Virulence of nematodes against larvae of the south-American fruit fly in laboratory using soil from Porto Amazonas, Paraná, Brazil, as substrate

Virulência de nematoides contra larvas de mosca-das-frutas-sulamericana em laboratório, utilizando solo de Porto Amazonas, Paraná, Brasil, como substrato

ABSTRACT:

Anastrepha fraterculus is an important orchard pest. Its management has been based in chemical sprays, but biological control is a growing demand. The objective of this work was to evaluate, under laboratory conditions, the virulence of eight nematode isolates to A. fraterculus in a soil collected in Porto Amazonas, Paraná, Brazil, and to estimate lethal doses (LD₅₀ and LD₉₀) for the more virulent isolate. Steinernema carpocapsae CB 02, three Heterorhabditis sp., two H. amazonensis and two Oscheius sp. isolates were tested in laboratory against A. fraterculus third-instar larvae using as substrate a loam Cambisol collected in an apple orchard. S. carpocapsae CB 02 isolate caused the higher percent mortality of A. fraterculus. Heterorhabditis sp. isolates and LAMIP 9 (Oscheius sp.) isolates were intermediate, while LAMIP 92 (Oscheius sp.) didn't differ from the control. S. carpocapsae CB 02 is able to kill 50% and 90% of A. fraterculus population with 96.3 and 314.7 infective juveniles per larva, respectively, in that soil. As a conclusion, CB 02 is the most virulent to A. fraterculus when the substrate is Porto Amazonas' apple orchard soil and it is able to kill 50 and 90% larval population with 96.3 and 314.7 infective juveniles per larva, respectively.

Key words:
Steinernema carpocapsae; Heterorhabditis; Oscheius; Anastrepha fraterculus

RESUMO:

Anastrepha fraterculus é uma importante praga em pomares. Seu manejo tem se baseado no uso de inseticidas químicos, porém, o controle biológico é uma demanda crescente. O objetivo do trabalho foi avaliar, em condições laboratoriais, a virulência de oito isolados de nematoides contra A. fraterculus em um solo coletado em Porto Amazonas, Paraná, Brasil, e estimar a dose letal do nematoide mais virulento. Steinernema carpocapsae CB 02, três isolados de Heterorhabditis sp., dois de H. amazonensis e dois de Oscheius sp. foram testados em laboratório contra larvas de terceiro instar de A. fraterculus, usando como substrato um Cambissolo franco coletado em um pomar de macieira. S. carpocapsae CB 02 provocou maior percentual de mortalidade de A. fraterculus. Isolados do gênero Heterorhabditis sp. e o LAMIP 9 (Oscheius sp.) foram intermediários, enquanto o LAMIP 92 (Oscheius sp.) não diferiu da testemunha. S. carpocapsae CB 02 é capaz de matar 50% e 90 % da população de larvas A. fraterculus com 96,3 e 314,7 juvenis infectivos por larva, respectivamente, naquele solo. Como conclusão, CB 02 é o mais virulento à A. fraterculus quando o substrato é solo de pomar de macieira de Porto Amazonas. Ele é capaz de controlar 50% e 90% da população de larvas com 96,3 e 314,7 juvenis infectivos por larva, respectivamente.

Palavras-chave:
Steinernema carpocapsae; Heterorhabditis; Oscheius; Anastrepha fraterculus

INTRODUCTION:

Apple [Malus domestica (Borkhausen)] is one of the most important fruit crop in Southern Brazil. In Paraná State, Porto Amazonas and Lapa counties are responsible for about 30% of apple production (IBGE, 2015). The South-American fruit fly Anastrepha fraterculus (Wied.) (Diptera: Tephritidae), is an apple and peach key pest in Southern Brazil and harms fruits by feeding and ovipositing on fruit (MONTEIRO & HICKEL, 2004). The extensive use of chemicals to control the pest has brought many environmental threats and human contamination. It has given rise to new researches on alternative control methods. Chemical-free or low-residue fruit are nowadays a demand. Biological control is an alternative measure, which includes the use of nematodes, since Anastrepha spp. have stages of larva and pupa in the soil (MONTEIRO & HICKEL, 2004).

