Virulence and Selection of Beauveria Bassiana for the Control of Thaumastocoris Peregrinus

Kamilla Emmanuelle Carvalho de Almeida Junia Grazielle Soares da Silva Janaína Fernandes Gonçalves Leonardo Rodrigues Barbosa Marcelo Luiz de Laia About the authors

Abstract

This work aimed to assess the virulence of Beauveria bassiana isolates in different concentrations on Thaumastocoris peregrinus (Hemiptera, Thaumastocoridae) and select them to control this insect pest. Suspensions of IBCB35, IBCB66, IBCB18, IBCB01 and JAB06 isolates were tested in the concentrations of zero, 1 × 104, 1 × 106, 1 × 108 conidia/ml, applied on the eucalyptus leaves used to feed the insects. Dead insects were disinfested daily and placed in a humid chamber to confirm the death by fungus. Mortality at the end of the experiment ranged from 12% for IBCB66 to 48% for IBCB18 at a concentration of 1 × 104 conidia/ml, between 32% for IBCB66 and 68% for JAB06 at a concentration of 1 × 106 conidia/ml, and between 60% for IBCB01 and 84% for JAB06 at the concentration 1 × 108 conidia/ml. The assessed isolates were efficient in controlling the insect, especially JAB06.

Keywords:
silviculture; forest entomology; entomopathogenic fungi

1. INTRODUCTION AND OBJECTIVES

Thaumastocoris peregrinus Carpinteiro & Dellapé 2006 (Hemiptera, Thaumastocoridae) (common name bronze bug) is a leaf-sucking insect of thin leaves and eucalyptus branches, capable of causing significant damage and making it impossible to maintain and manage new plantations in countries where its occurrence is recorded (Barbosa et al., 2010Barbosa LR, Santos F, Wilcken CF, Soliman EP. Registro de Thaumastocoris peregrinus (Hemiptera, Thaumastocoridae) no estado do Paraná. Pesquisa Florestal Brasileira 2010; 30(61): 75-77. 10.4336/2010.pfb.30.61.75
https://doi.org/10.4336/2010.pfb.30.61.7...
; Wilcken et al., 2010Wilcken CF, Soliman EP, Nogueira de Sá LA, Barbosa LR, Dias TKR, Ferreira Filho PJ, Rodrigues Oliveira RJ. Bronze bug Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae) on Eucalyptus in Brazil and its distribution. Journal of Plant Protection Research 2010; 50(2): 201-205. 10.2478/v10045-010-0034-0
https://doi.org/10.2478/v10045-010-0034-...
). Its rapid dissemination capacity and the severity of the injuries caused by the bronze bug in eucalyptus plantations have drawn the forest sector’s attention, which aims to develop efficient strategies to control this insect pest.

Among the methods recommended by Integrated Pest Management, control by biological agents is an important tactic against the eucalyptus bronze bug (Barbosa et al., 2010Barbosa LR, Santos F, Wilcken CF, Soliman EP. Registro de Thaumastocoris peregrinus (Hemiptera, Thaumastocoridae) no estado do Paraná. Pesquisa Florestal Brasileira 2010; 30(61): 75-77. 10.4336/2010.pfb.30.61.75
https://doi.org/10.4336/2010.pfb.30.61.7...
), and the use of entomopathogenic fungi is one of the best strategies. Records show fungi occurrence of the Entomophthorales order, and the Beauveria bassiana (Balsamo-Crivelli 1835) Vuillemin 1912 species associated with the bronze bug (Mascarin et al., 2012Mascarin GM, Duarte VS, Brandão MM, Delalibera I Jr. Natural occurrence of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), an invasive pest recently found in Brazil. Journal of Invertebrate Pathology 2012; 110(3): 401-404. 10.1016/j.jip.2012.03.025
https://doi.org/10.1016/j.jip.2012.03.02...
; Wilcken et al., 2010Wilcken CF, Soliman EP, Nogueira de Sá LA, Barbosa LR, Dias TKR, Ferreira Filho PJ, Rodrigues Oliveira RJ. Bronze bug Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae) on Eucalyptus in Brazil and its distribution. Journal of Plant Protection Research 2010; 50(2): 201-205. 10.2478/v10045-010-0034-0
https://doi.org/10.2478/v10045-010-0034-...
).

