The influence of mulch on the persistence of Steinernema brazilense(Nematoda: Steinernematidae) in sugarcane fields

Influencia del mantillo sobre la persistencia de Steinernema brazilense(Nematoda: Steinernematidae) en cultivo de caña de azúcar

LUIS GARRIGÓS LEITE^1,2, FABIO SILBER SCHMIDT^1,3, RICARDO HARAKAVA^1,4, ANTONIO BATISTA FILHO^1,5, FERNANDO HENRIQUE CARVALHO GIOMETTI^1,6, TATIANE DE CASTRO PIETROBON^1,7and JULIE CHACON-OROZCO<sup>8

1</sup> Instituto Biológico, CEIB. CP 70, 13001-970, Campinas, SP, Brazil.
² Ph. D. lgleite@biologico.sp.gov.br. Corresponding autor.
³ M. Sc. fabio@biocontrole. com.br.
⁴ Ph. D. harakava@biologico.sp.gov.br.
⁵ Ph. D. batistaf@biologico.sp.gov.br.
⁶ M. Sc. fernandohenrique@fermentec.com.br.
⁷ M. Sc. tatipietro@ hotmail.com.
⁸ M. Sc. Universidade Federal Rural do Rio de Janeiro, Programa de pós-gradua 9 ao em Fitossanidade e Biotecnologia Aplicada, Rodovia BR 465, Km 7, 23851-970, Seropédica, RJ, Brazil. jchaconorozco@gmail.com.

---

Abstract: Entomopathogenic nematodes (EPN's) were tested for sugarcane soil dwelling pests, including the billbug, Sphenophorus levis. The successful application of EPN's to protect sugarcane crops in Brazil requires the consideration of several aspects, including the widespread use of mechanical harvesting, which discards cane leaves in fields, resulting in a thick layer of mulch covering the ground. In this study, the influence of mulch on the persistence of Steinernema brazilense(strain IBCB n6) in sugarcane fields was examined. Nematode persistence in the soil was evaluated using Galleria mellonella larvae as an attractant. Results showed that S. brazilense can persists in sugarcane fields for at least 278 days after its application, and mulch does not affect its persistence, thus favoring its spread as compared to bare soil.

Key words: Entomopathogenic nematode. Biological control. Coleoptera.

---

Resumen: Los nematodos entomopatógenos (NEPs) son parásitos obligados de insectos, incluyendo al picudo de la caña Sphenophorus levis. Sin embargo, para la aplicación con éxito de NEPs en cultivos de caña en Brasil se debe tener en cuenta que la cosecha mecánica genera una capa de residuos de hojas (mantillo) en el suelo. En este estudio se evaluaron la influencia de la cobertura vegetal sobre la persistencia del nematodo Steinernema brazilense(cepa IBCB n6) en cultivo de caña, usando larvas de Galleria mellonella. S. brazilense puede persistir en el suelo de caña al menos durante 278 días después de su aplicación y el mantillo no afecta su persistencia, pero sí favorece su diseminación en comparación con el suelo desnudo.

Palabras clave Nematodos entomopatógenos. Control biológico. Coleoptera.

---

Introduction

Sugarcane crop in Brazil occupies > 9 million hectares of land, just Sâo Paulo state accounting for > 5 million hectares. Farmers expect that planted areas provide at least 5-6 ratton crops that mean the number of regrowth following a harvest. Meanwhile, several factors may affect the cane production in a way that the number of profiting ratton crops become much lower. Among these aspects, is notorious the importance of insect pests with emphasis on soil dwelling species that are the most diverse group, many of which are difficult to manage with chemical insecticides (Pinto et al. 2009). One promising alternative to this problem is to use entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis (Nematoda: Steinernematidae, Heterorhabditidae) which are soil dwelling organisms that kill these pests and recycle on the cadavers (Leite et al. 2012).

EPNs are mutualistically associated with bacteria of the generaXenorhabdus and Photorhabdus, which are voided from the nematode intestine into the hemolymph, propagate and kill the host insect by septicemia, usually within 48 h when infect susceptible hosts such as Galleria mellonella (Griffin et al. 2005). These nematodes possess a number of attractive qualities as biocontrol agents including a relative durable infective juvenile (IJ) stage, a broad host range, high virulence, great ability for host seeking, suitability to mass production and safety (Shapiro-Ilan and Gaugler 2002).

