Abstract: Background and Objective: Rice meal moth Corcyra cephalonica (C. cephalonica) and Red flour beetle Tribolium castaneum (T. castaneum) are the most common and cosmopolitan stored grain insect pests responsible for significant losses to stored wheat all over the world. The present study encompassed the laboratory determination of pathogenicity and virulence of entomopathogenic fungus, Beauveria bassiana formulation (Racer BBTM), against rice meal moth, Corcyra cephalonica larvae and red flour beetle, Tribolium castaneum adults. Materials and Methods: Three concentrations of B. bassiana i.e., 3×107, 6×107 and 9×107 conidia kg–1 of wheat grains were used. Mortality data was recorded at different post-treatment time intervals and were subjected to the analysis of variance followed by the separation of treatment means by least significant difference (LSD) test at standard significance level (α = 0.05). Results: Maximum larval and adult mortality was recorded, respectively for C. cephalonica (79.67%) and T. castaneum (70.00%) at higher concentration (9×107 conidia kg–1 wheat) of entomopathogenic fungus at 21 days post-treatment. Remaining concentrations of the fungus gave mortalities less than 50% for the treated stored grain pests. Conclusion: This study suggested that B. bassiana had the ability to be used as an effective biocontrol agent for the control of stored grain insect pests such as C. cephalonica (rice meal moth) and T. castaneum (red flour beetle).
INTRODUCTION
Grain storage is an essential and common activity that permits food grains to be utilized from weeks to months and years after crop harvest1. As production of grain crops is seasonal and their consumption is continuous throughout the year, their safe storage is inevitable in order to maintain their grains quality and quantity2. However, cereals, pulses and oilseed crops are normally infested by various insect pests which cause substantial quantitative and qualitative losses3,4. The most common insect pests of stored grain crops in Indo-Pak regionarerice meal moth (Corcyra cephalonica Staint.), red flour beetle (Tribolium castaneum Herbst), lesser grain borer (Rhyzopertha dominica Fab.) and angoumois grain moth (Sitotroga cerealella Olivier)5,6. Estimated about 10-40% losses were recorded in stored grain crops only due to the infestation of insect pests in developing countries7,8.
Among major stored grain insect pest species, C. cephalonica (Lepidoptera, Pyralidae) is a serious pest of wheat, rice, maize, groundnut, cocoa beans, sorghum and cotton9,10. Larval stage of these moths is the most damaging stage causing direct damage by feeding and indirect damage by spoiling stored grains with their fecal matter and silken cocoons11. Similarly, T. castaneum (Coleoptera, Tenebrionidae) is a cosmopolitan and polyphagous stored grain insect pest of stored grain crops8,12,13. The adults of this insect pestare not only responsible for the physical damage to grains but they also cause allergic diseases in human beings upon consumption of such infested grains14. Losses from 5-15% in grain weight incurred by C. cephalonica and T. castaneum have been reported during the storage of cereals, pulses and oilseed crops15,16.
As insect pests are one of the most significant and contemporary issues of storage sector all over the world, a number of synthetic chemicals have been registered and recommended for the control of stored grain pests in these countries including Indo-Pak region17,18. Although playing a vital role in lowering the insect pest infestations in stored grains, most of these synthetic pesticides are highly persistent and toxic16. Moreover, the indiscriminate and extensive use of these grain protectants (pesticides) have resulted in the development of pesticide resistance, resurgence of secondary pests and other environmental and human health hazards19-21.
Hence, there is a need to search for some alternative approaches for the control of these stored grain insect pests such as the use of biological control agents which have been as environment-friendly and safe pest control strategies22,23. For instance, a number of fungal species have been reported very effective against a wide variety of insect pests24-27. The entomopathogenic fungi have contact mode of action and usually grows through insect cuticle and penetrates into insect body, proliferates, produces toxins and finally causes death of the insect pest24,28,29. Many scientists have reported that optimum temperature for different entomopathogenic fungi ranges between 20-30°C30,31, therefore, their utilization for the control of stored grain insect pests is feasible and effective. Beauveriabassiana is one of the most widely used and effective entomopathogenic fungi against a large number of insect pest species including stored grain insects and mites11,32. The present study was aimed to evaluate the pathogenicity of a commercial formulation of B. bassiana against two major stored grain insect pests i.e., C. cephalonica and T. castaneum under laboratory conditions.
