Cordyceps cateniannulata and Cordyceps javanica: first report of pathogenicity to Glycaspis brimblecombei (Hemiptera: Aphalaridae)

Abstract

Cultivation of species of the genus Eucalyptus is important for the Brazilian economy, with 6.97 million hectares planted. Glycaspis brimblecombei Moore (Hemiptera: Aphalaridae), detected in Brazil in 2003, has dispersed and now damages Eucalyptus crops in all regions of this country. The location and identification of entomopathogenic fungi isolates may increase the options for integrated pest management. The objective of this research was to evaluate the pathogenicity of Cordyceps cateniannulata and Cordyceps javanica isolates to G. brimblecombei. Ten nymphs of G. brimblecombei, with or without lerps, were placed per Eucalyptus leaf cut with one of its edges on hydroretentive gel inside Petri dishes. The fungi isolates were suspended in a solution of Tween 80 (0.1%) at the concentration of 1.0 x 10⁸ conidia mL^-1 and sprayed on the G. brimblecombei nymphs. The mortality of this insect was evaluated daily for seven days, and the dead individuals were transferred to humid chambers. The conidia viability of the isolates was greater than 93%. The mortality of G. brimblecombei nymphs, seven days after the application of the fungi, was 100%. This is the first report of the pathogenicity of C. cateniannulata and C. javanica isolates, occurring naturally in the field, to G. brimblecombei.

Key words
biological control; entomopathogenic fungi; Eucalyptus; pest management

INTRODUCTION

The area of forest cultivation in Brazil reached nine million hectares in 2019, with an increase of 2.4% in relation to 2018, 77.4% of which planted with species of the genus Eucalyptus (IBÁ 2020). Native and exotic pests can reduce the productivity of Eucalyptus crops (Paine et al. 2011). The red gum lerp psyllid, Glycaspis brimblecombei Moore (Hemiptera: Aphalaridae), was reported in Brazil in 2003 in Mogi-Guaçu, São Paulo, in Eucalyptus camaldulensis and Eucalyptus tereticornis plantations, and has dispersed throughout the country, reducing Eucalyptus productivity. Biological control using the parasitoid Psyllaephagus bliteus Riek (Hymenoptera: Encyrtidae) (Berti-Filho et al. 2003) and entomopathogenic fungi, especially Beauveria bassiana and Metarhizium anisopliae (Dal-Pogetto et al. 2011), and the use of chemical products acetamiprid, acetamiprid + bifenthrin and etophenproxy (AGROFIT 2021), are the main strategies for managing this pest.

The search for and identification of entomopathogenic fungi isolates can complement the integrated management of insect pests in agricultural and forest crops. The objective of this research was to evaluate the pathogenicity of Cordyceps cateniannulata (LCBPF 17) and Cordyceps javanica (LCBPF 11) isolates, occurring naturally in the field, to G. brimblecombei.

MATERIALS AND METHODS

Site of study

The present research was developed in Botucatu, São Paulo, Brazil, in BOD-type incubators at a temperature of 25 ± 1 ºC, RH of 70 ± 10% and a photophase of 12 h.

Rearing Glycaspis brimblecombei (Hemiptera: Aphalaridae)

Glycaspis brimblecombei was reared in a laboratory at a temperature of 25 ± 2 ºC, 60 ± 10% RH and a photophase of 13 h on Eucalyptus camaldulensis and on saplings of the hybrid clone 3025 (Eucalyptus grandis x E. camaldulensis), both highly susceptible to this pest. Two Eucalyptus saplings planted per 1 L pot were placed in a standard cage (40 cm x 45 cm x 80 cm). A total of 80 to 100 G. brimblecombei adults were released per cage. These Eucalyptus saplings were irrigated daily, using a 500 ml laboratory wash bottle with water, and were changed at each insect life cycle, around 25 days (Wilcken et al. 2010).

Source of the fungi isolates

Isolates of the tested fungi Cordyceps javanica and Cordyceps cateniannulata were collected from soil in soybean crops (SO) and in native forest (NA), respectively, in the municipality of Botucatu, São Paulo state, Brazil. These fungi species were catalogued as LCBPF 11 (C. javanica) and LCBPF 17 (C. cateniannulata), and they were preserved in a freezer (-18 ^oC) in a Castellani medium.

Viability of the fungi isolates

The number of viable conidia was determined 14 days after the fungi cultivation at 25 °C in a Potato-Dextrose-Agar (PDA) medium. After this period, the conidia were suspended in 0.05% Tween 80 at a dilution of 1.0 x 10⁶ conidia mL^-1. The percentage of conidia viability, per isolate, was calculated by counting them after 18 hours in a Neubauer chamber (Wraight et al. 2007).

Pathogenicity of the fungi isolates to Glycaspis brimblecombei (Hemiptera: Aphalaridae)

The conidia were obtained by superficial scraping of the fungus colonies in the PDA culture medium 14 days after their onset. The material was suspended in Tween 80 (0.1%) and adjusted to the concentration of 1.0 x 10⁸ conidia mL^-1 in a hemocytometer with an optical microscope. A total of 125 µL of the conidia suspension was sprayed with a DB134K airbrush (Fenghua Bida Machinery Manufacture Co., China) mounted on top of acrylic cylinder tubes 25 cm apart and with a working pressure of 68.95 kilopascal on G. brimblecombei nymphs with or without G. brimblecombei lerps. The control was a spray of only water + Tween 80 (0.1%). The equipment was washed in 70% alcohol and autoclaved with distilled water after each application.