The B. bassiana species has been widely studied due to its wide action spectrum and easy laboratory productivity. It is also used as a biological agent to control several insect pests (Alves et al., 1998Alves SB, Almeida JEM, Moino JRA, Alves LFA. Técnicas de laboratório. In: Alves SB, editor. Controle microbiano de insetos. Piracicaba: FEALQ; 1998. p. 637-711.). Once in contact with the insect, the spores or hyphae of the fungus germinate and secrete enzymes that degrade the host’s cuticle, penetrating inside. After penetration, the fungus produces a toxin called Beauvericin, which decreases the insect’s immune defenses, causing its death (Azevedo et al., 1999Azevedo JL, Araújo WL, Maccheroni W Jr. Importância dos microrganismos endofíticos no controle de insetos. In: Melo IS, Azevedo JL, editors. Controle biológico. Jaguariúna: Embrapa Meio Ambiente; 1999. p. 57-93.).

Considering the scarce literature involving the relationships between B. bassiana and T. peregrinus fungi, in addition to the challenge of establishing effective programs in the biological control of this insect, studies conducted investigating the potential use of these agents in controlling the eucalyptus bronze bug are required. This study proposed to investigate the virulence of the B. bassiana fungus isolates in different concentrations on T. peregrinus adult insects under laboratory conditions, and then to select the best strains to control the eucalyptus bronze bug.

2. MATERIALS AND METHODS

The bioassay selection was conducted at the Genetics and Biotechnology Laboratory facilities of the Forest Engineering Department at the Federal University of Jequitinhonha and Mucuri, JK Campus, in Diamantina, Minas Gerais, Brazil.

The Beauveria bassiana fungal isolates used were JAB06, IBCB35, IBCB01, IBCB18 and IBCB66, provided by the São Paulo State University, Jaboticabal Campus. For the bioassay, the isolates were put into Petri dishes containing pre-autoclaved BDA culture medium under aseptic conditions. The plates were kept in air-conditioned BOD chambers (26 °C ± 0.5 °C, 12 h photophase and 70% ± 10% relative humidity) for 14 days for the pathogen growth and sporulation.

After the incubation period, hyphae and conidia formed on the surface of the culture medium were collected with the aid of a scalpel and transferred to Erlenmeyer flasks containing 50 ml of potato-dextrose (BD) liquid culture medium in triplicate. After inoculation, the flasks were kept in a BOD type air-conditioned chamber (26 °C ± 0.5 °C, 12 h photophase and 70% ± 10% relative humidity) for 14 days for developing the fungus.

To prepare the inoculum suspensions, the culture medium containing the fungal colonies was filtered and the mycelial mass together with the conidia were suspended in a sterile solution composed of 100 ml of sterile distilled water plus the adhesive spreader (Tween® 80) at 0.05% v/v. After grinding in an electric mixer, the inoculum concentration present in the initial suspension (adjusted to the concentration 1 × 108 conidia/ml) was quantified with the aid of a Neubauer chamber under optical microscopy, according to the quantification method adopted by Alves & Moraes (1998Alves SB, Moraes AS. Quantificação de inóculo de patógenos de insetos. In: Alves SB, editor. Controle microbiano de insetos. Piracicaba: FEALQ ; 1998. p. 765-777.). The other concentrations were then adjusted from the initial solution: 1 × 104 and 1 × 106 conidia/ml, plus the control composed of sterilized distilled water and the adhesive spreader at 0.05% v/v.

Microculture technique and direct examination in microscopy slides determined the viability of the spores to guarantee an experimental standard before applying suspensions to the bugs. The germination percentage was measured 12 h after inoculation, counting 150 conidia per field between germinated and non-germinated.

For the bioassay, healthy leaves of similar coloring and standardized size were collected from an Eucalyptus hybrid, whose identification could not be performed, but which was proven to be susceptible to the bronze bug’s attack. The leaves were immersed in the conidial suspensions and held in laminar flow chamber for 30 minutes to eliminate excess moisture. Then, five T. peregrinus adults were deposited on the leaves surface, which were placed in Petri dishes previously autoclaved.

