JOURNAL OF TROPICAL LIFE SCIENCE 2023, Vol. 13, No. 1, 1 – 12 http://dx.doi.org/10.11594/jtls.13.01.01 Research Article Survival of Mushrooms and Termites Upon Pesticide Exposure in the Cocoa Agro-ecosystem Eric Kumi Asare 1, Silas Wintuma Avicor 1, Yahaya Bukari 1, Ishmael Amoako-Attah 1, Solomon Agyare 1, Mabel Yeboah 1, Michael Wiafe-Kwagyan 2* 1 Cocoa Research Institute of Ghana, P. O. Box 8, New Tafo-Akim, Ghana 2 Department of Plant & Environmental Biology, School of Biological Sciences, College of Basic and Applied Sciences, University of Ghana, Legon – Ghana Article history: ABSTRACT Submission February 2022 Revised February 2022 Pesticides have become integral parts of cocoa cultivation for the management of in- Accepted August 2022 sect pests and fungal pathogens which cause significant damage to the crop. However, continuous pesticide usage in the cocoa agro-ecosystem is of concern due to perceived *Corresponding author: adverse effects on non-target organisms. In this study, mushrooms and associated ter- E-mail: mwiafe-kwagyan@ug.edu.gh mites were used to elucidate the possible effect of fungicides and insecticides on non- target organisms in the cocoa agro-ecosystem. The vegetative phase of Pleurotus sa- jor-caju (Oyster mushroom), Volvariella volvacea (Paddy straw mushroom), Termi- tomyces globulus, and Termitomyces robustus (Termite mushrooms) were subjected to concentrations of commercially formulated fungicides (metalaxyl 12 % + copper (I) oxide 60 % and cupric hydroxide 77 %) and insecticides (imidacloprid 20 % and bifenthrin 2.7 %) to observe their growth rates on Potato Dextrose Agar (PDA). Worker termites, Macrotermes bellicosus, were also exposed to the pesticides in Petri dishes for 24 h to observe their mortality. The manufacturer’s recommended concen- tration of 245 ppm for bifenthrin completely inhibited mycelial growth of all the mushrooms and caused 100% mortality of termites. At 0.0245 ppm, the insecticide caused 60% mortality of termites, but it had no inhibitory effect on the mushrooms. Except for P. sajor-caju, mycelial growth of all the other mushrooms was completely inhibited by metalaxyl + copper (I) oxide at the manufacturer’s recommended con- centration of 2400 ppm. However, mycelial growth rate of the mushrooms at 0.24 ppm of the fungicide was similar to the control plates. Although the recommended concentrations of the pesticides inhibited mushroom activity under controlled condi- tions, mushroom survival in the cocoa agro-ecosystem amidst pesticides could be due to diluted pesticide concentrations that result in the soil after application which is usu- ally directed at the pods, trunk, and foliage. Keywords: Agro-ecosystem, Cocoa, Fungicides, Insecticides, Mushrooms, Termites Introduction Mushrooms are fruiting bodies of macro- cine [4]. Commonly consumed in Africa and fungi, usually produced above the surface of sub- Southeast Asia are mushrooms in the genus Ter- strates [1]. They grow on diverse substrates, in- mitomyces [5-9]. They are probably the most pre- cluding deadwood, leaf litter and soil [2]. Mush- ferred mushrooms due to their health benefits. rooms play significant ecological and agricultural Fresh extract powder or paste of fruiting bodies roles in forests and other terrestrial ecosystems by of Termitomyces globulus is used for wound heal- providing food sources for wildlife, forming my- ing, whereas the syrup of Termitomyces ro- corrhizal associations with plants, and decompos- bustus is used as a remedy for constipation and in- ing dead organic matter [3]. Economically, they dolence [10]. According to a survey by are used for food, industrial purposes, and medi- Apetorgbor et al. [11], the consumption of some How to cite: Asare EK, Avicor SW, Bukari Y, et al. (2023) Survival of mushrooms and termites upon pesticide exposure in the cocoa agro- ecosystem. Journal of Tropical Life Science 13 (1): 1 – 12. doi: 10.11594/jtls.13.01.01. EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa edible mushrooms with medicinal and nutraceuti- cluding imidacloprid and thiamethoxam, are cur- cal properties lowers blood pressure, and they rently banned in the European Union (EU) be- serve as a blood tonic for children suffering from cause of the threat they pose to honeybees and kwashiorkor. Such haematinic properties have other pollinators [35]. Effective management also been observed in other mushroom species, of Phytophthora, the cause of black pod disease, such as Pleurotus tuber-regium [12]. Unlike P. depends on copper-based fungicides [32]. Phy- sajor-caju (Oyster mushroom) and Volvariella tophthora megakarya, which is noted for causing volvacea (Paddy straw mushroom), which can be the severe form of black pod disease, is effectively cultivated on agricultural wastes and industrial by- managed by combining copper and metalaxyl or products, the formation of fruiting bodies by Ter- its isomer, metalaxyl-M fungicides [36, 37]. mitomyces species is aided by termites in the sub- The aim of using pesticides in the cocoa agro- family Macrotermitinae [13] and hence, the name ecosystem is to boost cocoa yield by killing pests termite mushrooms. The mushroom exists in a and pathogens that destroy the crop. Unfortu- symbiotic relationship with the termites, and this nately, only 10% of the pesticides reach the target makes it difficult for domestication under labora- pests, while the remaining contaminates the envi- tory conditions. Generally, termites are described ronment [38, 39, 40]. These pesticides, including as ecosystem engineers due to their activities in cypermethrin, copper and glyphosate, have been the soil which result in the availability of resources reported to have adverse effects on beneficial in- for other organisms [14]. However, they attack sects and earthworms [41, 42]. Although fungi- and destroy cocoa trees [15, 16]. cides are known to reduce the population size of Cocoa is an important cash crop in the tropics, soil microflora [43], Kwodaga et al. [44] found no and its cultivation usually involves the conversion adverse effects of copper and metalaxyl-based of forests into agro-forests [17-19] which provide fungicides on mycoflora in cocoa soils. Even so, forest-like habitats for the survival of plants, ani- there is the perception that pesticides used in the mals and microorganisms interacting in an ecosys- cocoa agro-ecosystem negatively impact benefi- tem. In the cocoa agro-ecosystem are, edible cial organisms, including edible mushrooms and mushrooms collected, sold, and consumed by the associated termites. This perception was worth in- rural population [20]. Conservation of the cocoa vestigating, especially when residues of copper agro-ecosystem requires planting trees [21-23], have been detected in cocoa soils [45]. This study which may attract pests and pathogens to destroy aimed to investigate the effect of pesticides used the crop [24]. Notable among diseases of cocoa are against cocoa pests on mushrooms (Termitomyces black pod, witches’ broom, vascular streak die- globulus, Termitomyces robustus, P. sajor- back and monilia pod rot, which, together with the caju and V. volvacea) and termites (Macrotermes cocoa swollen shoot virus, account for 40 % of bellicosus). global annual crop yield losses [25]. Insect pests contribute significantly to worldwide yield losses Material and Methods of cocoa [26-28], and in Ghana, mirid damage is Pesticides estimated to reach 30-40 % [29, 30]. Two commercially formulated fungicides and Though there are many methods of managing insecticides commonly used to control black pod insect pests [31] and pathogens [32] in cocoa, pes- disease and mirids on cocoa in Ghana were se- ticides have become the preferred option among lected for this study. The fungicides containing farmers who consider them a quick solution to the metalaxyl 12 % + copper (I) oxide 60 % and cupric problems of insect pests and diseases. In this re- hydroxide 77 % as active ingredients were used gard, a number of insecticides and fungicides are according to the manufacturer’s recommended available to farmers for use in the cocoa agro-eco- rates of 50 (2400 ppm) and 100 g (5130 ppm) in system. Before using bifenthrin, imidacloprid and 15 L of water, respectively. The active ingredients thiamethoxam for the management of mirids, of the insecticides were imidacloprid 