is sugarcane, Saccharum spp., rice, Oryza sativa L., corn, the only viable approach to mitigating the pest’s impact Zea mays L., sorghum, Sorghum bicolor L., and many (Showler and Reagan 2012). noncrop grass species (Showler et al. 2011, 2012; The E. loftini female sex pheromone was isolated Beuzelin et al. 2011, 2013). E. loftini was first reported (Brown et al. 1988) and developed for use in phero- as a pest in the United States in 1980 on sugarcane in mone traps (Shaver et al. 1988, 1990, 1991), which are the Lower Rio Grande Valley of Texas (Johnson and deployed to monitor E. loftini range expansion (Reagan Van Leerdam 1981), where it now comprises >95% of et al. 2005, Reay-Jones et al. 2007b, Hummel et al. the sugarcane stem borer population (Legaspi et al. 2010) and for pest scouting in individual sugarcane 1999). E. loftini has since spread northeast through the fields (Wilson et al. 2012). The recent discovery of rice production area along the Texas Gulf Coast (Reay- E. loftini in Florida (Hayden 2012) highlights the Jones et al. 2007b) and was discovered in Louisiana in potential for rapid range expansion. Objectives of this December 2008 (Hummel et al. 2010). A quarantine work were to 1) monitor E. loftini distribution and was implemented during the 2005 crop production sea- expansion in southwestern Louisiana, 2) determine the son to prevent movement of infested Texas sugarcane severity of E. loftini infestations in rice and sugarcane, into Louisiana (Reagan et al. 2005), which is estimated and 3) evaluate the potential for use of pheromone to have saved the Louisiana sugarcane industry traps to improve scouting for E. loftini in rice. US$1.1–3.2 billion (Reay-Jones et al. 2008). Establish- ment of E. loftini throughout Louisiana is expected to cause annual revenue losses as great as US$220 and Materials and Methods US$40 million to the sugarcane and rice industries, Pheromone Trap Monitoring. Adult E. loftini respectively (Reay-Jones et al. 2008). Because E. loftini populations were monitored using pheromone traps in southwestern Louisiana from 2009 through 2013. 1 Department of Entomology, Louisiana State University Agricul- Standard green, yellow, and white bucket traps (Uni- tural Center, 404 Life Science Bldg., Baton Rouge, LA 70803. trap, Great Lakes IPM, Vestaburg, MI) were baited 2 Corresponding author, e-mail: [email protected] with synthetic E. loftini sex pheromone lures (Luresept, 3 Louisiana Department of Agriculture and Forestry, 5825 Florida Blvd., Baton Rouge, LA 70806. Hercon Environmental, Emigsville, PA). Each trap 4 Calcasieu Parish Extension Office, Louisiana State University contained an insecticidal strip (Vaportape II, Hercon Agricultural Center, 7101 Gulf Hwy, Lake Charles, LA 70607. Environmental). Traps were attached to metal poles

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 758 ENVIRONMENTAL ENTOMOLOGY Vol. 44, no. 3

1 m above the soil surface to maximize trap perform- parishes were located near sugar mills. Specimens ance (Shaver et al. 1991). Pheromone lures and insecti- which represented the possible initial detection of cidal strips were replaced every 4 wk according to label E. loftini in a parish were transported to the Louisiana instructions. State Arthropod Museum (Louisiana State University, The number and locations of traps in each parish Baton Rouge, LA) for identification (Reiss 1981, varied by year (Table 1). Trap locations were recorded Agnew et al. 1988). using a handheld GPS unit. The distance of each trap E. loftini Infesting Sugarcane and to its nearest neighboring trap was determined using Rice. Monitoring of E. loftini larval infestations in sug- GPS coordinates, and mean distance between traps arcane was conducted from May to October, 2013, in was calculated for each year. Mean distances between two fields in Calcasieu Parish (120 ha total; var. traps during each year were 2.7, 3.1, 5.6, 6.1, and L 99–226) and three fields in Jefferson Davis Parish 9.0 km in 2009, 2010, 2011, 2012, and 2013, respec- (220 ha total; var. L 99-226 and HoCP 96-540). A tively. Prior to 2013, trap locations were 5–15 km east pheromone trap was placed adjacent to each field of the eastern edge of the known E. loftini range as within 1 m of field edges to monitor adult populations. determined by monitoring in the prior years (Table 1). Larval infestations were assessed by monthly examina- Trapping was expanded in 2013 to include areas where tion (five sampling events per field) of 100 randomly the pest was already known to occur. Hence, monitor- selected stalks for the presence of E. loftini larvae. ing in 2013 involved Calcasieu, Cameron, Jefferson Stalks were considered infested if at least one E. loftini Davis, Beauregard, Allen, and Acadia parishes (Table larva was observed feeding within the stalk or on plant 1), >15,000 km2. Traps in southwestern Louisiana (Cal- surfaces. casieu, Cameron, Jefferson Davis, Beauregard, Allen, Surveys involved monitoring of adult population den- Acadia, and Vermilion parishes) were placed adjacent sities and larval infestations in rice fields treated with to rice fields, sugarcane fields, or pastures with wild chlorantraniliprole (Dermacor X-100, E.I. du Pont de hosts, and traps in St. Mary, St. Martin, and Iberia Nemours and Company, Wilmington, DE) applied to

Table 1. E. loftini pheromone trap monitoring in Louisiana, 2009–2013

Parish Year No. trap Date deployed Date retrieved No. times traps No. E. loftini- Total no. sites were sampled positive sites E. loftini captured Calcasieu 2009 40 30 Mar. 2009 19 Jan. 2010 13 0 0 2010 27 13 April 2010 12 Jan. 2011 12 1 7 2011 34 15 Mar. 2011 12 Dec. 2011 10 31 225 2012 0 NA NA NA NA NA 2013 18 28 Feb. 2013 4 Jan. 2014 14 18 8920 Cameron 2009 5 30 Mar. 2009 19 Jan. 2010 13 0 0 2010 4 13 April 2010 12 Jan. 2011 12 0 0 2011 14 15 Mar. 2011 12 Dec. 2011 10 6 43 2012 2 13 Feb. 2012 2 Jan. 2013 13 1 1 2013 8 7 Mar. 2013 18 Nov. 2013 12 8 3575 Jefferson Davis 2009 12 30 Mar. 2009 19 Jan. 2010 13 0 0 2010 11 6 April 2010 12 Jan. 2011 12 0 0 2011 6 15 Mar. 2011 12 Dec. 2011 10 4 11 2012 16 13 Feb. 2012 2 Jan. 2013 13 0 0 2013 22 7 Mar. 2013 4 Jan. 2014 14 20 4489 Beauregard 2009 3 30 Mar. 2009 19 Jan. 2010 13 0 0 2010 0 NA NA NA NA NA 2011 2 15 Mar. 2011 12 Dec. 2011 10 1 3 2012 0 13 Feb. 2012 2 Jan. 2013 13 0 0 2013 9 28 Feb. 2013 4 Jan. 2014 10 9 2024 Allen 2012 7 13 Feb. 2012 2 Jan. 2013 13 0 0 2013 5 14 Mar. 2013 4 Jan. 2014 10 5 187 Acadia 2013 5 22 May 2013 8 Jan. 2014 9 3 7 Vermilion 2011 1 15 Mar. 2011 12 Dec. 2011 10 0 0 2012 0 NA NA NA NA NA 2013 3 23 Oct. 2013 8 Jan. 14 3 2 4 Iberia 2009 8 10 July 2009 19 Jan. 2010 14 0 0 2010 8 21 June 2010 3 Jan. 2011 14 0 0 2011 8 18 July 2011 10 Jan. 2012 10 0 0 2012 7 27 Aug. 2012 16 Jan. 2013 10 0 0 2013 7 19 Sept. 2013 16 Feb. 2013 4 0 0 St. Mary 2009 3 10 July 2009 19 Jan. 2010 14 0 0 2010 3 21 June 2010 3 Jan. 2011 14 0 0 2011 3 18 July 2011 10 Jan. 2012 10 0 0 2012 3 27 Aug. 2012 16 Jan. 2013 10 0 0 2013 3 19 Sept. 2013 16 Feb. 2013 4 0 0 St. Martin 2012 1 27 Aug. 2012 16 Jan. 2013 10 0 0 2013 1 19 Sept. 2013 16 Feb. 2013 4 0 0 NA, not applicable. June 2015 WILSON ET AL.: MEXICAN RICE BORER EXPANSION IN LOUISIANA 759 seed at 80 g a.i./ha and fields without insecticidal seed as a random effect. Tukey’s honestly significant differ- treatments. No other insecticides were used on experi- ence test (a ¼ 0.05) was used for mean separations, and mental fields during the growing season. Eleven fields Kenward–Roger method was used for calculation of were surveyed in 2012 (four treated and seven non- error degrees of freedom (PROC MIXED, SAS Insti- treated) and 12 (six treated and six nontreated) in 2013. tute 2008). A multiple linear regression was conducted A replication consisted of a pair of one treated and one with capture per trap per day as the dependent variable nontreated field, each 30–165 ha, within 3.6 km of each and percentage injured tillers as the independent varia- other. Rice varieties reflected those most commonly ble (PROC REG, SAS Institute 2008). A qualitative grown in Louisiana and included CL111, CL151, dummy variable, z1, was used to differentiate between XL745, XL729, Cheniere, Cocodrie, and Mermentau. years (if year ¼ 2012 then z1 ¼ 0, if year ¼ 2013 then Due to lack of replication of varieties in the experimen- z1 ¼ 1) because trap captures were significantly higher tal design, rice variety was not included as an effect in (P < 0.05)in2013thanin2012. the statistical analysis. All fields surveyed were located in areas of Calcasieu Parish where development of sub- stantial E. loftini populations was anticipated based on Results pheromone trap captures during the previous spring. A single pheromone trap was placed directly adjacent to Pheromone Trap Monitoring. E. loftini phero- each field and monitored throughout the growing sea- mone trap monitoring efforts from 2009 to 2013 cap- son. These traps were monitored for the duration of tured a total of 19,496 moths at >100 trap locations the rice-growing season (May through August) and throughout seven parishes in southwest Louisiana augmented the traps used in the pheromone trap mon- (Table 1). Although moths were not detected in 2009, itoring described in the previous section. Fields were two specimens were trapped in November–December sampled for larval infestations six times in 2012 (30 of 2010 in noncrop habitat south of Vinton (30 May, 14 June, 7 July, 28 July, 12 August, and 26 5044.901000 N, 93 3105.901000 W) 22 km to the south August) and eight times in 2013 (21 June, 27 June, 3 and 7 km to the east of the 2008 detection (30 July, 10 July, 18 July, 29 July, 6 August, and 15 August). 17054.348000 N, 93 35021.330000 W, Hummel et al. On each sampling date, 25 rice tillers were randomly 2010). In 2011, E. loftini males were captured at 42 selected within a 50-m radius of the pheromone trap, new locations in three additional parishes (Cameron, observed for stem borer injury, and dissected. No other Jefferson Davis, and Beauregard; Fig. 1). The eastern- species of stem borer was recovered during the surveys most E. loftini detection in 2011 was near a sugarcane in either year, and all injury by stem borer larval feed- field 26 km south of Welsh (Cameron Parish; 30 ing was assumed to have been caused by E. loftini. 11010.715400 N, 92 51019.691400 W) and 64 km east Once rice fields neared maturity (hard-dough stage, 29 of the 2010 detection site. Deployment of traps in 2012 July–26 August), they were sampled by counting the revealed little eastward expansion with a single speci- total number of rice tillers and the number of tillers men captured in northwestern Cameron Parish (29 with “whiteheads” (incompletely emerged panicles or 59057.731400 N, 92 47029.43600 W) 4km east of the panicles which do not produce grain, resulting from 2011 easternmost edge. Expanded pheromone trap insect injury to vascular tissue during plant growth; monitoring in 2013 resulted in the capture of Pathak 1968) within a randomly positioned 1-m2 quad- >19,000 E. loftini adults. Adults were detected at 65 rat. Whiteheads present in each sample were dissected trapping sites, 30 of which were located in areas where to verify injury was caused by stem borer feeding, and E. loftini had not previously been found (e.g., Allen, all recovered larvae were verified as E. loftini. White- Acadia, and Vermilion parishes; Fig. 1). The range of head data were not collected in one treated and two E. loftini in 2013 extended eastward to traps positioned nontreated fields in 2012 because the fields were har- south of Estherwood (Acadia Parish; 30 7028.379400 N, vested before sampling. 92 27020.440800 W), 32 km further east than in 2012. Trap capture converted to daily estimates and Detection of E. loftini in Beauregard and Allen par- percentage of injured tillers for each growing season ishes as well as southern Cameron Parish revealed sub- were analyzed using a three-way analysis of variance stantial north–south range expansion since 2009. The (ANOVA; PROC MIXED, SAS Institute 2008)with southernmost detection site (29 46029.55300 N, 93 year, treatment, sampling date, year treatment, 27048.052200 W) was <1 km from the coast of the sampling date year, sampling date treatment, Gulf of Mexico, while the northernmost detection site and sampling date year treatment as fixed effects. (30 51040.243800 N, 93 19027.692400 W) was ANOVA models comparing trap captures and percent- 120 km north of the coast in northern Beauregard Par- age injured tillers included replication(year) and ish. Between 2009 and 2013 the eastern edge of the treatment replication(year) as random effects to range of E. loftini moved a total of 111 km east from account for the effects of the design of the study and the 2008 detection site (22.2 km per yr). The range of repeated measures (variance component covariance E. loftini encompassed all of Calcasieu, Beauregard, structure). The number of whiteheads per square Cameron, and Jefferson Davis parishes and regions of meter and the percentage of tillers with whiteheads Allen, Vermilion, and Acadia parishes. No E. loftini were compared using a two-way ANOVA (PROC adults have been captured near sugarcane mills in MIXED, SAS Institute 2008) with year, treatment, and Iberia, St. Mary, or St. Martin Parish as of December year treatment as fixed effects and replication(year) 2013. 760 ENVIRONMENTAL ENTOMOLOGY Vol. 44, no. 3

Fig. 1. E. loftini range expansion in Louisiana, 2008–2013.