Several studies have tested EPNs against sugarcane pests, especially against the sugarcane billbug, Sphenophorus levis (Vaurie, 1978) (Coleoptera: Dryophthoridae), the larvae of which feed on sugarcane underground stems + rhizomes, damaging up to 80% of the stems and reducing cane productivity by 30%. Adults usually remain underground becoming also potential target for EPNs. Previous field trials applying Steinernema brazilense(strain IBCB n6) in the sugarcane rows (1 x 10⁸ infective juveniles ha^-1), demonstrated that this nematode controls the billbug and enhance cane productivity (Leite et al. 2012).

The successful application of EPNs to protect sugarcane crops in Brazil requires consideration of several aspects, including the widespread use of mechanical harvesting, which discards the cane leaves in the field, resulting in a thick lay of mulch covering the ground. In this study, the influence of mulch on the persistence of S. brazilense (strain IBCB n6) in a sugarcane field was examined.

Methods

The nematodes were obtained by in vitro production using the sponge method (Bedding 1984) and were used soon after being collected, with the infective juveniles (IJs) showing more than 90% viability.

The study was done in a second ratton of a mechanically harvested sugarcane crop (variety SP 803280, planted in rows 1.5 m apart) located in the municipality of Santo Antonio de Posse in Sao Paulo state. Two identical experiments located 400 m apart were carried out, both starting on 10 December 2007, 91 days after the first harvest on 10 September 2007, when the plants were on average 40 cm height. For each experiment, four treatments were considered: (1) soil ground treated with nematodes and covered with mulch, (2) soil ground not treated with nematodes, but covered with mulch (control), (3) bare soil treated with nematodes, and (4) bare soil not treated with nematodes (control). Each treatment was replicated five times and each replicate consisted of a 10 x 7.5 m plot containing five rows 10 m long, randomly distributed in blocks. The plots were 5 m apart within blocks, and 4.5 m apart (three rows) between blocks. Both experiments were set up in an area covered with mulch, and plots without mulch (bare soil groups) were raked to remove any covering. The nematodes were applied just once, at a dose of 1 x 10⁸ IJs ha^-1, on 24 December 2007, using a manually operated backpack sprayer fitted with a jet tip that delivered 3 L plot^-1 (400 L ha') in a 15 cm wide strip along both sides of each row. For the mulch group, a 15 cm wide strip along both sides of each row was uncovered to allow the nematodes to be applied directly to the soil. After application, the sprayed area was covered with mulch again.

Nematode persistence in the soil was evaluated using Galleria mellonella (great waxmoth) larvae to bait the nematode. Evaluations were done 14 (10 December 2007) and 7 (17 December 2007) days before nematode application, and 3 (27 December 2007), 15 (8^th January 2008), 32 (25 January 2008), 76 (9^th March 2008), 105 (7^th April 2008), 160 (1^st June 2008), 225 (5^th August 2008) and 278 (27 September 2008) days after nematode application. The last evaluation was done immediately after sugarcane harvesting. For each evaluation, three soil samples were randomly collected from each plot, from the 20 cm wide strip along each side of the three central rows. From each spot surveyed, 1 kg of soil was collected and placed in a plastic chamber (15 cm diameter x 10 cm high) and transported to the laboratory, were five artificially reared larvae of G. mellonella were buried in each chamber. Before burying, the larvae were held inside a 10 cm x 6 cm metal-screen cage (aperture: 1 mm), together with 35 g of soil, and then buried in the chambers at a depth of 5 cm. To improve conditions for the EPNs, ~100 mL of water was added to each chamber. The chambers were incubated for five days at 25 °C on the dark. At the end of this period, the metal-screen cages were removed, opened and the insect mortality due to Steinernema brazilensewas assessed. Larvae killed by ENPs had a flaccid body, with worms leaving the cadaver after being placed in a white trap. If the ENPs isolated belonged to the genus Steinernema they were cross-bred (Kaya and Stock 1997) to confirm whether they belonged to the same species as that used in the experiments (S. brazilense). However, if the EPNs belonged to the genus Heterorhabditis, they were promptly identified by the red color of the cadavers. Molecular analysis through sequencing of 16S ribosomal RNA gene was done to confirm species identity. These identifications were necessary since the experiments were conducted at field conditions, where some other EPN species may be inhabiting the soil and infect the G. mellonella larvae.

