Methods
Plant material
The local tomato variety “Tounvi” and the improved variety “Padma” were used during the bioassays. Both varieties are semi-erect, with a development cycle that elapses from 65 to 90 and 60 to 70 days for local and improved varieties, respectively. The average weights of a tomato fruit is 24 g and 120–130 g for local and improved varieties, in that order. Tounvi and Padma are the 2 most produced and marketed varieties in Benin according to their agronomic quality, their color, and resistance to pests such as H. armigera (Assogba Komlan et al. 2016).
Rearing of Helicoverpa armigera
A rearing colony of H. armigera was established at the laboratory by caterpillars collected from tomato fields at different localities in major tomato production areas in Benin. The larvae were placed in plastic containers (6.5 × 19.5 cm) and reared on an artificial diet under controlled conditions (70 ± 5% RH, 26 ± 2 °C, with a photoperiod of L: D 12:12) until pupation (Douro Kpindou et al. 2012). In order to prevent cannibalism, third-instar caterpillars were transferred individually in Petri dishes provided with artificial diet. The artificial diet consisted of bean flour, beer yeast, methylparaben, ascorbic and sorbic acids, streptomycin, formaldehyde, vitamin complex, agar, and distilled water. Diet was replaced every 2 days in order to avoid desiccation; moistened filter paper was placed in each Petri dish (Barrionuevo et al. 2012). Pupae were collected and placed in polypropylene containers (6 × 12 cm) until adult emergence. Folded paper was placed inside the cages for egg deposition. Collected eggs were kept until hatching, and then the larvae were reared as described above. Third-instar larvae (L3; 7.4 ± 0.1 days) were used in all bioassays.
Source and production of the entomopathogenic fungi
The isolates Bb 11 and Bb 115 were obtained from the entomopathogenic fungi (EPF) collection of the Applied Entomology Laboratory and were selected in previous studies for their pathogenicity towards H. armigera (Douro Kpindou et al. 2012). The endophytic characters of the isolates remained to be demonstrated on different varieties of tomato most consumed in Benin.
Conidia from the 2 fungal isolates were picked from the stock culture and placed onto standard Potato Dextrose Agar (PDA) in Petri dishes (Ø = 9 cm) (Becton, Dickinson and Company; Sparks, MD 21152 USA) for subculture and incubated for 14 days at 26 ± 2 °C and a photoperiod of 14:10 h (L: D). Then, conidia of each isolate were harvested by scraping them from the PDA, using a sterilized scalpel and suspended in 0.01% (w / v) Tween 80®. Conidial concentrations were estimated, using a Neubauer hemocytometer and adjusted to 1 × 107conidia/ml and 1 × 109conidia/ml for isolates (Posada and Vega 2005).
The concentration of conidia to be used was calculated as follows:
$$ C\hbox{'}=\left(C\mathrm{o}\times {V}_{\mathrm{O}}\right)/\left(V\mathrm{o}+V\hbox{'}\right) $$
where C’ = concentration to be tested, Co = concentration of initial conidial suspension, Vo = volume needed, and V’ = volume to be added.
The viability of conidia after 24 h incubation on PDA was 89 ± 3.7% and 92 ± 1.5% for Bb 11 and Bb 115, respectively.
Treatment with B. bassiana through seed coasting
The seeds were sown and monitored under greenhouse conditions (26 ± 5 °C, 14:10 h photoperiod) until use of the tomato plants. On the other hand, before sowing, the seeds were mixed with a suspension of fungal conidia with a fungal inoculum of 1 × 107conidia/ml and 1 × 109conidia/ml of each isolate. Then, they were placed on filter paper and kept for 24 h before sowing (Russo et al. 2015). Experiment consisted of 5 treatments: (i) tomato seeds soaked in Bb 11 at 1 × 107conidia/ml; (ii) tomato seeds soaked in Bb 11 at 1 × 109conidia/ml; (iii) tomato seeds soaked in Bb 115 at 1 × 107conidia/ml; (iv) tomato seeds soaked in Bb 115 at 1 × 109conidia/ml; and (v) untreated control. Seeds were sown in 10 pots for each treatment. All treatments were replicated 3 times for each of the 2 tomato varieties.
Assessment of the endophytic colonization B. bassiana
The methods of Arnold et al. (2000) and Kambrekar and Aruna (2018) were used to re-isolate B. bassiana from inoculated plant organs. For this purpose, all the glassware were sterilized, using an autoclave at 121 °C for 15 min and then kept in a hot air oven at 55 °C for 1 h. Then, 10 leaves and roots randomly sampled from inoculated tomato plants were cut in 5 pieces with a sterilized knife under a laminar air flow chamber. The 5 pieces of each organ (leaf, root) variety and 3 replicates per treatment and per variety pieces (3 cm2) were sterilized in 0.5% sodium hypochlorite for 3 min, then with 70% ethanol for 2 min, and then washed with sterile water and dried before placing it onto PDA in Petri dishes (9 cm diameter). Petri dishes were incubated at room temperature (28 ± 2 °C) and periodically checked for fungal growth. The purity and sporulation of the culture were checked using a microscope. Colonization of B. bassiana was confirmed by microscopic observations (Ma et al. 2008). Petri dishes with B. bassiana were counted for each organ per treatment and per variety.
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{colonization}=\mathrm{no}.\mathrm{of}\ \mathrm{segments}\ \mathrm{colonized}/\left(\mathrm{total}\ \mathrm{no}.\mathrm{of}\ \mathrm{plant}\ \mathrm{sampled}\ \mathrm{segments}\times 100\right) $$
Percent colonization was determined for different plant organs and the most virulent B. bassiana isolate with the highest endophytic colonization was determined.
Assessment of leaf consumption and larvae survival in inoculated and untreated tomato plants
Leaf consumption was assessed by measuring leaf area consumed by each larva in each treatment. The test consisted of feeding 10 third-instar larvae of H. armigera for 24 h on tomato leaves sampled from inoculated and untreated tomato plants of both local and improved varieties. Ten discs of tomato leaves (3 cm in diameter) were obtained by cutting sampled leaves and offered each to the 10 H. armigera larvae placed in Petri dish (90 mm) (Magrini et al. 2015). The Petri dishes were then incubated for 24 h at 25 °C, 60% RH. Thus, using a graduated paper, the consumed area was estimated per treatment and per variety (Russo et al. 2015). Experiments were replicated 3 times. In parallel, larval survival and mortality of H. armigera larvae was checked daily per treatment and per variety. Leaves were replaced every 2 days until the 10th day after treatment (Ma et al. 2008).
Data analysis
The percentage of plants colonized by B. bassiana was compared using the chi-square test. Data on larval mortality and sporulation rates were processed by analysis of variance (ANOVA), using the general linear model (GLM) procedure of SAS (SAS Institute Inc 2003). Percentages were based on the initial number of larvae exposed. In case of significant F values, means were compared by using SNK (Student-Newman-Keuls). The modeling of the time-dose-mortality data was carried out, using the “Cox regression” model (Statistical Package for Social Science (SPSS) Inc. 1989-2003). Data on leaf area consumed by larva was compared by applying ANOVA, followed by SNK.