Larvicidal Efficacy of Entomopathogenic Fungi Isolated from the Farmland Areas in Osogbo South-West Nigeria
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
http://jmscr.igmpublication.org/home/
ISSN (e)-2347-176x ISSN (p) 2455-0450
DOI: https://dx.doi.org/10.18535/jmscr/v9i3.38
Larvicidal Efficacy of Entomopathogenic Fungi Isolated from the Farmland
Areas in Osogbo South-West Nigeria
Authors
Abideen Akinkunmi Wahab , Busayo Janet Ayanwola2, Abolaji Tolulope Adeyemo2,
1
Ojevwe Harrison Egbo3, Babatunde Adeola Afolabi4, Adeyemi Temitayo Adeyemo5*
1
Department of Microbiology, Osun State University, Osogbo, Nigeria
2
Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology
Teaching Hospital, Osogbo, Nigeria
3
Department of Morbid Anatomy and Histopathology, Edo State University, Uzairue, Nigeria
4
Department of Family Medicine, Lautech Teaching Hospital, Osogbo, Osun State, Nigeria
5
Department of Medical Microbiology and Parasitology, Obafemi Awolowo University Teaching Hospitals
Complex, Ile-Ife, Nigeria
*Corresponding Author
Adeyemi Temitayo Adeyemo
Department of Medical Microbiology and Parasitology, Obafemi Awolowo University Teaching Hospitals
Complex, Ile-Ife, Nigeria
Abstract
Thousands of entomopathogenic fungi are in existence, unexploited and it is important to identify and use
for biocontrol of mosquito larva. The aim of this study was to determine the larvicidal efficacy of newly
identified entomopathogenic fungi. Soil samples were taken from three different locations in a farmland at
Osun State University (UNIOSUN),Osogbo, Nigeria for isolation and identification of entomopathogenic
fungi and test its larvicidal effect on mosquito larvae. The samples were analyzed using spread plate
technique which include serial dilution of samples, inoculation on prepared Saboraud Dextrose Agar
using three dilutions (10-1, 10-3, 10-10) and incubation at 270c for 72 hours after which the plates were
examined for growth followed by sub-culturing. Mosquito larvae were breeded, picked into sterile distilled
water in Petri dishes. The spores of the fungi were scraped on the larva to determine the larvicidal effect
of the fungal isolates at varied interval: 40 minutes, 2 hours, 24hours, and 48hours. A total of seven fungi
were isolated two (Aspegillus flavus and Aspergillus niger) of which were able to show larvicidal effect on
mosquito larvae. Some larvae died at exactly 40 minutes of inoculation when tested with Aspergillus
niger, some took hours (48hours) while others remain viable. This study concludes that Aspergillus niger
and Aspergillus flavus isolated from soil samples exhibited biolarvicidal efficacy against mosquito larvae
and the mortality rate increases with time of exposure. Entomopathogenic fungi should therefore be
explored for control of mosquitoes in our environment.
