Steinernema diaprepesi Nguyen & Duncan (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Brazil

Abstract

Entomopathogenic nematodes (EPNs) can control pests due to their mutual association with bacteria. The use of these biological control agents is increasing worldwide due to advances in research about its control efficiency, range of action and mass production. The identification of EPNs adapted to specific environmental and climatic conditions is important for sustainable pest suppression in integrated management (IPM) programs. The objective is to report, for the first time, the occurrence of the Steinernema diaprepesi in Brazil. Steel mesh traps with Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae were buried in red latosol cultivated with Eucalyptus. Infective juveniles (IJs) were isolated from dead larvae and multiplied in healthy ones of this host to confirm its pathogenicity and to start a laboratory population from the strain found in the field. The DNA of the IJs was extracted and amplified using PCR technique with the universal primers D2A and D3B. The detection of S. diaprepesi is the first report of this nematode in Brazil, increasing the knowledge about its distribution in the world and the diversity of EPNs that must be considered as agents of biological pest control in the country.

Key words
Biological control; crop protection; Galleria mellonella; infective juveniles; PCR

INTRODUCTION

Steinernematidae nematodes can control important pests in the world (Javal et al. 2019, Salgado-Morales et al. 2019, Mbata et al. 2018). Entomopathogenic nematodes (EPNs) live in symbiotic association with bacteria of the genus Xenorhabdus (Poinar & Grewal 2012), responsible for host death and decomposition of their tissues which are used as food for the development of EPNs (Nermut et al. 2019). The use of these biological agents is increasing due to advances in the mass production, application technology and their range of action (Dolinski et al. 2012, Lacey & Georgis 2012, Gumus et al. 2015).

Research on EPNs in most South American countries is scarce (Nguyen et al. 2010, De Brida et al. 2017). Furthermore, many studies are published in non-English journals as thesis dissertations, conference proceedings and other non-readily available sources (San-Blas et al. 2019), limiting the access and comparison of data.

Steinernema glaseri (Steiner 1929), a species originally reported in North America, was the first Steinernematidae reported in Brazil, isolated from the eggs of Migdolus fryanus Westwood (Coleoptera: Cerambycidae) in the São Paulo state (Pizano et al. 1985). Steinernema brazilense Nguyen, a species native to Brazil, was identified in 2010 (Nguyen et al. 2010). Steinernema carpocapsae (Hominick 2002) and Steinernema rarum (De Brida et al. 2017) were isolated from collections in agricultural areas in the country.

The identification of EPNs adapted to local environmental and climatic conditions is important because some factors may interfere on their survival, behavior and development, such as predators, ultraviolet radiation, temperatures, soil moisture and texture, osmotic stress and pesticides (Andaló et al. 2018, Glazer 2002, Brown & Gaugler 1997) that can affect sustainable pest suppression in integrated pest management programs (De Brida et al. 2017).

The objective of this study is to report, for the first time, the occurrence of the entomopathogenic nematode, Steinernema diaprepesi Nguyen & Duncan (2002) in Brazil.

MATERIALS AND METHODS

Nematode survey at field

Twenty steel mesh traps, with two seventh instar Galleria mellonella L. (Lepidoptera: Pyralidae) larvae each, were buried in Red Latosol at 5 cm depth in a commercial plantation (22º 59’ 49’’ S, 48º 29’ 57’’ W, 870 m) of Eucalyptus grandis x Eucalyptus urophylla clones. The relative humidity, atmospheric and soil temperature were measured with thermo hygrometer and soil thermometer during the period on what the traps were installed in the field and these factors ranged from 50 to 98%, 15 to 36°C and 20 to 25°C, respectively. After seven days, the traps were removed from the soil, packed in plastic bags and transported to the FCA/UNESP Nematology Laboratory for isolation and identification of entomopathogenic nematodes from the field. Dead G. mellonella larvae were removed from the traps, washed in sodium hypochlorite solution (1%) and transferred to White traps (White 1927) and maintained in an incubator chamber (B.O.D.) at 25 °C (mean soil temperature at which the nematodes were collected). The IJs emergence from the White traps, multiplied from the primary infection by EPNs in the field, was observed daily during 21 days in laboratory. The live larvae were observed on until adult emergence to confirm non-infection by EPNs.

Multiplication of the isolates

Infective juveniles (IJs) obtained from dead G. mellonella larvae of field traps were multiplied in healthy larvae of this host in Petri dishes (9 cm in diameter x 1.5 cm in height) coated with filter paper moistened with a suspension of 500 IJs/cm². Galleria mellonella larvae dead inoculated with IJs were transferred to White traps (White 1927) and maintained in an incubator chamber (B.O.D.) at 25 °C for three days to establish the first population of these nematodes in the laboratory (LP1).