E. loftini Infesting Sugarcane and Rice. The Table 2. Larval E. loftini infestations and pheromone trap cap- first larva found to be infesting sugarcane was collected tures in Louisiana sugarcane, 2013 in a field of first season sugarcane (var. L 99-226) near Iowa (Calcasieu Parish; 30 14049.178400 N, 93 Parish Month 0 00 3 13.7946 W) on 29 March 2013. Season-long scout- May June July Aug. Sept. Oct. ing for E. loftini larvae in sugarcane revealed infesta- Calcasieu tions in 120 ha of sugarcane in Calcasieu Parish and No. E. loftini (total, 2 traps) 12 31 56 156 65 16 220 ha in Jefferson Davis Parish. The mean percentage Mean % infested stalks 0 0 0 11 8 8 of stalks with E. loftini larvae feeding on plant surfaces Jefferson Davis or inside stalks peaked in August with 11 and 8% of No. E. loftini (total, 3 traps) 4 40 28 39 68 51 Mean % infested stalks 0 0 2 8 4 5 stalks infested in Calcasieu and Jefferson Davis par- ishes, respectively (Table 2). Adult trap captures near rice fields varied through- out the growing season in both years (Fig. 2), although differences between sampling dates were not detected (Fig. 3). Late-season injury in nontreated rice fields in (Table 3). Daily trap captures were more than 3.5-fold 2013 rose to 21.2% 6 2.7(SEM) of tillers with E. loftini greaterin2013thanin2012(Table 3, Fig. 2). However, injury, while levels in 2012 peaked at 9.7% 6 2.1(SEM). differences were not detected among the traps near The percentage of tillers with E. loftini injury in chlorantraniliprole-treated and nontreated fields, nor chlorantraniliprole-treated fields remained below 5% were interactions detected (Table 3). Differences in throughout the growing season in both years. A linear percentage of tillers with larval injury were detected relationship occurred (F ¼ 8.79, df ¼ 2,66, P < 0.001, among treatments, sampling dates, and for the sam- R2 ¼ 0.2103, Root MSE ¼ 6.601) between the number pling date treatment interaction, but not for other of E. loftini per trap per day and the percentage of till- interactions (Table 3). The percentage of injured tillers ers with stem borer injury in nontreated rice fields. increased throughout most of the growing season, The dummy variable, z1, improved the regression peaking in late July and early August in both years model (t ¼ 3.08, P ¼ 0.003) by increasing the coefficient June 2015 WILSON ET AL.: MEXICAN RICE BORER EXPANSION IN LOUISIANA 761

Fig. 2. E. loftini daily pheromone trap captures (LS means 6 SEM) in chlorantraniliprole-treated and nontreated rice in (A) 2012 and (B) 2013. Differences were detected (P < 0.05) between years, but not among treatments, sampling dates, or the interactions.

Table 3. Statistical comparisons of E. loftini daily trap captures and percentage of injured tillers from chlorantraniliprole-treated and nontreated rice fields in Calcasieu Parish, Louisiana, 2012–2013

Fixed effect Daily trap capture Percentage injured tillers

F df P > FF df P > F Sampling date 1.22 7,94.9 0.298 4.99 7,84.9 <0.001 Treatment 0.02 1,16.5 0.880 20.93 1,16.4 <0.001 Year 17.41 7,12 0.001 0.01 1,12.6 0.906 Year treatment 2.68 1,10.8 0.130 0.0 1,12.7 0.950 Treatment sampling date 1.03 7,94.7 0.415 3.45 7,84.9 0.003 Year sampling date 1.23 5,93.1 0.300 0.58 4,83.6 0.681 Year treatment sampling date 1.35 5,93.2 0.181 0.81 4,83.6 0.524 of the explanatory variable by 4.9288 for 2013 data whiteheads between years, treatments, and a (slope ¼ 0.2817; intercepts ¼ 2012: 2.160 and 2013: year treatment interaction occurred (Table 4). The 7.085, Fig. 4). mean number of whiteheads per square meter ranged Differences were detected in the number of white- from 1.00 (chlorantraniliprole treated) to 12.67 heads per square meter, and the percentage of (nontreated). 762 ENVIRONMENTAL ENTOMOLOGY Vol. 44, no. 3

Fig. 3. Percentages of tillers with E. loftini injury (LS means 6 SEM) in chlorantraniliprole-treated and nontreated rice in (A) 2012 and (B) 2013. Differences were detected (P < 0.05) among sampling dates, treatments, and the sampling date treatment interaction, but not among years or other interactions.

Discussion expansion in 2012. Variation in population distribution This study documents the occurrence of E. loftini in between years has been reported for other lepidop- seven Louisiana parishes, and provides the first record teran species, and periods of reduced population of infestations in Louisiana field crops. The estimated growth of invasive species (Byers et al. 2002, Augustine rate of E. loftini range expansion during the 5-yr moni- et al. 2004) caused by biotic and abiotic factors likely toring period (22 km per yr) is consistent with previous have roles in range expansion. estimates (23 km per year from 1980 to 2005; A quarantine was initiated in 2005 by the Louisiana Reay-Jones et al. 2007b); however, eastward movement Department of Agriculture and Forestry and the Texas was sporadic and varied greatly between years. Chang- Department of Agriculture to prevent the transport of ing weather conditions and host availability probably sugarcane from Texas into Louisiana for processing, influenced E. loftini range expansion, and widespread with the aim of reducing human-aided movement of removal of rice straw after the 2011 growing season E. loftini into Louisiana (Reagan et al. 2005). However, (Schultz 2011, Beuzelin et al. 2012) and above average at present no regulations are in place preventing move- rainfall (U.S. Dept. of Commerce National Oceanic ment of E. loftini-infested sugarcane from western and Atmospheric Administration 2014) might have Louisiana parishes to sugarcane mills further east. reduced E. loftini populations and slowed range Based on its current distribution as estimated by June 2015 WILSON ET AL.: MEXICAN RICE BORER EXPANSION IN LOUISIANA 763

Fig. 4. Relationship between daily adult trap captures and larval injury in nontreated rice fields. Multiple linear regression (F ¼ 8.79; df ¼ 2, 66; P < 0.001; R2 ¼ 0.2103).

Table 4. E. loftini infestations (LS means 6 SEM) as affected by northernmost trap (120 km north of the Gulf Coast), insecticidal seed treatments in rice in Calcasieu Parish, Louisiana, indicating that the species may occur further north. 2012–2013 Overwintering survival of E. loftini is greater than that of the sugarcane borer, Diatraea saccharalis (F.) Fixed Effect Whiteheads/m2 Percentage Whiteheads (Rodriguez-del-Bosque et al. 1995), which occurs as far north as southern Arkansas (Lorenz and Hardke 2014). Year Hence, expanding pheromone trap monitoring might 2012 2.90 6 0.98a 0.90 6 0.25a 2013 5.67 6 0.81b 2.13 6 0.20b reveal a larger range for E. loftini than is currently F 4.72 14.66 known (Showler et al. 2012, Showler and Reagan P 0.046 0.002 2012). Treatment The increase of infestations from 2012 to 2013 in Chlorantraniliprole 1.00 6 0.98a 0.34 6 0.25a Nontreated 7.57 6 0.81b 2.69 6 0.20b Calcasieu Parish suggests that the invasive pest has F 26.59 53.75 become established there. Abundances of whiteheads P <0.001 <0.001 indicate substantial yield losses attributable to E. loftini Year Treatment in fields that were not protected with insecticides dur- 2012 Chlorantraniliprole 1.00 6 1.55a 0.27 6 0.39a Nontreated 4.80 6 1.20a 1.53 6 0.30b ing 2013. Yield losses of 1.0–4.2% have been reported 2013 Chlorantraniliprole 3.56 6 1.20a 1.05 6 0.30b for every percentage increase in whiteheads caused by Nontreated 12.67 6 1.10b 3.85 6 0.27c a complex of stem borers in Asia (Pathak 1968, F 4.72 11.77 Muralidharan and Pasalu 2006). Reay-Jonesetal. P 0.046 0.004 (2007a) estimated a 2.28% decrease in yield results Means in the same column for each fixed effect which share a low- from every whitehead per square meter caused by ercase letter are not significantly different (P > 0.05; Tukey’s HSD mixed infestations of D. saccharalis and E. loftini. test). For all tests, df ¼ 1,15. Hence, we estimate yield losses of 4–28% in unpro- tected rice fields in 2013. The injury levels in Louisiana were comparable with those reported in Texas (1–20 pheromone trapping, it is likely that E. loftini infesta- whiteheads per square meter) where E. loftini has tions are present throughout the 600 ha of sugarcane been present in rice for more than 10 yr (Way et al. in Calcasieu and Jefferson Davis parishes. Although the 2006, Reay-Jones et al. 2007a). Chlorantraniliprole pest’s range expansion through Texas and into Louisi- seed treatments provided season-long protection from ana is not known to have been a result of human-aided E. loftini injury. The similar numbers of adult E. loftini movement, movement of infested sugarcane could near chlorantraniliprole-treated rice fields and non- introduce E. loftini into the 45,000 ha of sugarcane in treated fields suggests that noncrop hosts harbor sub- Iberia, St. Mary, and St. Martin parishes. stantial populations. On an area-wide scale, pheromone With expanded pheromone trap monitoring in 2013, traps are useful (Reay-Jones et al. 2007b, Wilson et al. substantial increases in the northern range limit of 2012), and the correlation between trap captures and E. loftini were observed. Males were captured in the larval infestations in nontreated rice suggests the traps 764 ENVIRONMENTAL ENTOMOLOGY Vol. 44, no. 3 have potential to alert growers of increasing popula- 1981; Showler et al. 2011, 2012; Beuzelin et al. 2011, tions and the need to monitor larval infestations. Cur- 2013), reducing human-aided movement and imple- rently, insecticidal seed treatments including menting effective area-wide management tactics will be chlorantraniliprole and neonicotinoids are applied to critical to mitigating the impact of E. loftini in Louisi- most rice planted in Louisiana for control of the rice ana rice and sugarcane. As E. loftini expands its geo- water weevil, Lissorhoptrus oryzophilus Kuschel (Stout graphical range, susceptible varieties of corn and and Gore 2014). Neonicotinoid seed treatments are rel- sorghum grown in the central and northern regions of atively ineffective against stem borers (Way et al. 2011). Louisiana will also be affected (Showler et al. 2012, Chlorantraniliprole seed treatments, in contrast, are 2013). The availability of corn and sorghum is also effective against D. saccharalis (Sidhu et al. 2014)and anticipated to influence area-wide populations (Showler can be used to control a complex of stem borers. Addi- and Reagan 2012). Widespread monitoring through tionally, widespread adoption of chlorantraniliprole pheromone trapping and larval surveys will help to seed treatments in rice may contribute to reduced understand E. loftini population dynamics and range area-wide pest populations. Rice varieties have varying expansion. Combining climate data and host plant dis- levels of susceptibility to stem borers (Way et al. 2006), tributions might facilitate a role for geographical infor- and the effect of varieties used in this study is mation systems for projecting further range expansion, unknown. identifying high economic risk areas, and enhancing Surveys of sugarcane in Calcasieu and Jefferson integrated pest management strategies. Davis parishes in 2012 and 2013 led to detection of the first E. loftini larval infestation in Louisiana sugarcane. Acknowledgments While infestations did not reach economically damag- ing levels in 2013, the pest is capable of inflicting reve- We express gratitude to Craig Callahan, Brian Carroll, Jer- nue losses of up to US$220 million annually to the emi Rabalais, Michelle Poole, and other LDAF personnel for sugarcane industry (Reay-Jones et al. 2008). Infesta- efforts in helping to monitor and assist with quarantine activ- tions in sugarcane in the Lower Rio Grande Valley con- ities. Appreciation is expressed to Natalie Hummel for her sistently cause >20% bored internodes when left role in project conceptualization. Additional thanks are given unmanaged (Legaspi et al. 1999, Reay-Jones et al. to Tyler Torina, Najib Mahmud, and Rachel Anderson for technical assistance. This work would not have been possible 2005, Wilson et al. 2012), and severe infestations have without the cooperation of Habetz Farms, Sweet Lake Farms, caused fields to be unharvestable (Reagan et al. 2005, and other growers. This work was sponsored by grants from Showler and Reagan 2012). Although judiciously timed the U.S. Environmental Protection Agency Ag IPM Program, insecticide applications might reduce sugarcane yield the American Sugar Cane League, and U.S. Department of losses from E. loftini (Wilson et al. 2012), adequate Agriculture–National Institute of Food and Agriculture Grant control in sugarcane is difficult to achieve because of 2011-67009-30132. This paper has been approved by the insufficient exposure of larvae to insecticides (Johnson director of the Louisiana Agricultural Experiment Station as 1985, Meagher et al. 1994, Legaspi et al. 1999, Showler manuscript no. 2014-234-15628. and Reagan 2012, Wilson et al. 2012). Reductions in populations of the stem borers D. sac- References Cited charalis, Diatraea lineolata (Walker), and Diatraea magnifactella Dyar have been observed following the Agnew, C. W., L. A. Rodriguez-Del-Bosque, and J. W. Smith Jr. 1988. 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BLAKE E. WILSON,1,2 MATTHEW T. VANWEELDEN,1 JULIEN M. BEUZELIN,3 THOMAS E. REAGAN,1 MICHAEL O. WAY,4 WILLIAM H. WHITE,5 4 6 LLOYD T. WILSON, AND ALLAN T. SHOWLER

J. Econ. Entomol. 108(3): 1363–1370 (2015); DOI: 10.1093/jee/tov076 ABSTRACT The Mexican rice borer, Eoreuma loftini (Dyar), is a major pest of sugarcane (hybrids of Saccharum spp.) in Louisiana and Texas. Resistance to E. loftini was evaluated in 51 commercial and ex- perimental cultivars of sugarcane, energycane (hybrids of Saccharum spp.), and sorghum [Sorghum bi- color (L.) Moench and hybrids of Sorghum spp.] in four replicated small plot field experiments from 2009 to 2012. A relative resistance ratio was developed to compare levels of susceptibility among cultivars based on the percentage of bored internodes and survival to adulthood. This index was able to separate cultivars into five resistance categories and provides a new method for comparing levels of resistance among cultivars. E. loftini pest pressure in 2009 was among the highest recorded with injury ranging from 55 to 88% bored internodes. Commercial sugarcane cultivar HoCP 85-845 was identified as resis- tant in three of four experiments, whereas HoCP 04-838 was identified as susceptible in all experiments. Of the five sugarcane cultivars in commercial production in the Rio Grande Valley of Texas, only TCP 87-3388 was categorized as resistant. Of the cultivars with potential for bioenergy production, all of the energycane cultivars demonstrated higher levels of resistance than high-biomass and sweet sorghum cultivars. Continued evaluation of cultivar resistance to E. loftini is important to development of effective integrated pest management strategies for this pest.