Introduction by mosquito-borne diseases every year such as
Mosquitoes are well known vector of human and malaria dengue, chikungunya, Zuka, yellow fever,
animal pathogens and millions of people are killed encephalitis and filariasis.1 Vector control,
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 224
JMSCR Vol||09||Issue||03||Page 224-232||March 2021 sanitation, habitat disruption or personal host, acting as regulators for numerous harmful protection from mosquito bites are the most insects including both domestic and forest widely measures employed to control and protect insects.7 people from infection of these diseases.2 Over the In general mosquitoes show susceptibility towards past few decades, many countries organized entomopathogenic fungi and its derived products. official programs of mosquito vector control. They have low toxicity to non-target organisms Currently, synthetic chemical pesticides against and using entomopathogenic fungi as larvicides adults or larva have been the mainstay and are the may be a promising approach for biological most widely used for control of Mosquitoes. control of mosquitoes due to their selective Mosquito larvae are the attractive targets for these toxicity and ready decomposability in the pesticides because mosquitoes breed in water and ecosystem.8 Also, unlike the inherent dangers thus, it is easy to deal with them in this habitat3 which are associated with the process of The indiscriminate use of chemical insecticides to production of synthetic insecticides, the process target adult mosquitoes has given rise to problems for the manufacture of microbial products is safe such as mosquito resistance, environmental and less pollutant.9 contamination, and health risk to humans and non- This study therefore aimed at determining the target organisms. To mitigate these problems, larvicidal activities of fungal isolates with a view there is an urgent need to develop alternatives to to exploring their potential for biological and conventional chemical insecticides, which are environmental control of mosquito-borne diseases. safe, effective, biodegradable and highly selective. There has been an increasing awareness in the Materials and Methods possibility of using biological control agents as Collection of Soil Samples alternative to chemical control of mosquitoes. The study area was a farmland within Osun State Among the eminent biological control agents are University, Osogbo, Nigeria. About 100g of soil entomopathogenic microorganisms such as fungi samples from 10 – 15cm deep were aseptically as larvicides.4 Fungal biocontrol agents are the collected with sterilized hand trowel into sterile most important among all the Entomopathogenic Aluminum foil paper from three different microorganisms due to easy delivery, chances to locations on the same farmland and transported to improve formulation, vast number of pathogenic the laboratory for further analysis. strains known, easy engineering techniques and its Isolation of Fungi in the Soil Samples ability to control both sap sucking pests such as Fungi were isolated from soil samples using serial mosquito and aphids as well as pest with chewing dilution method; one gram (1g) of soil sample was mouth parts. They include numerous suspended in 9ml of sterile diluents and serially phylogenetically. morphologically and diluted up to tenth factor (10-10). Dilutions of 101, ecologically diverse fungal species which evolve 105 and 1010 was used to isolate the fungi. With to exploit insects with their main route of entry sterile syringe, 0.1ml of each dilution was drawn being through the insect’s integument, by and inoculated into the plates which have been ingestion or via wounds or trachea.5 Most labeled and arranged according to dilution, then entomopathogenic fungi can be grown on artificial glass spreader dipped into 100% ethanol and media (shin et al., 2009); being natural mortality flamed was used to spread the inoculums in the agents which are environmentally safe, there is a petri dish by slightly opened beside the flame to worldwide interest in the use and manipulation of avoid contamination. All plates were incubated at entomopathogenic fungi for biological control of room temperature (25oC – 27oC) and observed for insects and other arthropod pests.6 They display a growth between 3 to 5 days i.e. (72 hours – 120 higher degree of effectiveness in infecting their hours). The process was carried out in duplicate. Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 225
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
Sub culturing of Fungal Isolates picked and transferred into the spore suspension in
Distinct fungal colonies were aseptically picked the petri dishes. A lower spore concentration was
from the mixed culture using sterile inoculating prepared by further diluting 1ml of the standard
needle and transferred onto a freshly prepared concentration (prepared as above) with 1ml of
sterile SDA plates and incubated for 3 – 5 days at distilled water to decrease the concentration to
250C. This process was repeated until pure half (labeled as lower concentration), the same
cultures were obtained pure culture was quantity of larvae (four larvae) were then
maintained on SDA slant and stored at 4oC in the introduced into this spore suspension. Control
refrigerator for further analysis. treatment contains sterile distilled water and
mosquito larvae in a Petri dish. Death or viability
Macroscopic and Microscopic Identification of of mosquito larvae was monitored and recorded at
Isolated Fungi room temperature (25oC – 27oC) from the point of
Each pure culture plate was observed for inoculation to three days.
morphology characteristics of the isolates
including color, growth rate and texture. Confirmation Test
For presumptive identification of isolated fungi To confirm that the fungal isolates were
from soil samples, a drop of lactophenol blue was responsible for the mortality of mosquito larvae,
placed on a clean glass slide, and an inoculating the dead larvae were picked and inoculated on
needle was used to pick from the pure culture freshly prepared Saboraud Dextrose Agar. The
plate onto the stain on glass slide. The cover slip growth of the fungal isolates was shown on the
was gently placed on the prepared smear, mounted plates after 72hrs.