Molecular identification

The genomic DNA of 50 IJs from LP1 was extracted in 50 μl of 0.85% NaCl solution by Worm Lysis Buffer (WLB) extraction method (Carvalho et al. 2018, Williams et al. 1992). These samples were left at -70 °C for 15 minutes, incubated for one hour at 60 °C, another 15 minutes at 95 °C and stored at -20°C.

The DNA extracted was amplified using the polymerase chain reaction (PCR) technique with the universal primers D2A and D3B for the expansion of the 28S rDNA sequence (Al-Banna et al. 2004). The reactions were performed in an INFINIGEN thermal cycler (model TC-96CG) with 12.5 μl of Polymerase Mix Master Red (Neobio), 7.5 μl of NucleaseFreeWater (Promega), 1 μl of each primer (10 mM) and 3 μl of genomic DNA per sample, totaling 25 μl of solution. The cycles for the universal primers D2A and D3B were done (Mracek et al. 2006). Negative controls with 3 μl of water were added in the assays to check for possible PCR reagent contaminations. PCR amplification products were visualized by 1% agarose gel electrophoresis with marker (Norgen) and UV light transilluminator (Major Science).

The PCR product was purified according to the recommendations of the Celcco PCR Purification Kit (Qiagen, Cat#14400) and sequenced in a DNA Sanger automated sequencer (Model: ABI 3500, Applied Biosystems) at the Institute of Biotechnology (IBTEC-UNESP). Sequences were compared on BLAST, with data deposited at GenBank (http://www.ncbi.nlm.nih.gov).

RESULTS AND DISCUSSION

Galleria mellonella dead larvae were found in four traps in the field with EPNs infection symptoms (Figure 1). The mortality of this host at the concentration 1.000 of JIs was 100% after three days of inoculation. The IJs emergence from larvae of this host was observed confirming the virulence of this nematode.

Figure 1
Dead Galleria mellonella larvae collected with steel mesh trap showing symptoms of entomopathogenic nematode infection.

The nucleotide sequences obtained were 100% similar to those of Steinernema diaprepesi (Rhabditida: Steinernematidae) (accession number GU173994.1) (Figure 2).

Figure 2
1% agarose gel. MM= 100 bp molecular weight, 1: amplification of the 28S rDNA region (800 bp) using primers D2A and D3B, NC: Negative control.

The pathogenicity of S. diaprepesi was previously reported important pests as Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) (Geisert et al. 2018), Diaprepes abbreviates Linnaeus (Coleoptera: Curculionidae) (Nguyen & Duncan 2002, El-Borai et al. 2007, Ali et al. 2010) and Helicoverpa gelotopoeon Dyar (Lepidoptera: Noctuidae) (Caccia et al. 2017). High virulence of this nematode was confirmed for Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) with mortality greater than 93% when exposed to doses of at least 50 IJs/insect (Caccia et al. 2017). This insect is one of the most important pest in Brazilian maize fields (Garcia et al. 2019) causing significant economic losses. The virulence of the nematode S. diaprepesi is higher for lepidopteran larvae than for some species of the orders Coleoptera and Diptera, and null for Orthoptera (Del Valle et al. 2014). The S. diaprepesi efficacy against pests is attributed to its symbiotic bacterium, Xenorhabdus doucetiae Tailliez et al. (2006) (Caccia et al. 2017), with capacity to develop and reproduce at temperatures above 35 ºC (Tailliez et al. 2006).

The detection of S. diaprepesi in Eucalyptus plantations is the first report of this nematode species in Brazil. This species was first described in Florida, USA, isolated from Diaprepes abbreviatus Linnaeus (Coleoptera: Curculionidae) (Nguyen & Duncan 2002) and later in Venezuela (Spiridonov et al. 2004) in the Caribbean islands Martinique and Guadeloupe (Tailliez et al. 2006), in Mexico (Molina-Ochoa et al. 2009) and in Argentina (Caccia et al. 2017). The diversity of hosts and predators, the incidence of ultraviolet radiation on the soil surface, temperature, soil moisture and texture, osmotic stress and insecticides may interfere with the survival, behavior and development of nematodes (Brown & Gaugler 1997, Glazer 2002, Andaló et al. 2018). Therefore, detection of new EPNs species, which can be used as biological control agents, adapted to the different environments and edaphoclimatic conditions are fundamental for the IPM success programs (Andaló et al. 2014, Brown & Martin 2014).

