The white grub Laniifera cyclades Druce of prickly pear cactus or nopal is a pest that limits the commercial production of Opuntia. The gregarious larvae perforate the cladodes devouring the inner part, thereby forming large galleries until reaching the central axis of the plant; during their movement through the inner part of the cactus, the larvae make orifices to the exterior to expel their excrements. In this investigation, the virulence of strains BbZ3 and BbZ4 of the entomopathogenic fungus Beauveria bassiana was determined by introducing infested cadavers of Galleria mellonella L. through the orifices on the stem pads of the nopal plant. Both stains of B. bassiana were highly pathogenic causing 100% mortality in the larvae of L. cyclades inside the nopal cladodes in the greenhouse as well in the field. BbZ3 was more virulent with a LT50 of 5.1 d in the greenhouse and 6.4 d in the field, while the LT50 of BbZ4 was 6 and 7.5 d, respectively. The application of larval cadavers of G. mellonella infested with the fungus B. bassiana was an effective control strategy against larvae of L. cyclades.
The prickly pear cactus or nopal Opuntia spp. is one of the most important plants of Mexico, especially in semi-arid and arid regions where few crops can be cultivated. The main production of the cactus is for fruits and vegetables for human consumption, forage for livestock, and for industrial products such as cosmetics and dyes (Vigueras & Portillo 2001). The white grub Laniifera cyclades Druce of nopal is one of the pests limiting the production of Opuntia spp. (Badii & Flores 2001). The adults deposit eggs in groups of 30 to 50 on the cladodes, and the gregarious larvae perforate the cladodes devouring the inner part and gradually penetrating the tissue forming large galleries until reaching the central axis of the plant, where inside they pass through larval stages and pupate. During their movement through the inner part of the nopal, the larvae make orifices to the exterior in order to expel their excrements, forming on the ground what growers call “little mountains of rice.” These wastes serve as signs for detecting the presence of the pest, which enables destroying larvae mechanically, but often a major part of the plant must be destroyed. Strategies for control of L. cyclades recommended by governmental institutions consist in application of chemical insecticides through the orifices made by the larvae (Saenz 1998), thereby contaminating the whole plant and its fruits. Thus, an alternative biological control would be of great utility. Microbial pathogens offer possibilities as biopesticides, but little is known about the microbial enemies of L. cyclades. Beauveria bassiana Vuillemin is the entomopathogenic fungus most widely distributed in the world, and it infects insects in tropical, temperate, humid, and desert areas (Zimmermann 2007). Various products based on B. bassiana are commercially available for controlling insect pests of agricultural importance, such as the coffee berry borer Hypothenemus hampei Ferrari and various species of Curculionidae (Adane et al. 1996; de la Rosa et al. 1997). Lepidoptera are also important hosts of this fungus, including several species of agricultural importance (Abdel-Razek et al. 2006). One factor that affects the efficacy of B. bassiana is sunlight; persistence and infectivity are reduced within a few minutes after exposure to sunlight (Fargues et al. 1996). Reduced environmental humidity also affects the efficacy and survival of the fungus because the most effective germination of the spores on the insect cuticle requires a relative humidity (RH) range of 92 to 100%, but there are reports of B. bassiana infection at 60-70% RH (Zimmermann 2007). The relative humidity of the semi-arid region in the municipality of Noria de Ángeles, Zacatecas is on average 49%, but the use of infested cadavers might afford protection to B. bassiana. Koppenhofer et al. (1997) and Shapiro-Ilan et al. (2001) reported that entomopathogenic nematodes could survive under dry conditions for long periods of time if they remained inside cadavers of host insects. Larvae of the wax moth Galleria mellonella L. have been used as cadavers infested with the entomopathogenic nematode Heterorhabditis bacteriophora Poinar for controlling the sweetpotato weevil Cylas formicarius (Fabricius) (Jansson & Lecrone 1994). Cadavers of G. mellonella infested with B. bassiana and placed in the holes of nopal plants infested with L. cyclades could prevent exposure to sunlight and protect the fungus from the low RH outside the plant. The aim of this investigation was to evaluate the pathogenicity of the strains BbZ3 and BbZ4 of Beauveria bassiana to L. cyclades after introducing infested cadavers of G. mellonella into the excretion orifices in the nopal stem pads, under greenhouse and field conditions.