Both experiments were combined as single one to result 10 replications. The mortality of G. mellonella was analyzed by one-way analysis of variance (ANOVA) followed by the Tukey studentized range tes t for multiple comparisons. Mortality rates were arcsine VxJ100 transformed prior to analysis and all means were transformed back to the original units for presentation. All statistical comparisons were done using SPSS version 10.0 software, with a p value < 0.05 indicating significance.

Results and discussion

No Steinernema were found in the two surveys done before application, indicating that this genus did not occurred naturally in both experimental areas (Table 1). In contrast, indigenous Heterorhabditis sp. was detected at a low density and accounted for <16% of the mortality of G. mellonella larvae. Three days post application (PA), S. brazilense accounted for 49.3% of the mortality in G. mellonella larvae in both nematode treated groups (with and without mulch), indicating that S. brazilense IBCB n6 was introduced successfully in the sugarcane field. Several factors may have contributed to this introduction, including the high rainfall that helped to maintain soil moisture and the soil type, which was slightly sandy (clay = 23.8%, silt = 22.8%, total sand = 53.4%) favoring the action of the nematodes. In agreement, Susurluk (2009) reported that colonization and persistence of the nematodes S. feltiae and H. bacteriophora in different field crops and rotation regimes were correlated with the weekly precipitation following nematode release.

After introduction, there was a decreasing in G. mellonella infection rates that was not significantly affected by the presence or absence of mulch. In our study, the soil samples were rehydrated for baiting with G. mellonella, implying that the decrease in the mortality of the greater waxmoth was directly related to the decrease in the nematode population. Bednarek and Gaugler (1997) found that increased organic matter (organic manure) appeared to stimulate nematode establishment and recycling. The progressive decrease in the percentages of G. mellonella infected by S. brazilense, with no significant difference (F_{39 360} = 29.145; P = 0.124) up to the 32^th day PA in the nematode-mulch group, and up to 76^th day in the nematode-soil, suggests that this nematode persisted for about one to two months before losing its effectiveness as an agent for controlling sugarcane pests. However, despite this decrease, S. brazilense remained the main nematode responsible for G. mellonella mortality, except prior to application, when the indigenous Heterorhabditis sp. was the only nematode present. According to Kung et al. (1991), S. carpocapsae and S. glaseri survived for 32 days at low soil moistures of 2 and 4%, when soil samples were kept at RH of 100%.

By 76 days PA, the nematode S. brazilense was also found in the control plots (confirmed by cross-breeding identification), which were located 4.5 - 5 m from the neighboring plots where the nematodes were sprayed. This finding indicates that the nematode spread throughout the experimental area, migrating at least 5 m in 76 days, i.e., at least 1 m every 15 days. According to Weischer and Brown (2001), some species can move 3-6 m in approximately two months, or 1.5-2.0 m per month, which is similar to the rate observed in the current study. Passive dispersion by rain, wind, soil, humans or insects may occur over kilometers whereas active dispersal generally occurs over only a few centimeters (Smart and Nguyen 1994).

The high percentage of G. mellonella infected by S. brazilense in control-mulch group, not significantly different (F_{39 360} = 29.145; P = 0.124) from the nematode-treated groups at 3^th day PA (49.3%), but significantly greater (F_{39 360} = 29.149; P < 0.001) compared to the control-soil group at 76^th day PA (4,7%), probably reflected the greater suitability of mulch plots to enhance the nematode movement as well as to retain rainwater containing the nematodes from the treated plots, in addition to keeping the soil moist for a longer time. This result is in agreement to the study by Hsiao and All (1998), who showed that mulch can enhance the movement of some entomopathogenic nematodes in agricultural systems, with S. carpocapsae moving 3.5 cm/day on bare soil and 7.5 cm in rye mulch-covered soil.

Beyond 76 days PA, the percentages of G. mellonella infected by S. brazilense decreased in almost all treatments and remained low from 160 to 225 days PA, when the larva mortality was < 3.0%. From June to August (160-225 days PA), rainfall was low and may have accelerate a decreasing in the nematode population by drying the soil. This time has been a drought season for this region in Brazil. The level of moisture is one of the most important factors in the soil environment to influence the survival, virulence and persistence of nematodes (Klein 1990).

Beyond 225 days PA, the nematode population started to increase again, resulting in 9.3% mortality of G. mellonella larvae in both control-mulch and nematode-mulch groups by 278 days PA (last evaluation). The increase in the nematode population coincided with the beginning of the rainy season. Our results show that S. brazilense survived the unfavorable conditions associated with winter dry season, persisting in the soil by surviving as IJs or by recycling in the insect cadaver, for at least 278 days since its application.