KEY WORDS Saccharum spp., Eoreuma loftini, cultivar resistance

The Mexican rice borer, Eoreuma loftini (Dyar), is the dominant pest of sugarcane, Saccharum spp. hybrids, sugarcane borer, Diatraea saccharalis (F.) (Reagan and in Texas (Legaspi et al. 1997) and has recently moved Martin 1989, Bessin et al. 1990, Posey et al. 2006, into Louisiana’s sugarcane and rice, Oryza sativa L., Showler and Reagan 2012). For both stem borers, the producing regions (Wilson et al. 2015),whereitisex- most common parameter for measuring injury and esti- pected to cause substantial economic impact (Reay- mating yield loss is percentage of bored internodes Jones et al. 2008). Because of limited exposure of E. (Mathes and Charpentier 1969, White and Hensley loftini larvae to insecticides, chemical control of this 1987, White et al. 2008). Percentage of bored inter- pest is often not economical and has been largely aban- nodes has been shown to be inversely correlated with doned by Texas sugarcane growers (Meagher et al. yield parameters including juice purity, tonnage of sug- 1994). Thus, alternative control methods are needed to arcane, sugar per ton of cane, and sugar per ha reduce pest losses. Resistant cultivars are a major com- (Legaspi et al. 1999, Reay-Jones et al. 2005, White ponent of integrated pest management (IPM) in sugar- et al. 2008). This parameter is used to identify cultivar cane for E. loftini (Meagher et al. 1996; Reay-Jones resistance as expressed by reduced oviposition prefer- et al. 2003, 2005; Wilson et al. 2012), as well as for an- ence and low establishment of early instar larvae. How- other economically important stem borer, the ever, percentage of bored internodes does not address potential mechanisms which influence host suitability after young larvae have bored into stalks. Cultivars with reduced suitability can decrease survival to adulthood 1 Department of Entomology, Louisiana State University Agricul- and reduce area-wide pest populations (Bessin et al. tural Center, 404 Life Science Bldg., Baton Rouge, LA 70803, USA 1991). Bessin et al. (1990) developed a relative survival 2 Corresponding author, e-mail: [email protected] index based on the number of adult emergence holes 3 Dean Lee Research Station, Louisiana State University Agricul- tural Center, 8105 Tom Bowman Dr., Alexandria, LA 71302, USA relative to the number of bored internodes as an addi- 4 Texas A&M AgriLife Research and Extension Center, 1509 Aggie tional tool to evaluate D. saccharalis resistance among Dr., Beaumont, TX 77713, USA sugarcane cultivars. This index has also been used to 5 USDA-ARS Sugar Cane Research Unit, 5883 USDA Rd., Houma, assess survival to adulthood of E. loftini on sugarcane LA 70360, USA 6 USDA-ARS Knipling-Bushland U.S. Livestock Insects Research cultivars (Reay-Jones et al. 2003, 2005). However, un- Laboratory, 2700 Fredericksburg Rd., Kerrville, TX 78028, USA like the percentage of bored internodes, the relative

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 1364 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 3 survival index does not assess total injury and its rela- the Louisiana State University Agricultural Center tionship to yield loss has not been documented. Thus, (LSU AgCenter) Sugar Research Station in St. Gabriel, percentage of bored internodes and relative survival LA (L cultivars) and the United States Department of provide two distinct ways of assessing levels of stem Agriculture (USDA), Agricultural Research Service borer resistance in sugarcane. (ARS), Sugar Cane Research Unit in Houma, LA (Ho Bessin et al. (1990, 1991)andReay-Jonesetal. cultivars). Each experiment consisted of a randomized (2003) noted discrepancies among levels of resistance complete block design with five blocks (one replication based on percentage of bored internodes and those per block). Cultivars were planted by hand to one-row based on survival to adulthood. Development of an in- plots (2009: 4.6 m in length, 1.6-m row spacing and 1.5- dex, which compares stem borer resistance based on m alleys; 2010–2012: 3.7 m in length, 1.6-m row spac- host preference and suitability, would provide research- ing, and 1.5-m alleys) during the fall. ers with a more comprehensive tool for evaluating cul- For the 2009 experiment, six sugarcane cultivars tivars. The objective of the research provided herein is commonly grown in Louisiana were evaluated (Table to assess resistance to E. loftini among commercial 2). In spite of three irrigations of this experimental field and experimental sugarcane cultivars using a relative on 27 June, 24 July, and 7 August 2009, severe drought resistance ratio that incorporates percentage of bored caused significant reductions in stalk populations and internodes and relative survival to adulthood into a sin- stunted growth in all plots. gle index. Because stem borers are expected to impact For the 2010 experiment, sugarcane cultivars eval- production of dedicated bioenergy crops, energycane, uated included seven for commercial production in and high-biomass sorghum cultivars are evaluated in Louisiana, nine experimental clones from USDA and addition to conventional sugarcane cultivars. LSU AgCenter breeding programs, three germplasm clones derived from a recurrent selection program for D. saccharalis resistance at the USDA-ARS Sugarcane Materials and Methods Research Unit (US 93-15, US 01-40, and Ho 06-9610), Field Experiments. A series of four field experi- two clones derived from sugarcane progenitors with ments was conducted at the Texas A&M AgriLife lowlevelsofantibiosistoD. saccharalis (US 08-9001 Research facilities in Ganado (Jackson County, TX) and and US 08-9003) (White et al. 2011), and four cultivars Beaumont (Jefferson County, TX) from 2009 to 2012 obtained from the South African Sugar Experiment (Table 1). In each study, commercial sugarcane cultivars Station in Natal, South Africa (“N” cultivars) (Table 3). and breeding material were evaluated for resistance to Only four plots were harvested for L 07-57 and US stem borers under natural pest pressure. Known stem 93-15 because stalks of these two cultivars failed to borer-resistant, HoCP 85-845 (Reay-Jones et al. 2003, emerge in one block. Wilson et al. 2012), and susceptible, HoCP 04-838 The 2011 experiment included 5 commercial sugar- (Reagan et al. 2008) cultivars were included as stand- cane cultivars, 11 experimental sugarcane cultivars, and ards in each experiment. Seed cane for each cultivar 2 energycane lines (Table 4). Energycane cultivars was obtained from the sugarcane breeding programs at L 79-1002 and Ho 02-113 are hybrids of Saccharum

Table 1. Summary of field evaluations screening for cultivar resistance to E. loftini

Year Location No. cultivars evaluated Planting date Harvest date Sample size (stalks/plot) Injury data 2009 Jackson County, TX 6 18 Oct. 2008 19 Aug. 2009 12 Internal and external 2010 Jefferson County, TX 25 21 Oct. 2009 7 Sept. 2010 10 Internal and external 2011 Jefferson County, TX 19 28 Oct. 2010 5 Sept. 2011 10 Internal and external 2012 Jefferson County, TX 24 26 Oct. 2011 22 Oct. 2012 12 External

Table 2. Screening for cultivar resistance to E. loftini, Jackson Co., TX, 2009

Cultivara Percentage of No. emergence Relative survival Relative resistance ratio Resistance bored internodes holes/stalk (LS means 6 0.033 [SE]) (LS means 6 0.096 [SE])* categoryc (LS means 6 SE)b,* (LS means 6 0.30 [SE]) L 03-371 88.0 6 3.6a 1.04 0.148 0.850a Highly susceptible HoCP 04-838 68.2 6 6.9ab 1.43 0.157 0.834a Highly susceptible Ho 95-988 64.9 6 7.3ab 0.90 0.154 0.683ab Susceptible HoCP 85-845 61.3 6 7.6b 0.53 0.082 0.500ab Intermediate HoCP 05-961 55.4 6 7.9b 0.39 0.057 0.417b Intermediate HoCP 05-902 57.6 6 7.9b 0.38 0.076 0.383b Resistant F 3.8 2.2 3.7 5.7 df 5, 23.6 5, 20.0 5, 20.0 5, 24.0 P 0.012 0.097 0.015 0.001 a All cultivars are commercial sugarcanes grown in Louisiana. b Standard errors for individual means reported for data analyzed with a binomial distribution. c Resistance category based on relative resistance ratio values. *Means within a column which share a letter are not significantly different (Tukey’s HSD, a ¼ 0.05). June 2015 WILSON ET AL.: SUGARCANE CULTIVAR RESISTANCE TO E. loftini 1365

Table 3. Screening for cultivar resistance to E. loftini, Jefferson Co., TX, 2010

Cultivar Descriptiona Percentage of No. emergence Relative Relative resistance Resistance categoryc bored internodes holes/stalk survival ratio (LS means 6 SE)b,* (LS means 6 0.09 [SE]) (LS means 6 0.075 [SE]) (LS means 6 0.066 [SE])* Ho 06-563 ES 20.3 6 4.7a 0.38 0.148 0.724abc Susceptible HoCP 05-902 CS 14.4 6 3.7ab 0.32 0.150 0.756ab Susceptible HoCP 04-838 CS 10.9 6 2.9bc 0.20 0.134 0.712abc Susceptible Ho 07-612 ES 10.0 6 2.7bcd 0.18 0.130 0.700abc Susceptible L 03-371 CS 9.5 6 2.7bcd 0.14 0.174 0.764ab Susceptible HoCP 96-540 CS 7.8 6 2.2b–e 0.08 0.060 0.516a–f Intermediate L 07-57 ES 7.1 6 2.1c–f 0.31 0.206 0.785ab Susceptible Ho 07-604 ES 6.3 6 1.9c–f 0.04 0.026 0.440c–h Intermediate US 01-40 SCB-R 5.8 6 1.8c–g 0.06 0.052 0.460b–h Intermediate N-27 SAS 5.7 6 1.7c–g 0.12 0.226 0.796a Susceptible Ho 06-537 ES 5.7 6 1.7c–g 0.18 0.194 0.716abc Susceptible Ho 07-613 CS 5.4 6 1.6c–g 0.02 0.008 0.288e–i Resistant N-17 SAS 5.4 6 1.6d–g 0.08 0.050 0.428c–h Intermediate HoCP 05-961 CS 5.2 6 1.6d–g 0.12 0.196 0.724abc Susceptible US 08-9001 SCB-R 5.2 6 1.6d–g 0.04 0.024 0.312e–i Resistant Ho 06-9610 SCB-R 4.9 6 1.5d–g 0.04 0.110 0.472b–g Intermediate HoCP 00-950 CS 4.5 6 1.3d–h 0.04 0.200 0.664a–d Susceptible L 07-68 ES 4.0 6 1.3e–h 0.12 0.162 0.560a–e Intermediate Ho 07-617 ES 3.9 6 0.9e–h 0.06 0.082 0.420c–i Intermediate US 08-9003 SCB-R 2.6 6 0.9fgh 0.06 0.098 0.412c–i Intermediate N-24 SAS 2.4 6 0.9fgh 0.00 0.000 0.148hi Highly resistant L 01-299 CS 2.2 6 0.6fgh 0.04 0.080 0.352d–i Resistant US 93-15 SCB-R 1.2 6 0.4gh 0.00 0.005 0.225f–i Resistant HoCP 85-845 CS 1.0 6 0.4h 0.00 0.000 0.164ghi Highly resistant N-21 SAS 1.0 6 0.4h 0.00 0.000 0.112i Highly resistant F 17.7 1.6 1.0 13.4 df 24, 98.0 24, 94.0 24, 94.1 24, 98.0 P <0.001 0.067 0.457 <0.001 a CS, commercial sugarcane; ES, experimental sugarcane; SAS, South African sugarcane; SCB-R, D. saccharalis resistant. b Standard errors for individual means reported for data analyzed with a binomial distribution. c Resistance category based on relative resistance ratio values. *Means within a column which share a letter are not significantly different (Tukey’s HSD, a ¼ 0.05).

officinarum L., Saccharum spontaneum L., Saccharum stem borer larvae in 2009 (Showler and Reagan 2012). barberi Jeswiet, and Saccharum sinese Roxb. Amend. A specially designed stalk splitter (Tilby Technologies, Jeswiet, which are characterized by their low sucrose Victoria, British Columbia, Canada) was used in 2010 and high fiber content (Bischoff et al. 2008, Hale et al. and 2011 to split stalks down the middle for internal 2012b). examination. An internode was considered to be bored The 2012 experiment included three commercial if either an external entry hole or a larval tunneling was sugarcane cultivars grown in Louisiana, as well as five observed. Season-long monitoring of regional stem cultivars commonly grown for commercial sugar pro- borer infestations in 2012 revealed stem duction in the Rio Grande Valley of Texas, and seven borer species composition to be >95% E. loftini; thus, experimental cultivars. In addition, cultivars with only external examination of injury was conducted. The potential for bioenergy production including six energy- numbers of internodes, bored internodes, and adult cane lines, two high biomass sorghum cultivars emergence holes were totaled for each sample. (ES 5200 and ES 5140; Blade Energy Crops, Thousand Relative Survival and Relative Resistance Oaks, CA), and one sweet sorghum cultivar (M81E; Ratio. Relative survival (Bessin et al. 1990)for MAFES Foundation Seed Stocks, MS State, MS) were each sample was calculated using equation 1: evaluated (Table 5). Sorghum cultivars were planted on 19 April 2012 using hand planters (Earthway Precision Relative survival ¼ no: emergence holes= Garden Seeders, Earthway, Southbend, IN) calibrated (1) to deliver approximately 210,000 seeds/ha. no: bored internodes Sugarcane plots were maintained according to stand- ard production practices recommended by the LSU Because there are discrepancies between levels of AgCenter (Gravois 2014). Following season-long expo- resistance based on percentage of bored internodes sure to natural stem borer infestations, stalk samples and those based on survival to adulthood (Bessin et al. (Table 1) were randomly selected, and borer injury 1990, Reay-Jones et al. 2003), a relative resistance ratio data were collected by recording number of internodes, was developed to provide a single index which incorpo- number of bored internodes, and number of adult rates both measures of resistance. The proportion of emergence holes in each stalk. Stalks were manually bored internodes and relative survival were ranked split to assess internal injury and identify species of within each replication; these rankings were used to 1366 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 3

