This is the report from a BSPP Lockdown Bursary.
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The general idea of my research involved using entomopathogenic fungi as a component of integrated pest management (IPM) to combat potato aphids. Entomopathogenic fungi (EPF) are fungal species which cause harm to insects. They have been considered as the best replacement for synthetic chemicals. Aphids are very susceptible to entomopathogenic fungi and epizootics of fungal diseases of diseases affecting them, have been noted.
Some important genera of entomopathogenic fungi are, Beauveria, Metaarhizium, Lecanicillium. Entomopathogenic fungi has also been used to successfully control the cowpea aphid (Aphis craccivora) specifically M. aniopliae and B. bassiana in Nairobi, Kenya. Some species of entomopathogenic fungi such as Beauveria produce various secondary metabolites including Beauvericin, beauveriolides, bassianolides and Oxalic acid which are responsible in their pathogenic effect and virulence. Bio control agents of entomopathogenic fungi are applied as spore suspensions, when the fungal spores come in contact with the insect, it germinates and grows directly through the cuticle of the insect to the inner body, produce toxins and finally kills the insect. Once the insect is dead, the fungus grows back through the softer body portions with a layer of white mold commonly called “white muscadine disease”. There are about 100 commercial products that have been produced from entomopathogenic fungi and have been used in the control of pests. There are many ways of obtaining entomopathogenic fungi such as, direct isolation from insect cadavers showing signs of fungal infestation, isolation from the soil and isolation from associated plants where some can establish an endophytic relationship with their host.
Potato (Solanum tuberosum L.) is one of the world’s most important cultivated tuber crop. In Cameroon, it ranks among the major crops in tons produced after cassava, plantain, cocoyam, and maize, with majority production done by small holders mostly women, with most marketing done locally. Potato production is therefore still constrained by poor farming practices, pests, inefficient use of available technologies, poor soil fertility, high cost of inputs like fertilisers, seed and fungicides, lack of access to credit, and a lot more despite government subsidisation. The insect pests of potatoes are cutworms (the main pest attacking all the garden crops), fruitworms, leafminers, white flies and aphids experienced at various growth stages. The potato aphids known as green peach aphid and white flies being prominent at the maturity stage. Studies have identified various entomopathogenic fungi against the potato aphid including M. aniopliae and B. bassiana in Sargodha (Pakistan).
The objectives of this study were as follows; i) to identify species of entomopathogenic fungi and select active isolates from potato aphids, ii) to determine the toxicity of the selected isolates and their formulations on potato aphids under laboratory conditions, iii) to evaluate the effects of different formulations of the selected isolates of entomopathogenic fungi for the management of potato aphids under field conditions, iv) to determine the efficacy of intercropping potato with beans and the selected EPF for the management of potato aphids.
With the COVID-19 grant that I won from the BSPP, I conducted a survey in Santa and Small Babanki, in Bamenda, Cameroon. Insect cadavers suspected to have fungal infection were collected from the field (50 samples from Santa and 50 from Small Babanki). These samples were put in coolers and transported by means of public transport to the Life Science Laboratory in the University of Buea for isolation and morphological identification of entomopathogenic fungi.
The working environment was surface sterilised with 70% alcohol to reduce chances of contamination. Potato dextrose agar (PDA) medium with gentamycin and penicillin was prepared, the antibiotics added to prevent the growth of other microbes such as bacteria. The insect cadavers were put in small nets, surface sterilised by immersing in 10 % sodium hypochlorite solution, rinsed in distilled water, followed by rinsing with 70 % alcohol, rinsed in sterile distilled water and finally tap water, then plated upon the PDA medium and incubated at room temperature (25 °C) in the dark for 7 days. After 7 days, fungi that grew out of the inoculants were sub-cultured on fresh PDA plates. The distinct fungi colonies from primary cultures were cut out using a sterile scalpel, and then transferred to the fresh PDA plates to obtain pure cultures. Macroscopic features such as colony diameter, colony colour, texture, margin, form, elevation and aerial hyphae were recorded by observing the growth forms on PDA physically. The mycelium was observed under a light microscope and crosschecked with those in literature. Out of the 100 samples, I ended up with 44 clean samples. The mycelia of these pure cultures were harvested and stored in 10% glycerol and sent to Germany for molecular identification.
DNA extraction was done following a Sorbitol-CTAB protocol, purity was checked in a Nanodrop spectrophotometer and Sanger sequencing method was used to produce ITS and TEF sequences. When these sequences were obtained, some that were not really good were edited, after which all the sequences were subjected to NCBI to compare with already published sequences in the gene bank and identified using the Basic Local alignment Search Tool (BLAST).
Results of cultural identification gave three different groups of fungi, identified as Fusarium species, different species of Aspergillus including A. flavus, A. niger and A. fumigatus and Penicilium species (Table 1). After performing the BLAST in NCBI, the results gave 10 different groups of fungi which were, 3 species of Fusarium, one species of Chaetomium, one species of Trichoderma, one species of Aspergillus, 6 species of Cladosporium, one species of Periconia, one species of Claviceps, one species of Curvularia, one species of Microascus and 2 species of Penicilium. Therefore, a total of 18 species were obtained (Table 2).
