Whole genome comparisons reveal panmixia among fall armyworm (Spodoptera frugiperda) from diverse locations.

BACKGROUND: The fall armyworm (Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of > 80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat. RESULTS: In this study, the population structure and genetic diversity in 55 S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa. CONCLUSIONS: Our research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.

Whitefly-induced tomato volatiles enhance the virulence of Lecanicillium lecanii.

Entomopathogenic fungi (EPF) are a group of microorganisms that have potential for replacing synthetic chemical pesticides. However, EPF virulence is often insufficient, and therefore adoption of EPF biopesticides has been relatively limited. Previous studies have shown that herbivore-induced plant volatiles (HIPVs) promoted the virulence of Lecanicillium lecanii, and that extracellular proteases and chitinases are important virulence factors of entomopathogenic fungi. We therefore put forward the hypothesis that HIPVs promote L. lecanii virulence by inducing extracellular protease or/and chitinase activity. Lecanicillium lecanii was treated with tomato HIPVs induced by various numbers/densities of Bemisia tabaci, the effect of HIPVs on L. lecanii virulence and activities of virulence-related enzymes (extracellular proteases and chitinases) was analyzed. Changes in enzyme activities were confirmed by measuring the variations in Pr1 and Chit II genes expression of virulence-related enzymes under the influence of HIPVs. Finally, the correlation between virulence and virulence-related enzymes or genes was analyzed to explore the mechanism of promoting HIPVs in L. lecanii virulence enhancement. The results showed that volatiles produced by the tomato plants induced by 50 B. tabaci adults (50-HIPVs) significantly enhanced the virulence of L. lecanii. When exposed to both 50-HIPVs and L. lecanii, the mortality rate of B. tabaci reached 79.0% within 7 days compared to 28.87% in the control group. Additionally, 50-HIPVs significantly promoted the activity of chitinase and the relative expression of Chit II genes, which consequently increased by 62.74% and 48.55%, respectively. However, each group of HIPVs had no promoting effect on extracellular protease and Pr1 gene expression. Correlation analysis results showed that the virulence of L. lecanii is significantly correlated with the activity of chitinase and relative expression of Chit II genes. Thus, HIPVs induced virulence of L. lecanii by increasing chitinase activity. This study demonstrates important methods to enhance the virulence of L. lecanii. Moreover, from the perspective of chemical ecology, the findings provide theoretical support for field application of EPF, and also reveal the impact of HIPVs on EPF from a biochemical perspective.

Combining insect pathogenic fungi and a pheromone trap for sustainable management of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is a key invasive pest of maize and other crops in Africa. Entomopathogenic fungi play an important role in regulating the immature stages of this invasive pest as opposed to synthetic pesticides that are hazardous to human, environment and biodiversity. To tackle the adult stage of the pest (the moth) and to improve on the application strategy of the fungal-based biopesticides, this study evaluated the effect of various entomopathogenic fungi isolates on S. frugiperda moths. Twenty-two isolates (16 Metarhizium anisopliae and 6 Beauveria bassiana) were screened in the laboratory to assess their pathogenicity and virulence against S. frugiperda moths. The compatibility of the most pathogenic isolates with S. frugiperda pheromone FALLTRACT lure, the horizontal transmission of the inoculum among S. frugiperda moths, and the effect on oviposition were also determined under laboratory conditions. All 22 fungal isolates screened were pathogenic to the moths, but the mortality varied significantly among the isolates (P < 0.0001) seven days post-treatment. Beauveria bassiana ICIPE 621 and M. anisopliae ICIPE 7 outperformed all the other isolates by causing 100% mortality of the moths with the lowest LT50 values of 3.6 ± 0.1 and 3.9 ± 0.0 days, respectively. Both isolates were also found compatible with FALLTRACT lure, as the lure had no effect on the conidial germination in the laboratory. Male and female moths were able to horizontally transmit conidia of both fungal isolates to untreated moths, causing high mortality of S. frugiperda in 'donor' and 'recipient' groups. In addition, the oviposition, hatchability of eggs and longevity of larvae were significantly affected on the fungal infected females. Although single moths still retained high conidial numbers 72 h post-inoculation, the number of conidia decreased with time. These results suggest that ICIPE 7 and ICIPE 621 could be used in combination with S. frugiperda pheromone in an autodissemination approach to suppress S. frugiperda population.

Horizontal transmission of Metarhizium anisopliae between Spoladea recurvalis (Lepidoptera: Crambidae) adults and compatibility of the fungus with the attractant phenylacetaldehyde.

