The application of enzymatic extracts and conidia of Beauveria bassiana in Metamasius spinolae and Cyclocephala lunulata was evaluated. The variables were mortality and time of death. In M. spinolae, mortality with extracts 29%, conidia 27% and the combination of both 31%, all had a time of death of four days. Although with different symptoms, used enzymatic extracts: contraction and softening of the joints; by conidia: mycelium in the joints; in the combination of conidia and enzymatic extracts: abundant aerial mycelium. In C. lunulata, 100% mortality in all treatments; Time of death: enzymatic extracts and extracts with conidia 1.2 days; conidia 2.8 days. Symptoms were different, enzymatic extracts: melanization and internal tissue lysis; enzymatic extract and conidia: mycelium emerged and melanization; conidia: mycelium emerged. Enzymatic extracts showed insecticidal activity in M. spinolae and C. lunulata. These results suggest the potential of enzymatic extracts as biocontrol agents to improve the use of entomopathogenic fungi.
Metamasius spinolae (Gyllenhal, 1838) (Coleoptera: Curculionidae), is a main pest of the cacti crop (Opuntiasp) in Mexico. It reduces crop yield [
An isolate (11) of B. bassiana was cultured in potato dextrose agar (PDA) at 25˚C for 15 days. The spores were suspended in a solution of Tween 80 (0.05%) in sterile distilled water and stored at 4˚C.
Solid-state fermentation of B. bassiana was performed in 250 ml Erlenmeyer flasks. Culture medium was prepared as described by Barranco et al. [
A solution Tween 80 (0.05%) was added to a solid-state fermentation from 15 days in sterile distilled water at a ratio of 1:1 (w/v). The mixture was homogenized, filtered and the suspension was stored at 4˚C in sterile tubes. The conidia were counted using a hemocytometer (Neubauer, United States Patent PLANE Charles A. Neubauer, Baltimore, Md, 1970). The suspension was adjusted to a concentration of 1 × 108 conidia mL−1 [
Distilled water was added to a solid-state fermentation for five days at a ratio of 1:1 (w/v). The mixture was homogenized, pressed and centrifuged at 10,000 rpm for ten minutes at 4˚C. The supernatant was filtered through a membrane Millipore of 0.45 µm and the extract was stored at 4˚C in sterile tubes.
For the enzyme assay, the substrate was an emulsion of olive oil and gum arabic at 10% (1:1), 1 mL of this substrate, 2 mL of phosphate buffer 50 mM, pH 6.8 and 1 mL of crude enzyme extract were mixed. The reaction mixture was incubated one hour at 37˚C under constant stirring. The reaction was stopped by adding 4 mL of a solution of acetone ethanol 1:1 containing 0.09% of phenolphthalein. Enzyme activity was determined by titrating free fatty acids using 5 mL of NaOH [
Protease assay: 1) subtilisin (Pr1) succinyl-alanina 2 proline-phenylalanine-p-nitroani- line (Sigma-Aldrich, Inc. 3050 Spruce Street , St. Louis , USA ); 2) trypsin (Pr2) Benzoyl- phenylalanine-valine-arginine-p-nitroaniline (Sigma-Aldrich, Inc. 3050 Spruce Stree t , St. Louis , USA ). Reaction mixture: 0.05 mL of substrate (1 mmol), 0.85 mL of buffer Glycine-NaOH pH 8.5 and 0.1 mL of crude enzyme extract were mixture. The mixture was incubated at 50˚C for one hour and the reaction was stopped by adding 0.25 mL of 30% acetic acid. The mixture was allowed to stand 15 min on ice, and then centrifuged at 5000 rpm for 5 min at 4˚C. It was read at 410 nm [
0.5 ml of chitin colloidal 0.5% (w/v) was used as substrate along with 0.5 mL of enzyme extract and 1.0 mL phosphate buffer citrate 0.2 M, pH 5.6. The mixture was incubated at 50˚C for one hour, and the reaction was stopped adding 3 mL of dinitrosalicylic acid (DNS). The mixture was heated in a water bath for five minutes. The solution was centrifuged at 10,000 rpm and read at 575 nm [
The substrate used was p-nitrophenyl-N-acetyl-β-D-glucosamine (Sigma-Aldrich, Inc. 3050 Spruce Street , St. Louis , USA ). 200 μL of a solution of 1.0 mg∙mL−1 substrate, 200 μL of the enzyme extract and 200 μL of citrate phosphate buffer 0.2 M, pH 5.6 were taken and incubated at 37˚C for one hour under constant stirring at 180 rpm; the reaction was stopped with 1 mL of 0.02 M NaOH. It was read at 400 nm [
Adults of M. spinolae were collected in San Juan Tepenahuac, Milpa Alta, México, and moved to the insectarium laboratory of the Xochimilco Metropolitan Autonomous University (UAM-X). They were kept individually in plastic cups 1 L. They were fed 20 g of spiny tender cactus, changing every two days. The larvae of C. lunulata were collected on the lawn of the sports area of the UAM-X, by sampling five golds, on area of 50 × 50 cm2. The material was placed individually in plastic cups 1 L with 200 g of soil from the site of collection and were fed with a grass root ball of garden, changing according to the degree of wilting showed that each root ball. 20 larvae were separated until complete their development. With adults, taxonomic determination was made by Roberto Alejandro Terron Sierra from UAM-X. The individuals were acclimatized for seven days with a photoperiod of 12:12 L/D, 25˚C ± 2˚C and 50% RH in the bioclimatic chamber LUMISTELL®.