The petiole of each leaf was arranged in Eppendorf-type polypropylene tubes containing 1.5 ml of distilled water through a small hole in the tube cap. This procedure enabled maintenance of foliar turgidity and prevented the insects from having contact with water.

After inoculation, the plates were sealed with PVC film and kept in a BOD-type heated chamber at 26 °C ± 0.5 °C, 12 h photophase and 70% ± 10% relative humidity for 10 days. Daily evaluations were performed throughout the experimental period, observing the presence of dead individuals.

To confirm insect mortality by the pathogen, daily bronze bugs in each replicate were disinfested by immersion in 70% ethanol, 2% sodium hypochlorite and distilled water for 2 minutes each, and then transferred to chambers prepared in autoclaved Petri dishes kept in a BOD-type heated chamber at 25 °C ± 0.5 °C and relative humidity of 70% ± 10% in the dark in order to allow the pathogen’s sporulation on the insects. The entire procedure was performed aseptically in a laminar flow chamber to avoid contamination by microorganisms. The obtained spores were aseptically transferred to Petri dishes containing sterilized BDA culture medium and grown in an air-conditioned room under optimal conditions to develop the isolates for the fungus recovery and confirmation of its pathogenicity to the insect.

The selection of B. bassiana isolates was based on the T. peregrinus mortality percentage by the virulence assessment of each isolate at different concentrations.

The bioassay was organized in a completely randomized design in a factorial scheme with two factors + one control, with one factor being the isolate (IBCB35, IBCB66, IBCB18, IBCB01 and JAB06) and the other the concentration (control, 1 × 104, 1 × 106, 1 × 108 conidia/ml). The experimental unit consisted of a Petri dish containing a eucalyptus leaf with five adult T. peregrinus insects. Five replicates were established for each treatment.

Results were analyzed considering the different conidial concentrations of assessed isolates and the mortality rates of T. peregrinus adults submitted to the treatments. Data were previously normalized and then subjected to analysis of variance (Anova) and the means compared by the Tukey test at 5% significance. The analyses of the obtained data were carried out using the R statistical software program (R Core Team, 2014R Core Team. R: A language and environment for statistical computing. Vienna: The R Foundation; 2014.).

3. RESULTS

The symptoms caused by the bronze bug’s sucking habit were perceptible on the second day after transferring the insects to the eucalyptus leaves, observing the occurrence of chlorosis caused by the insect attack.

The longer assessment period enabled observing the action spectrum of the B. bassiana isolates, as well as the behavioral characteristics of the bronze bug, such as egg deposition, development of hatched nymphs and insect-plant relationships during the experimental period.

The low spore viability in the assessed suspensions reached 68.59% for IBCB35, 68.81% for IBCB01, 69.33% for IBCB66, 69.70% for IBCB18 and 69.92%% for JAB06, which ensured the ability of the suspensions to cause infection in the insects and standardization between them. Thus, it was possible to assure the same action conditions for the different isolates used in the tests. Mortality was confirmed by visualizing pronounced bowing of the bronze bugs’ legs, which were no longer fixed on the eucalyptus leaves, and also by total lack of mobility.

In relation to the mortality percentage of T. peregrinus over time, 100% of dead insects were recorded from the fifth day of assessment for IBCB66 and IBCB18 isolates, when 1 × 108 conidia/ml was considered. The IBCB66 isolate was tested at concentrations of 1 × 104 and 1 × 106 conidia/ml, and only reached this result on the ninth day, while total insect mortality was only observed on the tenth day for the IBCB18 isolate at a concentration of 1 × 106 conidia/ml, when 92% of dead insects were also observed for the concentration of 1 × 104 conidia/ml. The IBCB35 and JAB06 isolates were tested at the highest concentration and reached 100% mortality for T. peregrinus on the sixth day of assessment. However, both concentrations of 1 × 104 and 1 × 106 conidia/ml presented total insect mortality on the seventh day. The IBCB01 isolate also only promoted 100% accumulated mortality on the seventh day of assessment for the concentrations of 1 × 108 and 1 × 106 conidia/ml, and at the end of the experimental period, the concentration of 1 × 104 registered 88% of dead insects. Ten days after application of treatments, insects in the control group had accumulated mortality rates between 84% and 92% (Figure 1).

