CARIBBEAN FOOD CROPS SOCIETY 42

Forty Second Annual Meeting 2006

Carolina, Puerto Rico

Vol. XLII-Number 2 PROCEEDINGS

OF THE

42th ANNUAL MEETING

Caribbean Food Crops Society 42th Annual Meeting

July 9-15, 2006

Intercontinental Hotel Carolina, Puerto Rico

"Food Safety and Value Added Production and Marketing in Tropical Crops"

Edited by Héctor L. Santiago and Wanda I. Lugo

Published by the Caribbean Food Crops Society Use of a beneficial nematode against pest mole crickets in Puerto Rico

Ν. E. Vicente1, J. H. Frank2, and N. C. Leppla2

ABSTRACT

A beneficial nematode, Steinemema scapterisci (Rhabditida: Steinernematidae), has been researched at the University of Florida and is now marketed as a biopesticide. Its primary target is the mole cricket, Scapteriscus vicinus Scudder (Orthoptera: Gryllotalpidae), which has been a pest in Florida for about 100 years. The nematode survives so well in Florida's sandy, low-organic soils that it serves as a classical biological control agent to provide permanent suppression of pest mole cricket populations in places to which it has spread. In Puerto Rico and islands of the Lesser Antilles, where there is a much wider range of soils, the primary mole cricket pest is Scapteriscus didactylus (Latreille). Our research evaluates the use of the nematode to control Scapteriscus didactylus. Our earlier results in the field showed establishment and survival for up to seven months in a sandy loam with mole crickets, but no establishment in a highly organic sandy soil with mole crickets. A laboratory bioassay in sterilized sand showed 100% survival of controls, but up to 100% mortality of mole crickets within 168 h dependent upon number of nematodes applied (dose or 'rate'). Laboratory survival of nematodes was poorer in non-sterilized soils than in sterilized soils.

Key words: Gryllotalpidae, Scapteriscus didactylus, Steinemema scapterisci, Biological control

RESUMEN

El nematodo beneficioso, Steinemema scapterisci (Rhabditida: Steinernematidae), ha sido evaluado en la Universidad de Florida, y es mercadeado en la actualidad como un bioplaguicida. Su foco principal es la changa ο grillotopo Scapteriscus vicinus Scudder (Orthoptera: Gryllotalpidae), la cual ha sido una plaga en la Florida por aproximadamente 100 anos. El nematodo sobrevive muy bien en los suelos arenosos y con bajo porcentaje de materia orgânica de la Florida, de forma tal, que sirve como un agente de control biolôgico clâsico para suprimir de manera permanente las poblaciones de la changa en los lugares en donde ésta se ha diseminado. En Puerto Rico y en las islas de las Antillas Menores, en donde la diversidad de suelos es mâs amplia, Scapteriscus didactylus (Latreille) es la especie mâs importante. Nuestra investigaciôn évalua el uso del nematodo para el control de Scapteriscus didactylus. Resultados previos a nivel de campo muestran el establecimiento y supervivencia del nematodo hasta por siete meses en un suelo franco arenoso infestado con changas, pero no se ha logrado lo mismo en un suelo arenoso altamente orgânico. Un bioensayo de laboratorio en arena esterilizada mostrô 100% de supervivencia de los contrôles, y

1 Crop Protection Department, University of Puerto Rico, Mayagüez, PR 00681-9030. 2 Entomology & Nematology Department, University of Florida, Gainesville, FL 32611-0630.

180 hasta 100% de mortalidad de las changas en un periodo de 168 h dependiendo del nûmero de nematodos aplicados (dosis). La supervivencia de los nematodos fue menor en suelos no esterilizados que en ios que fueron previamente esterilizados.