There are already some studies on the main entomopathogenic nematode (EPN) genera Steinernema and Heterorhabditis as fruit flies biocontrolers. LEZAMA-GUTIÉRREZ et al. (2006) and TOLEDO et al. (2014) studied EPNs against A. ludens Loew; TOLEDO et al. (2005), in A. obliqua (Macquart); and TOLEDO et al. (2006) in A. serpentina (Wied). BARBOSA-NEGRISOLI et al. (2009) and RODRIGUES-TRENTINI (1996) tested the virulence of some EPNs against A. fraterculus, using sterile substrates. Oscheius genus contain species recently found to be facultative entomopathogenic nematodes (FEPNs): O. carolinensis (TORRES-BARRAGAN et al., 2011), O. chongmingensis (LIU et al., 2012) and O. gingeri (PERVEZ et al., 2014), but they were not reported infecting A. fraterculus yet.

Few studies about EPNs and FEPNs against A. fraterculus were made under soil influence. Soil characteristics as organic matter content, moisture, texture and biotic antagonists can affect nematode action (KAYA, 1990; LEZAMA-GUTIÉRREZ et al., 2006). Therefore, trials in sterile substrate like sand or Petri dishes with paper can estimate the potential efficiency of EPNs but trials in not sterilized soil reflects more closely the answer at field conditions for that specific soil (VOSS et al., 2009).

Thus, the objective of this work was to evaluate the virulence of nematode isolates of the genera Steinernema, Heterorhabditis and Oscheius to A. fraterculus larvae and estimate lethal doses for the more efficient nematode, having as substrate a loam Haplic Cambisol from an apple orchard of Porto Amazonas (Paraná State, Brazil), in laboratory condition.

MATERIAL AND METHODS:

A. fraterculus utilized in the trial were obtained from laboratory rearing from the CENA (Centro de Energia Nuclear na Agricultura), São Paulo - Brazil, started eight years before in Piracicaba, SP. The insects were reared according to SALLES (1992).

Five Heterorhabditis sp. isolates were obtained from UENP (Universidade Estadual do Norte do Paraná) and from IB (Instituto Biológico), and also one Steinernema carpocapsae from IB (Table 1). Two Oscheius sp. strains were isolated in Porto Amazonas (-25º32'07'', -49º54'41''), Paraná State, BR, by the authors, from a loam soil of an apple orchard, and identified by genetic markers at Florida University (unpublished results). After the initial purification, all the nematodes were inoculated on late-instar Galleria mellonella L. (Lepidoptera: Pyralidae) larvae and maintained in culture tissue bottles, with sponge, at 15°C (VOSS et al., 2009).

Table 1
Specie, source and origin of the entomopathogenic nematode isolates used in the experiment against A. fraterculus larvae.

• ^¹
  Universidade Estadual do Norte do Paraná, Cornélio Procópio, PR, Brazil, kindly provided by Professor Viviane Sandra Alves.
• ^²
  Instituto Biológico (Campinas, SP, Brazil), kindly provided by Dr. Luis Garrigós Leite.
• ^³
  Universidade Federal do Paraná - Laboratório de Manejo Integrado de Pragas (LAMIP).

Eight nematode isolates (Table 1) were experimented against A. fraterculus late third-instar larvae in a completely random design with ten repetitions (two sets of five repetitions at different times). Each plot consisted of 12 A. fraterculus larvae disposed in a 50-mL plastic container with 14.4g sieved (2mm) loam Haplic Cambisol from a Porto Amazonas orchard (22% clay, 41% silt; 37% sand, pH 5.9, 255mg dm^-3 P, 462mg dm^-3 K, 3.8% organic matter, 22% gravimetric moisture), equivalent to 13,04cm² of soil surface. The plastic cup was covered with parafilm to avoid loss of soil moisture. The soil was collected as VOSS et al. (2009) and the samples were tested for EPNs and FEPNs absence by exposing Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae during 15 days (VOSS et al., 2009). After A. fraterculus larvae had reached late third instar, they were removed from the semiliquid diet and mixed to an EPN-free soil for at least 30 minutes before use them in the bioassay, to avoid carrying excess of moisture and diet residues to the experimental plots. Then, 12 larvae were placed on the plots to penetrate the soil by themselves. Those that didn't penetrate the soil after 30 minutes were replaced (VOSS et al., 2009). EPN infective juveniles (IJ) were obtained from inoculated G. mellonella larvae and had less than 20 days since its production. The IJ concentration was determined in 20-microliter EPNs suspension drop with 5 replicates, with micropipette, using a stereomicroscope, as described by VOSS et al. (2009). After soil larvae penetration, 1,200 IJ (100IJ larva^-¹) were distributed on the soil surface suspended in 1.2mL distillated water, after homogenized, using a micropipette. The control plots received only distilled water in same volume. Plots were incubated in a controlled chamber at 25±2°C, RH 70±10 % (BARBOSA-NEGRISOLI, 2009).