and view under X40 objective on Microscope. The
Microscopic appearance was captured and Results
compared with the structures in soil fungi Fungi species Isolated from Soil Samples
Compendium.10 A total of seven (7) fungi were isolated from the
Breeding of Mosquito Larvae soil samples and they included Microsporum spp.,
Buckets containing half-filled dechlorinated water Fusarium spp., Malbranchea entero arthric,
were placed under shade at different site locations Aspergillus niger, Aspergillus flavus, Onychocola
for 7 days around the school General Laboratory Canadensis, and Arthrinium spp.
of the institution. After breeding, the buckets were Table 1 and Figure 1a and 1b show the
transported into the laboratory. morphological characteristics and microscopic
Rejuvenating of the Isolates appearances of the isolates through direct
Each fungal isolate was taken from the stock observation of colonies on solid media plates as
culture and inoculated on freshly prepared well as viewing under x40 objectives.
sterilized Saboraud Dextrose agar plates, Result of Biolavicidal Effect of Fungi
incubated at room temperature (25o – 27oC) for 72 Biolarvicidal potential of isolated fungi was
hours. carried out and it revealed that Aspergillus niger,
Procedure of Biolarvicidal Efficacy Aspergillus flavus have efficacy against the
One milliliter of sterile distilled water was mosquito larvae, but the rate at which death occur
dispensed into each petri dish onto which the in each isolate is different. Result is shown in
spores of fungal spores were scraped and the Table 2 and 3.
concentration standardized and determined by Confirmation of Biolavicidal Effect of Fungi
measuring its optical density (turbidity) (labeled This shows that the fungi isolates were
as higher concentration) by previously responsible for the mortality of mosquito larvae
documented protocols,11,12 then four larvae were
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 226
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
by observing growth of fungi on solid media isolate B-5d (Aspergillus flavus) shows no
plates after incubation. mortality at 2hours but some effect at 24hours in
Mortality Rate of Mosquito Larvae after which two larvae were viable while two were dead
Treatment with Fungi (50% mortality rate). At 48 hours all the four
Table 2 shows that isolate A-1a (Microsporum larvae were dead (100% mortality rate).
spp.) had no larvicidal effect on the all the four Table 3 shows mortality rates at a higher spore
larvae treated at lower spore concentration concentration (50.5mg/L). Aspergillus niger
(25.25mg/L), the same effect was observed for showed 100% mortality at different time of
isolates A-1b (Fusarium spp.), B-1c exposure ranging from 40 minutes to 24hours.This
(Malbranchea entero arthric), C-5a (Onychocola implies that Aspergillus niger has high larvicidal
canadensis) and C-5c (Arthrinium spp.) at the potency regardless of time of exposure. On the
same spore concentration. other hand, at the same spore concentration,
However, at the same spore concentration, isolate Aspergillus flavus showed no larvicidal effect on
B-5c (Aspergillus niger) had no effect at 2hours the mosquito larvae at 40 minutes, 50% mortality
on the four larvae but some effect at 24hours in at 2hours and 100% mortality at 24hours implying
which two larvae were viable while two were dead that the mortality rate with Aspergillus flavus
(50% mortality rate). At 48 hours all the four increases with time of exposure as represented in
larvae were dead (100% mortality rate). Likewise, Figure 3.
Table 1: Cultural morphology and characteristics of the isolates
SAMPLE SURFACE/REVERSE GROWTH RATE TEXTURE
CODE
A-1a White wooly surface/rusty brown at the reverse Grow slowly Velvety to wooly
A-1b Whitish and brown at the reverse Rapid growth Wooly to cottony
B-1c White wooly Moderately wooly Wooly
B-5c Black surface, yellowish filament and reverse Rapid growth Powdery
B-5d Green with whitish wooly growth/yellowish at the base Rapid growth Powdery and wooly
C-5a White becoming yellowish/pale Moderately rapid Smooth to slightly
floccose
C-5c White wooly surface and brown at the reverse Moderate growth Wooly to cottony
Isolate Surface picture Reverse picture Microscopic view Presumptive
code identification
A-1a Microsporum spp.
