The experiments were carried out in the 2017–2018 and 2019–2020 soybean seasons in the experimental area of the Department of Plant Health and the Laboratory of Microbial Control of Arthropods Pests (LCMAP), Paulista State University (FCAV/UNESP), Jaboticabal, São Paulo.
Helicoverpa armigera rearing conditions
Approximately, 100 H. armigera larvae were obtained from the company Ouro Fino Agrociência, São Paulo, Brazil. The larvae were reared individually in a transparent plastic container (50 ml) containing approximately 50 g of an artificial diet (Greene et al. 1976) until they reached the pupal stage. The pupae were kept in plastic containers (11.5 cm in diameter and 3.5 cm in height) with filter paper until adult emergence (males and females). Subsequently, they were transferred to PVC cages (20 cm in diameter and 30 cm in height). The upper parts of the cages were sealed with voile fabric, and the lowest parts were supported on a plastic plate (28 cm in diameter) covered with filter paper. Internally, the cage was lined with paper towels, which were used as a substrate for oviposition. Adult insects were fed with a 10% honey solution soaked in cotton.
The eggs were collected from the cages every 3 days and transferred to a transparent plastic container (500 ml) containing approximately 50 g of the artificial diet. The larvae were separated and kept individually after 10 days when they reached the third instar larvae to avoid cannibalism. The larvae were kept in an air-conditioned room at a temperature of 25 ± 2 °C, 70 ± 10% RH, and 12-h.
Field trials photophase
An area of 1700 m2 with plots subdivided in a completely randomized design was used for the field trials. Each plot (560 m2) was sprayed with a suspension of B. bassiana IBCB-1363 and another one from M. anisopliae IBCB-425, and 0.003 µl of Tween 80®. The control was sprayed with distilled water. Fungal suspensions were prepared by washing rice grains that were used as a solid substrate for the production of both fungi with 15 days of cultivation. A 5 l of water were used to wash 1.5 kg of rice, after washing the suspension was filtered to determine the concentration.
The two fungal isolates were chosen to be taken to the field based on preliminary tests in the laboratory, where the isolates that showed the greatest virulence (100% mortality) against H. armigera larvae were selected. Parcels were sampled for eight collection periods after fungal application. The applications were conducted at 7:00 AM using a hand sprayer, with a CO2 pressure of 1 bar and a flow rate of 70 l ha−1, for 15 s per line, shaking at the end of each line to maintain a uniform suspension of the conidia. At the time of spraying, the fungal suspension had a concentration of 108 conidia ml−1, as determined by a Neubauer chamber (400× magnification) under a Carl Zeiss-Axio LabA1 microscope.
Leaf disk collection and evaluation
After spraying the fungi, soybean leaflets were collected at eight different times (0.5, 1, 2, 3, 4, 5, 6, and 7 h after application) and taken to the laboratory. The leaflets were cut into disks (3 cm ø). Each treatment evaluation consisted of 55 disks (50 disks for the mortality test and 5 disks for the persistence test), totaling 400 replicates for each evaluation of mortality and 40 for persistence. The local temperature was recorded during the collection of the leaflets using a thermometer (Tecpel®). At the farm's meteorological station where the experiment was carried out, the relative humidity was measured over the two seasons (harvests of 2018 and 2019). There was no rain during the experimental period. Each experiment was repeated twice each season.
Fungal persistence and mortality evaluation
For the persistence trials, fungal suspensions were prepared with leaflet disks shaken in Falcon tubes (15 ml) that contained 10 ml of distilled water and 0.001 µl of Tween 80® (Sosa-Gómez et al. 2010). 150 µl of the suspension was then removed and poured into a Rodac® plate (Replicate Organism Detection and Counting), with 5 ml of the potato dextrose agar (PDA) culture medium. The millimeter face of the plate was reversed, and the plates were sealed with Parafilm M®. They were incubated at 26 ± 1 °C over 12 h photoperiod. Each plate was considered a replicate, totaling five plates for each sampling time (0.5, 1, 2, 3, 4, 5, 6, and 7 h).
After 72 h of inoculation, the colony-forming units (CFUs) were directly counted using an optical microscope (ZEISS AX 10®) and a manual counter, which considered the standard plate count (the number of CFUs on the plate were counted and multiplied by the corresponding dilution).
For the mortality test, the disks were placed individually in bioassay pots (18 mm × 30 mm), and the surface of the pots was covered with a gelled mixture of 2.5% agar-water and filter paper in order to maintain the turgor of the leaves during the evaluation period. A second instar H. armigera larvae was transferred in each pot, totaling 400 larvae per fungal treatment. The trials were maintained under controlled conditions (RH: 75% ± 12%, T: 25 ± 2 °C, and 12-h photoperiod). Mortality was assessed 3 days after inoculation, for 7 days. The larvae were considered dead if they did not move when touched with a fine bristle brush.
Insect death due to the pathogen was confirmed as follows: the insects were washed in 70% alcohol for 10 s, rinsed in distilled water for 20 s for external decontamination, placed in plates, and left in a wet chamber for 15 days, making it possible to observe the extrusion of the pathogen, thereby confirming infection.
Sublethal effects on Helicoverpa armigera
The larvae that survived fungal exposure in the leaf disks were monitored daily in each treatment (collection time). The parameters of instar duration (days), larval weight (mg), pupal weight (mg), fecundity, egg viability (%), sex ratio, and adult longevity (male and female) were then evaluated.
Surviving larvae, pre-pupae, and pupae were weighed using an analytical balance (SHIMADZU-ATY224®). Pupae were sexed using a magnifying glass (ZEIZZ-STEMI 508®), with the males separated from the females. With these data, it was possible to establish breeding couples to assess the pre-oviposition period (APOP: Adult pre-oviposition period) and total pre-oviposition (TPOP: Total pre-oviposition period). The breeding couples were kept under the same conditions described for rearing. Oviposition and adult longevity were recorded daily. The larvae used in the experiments were not subjected to stresses of different natures that could interfere with their susceptibility to the isolates of the tested fungi.
For the analysis of the biological parameters of H. armigera, the raw data containing development time, survival, daily fertility, and male and female longevity were analyzed based on the constructed life table. In these assessments, all tested individuals were considered, including those that died during the immature stages (Chi and Liu 1985; Chi 1988). The parameters of intrinsic population growth rate (r), finite population growth rate (λ), net reproduction rate (R0), average generation duration (T), and gross reproduction rate (GRR) were estimated using methodology of Chi and Liu (1985).
For all variables, the homogeneity of variance test was performed, using the Shapiro–Wilk test. All variables were normally distributed. To assess H. armigera mortality, an analysis of variance (ANOVA) was performed in the software Statistical Analysis System (SAS®) in a completely randomized design (CRD), with the treatments considered in a factorial arrangement. When a significant result was detected, the means were compared using Tukey's test at 5% probability. To assess the relationship between mortality and the number of colony-forming units, Pearson's correlation coefficients were determined using the R statistical software program (R Core Team 2018).
The sublethal effects were analyzed with the bootstrap procedure, which was used to estimate the variations, means, and standard errors of the biological parameters of development time, fertility, and longevity using 100,000 bootstraps Reference. The comparison of population parameters of H. armigera that received fungal treatments with controls was performed using the paired bootstrap test (p < 0.05) based on the difference confidence interval (CI 95%) in the TWOSEX-MS-Chart software (2021) (Chi 2016).