Seven days later, the soil was sieved and each larva or pupa (since some larvae turned to pupae) was placed in a 1.1-cm² well of a tissue culture plate (Kasvi^(r), model K12-024), with two layers of filter paper. They were observed every two days to recover moisture of the filter paper. Twenty days after inoculation individuals not giving rise to an adult were dissected and observed under stereomicroscope, to verify the presence/absence of EPNs. To access virulence, only insect individuals infected with nematode were counted for the mortality data (LEZAMA-GUTIÉRREZ et al., 2006). The virulence was calculated by the equation: V = (Y - X)/(1-X)*100, where V is the virulence, X is the mean mortality in the control and Y is the mortality observed in each plot (ABBOTT, 1925). Data on mortality of A. fraterculus were transformed [(y+1)^0.5] to normalize and submitted to analysis of variance and a Tukey tests using R 3.0.2 (R DEVELOPMENT CORE TEAM, 2008).

Lethal doses (LD₅₀ and LD₉₀) were determined for the more virulent isolate, using the previous methodology for nematode inoculum, insect larvae preparation, EPN-free soil, arena dimensions, incubation time, mortality determination and experimental design. The treatments were 0 (control), 75, 150, 225, 300, 375IJ larva^-1. Lethal concentrations were estimated submitting the mean corrected mortality for each dose to a Probit regression using PoloPlus 1.0. (ROBERTSON et al., 1980).

RESULTS AND DISCUSSION:

Nematodes virulence against A. fraterculus

All isolates of nematodes tested were able to infect A. fraterculus. Infection rates ranged from 28 to 84.2% among the treated larvae, significantly higher than the control (F=19.26; P<0.05). Irrespective to the treatment, most of the infected insects (at least 79%) reached pupal phase during the first week after inoculation (Figure 1A). The methodology couldn't define the time of infection: pupal or larval stage. The higher amount of pupae indicates infected insects died slowly and could reach pupal phase before, probably because A. fraterculus larva had active defenses against nematode by retarding its development on hemolymph (RODRIGUES-TRENTINI, 1996). However, the insect died anyways.

Figure 1
(A) Percentage of A. fraterculus deaths occurred at larval or pupal phase following infection by entomopathogenic nematode isolates. (B) Mortality caused by entomopathogenic nematodes isolates (100IJ.larva^-1) on A. fraterculus. In B, statistical analysis was performed in transformed data [y'=(y+1)^0.5], and columns with the same letter do not differ (Tukey test, α=0.05). All data were obtained in a loam soil from an apple orchard of Porto Amazonas, PR, under laboratory condition (25±2ºC, RH 70±10%).

The virulence of the treatments against A. fraterculus in the loam soil is showed in figure 1B. CB 02 had the highest virulence (90.5%). CB 24 and JPM4 reached 47.4 and 45.3% mortality of A. fraterculus, respectively. Moreover, Alho, LAMIP 9, RSC 05 and NEPET 11 formed another group with lower virulence, but still different from the control (Figure 1B). However, LAMIP 92 not surpassed the control.

S. carpocapsae CB 02 was highly virulent, killing 90.5% of the larvae treated. As a comparison, using an unspecified isolate of S. carpocapsae, RODRIGUES-TRENTINI (1996) observed insect mortality lower than 31% using clay loam soil (100IJ larva^-1). Five times more IJ larva^-1 were necessary to cause 91.7% mortality. Besides the possible inherent difference in virulence among the two isolates, the higher clay content of the soil can have influenced negatively the infection by the nematodes, especially because clay reduces soil pore size and aeration which hampers host finding by the EPN (KAYA, 1990). The present experiment was performed in a loam soil, with less clay, and probably enhanced CB 02 action. A similar but sandier soil (sandy loam) was found to be more appropriated than sand, clay loam, and clay soils to S. carpocapsae survival and pathogenicity (KUNG et al., 1990). Beyond the differences in soil texture, RODRIGUES-TRENTINI (1996) used a bigger soil volume per larva, compared to this work, what caused dilution of the pathogen in the soil. In Petri dishes (best substrate for EPNs), 316IJ larva^-1 were needed to kill 90% of A. fraterculus larvae, what suggests CB 02 is more virulent.