Table 4. Screening for cultivar resistance to E. loftini, Jefferson Co., TX, 2011

Cultivar Descriptiona Percentage of No. emergence Relative survival Relative resistance Resistance bored internodes holes/stalk (LS means 6 0.072 [SE]) ratio categoryc (LS means 6 SE)b,* (LS means 6 0.09 [SE]) (LS means 6 0.095 [SE])* HoCP 08-726 ES 18.6 6 2.4a 0.48 0.214 0.705a Susceptible HoCP 04-838 CS 15.3 6 2.3ab 0.35 0.164 0.600ab Susceptible Ho 08-711 ES 14.5 6 2.1ab 0.46 0.322 0.705a Susceptible L 08-090 ES 13.9 6 2.1ab 0.36 0.259 0.711a Susceptible HoL 08-723 ES 13.6 6 2.0ab 0.10 0.086 0.526ab Intermediate Ho 08-717 ES 12.3 6 1.9ab 0.21 0.165 0.563ab Intermediate Ho 07-613 CS 9.7 6 1.7b 0.28 0.210 0.568ab Intermediate Ho 08-706 ES 9.5 6 1.7bc 0.18 0.211 0.521ab Intermediate L 07-57 ES 9.0 6 1.5bc 0.22 0.168 0.500ab Intermediate HoCP 00-950 CS 8.9 6 1.7bc 0.08 0.118 0.426ab Intermediate L 79-1002 EC 8.8 6 1.6bc 0.18 0.234 0.490ab Intermediate Ho 08-709 ES 8.6 6 1.6bc 0.08 0.044 0.279ab Resistant HoCP 91-552 CS 8.2 6 1.6bc 0.22 0.299 0.558ab Intermediate HoCP 05-961 CS 8.1 6 1.6bc 0.26 0.368 0.563ab Intermediate L 08-088 ES 8.0 6 1.4bc 0.24 0.275 0.532ab Intermediate L 08-092 ES 7.8 6 1.5bcd 0.12 0.140 0.400ab Intermediate Ho 02-113 EC 7.7 6 1.5bcd 0.08 0.127 0.410ab Intermediate HoCP 85-845 CS 3.7 6 0.1cd 0.10 0.213 0.305ab Resistant L 08-075 ES 2.8 6 0.1d 0.02 0.100 0.200b Resistant F 8.6 1.9 1.4 2.2 df 18,76.0 18,76.0 18,76.0 18,76.0 P <0.001 0.025 0.162 0.009 a CS, commercial sugarcane; ES, experimental sugarcane; EC, energycane. b Standard errors for individual means reported for data analyzed with a binomial distribution. c Resistance category based on relative resistance ratio values. *Means within a column which share a letter are not significantly different (Tukey’s HSD, a ¼ 0.05).

Table 5. Screening for cultivar resistance to E. loftini, Jefferson Co., TX, 2012

