African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5233

Full Length Research Paper

In vitro efficacy of Trichoderma asperellum and detached leaflet assay on late blight pathogen: Phytophthora infestans

Kilonzi J. M.
  • Kilonzi J. M.
  • Department of Crops, Horticulture and Soil, Egerton University, P. O. Box 536-0000, Njoro, Kenya.
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Mafurah J. J.
  • Mafurah J. J.
  • Department of Crops, Horticulture and Soil, Egerton University, P. O. Box 536-0000, Njoro, Kenya.
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Nyongesa M. W.
  • Nyongesa M. W.
  • Kenya Agricultural Livestock and Research Organization-Tigoni, P. O. Box 338-0217, Limuru, Kenya.
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  •  Received: 20 August 2019
  •  Accepted: 24 October 2019
  •  Published: 31 May 2020

 ABSTRACT

Late blight is highly variable adapting to new fungicides and overcoming host resistance. The objective of the study was to determine efficacy of Trichoderma asperellum against Phytophthora infestans and its compatibility with fungicides. T. asperellum at 33% (3 × 106), 66% (7 × 106) and 100% (1 × 107 CFU/mL; g/L),   Ridomil® (Metalaxyl 4% + Mancozeb 64%) and Mistress 72® (Cynamoxil 4% + Mancozeb 64%) were plated alongside P. infestans in vitro and detached leaflets assay. Results indicated that Ridomil® and Mistress 72® completely inhibited mycelial growth of P. infestans in vitro and in detached leaves. The 33% T. asperellum concentration had the lowest inhibitory power (38.0%) while 66% (91.10%) and 100% (91.30%) T. asperellum concentrations were not significantly different (P=0.05). Lesion sizes were not significantly different in 66% T. asperellum (1.91 cm2) and 100% (1.89 cm2) concentration while 33% concentration (3.5 cm2) and untreated (3.55 cm2) did not differ significantly. Ridomil® and Mistress 72® had no significant effect on T. asperellum mycelial growth. The results suggest that T. asperellum at 66% was effective in managing late blight. Results further indicate that T. asperellum could be used in combination with fungicides for effective and economical option.
 
Key words: Trichoderma asperellum, Phytophthora infestans, detached leaf assay, in vitro.


 INTRODUCTION

Potato has high potential to  address the food insecurity and low income due to its high yield per unit area and relatively high nutritional value as compared to cereal grains (Azimuddin et al., 2009). The global and Africa annual potato production is estimated to be 377 and 25 metric tonnes, respectively. Kenya is ranked fifth in Africa with production of 1.35 metric tonnes annually (FAOSTAT,   2018).   Potato   demand   is   projected    to increase by 250% by 2020 with an annual demand increase of about 3.1% (Scott et al., 2010). In Kenya, potato yield per hectare ranges between 8 and 10 tonnes compared to a potential of 40 tonnes. This low yield is attributed to diseases as well as poor seed quality (Muthoni et al., 2013)with late blight being the major that reduce yield  (Were et al., 2014).
 
Potato  late  blight  caused  by  Phytophthora  infestans
 
can cause up to 100% yield loss depending on weather conditions and variety susceptibility (Mariita et al., 2016). Globally, late blight is responsible for 6.7 billion USD worth of potato yield loss annually (Nowicki and Majid, 2012)and therefore threatens food security globally (Cooke et al., 2012). Late blight epidemics is accelerated by shortage of seed experienced in most sub Saharan countries that has led to adoption of farm saved seed by farmers (Okello et al., 2017). Potato blight occurs in all potato production regions worldwide and is considered as the world’s costly disease because it is managed by extensive use of fungicides that cost about 1 billion USD (Haverkort et al., 2008). Late blight can rapidly defoliate a whole field within a week if unchecked resulting to tuber infection that lowers both tuber quality and quantity (Gigot et al., 2009). The oomycete survives well in potato seed (Johnson and Cummings, 2009)and serves as source of primary inoculum to new crops resulting into early late blight epidemics (Runno-Paurson et al., 2013). Seed tubers available to farmers may have latent infection that produce viable sporangia that cause disease epidemics  in the new crop (Johnson and Cummings, 2013).
 