INTRODUCTION

The changa or West Indian mole cricket, Scapteriscus didactylus (Latreille) (Orthoptera: Gryllotalpidae), is the worst of a trio of invasive pest mole crickets of crop plants and turf in Puerto Rico, the Dominican Republic, and many of the Lesser Antilles, especially in irrigated, tilled, and sandy soils. It probably invaded Puerto Rico hundreds of years ago (JHF, unpublished). Introduction of biological control agents into Puerto Rico before 1940 may have partially solved the mole cricket pest problem (Cruz and Segarra, 1992), but not totally so. Those authors assumed that the specialist parasitoid wasp Larra bicolor (F.) (Hymenoptera: Sphecidae), introduced from South America in 1938 (Wolcott, 1938), caused a reduction in mole cricket populations. That may be so, but nobody seems to have evaluated absolute or even relative effects of L. bicolor. The tawny mole cricket, Scapteriscus vicinus (Scudder), is the worst of a trio of invasive pest mole crickets in Florida. It, too, was combated by introduction of L. bicolor from South America, but in 1988 (Frank et al., 1995). It also was combated by successful introduction of Ormia deplete (Wiedemann) (Diptera: Tachinidae) and Steinernema scapterisci Nguyen and Smart (Rhabditida: Steinernematidae) from South America in 1988 and 1985, respectively (Nguyen and Smart, 1990; Frank et al., 1996; Frank and Walker, 2006). Steinernema scapterisci was introduced into Florida as a classical biological control agent. However, it caught the attention of industry and is now marketed in the continental USA as a biopesticide under license from the University of Florida. Our objective was to evaluate how it might function in Puerto Rico as a classical biological control agent or as a biopesticide against Scapteriscus didactylus or the other two pest mole crickets that occur there. We have not attempted to introduce Ormia depleta into Puerto Rico. But in 2001-2004 we applied S. scapterisci at four field sites in western Puerto Rico. In November 2001 we applied S. scapterisci at (a) a sod farm near San German and (b) Punta Borinquen golf course (PB) in Aguadilla. Areas treated were in each case 0.2 ha. Applications were made in low evening light during light rain at the level of 2.47 billion infective-stage nemas per ha. Equipment was a hand-held sprayer nozzle attached to a truck-mounted pump and 95 L tank containing the nemas suspended in water. In May 2002, and again in August 2004, nemas were applied by sprinkling can, preceded and followed by copious irrigation, from a garden hose, to six (of thirteen) small plots of organically-grown vegetables in an organic sandy soil at Instituto Ann Wigmore in Aguada (AW). In February 2002, nemas were applied on two fairways of a golf course in south-central Puerto Rico in light rain in the early evening following irrigation and using a 568 L spray tank belonging to the golf course. In the final trial in August 2004 at PB, nemas were applied to five 0.2-ha plots (with five untreated plots) in an attempt to evaluate treatment effects; the manager/lessee insisted that all plots be in unirrigated areas (roughs); the golf course's spray equipment was used, but the manager/lessee would not allow application in the evening and insisted it be made