Inoculation. Adults of M. spinolae were immersed in each treatment for 10 s. In order to remove excess water, they were placed on absorbent paper for 10 s [
Experimental Design. Completely randomized, with six treatments: T1: negative control (0.05% Tween 80); T2: crude enzymatic extract; T3: 1 × 108 conidia; T4: conidia more crude enzymatic extract; T5: positive control (Bea Tron®) 5 × 108; T6: enzymatic extract more positive control. M. spinolae had 48 replications with a total of 288 insects per treatment and C. lunulata had 10 replications per treatment with a total 60 insects. The observations were made every 24 hours for seven days. The experiments were established in the bioclimatic chamber LUMISTELL® under the conditions described above.
Data were analysed with the software JMP® 8. The independent analyses were made for the bioassay in M. spinolae. Chi square tests to mortality rate and Kruskal-Wallis to average time death, because the model did not meet the assumptions of normality and homoscedasticity. In C. lunulata larvae, analysis of variance and post hoc multiple comparison of mean values were carried out by Tukey test. All experiments described in this paper were for triplicate. For all analysis, differences of means were considered significant at p values <0.05.
The enzymatic activities obtained of the crude extract of B. bassiana from five days correspond to lipases (820.83 U), subtilisin-type protease Pr1 (0.422 ± 0.077 U), trypsin-type protease Pr2 (0.095 ± 0.029 U), endochitinases (3.239 ± 0.382 U) and exoquitinases (2.41 ± 0.07 U). These enzymes are responsible for breaking the integument of insects. The production of the subtilisin-type protease (Pr1) and trypsin (Pr2) was high: Pr1 activity is 4.4 times greater than the activity of Pr2. This is the first report of the use of enzymatic extracts and conidia of B. bassiana together to reduce the time of death of M. spinolae and Clunulata under controlled conditions.
Mortality. M. spinolae: there are significant differences among treatments, χ2 (5) = 91.271, p < 0.001. The best results were obtained by the positive control treatment Bea Tron® with crude enzymatic extract (T6) with 77% mortality; 75% in the positive control Tron® Bea (T5), followed by the crude enzymatic extract (T2), conidia (T3) and conidia with crude enzymatic extract (T4) with 29, 27 y 31% (
Mortality. In larvae C. lunulata, the results showed significant difference, χ2 (5) = 46.614, p < 0.001, among treatments: crude enzymatic extract (T2), conidia (T3) and conidia more crude enzymatic extract (T4) had 100% mortality, the positive control with crude enzymatic extract (T5) had 90% and positive control (T6) had 50% (
Time of Death. M. spinolae: The results showed significant differences between treat- ments by applying the Kruskal-Wallis test: χ2 (5) = 78,548, p < 0.0001. In the treatments: crude enzymatic extract (T2), conidia (T3) and conidia more crude enzymatic extract (T4) observed that there were shorter times of death of four days on average. While, in treatments: positive control (Bea Tron®) (T5) and positive control (Bea Tron®) more crude enzymatic extracts (T6) were nine days on average (
Time of Death. C. lunulata: In this case, the results showed significant differences between treatments by applying analysis of variance (F (5, 298.84) = 35.93; p < 0.0001). The treatments: crude enzymatic extract (T2) and conidia more crude enzymatic extract (T4) caused the death of the larvae in a time of 1.2 days on average, Respect to
Treatment | Mortality (%) | Time (days 9) |
---|---|---|
Ti. Negative control (Tween 80 0.05%) | 0 | N.D. |
T2. Enzymatic extract | 29.17a | 3.87 ± 1b |
T3. Conidia 1 × 108 | 27.08a | 3.93 ± 1b |
T4. Enzymatic extract + Conidia 1 × 108 | 31.25a | 4.3 ± 1b |
T5. Positive control ( Bea Tron®) 5 × 108 | 75.00b | 9.22 ± 1a |
T6. Positive control + Enzymatic extract | 77.08b | 8.89 ± 1a |
N.D. It was not determined. Means signed by the same letter differ not significantly according to Tukey. N = 48.
