Entomopathogenicity Potentials of Molds Isolated from Soil and Plant Debris on Comparative Mortality of Bean Weevil (Acanthoscelides Obtectus)

Entomopathogenicity Potentials of Molds Isolated from Soil and Plant Debris on Comparative Mortality of Bean Weevil (Acanthoscelides Obtectus)

Aniekwu. C. C^1*; Agu, K. C.¹; Uwanta, L.I¹; Madubuko, S. E²; Eleh. E. E¹; Okafor. U. C.¹

¹ Departmental of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Anambra State,

² Department of Microbiology, University of Benin, Benin City.

*Correspondence Author

DOI: https://doi.org/10.51584/IJRIAS.2024.908018

Received: 22 July 2024; Accepted: 05 August 2024; Published: 02 September 2024

ABSTRACT

Entomopathogenic fungi, with their eco-friendly nature, have emerged as promising biocontrol agents for pest management. This study explores the entomopathogenic potential of various isolates, including Aspergillus niger, Aspergillus flavus, Rhizomucor spp., Fusarium spp., and Aspergillus terreus, obtained from soil and plant debris. Confirming their entomopathogenic properties through morphological characteristics, the study aligns with previous research findings on Fusarium spp. and Aspergillus sp. isolation from diverse sources. In entomopathogenic assays against the Beans Weevil, all tested isolates exhibited efficacy, with Rhizomucor spp. demonstrating rapid and sustained mortality rates, reaching 100% within the initial day. Fusarium spp. displayed a cumulative impact, reaching full efficacy by the fourth day. Aspergillus terreus and Aspergillus flavus exhibited robust potential, achieving 100% mortality by the fourth day, while Aspergillus niger displayed slightly delayed onset but reached full efficacy by the third day. These results parallel findings on the effectiveness of Aspergillus species against various insects. Comparative analysis against conventional insecticides revealed entomopathogenic fungi’s superior efficacy, with faster mortality rates than different concentrations of the insecticide. The water control group exhibited a gradual weevil mortality rate, emphasizing the natural course of mortality. The insecticide treatments, even at lower concentrations, consistently outperformed the water control group, highlighting the nuanced relationship between insecticide concentration and efficacy.

 Key words: Entomopathogenicity, Fungi, Soil, Comparative mortality, Bean weevil

INTRODUCTION 

The Bean Weevil, Acanthoscelides obtectus (Say) commonly known as bruchids is a significant pest of legumes, especially beans, (Rugumamu, 2014: Nta et al.,2019). They are light olive coloured and mottled with dark brown or grey reddish legs. They are widely distributed in almost all continents. The bean weevils are granivores, seen in stored products and typically infest various kinds of bean species particularly the species Phaseolus vulgaris, where they live for most of their lives inside the seed feeding on the seeds and leaving perfectly round holes in the beans. This reduces the food value as well as the germination potential of the seeds (Edet et al., 2011), infestation of beans by A. obtectus begins in the field in drying beans pods and continues in the stored beans. They cause major losses in the field before harvest as well as in storage and has been classified as a primary pest with status 4, “destructive pest – a major pest rapidly capable of extensive damage”(Kljajić et al., 2022). Seed losses can range from 7–40%, which means 1.59–9.12 million tons of damaged seeds each year in the world caused by bruchid. This equates to a loss of 1.59–9.12 million tons each year in the world (Mbogo et al., 2009).

Chemical control is the most common practice of managing bean weevils, (Sağlam et al., 2022), which is traditionally achieved with pyrethroids, organophosphates, and the fumigant phosphine (PH₃) and other synthetic insecticides (Lopes et al., 2018).  The increased use of chemical pesticide in the control of vector filthy insects has prompted some concern over the adverse effect of these chemical substances on living organisms and their environment. The harmful nature of these synthetic chemical pesticides leads to the inhibition of actions of enzymes and blocking of essential processes in organisms. There is increasing use of harmful organophosphates such as Actellic dust and Sniper (2, 2 dichlorovinyl-dimethyl phosphate) in the preservation of beans. Ideally, a pesticide must be lethal to the target species, but not to non-target species, including man, but unfortunately, this is not the case, hence the controversy and problem (Okafor et al., 2019).

