Evaluation of D. citri fungal infection in citrus seedlings under controlled conditions
Two strains of B. bassiana (2067 and 2121) and one of M. anisopliae (2411) were evaluated. The virulence of each fungal strain on D. citri was performed, using a suspension of 1 × 107 viable conidia/ml 0.02% (v/v) Tween 80. Application of fungal suspension was carried out on 7-month-old citrus seedlings, artificially infected with different nymphal instars of D. citri. A total of 2132 individuals were obtained from the mass rearing of the Instituto Nacional de Investigación Agropecuaria (INIA) Salto Grande. Twenty-four pots, with 3 seedlings each, were used. For each selected fungal strain, 30 ml of the conidial suspension was sprayed on the foliar surface of seedlings in each pot. Negative controls were sprayed only with 0.02% (v/v) Tween 80. Six replicates (pots) were done per treatment. Each pot was covered with fine mesh that prevents the movement of insects from one pot to another. Plants were incubated at 25 ± 2 °C for 5 days and then buds of treated and control seedlings were cut and investigated with a stereomicroscope. Living and dead individuals of nymphs were counted, and the presence of mycelium emerging from insects was recorded. Dead insects without evident presence of mycelium were transferred to individual moist chambers to determine if fungal infection had occurred. Insects were individually placed to avoid fungal transmission between samples and the overestimation of infection, as suggested by Hesketh et al. (2010). The percentage of insect mortality caused by fungi was calculated as the number of dead insects with the EPF/total of treated insects × 100.
To evaluate if there are significant differences between insect mortalities, data were transformed as arcsen√x and Tukey’s test was performed considering α = 0.05.
Evaluation of insect infection under semi-field conditions
To evaluate the insect infection under field conditions, a similar assay as mentioned before under controlled conditions was carried out. Seven-month-old citrus seedlings were artificially infected by nymphs of D. citri at different stages of development. A total of 1657 individuals were obtained from the mass rearing of INIA Salto Grande. For each fungus strain, 10 pots containing 3 seedlings each were inoculated. Surface of seedling leaves was sprayed by 30 ml suspension of 1 × 107 viable conidia/ml 0.02% (v/v) Tween 80. The control was performed by applying 0.02% (v/v) Tween 80 (10 pots with 3 infected seedlings each). Each pot was covered by a fine mesh to prevent exit of insects. All pots were placed randomly under the trees canopy interspersed inside a citrus plantation. After 5 days, the effect of the inoculation with EPF on D. citri infection was evaluated.
All living and dead individuals were counted by using a stereomicroscope. Dead insects were placed in a humid chamber to analyze fungal infection. Percentage of insect mortality caused by the fungi was calculated as the number of dead insects with EPF evidence/total of treated insects. To determine if there were significant differences in the mortality of D. citri between treatments, data were transformed as arcsen√x and Tukey’s test was performed considering α = 0.05.
Effect of temperature on EPF
Since the highest infection occurs in summer, where the temperature is highly variable, the conidial viability at different temperatures were evaluated. A conidial suspension of 1 × 103 conidia/ml 0.02% (v/v) Tween 80 was inoculated on potato dextrose agar (PDA) culture medium (OxoidTM) and incubated at 15, 20, 25, 30, and 35 °C for 20 h in the dark. Five replicates were tested, and 200 spores were counted per plate. Viability of spores was calculated as the germinated conidia/total conidia × 100. A spore was considered viable, if the germinated tube reached ¾ the size of the spore. To evaluate if there were significant differences in spore germination at different temperatures, the transformation of the data was done as arcsen√x and Kruskal-Wallis test was performed considering α = 0.05.
Evaluation of EPF incidence on the predators of D. citri
Breeding units of C. externa and H. axyridis were established in the Laboratorio de Entomología, Facultad de Agronomía-Universidad de la República, to supply individuals for the assays.
Effect of EPF on H. axyridis
Suspensions of spores of B. bassiana 2067 or M. anisopliae 2411 of 1 × 107 viable conidia/ml 0.02% (v/v) Tween 80 were applied on the insects. The control was carried out by applying only 0.02% (v/v) Tween 80. The individuals were placed separately in cages provided with specific food. The larvae were placed individually to avoid cannibalism. The bioassay was kept for 7 days under controlled conditions of temperature (24 ± 2 °C). Three replicates of each assay were performed. A total of 207 individuals of H. axyridis were analyzed, 41 larvae and 166 adults. The insects that died in the trial were placed in a humid chamber to observe fungal infection. If fungal mycelium was present, microscopic observation was performed to confirm the presence of EPF. To determine if there were significant differences in the mortality rates of H. axyridis between treatments, data were transformed as arcsen√x and Dunn’s test was performed considering α = 0.05
Effect of EPF on C. externa
Cages of C. externa larvae, with one individual per cage, were performed to avoid cannibalism. On the other hand, groups of adult individuals were placed in cages. In each cage, a wet cotton and yellow paper with eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) as food was placed. Suspensions of 1 × 107 viable conidia/ml 0.02% (v/v) Tween 80 of B. bassiana and M. anisopliae were applied on the larvae and adult individuals of C. externa. In control cages, only 0.02% (v/v) Tween 80 was applied on the insects. The bioassay was kept for 7 days under controlled conditions of temperature (24 ± 2 °C). Three replicates of each assay were performed. A total of 321 individuals of C. externa were analyzed, 183 larvae and 138 adults. Dead or alive individuals were counted daily, and dead insects were placed in a humid chamber to corroborate the fungal infection. To determine if there were significant differences in the mortality rates of C. externa between treatments, data were transformed as arcsen√x and a Dunn’s test was performed considering α = 0.05.
Effect of pesticides on selected EPF
The effect of copper oxychloride, abamectin, and mineral oil products, the most frequently used in citrus plantations, on conidial germination and mycelial growth, was evaluated. Three concentrations (350, 500, and 650), (1.75, 2.50, and 3.25), and (12.25, 17.50, and 22.75) for Abamectin (μl/l), copper oxychloride (g/l), and mineral oil (ml/l), respectively, were used, based on the concentration usually applied in the field and additionally + a concentration 30% high and 30% low.
Both copper oxychloride and mineral oil were incorporated into the PDA culture medium before autoclaving. Since abamectin is a very volatile compound, it was incorporated by filter-sterilization into the medium after the sterilization process. To determine the viability of the conidia in presence of copper oxychloride and abamectin, conidia germination was evaluated (germinated conidia/total conidia × 100). Viability with mineral oil was determined by means of colonies quantification after 3 days of incubation. The effect of pesticides on conidial germination was determined by plating 200 μl of a suspension of 1 × 103 conidia/ml 0.02% (v/v) Tween 80, onto 9-cm Petri dish diameter with 2% PDA culture medium (OxoidTM), which containing pesticides in the concentrations was indicated. Plates were incubated at 25 °C for 24 h in the dark. Four replicates were performed and 200 spores per plate were counted.
The effect of pesticides on mycelial growth was evaluated by placing a 0.7-cm disc with a mycelium 4 days old onto 9-cm Petri dish diameter with PDA solid medium (OxoidTM) containing pesticides in the concentrations indicated. Inoculated media were incubated at 25 °C for 8 days, and the diameter of the colony was daily recorded. Four replicates of each assay were performed. Kruskal-Wallis Test (α = 0.05) was used to evaluate the effect of pesticides on conidial germination and mycelial growth.