Cultivar Descriptiona Percentage of No. emergence Relative survival Relative Resistance bored internodes holes/stalk (LS means 6 0.05 [SE]) resistance ratio categoryc (LS means 6 SE)b (LS means 6 0.19 [SE]) (LS means 6 0.091 [SE]) L 08-090 ES 26.5 6 1.7a 1.43a 0.374ab 0.833a Highly susceptible CP 79-1210 RGV-S 22.8 6 2.0ab 0.98ab 0.458a 0.821ab Highly susceptible M81E SS 20.5 6 1.6abc 0.82ab 0.304a–d 0.750abc Susceptible CP 89-2143 RGV-S 19.3 6 1.5a–d 0.87ab 0.274a–d 0.625a–d Susceptible Ho 08-717 ES 18.3 6 1.4b–e 0.70ab 0.220a–d 0.579a–d Intermediate HoCP 04-838 CS 17.2 6 1.3b–f 0.95ab 0.328a–d 0.675a–d Susceptible ES 5140 HBS 16.8 6 1.2b–g 0.77ab 0.224a–d 0.571a–d Intermediate Ho 05-961 CS 16.5 6 1.3b–g 0.72ab 0.240a–d 0.567a–d Intermediate L 08-088 ES 16.4 6 1.3b–g 1.00ab 0.352abc 0.704a–d Susceptible ES 5200 HBS 15.3 6 1.2b–h 0.98ab 0.296a–d 0.625a–d Susceptible TCP 99-4474 RGV-S 14.8 6 1.3c–i 0.67ab 0.294a–d 0.596a–d Intermediate L 08-092 ES 14.5 6 1.4c–j 0.47ab 0.176bcd 0.463a–d Intermediate Ho 08-709 ES 13.4 6 1.3d–j 0.55ab 0.246a–d 0.513a–d Intermediate Ho 07-613 CS 13.4 6 1.2d–j 0.55ab 0.268a–d 0.508a–d Intermediate Ho 08-711 ES 13.2 6 1.3d–j 0.63ab 0.252a–d 0.417a–d Intermediate Ho 07-9014 EC 12.9 6 1.2d–j 0.32b 0.160bcd 0.400a–d Intermediate TCP 87-3388 RGV-S 12.2 6 1.1e–j 0.28b 0.152bcd 0.363a–d Resistant L 79-1002 EC 11.2 6 1.2f–j 0.20b 0.126bcd 0.342abc Resistant Ho 07-9017 EC 11.1 6 1.1g–j 0.11b 0.062d 0.250d Resistant TCP 99-4480 RGV-S 11.0 6 1.1g–k 0.46ab 0.273a–d 0.500a–d Intermediate Ho 07-9027 EC 10.0 6 1.0h–k 0.23b 0.152bcd 0.333cd Resistant Ho 02-113 EC 9.6 6 1.0ijk 0.28b 0.208a–d 0.421a–d Intermediate Ho 07-9076 EC 9.0 6 0.9jk 0.14b 0.094cd 0.246d Resistant HoCP 85-845 CS 6.0 6 0.8k 0.23b 0.196a–d 0.308cd Resistant F 14.5 3.1 3.2 3.5 df 23,96.0 23,96.0 23,92.0 23,96.0 P <0.001 <0.001 <0.001 <0.001 a CS, commercial sugarcane; ES, experimental sugarcane; EC, energycane; RGV-S, Rio Grande Valley sugarcane; SS, sweet sorghum; HBS, high-biomass sorghum. b Standard errors for individual means reported for data analyzed with a binomial distribution. c Resistance category based on relative resistance ratio values. Means within a column which share a letter are not significantly different (Tukey’s HSD, a ¼ 0.05). June 2015 WILSON ET AL.: SUGARCANE CULTIVAR RESISTANCE TO E. loftini 1367 calculate a relative resistance ratio for each cultivar most resistant, whereas L 03-371 was the most suscep- within each replication. The relative resistance ratio for tible (Table 2). a particular cultivar in a particular replication was cal- In 2010, differences in the percentage of bored culated using equation 2: internodes, which ranged from 1.0 to 20.3%, and rela- tive resistance ratio, which ranged from 0.112 to 0.764, RR1 þ RR2 were detected among cultivars (Table 3). However, dif- Relative resistance ratio ¼ (2) 2n ferences in emergence per stalk and relative survival among cultivars were not detected. Of the South Afri- Where n is the number of cultivars evaluated, RR1 can cultivars, N-21 and N-24 were categorized as is the relative resistance ranking based on proportion of highly resistant, whereas N-27 was considered suscepti- bored internodes (the lowest proportion has a RR1 ¼ 1, ble. Based on the relative resistance ratio, N-21 was the highest has a RR1 ¼ n), and RR2 is the relative the most resistant, whereas L 03-371 was the most sus- resistance ranking based on relative survival (the lowest ceptible (Table 3). relative survival has a RR2 ¼ 1, the highest has a In 2011, differences in the percentage of bored RR2 ¼ n). internodes, which ranged from 2.8 to 18.6%, and rela- Statistical Analyses. Data from each of the four tive resistance ratio, which ranged from 0.200 to 0.711, field experiments were analyzed separately using gener- were detected (Table 4). Although the effect of cultivar alized linear mixed models (PROC GLIMMIX, SAS on the number of emergence holes per stalk was signif- Institute 2008). Models included “cultivar” as a fixed icant (P < 0.05), differences among cultivars were not effect and “block” as a random effect. Proportion of detected when separated by Tukey’s HSD. Differences bored internodes data were compared with models in relative survival were not detected among cultivars. with a binomial distribution and were reported as per- The most resistant cultivar based on the relative resist- centages of bored internodes. Adult emergence per ance ratio was L 08-075 and the most susceptible was stalk, relative survival, and the relative resistance ratio L 08-090; however, no cultivars were categorized were analyzed with models with a Gaussian distribu- as either highly susceptible or highly resistant. Energy- tion. In addition, the relative resistance ratio for HoCP cane cultivar L 79-1002 was intermediate, whereas Ho 85-845, HoCP 04-838, and HoCP 05-961 across the 02-113 was resistant. four experiments was compared with a model with a Differences among cultivars were detected in 2012 Gaussian distribution. The model included “cultivar” as with percentage of bored internodes ranging from 6.0 a fixed effect and “experiment” as a random effect. The to 26.5% (Table 5). In addition, differences in emer- Kenward–Rogers method was used to estimate degrees gence per stalk ranging from 0.11 to 1.43, relative sur- of freedom and Tukey’s Honest Significant Difference vival ranging from 0.062 to 0.458, and the relative (HSD) (a ¼ 0.05) was used for mean separations for all resistance ratio ranging from 0.246 to 0.833 were models. detected. Based on the relative resistance ratio, Ho 07- The following resistance categories were developed 9076 was the most resistant, whereas L 08-90 was the based on the mean relative resistance ratios of all culti- most susceptible. Of the experiments conducted in Jef- vars within each year: highly resistant (0.000–0.199), ferson County (2010, 2011, and 2012), percentage of resistant (0.200–0.399), intermediate (0.400–0.599), bored internodes, emergence per stalk, and relative susceptible (0.600–0.799), and highly susceptible survival were greatest in 2012. Of the cultivars grown (0.800–0.999). in the sugarcane production regions of South Texas, only TCP 87-3388 was classified as resistant. All of the energycane cultivars demonstrated moderate to high levels of resistance. Sorghum cultivars were either Results susceptible (ES 5200 and M81E) or intermediate E. loftini accounted for >90% of stem borer (ES 5140). infestations in each of the four experiments although Commercial cultivars evaluated in all four experi- D. saccharalis is present in Jefferson and Jackson ments included HoCP 04-838, HoCP 85-845, and Counties. In the 2009 experiment, a single D. sacchara- HoCP 05-961. HoCP 85-845 was categorized as inter- lis larva was recorded compared with >1,000 E. loftini mediate based on the relative resistance ratio in 2009. larvae. Because of low numbers of D. saccharalis in However, in 2010, 2011, and 2012, this cultivar was these experiments, all injury characteristic of stem among the most resistant in terms of percentage of borer feeding was considered to have resulted from bored internodes, emergence per stalk, and the relative E. loftini. resistance ratio. HoCP 04-838 was consistently among The experiment conducted in 2009 in Jackson the most injured cultivars in terms of percentage of County had the highest level of E. loftini pest pressure bored internodes and was classified as either suscepti- of the four experiments. Each cultivar sustained severe ble or highly susceptible in all four experiments. HoCP injury, ranging from 55 to 88% bored internodes (Table 05-961 was categorized as intermediate in 3 of 4 yr 2). Differences in the percentage of bored internodes but was categorized as susceptible in 2010. Differences and relative resistance ratio were detected among culti- were detected (F ¼ 4.28, df ¼ 2, 9; P ¼ 0.0495) in the vars (Table 2). Relative survival ranged from 0.057 to relative resistance ratio among these three cultivars 0.157, which was lower than in the other years. Based when compared across the four experiments. HoCP on the relative resistance ratio, HoCP 05-902 was the 85-845 was resistant (mean relative resistance 1368 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 108, no. 3 ratio ¼ 0.385), HoCP 05-961 was intermediate (0.515), to cultivar resistance evaluations which incorporate and HoCP 04-838 was susceptible (0.708). known resistant and susceptible cultivars along with large numbers of cultivars with diverse genetic back- grounds with varying levels of resistance. Although it is Discussion not ideal for all analyses of injury and survival data, the This research assessed resistance to E. loftini in 51 relative resistance ratio provides a useful index, which commercial and experimental cultivars over 4 yr and can be adapted to a variety of plant-insect systems. two locations based on host preference (plant injury) Additionally, incorporation of yield response rankings and suitability (relative survival). Host preference of could further expand the ratio to account for differen- E. loftini is most commonly measured in terms of selec- ces in tolerance to insect feeding among cultivars. tion of oviposition sites (Reay-Jones et al. 2007; Show- Cultivar resistance experiments conducted under ler and Castro 2010a,b; Showler et al. 2011; Beuzelin conditions of uncharacteristically high or low insect et al. 2013). In our study, host preference was meas- pressure may not be applicable in other environments ured in terms of early instar larval injury recorded as (Painter 1951, Smith 1989). The unusually high E. lof- percentage of bored internodes. This parameter tini pest pressure observed in the 2009 experiment reflects oviposition preference and establishment of may largely be attributed to generally high stem borer early instar larvae. Percentage of bored internodes is populations in the region (Beuzelin et al. 2011)and a widely used index to measure early instar injury in extreme drought during the growing season, a condi- sugarcane and is directly correlated to yield reductions tion conducive to greater E. loftini infestations (Reay- (Hensley and Long 1969, Reay-Jones et al. 2005, White Jones et al. 2005, Showler and Castro 2010b, Showler et al. 2008). However, Bessin et al. (1990) asserted the and Reagan 2012). Cultivar resistance observed in the need to assess cultivar resistance based on survival to 2009 study may not be representative of resistance adulthood. The effect of resistance mechanisms affect- levels under less extreme conditions as high insect ing host suitability in our study is represented by rela- pressure and drought stress can mask some resistance tive survival to adulthood based on bored internodes mechanisms (Smith 1989). and adult emergence holes (Bessin et al. 1990). The relative resistance ratio consistently identified A decreased concentration of nutrients including free commercial cultivar HoCP 04-838 as E. loftini suscepti- amino acids (FAA) can inhibit larval development and ble and HoCP 85-845 as resistant, the latter being con- reduce host suitability (Smith 1989, Reay-Jones et al. sistent with previous research involving HoCP 85-845 2005, Showler et al. 2012, Showler and Reagan 2012). (Reay-Jones et al. 2003, 2005; Wilson et al. 2012). Our Additionally, high levels of FAAs, particularly histidine, findings suggest that HoCP 85-845 can continue to be are associated with increased E. loftini oviposition pref- viewed as a standard for stem borer resistant sugarcane erence (Reay-Jones et al. 2007, Showler and Castro cultivars. However, results from the 2009 experiment 2010b, Showler and Moran 2014) indicating resistance are also consistent with previous documentation that mechanisms influencing host preference and those under severe E. loftini pressure, the level of resistance affecting suitability may be related. Additionally, rela- in HoCP 85-845 is reduced relative to other cultivars tively high concentrations of fructose have been associ- (Reay-Jones et al. 2003). Interestingly, HoCP 04-838 ated with increased E. loftini oviposition in crop and was developed from a cross of HoCP 85-845 and LCP non-crop hosts (Showler and Moran 2014). 85-384 and is considered to be resistant to D. sacchara- Discrepancies between levels of resistance based on lis (Hale et al. 2012a). HoCP 85-845 is resistant to both percentage of bored internodes and those based on rel- E. loftini (Reay-Jones et al. 2003, 2005; Wilson et al. ative survival to adulthood (Bessin et al. 1990, 1991; 2012)andD. saccharalis (Posey et al. 2006), whereas Reay-Jones et al. 2003) create difficulty in interpreta- LCP 85-384 is susceptible to both (Reay-Jones et al. tion of cultivar resistance data. The relative resistance 2003, Posey et al. 2006). Differences in cultivar sus- ratio presented here allows for categorization of a large ceptibility to the two stem borer species among culti- number of cultivars into resistance groups based on rel- vars may become more apparent as the range of ative levels of both preference (percentage of bored E. loftini further overlaps with that of D. saccharalis in internodes) and suitability (survival to adulthood). sugarcane production areas of Louisiana. Because the index uses rankings relative to other culti- Cultivars N-24 and N-21 show potential for high lev- vars evaluated, it consistently identified susceptible and els of resistance and may provide unique resistant resistant cultivars. Pest pressure was fairly consistent germplasms for incorporation into other breeding pro- during all 3 yr of research in Jefferson County and grams. Common cultivation of susceptible cultivars is repeatedly demonstrated a wide range in susceptibility likely a key factor, which led to increased E. loftini pest of sugarcane cultivars to E. loftini. Nevertheless, the problems in the Rio Grande Valley (Meagher et al. application of the relative resistance ratios to compare 1994, 1996, 1998). Increased production of resistant resistance levels across experiments may be limited cultivars can improve the success of IPM programs by because it is based on relative rankings rather than on reducing area-wide populations. Additionally, cultivars actual injury ratings. Another limitation of the relative which impede stalk entry may improve the efficacy of resistance ratio is that it may over-estimate differences insecticide applications against E. loftini (Wilson et al. in resistance levels in evaluations containing a small 2012). number of cultivars or if a wide range of injury is not With increased demand for renewable fuel sources, present. The relative resistance ratio is most applicable production of dedicated bioenergy feedstocks for June 2015 WILSON ET AL.: SUGARCANE CULTIVAR RESISTANCE TO E. loftini 1369 lignocellulosic biomass, such as energycane, high-bio- Bischoff,K.P.,K.A.Gravois,T.E.Reagan,J.W.Hoy,C.A. mass sorghum and switchgrass, Panicum virgatum L., Kimbeng,C.M.LaBorde,andG.L.Hawkins.2008. is expected to increase along the U.S. Gulf Coast (Solo- Registration of ‘L 79-1002’ sugarcane. J. Plant Regist. 2: man et al. 2007); however, the impact of stem borers 211–217. such as E. loftini and D. saccharalis remains largely Coburn,G.E.,andS.D.Hensley.1972.Differential survival of Diatraea saccharalis larvae on two varieties of sugarcane. unknown. The six energycane cultivars evaluated Proc. Int. Soc. Sugarcane Technol. 14: 440–444. herein were identified as being either resistant or Gravois, K. 2014. Sugarcane production handbook 2014. LSU intermediate. High fiber content and increased rind AgCenter Publication #2859, Baton Rouge, LA. hardness may convey higher levels of stem borer resist- Hale, A. L., W. H. White, E. O. Dufrene, T. L. Tew, M. P. ance to energycane cultivars (Coburn and Hensley Grisham, Y. B. Pan, and J. D. Miller. 2012a. HoCP 04- 1972, Martin et al. 1975). The relative susceptibility of 838—a new sugarcane variety for Louisiana. J. Am. Soc. high-biomass and sweet sorghum cultivars suggest Sugar Cane Technol. 32: 84–85. these crops may sustain heightened levels of stem Hale,A.L.,E.O.Dufrene,T.L.Tew,Y.Pan,R.P.Viator, borer injury under conditions of high pest pressure. P. M. White, J. C. Veremis, W. H. White, R. Cobill, E. P. Richard, H. Rukavina, and M. P. Grisham. 2012b. Because of the limitations of chemical and biological Registration of ‘Ho 02-113’ sugarcane. J. Plant Regist. 7: control against E. loftini (Meagher et al. 1994, 1998; 51–57. Legaspi et al. 1997), host plant resistance will continue Hensley, S. D., and W. H. Long. 1969. Differential yield re- to be an important facet of management. As a control sponses of commercial sugarcane varieties to sugarcane borer tactic, host plant resistance not only aids in reducing damage. J. Econ. Entomol. 62: 620–623. stem borer injury but also reduces area-wide popula- Legaspi,J.C.,B.C.Legaspi,Jr.,E.G.King,andR.R.Sal- tions and has potential to slow the spread of invasive dan˜ a. 1997. Mexican rice borer, Eoreuma loftini (Lepidop- species. The relative resistance ratio provides a single tera: Pyralidae) in the Lower Rio Grande Valley of Texas: its index to measure resistance based on both plant injury history and control. Trop. Plant Sci. 49: 53–64. Legaspi,J.C.,B.C.Legaspi,Jr.,andR.R.Saldan˜ a. 1999. and pest survival to adulthood. Continued evaluation of Laboratory and field evaluations of biorational insecticides commercial and experimental sugarcane cultivars for against the Mexican rice borer (Lepidoptera: Pyralidae) and a stem borer resistance is necessary as host plant resist- parasitoid (Hymenoptera: Braconidae). J. Econ. Entomol. 92: ance remains a valuable tool in sugarcane IPM. 804–810. Martin,F.A.,C.A.Richard,andS.D.Hensley.1975.Host Acknowledgments resistance to Diatraea saccharalis (F.): relationship of sugar- cane internode hardness to larval damage. Environ. Entomol. Mark Nunez, Rebecca Pearson, and other Texas A&M 4: 687–688. AgriLife Research Beaumont staff provided technical sup- Mathes, R., and L. J. Charpentier. 1969. Varietal resistance in port. Randy Richard (USDA-ARS) assisted in obtaining the sugar cane to stalk moth borers, pp. 175–188.InJ. R. Wil- Louisiana cultivars and participated in planting and harvest- liams, J. R. Metcalfe, R. W. Montgomery, and R. Mathes ing activities. Tony Prado with the Rio Grande Valley Sugar (eds.), Pests of sugar cane. Elsevier, New York. Growers, as well as Andy Scott and Eddie Hernandez of Rio Meagher,R.L.,Jr.,J.W.Smith,Jr.,andK.J.R.Johnson. Farms, provided Rio Grande Valley sugarcane cultivars. Ken- 1994. Insecticidal management of Eoreuma loftini (Lepidop- neth Gravois provided cultivars from the LSU AgCenter tera: Pyralidae) on Texas sugarcane: a critical review. J. Econ. breeding program. This work was supported in part by USDA Entomol. 87: 1332–1344. National Institute of Food and Agriculture (NIFA) bioenergy Meagher, R. L., Jr., J. E. Irvine, R. G. Breene, R. S. Pfan- grant 2011-67009-30132 and the American Sugar Cane nenstiel, and M. Gallo-Meagher. 1996. Resistance mech- League. This manuscript was approved by the director of the anisms of sugarcane to Mexican rice borer (Lepidoptera: Louisiana Agricultural Experiment Station as manuscript no. Pyralidae). J. Econ. Entomol. 89: 536–543. 2014-234-15668. Meagher,R.L.,Jr.,J.W.Smith,Jr.,H.W.Browning,and R. R. Saldan˜ a. 1998. Sugarcane stemborers and their para- References Cited sites in southern Texas. Environ. Entomol. 27: 759–766. Painter, R. H. 1951. 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W. Akbar1*, A.T. Showler2,T.E.Reagan1,J.A.Davis1 & J.M. Beuzelin3 1Department of Entomology, Louisiana State University Agricultural Center, 404 Life Sciences Bldg, Baton Rouge, LA 70803, USA, 2Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX 789028, USA, and 3Dean Lee Research Station, Louisiana State University Agricultural Center, 8105 Tom Bowman Dr, Alexandria, LA 71302, USA Accepted: 18 September 2013

Key words: EPG, feeding behavior, free amino acids, honeydew, phloem sap, Saccharum, Hemiptera, Aphididae, Poaceae

Abstract Feeding behavior of Melanaphis sacchari Zehntner (Hemiptera: Aphididae) was studied on sugarcane, Saccharum spp. (Poaceae), cultivars HoCP 91-555 (resistant), LCP 85-384 (moderately resistant), and L 97-128 (susceptible) using the electrical penetration graph (EPG) technique. Constitutive con- centrations of total phenolics and available carbohydrates, water potential at the whole-leaf tissue level, and free amino acids (FAAs) in phloem sap extracts, and in honeydew produced by aphids fed on L 97-128 and HoCP 91-555 were determined. Cultivar did not influence time for M. sacchari to access phloem sieve elements. Total time in sieve elements was ca. two-fold greater on L 97-128 than on HoCP 91-555, whereas it did not differ from LCP 85-384 in either cultivar. The mean duration of individual events associated with phloem sap ingestion was ca. 50% shorter on both HoCP 91-555 and LCP 85-384 than on L 97-128. Although cultivar effects were not detected for levels of total phen- olics, available carbohydrates, and water potential, two free essential amino acids, histidine and argi- nine, were absent from phloem sap in HoCP 91-555. Two free essential amino acids, leucine and isoleucine, and two free non-essential amino acids, tyrosine and proline, were absent from honeydew of aphids fed on HoCP 91-555. These results suggest that despite apparent biosynthesis of some FAAs, the absence of important FAAs in the phloem sap of HoCP 91-555 and the inability of M. sac- chari and its endosymbionts (e.g., Buchnera) to derive specific free essential and non-essential amino acids from other ingested molecules, possibly along with other unidentified factors, underlie the pest’s decreased phloem sap ingestion and consequently reduced growth potential on HoCP 91-555.

compounds, such as phenolics, in leaf tissues (Fraenkel, Introduction 1969; Todd et al., 1971; Risebrow & Dixon, 1987) can be Host plant resistance or susceptibility to herbivores can physical barriers that deter aphids (Roberts & Foster, 1983; depend on accessibility and nutritional value of host Jackson & Sisson, 1990; Sosa, 1990). Phloem sap is par- tissues. Aphid feeding occurs primarily on phloem sap tially comprised of amino acids, which are considered lim- (Douglas, 1998), but chemical or physical factors within iting factors to aphid growth, development, and survival the leaf can impede access to it (Mayoral et al., 1996). (Douglas, 1998; Karley et al., 2002; Wilkinson & Douglas, Cuticular components and leaf pubescence in wheat, 2003), although aphid endosymbionts in many instances Triticum aestivum L., and sugarcane, Saccharum spp. (both can synthesize some amino acids when they are not Poaceae), for example, as well as secondary plant defensive supplied in food (Wernegreen & Moran, 2000). In Louisiana, the sugarcane aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae), has become the most *Correspondence and current address: Waseem Akbar, Monsanto abundant aphid species on sugarcane (Akbar, 2010). Feed- Company, 700 Chesterfield Pkwy West, Chesterfield, MO 63017, ing by M. sacchari on sugarcane causes a fading of leaf USA. E-mail: [email protected] greenness, and heavily infested leaves are covered with a