Entomopathogenic microorganisms such us fungi, bacteria, viruses, and protozoa are among the most promising alternatives to chemical insect control because of their high efficacy and compatibility (Mantzoukas et al., 2022).

Numerous fungal microorganisms are pathogenic for many insect species and are able to control their natural populations by limiting their spread (Islam et al., 2021). Aspergillus niger, Aspergillus flavus, Rhizomucor spp , Fusaruim spp and Aspergillus terreus are group of fungi, have been reported to efficiently control insects, demonstrating attributes such as high mortality rates, swift action, and abundant sporulation, which are valuable in insect control for agricultural purposes (Da Silva Santos et al., 2020).

These species are generally considered soil borne fungi because of their abundance in the soil and their frequent association with plant roots as parasites or saprophytes. These species have usually been reported as plant pathogens causing serious diseases on many plant species (e.g., vascular wilts on a wide range of horticultural crops, crown rot and root rot diseases on many crops, head blight on cereal grain and other plant species) However, some of these species may act as weak to virulent entomopathogens and may live as saprophyte on dead insects (Batta, 2012).

An increasing number of studies demonstrating the potential of little known and explored Fusarium species as biological control agents of insects, Fusarium avenaceum was one of the entomopathogenic species of Fusarium that has been isolated from a number of insect species, and its pathogenicity to insects has been demonstrated in few species, such as greenhouse whitefly, Trialeurodes vaporariorum pruce budworm, Choristoneura fumiferana, and wheat stem sawfly, Cephus cinctus,  However, no reports are yet available on the entomopathogenic activity of any Fusarium species against The Bean Weevil, Acanthoscelides obtectus  This research study focuses on emphasizing the ecological and sustainable attributes of entomopathogenic fungi as effective alternatives for comparative  Beans Weevil mortality and for a sustainable insect pest management.

MATERIALS AND METHODS

Sample Collection

Various samples were obtained from diverse sources, including soil, plant debris, and fruits was done for Fusarium species isolation. Selection criteria prioritized isolates with documented entomopathogenicity or recognized insecticidal properties. One hundred and fifty (150) adult Beans Weevils were collected from infested bean storage facilities to ensure a diverse representation of weight, life cycle developmental stage (age) and sex.

Isolation, Characterization and Identification of Entomopathogenic Fungi 

Small portions of the fruit tissues harboring the spoilage organisms and connecting healthy tissue were surface disinfected by cleaning with cotton wool soaked in 0.1% silver nitrate solution. The excess disinfectant was then blotted out with sterile cotton wool. With the aid of a sterile knife and forceps the spoilt fruits portions were obtained and plated aseptically onto Sabouraud dextrose agar (SDA) in duplicates (replicate-plating). Incubations were done at room temperature until there were visible growths on the agar plates. The developing fungi were further purified by sub culturing onto freshly prepared SDA plates. Preliminary fungal characterization were done by studying the cultural characteristics and the slide culture technique for evaluating the fungal microscopic features with reference to the Manual of Fungal Atlases according to Frey et al. (1997); Barnett and Hunter, (2000); Watanabe, (2002) and Ellis et al. (2007).

Fungal Identification

This was done based on the description of the gross morphological appearance of fungal colonies on the SDA culture medium and the modified slide culture technique using lactophenol cotton blue stain for the microscopic evaluation under X10 and X40 magnification of the microscope.

Entomopathogenicity Assay

Selected number (15) of the weevil was introduced into petri-dishes containing pure culture of the fungal isolates and was incubated at room temperature for a 5 day period to monitor the kinetics of the death rate by entomopathogenicity. A control set-up was done using 10-folds serial dilution of a commercially sold pesticide (kill all) at concentration 0.1, 0.01, 0.001, 0.0001 and 0.00001 at the same incubation parameters.

 Safety Considerations

Safety protocols for handling fungal cultures were strictly adhered to, including the use of personal protective equipment (PPE) and work within designated laboratory areas.