Figure 1
Daily mortality percentage observed in Thaumastocoris peregrinus for treatments with the IBCB66, IBCB35, JAB06, IBCB18, and IBCB01 Beauveria bassiana isolates at concentrations 1 × 104, 1 × 106, 1 × 108 conidia/ml and the control concentration.

The dead insects representative of the control group of each isolate did not demonstrate conidiogenesis or contamination by other fungi, thus indicating that the superficial disinfestation was in fact efficient. Conversely, five days after being subjected to the humid chamber, sporulation was visible in most of the dead bugs that were exposed to fungal spores. The fungi spores developed on the cadavers and transferred to Petri dishes containing BDA culture medium enabled by multiplying the isolates without having contaminants, confirming their virulence to the insect. The colonies obtained in BDA culture medium showed the mycelium with yellowish-white and cotton-like appearance, with aerial and regular growth. These morphological characteristics are identical to those found in the literature for other B. bassiana isolates (Fernandes et al., 2006Fernandes EKK, Costa GL, Morales AML, Zahner V, Bittencourt VREP. Study on morphology, pathogenicity, and genétic variability of Beauveria bassiana isolates obtained from Boophilus microplus tick. Parasitology Research 2006; 98: 324-332. 10.1007/s00436-005-0058-y
https://doi.org/10.1007/s00436-005-0058-...
; Varela & Morales, 1996Varela A, Morales E. Characterization of some Beauveria bassiana isolates and their virulence toward the coffee berry borer Hypothenemus hampei. Journal of Invertebrate Pathology 1996 67(2): 147-152.).

For confirmed mortality, even though the JAB06, IBCB18, IBCB35, IBCB66, IBCB01 isolates tested at the concentration of 1 × 108 conidia/ml, JAB06, IBCB18, IBCB01, and IBCB35 at 1 × 106 conidia/ml, and IBCB18 and JAB06 at 1 × 104 conidia/ml did not present significant differences, the JAB06 isolate (1 × 108 conidia/ml) presented the highest confirmed mortality rate in T. peregrinus. This isolate was significantly different from the IBCB01 and IBCB35 isolates tested at the concentration 1 × 104 conidia/ml, and IBCB66 tested at concentrations of 1 × 104 and 1 × 106 conidia/ml, which, in turn, were statistically the same as all isolates tested at the control concentration. The worst confirmed mortality response in T. peregrinus was observed for the IBCB66 isolate tested at the concentration 1 × 104 conidia/ml (Table 1).

Table 1
Mortality percentage of the Thaumastocoris peregrines adults by the Beauveria bassiana fungus isolates tested at different concentrations.

4. DISCUSSION

Fungus virulence may vary depending on several factors, including environmental conditions or the presence of elements in the insect cuticle, which may inhibit the action of the pathogen (Boucias & Pendland, 1998Boucias DG, Pendland JC. Principles of insect pathology. Massachusetts: Kluer Academic; 1998.). Generally, the rapidity of insect mortality is directly proportional to the conidia concentration (Fargues & Rodrigues-Rueda, 1980Fargues J, Rodrigues-Rueda D. Sensibilitè dês larves de Spodoptera litoralis (Lepidoptera: Noctuidae) aux hyphomycètes entomopathogènes Nomureae rileyi et Paecilomyces fumosoroseus. Entomophaga 1980; 25(1): 43-54.) and to the inoculation mode of the pathogen. The IBCB66, IBCB18, IBCB01, IBCB35 and JAB08 B. bassiana isolates were pathogenic at all concentrations and the mortality rate increased with increasing concentrations. In general, the JAB06 isolate had higher efficiency in T. peregrinus.