Ten G. brimblecombei nymphs per replication in a Petri dish (90 x 15mm) were sprayed with the treatments and control and kept in BOD-type incubators (Eletrolab, model EL202/4), at the temperature of 25.0 ± 1.0 °C, relative humidity of 83.0 ± 2.0% and 12 h photophase with a total of 10 replications. A piece of approximately 5 cm² was removed from each leaf of the clone 433 (E. urophylla var. platyphylla) and placed on hydroretentive gel for each replication, reducing the loss of turgor and preventing G. brimblecombei from escaping. Each replication (a Petri dish) was evaluated daily for seven days, and the dead insects were counted and transferred to moist chambers to stimulate the fungi development and to evaluate the insect mortality. The mortality values were corrected using the Schneider-Orelli formula. The equation (r²) was estimated by adjusted polynomial trendline.

The datasets generated during and/or analyzed during the current study are available in the UNESP repository [https://repositorio.unesp.br/handle/11449/204462].

RESULTS

The C. cateniannulata and C. javanica isolates were identified by BLAST search using GenBank, with 100% homology. The viability of the conidia of the isolates, used in the pathogenicity bioassay for G. brimblecombei, was higher than 93% (Table I).

Table I
Molecular identification code (Code), species, host, culture (Cul.), coverage (Cov.), identity (Ident.), genBank access code (AC) and percentage viability (Viab.) (Mean±^SE) of the entomopathogenic fungi Cordyceps javanica isolates collected in soybean (SO) and Cordyceps cateniannulata in native forest (NA) in Botucatu, São Paulo, Brazil.

Code	Species	Host	Cul.	Cov.	Ident.	AC	Viab.(%)
LCBPF 11	C. javanica	B. tabaci	SO	100%	100%	MW138089	97.63±0.65
LCBPF 17	C. cateniannulata	T. molitor	NA	100%	100%	MW131688	95.61±3.06

The virulence, conidia production and infection rate of G. brimblecombei nymphs were similar between the C. cateniannulata and C. javanica isolates, causing mortality of this insect mainly from the third day after their application (Figures 1 and 2). Mortality in the control was low, starting on the third day after application, with an average of two nymphs killed per replication, without evidence of fungal infection. The mortality of G. brimblecombei nymphs, seven days after the fungi application, was 100% (Figure 2).

Figure 1
Nymphs of Glycaspis brimblecombei (Hemiptera: Aphalaridae) infected by Cordyceps cateniannulata (a) and Cordyceps javanica (b); Healthy nymph (c).

Figure 2
Accumulated corrected mortality of Glycaspis brimblecombei (Hemiptera: Aphalaridae) nymphs with (C/) or without (S/) lerp, over the days after application of the LCBPF 11 Cordyceps javanica (a) and LCBPF 17 Cordyceps cateniannulata (b) isolates with trendline.

DISCUSSION

The high viability of the conidia of the fungus isolates is similar to that observed for those of B. bassiana and C. javanica, above 92%, for Duponchelia fovealis Zeller (Lepidoptera: Crambidae) (Baja et al. 2020). The infection and sporulation of C. cateniannulata and C. javanica on G. brimblecombei nymphs agrees with that reported for C. javanica on Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) (Scorsetti et al. 2008) nymphs and adults, and on Diaphorina citri Kuwayama (Hemiptera: Liviidae) (Ou et al. 2019), C. cateniannulata on Tetranychus urticae Koch (Acari: Tetranychidae) (Zhang et al. 2016) and Beauveria bassiana on Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Aphalaridae) (Alizadeh et al. 2007), indicating that these fungi can be used as mycoinsecticides.

The high mortality of G. brimblecombei nymphs, seven days after the application of the C. cateniannulata and C. javanica isolates, confirms their pathogenicity to this pest, with better results than those of B. bassiana and Metarhizium anisopliae, causing mortality above 90% of the G. brimblecombei nymphs (Dal-Pogetto et al. 2011), of B. tabaci by B. bassiana, Cordyceps sp. and M. anisopliae (Sani et al. 2020), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) by C. cateniannulata (Zhou et al. 2020) and D. fovealis larvae by Cordyceps javanica (Baja et al. 2020). The pathogenicity of these fungi is due to the production of mycotoxins that affect the host immune system, such as the bassianolide by C. fumosorosea, with mortality of D. citri nymphs and adults of 70% and 80%, respectively (Qasim et al. 2020).

This is the first report of pathogenicity of isolates of the fungi C. cateniannulata and C. javanica, of natural occurrence, to G. brimblecombei. This research is the initial step towards new formulations and products for the management of this forest pest, indicating the potential for using isolates of entomopathogenic fungi of the genus Cordyceps as a new tactic for the integrated management of G. brimblecombei.

ACKNOWLEDGMENTS

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. This study was funded by the Brazilian institutions Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (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.

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Publication Dates

• Publication in this collection
  21 Nov 2022
• Date of issue
  2022

History

• Received
  7 Dec 2021
• Accepted
  25 Feb 2022