Materials and Methods
Biological Material
Third instars of L. cyclades to be used in the bioassays in the greenhouse were collected directly from nopal plants in Noria de Ángeles, Zacatecas, Mexico. These were transported to the Entomology and Biological Control Laboratory of the Academic Unit of Agronomy at the Universidad Autónoma de Zacatecas, maintained for a week at 23 ± 1°C, 33 ± 5% RH in Petri dishes, and fed daily with pieces of fresh nopal. Holding the larvae is this manner enabled us to rule out the presence of disease.
Strains BbZ3 and BbZ4 of B. bassiana were originally isolated from soil samples from 2 orchards of “nopal tunero” in the municipalities of Noria de Ángeles and Pinos, Zacatecas, Mexico, and they are part of the collection of entomopathogenic fungi of the Academic Unit of Agronomy at the Universidad Autónoma de Zacatecas. Pathogenicity bioassays were conducted on third instars of L. cyclades. The isolated strains of B. bassiana were grown in Sabouraud dextrose agar (SDA) with yeast extract (2g/L) (SDAY) at 24 ± 1°C. After 3 weeks, the conidia were collected in 10 mL sterile distilled water with 0.01% Tween-80 to reduce surface tension. Conidia were counted in a Neubauer chamber and dilutions were made to a final concentration of 1 ×108 conidia/mL. Whatman No.1 filter paper was placed on the bottom of 40 Petri dishes and 10 fifth instars of G. mellonella were inoculated in each Petri dish with 1 mL spray of a conidial suspension of each fungal isolate. Two weeks later, 100% of the larvae were dead and infested, and each of the cadavers of G. mellonella was utilized for application of the fungus during the bioassays with L. cyclades in the field and in the greenhouse. The method for applying the fungus consisted of the introduction of a G. mellonella cadaver into the orifices made by the L. cyclades larvae in the nopal plants. In the greenhouse, the orifices were made manually and larvae of L. cyclades were inserted into the orifices. The conidial density applied per orifice was estimated by vortexing an infested G. mellonella larva with conidia in 10 mL of 0.01% Tween-80 solution for 3 min, and then counting spores and colony forming units (Tang & Hou 2001). The dose employed was 1013 conidia or CFU per cadaver.
Bioassays in the Greenhouse
For the inoculation of fungus in the greenhouse, 30 healthy nopal plants were selected, and in 1 cladode per plant an orifice was made that was 20 cm long and 1 cm in diameter, similar to those made by the larvae in the field. Two days later, 10 third instars of L. cyclades were introduced, and after a week the larvae were established as evidenced by the external mounds of excrement. At that time an infested cadaver of G. mellonella was introduced, so that L. cyclades larvae had to crawl over the wax moth larva when exiting to expel excrements. Starting on the fourth day, mortality was recorded daily. On ninth d, larvae were extracted from all the plants and placed in Petri dishes to continue to monitor them for mortality. For control treatments, 10 plants were selected that had mounds of excrement, and maintained without introduction of wax moth cadavers.
Bioassays in the Field
In an orchard of nopal of 2,500 m2 situated in the municipality of Noria de Ángeles, Zacatecas, 30 plants with orifices and “little mountains of rice” indicating the presence L. cyclades larvae were selected. Each treatment was evaluated in 10 isolated plants. A B. bassiana-infested cadaver of G. mellonella was introduced into the natural excretion orifices, and starting on the fourth day and continuing to the eighth day, the diseased larvae of L. cyclades that exited to die outside of the colony were collected. On the ninth d all remaining larvae were extracted from the plant and held in the laboratory in Petri dishes to continue to monitor mortality. Dead larvae were placed in moist chambers to determine if sporulation of the fungus occurred. For control treatments, 10 plants showing evidence of excrement from larvae of L. cyclades were selected but were not inoculated with a waxmoth larval cadaver.
Statistical Analyses
The percentages of mortality were arcsine transformed and analyzed in a complete randomized block design (each repetition considered as a block), and analysis of variance (ANOVA) was utilized, followed by means separations by Tukey’s test (P < 0.05) (SAS Institute 1998). The means of mortality are presented with the original data. The LT50 was determined by probit analysis in the program ED50 plus v1.0.