MATERIALS AND METHODS
The present study was conducted at the Grain Research, Training and Storage Management Cell, Department of Entomology, University of Agriculture, Faisalabad, Punjab, Pakistan during spring, 2017. Populations of T. castaneum and C. cephalonica were collected from the grain market of Faisalabad (Punjab, Pakistan) and were brought to the laboratory under cool conditions. Both insect pests were reared in sterilized clear plastic jars (1 L) in a controlled incubator (Sanyo MIR-254, Japan) at 30±2°C and 60±5% relative humidity. Sterilized wheat grains and wheat flour were used as a culture media for C. cephalonica and T. castaneum, respectively. The adults of C. cephalonica and T. castaneum were released on wheat grains (500 g) and fresh wheat flour (500 g), respectively. Jars were then covered with fine muslin cloth for aeration and tightened up by rubber bands to avoid the escape of insects. The larvae of C. cephalonica and adults of T. castaneum emerged from these stock colonies were used for further experiments.
Bioassays were conducted under laboratory conditions following complete randomized design (CRD) according to the protocol described by Dal Bello et al.33. The commercial formulation of entomopathogenic fungus Beauveria bassiana (Racer®BB1.15% WP, AgriLifeTM, Hyderabad, India) was evaluated against these insect pests. This formulation had been derived from two naturally occurring soil strains i.e., ATCC and NCIM 1216. Bioassays were conducted using three different concentrations of B. bassiana as independent treatments i.e., T1 = 3×107, T2 = 6×107 and T3 = 9×107 conidia kg–1 of wheat grains with three replications for each treatment. Sterilized food was supplied to both insect pests during the experimentation. Samples of 50 g of treated commodity for each concentration were taken into three separate sterilized plastic jars. Three jars with untreated food were set-up as control. Thereafter, one hundred active adults of T. castaneum and larvae of C. cephalonica were released in each jar separately and jars tops were closed with muslin cloth to ensure proper aeration. The treatments were kept in an incubator at 30±2°C and 60±5% relative humidity. With weekly intervals, the mortality of test insects was recorded at 7, 14 and 21 days post-release of insect pests. Dead insects having fungal growth on their body were removed from the jars and counted and considered dead as a result of infection caused by B. bassiana.
Statistical analysis: Of data was performed using software Statistic V. 8.1 (Analytical Software)34. Mortality data recorded for different concentrations at different post-treatment time intervals were subjected to the analysis of variance (one-way ANOVA), followed by the separation of treatment means by least significant difference (LSD)test at standard significance level (α = 0.05).
RESULTS
According to statistical analysis, there was significant difference for all treatments as compared to control treatment for all observation times (Table 1 and 2). After 7 days interval, a significant difference (p>0.05) was observed for each treatment in reference to control treatment. Maximum mortality of C. cephalonica was observed in treatment 3 (9×107 conidia kg–1 of grains). After 21 days, cumulative mortality percentages of C. cephalonica larvae were recorded as 79.67for treatment 3, 49.33 for treatment 2 and 36.67 for treatment 1 (Fig. 1).
Similar trend of mortality was found in case of T. castaneum adults. At all the time intervals, mortality of T. castaneum adults was significantly higher as compared to control treatment. Maximum mortality was observed for treatment 3 (9×107 conidia kg–1 of grains) suggested that higher was the B. bassiana conidial concentration, greater will be efficiency. Maximum mortality of T. castaneum for all treatments was observed at 21 days post-treatment. Cumulative mortality percentages of T. castaneum adults after 21 days were 70.03 for treatment 3, 49.67 for treatment 2 and 29.67 for treatment 1 (Fig. 2). Among both insect pest species, C. cephalonica larvae were more susceptible to B. bassiana formulation rather than T. castaneum adults because the highest mortalities of C. cephalonica larvae at all the exposure times and concentration rates were observed for this species (Fig. 1 and 2).
| Fig. 1: | Mortality ± SEM (%) of Corcyra cephalonica 3rd instar larvae recorded for three different concentrations of Beauveria bassiana at time intervals. T1 = 3×107 conidia kg–1 grains, T2 = 6×107 conidia kg–1 grains, T3 = 9×107 conidia kg–1 grains, TC = Control grains food. For each time interval, columns bearing different alphabet letters are significantly different from other treatments |
| (LSD test at p = 0.05) | |
| Table 1: | Analysis of variance for percent mortality in C. cephalonica larvae bioassayed against different concentrations of B. bassiana at different exposure periods |
| One-way ANOVA (p<0.05) taking concentration as factor | |
| Table 2: | Analysis of variance for percent mortality in T. castaneum adults against different concentrations of B. bassiana at different exposure periods |
| One-way ANOVA (p<0.05) taking concentration as factor | |
Average mortality was recorded as 55 and 49% for C. cephalonica and T. castaneum, respectively.