181 beginning at dawn to match his crew's normal work schedule. Although the employee who made the applications first applied 568 L of water from the tank (irrigation), and then applied the nematodes, the poor results in most plots suggest that inadequate irrigation and excessive exposure to ultraviolet radiation plus the hard-dried unirrigated soil caused rapid death of virtually all of the applied nematodes. Pitfall traps, modified from Lawrence (1982), were installed in all plots at all sites the day before application to collect mole crickets. Traps were emptied twice per week and all mole crickets were carried to the laboratory and held until death. All mole crickets that produced nematodes (or nematodes therefrom) were shipped in 5% formalin to Florida for nematode identification by Κ. B. Nguyen. Some of the applications were wasted effort. The sod farm near San German produced mole crickets parasitized by S. scapterisci for a few weeks until the entire area was flooded by a rising creek, after which no more nematode-parasitized mole crickets were obtained. The superintendent of the south-central golf course, who had been asked to check the pitfall traps twice weekly and send us mole crickets thus trapped, claimed that no mole crickets were ever trapped; we thought that was strange because we collected mole crickets on a pre-application visit to the site. The August 2004 application at PB was wasted effort when the manager/lessee insisted that we remove the pitfall traps after six months because they interfered with play - until then we had been collecting nematode-parasitized mole crickets. The most interesting applications were those of November 2001 at PB and in May 2002 and August 2004 at AW, but for disparate reasons. The soil at PB was a sandy loam of pH 6.87, which dried hard in the absence of rain or irrigation. The soil at AW was sand of pH 8.06 with a high organic content formed by addition of compost, and was kept moist by rain or irrigation. All mole crickets trapped at PB were S. didactylus; from November 2001 through June 2002 (7 mo) considerable numbers were trapped and a considerable proportion was parasitized by S. scapterisci. Then, the part-time student employee performing the field work left, another was hired in replacement, and the number of mole crickets recorded fell rapidly, with none recorded as parasitized. We may at least conclude that S. scapterisci became established for seven months after application in 2001. Mole crickets trapped at AW were a mixture of S. didactylus and S. abbreviatus (Scudder); none was ever recorded as parasitized despite the two applications of nematodes in seemingly ideal conditions. Because of this disparity and because we had read about nematode-suppressive soils (e.g., Kopppenhöfer et al., 1996; Gao and Becker, 2002) we resolved to test survival of S. scapterisci in soils of those two localities, as well as the dose of nematodes required to kill S. didactylus.

MATERIALS AND METHODS

Steinemema scapterisci bioassay against mole crickets. A bioassay was set up in plastic cups (120 ml, 7 cm depth) with perforated lids (to allow air circulation) to assess the effects of a range of closes of these beneficial nematodes on mortality of field-collected adult S. didactylus mole crickets. Doses of 0, 500, 1000 and 2000 nematodes per mole cricket were applied. Forty mole crickets were placed at 5° C before setting up the bioassay. Fifteen grams of sand were placed in each of 40 plastic cups. Seventy five grams of formulated S. scapterisci (Nematac®S, Becker Underwood,

182 Ames, ΙΑ) were mixed with water to form a suspension and made up to 2000 ml in a measuring cylinder. Nematodes were counted to calculate the number of milliliters of the original suspension that would give the following numbers of nematodes: 500, 1000 and 2000 nematodes per milliliter. One milliliter of nematode suspension containing 500, 1000 or 2000 nematodes per ml was added to the sand in each plastic cup. Controls (no nematodes) used 0.5 ml of tap water followed by 0.5 ml of 0.02% Tween 20™ (a surfactant, Sigma Chemicals, St Louis, MO). Ten replicates were used for each treatment. One mole cricket was placed in each cup. Cups were placed in large plastic containers with damp tissue (to maintain humidity) and incubated at 25° C for 7 d. Data on mole cricket mortality were recorded at 48, 72, 120 and 144 and 168 h. Survival of Steinernema scapterisci in organic soils vs. loam soils. Soil samples, a little larger than optimal size should be, were taken from plant beds (a) at AW and (b) at PB. We took 32 samples at each locality (32 at AW, 32 at PB), placed them into plastic bags, placed the bags into a cooler chest, and took them to the laboratory. Each sample was from the surface layer, not deeper than 2.5 cm. Two of the soil samples from AW and two from PB were used to measure soil moisture. Deep Petri dishes with lids (150 mm diam. χ 20 mm depth) were used for this experiment. Surface area was calculated, and then we calculated the appropriate number of Steinernema scapterisci to apply to the surface of one Petri dish (on the basis of 2.47 billion per ha), then estimated what proportion of a packet of Nematac®S to apply (on the basis of 250 million per packet). We used 3,800 nematodes per dish. Thirty of the Petri dishes were filled with soil from AW, and 30 with soil from PB. Half of them were autoclaved (15 from each location). Dishes were labeled with number, and 3,800 nematodes were applied per dish. The surface of the soil was sprayed daily in each dish with a mist of distilled water from a wash bottle. Each dish was weighed. The soil surface of each dish was sprayed daily with water for the duration of the experiment to prevent desiccation. The results were analyzed by Fisher's exact method because the chi-square method may not have been valid because some of the cells (Table 1 ) had expected counts of less than five.

