Potential biological control of the pupal stage of the European grapevine moth by the entomopathogenic fungus Beauveria pseudobassiana in the winter season in Chile CURRENT STATUS:

Objective: Lobesia botrana, the European grapevine moth, affects Vitis vinifera L. and other species of economic importance in a number of countries through damage caused by its larvae in berries and associated secondary diseases such as Botrytis cinerea. Control of the moth in urban areas is difficult due to poor chemical management of infested plants in houses. Additionally, in winter, L. botrana is in its pupal stage covered with a cocoon that prevents the penetration of chemical pesticides. For this reason, the objective of this work was to control the pupal stage with a formulation based on the entomopathogenic fungus Beauveria pseudobassiana in urban areas. Results: The present study of the biocontrol activity of the formulated fungus against the infestation of vines with breeding pupae without cocoons showed 100% infection 21 days after inoculation under winter conditions. Finally, the biocontrol activity of the formulated fungus against natural infestations of L. botrana in winter in urban areas reached an efficacy of 51%. This result suggests that the B. pseudobassiana formulation is able to penetrate the cocoon and contributes to the integrated pest management of L. botrana.


In vitro EPF formulation evaluation against L. botrana
The assessment of the formulation was carried out in in vitro assays at 25°C. One milliliter of the formulation mix was placed in the center of a paper disk that covered the bottom of a plate, and ten moth pupae were deposited on the paper. As a control, water was used instead of the formulation.
The infection of pupae with EPF was monitored every day for eight days. The experiment included three replicates.

Field trial
The assay was developed in the V. vinifera ´Red Globe´ field at La Platina research station in the Metropolitan region of Santiago, Chile, in natural environmental conditions in July, during the winter.
The field trial was arranged in a randomized block design with four treatments, each with four replications with three vines each (4x4x3). To infest the vines, ten pupae without cocoons per plant were deposited under the rhytidome, covered with a tulle mesh and fixed with adhesive tape to avoid dispersion of the pupae and the entry of other arthropods. The first treatment was performed using one application of the formulation. In the second treatment, we used two applications on days one and seven. In the third treatment, we performed applications during days one, seven and fourteen.
The fourth treatment was the control using water. The EPF concentration for treatments one, two and three was 10 9 CFU/L. The applications were performed with a hand pump, and wetting was performed with a volume of 500 mL per vine. Each treatment was inspected seven days after each application.
For the inspection of the infected pupae, we removed the pupae from under the rhytidome and deposited them in a plate containing a humid paper disk when the EPF had completed colonization.
The evaluation of efficacy was performed over 48 h at 25°C.

Urban trial
The assay was developed in V. vinifera plants located in urban residences in the Metropolitan region of Santiago, Chile, under natural environmental conditions in August, during the winter diapause of pupae with cocoons. The urban trial was arranged in a randomized block design with four treatments, each with three replications with three vines each (4x3x3). The distribution of the blocks in the urban area was set up according the captures of the moths in pheromone (E7, Z9-dodecadienyl acetate) traps during the spring and summer by SAG. The treatments and the methodology for the application of the formulation and inspection of the vines were the same as those described for the field trial.
Finally, the pupae grown in cocoons with EPF were dissected to determine infection by the fungus.
The pupae were dyed with lactophenol blue (Merck) to observe the penetration of the fungus into their structure.
Temperature and humidity data during the field and urban trials were collected through the weather station network (http://agromet.inia.cl/).

Statistical analysis
The efficacy of the formulation in vitro and in field trials was determined for uniform populations [11], and the efficacy in the urban trials was determined for nonuniform populations [12]. The percentages of efficacy in the field test and urban test were compared by Tukey´s HSD test (a=0,05). All the experiments were analyzed using the software Statgraphics Centurion XVII (Statpoint Technologies Inc., VA, USA).

Molecular species identification of an EPF isolate
The Beauveria sp. RGM 1747 strain was subjected to molecular characterization using MLSA methodology, which indicated that this strain belongs to the clade of Beauveria pseudobassiana, as shown in Fig. 1 and Table 1S. It was grouped with other strains that have been isolated from different insect orders and countries such as Hymenoptera in South Korea and Lepidoptera and Thysanoptera in the USA.