20 % and many insecticides were used but banned due to bifenthrin 2.7 %. They were respectively used at their adverse effects on the crop and the environ- rates of 30 and 100 mL in 11 L of water as recom- ment [30, 33, 34,]. These insecticides formed the mended by the manufacturers, thus 545 and 245 basis for preparing other insecticide cocktails ppm. The pesticides were further tested at lower against mirids [31]. However, neonicotinoids, in- rates of 10-1 to 10-4 folds of their recommended JTLS | Journal of Tropical Life Science 2 Volume 13 | Number 1 | January | 2023 EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa rates corresponding to 240, 24, 2.4 and 0.24 ppm uate the effect of the pesticides on the vegetative for metalaxyl + copper (I) oxide; 513, 51.3, 5.13 growth of the mushrooms [49]. The pesticide con- and 0.513 ppm for cupric hydroxide; 54.5, 5.45, centrations as stated above were prepared in 100 0.545 and 0.0545 ppm for imidacloprid; 24.5, mL of molten PDA (Oxoid, UK) at 50℃ and 2.45, 0.245 and 0.0245 ppm for bifenthrin. poured into Petri dishes in a laminar flow cabinet (ESCO, Heal Force Bio-Meditech Holdings Lim- Collection of mushrooms ited, China). The plates were allowed to set and Termitomyces globulus and T. robustus were left overnight in the cabinet for a sterility test. The collected from the floor of a cocoa farm (Plot D20) centre of the plates was inoculated with 5 mm my- at the Cocoa Research Institute of Ghana (CRIG), celial disc plugs taken from the periphery of 5- New Tafo-Akim in March 2020 when few drops day-old cultures of P. sajor-caju and V. volva- of rain were experienced after a long drought. This cea whereas 21 days old cultures were used for T. plot is routinely sprayed with fungicides and in- globulus and T. robustus. Non-amended PDA secticides for the control of black pod disease and plates were similarly inoculated to serve as con- mirids, respectively. P. sajor-caju was collected trol. There were 3 replicated plates per treatment from a local mushroom farm at New Tafo-Akim for each mushroom and they were incubated in the whereas V. volvacea was harvested from a dead oil dark at 25°C. Growth diameter was measured at palm tree at Old Tafo-Akim, all in the Abuakwa right angles to each other 5 days after incubation North Municipality of the Eastern Region of of P. sajor-caju and V. volvacea. Diameter Ghana. Both mushrooms were collected in June growth for T. globulus and T. robustus was rec- 2020. The mushrooms were identified based on orded after 21 days of incubation. Radial growths macro-morphological characteristics such as sha- of the mushrooms were calculated by dividing di- pe, colour and position of cap, stipe, gills, volva, ameter growths by two. and annulus [46, 47]. They were photographed and harvested into sterile transparent ziplock bags for Pesticide toxicity on termite culturing on agar media in the laboratory. The test termites were collected from experi- mental plots of CRIG at New Tafo-Akim. The test Culturing of mushroom concentrations were obtained by diluting the pes- The isolation technique described in Tudses ticides with distilled water. Filter paper was mois- [48] was used to obtain cultures of the mushrooms tened with 1 ml of pesticide concentration and on Potato Dextrose Agar (PDA) (Oxoid, UK). used to line the base of a Petri dish. Ten worker Small pieces (2×2 mm2) of inner tissues were re- termites (Macrotermes bellicosus) were placed on moved from the caps with sterile sharp blades and the filter paper. The setup was allowed to stand for placed on PDA amended with chloramphenicol at 24 h to observe termite mortality. This was repli- 100 mg.L-1 to inhibit bacterial growth. The plates cated thrice for each pesticide concentration and were incubated in the dark at 25°C for 5 days for the controls. Distilled water was used for the con- the mycelial growth of P. sajor-caju and V. volva- trols. The test was done under the following labor- cea. Plates of T. globulus and T. robustus were in- atory conditions; a temperature of 25-29°C, rela- cubated for 10 days. The cultures were purified tive humidity of 75-86%, and a photoperiod of 12 through