Chemical vs entomopathogenic control of Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae) via aerial application in eucalyptus plantations

The Thaumastocoris peregrinus spread to eucalyptus plantations in many countries. Chemical control is a questionable measure, mainly due to the environmental impact, high cost and moreover has the use restricted by the forest certifications. Bio-insecticides may have similar efficiency to chemical products to control T. peregrinus. The chemical thiamethoxam, thiamethoxam + lambda-cyhalothrin, acephate and the microbial Beauveria bassiana and Metarhizium anisopliae insecticides were tested at different doses to manage T. peregrinus. The products were sprayed on eucalyptus plants using aircraft and populations of this insect were counted before application and at 1, 14 and 21 days afterwards (DAA). Ten eucalyptus trees were evaluated per plot, with the collection of ten leaves from the middle third of the crown of each tree, and the number of T. peregrinus nymphs and adults obtained per leaf was determined. All the chemical insecticides had similar control at 1 DAA for T. peregrinus nymphs and adults. At 14 DAA, the number of T. peregrinus nymphs and adults on eucalyptus leaves was similar for the chemical and microbial insecticide treatments. At 21 DAA the control efficiency of T. peregrinus nymphs and adults was higher than 80% with all insecticides. The entomopathogenic insecticides have potential for aerial application to control T. peregrinus nymphs and adults and provide viable and environmentally-friendly alternative to manage this pest.


Results
The number of T. peregrinus nymphs and adults per eucalyptus leaf, before application, was similar between treatments ( Table 1).
The number of T. peregrinus nymphs and adults at 21 days after application was lower for B. bassiana with the lowest doses and similar to the treatments with Engeo Pleno, Orthene and Actara (0.15 Kg/ha) ( Table 1). The control was above 80% in all treatments, with greater efficiency for B. bassiana 0.5 kg/ha (99%) and acephate 0.5 kg/ha (97%) (Fig. 1).

Discussion
Biological insecticides, such as entomopathogenic fungi, are safer and have lower health risks than chemicals in pest control 45,46 . The bronze bug mortality by B. bassiana and M. anisopliae in the field is poorly studied but microbial products may have high efficiency in the integrated management of this pest in forest crops. Temperatures of 27, 28 and 29 °C and precipitation of 20; 25 and 63 mm in August, September and October were adequate for sporulation and favored the control efficiency of T. peregrinus by B. bassiana and M. anisopliae, as observed in other works 46,47 . These entomopathogens have a wide host range, but their germination, conidia persistence, host mortality and sporulation depend on adequate environmental conditions such as temperature and humidity 48,49 . Beauvaria bassiana and M. anisopliae can grow between 5 to 30 and 5 to 40 °C respectively, but showing optimal growth at temperatures of 25 and 30 °C 50 , determining their efficacy in biological control. This makes necessary an adequacy between the temperature and humidity for the efficiency of these fungi. Entomopathogen applications in the field are preferable in the late afternoon to avoid the negative impact of abrupt changes in temperature 51 . The low precipitation level may initially compromise the spore penetration and survival even at adequate temperatures. However, increased precipitation favored conidiogenesis in the first dead individuals, with horizontal www.nature.com/scientificreports www.nature.com/scientificreports/ transmission and dissemination of the disease throughout the populations. This is important because the potential for fungus conidiogenesis is determinant in the pathogen spread among pest individuals.