BARBOSA-NEGRISOLI et al. (2009) tested other EPN species, H. bacteriophora and S. riobrave (100IJ larva^-1), against A. fraterculus and found 55 and 58% of larvae mortality, respectively, using sterile sand as substrate. These results are better than the virulence of the most part of the EPNs tested in this work. Sand is better than fine-textured soils to general EPNs action (KAYA, 2009), what helps explain the difference. However, the S. carpocapsae CB 02 was still more virulent (Figure 1B) even in loam soil.

Heterorhabditis sp. isolates performed worse than S. carpocapsae in the trials now presented. Heterorhabditis differs from Steinernema by its searching behavior that helps in the pray finding (KAYA, 1990). S. carpocapsae is ambusher, whilst most of the Heterorhabditis species are cruisers. However, in the present work, this characteristic could have been not important. The passive dispersal of the nematodes by the water through the soil pore space and the small soil depth could have helped S. carpocapsae to spread in the soil. The temperature is another possible explanation, since Steinernema genus was observed to be more efficient at 25°C (temperature of this experiment) while Heterorhabditis showed higher efficiency at 30°C (ROHDE et al., 2010). Furthermore, Steinernematids seem to be more adaptable to different textures of soil than Heterorhabditis sp. (KAYA, 1990).

The Oscheius sp. isolate LAMIP 9 was able to infect and kill A. fraterculus larvae (Figure 1). This pathogenic relationship has not been previously reported. Both Oscheius sp. were able to develop and kill G. mellonella. Some Oscheius sp. are known to be facultative parasites like: O. carolinensis (TORRES-BARRAGAN et al., 2011); O. chongmingensis (LIU et al., 2012); and O. gingeri (PERVEZ et al., 2013). Difference between LAMIP 9 and LAMIP 92 virulence, considering they are the same species, can be explained by the amount and kind of bacteria they are associated. An unique Oscheius species can carry more than one bacteria outside its body (TORRES-BARRAGAN et al., 2011), with different virulence.

LAMIP isolates performed as well or worse than other-origin ones. In agreement with this result, GREWAL et al. (2002) found that it was common the EPNs from an insect species to be less virulent than isolates from other insect and/or geographical origin. Specificity seems to be a more important factor in EPN virulence than adaptation to the environment. It's possible that LAMIP isolates facultative parasitic behavior allows them to survive on other suitable food sources than A. fraterculus, since this insect is not present in the orchards during the entire year.

Lethal doses of CB 02

S. carpocapsae CB 02 lethal doses are presented in figure 2. Its virulence rose as the dose of IJ increased until 300IJ larva^-1, and reached 92.2%. The lethal doses, LD₅₀ and LD₉₀, of S. carpocapsae CB 02 on A. fraterculus larvae were estimated to be 96.3IJ larva^-1 (confidence interval: 80.6 to 110.2IJ larva^-1) and 314.7IJ larva^-1 (confidence interval: 272.7 to 380.9IJ larva^-1), respectively (χ²=2.47, heterogeneity=0.82).

Figure 2
Probit regression (dark line) for observed mortality [corrected according ABBOTT (1925)] of the fruit fly A. fraterculus following increasing doses of S. carpocapsae CB 02, in a loam soil of an apple orchard of Porto Amazonas (PR, Brazil) as substrate, at laboratory condition. LD50 and LD90 are indicated by dash lines.

RODRIGUES-TRENTINI (1996) estimated the confidence interval of LD₅₀ for S. carpocapsae against A. fraterculus, in a loam clay soil, to be 261.5 to 429.0IJ cm^-², similar to LD₉₀ of the S. carpocapsae CB 02. Besides differences of inherent virulence among the isolates tested, the lower LD₅₀ estimated in this work for S. carpocapsae CB 02 can be due to the substrate used. Comparing to other studies (RODRIGUES-TRENTINI, 1996; BARBOSA-NEGRISOLLI et al., 2009), the relative low LD of S. carpocapsae CB 02 in the loam soil is a suitable characteristic for its possible use as a biocontrolers in the orchard.

Insect mortality usually falls down after EPN concentration over an optimal point because of IJ competition (ROHDE et al., 2012). However, this work did not report decrease in control efficiency of S. carpocapsae CB 02 against A. fraterculus larvae, corroborating the observation by RODRIGUES-TRENTINI (1996).

CONCLUSION:

All Heterorhabditis sp. isolates, S. carpocapsae CB 02 and Oscheius sp. LAMIP 9 are virulent to A. fraterculus larvae when the substrate is Porto Amazonas' apple orchard loam soil. Oscheius sp. LAMIP 9 and LAMIP 92 are less effective than EPNs from other origins tested in this trial. CB 02 is the most efficient on A. fraterculus control, and is able to kill 50% and 90% of A. fraterculus population with 96.3 and 314.7 infective juveniles per larva, respectively, in that soil.

