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Rhabditida: Steinernematidae, Heterorhabditidae) 342 Andaló et al. Note Substrates for storing entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae) Vanessa Andaló1; Ricardo Sousa Cavalcanti1; Juan Pablo Molina2; Alcides Moino Jr.3* 1 UFLA – Programa de Pós-Graduação em Entomologia. 2 Corpoica C.I Turipana, km 13 via Cereté – 05747 – Colômbia. 3 UFLA – Depto. de Entomologia, Lab. de Patologia de Insetos, C.P. 3037 – 37200-000 – Lavras, MG – Brasil. *Corresponding author <[email protected]> ABSTRACT: The survival of entomopathogenic nematodes under laboratory conditions is low. With the aim of evaluating substrates to extend the survival of entomopathogenic nematodes, suspensions of Heterorhabditis sp. JPM4 and Steinernema carpocapsae All (3,000 IJ mL–1) were added to dirt, fine sand, coarse sand, foam, expanded clay, phenolic foam, agar, corn starch, Plantmax®, and water. The substrates were placed on Petri dishes (5 cm) and kept at 16 ± 1°C. Survival evaluations were made after 30, 60, 90, 120, 150, and 180 days, with three replicates. After 180 d, a greater percentage of S. carpocapsae infective juveniles (IJs) were still alive in the foam treatment (57.5%) as compared to other treatments, while expanded clay (28.4%), Plantmax® (9.3%) and phenolic foam (11%) were not effective in maintaining the survival rate. Foam (55.6%), coarse sand (53.1%), and fine sand (50.6%) provided greater Heterorhabditis sp. JPM4 IJ survival at 180 days. Agar (19.3%), phenolic foam (11.6%), and Plantmax® (10.7%) had lower survival indices than the control (29.7%). The use of an appropriate substrate can provide greater IJ survival. Key words: Heterorhabditis, Steinernema, biological control, persistence, survival Substratos para armazenar nematóides entomopatogênicos (Rhabditida: Steinernematidae, Heterorhabditidae) RESUMO: Os nematóides entomopatogênicos apresentam baixa viabilidade em condições de laboratório. Com o objetivo de avaliar substratos para prolongar a sobrevivência dos nematóides entomopatogênicos, suspensões de Heterorhabditis sp. JPM4 e Steinernema carpocapsae All (3.000 JI mL–1) foram adicionadas aos substratos solo, areia fina, areia grossa, espuma, argila expandida, esponja fenólica, ágar, amido de milho, Plantmax® e água. Estes foram colocados em placas de Petri (5 cm) e mantidos a 16 ± 1°C. As avaliações foram feitas após 30, 60, 90, 120, 150 e 180 dias, com três repetições para cada dia. Após 180 dias, para S. carpocapsae All o substrato espuma (57,5%) manteve maior porcentagem de juvenis infectantes (JI) vivos; argila expandida (28,4%), Plantmax® (9,3%) e esponja fenólica (11%) não foram eficientes para manutenção da sobrevivência. Para Heterorhabditis sp. JPM4, espuma (55,6%), areia grossa (53,1%) e areia fina (50,6%) proporcionaram maior sobrevivência dos JI ao final de 180 dias. Ágar (19,3%), esponja fenólica (11,6%) e Plantmax® (10,7%) tiveram índices de sobrevivência inferiores ao da testemunha (29,7%). O uso de substrato adequado pode propiciar maior sobrevivência de JI. Palavras-chave: Heterorhabditis, Steinernema, controle biológico, persistência, sobrevivência Introduction in water (Grewal, 2000). In addition to aqueous suspen- sion, EPNs can be stored in several substrates such as Entomopathogenic nematodes (EPNs) have been activated charcoal, alginate, vermiculite, clay, and po- more and more studied for pest control purposes. They rous foam, which provide a high surface area to volume are compatible in relation to many phytosanitary prod- ratio, as well as adequate interstitial space (Kaya and ucts, allowing their use in integrated management pro- Stock, 1997); however, survival in these substrates varies grams, and present synergism with other according to species. entomopathogenic agents, increasing the cost-effective- Many factors can influence the survival of these or- ness and efficiency of the adopted method (Ferraz, 1998). ganisms. The use of clay and silt results in decreased Storage difficulties constitute one of the major ob- nematode movement when compared with sand stacles to expand the use of EPNs as bioinsecticides. (Woodring and Kaya, 1988). Thus, EPN survive better High demand for oxygen, sensitivity of some species to in sandy soils, which allow better movement and oxy- temperature variations, susceptibility to microbial con- genation than clayey soils (Burman and Pye, 1980; Kung taminants, and toxicity from antimicrobial agents are et al., 1990). Another factor that influences nematode ac- factors that influence the quality of nematode storage tivity is represented by changes in the chemical or mi- Sci. Agric. (Piracicaba, Braz.), v.67, n.3, p.342-347, May/June 2010 Substrates for storing entomopathogenic nematodes 343 crobial composition of the storage medium (Dempsey clay is submitted to