32 © 2013 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 150: 32–44, 2014 Sugarcane resistance to the sugarcane aphid 33 black layer of sooty mold growing on deposits of honey- fields (Anonymous, 2007, 2008). The cuttings were heat dew (Hall & Bennett, 1994). A serious problem associated treated in water at 50 °C for 2 h for control of ratoon with M. sacchari is transmission of the persistent Sugar- stunting disease (Comstock, 2002) and planted, one per cane yellow leaf virus (ScYLV; Luteoviridae, genus Polerovi- 1.9-l pot of Sunshine mix no. 1 nursery potting soil (75% rus) (Schenck & Lehrer, 2000). Absence of ScYLV has been sphagnum peat moss, perlite, dolomitic limestone, and added to certification standards for micropropagated gypsum; Sungro Horticulture, Bellevue, WA, USA) with seedcane to minimize virus spread in Louisiana, USA 0.5 g 19:6:12 (N:P:K) controlled release fertilizer (Osmo- (McAllister et al., 2008). Field surveys in Louisiana identi- cote; Scotts Miracle-Gro, Marysville, OH, USA). Irrigation fied ScYLV infections in all sugarcane growing areas with was provided to soil saturation every 2 days. Sugarcane some fields having up to 27% infected plants (McAllister plants at the 4–6 leaf stage (60–75 cm high to the bottom et al., 2008), and ScYLV-related sugar yield losses have of the whorl leaf) were used in the experiments. For phys- reached 14% (Grisham et al., 2001). Aphid population iochemical sampling, 30 cuttings of each cultivar were growth studies on currently important commercial sugar- similarly planted in separate pots. cane cultivars in Louisiana have shown that HoCP 91-555 is resistant and L 97-128 is susceptible to M. sacchari, Electrical penetration graph setup and data recording whereas LCP 85-384 shows intermediate levels of resis- EPG experiments were conducted in a Faraday cage using tance (Akbar et al., 2010). a Giga 8DC EPG amplifier with 1-gigaohm input resis- Because the physiological basis for resistance is not well tance and an AD conversion rate of 100 Hz (Wageningen understood, the electrical penetration graph (EPG) tech- Agricultural University, Wageningen, The Netherlands). A nique was used to detect differences in feeding behavior of DAS-800 Digital Acquisition Card (Keithley Instruments, M. sacchari on HoCP 91-555 (resistant), LCP 85-384 Cleveland, OH, USA) digitalized the analog signals, which (moderately resistant), and L 97-128 (susceptible) culti- were displayed and recorded using WinDaq/Lite software vars. In addition, quantity and composition of free amino (DATAQ Instruments, Akron, OH, USA). A 4-cm-long, acids (FAAs) in the phloem sap and honeydew of aphids 25-lm-diameter gold wire (GoodFellow Metal, Cam- fed on L 97-128 and HoCP 91-555 were determined, as bridge, UK) was attached to the aphid dorsum using silver were leaf tissue concentrations of total phenolics, total conductive paint (Pelco Colloidal Silver no. 16034; Ted available carbohydrates, and water potential, each of which Pella, Redding, CA, USA). The other end of the gold wire has been associated with aphid growth, development, and was connected by silver paint to a copper wire soldered to survivorship (Maltais, 1959; Auclair, 1963, 1967; Cole, a copper nail. Aphids were allowed to acclimate to walking 1984; Leszczynski et al., 1989; Lattanzio et al., 1990; Will with the wire for 1 h. One of the lower five leaves of a sug- & van Bel, 2006). arcane plant was turned abaxial surface face up. Aphids were lowered to contact the abaxial surface and EPG mon- itoring commenced. There were three aphids per record- Materials and methods ing and 10 recordings were made; hence, 30 aphids were Aphid colonies used per cultivar. Feeding behavior was recorded on each The M. sacchari colony used in these experiments was set of three aphids for 4 h, based on preliminary tests indi- founded from a single aphid collected from a sugarcane cating that it was sufficient time for M. sacchari to reach field at the Louisiana State University Agricultural Center leaf sieve elements. (LSU AgCenter) Sugar Research Station, St. Gabriel, LA, A probe was defined as all behaviors occurring from USA (30°26′80″N,91°09′81″W), during July 2007. The col- start of stylet penetration into plant tissue until stylet onies were maintained in the greenhouse on sorghum withdrawal (Backus, 2000). Feeding behavior waveforms plants at 25–30 °C and natural photoperiod. identifying specific aphid probing activities were identi- fied using predefined characteristics (Reese et al., 2000). Plant material Measured parameters included duration to initiate the Cuttings of commercial sugarcane cultivars HoCP 91-555, stylet pathway phase (i.e., contact with the host plant, LCP 85-384, and L 97-128 (Akbar et al., 2010) used in stylet sheath formation, and pathway events exhibited by these studies were obtained from seedcane fields at the waveform A, B, and C), the xylem phase (i.e., ingestion LSU AgCenter Sugar Research Station. Because ScYLV from xylem exhibited by waveform G), and the sieve ele- poses a serious threat to Louisiana’s sugarcane industry, a ment phase (i.e., salivation into and ingestion from sieve comprehensive program is in place to detect its presence at elements exhibited by waveform E1 and E2, respec- the Sugarcane Research Station and there were no reports tively). Stylet penetration difficulties (exhibited by wave- of its occurrence when cuttings were collected from these form F), when observed, were also included in the stylet 34 Akbar et al. pathway phase (Diaz-Montano et al., 2007). Total num- cultivar was excised and freeze dried for 24 h. Each leaf bers of each phase, potential drops (when the stylets was then ground into powder using a Wiley Mini Mill puncture a cell membrane), and the numbers of poten- (Thomas Scientific, Swedesboro, NJ, USA). Total available tial drops to reach the sieve element phase were assessed. carbohydrates were extracted from 30 mg of the lyophi- Computations of the EPG-generated data included lized tissue with 1 ml deionized water while stirring for non-probe time, total time spent probing, duration of 30 min at 25 °C. The mixture was then incubated at 4 °C individual probing events (probing is not a continuous for 16 h, followed by 15 min of centrifugation at process as the aphid removes its stylets from time to 37 788.4 g force. Fifty microlitre of extract from each sam- time; hence, each separate probing event has its own ple was mixed with 15 ll anthrone-sulfuric acid reagent duration), and time in the stylet pathway, xylem, and [12.7 M H2SO4 in water containing 0.1% (wt:vol) sieve element phases within 4 h. Because we were inter- anthrone and 0.1% (wt:vol) thiourea] and incubated at ested in time spent with stylets in the sieve elements 60 °C for 20 min, 0 °C for 3 min, and 25 °C for 20 min. from initial contact to the end of ingestion, waveform Reactions were quantified at 625 nm on a GeneSys-2 spec- E1 and E2 were combined and labeled as waveform E trophotometer (Spectronic, Rochester, NY, USA). Glucose for calculating total time spent in sieve element phase was used as a standard to calculate total available carbohy- and the mean duration of each discrete sieve element drate content in mg gÀ1 dry weight. A linear regression of phase (Diaz-Montano et al., 2007). In instances where dry weight on fresh weight (fresh weight = 3.61348 9 dry some probing behaviors (xylem or sieve element phases) weight + 0.07665; R2 = 0.99) was used to convert total were not detected, data for the time to reach each phase available carbohydrate values into mg gÀ1 fresh weight and the total time and mean duration of each discrete (Moran & Showler, 2005). For water potential measure- phase event were entered without adjustment, and unob- ment, one of the four lower leaves from each of 15 plants served probing behavior was not included in the analysis of both cultivars was excised to measure water potential (Brewer & Webster, 2001). Measured feeding behavior using a Model 610 pressure chamber (PMS Instrument, parameters were not normally distributed and were Corvallis, OR, USA). therefore analyzed using Kruskal–Wallis tests (Proc For quantifying FAAs, phloem sap was obtained using NPAR1WAY, SAS, version 9.1.3; SAS Institute, Cary, the ethylenediaminetetraacetic acid (EDTA)-exudation NC, USA). technique (King & Zeevaart, 1974). One leaf from each of 15 plants of both cultivars was excised at a ligule Plant and aphid honeydew biochemical analyses with scissors, the cut end immediately immersed in a Fifteen plants of HoCP 91-555 and L 97-128 each were 1.5-ml solution of 5-mM EDTA at pH 7 in a 15-ml used for honeydew collection, and one of the four lowest vial. The gap between the leaf and vial opening was leaves (favored for feeding) on the remaining 12–15 plants sealed with Parafilm to avoid water loss through evapo- per cultivar was used for measurements of water potential, ration. The vials were immediately placed in a dark total phenolics, total available carbohydrates, and FAAs. incubator at 25 °C and more than 90% r.h. for 1 h. For total phenolic extraction, excised leaf tissue (ca. Then the leaves were discarded and EDTA with the exu- 4cm2) from each of 15 plants of both cultivars was date was pipetted into 1.5-ml Eppendorf tubes and chopped into small pieces, weighed, submerged in 5 ml of stored at À80 °C until the samples were prepared for 50% methanol, and kept at room temperature for 1 week. FAA analysis using a high-performance liquid chro- A100-ll aliquot of each methanol extract was diluted to matograph (HPLC). 2.75 ml with distilled water in a test tube and vortexed for Because of M. sacchari’s small size (typically <2mm 5 min. Folin-Ciocalteau reagent (0.5 ml of 1 N solution; long) and low amounts of excreted honeydew, it was not Sigma-Aldrich, St. Louis, MO, USA) was then added to possible to determine the composition of honeydew the diluted extract (Stout et al., 1998). After 5 min, 0.5 ml excreted by individual aphids. Instead, each sample con- of 20% sodium carbonate was added, vortexed for 5 min, sisted of honeydew collected from 10 late instars confined and stored for 90 min at room temperature. Absorbance for 3 days within a 2 9 0.6-cm double-sided adhesive was measured at 720 nm with a Shimadzu UV-1601 spec- cage (Scotch mounting tape; 3M, St. Paul, MN, USA) cov- trophotometer (Shimadzu Scientific Instruments, Colum- ered with Parafilm on the abaxial surface of a leaf. Aphids bia, MD, USA). Total phenolic concentration in each were then removed from the plant and the Parafilm with sample was calculated based on a standard ferulic acid the honeydew drops was weighed. Honeydew from aphids curve. feeding on three plants of the same cultivar was pooled For determining total available carbohydrate content, and replicated five times per cultivar. Honeydew was one of the lower four leaves from each of 12 plants of each washed from the Parafilm with 1 ml of distilled water and Sugarcane resistance to the sugarcane aphid 35 stored in 1.5-ml Eppendorf tubes. The Parafilm was allowed to dry and weighed again to determine the Results amount of honeydew dissolved in 1 ml of distilled water. Electrical penetration graph analyses These samples were immediately stored at À80 °Cuntil Melanaphis sacchari spent 26, 17, and 20% of the 4-h analyzed in the HPLC. experimental period without probing on LCP 85-384, For measuring FAA concentrations, 1-ml aliquots of HoCP 91-555, and L 97-128, respectively, but statistically supernatant from phloem sap and from honeydew sam- significant cultivar differences were not detected (Table 1). ples were filtered through a 0.5-ll filter fitted to a 5-ml However, both total probe time and duration of individual plastic syringe. Samples were placed in the autosampler of probing events differed among cultivars, with 1.13-fold an 1100 Series reversed-phase HPLC (Agilent Technolo- longer total probe time on HoCP 91-555 than on LCP gies, Atlanta, GA, USA) with a binary pump delivering 85-384, and 1.54-fold longer probe duration on L 97-128 solvent A [1.36 g sodium acetate trihydrate + 500 ml than on LCP 85-384 (Table 1). The mean total numbers of purified HPLC grade water + 90 ll triethylamine stylet pathway and sieve element phases throughout the (TEA) + sufficient acetic acid to bring the pH to experiment were not influenced by cultivar. Although E2 7.2 Æ 0.05 (95% confidence interval)] and solvent B followed E1 in all three cultivars, only 53, 43, and 73% [1.36 g sodium acetate trihydrate + 100 ml purified resulted in ingestion lasting >10 min on LCP 85-384, HPLC grade water (acetic acid added to this mixture to HoCP 91-555, and L 97-128, respectively (Table 1). bring the pH to 7.2 Æ 0.05 95% CI + 200 ml acetoni- Total numbers of potential drops were not affected by trile + 200 ml methanol] at 100 and 1.0 ml per min on a cultivar, and proportions of aphids with at least one suc- Zorbax Eclipse AAA 4.6 9 150 mm 3.5 l column cessful probe were ≥75% among the three cultivars (Agilent Technologies). Absorbances at 262 and 338 nm (Table 1). An average of 22 min was required to initiate were monitored on a variable wavelength detector for the stylet pathway phase regardless of cultivar, and the 26 min per sample. The autosampler measured and mixed time to contact with xylem and phloem vessels after onset 6 ll sodium borate buffer (0.4 N, pH 10.2 in water), 1 ll of the first probe was also not affected (Table 1). While 9-fluorenylmethyl chloroformate (FMOC), and 1 ll M. sacchari probed, the greatest amount of time was in o-phthalaldehyde (OPA) derivitizing agents, and 2 llof stylet pathway phase in all three cultivars, but the total sample, then injected 2 ll for chromatographic separation time spent ingesting from xylem vessels was ca. two-fold of FAAs. Identification and quantification of 17 derivitized longer on HoCP 91-555 than on L 97-128 (v2 = 8.55, FAAs were achieved by calibrating with a standard mixture d.f. = 2, P = 0.014) (Figure 1), whereas the total time in of amino acids. Peak integration accuracy was enhanced sieve elements was ca. 50% shorter on HoCP 91-555 than by manual establishment of peak baselines using Agilent on L 97-128 (v2 = 7.31, d.f. = 2, P = 0.026) (Figure 1). software (Chemstation rev. A.10.02 1757) (Showler & Cultivar effects were not found for total duration of indi- Castro, 2010), and this method could detect FAA concen- vidual stylet pathway and xylem phases, but the mean trations to the picomole level. duration in sieve elements was ca. two-fold longer on The concentrations of total FAAs in each sample of L 97-128 than on LCP 85-384 (v2 = 5.68, d.f. = 2, phloem sap (pmol llÀ1 phloem sap exudate) and honey- P = 0.017), and 2.3-fold longer on L 97-128 than dew (pmol mgÀ1 honeydew) were calculated by combin- on HoCP 91-555 (v2 = 9.25, d.f. = 2, P = 0.0023) ing individual concentrations of all detected FAAs in that (Figure 2). sample. The concentrations (%) of individual FAAs were calculated by using the following formula (with arginine Plant biochemicals and aphid honeydew analyses selected for illustrative purposes): (pmol arginine/total Cultivar effects between L 97-128 and HoCP 91-555 were pmol FAAs) 9 100%. Because the amount of honeydew not detected for levels of total available carbohydrates, dissolved in each sample varied, the concentrations of total phenolics, and water potential (Table 2). Free essen- FAAs were adjusted for weight of honeydew in each sam- tial amino acids (critical for insect growth and develop- ple by dividing total concentration by respective sample ment; Gilmour, 1961; Dadd, 1985) detected using our weight. Treatment differences in terms of total FAA con- system were comprised of arginine, histidine, isoleucine, centrations in phloem sap and honeydew, concentrations leucine, lysine, methionine, phenylalanine, threonine, and of total available carbohydrates and phenolics, and valine (tryptophan, the only remaining essential amino measurements of water potential were detected using the acid, could not be detected using our system), and free Student’s t-test (SAS, v9.1.3). Percentage concentrations non-essential amino acids were alanine, aspartic acid, cys- of individual FAAs were arcsin √-transformed before tine, glutamic acid, glycine, proline, serine, and tyrosine. analysis (SAS, v9.1.3). Free essential amino acids in phloem sap comprised 22 36 Akbar et al.