Ridomil® and Mistress 72® are among the most widely used fungicides in Kenya to manage late blight due to their curative, preventive and systemic modes of action (Nyankanga et al., 2007). However, dependence on chemical application has raised environmental and human health concerns and emergence of fungicide insensitive strains including metalaxyl insensitive isolates threatening the efficacy of fungicides (Matson et al., 2015). In addition, emergence of new strains of P. infestans that adapt to new chemical fungicides and host resistance has led to reduced spray intervals (three to five days interval) resulting into 10 to 15 sprays per cropping season (Njoroge et al., 2019). Use of biocontrols could offer the best sustainable and ecofriendly alternative to chemical application (Yao et al., 2016). Biological agents including Trichoderma species, Bacillus species and Pseudomonas fluorescents and plant extracts have been explored in managing late blight on Solanaceae plants (An et al., 2010;Chowdappa et al., 2013; Kabir et al., 2013). Trichoderma spp. is one of the most studied fungi that is widely used in management of diseases. The fungus is known to induce systemic disease resistance in plants as well as offer prior protection by activating enzymes that degrade cell walls in the pathogen (Yao et al., 2016). Saravanakumar et al. (2016)reported that suppression mechanisms of Trichoderma harzianum on P. infestans through competition, antibiosis, promotion of crop growth and mycoparasitism while Wu et al. (2017)reported enzymatic activities against plant and soil borne pathogens. Various studies have shown that Trichoderma asperellum could manage a number of plant diseases. Patel and Saraf (2017), Carrero-carr et al. (2016), and Kipngeno et al. (2015)reported that T. asperellum could manage Fusarium wilt in tomato, Verticillium wilt in olive and  Pythium   in   tomato.   However,   there   is    limited information on the efficacy and potential of T. asperellum to manage late blight on potato.
 
The use of biocontrols is yet to be fully exploited in managing late blight, because synthetic fungicides act faster than biocontrols against disease causing agents (Xu et al., 2011). The quick action of fungicides against plant pathogens can be effectively tapped and combined with biocontrols to reduce overuse that has raised economical, human and environmental concerns. However, little is known on possibility of combining the two in managing P. infestans on potato that could result to reduced chemical applications. Therefore, the objective of the study was to determine the antifungal activity and efficacy of T. asperellum against P. infestans in vitro and detached leaflets and to assess the compatibility of Ridomil® and Mistress 72® with T. asperellum.


 MATERIALS AND METHODS

Isolation, culturing and bulking of P. infestans inoculum
 
Thirty freshly blighted potato leaves samples were collected randomly from Kenya Agricultural Livestock and Research Organization (KALRO) Tigoni fields. The centre is located at longitude 36 4’ 72’’ east and latitude 10° 9ËŠ 22˝ south and located at an altitude of 2300 m above sea level (Jaetzold et al., 2006). Isolation of associated fungi and pure culture on Pea Agar amended with rifampicin antibiotics (50 µg/mL) was obtained according to Rhouma et al. (2016). Mycelial plug of about 80 mm in diameter of the pure culture from Pea Agar plates was obtained using a sterilized spatula and put in 20 mL Eppendorf tube containing 10 mL of sterilized distilled water. The suspension was vortexed for 2 min using electric vortex model VM-1000 of MRC Laboratory Equipment Company at 3000 revolution per minutes (rpm), filtered through sterilized four layered muslin cloth and incubated in refrigerator for 4 h at 4°C to enhance sporangia and zoospore formation. Identification and pathogenicity test was done on healthy test potato seedling and tuber slices of  Asante varieties using Koch’s postulates (Forbes, 1997). This was used for the detached leaf assay.
 