several transfers on PDA plates at 25°C. L: 12 D. The number of dead termites in each Colony morphology was recorded as growth pat- treatment was counted and converted to percent- tern and growth diameter was measured at right age mortality. angles to each other after 5 days of incubation for P. sajor-caju and V. volvacea and 21 days Data analysis for Termitomyces. The growth diameter was con- Each experiment was repeated twice and ho- verted to radial growth by dividing it by two. The mogeneity of variances for datasets was confirmed cultures were stored in sterile distilled water at by F-tests. Data was pooled together for transfor- 25°C until needed. mation. Data for mycelial growth rate of mush- rooms was square root transformed. Arcsine Effect of pesticides on vegetative growth of square root transformation was performed on in- mushrooms sect mortality data. Effect of different concentra- Poisoned agar technique was used to eval- tions of pesticides on mycelial growth rate of JTLS | Journal of Tropical Life Science 3 Volume 13 | Number 1 | January | 2023 EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa mushrooms and percentage mortality of termites [56]. Moniliophthora perniciosa, the mushroom was statistically analysed by one-way analysis of that causes witches’ broom disease of cocoa, is a variance (ANOVA) using GenStat (11th edition, major problem in Brazil [57]. In Ghana, Marasmi- VSN International Ltd.) at 5% probability level. ellus scandens has been identified to cause thread blight disease [58]. The destructive nature of these Results and Discussion pathogens requires a combination of phytosanitary Mushrooms in the cocoa agro-ecosystem practices and fungicides for effective manage- The collection of T. globulus and T. ro- ment. Amoako-Attah et al. [59] recommended bustus from the floor of a cocoa farm indicates the copper and metalaxyl-based fungicides for the conduciveness of agro-ecosystem as a habitat for management of M. scandens. These fungicides are the survival of edible mushrooms [20]. The floor also effective against Phytophthora, the pathogen was the best place to find the Termitomyces be- of black pod disease. However, these fungicides cause soil covered with rich tree leaf litter presents are perceived to have a negative effect on benefi- a good substrate for their growth [50]. The collec- cial fungi including edible mushrooms in the co- tion of T. globulus and T. robustus at the onset of coa agro-ecosystem. This perception was investi- the rainy season in March confirms their period gated by growing the vegetative phase of T. glob- (February – April) of occurrence in Ghana [11]. In ulus, T. robustus, P. sajor-caju, and V. volva- Côte d’Ivoire, fruiting bodies of Termitomyces are cea on PDA amended with the manufacturers’ rec- usually observed in the rainy season [8]. Fructifi- ommended and lower concentrations of metalaxyl cation of Termitomyces is often associated with + copper (I) oxide and cupric hydroxide fungi- termites in the subfamily Macrotermitinae [9, 13, cides. The study was expanded to include im- 51] as was evidenced in the collection of T. glob- idacloprid and bifenthrin insecticides used for the ulus where the worker termites, M. bellicosus, control of mirids on cocoa. emerged from the holes. Macrotermes belli- cosus is among a group of termites reported to be Macro-morphological characteristics of mush- associated with cocoa farms in Ghana [52]. How- rooms ever, none of the cocoa trees was attacked by ter- Macro-morphological characteristics of P. sa- mites in the cocoa farm where T. globulus and T. jor-caju, V. volvacea, T. globulus, and T. ro- robustus were collected. bustus are summarized in Table 1. Typical of P. The limited period of survey for mushrooms in sajor-caju was the grey to a cream oyster or sea cocoa farms might have prevented the collection seep-shaped cap (Figure 1a). Under the cap were of Lentinus squarrosulus and V. volvacea which crowded, decurrent, soft, and white gills. The solid have been reported to grow in cocoa plantations in stipe lacked annulus and it attached to the cap at Cameroon [20]. To increase the number of edible its margin. There