high temperatures, generating an and Griffin, 2002; Fitters and Griffin, 2004). Conse- internal space containing micropores and a hard and quently, investigating the parameters that influence EPN resistant shell); phenolic foam (substrate used in floral survival under storage is an important aspect to be con- centerpieces in order to retain water); agar (28°C); corn sidered for their release in the field in biological con- starch (10 g); and the substrate Plantmax® (9 g) (con- trol programs (Brown and Gaugler, 1997). Therefore, the sisting of Vermiculite® and Pinus sp. bark chips). The objective of this study was to evaluate the use of sub- treatments were placed in Petri dishes (5 cm diameter), strates for two species of entomopathogenic nematodes with three replicates for each evaluation day; each dish during the storage period. represented one replicate. Sterilized distilled water was added to maintain the substrates at a saturation value Material and Methods of 11% (weight volume–1). For treatments involving foam and phenolic foam, Multiplication and maintenance of entomopathogenic the substrates were cut to a 4.5 cm diameter and 1.0 cm nematodes height. The agar was prepared at a concentration of 2% The nematodes Steinernema carpocapsae All (Weiser, and, before becoming completely solidified, the nema- 1955) Wouts, Mracek, Gerdin and Bedding 1982 and tode suspension was added to the Petri dishes, totaling Heterorhabditis sp. JPM4 were grown in Lavras, state 12 mL. The fine sand treatment was mounted by sifting of Minas Gerais, Brazil. The nematodes were main- the sand through a 24 mesh sieve (0.71 mm). For the tained in Erlenmeyer flasks in a BOD incubator ad- coarse sand treatment, a 16 mesh sieve (1 mm) was used. justed to 16 ± 1°C, in an aqueous suspension contain- –1 For the dirt treatment, a 5 mesh (4 mm) sieve was used ing 500 to 1,000 IJ mL . Nematodes were multiplied to remove undesired material, such as branch parts, on Galleria mellonella (L.) (Lepidoptera: Pyralidae) lar- leaves, or stones. The control consisted of the nematode vae reared according to the methodology described by suspensions alone in the Petri dish (12 mL), without the Dutky et al. (1964), using the artificial diet modified addition of any substrate. Evaluations were made at 30, by Parra (1998). 60, 90, 120, 150, and 180 d, counting the numbers of live Ten last-instar G. mellonella larvae were transferred infective juveniles. The nematode suspensions recovered to Petri dishes (9 cm diameter) with the interior lined from the substrates were obtained using different meth- with filter paper to multiply the two EPN species used ods, according to each treatment. in this study. Simultaneously, 1mL of nematode suspen- A 150 mesh sieve was used over a 500 mesh sieve to sion at the concentration of 20 infective juveniles (IJ)/ obtain the IJ suspensions maintained in fine sand and larvae was added. The dishes were maintained in a BOD coarse sand; the nematodes were retained on the bottom incubator for 72h at 24°C ± 1°C and a 24h scotophase. sieve and then collected to calculate survival. The pro- Upon confirmation of the characteristic symptomatol- cedure used in the treatments involving dirt, Plantmax®, ogy of larva death, the larvae were transferred to a dry and corn starch was identical to the procedure de- chamber (Molina and López, 2002) for four days. scribed above, but a 325 mesh sieve was used on top, After that period, the larvae were transferred to modi- instead of the 150 mesh sieve. The IJs obtained from the fied White traps (White, 1927) to collect IJs. The traps storage in corn starch produced a cloudy suspension due were maintained in a BOD incubator for a period of to the passage of starch particles; nevertheless, this did three to seven days. The nematode suspension collected not prevent nematode viability evaluation. In the case daily was transferred to 1,000 mL capacity graduated cyl- of expanded clay, the substrate was washed with water inders containing 800 mL distilled water, allowing the through a 16 mesh sieve and the nematodes were col- IJs to settle within 24 h. The settling process was con- lected in the resulting aqueous suspension. A 325 mesh ducted to separate the fat insect bodies from the IJs. sieve was used with both the phenolic foam and the foam Upon completion of the nematode purification process, treatments, for which samples were placed into the sieve the nematode suspensions were diluted and quantified and squeezed through manually, adding water as an ex- in plastic dishes of the type used for serological tests (“Elisa”) under the stereoscopic microscope. traction aid. Therefore, pieces of the substrate were re- tained on the sieve while the nematodes passed through Evaluation of different substrates for the storage
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