Table 1 Mean (Æ SE) feeding behavior parameters of Melanaphis sacchari during a 4-h period on three sugarcane cultivars, LCP 85-384 (moderately resistant), HoCP 91-555 (resistant), and L 97-128 (susceptible)

Cultivar Parameter LCP 85-384 HoCP 91-555 L 97-128 v2 P Non-probe time (min) 62.6 Æ 10.5 43.0 Æ 12.5 47.6 Æ 10.0 1.82 0.40 Total probe time (min)1 180.0 Æ 10.1b 203.7 Æ 12.5a 192.4 Æ 10.4ab 10.23 0.0059 Individual probe duration (min)1 62.3 Æ 10.4b 91.9 Æ 15.7ab 96.4 Æ 12.2a 8.43 0.015 Time (min) to first stylet pathway phase 21.7 Æ 7.3 27.3 Æ 10.0 18.2 Æ 4.5 1.27 0.53 Time (min) to first xylem phase2 79.3 Æ 19.2 35.1 Æ 9.9 83.4 Æ 15.9 5.41 0.067 Time (min) to first sieve element phase3 80.5 Æ 10.2 105.7 Æ 14.4 101.2 Æ 14.0 1.32 0.52 No. stylet pathway phases 6.2 Æ 0.6 6.0 Æ 0.6 4.7 Æ 0.5 2.89 0.24 No. xylem phases 0.4 Æ 0.2b 1.2 Æ 0.3a 1.2 Æ 0.3a 7.81 0.020 No. E1 (salivation into sieve elements) 1.7 Æ 0.3 1.4 Æ 0.2 1.1 Æ 0.2 1.53 0.47 No. E2 (ingestion from sieve elements) 1.7 Æ 0.3 1.4 Æ 0.2 1.1 Æ 0.2 1.53 0.47 No. E2 <10 min 0.8 Æ 0.2 0.8 Æ 0.2 0.3 Æ 0.1 4.68 0.096 No. E2 >10 min 0.9 Æ 0.2 0.6 Æ 0.2 0.8 Æ 0.1 1.69 0.43 Total no. pds 30.6 Æ 3.6 43.2 Æ 5.8 36.6 Æ 3.6 2.83 0.24 No. pds to sieve element phase 16.4 Æ 2.1 22.8 Æ 2.7 22.4 Æ 2.4 5.77 0.056 % successful probes4 81.2 75.0 76.6

Means within rows followed by different letters are significantly different (Kruskal–Wallis test: d.f. = 2, P<0.05). 1Total probe time is the cumulative time an aphid spends probing a plant; because probing is not a continuous process and the aphid removes its stylets from time to time, each individual probing event has its own duration. 2Aphids that made contact with xylem on LCP 85-384, n = 7; HoCP 91-555, n = 13; L 97-128, n = 17. 3Aphids that made contact with sieve elements on LCP 85-384, n = 26; HoCP 91-555, n = 21; L 97-128, n = 23. 4At least one ingestion event from sieve elements lasted >10 min. Data were not statistically analyzed because of lack of replication.

180 LCP 85-384 100 LCP 85-384 160 a HoCP 91-555 HoCP 91-555 L 97-128 a L 97-128 140 a a 80 a 120 a 100 a 60 80 ab ab b 60 b a b b 40 a a

Total time (min) a 40 Duration (min) a 20 20 0 Stylet pathway Xylem Sieve element 0 Phase Stylet pathway Xylem Sieve element Phase Figure 1 Mean (Æ SE) total time Melanaphis sacchari spent stylet probing in each phase on three sugarcane cultivars. Bars within Figure 2 Mean (Æ SE) duration of individual stylet probing – each phase bearing different letters differ significantly (Kruskal events in each phase by Melanaphis sacchari on three sugarcane < Wallis test: P 0.05). cultivars. Bars within each phase bearing different letters differ significantly (Kruskal–Wallis test: P<0.05). and 5% of the total FAAs in L 97-128 and HoCP 91-555, respectively (t = 2.09, d.f. = 23, P = 0.048) (Table 2). The full spectrum of detectable FAAs was not found in any cultivars was predominantly comprised of non-essential of the phloem sap samples, and the arrays of FAAs also amino acids, the most abundant of which were alanine, varied. Eight FAAs were detected in the phloem sap of accounting for 26 and 35% of total FAAs in L 97-128 and L 97-128, whereas seven were found in HoCP 91-555 HoCP 91-555, respectively, and glutamic acid, accounting (Figure 3). The FAA profile of phloem sap of both for 19 and 22% of total FAAs in L 97-128 and HoCP Sugarcane resistance to the sugarcane aphid 37

Table 2 Mean (Æ SE; sample sizes in parentheses) total available carbohydrates, water potential, and total phenolics in leaf tissue and total free amino acids in phloem sap of Melanaphis sacchari susceptible (L 97-128) and resistant (HoCP 91-555) sugarcane cultivars

Sugarcane cultivar Student’s t-test Measurement L 97-128 HoCP 91-555 P Whole-leaf tissue Total available carbohydrates (mg gÀ1 fresh weight) 214.5 Æ 24.6 (12) 250.0 Æ 12.2 (12) 0.21 Total phenolics (lmol gÀ1 fresh weight) 15.9 Æ 0.7 (15) 14.7 Æ 0.9 (15) 0.31 Water potential (bar) 5.4 Æ 0.6 (15) 3.9 Æ 0.5 (15) 0.074 Phloem sap Total free amino acids (pmol llÀ1)688.1Æ 73.2 (11) 781.4 Æ 117.3 (14) 0.54 Total free essential amino acids (pmol llÀ1)152.0Æ 56.3 (11) 39.6 Æ 20.0 (14) 0.048 Total free non-essential amino acids (pmol llÀ1)536.4Æ 51.4 (11) 742.3 Æ 102.1 (14) 0.11

45 L 97-128 40 HoCP 91-555 35 * 30 25 20 15 *

% concentration 10 Figure 3 Mean (Æ SE) percentages of free 5 * non-essential and essential amino acids in 0 * the phloem saps of Melanaphis sacchari susceptible (L 97-128) and resistant Valine Lysine Serine Proline Cystine Glycine Alanine Leucine Arginine

(HoCP 91-555) sugarcane cultivars. The Tyrosine Histidine Threonine Isoleucine asterisks indicate significant differences Methionine Aspartic acid Glutamic acid between cultivars (Student’s t-test: Phenylalanine Free non-essential amino acids Free essential amino acids P<0.05).

91-555, respectively. Alanine and glutamic acid were also phloem sap and honeydew of aphids feeding on that culti- the only two FAAs detected in every sample, and alanine var (Figure 4A). However, there were seven FAAs detected was 1.3-fold more concentrated in HoCP 91-555 than in in the honeydew of aphids feeding on L 97-128 that were L97-128(t= 3.24, d.f. = 23, P = 0.0036) (Figure 3). not found in phloem sap, five of them essential: isoleucine Aspartic acid and serine, although not ubiquitous, were (t = 3.76, P = 0.0021), leucine (t = 3.50, P = 0.0035), commonly found, but cystine, proline, and tyrosine were lysine (t = 3.59, P = 0.0029), phenylalanine (t = 2.50, not detected in either cultivar. Among free essential amino P = 0.025), and valine (t = 2.45, d.f. = 14, P = 0.028; all acids, histidine (t = 2.87, d.f. = 23, P = 0.0086) and argi- d.f. = 14) (Figure 4A). Free non-essential amino acids nine (t = 3.18, d.f. = 23, P = 0.0042) were present only in tyrosine (t = 3.69, d.f. = 14, P = 0.0024) and proline the phloem sap of L 97-128, whereas valine (t = 3.05, (t = 3.73, d.f. = 14, P = 0.0022) were also present in hon- d.f. = 23, P = 0.0061) was detected only in the phloem eydew but not in phloem sap of L 97-128 (Figure 4A). For sap of HoCP 91-555 (Figure 3). aphids feeding on HoCP 91-555, the FAAs detected in Comparison of FAAs in phloem sap and honeydew honeydew but not in phloem sap, arginine (t = 5.99), revealed different compositions and concentrations in the histidine (t = 6.10), lysine (t = 6.37), and phenylalanine two cultivars. Free alanine was the most abundant FAA in (t = 6.92; all d.f. = 17, P<0.0001) were all essential phloem sap, whereas free glutamic acid and aspartic acid (Figure 4b). predominated in honeydew regardless of host cultivar Differences between the two cultivars in terms of total (Figure 4). Free arginine and histidine were the most free essential amino acids in honeydew excreted by abundant essential amino acids detected in both L 97-128 M. sacchari were not detected, but honeydew from aphids 38 Akbar et al.

45 A L 97-128 Sap 40 L 97-128 Honeydew 35 30 25 * 20 * 15 10

% concentration 5 0 * * * * * * 45 B HoCP 91-555 Sap 40 HoCP 91-555 Honeydew 35 30 * 25 20 15 * Figure 4 Mean (Æ SE) percentages of free 10 non-essential and essential amino acids in 5 * % concentration * 0 * phloem sap and in excreted honeydew of Melanaphis sacchari feeding on (A) susceptible (L 97-128) and (B) resistant Valine Lysine Serine Proline Cystine Glycine – Alanine

Leucine (HoCP 91 555) sugarcane cultivars. The Arginine Tyrosine Histidine Threonine Isoleucine

Methionine asterisks indicate significant differences Aspartic acid Glutamic acid Phenylalanine between sap versus honeydew (Student’s Free non-essential amino acids Free essential amino acids t-test: P<0.05). feeding on L 97-128 had 4.1-fold (t = 4.37, d.f. = 8, sugarcane cultivars with a differential susceptibility. Some P = 0.0024) and 5.1-fold (t = 3.77, d.f. = 8, P = 0.0054) particular parameters of interest were as follows: time more total FAAs and total non-essential amino acids, required for aphid stylets to reach sieve elements, a mea- respectively, than HoCP 91-555 (Figure 5). There were 15 sure of accessibility, and recognition of the target feeding FAAs detected in the honeydew of aphids feeding on site (Reese et al., 1994; Tjallingii, 2006); relative incidence L 97-128 as compared to 11 from aphids feeding on HoCP of successful probes (sustained ingestion of >10 min), a 91-555 (Figure 6). The four FAAs detected only in the measure of phloem acceptance (Tjallingii, 1990; Davis & honeydew of aphids feeding on L 97-128 were comprised Radcliffe, 2008); and length of time an aphid continuously of the free essential amino acids isoleucine (t = 2.42, ingests sap, a measure of phloem-based resistance P = 0.042) and leucine (t = 2.26, P = 0.044), and the free (Zehnder et al., 2001; Klingler et al., 2005; Diaz-Montano non-essential amino acids tyrosine (t = 2.38, P = 0.045) et al., 2007). Although differences were detected in total and proline (t = 2.41, P = 0.043; all d.f. = 8) (Figure 6). probe time, individual probe duration, total time spent in xylem vessels, and total numbers of xylem phases, those measurements appear to be of little value in terms of Discussion differentiating resistant and susceptible cultivars, in con- The EPG technique was used in this study to detect possi- trast with time required to reach the sieve elements and ble differences in feeding behavior of M. sacchari among duration of time spent ingesting sap (Kennedy et al., 1978;

600 L 97-128 ) HoCP 91-555 –1 500 * 400 * Figure 5 Mean (Æ SE) concentrations 300 (pmol mgÀ1) of free total, essential, and 200 non-essential amino acids in honeydew of Melanaphis sacchari fed on susceptible 100 (L 97-128) and resistant (HoCP 91-555) Concentration (pmol mg sugarcane cultivars. The asterisks indicate 0 significant differences between cultivars Total FAAs Free essential amino acids Free non-essential amino acids (Student’s t-test: P<0.05). Sugarcane resistance to the sugarcane aphid 39

45 L 97-128 Honeydew 40 HoCP 91-555 Honeydew 35 30 25 20 * 15 10 % concentration Figure 6 Mean (Æ SE) percentages of free 5 non-essential and essential amino acids in 0 * * * honeydew of Melanaphis sacchari fed on susceptible (L 97-128) and resistant Valine Lysine Serine Proline Cystine Glycine Alanine Leucine Arginine Tyrosine (HoCP 91-555) sugarcane cultivars. The Histidine Threonine Isoleucine asterisks indicate significant differences Methionine Aspartic acid Glutamic acid between cultivars (Student’s t-test: Phenylalanine Free non-essential amino acids Free essential amino acids P<0.05).