were tiny white mycelial threads mushrooms used in this study, P. sajor- at the base of the stipe without volva. On the con- caju and V. volvacea were added even though they trary, V. volvacea produced a brown volva at the were not collected from the cocoa agro-ecosys- base of the stipe. It had long, thick, and numerous tem. These mushrooms were considered because mycelial threads which supported its growth on a of their availability and consumption in dead oil palm tree. The off-white solid stipe with- Ghana. Pleurotus sajor-caju was collected from out annulus was attached to the centre of a pinkish- prepared sawdust bags whereas V. volvacea was brown umbonate-shaped cap (Figure 1b) with harvested from a decaying oil palm tree. They are crowded, free, and pinkish-brown gills under- the most preferred mushrooms in Ghana [7, 53] neath. On the white solid stipe of T. globulus was which grow on wood logs, forest floors, and de- a large, white pendant annulus. The stipe was at- caying oil palm [11] but Pleurotus species are do- tached to the centre of a white umbonate cap with mestically cultivated on a wide range of substrates brown colouration at the centre (Figure 1c). The including sawdust [7, 54]. stipe tapered at the soil level, and it was deeply In addition to edible mushrooms in cocoa, rooted. Worker termites, M. bellicosus, emerged agro-ecosystems are pathogenic ones. Those that from the holes of the mushroom when uprooted. are pathogenic to cocoa are mostly not edible. For The mycelial thread of T. robustus was relatively instance, Armillaria mellea causes root rot in co- short. It had a white scaled stipe, and it was at- coa [55] and its consumption causes stomachache tached to the centre of a tawny brown and flat cap JTLS | Journal of Tropical Life Science 4 Volume 13 | Number 1 | January | 2023 EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa Table 1. Macro-morphological characteristics of mushroom Mushroom Cap Gills Stipe ring Stipe Cup Mycelial (Annulus) (Volva) threads P. sajor-caju Grey to cream Crowded, Absent White and Absent White and and oyster or decurrent, attached at tiny sea seep- soft and cap margin shaped white V. volvacea Pinkish-brown Crowded, Absent Off-white Brown Long, thick and umbonate free and and numer- with cracks pinkish- ous brown T. globulus White umbo- Crowded, White pen- White and it Absent Long and nate cap with free and dent tapers at the deeply brown coloura- white soil level rooted tion at the cen- tre T. robustus Tawny brown Crowded, Absent White with Absent Short and and flat with free and scales tiny cracks white (a) (b) (c) (d) Figure 1. (a) P. sajor-caju on sawdust (b) V. volvacea on dead oil palm tree (c) T. globulus on the floor in a cocoa farm (d) T. robustus on the floor in a cocoa farm. with cracks (Figure 1d). Under the cap were Effect of pesticides on vegetative growth of crowded, free, and white gills. mushrooms There were significant (p < .001) differences Mushroom cultures in radial growths of the mushrooms when they Table 2 summarizes the colony characteristics were grown on only PDA or when grown on PDA and growth rate of P. sajor-caju, V. volvacea, T. amended with different concentrations of the pes- globulus, and T. robustus. The culture of P. sajor- ticides. Mycelial growths of the mushrooms were caju produced white felty, circular, and flat colo- reduced at the manufacturer’s recommended con- nies with an entire margin on PDA (Figure 2a). centration of the pesticides, but they increased as The colonies of V. volvacea were cottony and concentrations of the pesticides reduced. Bifen- creamy white in appearance (Figure 2b). The thrin completely inhibited mycelial growths of all growths of cultures of P. sajor-caju and V. volva- the mushrooms at the manufacturer’s recom- cea were rapid, attaining radial growths of 42.9 ± mended concentration of 245 ppm for the control 0.1 and 44.5 ± 0.5 mm respectively, on PDA after of mirids on cocoa. At 10-fold reduced concentra- 5 days of incubation at 25°C. On the other hand, tion of 24.5 ppm, mycelial growths ranging from cultures of T. globulus and T. robustus were slow 3.0 mm for P. sajor-caju and T. robustus to 