The S. diaprepesi detection in Brazil increases the knowledge about the distribution of this nematode in the world and the diversity of EPNs that should be considered as agents of biological control of pests. Future studies will show the potential of S. diaprepesi as new alternative biological agent into the IPM programs in Brazil.

ACKNOWLEDGMENTS

We thank to the Brazilian institutions Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES)- Finance Code 001, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Programa Cooperativo sobre Proteção Florestal (PROTEF) do Instituto de Pesquisas e Estudos Florestais (IPEF) for financial support. David Michael Miller, a professional editor and proofreader and native English speaking, has reviewed and edited this article for structure, grammar, punctuation, spelling, word choice, and readability.

REFERENCES

• AL-BANNA L, PLOEG AT, WILLIAMSON VM & KALOSHIAN I. 2004. Discrimination of six Pratylenchus species using PCR and species-specific primers. J Nematol 36: 142-146.
• ALI JG, ALBORN HT & STELINSKI LL. 2010. Subterranean herbivore induced volatiles released by citrus roots upon feeding by Diaprepes abbreviatus recruit entomopathogenic nematodes. J Chem Ecol 36: 361-368.
• ANDALÓ V, MIEKO J, CARVALHO FJ, ASSIS GA, FARIA LS, ASSIS FA, SANTOS RA & ROSA F. 2018. Entomopathogenic nematode distribution and edaphoclimatic conditions in the Cerrado of Minas Gerais, Brazil. Appl Entomol Zool 53: 129-136.
• ANDALÓ V, MOREIRA GF & MOINO JR A. 2014. Heterorhabditis amazonensis RSC5 (Rhabditida: Heterorhabditidae) movement and host recognition. Rev Colomb Entomol 40: 91-97.
• BROWN AP & MARTIN G. 2014. Commercial examples of the use of entomopathogenic nematodes in integrated pest management (IPM) programs. J Nematol 46: 141-142.
• BROWN IM & GAUGLER R. 1997. Temperature and humidity influence emergence and survival of entomopathogenic nematodes. Nematologica 43: 363-375.
• CACCIA M, DUEÑAS JR, DEL VALLE E, DOUCET ME & LAX P. 2017. Morphological and molecular characterization of an isolate of Steinernema diaprepesi Nguyen & Duncan 2002 (Rhabditida: Steinernematidae) from Argentina and identification of its bacterial symbiont. Syst Parasitol 94: 111-122.
• CARVALHO VR, WILCKEN SRS, WILCKEN CF, CASTRO BMC, SOARES MA & ZANUNCIO JC. 2018. Technical and economic efficiency of methods for extracting genomic DNA from Meloidogyne javanica. J Microbiol Meth 157: 108-112.
• DE BRIDA AL, ROSA JMO, DE OLIVEIRA CMG, CASTRO BMC, SERRÃO JE, ZANUNCIO JC & WILCKEN SRS. 2017. Entomopathogenic nematodes in agricultural areas in Brazil. Sci Rep-Uk 7: 45254.
• DEL VALLE EE, BALBI EI, LAX P, DUEÑAS JR & DOUCET ME. 2014. Ecological aspects of an isolate of Steinernema diaprepesi (Rhabditida: Steinernematidae) from Argentina. Biocontrol Sci Techn 24: 690-704.
• DOLINSKI C, CHOO HY & DUNCAN LW. 2012. Grower acceptance of entomopathogenic nematodes: case studies on three continents. J Nematol 44: 226-235.
• EL-BORAI FE, ZELLERS JD & DUNCAN LW. 2007. Suppression of Diabrepes abbreviatus in potted citrus by combinations of entomopathogenic nematodes with different lifespans. Nematropica 37: 33-41.
• GARCIA AG, FERREIRA CP, GODOY WAC & MEAGHER RL. 2019. A computational model to predict the population dynamics of Spodoptera frugiperda. J Pest Sci 92: 429-441.
• GEISERT RW, CHERUIYOT DJ, HIBBARD BE, SHAPIRO-ILAN DI, SHELBY KS & COUDRON TA. 2018. Comparative assessment of four Steinernematidae and three Heterorhabditidae species for infectivity of larval Diabrotica virgifera virgifera. J Econ Entomol 111: 542-548.
• GLAZER I. 2002. Survival biology. In: Gaugler R (Ed). Entomopathogenic nematology. CABI Publishing: New York, p. 169-187.