In vitro evaluation
Once the EPF formulation was prepared, in vitro infestation was carried out at 25°C under high humidity. All the pupae were infected by the EPF with 100% efficacy by four dpa (Fig. 2a).

Field trial evaluation
The vineyard infestations were performed under controlled conditions with an average temperature of 9.1°C and relative humidity of 78.3%. In the three treatments with the application of the EPF formulation, the development of incipient mycelia and conidia of B. pseudobassiana in different areas on the head and sutures of the pupa was observed. All the examined pupae were infected by B.
pseudobassiana, which showed the capacity of the EPF to achieve colonization in winter conditions with an efficacy of 100% at 21 dpa (Fig. 2b).

Urban trial evaluation
We used the same formulation treatment as in the field trial, but the infestations of L. botrana were natural in this case. The average temperature and relative humidity in the assay were 8.4°C and 73.7%, respectively. In these assays, not all the examined pupae were infected by B. pseudobassiana.
The efficacy reached 51% in the first treatment (Fig. 2c). The efficacy in the second and third treatments reached 15% and 13%, respectively. The pupae colonized by the EPF showed development of different levels mycelia and conidia of B. pseudobassiana in several areas on the head and sutures of the pupa when the cocoon was removed (Fig. 3). These results indicate the ability of the EPF formulation to penetrate the cocoons and colonize the pupa.

Discussion
Based on the increasing interest in biological control, we identified and classified B. pseudobassiana RGM 1747 and compared its activity with that of other isolates from EPF-infected insects around the world that had already been sequenced. In Chile, B. pseudobassiana was isolated from P. gallicus and showed activity against L. botrana in this work. In addition, Gerding et al. (2008) reported that this EPF was assessed in A. mellifera to understand its potential adverse effects on this pollinizer. These authors showed no negative effect in bees and found that the fungus achieved control over V.
germanica [13]. Furthermore, the use of biological products in winter stages reduces the possibility of cross-activity with beneficial arthropods due to a decrease in their activity during winter because of low temperatures and a lack of flowers. In Chile, there are no chemical and biological products for controlling the pupae during the winter. Therefore, the application of EPF in winter could be compatible with application of fungicides in spring within an IPM program for fungal disease control in grapes.
The in vitro formulation evaluation achieved 100% efficacy in L. botrana due to the occurrence of all the conditions required for the EPF to colonize the pupae. However, we considered it necessary to perform field trials under the temperature and humidity conditions of July. This trial reached an efficacy of 100% and was considered representative of the behavior of the fungi because under the environmental conditions and oscillations in temperature and humidity that occurred in this trial, hypha and conidia were observed between the sutures of the pupae, and the viability of the fungi was assessed and confirmed in cultures of different pupae from the field.
On the basis of the results of the field trial, we assessed an assay conducted in urban zones during August. The primary barrier to the EPF is the cocoon, which provides natural protection against pathogens and arthropods. In this case, the efficacy reached 51% using only one application. The other treatments in which we performed two and three applications did not show significant differences (resulting in 15% and 13% efficacy, respectively), but there was a significant difference compared with the first treatment. In addition to the decrease in efficacy caused by the cocoon in all the treatments, another possible explanation for this difference is that during the first application, the EPF consumes most of the carbon sources and proteins from the pupae and the formulation, but after seven dpa, opportunistic fungi such as Aspergillus sp. Penicillium sp. and Rhizopus sp. begin to appear (observations of urban samples). These fungi may have taken advantage of the components of the formulation and reduce the efficacy of the product.
In conclusion, we achieved an efficiency of 51% with one application of EPF formulation in urban areas to control the pest during its winter diapause. Additionally, the formulation is useful in fruit crops because pollinators and antifungal chemical applications are not encountered in the field. Finally, possible annual applications in urban and rural zones contribute to the control of L. botrana in the country in combination with other IPM strategies.

Limitations
To improve the formulation, it will be necessary to examine its components and different concentrations thereof to increase their efficacy. We will need to perform a series of urban trials over time to optimize the use of the EPF.