The susceptibility of the insect to entomopathogenic fungi may vary depending on their development stage (Alves et al., 1998Alves SB, Almeida JEM, Moino JRA, Alves LFA. Técnicas de laboratório. In: Alves SB, editor. Controle microbiano de insetos. Piracicaba: FEALQ; 1998. p. 637-711.). In tests with B. bassiana on the bronze bug predator Podisus nigrispinus Dallas 1851, França et al. (2006França IWB, Marques EJ, Torres JB, Oliveira JV. Efeitos de Metarhizium anisopliae (Metsch.) Sorok. e Beauveria bassiana (Bals.) Vuill. sobre o percevejo predador Podisus nigrispinus Dallas (Heteroptera: Pentatomidae). Neotropical Entomology 2006; 35(3): 349-356. 10.1590/S1519-566X2006000300009
https://doi.org/10.1590/S1519-566X200600...
) recorded a higher percentage of average mortality for nymphs than for adults.

França et al. (2006França IWB, Marques EJ, Torres JB, Oliveira JV. Efeitos de Metarhizium anisopliae (Metsch.) Sorok. e Beauveria bassiana (Bals.) Vuill. sobre o percevejo predador Podisus nigrispinus Dallas (Heteroptera: Pentatomidae). Neotropical Entomology 2006; 35(3): 349-356. 10.1590/S1519-566X2006000300009
https://doi.org/10.1590/S1519-566X200600...
) tested the methods of direct spraying of insects and surface treatment of the leaves used in the bioassay, obtaining 59% and 8% of confirmed mortality, respectively. In this work, suspensions of B. bassiana isolates were applied indirectly by treating the surface of eucalyptus leaves used as food substrate for the bronze bugs. In treating the leaf surface, the number of conidia that had contact with the insect tends to be less because they are restricted to some regions of the body (Santoro et al., 2007Santoro PH, Neves PMOJ, Alexandre TM, Alves LFA. Interferência da metodologia nos resultados de bioensaios de seleção de fungos entomopatogênicos para o controle de insetos. Pesquisa Agropecuária Brasileira 2007; 42(4): 483-489. 10.1590/S0100-204X2007000400005
https://doi.org/10.1590/S0100-204X200700...
). However, the bronze bug’s sucking and behavioral feeding habit on the eucalyptus leaves facilitated the insect infection by the fungus, even if it was only applied on the food substrate.

The application method and the fungal suspension concentrations used enabled virulence expression of the B. bassiana isolates on T. peregrinus, demonstrating that the application method of the isolates with the surface treatment of the leaves was efficient. In this sense, application by substrate treatment can effectively represent action potential of the fungus.

In field conditions, spraying mycoinsecticides on insect pests can be prevented by the difficulty of direct application to the host, especially in large areas such as those used to plant eucalyptus. In addition, infestation control occurring in the canopy can be difficult and expensive given the height and location of trees in the forest. When sprayed on the plantation, mycoinsecticides can reach a large part of the population of insect pests when they feed on the product deposited on the leaves in the tree canopy. According to Wright (1993Wright JE. Control of the boll weevil (Coleoptera: Curculionidae) with Naturalis-L: a mycoinsecticide. Journal of Economic Entomology 1993; 86(5): 1355-1358. 10.1093/jee/86.5.1355
https://doi.org/10.1093/jee/86.5.1355...
), spraying applications of the mycoinsecticides can present similar efficiency to chemical insecticides. Therefore, it is a potential technique to be considered in pest control programs in forest plantations.

Results found in this study corroborate those previously observed by Lorencetti et al. (2011Lorencetti GAT, Menezes MJS, Jung PH, Gonçalves TE, Barbosa LR, Potrich M et al. Análise do potencial de Beauveria bassiana vuill (Ascomycetes: Clavicipitaceae) para o controle de Thaumascocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae). In: I Congresso de Ciência e Tecnologia da UTFPR; 2011; Dois Vizinhos. [place unknown: publisher unknown]; 2011. p. 94-97.) in adopting a similar methodology when studying the potential of B. bassiana for T. peregrinus adults control. The obtained mortality percentage results ranged from 46.8% for the IBCB440 isolate to 78.6% for isolate 384, both in concentrations 1 × 108 conidia/ml.