ACKNOWLEDGEMENTS

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

REFERENCES:

• ABBOTT, W.S. A method of computing effectiveness of an insecticide. Journal of Economic Entomology, v.18, p.265-267, 1925.
• BARBOSA-NEGRISOLI, C.R.C. et al. Efficacy of indigenous entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae), from Rio Grande do Sul Brazil, against Anastrepha fraterculus (Wied.) (Diptera: Tephritidae) in peach orchards. Journal of Invertebrate Pathology, v.102, p.6-13, 2009. Available from: <Available from: http://dx.doi.org/10.1016/j.jip.2009.05.005 >. Accessed: Jan. 21, 2015. doi: 10.1016/j.jip.2009.05.005.
  » https://doi.org/10.1016/j.jip.2009.05.005.» http://dx.doi.org/10.1016/j.jip.2009.05.005
• IBGE (2015). Produção Agrícola Municipal. Available from: <Available from: http://www.ibge.gov.br/home/estatistica/economia/pam/2013/default_perm_xls.shtm >. Online. Accessed: Feb. 02, 2015.
  » http://www.ibge.gov.br/home/estatistica/economia/pam/2013/default_perm_xls.shtm
• GREWAL, P.S. et al. Differences in susceptibility of introduced and native white grub species to entomopathogenic nematodes from various geographic localities. Biological Control, v.24, p.230-237, 2002. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1049964402000257 >. Accessed: Feb. 22, 2015. doi: 10.1016/S1049-9644(02)00025-7.
  » https://doi.org/10.1016/S1049-9644(02)00025-7.» http://www.sciencedirect.com/science/article/pii/S1049964402000257
• KAYA, H.K. Soil ecology. In: GAUGLER, R.; KAYA, H.K. (Eds.). Entomopathogenic nematodes in biological control. Boca Raton, FL: CRC, 1990. Cap.5, p.93-116.
• KUNG, S.P. et al. Soil type and entomopathogenic nematode persistence. Journal of Invertebrate Pathology, v.55, p.401-406, 1990. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/002220119090084J# >. Accessed: Jan. 19, 2015. doi: 10.1016/0022-2011(90)90084-J.
  » https://doi.org/10.1016/0022-2011(90)90084-J.» http://www.sciencedirect.com/science/article/pii/002220119090084J#
• LEZAMA-GUTIÉRREZ, R. et al. Efficacy of Steinernematid nematodes (Rhabditida: Steinernematidae) on the suppression of Anastrepha ludens (Diptera: Tephritidae) larvae in soil of differing textures: laboratory and field trials. Journal of Agricultural and Urban Entomology, v.23, p.41-49, 2006.
• LIU, Q. et al. Re-description of Oscheius chongmingensis (Nematoda: Rhabditidae) and its entomopathogenicity. Nematology, v.14, p.139-149, 2012. Available from: <Available from: http://dx.doi.org/10.1163/138855411X580777 >. Accessed: Jan. 12, 2015. doi: 10.1163/138855411X580777.
  » https://doi.org/10.1163/138855411X580777.» http://dx.doi.org/10.1163/138855411X580777
• MONTEIRO, L.B.; HICKEL, E. Pragas de importância econômica em fruteiras de caroço. In.: MONTEIRO, L.B. et al. (Ed.). Fruteiras de caroço: uma visão ecológica. Curitiba: Reproset I, 2004. Cap.11, p.223-261.
• PERVEZ, R. et al. Penetration and infectivity of entomopathogenic nematodes against Lema sp. (Chrysomelidae: Coleoptera) infesting turmeric (Curcuma longa L.) and their multiplication. Journal of Spices and Aromatic Crops, v.23, p.71-75, 2014. Available from: <Available from: http://www.indianspicesociety.in/josac/index.php/josac/article/view/143/139 >. Accessed: Feb. 02, 2015.
  » http://www.indianspicesociety.in/josac/index.php/josac/article/view/143/139
• RODRIGUES-TRENTINI, R.F. Mecanismos de defesa e controle de Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae) expostas a nematoides entomopatogênicos. 1996. 73f. Tese (Mestrado em Ciências Biológicas) - Curso de Pós-graduação em Ciências Biológicas, Universidade Federal do Paraná, PR.