The higher efficiency of the chemical insecticides, 1 day after application, shows its faster impact due to the pyrethroid and neonicotinoid knock-down effect. This is similar to other neonicotinoids and pyrethroids such as imidacloprid and lambda-cyhalothrin against Bucephalogonia xanthophis (Berg 1879) (Hemiptera: Cicadellidae) with mortality above 90% in 24 h and near 100% within 48 h 52 . The neonicotinoid-pyrethroid mixture (thiamethoxam + lambda-cyhalothrin) increased the control efficiency of T. peregrinus with results similar to that observed for Myzus persicae nicotianae Blackman 1987 (Hemiptera: Aphididae) up to 70 DAA in tobacco crop 53 . Imidacloprid and thiamethoxam are systemic insecticides of the neonicotinoid group that act as an acetylcholine agonist in the synapses of the central nervous system 54 . The pyrethroid lambda-cyhalothrin is a sodium channel modulator, causing repetitive and uncontrolled impulses, hyperexcitation and death 55 . However, these insecticides have rapid action and often are toxic to beneficial organisms. Bees may come into direct contact during pollen and nectar collection or through contaminated water 32,33 . Thiamethoxam is toxic to parasitoids and predators in forest environments and agricultural crops 56 . In addition, airborne application with chemical insecticides may aggravate the situation by contaminating nearby wild and cultivated plants 33 .
The similar control efficiency of B. bassiana and M. anisopliae entomopathogens and chemicals for the T. peregrinus nymphs and adults after 14 DAA suggests efficiency of the microbial control. The horizontal transmission and dissemination of diseases in pest populations determine the pathogen efficiency being favored by greater humidities 57 . Delayed effects via horizontal transmission for entomopathogens have been reported for M. anisopliae on Oncometopia facialis (Signoret) (Hemiptera: Cicadellidae) 58 and B. bassiana in Bemisia tabaci (Gennadius, 1889) (Hemiptera: Aleyrodidae) 45 . Epizootic occurrences of Entomophtorales fungi were reported for T. peregrinus nymphs and adults in a Eucalyptus plantation in São Paulo state, Brazil 44 . Entomopathogen use in integrated pest management is a viable, low-risk technique and has the capacity to exploit a wide host  www.nature.com/scientificreports www.nature.com/scientificreports/ range through different action modes 46 . Fungi cause death by penetrating and destroying the external arthropod cuticle 59,60 . The fungi B. bassiana and M. anisopliae have rapid dispersion in the field with potential to control forest insect outbreaks 40,41 , and sucking insects such as Nilaparvata lugens Stål (Hemiptera: Delphacidae) 61 and Diaphorina citri Kuwayama (Hemiptera: Liviidae) 62 . The gregarious behavior of T. peregrinus 25,28,63 may facilitate entomopathogenic fungal epizootics in the field. The entomopathogenic fungi such as M. anisopliae and B. bassiana are effective against pest insects but they can affect natural enemies. This suggests a careful criterion in using these fungi to maintain the effectiveness of the control exerted by both.
The control efficiency of T. peregrinus, at 21 days, with the entomopathogen products, mainly for B. bassiana, shows residual effects and the horizontal dispersion capacity of this fungus as reported against Bucephalogonia xanthophis (Berg) (Hemiptera: Cicadellidae) 45 . Entomopathogenic fungi are slower acting and need higher relative humidity and/or rainy periods. Additionally, they require longer periods to cause mortality compared to synthetic chemical products 64 , but side-effects in infected insects reduces feeding and damage 65 . The pathogenicity and virulence of the mycoinsecticides indicate that the fungi overcome the physical barriers such as insect sclerotic exoskeleton as found for the natural occurrence of a fungus from the order Entomophthtorales on T. peregrinus in São Paulo state, Brazil 44 . The tegument may act as a physical barrier to the penetration and the germinative tube or may have chemical properties inhibiting conidia germination. Thaumastocoris peregrinus control over 80% at 21 DAA using entomopathogenic fungi indicates the delayed effect of this product as found for B. bassiana surviving and colonizing foliar tissues 30 days after inoculation without damaging plants 66 .
Beauveria bassiana and M. anisopliae have with potential to control T. peregrinus as found against Hemiptera pests such as aphid 67 , Riptortus pedestris (Fabricius, 1775) 68 , Diaphorina citri Kuwayama 69 , Bemisia tabaci 70 . Certified forest companies seek practices that conserve the environment, such as integrated pest management, giving preference to biological, cultural control and the use of less toxic products. Biological control is the only viable option to manage T. peregrinus in commercial eucalyptus plantations reducing the toxicity drift caused by the pyrethroid and the neocotinoid in aerial applications. The compatibility of chemicals with microbial agents and the effect of these products on natural enemies need better studies for integrated pest management.