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 227
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
A-1b Fusarium spp.
B-1c Malbranchea spp.
Figure 1a: Macroscopic and microscopic appearances of the isolates
B-5c Aspergillus niger
B-5d Aspergillus flavus
C-5a Onychocola
canadensis
C-5c Arthrinium spp.
Figure 1b: Macroscopic and microscopic appearances of the isolates
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 228
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
Table 2: Biolarvicidal efficacy of lower concentration of fungi isolates on Mosquito larvae
ISOLATE CODE NUMBER OF LARVAE IN 2MLS OF PERIOD OF INNOCULATION
LOWER CONCENTRATION FUNGAL
SUSPENSION
2 hrs. 24 hrs. 48 hrs.
A- 1a 4 4V 4V 4V
A-1b 4 4V 4V 4V
B-1c 4 4V 4V 4V
B-5c 4 4V 2D 2V 4D
B-5d 4 4V 2D 2V 4D
C-5a 4 4V 4V 4V
C-5c 4 4V 4V 4V
V= Viable
D=Dead
Table 3 Biolarvicidal efficacy of higher concentration of fungi Isolates on Mosquito larvae
ISOLATE CODE NUMBER OF LARVAE IN PERIOD OF INNOCULATION
2MLS OF HIGHER
CONCENTRATION OF
FUNGAL SUSPENSION
40 minutes 2hrs 24hrs
A- 1a 4 4V 4V 4V
A-1b 4 4V 4V 4V
B-1c 4 4V 4V 4V
B-5c 4 4D 4D 4D
B-5d 4 4V 2D 2V 4D
C-5a 4 4V 4V 4V
C-5c 4 V V V
V= Viable
D=Dead
Sample Code Name of the Isolates Picture of the Isolates
B – 5c Aspergillus niger
B – 5d Aspergillus flavus
Figure 2: Confirmation test result showing growth of mosquito larvae on solid media
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 229
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
100%
90%
80%
70%
60%
Aspergillus niger
50%
Aspergillus flavus
40%
30%
20%
10%
0%
40 mins 2hrs 24hrs
Figure 3: A chart showing the mortality rate of mosquito larvae at a concentration of 2 larvae per milliliter
(4 per 2 millilitres) at different durations
Discussion large-scale field trial conducted in the United
The researches on the larvicidal potential of States, mycelium of Lagenidium giganteum
microbes particularly fungi for controlling the caused 40-90% infection rates in Culextarsalis
mosquito larvae have only been extensively and Anopheles freeborn sentinel larvae.16
carried out on adult mosquito for several decades. The potentials of many fungi have been
Entomogenous fungi have caused high mortalities established for mosquito control, nevertheless,
in mosquito population in many laboratories only a few have received commercial attention
including Aedes and Anopheles species.13 and are marketed for use in vector control
However, a review of studies by elucidates the programmes globally. It is therefore becoming
larvicidal potentials of fungi and revealed that the obvious that entomopathogenic fungi promises a
fungi could successfully infect and kill larvae of a greater roles in expansion of the limited options
wide range of mosquito genera with varying rates available for effective mosquito control, thereby
of mortality.6 Furthermore, a research conducted contributing in a substantial and sustainable
in East Africa to determine the pathogenicity of manner to prevention and control of vector-borne
entomopathogenic fungi against several strains of diseases.