• GUMUS A, KARAGOZ M, SHAPIRO-ILAN D & HAZIR S. 2015. A novel approach to biocontrol: release of live insect hosts pre-infected with entomopathogenic nematodes. J Invertebr Pathol 130: 56-60.
• HOMINICK WM. 2002. Biogeography. In: Gaugler, R. (Ed). Entomopathogenic Nematology. CABI Publishing, Wallingford: UK, p. 115-143.
• JAVAL M, TERBLANCHE JS, CONLONG DE & MALAN AP. 2019. First screening of entomopathogenic nematodes and fungus as biocontrol agents against an emerging pest of sugarcane, Cacosceles newmannii (Coleoptera: Cerambycidae). Insects 10: 117.
• LACEY LA & GEORGIS R. 2012. Entomopathogenic nematodes for control of insect pests above and below ground with comments on commercial production. J Nematol 44: 218-225.
• MBATA GN, IVEY C & SHAPIRO-ILAN D. 2018. The potential for using entomopathogenic nematodes and fungi in the management of the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae). Biol Control 125: 39-43.
• MOLINA-OCHOA J, NGUYEN KB, GONZÁLEZ-RAMÍREZ M, QUINTANA-MORENO MG, LEZAMA-GUTIÉRREZ R & FOSTER JE. 2009. Steinernema diaprepesi (Nematoda: Steinernematidae): Its occurrence in western Mexico and susceptibility of engorded cattle ticks Boophilus microplus (Acari: Ixodidae). Fla Entomol 92: 661-663.
• MRACEK Z, NGUYEN KB, TAILLER P, BOAMARE N & CHEN S. 2006. Steinernema sichuanense n. sp. (Rhabditida, Steinernematidae) a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China. J Invertebr Pathol 93: 157-169.
• NERMUT J, ZEMEK R, MRÁCEK Z, PALEVSKY E & PUZA V. 2019. Entomopathogenic nematodes as natural enemies for control of Rhizoglyphus robini (Acari: Acaridae)? Biol Control 128: 102-110.
• NGUYEN KB & DUNCAN LW. 2002. Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a parasite of the citrus root weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae). J Nematol 34: 159-170.
• NGUYEN KB, GINARTE CMG, LEITE LG, DOS SANTOS JM & HARAKAVA R. 2010. Steinernema brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil. J Invertebr Pathol 103: 8-20.
• PIZANO MA, AGUILLERA MM, MONTEIRO AR & FERRAZ LCB. 1985. Incidence of Neoplectana glaseri Steiner 1929 (Nematoda: Steinernematidae) parasitizing Migdolus fryanus (Westwood, 1863) (Coleoptera: Cerambycidae). Entomol News 17: 9-10.
• POINAR GO & GREWAL PS. 2012. History of entomopathogenic nematology. J Nematol 44: 153-161.
• SALGADO-MORALES R, MARTÍNEZ-OCAMPO F, OBREGÓN-BARBOZA V, VILCHIS-MARTÍNEZ K, JIMÉNEZ-PÉREZ A & DANTÁN-GONZÁLEZ E. 2019. Assessing the pathogenicity of two bacteria isolated from the entomopathogenic nematode Heterorhabditis indica against Galleria mellonella and some pest insects. Insects 10: 83.
• SAN-BLAS E ET AL. 2019. Entomopathogenic nematology in Latin America: A brief history, current research and future prospects. J Invertebr Pathol 165: 22-45. https://doi.org/10.1016/j.jip.2019.03.010.
• SPIRIDONOV SE, REID AP, PODRUCKA K, SUBBOTIN SA & MOENS M. 2004. Phylogenetic relationships within the genus Steinernema (Nematoda: Rhabditida) as inferred from analyses of sequences of the ITS1-5.8S-ITS2 region of rDNA and morphological features. Nematology 6: 547-566.
• TAILLIEZ P, PAGES S, GINIBRE N & BOEMARE N. 2006. New insight into diversity in the genus Xenorhabdus, including the description of ten novel species. Int J Syst Evol Micr 56: 2805-2818.
• WHITE GF. 1927. A method for obtaining infective nematode larvae from cultures. Science 66: 302-303.
• WILLIAMS BD, SCHRANK B, HUYNH C, SHOWNKEEN R & WATERSTON RH. 1992. A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics 131: 609-624.

Publication Dates

• Publication in this collection
  27 Sept 2021
• Date of issue
  2021

History

• Received
  15 Aug 2019
• Accepted
  25 Oct 2019