Antifungal bioassay through co-inoculation of T. asperellum and P. infestans
 
Dual culture method was employed at the Tigoni laboratories of Kenya Agricultural Livestock and Research Organization (KALRO) to determine the inhibition of P. infestans caused by the biocontrol agent as described by Fatima et al. (2015). T. asperellum pure spores were obtained from Real IPM Company, Kenya and their viability confirmed on Potato Dextrose Agar (PDA). The PDA in 9 cm Petri dishes were inoculated with 0.5 × 0.5 cm P. infestans mycelial plug cut using sterilized surgical blades and incubated at 18°C for 48 h. This was followed by introduction of the biocontrol suspensions prepared as follows: 0.1 g of T. asperellum spores powder was weighed and placed in falcon tube containing 10 mL of distilled water and was adjusted to 1 × 107 CFU/mL (100%) using hemocytometer. 66% (7 × 106 CFU/mL) and 33% (3 × 105 CFU/mL) concentrations were achieved by varying the 0.1 g of T. asperellum that formed 100% concentration by 66 and 33%, respectively followed by adjustment using hemocytometer. This was mixed with sorghum coarse grains and incubated at  room  temperatures  for  3 days to initiate sporulation. Around 20 µL of the suspension was pipetted into PDA plates bearing P. infestans culture about 1 cm from the point of inoculation of the pathogen in the Petri Dishes. To prepare positive control plates, 20 µL droplet of positive controls (Ridomil® (2.5 g/L) and Mistress 72® (2 g/L)) were separately inoculated in similar way as the T. asperellum concentrations mentioned earlier. Negative control plates were inoculated with P. infestans only. The plates were incubated at room temperature (18 ± 2°C) under alternating lighting of 12-h light and 12-h darkness for 7 days (Goufo et al., 2017). The treatments were laid in completely randomized design with three replications. The experiment was repeated two times and observations were made using optical microscope on 3rd, 5th and 7th days after inoculation. Inhibition of the test phytopathogenic fungus and the control were determined by the percentage of mycelial growth inhibition in centimetres (cm) calculated by the formula of Yao et al. (2016):
 
I= (R1-R2/R1)×100
 
Where I represents the percentage reduction of growth (inhibition) of the fungi, R1 diameter of radial growth of pathogenic fungus in control plates and R2 diameter of radial growth of P. infestans in the presence of T. asperellum concentrations.
 
Antagonistic effects of T. asperellum on P. infestans in detached leaf assay
 
Approximately equal in size (6 cm long × 4.5 cm wide) healthy leaflets from middle canopy were detached from apical cuttings (6-7 weeks old) in glasshouse using sterilized office scissors. The leaflets were washed with sterilized distilled water and their bases covered with moist cotton to reduce desiccation (Goufo et al., 2008).  T. asperellum concentrations and positive controls (Mistress 72® and Ridomil®) suspensions were prepared and applied by dipping the leaves for 1 s on the abaxial side only in the suspensions under study in a shallow dish. The leaves were then placed upside down (abaxial surface up) in 20 cm (length) × 18 cm (width) × 6 cm (depth) plastic dishes lined with a wet serviette paper to create humidity (6 leaflets per dish). Pure culture of P. infestans mycelial plug from the incubator was scrubbed using a sterilized spatula and put in Eppendorf tube containing 10 mL of sterilized distilled water. The suspension was vortexed for 2 min using electric vortex and filtered through four layered cheese cloth. The suspension was incubated in the refrigerator for 4 h at 4°C to enhance sporangia and zoospore formation. The suspension was adjusted to 1 × 104 zoospores/mL using hemocytometer and 40 µL was applied on the abaxial side of leaf using a micropipette. The negative control included leaves inoculated with P. infestans alone.
 
The plastic dishes while open were placed in laminar flow hood for about 5 min to air dry the wet leaves and then incubated at room temperatures (20 ± 2°C) for 24 h. The treatments were laid in completely randomized design with three replications and measurements on lesion size taken after 3 days and then once after every two days for two weeks. The lesion size was measured using the formula of Fontem et al. (2005):
 
 
Where S, L and W represents the area, length and width of the lesion for each detached leaflet, respectively. π = 3.14
 
T. asperellum compatibility with Ridomil® and Mistress 72®
 
The mycelial plug of T. asperellum from  PDA  plate  was  scrubbed using a sterile spatula and placed in 10 ml of distilled sterilized water in falcon tube. About 40 µL (1 × 107 sporangia/mL) of the suspension was drawn and inoculated on fresh PDA plate and incubated for 48 h. About 40 µL of Ridomil® (2.5g/L) and Mistress 72® (0.5 g/L) was applied on the developing T. asperellum mycelia in PDA plates. Mycelial growth was observed daily under optical microscope for 7 days.
 