Table 1: Fungi species identified morphologically using cultural studies
|Code||Suspected fungus||Colony diameter||Colony form||Colony margin||Surface colony colour||Reverse colony colour|
|G42||Aspergillus fumigatus||78||Raised||Circular||Dark green||Black|
Table 2: Identified fungi from NCBI using the BLAST
|G1||Fusarium oxysporum||1007||1075||91%||0.0||99.82%||732||ON927009.1||Schltdl, 1824|
|G4||Fusarium oxysporum||1005||1005||98%||0.0||99.64%||865||MG574894.1||Schltdl, 1824|
|G5||Fusarium babinda||937||937||90%||0.0||100.00%||507||MT478951.1||Summerell et al., 1995|
|G6||Fusarium oxysporum||479||598||99%||2e-130||82.77%||607||MW497617.1||Schltdl, 1824|
|G9||Fusarium babinda||937||937||90%||0.0||100.00%||507||MT478951.1||Summerell et al., 1995|
|G10||Fusarium tricinctum||928||928||91%||0.0||99.41%||540||KR011974.1||Corda, 1838|
|G12||Fusarium babinda||937||937||79%||0.0||100.00%||507||MT478951.1||Summerell et al., 1995|
|G13||Chaetomium cochliodes||1029||1029||97%||0.0||99.65%||575||OW982621.1||Palliser, 1910|
|G14||Fusarium oxysporum||952||1006||99%||0.0||98.00%||1410||LT746251.1||Schltdl, 1824|
|G15||Trichoderma gamsii||1114||1114||100%||0.0||99.67%||857||KM491887.1||Samuels & Druzhinina, 2006|
|G19||Fusarium oxysporum||994||994||100%||0.0||99.45%||1419||LT970803.1||Schltdl, 1824|
|G21||Aspergillus sydowii||1048||1048||100%||0.0||99.65%||971||LN898726.1||Thom and church, 1926|
|G22||Aspergillus niger||1109||1109||100%||0.0||99.83%||76751||AM270051.1||Tieghem, 1867|
|G23||Cladosporium xanthochromaticum||1024||1024||98%||0.0||100.00%||1219||KY781767.1||Sandoval-Denis et al., 2016|
|G24||Cladosporium aciculare||983||983||97%||0.0||99.44%||547||MZ568163.1||Bensch et al., 2015|
|G26||Cladosporium anthropophilum||1024||1024||100%||0.0||100.00%||840||MF472932.1||Sandoval-Denis et al., 2016|
|G28||Periconia cookei||826||826||86%||0.0||96.78%||497||MG333490.1||Mason and Ellis, 1953|
|G29||Cladosporium xanthochromaticum||1005||1005||98%||0.0||100.00%||593||MK732115.1||Sandoval-Denis et al., 2016|
|G30||Cladosporium colombiae||1020||1020||97%||0.0||100.00%||553||ON920710.1||Schub and Crous, 2009|
|G32||Claviceps pupurea||163||163||19%||3e-35||91.53%||550||MN218708.1||Fries, 1823|
|G33||Curvularia affinis||1053||1053||96%||0.0||100.00%||714||HG778981.1||Boedijn, 1933|
|G34||Cladosporium cladosporioides||1005||1091||99%||0.0||99.28%||1651||KJ596320.1||Fresen, 1850|
|G35||NO SIGNIFICANT SIMILARITY|
|G36||Microascus murinus||979||979||89%||0.0||100.00%||530||MG457819.1||Sandoval-Denis et al., 2015|
|G37||Penicillium chrysogenum||1077||1077||99%||0.0||100.00%||608||MH127463.1||Thom, 1910|
|G40||Penicillium paxilli||368||368||48%||5e-97||90.18%||800||JN617709.1||Bainier, 1907|
|G42||Cladosporium dominicanum||959||959||93%||0.0||100.00%||520||OW986440.1||Zala et al., 2007|
|G43||Periconia cookie||937||937||100%||0.0||96.33%||584||OU989471.1||Mason and Ellis, 1953|
|G44||Curvullaria affinis||946||946||92%||0.0||98.15%||541||AB705141.1||Boedijn, 1933|
This grant has been of great help to me because I was able to accomplish one of my specific objectives of which I could not have been able to proceed with the remaining objectives (which are, to carry out a pathogenicity test on the identified fungi species so as to sought out which of them are entomopathogenic, determine the toxicity of the selected isolates and their formulations on potato aphids under laboratory conditions, evaluate the effects of different formulations of the selected isolates of entomopathogenic fungi for the management of potato aphids under field conditions and determine the efficacy of intercropping potato with beans and the selected EPF for the management of potato aphids) without the results of the first objective. This remaining part of the work will be completed in February 2023.
I thank the BSPP for their generosity because I wouldn’t have been able to go this far with my research if the grant was absent. I also wish to thank my supervisors Prof Tonjock Rosemary Kinge and Prof Tofel Haman Katamssadan in a special way for without them, no other person would have given me the wonderful training and follow up throughout this work and making it a success. Special thanks also go to the lab attendant Mr Mbabe Felix and the lab assistant Miss Moforcha Lilian for their time, patience and directives they offered to me while I was working at the Life Science Laboratory in the University of Buea.
CHIA Genevieve KAIN
The University of Bamenda, Cameroon
This is the report from a BSPP Lockdown Bursary.