adult Anopheles gambiae revealed a high infection In this study, seven fungi were isolated from soil
rates ranging from 46 to 88% with Metarhizium samples and been identified as: Microsporum
anisopliae being the most pathogenic strain.14 In spp., Fusarium spp., Malbranchea entero arthric,
recent years, there is a considerable amount of Aspergillus niger, Aspergillus flavus, Onychocola
attention focusing on identifying potential canadensis, Arthrinium spp. The larvicidal
mosquitocidal fungus from natural sources for potential of these fungi were tested at different
effective control of mosquitoes as a key measure concentration against mosquito larvae at different
to curtailing the vector borne diseases in human. period of exposure (40 minutes, 2hours, 24hours
A study done in Asia researching the larvicidal and 48hours). Two isolates which were
potential of Lagenidium giganteum, a water weed, Aspergillus niger, and Aspergillus flavus
noted its efficacies in killing the tested vectors exhibited larvicidal potential against mosquito
with appreciable safety to non-target organisms larvae while other five isolates showed no effect
and good biological stability.15 According to a on the larvae. At a lower fungal spore
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 230
JMSCR Vol||09||Issue||03||Page 224-232||March 2021 concentration (25.25mg/L), Aspergillus niger reduction in malaria transmission due to enhanced showed no mortality at 2hours, 50% mortality at larval mortality and promoted good larval control 24hours and 100% mortality at 48hours. At the at more moderate levels of coverage even in areas same spore concentration, Aspergillus flavus where insecticide resistance makes control showed similar mortality rates at the same time of ineffective.4 exposure as Aspergillus niger. At higher spore concentration (50.5mg/L), Aspergillus niger Conclusion and Recommendations showed a mortality rate of 100% mortality at 40 From the result of this study, Aspergillus niger minutes, 2hours and 48hours. This is similar to the and Aspergillus flavus isolated from soil samples study by Mauryaet al. who evaluated larvicidal exhibited biolarvicidal efficacy against mosquito potential of Aspergillus niger, Aspergillu sflavus, larvae. It was also shown that the mortality rate Aspergillus parasiticus against Anopheles increases with time of exposure. These isolates are stephensi and Culex quinque fascistus with therefore potential be deployed for mosquito Aspergillus flavus showing greatest bio efficacy at control programs singularly or in combination 50% lethal concentration (LC50) against larvae of with other control agents. Further studies on Anopheles stephensi and Culex quinquefasciatus larvicidal activity of fungi species should be at 24 hours of exposure and bio efficacy increased carried out in this environment for evidence-based in both larval species at 48 hours.17 information to further establish the usefulness of At the same higher concentration, Aspergillus these species as myco-insecticides to combat flavus showed no mortality at 40 minutes, 50% mosquito and other arthropods which are mortality rate at 2hours and 100% mortality rates causative agents of human diseases. at 24 hours which also similar to the research of Govindraja et al who evaluated larvicidal effect of Acknowledgements; The authors are grateful to Aspergillus flavus and other fungi against Culex the technical staff of Department of Microbiology, quinque fascistus with high mortality rate.18 Osun State University, Osogbo, Nigeria. Similarly, a study conducted in northern Nigerian Competing interests; The authors declare that showed that the fungal concentration required to they have no competing interests. kill 50% of larvae decreased as the exposure Authors’ contributions; BJA and AAW duration (in hours) increased. It also reported that conceived and designed the study. BJA and AAW larval mortality rates increased proportionately conducted laboratory experiments. ATA, OHE, with concentration of pathogenic fungi.8 BAO and ATA wrote the initial draft, ATA, OHE, According to a study by Bukari et al. fungal BAO and ATA edited and reviewed the final spores of entomopathogenic fungi, especially manuscript. All authors approved the final report. when formulated in synthetic oil as an effective Funding: This study was self-sponsored. carrier, provided a better lavicidal effect with Availability of data and material: The datasets significant reduction of vector by up to 50% in used and analysed during the current study are Kenya, emphasizing its promising role for available from the corresponding author on controlling larval populations of mosquitoes.9 reasonable request. The usefulness of fungal biolavicidal effects for Disclaimer: The views expressed in the submitted potentiating the synthetic (chemical) lavicides, manuscript are authors and not an official position even at low (environment-friendly) of the institution. concentrations, had recently been studied. An Indian study demonstrated the synergistic References potential of Aspergillus flavus and Temephos (an 1. Odalo J,Omolo M, Malebo H, Angira J, Njeru organophosphate lavicide) by showing dramatic P, Ndiege I. Repellance of essential oil of Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 231
JMSCR Vol||09||Issue||03||Page 224-232||March 2021
some plants from Kenya Coast against 11. Morris SC, Nicholls PJ. An evaluation of
Anopheles gambiae. Acta Tropica 2005; 95: optical density to estimate fungal spore
210-18. concentrations in water suspensions.