4.3 in growth, attaining radial growths of 15.5±1.0 mm for V. volvacea were recorded. There was a and 16.9±1.1 mm respectively, after 21 days of in- gradual increase in the growths of V. volvacea and cubation. Colonies of T. globulus were whitish- T. globulus as the concentration of bifenthrin re- brown and umbonate in appearance with an undu- duced (Figure 3). Unlike bifenthrin, imidacloprid lating margin (Figure 2c), whereas T. ro- did not completely inhibit mycelial growths of the bustus produced whitish-brown colonies on PDA mushrooms but significantly (p < .001) reduced (Figure 2d) after 21 days of incubation at 25°C. them at the manufacturer’s concentration of 545 JTLS | Journal of Tropical Life Science 5 Volume 13 | Number 1 | January | 2023 EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa Table 2. Colony characteristics and radial growth of Pleurotus sajor-caju, Volvariella volvacea, Termitomy- ces globulus and Termitomyces robustus on Potato Dextrose Agar at 25°C Mushroom #Doi Form Eleva- Margin Texture Colour Reverse *Radial tion growth (mm) P. sajor- 5 Circular Flat Entire Felty White White 42.9±0.1 caju V. volvacea 5 Filamen- Raised Filiform Cottony Creamy Creamy 44.5±0.5 tous white white T. globulus 21 Irregular Umbo- Undulate Com- Whit- Brownish- 15.5±1.0 nate pact ish- white brown T. robustus 21 Irregular Umbo- Undulate Com- Brown- Brown 16.9±1.1 nate pact ish- white #Doi: Number of days of incubation period *Each value is a mean of 3 replicated plates. (a) (b) (c) (d) Figure 2. Cultures of (a) Pleurotus sajor-caju (5 days old) (b) V. volvacea (5 days old) (c) T. globulus (21 days old) and (d) T. robustus (21 days old) on Potato Dextrose Agar at 25°C. 8.0 7.0 Pleurotus sajor-caju 6.0 Volvariella volvacea 5.0 Termitomyces globulus 4.0 3.0 Termitomyces robustus 2.0 1.0 0.0 Concentration of bifenthrin (ppm) Figure 3. Effect of different concentrations of bifenthrin on radial growth of P. sajor-caju, V. volvacea, T. glob- ulus and Termitomyces robustus on Potato Dextrose Agar. Square root transformed data and analyzed by one-way ANOVA at 5% probability level. Least significant difference (Lsd) = 0.5. Bars represent standard error of means. ppm (Figure 4). Mycelial growth of the Termito- tration of 5130 ppm and its 10-fold reduction (Fig- myces was completely inhibited by cupric hydrox- ure 5). Except P. sajor-caju, mycelial growth of ide at the manufacturer’s recommended concen- the other mushrooms was completely inhibited at JTLS | Journal of Tropical Life Science 6 Volume 13 | Number 1 | January | 2023 Radial growth of mushrooms (mm) EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa 8.0 7.0 Pleurotus sajor-caju 6.0 Volvariella volvacea 5.0 4.0 Termitomyces globulus 3.0 Termitomyces robustus 2.0 1.0 0.0 Concentration of imidacloprid (ppm) Figure 4. Effect of different concentrations of imidacloprid on radial growth of P. sajor-caju, V. volvacea, T. globulus and T. robustus on Potato Dextrose Agar. Square root transformed data and analyzed by one-way ANOVA at 5% probability level. Least significant difference (Lsd) = 0.5. Bars represent standard error of means. 8.0 7.0 6.0 Pleurotus sajor-caju 5.0 Volvariella volvacea 4.0 Termitomyces globulus 3.0 Termitomyces robustus 2.0 1.0 0.0 Concentration of cupric hydroxide (ppm) Figure 5. Effect of different concentrations of cupric hydroxide on radial growth of P. sajor-caju, V. volvacea, T. globulus and T. robustus on Potato Dextrose Agar. Square root transformed data and analyzed by one-way ANOVA at 5% probability level. Least significant difference (Lsd) = 0.4. Bars represent standard error of means. the manufacturer’s recommended concentration of sing. This is because of the known toxicity of 2400 ppm for metalaxyl + copper (I) oxide. How- some insecticides to mushrooms. Sharma and De- ever, radial growth of the mushrooms increased wangen [60] found cartap hydrochloride to be drastically at 10-fold reduced concentration of 240 toxic to Agaricus bisporus by inhibiting mycelial ppm below the recommended concentration (Fig- growth of the mushroom on PDA. Copper is a ure 6). broad-spectrum fungicide and hence, its effect on The effect of bifenthrin