Table 1. Effect of Steinernema scapterisci on Scapteriscus didactylus mole crickets in the laboratory.

Dead mole crickets Total alive Dose 48 h 72 h 120 h 144 h 168 h after 168 h Control 0 0 0 0 0 10 500 0 0 0 2 2 6 1000 0 0 0 2 2 6

2000 4 2 4 - - 0

After 24 h (1 d), we weighed 10 of the dishes from AW, and 10 from PB, and extracted (Baermann method) and counted the number of living nematodes from each. After 8 d, we repeated the weighing, extraction, and counting of nematodes from 10 dishes from each locality. After 15 d, we repeated the weighing, extraction, and counting of nematodes from the remaining dishes. Time intervals were thus 1-8-15 d. The results were analyzed using the GLM procedure for repeated measures analysis of variance.

183 RESULTS

The results of the bioassay show a dose-related response. Evaluated by Fisher's exact method, the overall results showed that survival was not independent of treatment, P=1.651E-05 (i.e., P«0.0001), with the highest dose (2000 nematodes) having an effect completely contrasting with the control treatment. The results of the survival test showed that the day χ treatment effect was significant (P=0.0101), that there was a quadratic decrease over time of the number of nematodes (P=0.0190), and that the quadratic effect differed among the four soils (P=0.0415).

DISCUSSION

In Florida, the invasive mole cricket Scapteriscus borellii Giglio-Tos is readily infected in field applications of S. scapterisci. Aguillera (1992) performed a laboratory biossay of the nematode on that mole cricket, finding that "At 72 and 96 hours percent mortality was significantly different from the 0 inoculum control at level of >2000 nematodes." Our laboratory bioassay (Table 1) with S. didactylus differed little from Aguillera's method and our results suggest that Puerto Rican S. didactylus is at least as susceptible to infection by this nematode. Field applications of S. scapterisci in Florida against Scapteriscus mole crickets have, to the best of our knowledge, all been made in low-organic sandy soils. Successful applications (few have failed to the best of our knowledge) have resulted in establishment of populations. Where the S. scapterisci population has been investigated in years subsequent to application, it has been found not only to have persisted but even to have spread to surrounding areas. Thus, applications on two Gainesville, Florida, golf courses in the 1980s resulted in populations still present 12 y later (Barbara and Buss, 2006). Application of S. scapterisci to a pasture in Hillsborough County Florida, in 1989, seems to have resulted in presence of the nematode in 2006 in pastures many kilometers distant in that county (JHF, unpublished). In Puerto Rico, there is now little agriculture using sandy soils because they are virtually restricted to coastal areas, and coastal areas have been very heavily urbanized. Agricultural areas have retreated to inland areas of other, heavier soils. Although sandy soils seem to be the preferred habitat of pest mole crickets, the inland soils are suitable when they are moist. Thus, irrigated areas of inland golf courses and sod farms may harbor pest mole crickets. Inland fields that have been tilled, irrigated, and planted with seedling crops may be invaded by mole crickets and the crops heavily damaged. It is such areas that represent many of the likely application sites for S. scapterisci. Our findings here that survival of S. scapterisci was poor in a highly organic sandy soil (as compared with the same soil sterilized) suggest the likely presence of antagonists in that soil, and that such soils may be inimical to establishment of the nematode. Our findings here that survival of S. scapterisci also was reduced in the sandy loam of the PB site (as compared with the same soil sterilized) suggest that sandy loam soils may not be as favorable as Florida's low organic sandy soils to survival of the nematode. However, survival of a nematode population over seven

184 months after application in that soil suggest that the nematode became established and recycled in the mole cricket population, so that it might still be used as a biopesticide with need for annual application. For coastal golf courses based on sandy soils, we see no current reason why survival of the nematode should not be as good as in Florida's sandy soils, nor why its use as a classical biological control agent should not be as good.