RESULTS AND DISCUSSION

Table 1. Assay of various fungi including Fusarium spp on efficacy as entomopathogen showing mortality rate of Weevil within the period of 5 days against the isolates

Number of Weevil plated for the assay = 15

Table 2: Mortality Response of Beans Weevils to (control) Water and Various Concentrations of Conventional Insecticide over Time This table presents the mortality response of beans weevils subjected to different treatments, including water and various concentrations of a conventional insecticide, over a twelve-day period. The numerical values represent the number of deceased weevils at each respective concentration and time point.

Table 3: Colonial and Microscopic Identifications of the Various Fungi Isolates.

Discussion

Entomopathogenic fungi are microorganisms that can infect and kill insects at different stages of their life-cycle. They have gained attention as potential biocontrol agents in pest management due to their environmentally more friendly nature. The effectiveness of entomopathogenic fungi depends on various factors, including the fungal species, application methods, environmental conditions, and the target pest species.

In this study, Aspergillus niger, Aspergillus flavus, Rhizobium spp, Fusaruim spp and Aspergillus terreus were isolated and characterized from soil, plant debris within Nnamdi Azikiwe University, Awka and screened for their entomopathogenic potentials against specific number of the Beans weevil (Acanthoscelides obtectus). The isolates were confirmed based on morphological and biochemical characteristics of the various isolates. The findings in this study are in accordance with Abrar et al. (2023) which isolated entomopathogenic Fusarium spp from soil and leaf samples in Manga Forest Pakistan. Yan et al. (2022) in in their study isolated Aspergillus sp. From plant debris Dong et al. (2023) also isolated Fusarium spp. From peach plant debris in China.

Table 1. Shows result the entomopathogenic assay of the various fungal isolates against beans weevil. The study reveals that all tested isolates, including Aspergillus niger, Aspergillus flavus, Rhizomucor spp., Fusarium spp., and Aspergillus terreus, exhibit entomopathogenic efficacy against Beans Weevils. Rhizomucor spp., exhibited rapid and sustained mortality rates, reaching 100% within the initial day (all 15 weevils died on the first day of exposure). Fusarium spp., consistent with its recognized entomopathogenic nature, displayed a gradual yet cumulative impact on Beans Weevils. Mortality rates steadily increased, reaching 100% by the fourth day. The observed pattern aligns with expectations, substantiating Fusarium spp.’s potential as a biocontrol agent.

Both Aspergillus terreus and Aspergillus flavus demonstrated robust entomopathogenic potential, achieving 100% mortality by the fourth day.

These Aspergillus species exhibited a rapid and sustained impact on Beans Weevils, indicating their efficacy in pest management strategies. Aspergillus niger, while effective, displayed a slightly delayed onset, with notable mortality observed by the second day and reaching 100% by the third day. This is consistent with the findings of Abrar et al. (2023) which showed that both fungi isolate (Fusarium spp.) were effective against A. aegypti population.  Numerous studies have reported that Aspergillus species have high pathogenicity against different insects, indicating that the fungus has promising biological control potential in pest management (Ibarra-Cortes et al., 2018; Zhang et al., 2015; Lin et al., 2021; Karthi et al., 2018). Assessing the mortality patterns over time Rhizomucor spp. stands out as the most efficient entomopathogenic organism in achieving rapid and sustained mortality rates (at 100% mortality rate on the first).

Table 2 represents the results for the comparative control groups for this study. The table provides a comprehensive overview of the mortality response of beans weevils to varying insecticide concentration treatments, using the control (water) over a twelve-day period. The water-insecticide dependent control exhibited a gradual weevil mortality rate indicative by reduced mobility of the bean weevil.  Conversely, the conventional insecticide at Concentration variations yielded diverse outcomes. The highest concentration (0.1) demonstrated indicated a rapid mortality rate, achieving complete weevil mortality by day 11. Similarly, the 0.01 concentration exhibited comparable effectiveness, reaching full weevil mortality by the same day. The 0.001 concentration, while displaying a delayed effect, eventually achieved complete weevil mortality by day 12. In contrast, the lower concentrations (0.0001 and 0.00001) exhibited a more gradual impact, with maximal mortality observed by day 15. This concentration dependent response highlights the nuanced relationship between insecticide concentration and efficacy.

Comparative Mortality analysis, between the control groups against the entomopathogenic fungi for entompathogenic efficacy against beans suggest Entomopathogenic fungi to possess a competitive efficacy against Beans weevils as compared to the carcinogenic conventional insecticide. This observation aligns with Douro et al. (2013) comparative study, which reported the efficacy of entomopathogenic fungi surpassing conventional insecticides in the management of cotton pests.