• ROHDE, C.R. et al. Influence of soil temperature and moisture on the infectivity of entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae) against larvae of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Neotropical Entomology, v.39, p.608-611, 2010. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S1519-566X2010000400022&script=sci_arttext >. Accessed: Jan. 19, 2014. doi: 10.1590/S1519-566X2010000400022.
  » https://doi.org/10.1590/S1519-566X2010000400022.» http://www.scielo.br/scielo.php?pid=S1519-566X2010000400022&script=sci_arttext
• ROHDE, C.R. et al. Selection of entomopathogenic nematodes for the control of the fruit fly Ceratitis capitata (Diptera: Tephritidae). Revista Brasileira de Ciências Agrárias, v.7, p.797-802. 2012. Available from: <Available from: http://www.redalyc.org/pdf/1190/119025455014.pdf >. Accessed: Jan. 19, 2014. doi: 10.5039/agraria.v7isa2217.
  » https://doi.org/10.5039/agraria.v7isa2217.» http://www.redalyc.org/pdf/1190/119025455014.pdf
• R DEVELOPMENT CORE TEAM. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2008. Available from: <Available from: http://www.R-project.org >. Online. Accessed: Nov. 20, 2013.
  » http://www.R-project.org
• ROBERTSON, J. et al. Polo Plus: a user's guide to probit or logit analysis. Berkeley: Pacific Southwest Forest and Range Experiment Station, 1980. Available from: <Available from: http://www.fs.fed.us/psw/publications/documents/psw_gtr038/psw_gtr038.pdf >. Online. Accessed: May 15, 2014.
  » http://www.fs.fed.us/psw/publications/documents/psw_gtr038/psw_gtr038.pdf
• SALLES, L.A.B. Metodologia de criação de Anastrepha fraterculus (Wied., 1830) (Diptera: Tephritidae) em dieta artificial em laboratório. Anais da Sociedade Entomológica do Brasil, v.21, p.479-486, 1992.
• TOLEDO, J. et al. Susceptibilidad de larvas de Anastrepha obliqua Macquart (Diptera: Tephritidae) a Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) en condiciones de laboratório. Vedalia, v.12, p.11-21, 2005.
• TOLEDO, J. et al. Efficiency of Heterorharbditis bacteriophora (Nematoda: Heterorhabditidae) on Anastrepha serpentina (Diptera: Tephritidae) larvae under laboratory conditions. Florida Entomologist, v.89, p.524-526, 2006. Available from: <Available from: http://dx.doi.org/10.1653/0015-4040(2006)89[524:EOHBNH]2.0.CO;2 >. Accessed: Jan. 19, 2015.
  » http://dx.doi.org/10.1653/0015-4040(2006)89[524:EOHBNH]2.0.CO;2
• TOLEDO, J. et al. Effect of soil moisture on the persistence and efficacy of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) against Anastrepha ludens (Diptera: Tephritidae) Larvae. Florida Entomologist, v.97, p.528-533, 2014. Available from: <Available from: http://www.bioone.org/doi/full/10.1653/024.097.0225 >. Accessed: Jan. 19, 2015. doi: 10.1653/024.097.0225.
  » https://doi.org/10.1653/024.097.0225.» http://www.bioone.org/doi/full/10.1653/024.097.0225
• TORRES-BARRAGAN, A. et al. Studies on the entomophathogenicity and bacterial associates of the nematode Oscheius carolinensis. Biological Control, v.59, p.123-129, 2011. Available from: <Available from: http://dx.doi.org/10.1016/j.biocontrol.2011.05.020 >. Accessed: Feb. 02, 2015. doi: 10.1016/j.biocontrol.2011.05.020.
  » https://doi.org/10.1016/j.biocontrol.2011.05.020.» http://dx.doi.org/10.1016/j.biocontrol.2011.05.020
• VOSS, M. et al. Manual de técnicas laboratoriais para obtenção, manutenção e caracterização de nematoides entomopatogênicos. Passo Fundo: Embrapa Trigo, 2009. 44p. Available from: <Available from: http://www.infoteca.cnptia.embrapa.br/handle/doc/853176 >. Online. Accessed: May 21, 2013.
  » http://www.infoteca.cnptia.embrapa.br/handle/doc/853176

• 1
  CR-2015-0370.R1

Publication Dates

• Publication in this collection
  27 Nov 2015
• Date of issue
  Mar 2016

History

• Received
  12 Mar 2015
• Accepted
  03 Aug 2015
• Reviewed
  29 Oct 2015