The T. peregrinus control was similar with entomopathogens and chemical insecticides. The efficiency of the fungi B. bassiana and M. anisopliae at lower concentrations and its high residual period shows the potential of these products to control T. peregrinus nymphs and adults in eucalyptus plantations with low impact on other organisms such as parasitoids and predators. The adoption of control measures may be part of integrated management programs, where other control strategies can be used in a joint manner.
Methods obtaining fungal spores. The fungus Beauveria bassiana (isolated ESALQ PL63-obtained from Atta spp. in Piracicaba, São Paulo, Brazil) was the active ingredient of the product Boveril and Metarhizium anisopliae (ESALQ E9 isolate -obtained from Mahanarva posticata in Boca da Mata, Alagoas, Brazil) that of the product Metarril. Both are deposited in the Bank of the Laboratory of Pathology and Microbial Control of Insects of ESALQ/USP Piracicaba, São Paulo, Brazil. These microorganisms were cultured by solid fermentation in rice and their conidia were dried and extracted for the assays. Spore production followed a methodology described 71 , with modifications. This methodology includes pre-baking the rice, packing it in polypropylene bags, closing the bags and sterilizing them for 20 minutes in an autoclave at 121 °C. After cooling the rice, the substrate is inoculated with microorganism strains, homogenized by manual shaking and stored in air-conditioned rooms with a controlled temperature of 25.5 ± 1.0 °C and 12 hour photoperiod and placed on shelves for four days. After this time, the rice with mycelium was spread in trays for another eight days until the conidia sporulation. After this process, the solid fermentation product is dried for three days under the same conditions of controlled temperature and photoperiod and sieved to extract the pure conidia (Personal communication, Luciano Koppert).  www.nature.com/scientificreports www.nature.com/scientificreports/ The pure spores of the entomopathogenic fungi were used in the same proportion of the active ingredient used in the commercial product Boveril and Metarril corresponding to 2.5 × 10 9 spores/ha and 6.9 × 10 9 spores/ ha respectively.
Conducting the experiment. The experiment was carried out in Pompéu, Minas Gerais state, Brazil in areas of the Vallourec & Mannesmann Florestal (V&M) with a randomized complete block design. The 12 treatments were conducted with chemical and biological insecticides with different concentrations (Table 2) with four replications and 48 plots, each 40 m wide and 500 m long, equivalent to 2 ha. The evaluation was done in the central area (2 ha) of each plot to avoid drift contamination between treatments. The clones VM01 (hybrids of E. urophylla and E. camaldulensis) with approximately 12 to 16 months old and spaced 2 × 3 m were used.
The products and water (control) were sprayed using an agricultural aircraft model Ipanema with Micronair AU 5000 rotary spray nozzles with electronic beacon DGPS in a round-trip evolution system with a diameter of 200 micrometer drops. After each spraying with the respective treatments, the tank was cleaned with 100 liters of water, with the aid of a tank kite. The temperature and humidity conditions in the field ( Table 2) were adequate for the B. bassiana and M. anisopliae survival and development.
Thaumastocoris peregrinus nymphs and adults were collected before and at 1, 14 and 21 days after spraying. Microbial insecticides were not evaluated 1 day after application due to their slower action.
Ten trees were evaluated per plot with the collection of ten leaves from the middle third of the crown of each one 43 . The leaves were removed from the plant and packed in sealed paper bags which were transported to the FCA/UNESP Biological Control of Forest Pests Laboratory in Botucatu, São Paulo state, Brazil, where live insects were counted.
The mean numbers of T. peregrinus nymphs and adults per eucalyptus leaf were submitted to variance analysis and compared using Tukey test (p < 0.05). The control efficiency of the products was corrected by the Henderson-Tilton's formula 72 , adequate to evaluate the number of live insects in a non-uniform population: efficiency (%) = [(numbers in the control before application x numbers in the treatment after application)/(number in the control after application x numbers in the treatment before application)] × 100}.