CONCLUSION 

This study sheds light on the entomopathogenic potential of Aspergillus niger, Aspergillus flavus, Rhizobium spp., Fusarium spp., and Aspergillus terreus against Beans Weevils. The findings underscore the efficacy of these fungi in exerting entomopathogenic activity, with Rhizobium spp. demonstrating exceptional efficiency by achieving rapid and sustained mortality rates. Comparative analysis with conventional insecticides reveals the superiority of entomopathogenic fungi, as they exhibit faster mortality rates than all concentrations of the insecticides. The study reaffirms the ecological and sustainable attributes of entomopathogenic fungi as viable alternatives for pest management.

REFERENCES

1. Abrar, A., Sarwar, S., Abbas, M., Chaudhry, H., Ghani, N., Fatima, A., and Tahira, A. (2023). Identification of locally isolated entomopathogenic Fusarium species from the soil of Changa Manga Forest, Pakistan and evaluation of their larvicidal efficacy against Aedes aegypti. Brazilian Journal of Biology, 83, e246230.
2. Al-Hatmi, A.M.S.; Meis, J.F.; and de Hoog, G.S (2016). Fusarium: Molecular diversity and intrinsic drug resistance. PLoS Pathogens12, e1005464.
3. Barnett, H.L. and Hunter, B.B. (2000). Illustrated genera of imperfect fungi. 4th edn. Laskin, A. I. and Lechevalier, H. A. (eds). CRC Press, West Palm Beach, Florida, pp 1- 197
4. Batta, Y. A. (2012). The first report on entomopathogenic effect of Fusarium avenaceum (Fries) Saccardo (Hypocreales, Ascomycota) against rice weevil (Sitophilus oryzae L.: Curculionidae, Coleoptera). Journal of Entomological and Acarological Research, 44(3), 51-55.
5. Da Silva Santos, A. C., Gonçalves Diniz, A., Vieira Tiago, P., and Tinti de Oliveira, N. (2020). Entomopathogenic Fusarium species: A review of their potential for the biological control of insects, implications, and prospects. Fungal Biology Reviews, 34(1), 41-57
6. Dong, J., Shi, H., Wu, Y., Yang, L., Zhu, F., & Ji, Z. (2023). Identification and pathogenicity analysis of Fusarium spp. on peach in China. BMC Microbiology, 23(1), 211.
7. Douro Kpindou, O., Djegui, D., Maffon, S., Paraïso, A., Glitho, I.A. and Tamò, M. (2013). Comparative study of the efficacy of entomopathogenic fungi, chemicals and botanical pesticides in the management of cotton pests and their natural enemies in Benin. International Journal of Science and Advanced Technology, 3 (1), 21-33.
8. Edet, E. A., Wilson, L. A., and Afiaglobal, U. U. (2011). Chemical composition of bean weevil (Acanthoscelides obtectus). Global Journal of Pure and Applied Sciences, 17(2), 179-188.
9. Ellis, D., Davis, S., Alexiou, H., Handke, R., and Bartley, R. (2007). Descriptions of Medical Fungi. www.mycology.adelaide.edu.au, accessed on September 13, 2013.
10. Ibarra-Cortes, K. H., Guzman-Franco, A. W., Gonzalez-Hernandez, H., Ortega-Arenas, L. D., Villanueva-Jimenez, J. A., & Robles-Bermudez, A. (2018). Susceptibility of Diaphorina citri (Hemiptera: Liviidae) and its parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) to entomopathogenic fungi under laboratory conditions. Neotropical Entomology, 47(2), 131–138.
11. Islam, W., Adnan, M., Shabbir, A., Naveed, H., Abubakar, Y. S., Qasim, M., Tayyab, M., Noman, A., Nisar, M. S., Khan, K. A., and Ali, H. (2021). Insect-fungal-interactions: A detailed review on entomopathogenic fungi pathogenicity to combat insect pests. Microbial Pathogenesis, 159, 105122.