Campbell et al., 1982; Reese et al., 2000; Brewer & Web- Koch, in sieve elements were lower on resistant than on ster, 2001; Lei et al., 2001). Because sieve elements are the susceptible lupin, Lupinus spp. (Zehnder et al., 2001), but main target site of aphid feeding, reaching sieve elements in our study, numbers of aphids engaged in ingestion for is important for host plant acceptance and colonization of >10 min were not influenced by cultivar, demonstrating a host plant (Davis & Radcliffe, 2008), but aphids might host acceptance (Davis & Radcliffe, 2008). Relatively short first have to contend with physical or chemical barriers. durations of ingestion from sieve elements has been For example, a longer sieve element access time and fewer reported to reduce population growth of the green peach aphids reaching there were attributed to high levels of 2,4- aphid, Myzus persicae Sulzer, on barley, Hordeum vulgare dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), L., and rye, Secale cereale L. (Davis & Radcliffe, 2008). a hydroxamic acid in leaves of wheat (Givovich & Niemey- Similarly, the relatively short ingestion time and reduced er, 1991). Gabrys & Pawluk (1999) showed that deterrent nutrient uptake of the foxglove aphid, Aulacorthum solani factors inside the leaf differ in activity and can hinder stylet Kaltenbach, from the sieve elements of resistant soybean, penetration of epidermal, parenchyma, and phloem cells Glycine max L. (Merr.), resulted in lower aphid survival by the cabbage aphid, Brevicoryne brassicae L. Leaf cells are and reproduction, and slower development than on held together by a layer of intercellular pectin called mid- susceptible varieties (Takahashi et al., 2002). Klingler et al. dle lamella, and duration of stylet pathway phase was cor- (2005) reported that the blue alfalfa aphid, Acyrthosiphon related with rate of pectin depolymerization by pectinase kondoi Shinji, spent less time ingesting phloem sap on an in aphid saliva injected into intercellular spaces as aphids aphid-resistant legume variety than on a susceptible vari- probe (Dreyer & Campbell, 1987). Morris & Foster (2008) ety, and concluded that the resistance occurred at the reported increased time between stylet insertion into the phloem sieve elements. In our study, more than two-fold epidermis and start of sieve element physiochemical resis- differences in the mean duration of individual sieve ele- tance in intercellular spaces. In our study, relatively high ment phases in sieve elements between HoCP 91-555, LCP percentages of aphids reached sieve elements on all three 85-384 (resistant and moderately resistant, respectively), cultivars and time required to reach there was not influ- and susceptible L 97-128 explain previously observed enced by cultivar, suggesting an absence of impediments cultivar-related differences in the biotic potential of to locating sieve elements (Reese et al., 1994). This was M. sacchari (Akbar et al., 2010, 2011) and suggest that the corroborated by lack of cultivar-associated differences in resistance is associated with sieve elements. the total numbers of potential drops and numbers of Aphid feeding from sieve elements triggers wound potential drops to reach sieve elements. Other sugarcane responses such as coagulation of p-proteins in the plant’s aphid antixenosis experiments also revealed a lack of culti- sieve elements and in the food canal of the aphid’s stylets, var-associated deterrence or repellency (Akbar et al., but aphids overcome coagulation responses by injecting 2010). watery saliva into the sieve elements during E1 and E2 Longer time spent ingesting phloem sap is an indication (Tjallingii, 2006). However, each E1 phase may or may not of host plant acceptance and suitability (Montllor & be followed by an E2 phase, depending on the difficulty of Tjallingii, 1989; Lei et al., 2001). The total and mean transitioning from E1 to E2. Resistance can result in cessa- durations spent by the cowpea aphid, Aphis craccivora tion of sieve element phase after a single E1, and duration 40 Akbar et al. of E1 can also be extended (Tjallingii, 2006). We did not forms (Risebrow & Dixon, 1987; Febvay et al., 1988). The detect cultivar effects on numbers and durations of E1, or availability of FAAs in the phloem sap is widely believed to on numbers of E2 followed by E1, indicating that M. sac- affect aphid performance and feeding behavior (Auclair, chari recognized sieve elements and initiated phloem sap 1963; Douglas, 1998; Karley et al., 2002). Positive correla- ingestion regardless of cultivar. van Helden & Tjallingii tions were reported between FAA levels in phloem sap of (1993) also documented similar numbers and durations of Brassica spp. and population increase in the B. brassicae E1 for the lettuce aphid, Nasonovia ribisnigri Mosley, on (Cole, 1997). Fecundity of the bird oat-cherry aphid, resistant and susceptible lines of lettuce, Lactuca sativa L. Rhopalosiphum padi L., was correlated with FAA levels in Tjallingii (2006) hypothesized that prolonged E1 and wheat phloem (Kazemi & van Emden, 1992). Weibull shortened E2 on resistant plants result from the aphid’s (1987) also documented that the growth rate of R. padi reduced ability to suppress phloem wound responses. was directly proportional to FAA concentrations in the Fartek et al. (2012) demonstrated strong reduction in sus- phloem saps of oat, Avena sativa L., and barley, Hordeum tained ingestion from sieve elements of a resistant sugar- vulgare L. A change in feeding behavior of R. padi with cane cultivar and concluded that a resistance factor was more time spent salivating before sustained ingestion has located in the phloem. Our findings also indicate that the been reported on nitrogen-deficient barley (Ponder et al., most likely basis of resistance in HoCP 91-555 and LCP 2001). Similarly, Hunt et al. (2010) recorded increased 85-384 is another phloem-associated mechanism. salivation by M. persicae into the sieve elements of mutant This study is the first to quantify selected primary and Arabidopsis plants that had reduced levels of essential and secondary metabolites in association with sugarcane resis- non-essential amino acids. Consistent with the changes in tance to aphids. Nutritional components, such as water feeding behavior, a small effect on aphid reproduction was and carbohydrates, are important to insect feeding and also reported. In our study, free non-essential amino acid survival (Chapman, 2003); however, in the context of our compositions in L 97-128 and HoCP 91-555 phloem sap study, the lack of cultivar effects suggests that their roles in were similar, and free non-essential amino acids predomi- governing M. sacchari population growth are negligible. nated in both cultivars, which have also been reported for Defensive phenolic compounds are widely distributed other graminous crops (Hayashi & Chino, 1986; Weibull among members of Poaceae, including sugarcane (God- et al., 1990; Wilkinson & Douglas, 2003). However, differ- shall & Legendre, 1988; Balcerek et al., 2009). Although ences recorded for free essential amino acids and presence aphid stylets penetrate epidermal and mesophyll tissues of histidine and arginine in L 97-128 were a likely cause for intercellularly, avoiding contact with vacuoles and other discrimination between the two cultivars for sustained organelles that can be high in phenolics (Dreyer & Camp- feeding. bell, 1987; Duarte-Almeida et al., 2006), plants with rela- The chemical composition of honeydew is not the same tively high concentrations of phenolics have been shown as that of phloem sap (Molyneux et al., 1990), suggesting to impair growth, development, and fecundity of aphids that the aphid’s metabolic processes and its endo- (Leszczynski et al., 1995; Kessler & Baldwin, 2002; Urban- symbionts alter composition of ingested phloem sap ska et al., 2002; Singh et al., 2004). However, lack of culti- before excretion (Douglas, 1998; Telang et al., 1999). var differences suggest that levels of these secondary Buchnera endosymbionts, for example, have been shown metabolites might not play a role in aphid resistance in to biosynthesize many amino acids, including some that HoCP 91-555. are essential (Mittler, 1971; van Ham et al., 1997; Douglas At a more fundamental level of the plant–insect rela- et al., 2001). In our study, the occurrence of free arginine, tionship, nitrogen is critical for growth because of its histidine, lysine, and phenylalanine in the honeydew of centrality to metabolic processes, cellular structure, and M. sacchari feeding on HoCP 91-555, each of which were genetic coding; therefore, it is potentially limiting to insect absent in the phloem sap, indicates that aphids were able development and reproduction (Mattson, 1980). Insects to derive these amino acids, rendering their involvement absorb nitrogen through the gut primarily in the form of in varietal resistance unlikely. Free leucine, isoleucine, FAAs or very small peptides; hence, the initial cost of pro- tyrosine, and proline, however, were absent only in the teolysis is saved if amino acids are ingested in this form, honeydew of aphids feeding on HoCP 91-555, which and the distinction between FAAs and those bound in pro- suggests that the inhibition of biosynthesis of these FAAs teins is particularly important for insects, such as aphids, might have roles in this cultivar’s resistance. Dadd & that are physiologically incapable of ingesting large Krieger (1968) found that dietary isoleucine was essential peptides (Brodbeck & Strong, 1987). Therefore, aphids for normal development of M. persicae, and Cole (1997) primarily target phloem sieve elements where FAAs are detected a positive correlation between rate of B. brassicae available in soluble, readily assimilable, and renewable population increase and four FAAs, including leucine and Sugarcane resistance to the sugarcane aphid 41 tyrosine. Tyrosine, needed for sclerotization of insect cuti- early stylet withdrawal from the phloem (Zehnder et al., cle after molting (Urich, 1994), is derived from phenylala- 2001), the similar numbers of potential drops in our study nine (Sandstrom€ & Moran, 1999), and both phenylalanine does not support that possibility. Instead, our findings and tyrosine were detected in the honeydew of aphids suggest that resistance in HoCP 91-555 was associated, feeding on the susceptible variety L 97-128, but only phen- without ruling out the role of other unidentified factors, ylalanine was found in the honeydew of HoCP 91-555, with its deficient nutritional quality and inability of suggesting the aphid’s inability to derive tyrosine on that M. sacchari to derive specific free essential and non-essen- cultivar. Biotic and abiotic stresses on sugarcane can result tial amino acids. in increased accumulation of proline (Showler et al., 1990; Singh et al., 1993; Reay-Jones et al., 2005; Showler & Castro, 2010). Proline detection only in the honeydew of Acknowledgments M. sacchari feeding on L 97-128 might have occurred Thanks are due to Jaime Cavazos (USDA-ARS, Weslaco, because aphids were confined in a small cage for 3 days, TX, USA) for processing the free amino acid and total possibly causing enough localized stress to elicit accumula- available carbohydrate samples, and Mike Stout (LSU tion of more proline in L 97-128 phloem sap. AgCenter) for guidance on total phenolics work. The Insect feeding behavior, total food consumption, and authors also thank two anonymous reviewers for thought- consumption rate are affected by nutritional suitability of ful comments on the manuscript. This study is approved host plants (Mattson, 1980). Differences in FAA accumu- for publication by the Director of the Louisiana Agricul- lations in sugarcane are linked to a number of stresses, tural Experiment Station as manuscript number 2013- some of which have been associated with Mexican rice 234-8086. borer, Eoreuma loftini (Dyar), oviposition preference, and populations of stunt nematodes, Tylenchorhynchus annu- latus (Casidy) Golden (Showler et al., 1990, 1991; Reay- References Jones et al., 2007; Showler & Castro, 2010). Studies on Akbar W (2010) Categorization and Identification of Biochemi- other plants have shown that host plant nutritional quality – – cal Bases of Sugarcane Resistance to the Sugarcane Aphid in terms of FAA concentrations and compositions has (Hemiptera: Aphididae). PhD Dissertation, Louisiana State a role in mediating aphid feeding behavior and perfor- University, Baton Rouge, LA, USA. mance. As an example, black bean aphids, Aphis fabae Akbar W, Showler AT, White WH & Reagan TE (2010) Scopoli, spent more time ingesting phloem sap from sus- Categorizing sugarcane cultivars for resistance to the sug- ceptible broad bean, Vicia fabae L., cultivars than from arcane aphid and yellow sugarcane aphid (Hemiptera: resistant lines, and susceptibility was associated with rela- Aphididae). Journal of Economic Entomology 103: 1431– tively high concentrations of FAAs (Cichocka et al., 2002). 1437. In soybeans, reduced nutritional quality in terms of amino Akbar W, Showler AT, Beuzelin JM, Reagan TE & Gravois KA acids composition was related to resistance against soy- (2011) Evaluation of aphid resistance among sugarcane culti- bean aphids, Aphis glycines Matsumura (Chiozza et al., vars in Louisiana. Annals of the Entomological Society of America 104: 699–704. 2010), and an EPG experiment showed that the time from Anonymous (2007) Pathology Research. Louisiana State Univer- the initiation of aphid stylet penetration of the plant to sity Agricultural Center, Sugarcane Research Annual Progress first phloem sieve element phase tripled in a resistant vari- Report 2006, pp. 148–160. ety (Crompton & Ode, 2010). Also, more feeding occurred Anonymous (2008) Pathology Research. Louisiana State Univer- in the xylem than in the phloem and the reduced ability to sity Agricultural Center, Sugarcane Research Annual Progress ingest the more nutritious phloem sap had an antibiotic Report 2007, pp. 138–148. effect (Crompton & Ode, 2010). The effects of phloem sap Auclair JL (1963) Aphid feeding and nutrition. Annual Review of amino acid composition on aphid reproduction and Entomology 8: 439–490. development have been well documented (Febvay et al., Auclair JL (1967) Effects of light and sugars on rearing the cotton 1988; Prosser & Douglas, 1992; Sandstrom€ & Pettersson, aphid, Aphis gossypii, on a germ-free and holidic diet. Journal – 1994). Differences in FAA concentrations in phloem sap of Insect Physiology 13: 1247 1268. Backus EA (2000) Our own jabberwocky: clarifying the terminol- likely contributed toward the observed differences in aphid ogy of certain piercing sucking behaviors of homopterans. feeding behavior on L 97-128 versus HoCP 91-555, and Principles and Applications of Electronic Monitoring and previously reported reduced population growth on HoCP Other Techniques in the Study of Homopteran Feeding Behav- 91-555 (Akbar et al., 2010). Although it is conceivable that ior (ed. by GP Walker & EA Backus), pp. 1–13. Thomas Say the presence of a feeding deterrent or lack of a feeding Publications in Entomology, Entomological Society of stimulant in the sap of HoCP 91-555 might have triggered America, Lanham, MD, USA. 42 Akbar et al.

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