DISCUSSION
Fungal mycelial growth on the tested insect body demonstrated that the target insects died due to the pathogenicity incurred by B. bassiana.
| Fig. 2: | Mortality ±SEM (%) of Tribolium castaneum adults recorded for three different concentrations of Beauveria bassiana at time intervals. T1 = 3×107 conidia kg–1 grains, T2 = 6×107 conidia kg–1 grains, T3 = 9×107 conidia kg–1 grains, TC = Control grains food. For each time interval, columns bearing different alphabet letters are significantly different from other treatments |
| (LSD test at p = 0.05) | |
In both bioassays, mortality was found directly proportional to the conidial concentration of B. bassiana formulation and to the exposure time intervals. The highest conidial concentration of B. bassiana, i.e., 9×107 conidia kg–1 grains, proved most effective against both tested insect pests than other lower concentrations. Larvae of C. cephalonica were more susceptible to B. bassiana infection as compared to adults of T. castaneum. It is most probably due to the more delicate and less sclerotized larval cuticle of C. cephalonica as compared to hard and rigid body wall of T. castaneum adults with only the intersegmental membranes as penetration sites rendering the larvae more susceptible to B. bassiana infection and proliferation35.
The application of B. bassiana formulation caused mortality more than 79% in C. cephalonica larvae and about 70% in adults of T. castaneum. Similar results for B. bassiana causing maximum mortality in the larvae of C. cephalonica at a concentration of 2.02×108 conidia mL–1 11. However, this mortality recorded for both insect pests is less than observed for a B. bassiana isolate bioassayed against S. oryzae, R. dominica and T. casteanesum 36. In another study conducted in which isolation of B. bassiana (BbGc and BbPs) and Metarhizium anisopliae (MaPs) caused mortality among larvae of C. cephalonica as a factor of dose and time37. Likewise, the efficacy of B. Bassiana isolates (ITCC No. 6628, ITCC No. 6645 and B. NCIPM) against C. cephalonica and found effective with mortality percentage of 31-98 achieved within 3 weeks of application38. Similar results are reported by Khashaveh et al.39, who revealed that B. bassiana is very efficient against T. castaneum, S. granarius and O. surinamensis exhibiting an average mortality of 64.99, 88.33 and 78.31%, respectively after 15 days of exposure to a concentration of 1 g kg–1 wheat or 2×109 conidia g–1 wheat which is higher than the application rates of 3×107 to 9×107 conidia kg–1 of wheat grains used in this study. High dose rates used by Khashaveh et al.39 would probably be responsible for a high mortality of test insects within a short exposure period. Hence, the results of this study revealed that the effectiveness of biocontrol fungi such as B. bassiana can be achieved even at lower conidial concentration by providing proper conditions. Although Padin et al.15 recorded similar findings that B. bassiana produced maximum mortality (55%) in T. castaneum (adults) under laboratory conditions after 21 days of inoculation, there was no significant difference found in this study regarding the percent weight loss among treated and untreated wheat grains infested by T. castaneum15. However, the results of this study corroborate the findings of many previous studies which have revealed the effectiveness of various isolates of B. bassiana against various insect pests species either alone or in combination of diatomaceous earth and synthetic insecticides either under laboratory or field conditions15,20,23,27,40-46.
CONCLUSION
In brief, B. bassiana has the ability to be used as an effective entomopathogenic fungus and to be utilized for the control of stored grain insect pests such as C. cephalonica (rice meal moth) and T. castaneum (red flour beetle). In grain storage, application of commercial formulations of B. bassiana Racer® BB 1.15% WP could be recommended as an alternate and environmental-friendly grain protectant.
SIGNIFICANCE STATEMENTS
Application of microbial biocontrol agents against stored grain insect pests can be an effective and environment-friendly alternate strategy. Most of entomopathogenic fungal formulations such as of Beauveria bassiana are more selective to target pests and less toxic to humans and hence can be efficiently utilized in post-harvest pest management programs. This laboratory study demonstrated the effectiveness of a commercial formulation of B. bassiana (Racer® BB 1.15% WP) against two most economic stored grain insect pests, i.e., rice meal moth (C. cephalonica) and Red flour beetle (T. castaneum).
ACKNOWLEDGMENT
The authors are grateful to Dr. Venkatesh Devanur, Managing Director, AgriLifeTM, SOM Phyto-pharma (India) Limited, Medak Dist. (Hyderabad) AP, India for providing the bio-insecticide B. bassiana (Racer BBTM). The authors are grateful to Dr. John Collins for his valuable comments and proof-reading of the manuscript.