Although they are pathogens of the same species, some B. bassiana isolates assessed in this bioassay presented different behavior in relation to the virulence of T. peregrinus adults. This frequently observed variation may be related to factors such as susceptibility and/or tolerance of the host insect, as well as characteristics of each isolate, such as virulence, specificity and the genetic variability of each of them (Alves et al., 1988Alves SB, Almeida JEM, Moino JRA, Alves LFA. Técnicas de laboratório. In: Alves SB, editor. Controle microbiano de insetos. Piracicaba: FEALQ; 1998. p. 637-711.; Vestergaard et al., 1995Vestergaard S, Gillespie AT, Butt TM, Schreiter G, Eilenberg J. Pathogenicity of the Hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to the western thirps, Frankliniella occidentalis. Biocontrol Science and Technology 1995; 5(2): 185-192. 10.1080/09583159550039909
https://doi.org/10.1080/0958315955003990...
).

In their studies, Gaitan et al. (2002Gaitan A, Valderrama AM, Saldarriaga G, Velez P, Bustillo A. Genetic variability of Beauveria bassiana associated with the Coffee Berry Borer Hypothenemus hampei and other insects. Mycological Research 2002; 106(11): 1307-1314. 10.1017/S0953756202006676
https://doi.org/10.1017/S095375620200667...
) observed the occurrence of genetic variation among isolates of native B. bassiana populations with the intraspecific distribution obtained from ITS (internal transcribed spacer) and β-tubulin sequence, and also AFLP markers (amplified fragment length polymorphism). Therefore, differences in confirmed mortality percentage of T. peregrinus adults found for different isolates can also be justified by the genetic variability among B. bassiana isolates.

The B. bassiana isolates assessed in this experiment are potentially efficient for T. peregrinus control, and the virulence of B. bassiana on T. peregrinus was generally evident. In addition to reaching 100% total mortality between the fifth and sixth day when tested in the highest concentration, some of the isolates had a high confirmed mortality rate, reaching values above 80%.

Choosing the correct strategy as well as adequate inoculum concentration is of great importance, being a fundamental factor in the successful development and use of entomopathogens as bioinsecticides (Alves et al., 1998Alves SB, Almeida JEM, Moino JRA, Alves LFA. Técnicas de laboratório. In: Alves SB, editor. Controle microbiano de insetos. Piracicaba: FEALQ; 1998. p. 637-711.). The search for new entomopathogenic fungi isolates is strategically important and may represent an alternative for the biocontrol of the eucalyptus bronze bug in confronting the need to develop efficient methods and low environmental impact for the management of this insect (Barbosa, Belinovski et al., 2012Barbosa LR, Santos F, Machado BO, Wilcken CF, Soliman EP, Zaché B. Percevejo bronzeado do eucalipto: reconhecimento, danos e direcionamentos para o controle [Internet]. 2012 [cited 2014 Jan. 24]. Available: Available: https://bit.ly/36Z5ZM6
https://bit.ly/36Z5ZM6...
; Barbosa, Santos et al., 2012Barbosa LR, Belinovski C, Machado BO, Franciscon L, Soliman EP, Wilcken CF. Efeito de Eucalyptus spp. e da superfície foliar na excreção de honeydew e sobrevivência de adultos do Percevejo bronzeado [Internet]. 2012 [cited 2014 Mar. 24]. Available from: Available from: https://bit.ly/2GUjsdn
https://bit.ly/2GUjsdn...
).

5. CONCLUSIONS

The B. bassiana isolates are efficient for T. peregrinus control, especially when tested in the highest concentrations, with the best responses being observed for the JAB06 isolate.

The methodology used to assess virulence and selection of B. bassiana isolates enables developing other studies that require creating T. peregrinus in vitro.

ACKNOWLEDGEMENTS

The authors are thankful for the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support given to develop this study.

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  • FINANCIAL SUPPORT Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant/Award Number: 001).

Publication Dates

  • Publication in this collection
    11 May 2020
  • Date of issue
    2020

History

  • Received
    26 June 2017
  • Accepted
    05 Dec 2018
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