Data analysis
 
Data on percentage inhibition of P. infestans induced by T. asperellum and lesion size on detached leaf assay (first transformed using the square root (x + 0.5) (Goufo et al., 2008)was subjected to analysis of variance (ANOVA) using Statistical Analysis System (SAS) version 8.2. Treatment means were separated using Tukey’s Honest Significant Difference (HSD) whenever ANOVA showed significant difference (p < 0.05) among the treatment means.

 


 RESULTS

Antifungal bioassay
 
The antagonistic activity of T. asperellum against P. infestans was observed in dual culture but depended on the biocontrol inoculum concentration. T. asperellum at 66 and 100%, positive control at manufacturer recommended rates (MRR), Ridomil® and Mistress 72® significantly (p=0.05) inhibited mycelial growth of P. infestans  in vitro (Figure 1). P. infestans mycelial growth inhibition was clearly observed on the third day reaching 89.7 and 89.3% but showed no observable change in growth after the fifth day on plates treated with Ridomil® and Mistress 72®, respectively. Higher mycelial growth rate of T. asperellum at 66% (64 mm) and 100% (65 mm) than P. infestans (42 mm) in the pure culture was observed from third day after inoculation (Figure 2). There was no significant difference (p<0.05) in rate of growth of P. infestans treated with T. asperellum concentrations at 66 and 100%, Ridomil® and Mistress 72® by third day after inoculation. T. asperellum concentration at 33% gave the lowest inhibition (37.3, 46.0 and 38.0 mm) in co-culture across all days, respectively after inoculation (Table 1). Figure 2 shows P. infestans mycelial growth continues even in the presence of T. asperellum at 33% concentration. P. infestans and 33% T. asperellum in the dual culture had a lower mycelial growth rate than any other treatment. This figure also shows that pure cultures of 33% T. asperellum concentration growth rate was similar to that of P. infestans in separate plates and indeed in 33% T. asperellum and P. infestans dual culture, P. infestans mycelial radius was higher than dual culture associated with 66 and 100% T. asperellum whose mycelial growth was higher than the pathogen in separate pure cultures. In addition, 66 and 100% T. asperellum co-culture shows a plateau at day three indicating successful inhibition of the   pathogen   while   for   33%  dual   culture    showed continuous growth. An increase in T. asperellum inoculum concentration (from 33 to 100%) resulted to enhanced growth restriction of pathogen from 3rd to 7th day after inoculation (Table 1).
 
Determination of antagonistic effects of T. asperellum against P. infestans in detached leaflet assay
 
T. asperellum and the fungicides influenced late blight lesion size on the detached leaf assay. Lesion size was progressively reduced from day 3 by 4, 14 and 16% to 11th day by 1, 47 and 49%) after inoculation when T. asperellum at 33, 66 and 100% concentrations were applied, respectively relative to P. infestans (Table 2). There was no significant difference (p<0.05) in lesion size between T. asperellum at 66% (1.91 cm2) and 100% (1.89 cm2) concentrations. Lesion expansion was curtailed in detached leaflets treated with Mistress 72® and Ridomil® (Figure 3). Initially (first 3 days after inoculation), T.  asperellum  at  33%  concentration    was able to manage lesion increase thereafter it was not significantly different with the negative control (P. infestans alone) indicating its inability to halt the pathogen growth and multiplication.
 
T. asperellum compatibility with Ridomil® and Mistress 72®
 
T. asperellum pure culture colony grew in Potato Dextrose Agar (PDA) forming a white concentric ring that changed to green as it matured after 5 days (Figure 4). Establishment and development of T. asperellum was not inhibited in anyway by Ridomil® nor by Mistress 72® in vitro. T. asperellum mycelia continued to grow in PDA over the incubation period neither in Ridomil® nor by Mistress 72®. Radial mycelial growth for pure T. asperellum culture, T. asperellum + Ridomil®  and T. asperellum + Mistress 72® were not significant different. However, Mistress 72® showed more T. asperellum mycelial growth suppression than Ridomil® across all days after inoculation (Figure 5).
 