S. brazilense can persist in the sugarcane field for at least 278 days after its application on the soil, and the mulch does not affect its persistence, favoring its spread compared to bare soil. The percentage of G. mellonella infected by the nematode decreased as the time approached to the draught season, dropping significantly after one - two month from the nematode application.

Acknowledgements

To Fundagao de Amparo a Pesquisa do Estado de Sao Paulo-FAPESP for financial support, and to Abengoa Bioenergia Corporation for the experimental support.

Literature cited

BEDDING, R. A. 1984. Large scale production, storage and transport of the insect nematodes Neoplactana spp. and Heterorhabditis spp. Annual Applied Biology 104: 117-120.         [ Links ]

BEDNAREK, A.; GAUGLER, R. 1997. Compatibility of soil amendments with entomopathogenic nematodes. Journal ofNematology 29: 220-227.         [ Links ]

GRIFFIN, C. T.; BOEMARE, N. E.; LEWIS, E. E. 2005. Biology and behavior. pp. 47-64. In: Grewal, P. S.; Ehlers, R. U.;Shapiro-Ilan, D. I. (Eds.). Nematodes as biocontrol agents. CABI Publishing, Cambridge.         [ Links ]

HSIAO, W. F.; ALL, J. N. 1998. Survey of the entomopathogenic nematode, Steinernema carpocapsae: (Rhabditida: Steinerne-matidae) natural populations and its dispersal in the field. Chinese Journal of Entomology 18: 39-49.         [ Links ]

KAYA, H. K.; STOCK, S. P. 1997. Techniques in insect nematology. pp. 281-324. In: Lacey, L. (Ed.). Manual of techniques in insect pathology. San Diego: Academic Press.         [ Links ]

KLEIN, M. G. 1990. Efficacy against soil-inhabiting insect pests. pp. 195-214. In: Gaugler, R.; Kaya, H. (Eds.). Entomopathogenic nematodes in biological control. Boca Raton, CRC Press.         [ Links ]

KUNG, S. P.; GAUGLER, R.; KAYA, H. K. 1991. Effects of soil temperature, moisture, and relative humidity on entomopathogenic nematode persistence. Journal of Invertebrate Pathology 57 (2): 242-249.         [ Links ]

LEITE, L. G.; TAVARES, F. M.; BOTELHO, P. S. M.; BATISTA FILHO, A.; POLANCZYK, R. A.; SCHMIDT, F. S. 2012. Eficiencia de nematoides entomopatogenicos e inseticidas químicos contra Sphenophorus levis e Leucothyreus sp. em cana-de-ajúcar. Pesquisa Agropecuária Tropical 42 (1): 40-48.         [ Links ]

PINTO, A. S.; BOTELHO, P. S. M.; OLIVEIRA, H. N. 2009. Guia ilustrado de pragas e insetos benéficos da cana-de-afúcar. CP 2, Piracicaba, 160 p.         [ Links ]

SHAPIRO-ILAN, D. I.; GAUGLER, R. 2002. Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology and Biotechnology 28: 137146.         [ Links ]

SMART JR., G. C.; NGUYEN, K. B. 1994. Role of entomopathogenic nematodes in biological control. pp. 231-252. In: Rosen, D.; Bennett, F. D.; Capinera, J. L. (Eds.). Pest management in the subtropics: Biological control - a Florida perspective. Andover, UK: Intercept.         [ Links ]

SUSURLUK, I. A. 2009. Seasonal and vertical distribution of the entomopathogenic nematodes Heterorhabditis bacteriophora (TUR-H2) and Steinernema feltiae (TUR-S3) in turf and fallow areas. Nematology 11 (3): 321-327.         [ Links ]

WEISCHER, B.; BROWN, D.J.F. 2001. Conhecendo os nematoides. Pensoft, Sofia, 209 p.         [ Links ]

Received: 26-Jun-2014
Accepted: 25-Nov-2015

Suggested citation:

LEITE, L. G.; SCHMIDT, F. S.; HARAKAVA, R.; BATISTA FILHO, A.; GIOMETTI, F. H. C.; PIETROBON, T. de C.; CHACON-OROZCO, J. 2015. The influence of mulch on the persistence of Steinernema brazilense(Nematoda: Steinemematidae) in sugarcane fields. Revista Colombiana de Entomologia 41 (2): 176-179. Julio - Diciembre 2015. ISSN 0120-0488.