Three strains of the entomopathogenic fungus Beauveria bassiana Vuill. were tested for pathogenicity against adults of Metamasius (=Cactophagus) spinolae Gyllenhal. M. spinolae is an important pest of cactus plants (Opuntia ficus indica), which are used as a food crop and to avoid erosion in Mexico. After inoculation in a spray tower, M. spinolae adults were susceptible to B. bassiana at concentrations of 1 × 108 conidia per milliliter. Female mortality was steadily higher than male mortality for all isolates. One of the three isolates caused significantly higher mortality (82%) in females, whereas male mortality was the same for all isolates. These results indicate for the first time the possible use of B. bassiana as biocontrol agent against this insect pest.
The cactus (Opuntia ficus indica) is a very important plant in Mexico, especially in semi-arid regions where few crops can be cultivated (Vigueras & Portillo 2001). The cactus weevil Metamasius (= Cactophagus) spinolae Gyllenhal is a limiting factor for commercial production of Opuntia spp. (Baddi & Flores 2001; Flores-Valdez 2001). The larvae, 25-35 mm long, tunnel into apparently healthy cactus pads, from joint to joint, where they cause disintegration of the cactus tissues (Granados & Castañeda 1991). Pupation takes place in the hollowed stem of the plant, which provides a protected environment for pupal overwintering. Adults can be found from May through September; they are relatively large weevils at 23-36 mm in length. They feed on the margins of the young pads causing additional damage. Control strategies for M. spinolae rely to a large extent on the use of chemical insecticides (Borrego & Burgos 1986; Baddi & Flores 2001). However, a biologically based control strategy that can be used on M. spinolae would be highly desirable.
Microbial insect pathogens may offer a strategy for use as localized biopesticides, but little is known about natural microbial enemies of M. spinolae. Several products based on Beauveria bassiana Vuill. are available for managing adults of other pest insect species, such as Hypothenemus hampei Ferrary (coffee berry borer), and various species of Curculionidae (Adane et al. 1996; de la Rosa et al. 1997; Rice & Cogburn 1999). So far, however, no entomopathogenic fungus has been evaluated for the control of M. spinolae. In this study we assess the susceptibility of adult M. spinolae to isolates of B. bassiana under laboratory conditions.
Materials and Methods
Biological Material
Isolates from the Insect Pathology Collection (Colegio Postgraduados Texcoco, Mexico) were used in bioassays with M. spinolae adults (Table 1). All isolates were cultured on Sabouraud dextrose agar (SDA) with yeast extract (2g/l) (SDA-Bioxon, Mexico) and incubated at 27°C, 70% RH for 2-3 weeks until conidia were produced. For bioassays, conidia were harvested into sterile 0.01% Tween 80 solutions to a final concentration of 1 × 108 conidia/ml. Conidia viability was determined by serial dilution plating onto SDA and colony forming units were counted six days after incubation at 27°C.
For bioassay experiments, M. spinolae adults were collected directly from cactus pads in Morelos, Mexico. They were sexed, held separately in screen cages (32 × 32 × 32 cm, 1.5 mm mesh size). Adults were stored in fresh cactus pads at 23 ± 1°C, 33 ± 5% RH, and a 12:12 (L:D) photoperiod, before use in bioassays.
Laboratory Test
For inoculation, M. spinolae adults were placed into 90-mm plastic Petri dishes. Groups of 10 adults of the same sex were stored at 4°C for 15 minutes to anesthetize them. The adults were exposed to a B. bassiana isolate by applying 10 ml of a 1 × 108 conidia/ml suspension in a spray tower (Altre et al. 1999) with constant pressure (0.7 kg/cm2). On average, each insect received 1 × 108 conidia, while control adults received 0.1% Tween 80. The density of conidia on the insects was estimated by counting the number of conidia in a sample area on five agar disks (1 cm dia.) placed in the Petri dish during inoculation.
After application, the adults were placed in separate screen cages, fed with fresh untreated cactus pads and kept for 16 days at 23 ± 1°C, 33 ± 5% RH, and a photoperiod of 12:12 (L:D) h. The bioassays were repeated three times. The adults were examined for mortality every 48 h for 16 d. Dead insects were removed and incubated at 25°C and 90% relative humidity to check for B. bassiana infection by direct visual observation.
Statistical Analyses
Analysis was performed on mean cumulative percentage mortality data. After correction for control mortality (Abbott 1925), percentage mortality and average time to death were subjected to a one-way analysis of variance (SAS Institute 1999). Treatment effects were tested by the Fisher protected least significant difference (LSD) test (Sokal & Rohlf 1995).
Results and Discussion
B. bassiana isolates were pathogenic to M. spinolae exposed to 1 × 108 conidial suspensions (Table 2). However, the three B. bassiana isolates used for this report differed in their virulence to M. spinolae adults. For all three, the females were more susceptible to fungal infection than males (F = 20.49; df = 1; P = 0.00005). B. bassiana isolate Bb88 was more virulent against females than Bb4 or Bb113 isolate (F = 5.55; df = 2; P = 0.04). Fungal virulence of the three isolates were similar against males (F = 1.18; df = 2; P = 0.36;Table 2). All females exposed to the fungi died within 8.5 to 10.6 days (F = 0.45; df = 2; P = 0.65) and males died within 7.5 to 11 days (F = 1.06; df = 2; P = 0.40). There were no significant differences in mean time to death (Table 2). Most cadavers supported fungal sporulation, indicating successful infection and the ability of the isolates to sporulate under low humidity. An increase in female mortality was noted by day four after inoculation and female cumulative mortality was steadily higher than male mortality for all isolates (Fig. 1).
Conidial dosages applied per mm2 ranged from 780 ± 43 (Bb4) to 1341 ± 86 (Bb113) (Table 2). The Bb4 dosage was lower than Bb88 and Bb113 dosages (F = 21.07; df = 2; P = 0.0003). Thus, the low virulence of the Bb4 strain could be related to the lower conidial dosage applied. However, previous work showed that the same strain was highly virulent to its original host H. hampei (de la Rosa et al. 1997). Hence, these two species appear to have differing susceptibility to Bb4. Similar comparisons for the M. spinolae isolate (Bb113) are not available.
The data in this report also demonstrate that it is feasible to contaminate adults by spraying conidia, and that most cadavers supported fungal sporulation. This may be important for any control strategy aimed at attracting beetles to fungus contaminated traps, and subsequent transfer to adults or larvae in cactus pads and tunnels. Fortunately, there is evidence of an aggregation pheromone (Tafoya et al. 2003) which can be used to attract the males to a contaminated trap with fungi and possibly transfer the infective conidia to adults or larvae in tunnels. Similar strategies of autodissemination have been developed for other insects (Furlong et al. 1995).
The results presented in this study demonstrate a pathogenic effect of B. bassiana on M. spinolae adults under laboratory conditions. To our knowledge, this is the first report on infection of this pest insect with an entomopathogenic fungus. Further research is necessary to determine the effectiveness of B. bassiana under field conditions and to examine its potential impact on non-target species.
Acknowledgments
The study was funded by a grant (34769-B) and a scholarship to Felipe Tafoya from Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico.