Figure 1. Laboratory survival of nematodes applied to soils, sterilized and unsterilized, from two localities where S. scapterisci nematodes were applied in the field. Treatments were AWster (sterilized soil from AW), AWnonst (non- sterilized soil from AW), PBster (sterilized soil from PB), and PBnonst (non-sterilized soil from PB). Readings were taken at 1, 8, and 15 d after application. Each column represents the mean of 5 samples, and SD is shown by an error bar.

Nematodes vs time - 2004

2000

m AWster HDAwnons t • PBster • PBnonst

8 15 days

ACKNOWLEDGEMENTS

We thank Khuong B. Nguyen (Entomology & Nematology Department, University of Florida) for identification of many nematode specimens shipped from Puerto Rico, and Janice Col and Meghan Brennan (IFAS Statistics Department, University of Florida) respectively for performing the GLM procedure for repeated measures analysis of variance for the laboratory survival data, and Fisher's exact test on results of the bioassay. We thank Becker Underwood of Ames, IA, especially Eda Reinot of that company, for supplying the S. scapterisci (Nematac®S) used in field and laboratory trials. Work was supported by a USDA-TSTAR grant 'Integrated management of pest mole crickets in Puerto Rico and Florida', UPN 04051952.

185 LITERATURE CITED

Aguillera, M.M. 1992. Steinernema scaptersci Nguyen and Smart 1990: Bacterial associates, culture, and pathogenicity. PhD dissertation, University of Florida. Barbara, K.A. and E.A. Buss. 2006. Augmentative applications of Steinernema scapterisci (Nematoda: Steinernematidae) for mole cricket (Orthoptera: Gryllotalpidae: Scapteriscus spp.) control on golf courses. Florida Entomol. 89: 257-262 Cruz, C. and A. Segarra. 1992 Potential for biological control of crop pests in the Caribbean. Flohda Entomol. 75: 400-408. Frank J.H., J.P. Parkman and F.D. Bennett. 1995. Larra bicolor (Hymenoptera: Sphecidae), a biological control agent of Scapteriscus mole crickets (Orthoptera: Gryllotalpidae), established in northern Florida. Florida Entomol. 7: 619-623. Frank J.H., J.P. Parkman and T.J. Walker. 1996. The introduction, establishment and spread of Ormia depleta in Florida. Biological Control 6: 368-377. Frank J.H. and T.J. Walker. 2006. Permanent control of pest mole crickets (Orthoptera: Gryllotalpidae: Scapteriscus) in Florida. American Entomol. (in press). Gao, X. and J.O. Becker. 2002. Population development of both sexes of Heterodera schachtii is diminished in a beet cyst nematode-suppressive soil. Biological Control 25: 187-194. Koppenhöfer, A.M., B.A. Jaffee, A.E. Muldoon, D.R. Strong, and H.K. Kaya. 1996. Effect of nematode-trapping fungi on an entomopathogenic nematode originating from the same field site in California. J. Invert. Pathol. 68: 246-252. Lawrence, Κ Ο. 1982. A linear pitfall trap for mole crickets and other arthropods. Florida Entomol. 65: 376-377. Nguyen .B. and G.C. Smart Jr. 1990. Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae). J. Nematol. 22: 178-199. Wolcott, G.N. 1938. The introduction into Puerto Rico of Larra americana Saussure, a specific parasite of the "changa" or Puerto Rican mole-cricket Scapteriscus vicinus Scudder. J. Agric. Univ. P. R. 22: 193-218 [in reality the wasp was Larra bicolor and the mole cricket was Scapteriscus didactylus].