positive control (Bea Tron®) more enzymatic extracts (T6) was in 1.7 days, these treatments showed reduced the time of death. The treatments conidia (T3) and positive control (Bea Tron®) caused death in a longer time with 2.8 and 8.8 days, respectively. In both bioassays, the negative controls (T1) not cause the death of any insect (
In entomopathogenic fungi, enzymes that degrade insect cuticle play an important role in initiating the infectious process [
Treatment | Mortality (%) | Time (days 9) |
---|---|---|
Ti. Negative control (Tween 80 0.05%) | 0 | N.D. |
T2. Enzymatic extract | 100a | 1.2 ± 0.4b,c |
T3. Conidia 1 × 108 | 100a | 1.2 ± 0.4b.c |
T4. Enzymatic extract + Conidia 1 × 108 | 100a | 2.8 ± 0.4b |
T5. Positive control ( Bea Tron®) 5 × 108 | 60b | 8.8 ± 0.057a |
T6. Positive control + Enzymatic extract | 90a | 1.7 ± 0.42b |
N.D. It was not determined. Means signed by the same letter differ not significantly according to Tukey. N = 10.
activity of Pr1 in relation to Pr2. These results are similar to those obtained by Dias et al. [
The insecticidal action was mainly due to softening of the integument by the action of enzymes contained in the crude enzymatic extract, an effect that allowed the germ tube to penetrate the structure more easily to finally reach the haemocoel and promote infection in less time. So, the enzymatic extracts of B. bassiana showed insecticide when applied topically to adult of M. spinolae, alone or together with conidia, which re- presents the first report for this pest. Enzymes have their specific action on the cuticle of each component. Lipases hydrolyze the ester bonds of lipoproteins, fats and waxes epicuticle [
The bodies of M. spinolae treated with conidia (T3) presented mycelium of B. bassiana, mainly on the coxa and intersegmental space pronotum and abdomen (
Use of enzymatic extracts with conidia of B. bassiana resulted in symptoms such as feeding activity inhibition: in three or four days the insect stops feeding and alterations in its locomotor system. Besides, the extracts could facilitate the emergence of mycelium, giving the fungus the greatest attributes in terms of persistence and spread. From
an agricultural point of view, this raises their viability in the field as it becomes a new source of infection to be dispersed by abiotic factors such as the air and the breeze or contamination between individuals [
Injection of the enzyme extract in larvae of C. lunulata caused a toxic effect. The enzymes Pr1 and Pr2 contained in the enzyme extract caused pigmentation of cuticles as a response of the immune system to the presence of foreign enzymes inside the bodies (
[
In larvae of C. lunulata, various pigmentary changes were observed on the tegument. Negative control larvae maintained their characteristic creamy white and soft texture and body (
In the bodies of larvae inoculated with conidia (T3), were observed changes in pigmentation to an orange color in the body. Tissue of the bodies also varied. It was observed with greater rigidity and later emerged mycelium (
The insecticidal activity of enzymes such as chitinases and proteases of B. bassiana has already been tested in some pests of agricultural importance when topically applied as a powder, it showed insecticidal activity on cotton aphid Aphis gossypii Glover [
The crude enzymes extract of B. bassiana obtained by solid-state fermentation have insecticidal potential to be used in the biological control of pests of agricultural importance, given that they are constituted by a mixture of lipases, proteases and chitinases, which caused a toxic effect on M. spinolae and C. lunulata. Also, to use it as an adjuvant with conidia reduced the time of death of the insects, opening new prospects for the development of bio-insecticides with less impact to the environment, because they are a sustainable option to replace and reduce the use of chemical insecticides. It is necessary to continue studies to assess the stability and effectiveness of the enzymatic extracts in other models and different biological states and perform histological studies of insects treated, to determine the effect of these biomolecules on insect’ physiology.
We are grateful to John A. Goolsby, Ph.D., Research Entomologist Biological Control and Integrated Pest Management of Ticks, Insects and Weeds. United States Department of Agriculture, Agricultural Research Service, for their valuable comments and correcting the manuscript.This research was support by Consejo Nacional de Ciencia y Tecnología (CONACYT), Lluvia de Carolina Sánchez Pérez beneficiary of a grant.
Sánchez-Pérez, L., Rodríguez-Navarro, S., Marín-Cruz, V.H., Ra- mos-López, M.Á., Ramos, A.P. and Barran- co-Florido, J.E. (2016) Assessment of Beauveria bassiana and Their Enzymatic Extracts against Metamasius spinolae and Cyclocephala lunulata in Laboratory. Advances in Enzyme Research, 4, 98-112. http://dx.doi.org/10.4236/aer.2016.43010