and imidacloprid on the macro-fungi is possible. According to Das vegetative growth of these four test mushrooms [61], mycelial growth of macro-fungi may be in- demonstrated in this study is the first time in hibited by heavy metals such as copper at low to Ghana. The inhibitory effect of the insecticides on medium concentrations, but death of mushrooms vegetative growth of the mushrooms is not surpri- may occur at higher concentrations. This was evi- JTLS | Journal of Tropical Life Science 7 Volume 13 | Number 1 | January | 2023 Radial growth of mushrooms (mm) Radial growth of mushrooms (mm) EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa 8.0 7.0 Pleurotus sajor-caju 6.0 5.0 Volvariella volvacea 4.0 Termitomyces globulus 3.0 Termitomyces robustus 2.0 1.0 0.0 Concentration of metalaxyl + copper (I) oxide (ppm) Figure 6. Effect of different concentrations of metalaxyl + copper (I) oxide on radial growth of P. sajor-caju, V. volvacea, T. globulus and T. robustus on Potato Dextrose Agar. Square root transformed data and analyzed by one-way ANOVA at 5% probability level. Least significant difference (Lsd) = 0.5. Bars represent standard error of means. 120 100 80 60 40 20 0 0.0 0.0245 0.245 2.45 24.5 245 (Water) Concentration of bifenthrin (ppm) Figure 7. Effect of different concentrations of bifenthrin on mortality of M. bellicosus. One-way ANOVA at 5% probability level. Least significant difference (Lsd) = 10.27. Bars represent standard error of means. d ent in this study where mycelial growth of T. did not significantly (p > 0.05) have inhibitory ef- globulus and T. robustus were completely inhib- fect on radial growth of the mushrooms. This sug- ited at the highest concentration of cupric hydrox- gests that the pesticides may not have a direct ef- ide. Vegetative growth of the Termitomyces was fect on mushrooms in the cocoa agro-ecosystem. mainly inhibited by the pesticides due to their slow This is because of the method of pesticide applica- growth. Radial growths of 15.5 and 16.9 mm were tion which is directed at tree trunks and cocoa can- respectively recorded for T. globulus and T. ro- opies where pests and pathogens attack the crop. bustus on PDA after 21 days of incubation. These The pesticides are washed down the cocoa trees growths were comparatively slower than 42.9 and during rainfall and hence diluted before reaching 44.5 mm which were respectively recorded for P. the soil where edible mushrooms thrive. Detecta- sajor-caju and V. volvacea after 5 days of incuba- ble residues of bifenthrin in cocoa soils range from tion. The synergistic effect of metalaxyl and cop- <0.01 – 0.03 ppm [62]. Kwodaga et al. [63] found per was demonstrated in the complete inhibition of residues of copper in cocoa soil to range from mycelial growth of V. volvacea, T. globulus and T. 14.90 – 27.50 ppm. Residues of bifenthrin in co- robustus at the manufacturer’s concentration. coa soils are less than or equal to 0.0245 ppm, However, lower concentrations of the fungicide where radial growth of V. volvacea, T. globulus JTLS | Journal of Tropical Life Science 8 Volume 13 | Number 1 | January | 2023 Radial growth of mushrooms (mm) Termite mortality (%) EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa 120 100 80 60 40 20 0 0.0 0.0545 0.545 5.45 54.5 545 (Water) Concentration of imidacloprid (ppm) Figure 8. Effect of different concentrations of imidacloprid on mortality of M. bellicosus. One-way ANOVA at 5% probability level. Least significant difference (Lsd) = 19.22. Bars represent standard error of means. 15 10 5 0 0.0 0.513 5.13 51.3 513 5130 (Water) Concentration of cupric hydroxide (ppm) Figure 9. Effect of different concentrations of cupric hydroxide on mortality of M. bellicosus. One-way ANOVA at 5% probability level. Least significant difference (Lsd) = 11.09. Bars represent standard error of means. 