Results
The strains BbZ3 and BbZ4 of B. bassiana produced 100% mortality in the larvae of L. cyclades inside the cladodes of nopal by d 9 in the greenhouse and by d 10 in the field. Daily mean mortality data from d 4 to d 12 in the laboratory and field are presented in Table 1and Table 2, respectively. The data for mortality in the greenhouse indicated that by d 4, and continuing through d 10, strain BbZ3 of B. bassiana was statistically more pathogenic than strain BbZ4 to the larvae of L. cyclades (F = 115.89; df = 2, 27; P = 0.0001) after exposure to 1 infested larva of G. mellonella. By d 11 and 12 in the greenhouse, essentially 100% mortality was caused by both strains of the fungus. Similarly, in the treatment in the field, strain BbZ3 was more pathogenic than BbZ4, with a statistically significant difference starting at d 5 (F= 174.87; df = 2, 27; P = 0.0001) and continuing through d 11 (F = 1699.68; df = 2, 27; P = 0.0001). On d 12 in the field both strains of the fungus had caused 100% mortality in L. cyclades larvae. All the cadavers of L. cyclades gave rise to sporulation of the fungus, indicating that death was caused by infection and that the strains were capable of sporulating under existing humidity conditions. The LT50 was determined by linear regression in which percentage of mortality with each treatment was plotted against time for the days of evaluation. The LT50 occurred at 5.1 and 6.4 d in the greenhouse and field, respectively, for strain BbZ3, and at 6.0 and 7.5 d, respectively, in greenhouse and field for strains BbZ4.
Discussion
Strains BbZ3 and BbZ4 of B. bassiana infesting cadavers of G. mellonella larvae were pathogenic to larvae of L. cyclades when the cadavers were placed inside nopal plants. The LT50 value was slightly lower for strain BbZ3 than for strain BbZ4, but both eventually killed 100% of L. cyclades larvae within 11 to 12 d in the greenhouse and field, respectively. Samuels et al. (1989) from their study noted that an LT50 longer than 14 d indicated non-pathogenicity. Sprenkel & Brooks (1975) demonstrated that the conidia of Nomuraea rileyi could remain infective on the surface of cadavers of the tobacco budworm Heliothis virescens (F.) for at least 256 d, causing epizootics and reduction in the populations of Lepidoptera pests in the soybean crop. The gregarious larvae L. cyclades have favorable conditions to develop induced epizootics inside the plants. Tanada & Kaya, (1993) argue that Infection and sporulation of several entomopathogenic fungi are influenced by environmental factors, especially temperature and humidity, and to lesser extent photoperiod. In this study, we have demonstrated that the introduction of sporulating G. mellonella cadavers into nopal plants infested with L. cyclades is an effective strategy to maintain pathogenicity of the fungus under the semi-arid conditions of the region. Movement of L. cyclades larvae over the infested G. mellonella cadavers may be an important factor in promoting infection with pathogens. For example, Baverstock et al. (2005) demonstrated that aphid movement could indirectly influence transmission of Pandora neoaphidis (Remaudière & Hennebert) to Acyrthosiphon pisum (Harris) by allowing the aphids to come into contact with the conidia. Shimazu (2004) noted that adhesion of dry conidia to the pine borer Monochamus alternatus from contact provided effective control of the insect. Our results demonstrate that it is easy to infect larvae of L. cyclades inside nopal plants with inoculation of B. bassiana sporulating in cadavers of G. mellonella larvae, and that it can be useful in controlling L. cyclades. Meyling et al. (2006) reported that, even though aphids Microlophium carnosum (Bukton) are likely to only contact inoculum briefly, they apparently become contaminated upon such encounters and distribute the inoculum on the host plant. Although the method we used of introducing an infected cadaver into an opening in nopal plant to control L. cyclades larvae is labor intensive, the use of infected insect cadavers may be a control strategy for other pests that develop inside cladodes such as Cactoblastis cactorum Berg (Lepidoptera: Pyralidae), the zebra worm Olycella nephelepasa (Lepydoptera: Pyralidae), and Moneilema variolaris Thompson (Coleoptera: Cerambycidae). Further investigations are necessary to evaluate the effectiveness of additional B. bassiana strains under laboratory and field conditions and to examine the potential impact on other species.
Acknowledgments
We thank Albert Leyva for assistance in the preparation of the manuscript and José Hernández Martínez for statistical support.
References Cited
Table 1.
Mean cumulative percent mortality ±SD of Laniifera cyclades larvae exposed to larval cadavers of G. mellonella infested with the fungus B. bassiana in the greenhouse.
Table 2.
Mean cumulative percent mortality ±SD of Laniifera cyclades larvae exposed to larval cadavers of G. mellonella infested with the fungus B. bassiana in the field.