12. Karthi, S., Vaideki, K., Shivakumar, M. S., Ponsankar, A., Thanigaivel, A., Chellappandian, M., Vasantha-Srinivasan, P., Muthu-Pandian, C. K., Hunter, W. B., and Senthil-Nathan, S. (2018). Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera litura Fab. (Lepidoptera: Noctuidae) larvae. Pesticide Biochemistry and Physiology, 149, 54–60.
13. Kljajić, P., Andrić, G., Pražić Golić, M., & Jovičić, I. (2022). Bean weevil Acanthoscelides obtectus (Say) survival and progeny production affected by residual insecticide deposits, and related damage of two types of bean. Journal of Stored Products Research, 98, 102004.
14. Kljajić, P., Andrić, G., Pražić Golić, M., and Jovičić, I. (2022). Bean weevil Acanthoscelides obtectus (Say) survival and progeny production affected by residual insecticide deposits, and related damage of two types of bean. Journal of Stored Products Research,98, 102004.
15. Lin, W. J., Chiu, M. C., Lin, C. C., Chung, Y. K., and Chou, J. Y. (2021). Efficacy of entomopathogenic fungus Aspergillus nomius against Dolichoderus thoracicus. BioControl, 66(4), 463–473.
16. Lopes, L. M., Nascimento, J. M., Santos, V. B., Faroni, L. R. D., and Sousa, A. H. (2018). Emergence rate of the Mexican Bean weevil in varieties of beans from the southwestern Amazon” Revista Caatinga, 31(4), 1048-1053.
17. Mantzoukas, S., Kitsiou, F., Lagogiannis, I., and Eliopoulos, P. A. (2022). Potential Use of Fusarium Isolates as Biological Control Agents: Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) Case Study. Applied Sciences, 12(17), 8918.
18. Mbogo, K. P., Davis, J., and Myers, J. R. (2009). Transfer of the arcelin-phytohaemagglutinin-a amylase inhibitor seed protein locus from tepary bean (Phaseolus acutifolius A. Gray) to common bean (P. vulgaris L.). Biotechnology, 8, 285–295.
19. Nta, A. I., Mofunanya, A. A. J., Ogar, V. B., Omara-Achong, T. E., and Azuike, P. A. (2019). Comparative Effect of Acanthoscelides obtectus (Say) Infestation on Nutrients of Phaseolus vulgaris (Linn.) and Phaseolus acutifolius (Gray). Pakistan Journal of Biological Sciences, 22, 494-501
20. Okafor, C. S., Ononuju, C. O., Eze, C. G., Nwosu, O. K., &Ifedilichukwu, N. H. (2019). Chromolaena odorata (Siam Weed) as Bio-pesticide Against Beans Weevils. Agricultural and Biological Sciences Journal, 5(3), 105-109.
21. Rugumamu, C.P. (2014) Potency of Traditional Insecticide Materials against Stored Bean Weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae) in Tanzania. Huria: Journal of the Open University of Tanzania, 16, 126-139.
22. Sağlam, Ö., Çelik, A., Işıkber, A. A., Bozkurt, H., Sakka, M. K., and Athanassiou, C. G. (2022). Efficacy of Spinetoram for the Control of Bean Weevil, Acanthoscelides obtectus (Say.) (Coleoptera: Chrysomelidae) on Different Surfaces. Insects, 13(8), 723.
23. Watanabe, T. (2002). Morphologies of cultured fungi and key to species. In: Pictorial atlas of soil and seed Fungi. 2nd edn. Haddad, S., Dery, E. Norwitz, B.E. and Lewis, R. (eds). CRC Press LLC, 2000 N. W. Corporate Blvd., Boca Raton, Florida 33431. pp 1-486.
24. Yan, J., Liu, H., Idrees, A., Chen, F., Lu, H., Ouyang, G., and Meng, X. (2022). First Record of Aspergillus fijiensis as an Entomopathogenic Fungus against Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Journal of Fungi, 8(11), 1222.
25. Zhang, P., You, Y., Song, Y., Wang, Y., and Zhang, L. (2015). First record of Aspergillus oryzae (Eurotiales: Trichocomaceae) as an entomopathogenic fungus of the locust, Locusta migratoria (Orthoptera: Acrididae Biocontrol Science and Technology), 25(11), 1285–1298.