2. World Health Organization (WHO), (2013). Phytopathology 1978; 68: 1240-42.
Global Programme to eliminate lymphatic 12. or e th rg ni tion .
filariasis. (Available on www.who.int). Guidelines for laboratory and field testing of
3. Arivoli S., Ravindran J.K., Tennyson S., mosquito larvicides. World Health
(2012). Larvicidal Efficacy of plant Extracts Organization.
against the malaria vector Anopheles https://apps.who.int/iris/handle/10665/69101
stephensis Liston (Diptera:Culicidae).World 13. Kerwin JL, Washino RK. Field evaluation of
J. MedSci 7:77-80. Lagenidium giganteum (Oomycetes
4. Bhan S, Shrankhla, Mohan L, Srivasstava :Lagendales) and description of a natural
CN. Larvicidal toxicity of Temephos and epizootic involving a new isolates of fungus.
entomopathogenic fungus, Aspergillus J. Med. Entomol 1988; 25: 452-60.
flavusand their synergistic activity against 14. Scolte EJ, Takken W, Knols BGJ.
malaria vector, Anopheles stephensi. Journal Pathogenicity of five East Africa
of Entomology and Zoology Studies 2013; 1 entomopathogenic fungi to Adult Anopheles
(6): 55-60. gambiae (Diptera: culicidae). Proc. Exper.
5. Watarabe T. Pictorial Atlas of soil and seed Appl. Entomol. Nev Amsterdam 2003; 1425-
fungi morphologies of cultured fungi 2010; 9.
P295-296. 15. Vyas N, Dua KK, Prakash S. Efficacy of
6. Scolte EJ, Knols BGJK, Samson RA,Takken Lagenidium giganteum metabolites on
W. Entomopathogenic fungi for Mosquito mosquito larvae with reference to non-target
control: A review.Journal Insect Science organism. Parasitol. Res 2007; 101: 385-90.
2004; 4:19. 16. Kerwin JL, Washino RK. Ground and aerial
7. Augustyniuk-Kram A, Kram K J, Lo JA. application of the asexual stage of
Entomopathogenic Fungi as an Important Coelomomyces giganteum for control
Natural Regulator of Insect Outbreaks in ofmosquitoes associated with rice culture in
Forests (Review). Forest ecosystem- more the central valley of California. Journal of the
than Just Trees 2012; P265-94. (Available on American Mosquito Control Association
www.intechopen.com). 1987; 3: 59-64.
8. Soni N, Soam P. Effect of Chrysosporium 17. Maurya P, Mohan L, Sharma P, Srivastava
keratinoplulum metabolites against Culex CN. Evaluation of larvicidal potential of
quinquefasciantus after chromatographic certain insect pathogenic fungi extracts
purification. Parasitol Res 2010; 107: 1329- against Anopheles stephensi and Culex
36 . quinquefasciatus. Entomological Research
9. Bukhari T, Willem T. Development of 2011; 41(5): 211-15.
Metarhizium anisopliae and Beauveria 18. Govindraja M, Jebamesan A, Reetha D.
bassiana formulations for control of malaria Larvicidal effect of extracellular secondary
Mosquito larvae. Parasites & Vectors 2011; metabolites of different fungi against the
4(1):23. DOI: 10.1186/1756-3305-4-23. mosquito, Culex quinquesfasciatus. Tropical
10. Ellis D, Davis S, Alexiou H, Handke R, Biomedicine 2005; 22: 1-3.
Bartley R. Descriptions of medical fungi. 2nd
ed. Adelaide, South Australia; 2007.
Abideen Akinkunmi Wahab et al JMSCR Volume 09 Issue 03 March 2021 Page 232
You can also read