16 14 12 10 8 6 4 2 0 0.0 0.24 2.4 24 240 2400 (Water) Concentration of metalaxyl + copper (I) oxide (ppm) Figure 10. Effect of different concentrations of metalaxyl + copper (I) oxide on mortality of M. bellicosus. One-way ANOVA at 5% probability level. Least significant difference (Lsd) = 12.58. Bars repre- sent standard error of means. and T. robustus on bifenthrin-amended PDA than the residues of copper in cocoa soils. This in- plates were similar to the control plates. The con- dicates that lower concentrations of the pesticides centration of 51.3 ppm for cupric hydroxide, at in cocoa soils have no effect on vegetative growth which the radial growth of P. sajor-caju and V. of mushrooms and hence their survival in the co- volvacea was similar to the control plates, is more coa agro-ecosystem. JTLS | Journal of Tropical Life Science 9 Volume 13 | Number 1 | January | 2023 Mortality (%) Termite mortality (%) Termite mortality (%) EK Asare, SW Avicor, Y Bukari, et al. 2023 / Post pesticide survival of mushrooms and termites in cocoa Effect of pesticides on termite organisms in the cocoa agro-ecosystem, strict The insecticides caused a mortality of 60 to compliance to good agricultural practices (GAP) 100% (bifenthrin) and 10 to 100 % (imidacloprid) with respect to pesticide usage should be adhered on the termites after 24 h while cupric hydroxide to. and metalaxyl + copper (I) oxide caused a termite mortality of 0 to 13.3% and 3.3 to 10%, respec- Acknowledgment tively. Higher termite mortality (100%) was rec- We are grateful to the staff of Plant Pathology orded at the manufacturer’s recommended con- and Entomology Divisions of Cocoa Research In- centration of 245 ppm for bifenthrin and its 1000- stitute of Ghana (CRIG) for their assistance in the fold reduced concentration of 0.245 ppm (Figure collection of mushrooms and termites. The assis- 7). Similarly, the manufacturer’s recommended tance of Mr. Prince Kwabena Kwakye and Miss. concentration of 545 ppm for imidacloprid caused Emma Korkor Anthonio in the laboratory inhibi- 100% mortality but significantly (<.001) declined tory and mortality tests for mushrooms and ter- at reducing concentrations of the insecticide (Fig- mites, is gratefully acknowledged. ure 8). Cupric hydroxide caused 13.3% mortality at the manufacturer’s recommended concentration References of 5130 ppm (Figure 9) whereas metalaxyl + cop- 1. Kumar K (2015) Role of edible mushroom as functional per (I) oxide caused 10% mortality at 2400 ppm foods: A review. South Asian Journal of Food Technol- ogy and Environment 1(3&4): 211 – 218. (Figure 10). doi:10.46370/sajfte.2015.v01i03and04.02. Bifenthrin and imidacloprid, commonly used 2. Ache NT, Tonjock RK, Eneke ET B et al. (2019) Mush- in the cocoa ecosystem for the control of mirids, room species richness, distribution and substrate specific- were also effective against termites by causing ity in the Kilum-Ijim forest reserve of Cameroon. Journal of Applied Biosciences 133: 13592 – 13617. 100% mortality at the manufacturers’ recom- doi:10.4314/jab.v133i1.11. mended concentrations. The recommended insec- 3. Marcot BG (2017) A review of the role of fungi in wood ticide for termite control in cocoa is fipronil [34]. decay of forest ecosystems. U.S. Department of Agricul- However, it was not included in this study because ture, Forest Service, Pacific Northwest Research Station. of its limited use in the cocoa ecosystem compared Research Note PNW-RN-575. https://www.fs.fed.us/pnw/pubs/pnw_rn575.pdf. Ac- to the test insecticides. Efficacies of bifenthrin and cessed date: 27th January, 2021. imidacloprid confirm previous reports [64-66] but 4. Lakhanpal TN (2014) Mushroom biodiversity in India: lower imidacloprid concentrations of 54.5 ppm Prospects and potential. Proceedings of the 8th Interna- and lower were less lethal to the termites. This tional Conference on Mushroom Biology and Mushroom Products (ICMBMP8). pp. 7 – 16. doi:10.2737/PNW- suggests that the less detectable residues of the RN-575. pesticides [62] in cocoa soils do not affect the ac- 5. Pegler DN, Vanhaecke M (1994) Termitomyces of South- tivity of termites in the cocoa agro-ecosystem. east Asia. Kew Bulletin 49(4): 717 – 736. doi:10.2307/4118066. 6. 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Journal of Economic Entomology M, Prempeh J (2018) Survey of mushroom consumption 113(1): 367 – 374. doi:10.1093/jee/toz293. JTLS | Journal of Tropical Life Science 12 Volume 13 | Number 1 | January | 2023