 
 
 


 DISCUSSION

The use of biological agents to suppress plant diseases has been demonstrated in previous studies to inhibit P. infestans (Miles et al., 2012; Fatima et al., 2015;Yao et al., 2016; Syed et al., 2018).  The  results  of  the  present study indicate that T. asperellum is pathogenic and aggressive against P. infestans but this inhibitory action is underpinned by the initial biocontrol inoculum concentration. These findings are consistent with previous study of Kipngeno et al. (2015)who reported the efficacy of Bacillus subtilis and T. asperellum on  Pythium  aphanindermatum in tomato. Positive results were also reported by Istv (2014), Widmer (2014),Fatima et al. (2015), Yao et al. (2016), and Bahramisharif and Rose (2018)during their studies in effort to manage potato diseases using biocontrols. Agbeniyi et al. (2014)reported that T. asperellum was able to reduce fungicide Cacao pod rot severity when combined with mancozeb fungicide. However, there is need for new strains of biological agents from the vicinity of host plant that may have a better biocontrol activity against phytopathogens to be explored to enhance effective disease control.  Therefore, this study attempts to explore the effects of T. asperellum on P. infestans to widen the biocontrol’s action spectrum against pathogens when combined with reduce fungicides application frequency.
 
Antifungal bioassay
 
In dual culture, T. asperellum at 66 and 100% concentration which were not significantly different had the highest inhibitory action in vitro. The treatments had a high sporulation and competing capacity filling up the PDA plates faster than P. infestans. This overwhelmed the pathogen and indeed the Trichoderma mycelia grew over the P. infestans. High rate of sporulation and colonization  are   the  key  important  traits  for  excellent biocontrol (Xu et al., 2011), zone of inhibition was observed between the two fungi which could be attributed to the effect of diffusible products released by the T. asperellum which suppressed further growth of the P. infestans. The continual growth of P. infestans mycelial in the presence of T. asperellum at 33% concentration (Figure 2) indicated that concentration of biocontrols is an important factor in their action against disease causing microorganisms. Similar results were reported by Patel and Saraf (2017)on efficacy of T. asperellum against Fusarium oxysporium on tomato (Lycospercum esculenta) that caused 85% wilting severity decrease. The mycoparasitic action of the T. asperellum suggests that the biocontrol has a high potential of managing late blight of potato. Similar mycoparasitism and competition by Trichoderma spp. against phytopathogens were reported by Sharma et al. (2017).  The inhibition zone observed (white mycelial growth) indicated release of metabolite products by the biocontrol which was also reported by Widmer (2014). The absence of inhibition zone observed in dual culture associated with 33% T. asperellum-P. infestans interaction suggests that the biocontrol concentration was low and therefore unable to release sufficient metabolites to overcome similar products released by the pathogen. This was consistent with Sharma (2011)study who reported Fusarium wilt-Trichoderma  chemical   signal  interactions.  Further,  the two increase in mycelial growth was low compared to other treatments in co-culture possibly due to ‘tag of war’ involving metabolites that have to be secreted first and in sufficient amounts before being released by the two fungi against each other where the strong one overcome the other. Leonetti et al. (2017)and Naglot et al. (2015)reported SA signaling pathway and enzymatic activities, respectively have to be started before mycelial growth. This shows that for a biocontrol to be effective it should have high reproduction and strong in releasing metabolites that cause antibiosis, cell wall degradation and mycoparasitism as reported by Wu et al. (2017). T. asperellum mycelia growth was directed towards the P. infestans indicating chemotropism towards the pathogen as observed bySharma (2011). However, the present study reports the antifungal activities by T. asperellum being slow allowing the pathogen to partially grow compared to Ridomil® and Mistress 72® that were effective in inhibiting P. infestans growth in 33% T. asperellum concentration suggesting fast growth and inhibitory is affected by concentration of the biocontrol. The effectiveness of Ridomil® and Mistress 72® could be attributed to the fact that their active ingredients act by targeting specific region of the pathogen as reported bySharma and Saikia (2013).
 
Antagonistic effects of T. asperellum against P. infestans in detached leaflet assay
 
The trend in slowed increase in lesion size from day three to eleven (Table 2) after inoculation indicate that T. asperellum are slow acting as reported by Lal (2017). The 66 and 100% T. asperellum inhibited late blight lesion increase while Ridomil® and Mistress 72® did not allow lesion establishment at all. The 33% T. asperellum lesion size was similar to that of P. infestans alone (control) providing further evidence that concentration of the biocontrol is a key trait. Even though their lesion development was observed in 66 and 100% T. asperellum, the treatments in the long run managed the disease lesion preventing further increase showing biocontrols have slow action against phytopathogens. The biocontrol required pathogen signal to secrete enzymes, pathogenesis related proteins and metabolites that may take some time giving the pathogen a chance to establish. The observed antifungal activity could be attributed to a faster growth of the T. asperellum compared to P. infestans (competition) and secondary metabolites (defense mechanisms) released by the biocontrol that have antagonistic activity against the pathogen (Amin et al., 2010). Schuster et al. (2010)reported the presence of cell wall degrading enzymes including the glucanases that degrade P. infestans cells affecting their growth. Further, the antagonistic activity of T. asperellum on P. infestans revealed mycoparasitism of P.  infestans  as  indicated   presence  of   white  mycelial between the two fungi that confirms the report of Itachi et al. (2007). Similar mycoparasitic actions were observed in Trichoderma viride antagonistic activities against P. infestans in vitro (Ephrem et al., 2011). This study provides evidence that a biocontrol concentration is an important characteristic in biological agents for them to be effective and this is observed missing in the literatures.
 
At 66 and 100% concentrations, T. asperellum provided excellent control of late blight. Thus, it appearance at 66% of T. asperellum concentration could be used to manage the P. infestans under field conditions. The implication is the slowing of the rate of evolution of new strains that adapt to new fungicides formulations and resistant varieties. Newly emerging strains tend to be aggressive and require increased fungicide application (Childers et al., 2014)to control. This poses a threat to environment, human population and increased cost of production (Cooke et al., 2011). The present work also reports unhindered mycelial growth of T. asperellum in the presence of Ridomil® and Mistress 72® indicating possibility of combining the two in integrated disease management program. This information could lead to adoption of effective rate of T. asperellum which offer safer option as they are eco-friendly and can be combined with fungicides in effort to reduce overuse of synthetic fungicides.
 
T. asperellum compatibility with Ridomil® and Mistress 72®
 
In Ridomil® and Mistress 72®-T. asperellum compatibility experiment, the biocontrol radial mycelial growth was not inhibited by Ridomil® and Mistress 72®. These results are in agreement with Aparecida et al. (2018)who reported that Trichoderma asperelloides reduced Sclerotinia minor growth more when combined with azoxystrobin. Thus, the results indicated possibility of combining T. asperellum with either of these two synthetic fungicides in alternation to effectively control late blight while reducing human, economic and environmental concerns. However, Mistress 72® had more suppression on T. asperellum mycelial than Ridomil®. The two fungicides have mancozeb in similar concentration but in addition Ridomil® has metalaxyl while Mistress 72® has cynamoxil. This suggests cynamoxil may have inhibition aspects if its concentration was increased while T. asperellum could tolerate metalaxyl better. Co-formulation of fungicides with metalaxyl aims at lower metalaxyl dose to reduce chances of resistance development by P. infestans strains (Muchiri et al., 2017).


 CONCLUSION

This   study   aimed   to   ascertain   effectiveness   of   T. asperellum in managing late blight and possibility of combining the biocontrol with fungicides under controlled conditions. Concentration of biocontrol is one of the most key characteristics in enhancing their effectiveness. Late blight development was influenced by change in concentration of the T. asperellum. In vitro and detached leaflet assay experiments demonstrate T. asperellum concentrations against P. infestans are key. However, the study did not establish mechanisms and defence metabolites expressed by T. asperellum which could be explored in the future studies. Although this biological agent was effective in controlling P. infestans in vitro and detached leaf assay conditions, their adoption could offer safer and sustainable alternative to synthetic fungicides and become a key component of Integrated Disease Management (IDM) system under field conditions that will ultimately reduce fungicides usage and their negative impacts, thereby contributing to increased national potato production. However, field trials of the biocontrol are required to determine consistency as well as possibility of managing other diseases of potato. Cynamoxil is one of the proposed fungicide molecules to replace metalaxyl due to emergence of metalaxyl resistant P. infestans strains, therefore further studies need to focus on increased cynamoxil dosage on T. asperellum mycelial growth.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

 


 ACKNOWLEDGEMENTS

The authors thank CESAAM Egerton University for providing funds and the Centre Director, Kenya Agricultural and Livestock Research organization (KALRO)-Tigoni for providing laboratory and technical staff. They also thank Real IPM Company, Kenya for providing the biocontrol used in this study.



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