The compatibility of the entomopathogenic fungus Metarhizium anisopliae ICIPE 30 which was proved to be pathogenic to adult Spoladea recurvalis, and phenylacetaldehyde (PAA) floral attractant for lepidopteran moths, was investigated under laboratory and field conditions through spatial and temporal separations. Horizontal transmission of M. anisopliae ICIPE 30 between adult S. recurvalis and the number of conidia picked up by a single moth from the autoinoculation device were also determined under laboratory tests. When freshly emerged moths were inoculated with fungal conidia ("donors") and maintained together with an equal number of untreated freshly emerged moths ("recipients") in the laboratory, they were able to transmit infection to untreated moths resulting to 76.9% mortality with an LT50 value of 6.9 days. The quantity of conidia a moth could acquire and retain from the autoinoculation device in the laboratory was assessed at 0, 24, 48, and 72 h post-inoculation. The overall mean number of conidia acquired by a single moth was significantly higher immediately after exposure (0 h) (14.3 ±â€¯2.5 × 105) than at 24, 48, and 72 h after inoculation (F = 10.26, Df = 3,8, P = 0.003), though a single moth still retained 4.6 ±â€¯0.9 × 105 conidia 72 h post inoculation. Laboratory results showed that PAA completely inhibited the germination of the conidia 8 days post exposure, while the conidial viability was not affected in the control treatment without PAA. Under field conditions, the inhibitory effects of PAA on conidial germination was minimized by placing it at a distance of 5-10 cm from M. anisopliae isolate ICIPE 30 conidia. There was no significant difference in conidial germination in the control treatment and in treatments where PAA was placed at 5 cm and 10 cm away from M. anisopliae isolate ICIPE 30. Conidial germination was low in the autoinoculation device that had PAA directly exposed to the fungus. PAA is therefore compatible with M. anisopliae ICIPE 30 for use in integrated management of S. recurvalis, if spatially separated 5 cm away from the fungus and could thus be combined in an autocontamination devices for the control of S. recurvalis.

Metarhizium anisopliae infection reduces Trypanosoma congolense reproduction in Glossina fuscipes fuscipes and its ability to acquire or transmit the parasite.

BACKGROUND: Tsetse fly-borne trypanosomiasis remains a significant problem in Africa despite years of interventions and research. The need for new strategies to control and possibly eliminate trypanosomiasis cannot be over-emphasized. Entomopathogenic fungi (EPF) infect their hosts through the cuticle and proliferate within the body of the host causing death in about 3-14 days depending on the concentration. During the infection process, EPF can reduce blood feeding abilities in hematophagous arthropods such as mosquitoes, tsetse flies and ticks, which may subsequently impact the development and transmission of parasites. Here, we report on the effects of infection of tsetse fly (Glossina fuscipes fuscipes) by the EPF, Metarhizium anisopliae ICIPE 30 wild-type strain (WT) and green fluorescent protein-transformed strain (GZP-1) on the ability of the flies to harbor and transmit the parasite, Trypanosoma congolense. RESULTS: Teneral flies were fed T. congolense-infected blood for 2 h and then infected using velvet carpet fabric impregnated with conidia covered inside a cylindrical plastic tube for 12 h. Control flies were fed with T. congolense-infected blood but not exposed to the fungal treatment via the carpet fabric inside a cylindrical plastic tube. Insects were dissected at 2, 3, 5 and 7 days post-fungal exposure and the density of parasites quantified. Parasite load decreased from 8.7 × 107 at day 2 to between 8.3 × 104 and 1.3 × 105 T. congolense ml- 1 at day 3 post-fungal exposure in fungus-treated (WT and GZP-1) fly groups. When T. congolense-infected flies were exposed to either fungal strain, they did not transmit the parasite to mice whereas control treatment flies remained capable of parasite transmission. Furthermore, M. anisopliae-inoculated flies which fed on T. congolense-infected mice were not able to acquire the parasites at 4 days post-fungal exposure while parasite acquisition was observed in the control treatment during the same period. CONCLUSIONS: Infection of the vector G. f. fuscipes by the entomopathogenic fungus M. anisopliae negatively affected the multiplication of the parasite T. congolense in the fly and reduced the vectorial capacity to acquire or transmit the parasite.

Impact of pre-harvest fungicide application on the storage performance of tomato fruits.

To examine the impact of pre-harvest fungicide applications on the postharvest storage performance of tomato fruits, we measured the lycopene content, hardness, soluble solids content, rotting rate, and weight loss rate of the fruits, as well as conducted a sensory assessment. Protective and systematic fungicides were sprayed on tomatoes 20 days before harvest in order to prevent rotting and weight loss during storage. Our findings showed that, the fungicide-treated tomatoes had a significantly lower rotting rate of 16.00% and a weight loss rate of 3.96%. However the control group experienced 65.33% rotting rate and 6.90% weight loss rate on 12th days of storage. Out of the pre-harvest applications, 'Zineb' a protective fungicide significantly delayed the loss of hardness and soluble solids accumulation in tomato fruits during storage, but it had no significant effect on lycopene content. On the other hand, when comparing to the examined treatment, the systemic fungicides did not have any significant effect on the postharvest storage performance of tomato fruits. Sensory evaluation results indicated that systemic fungicides improved the aroma of the fruits, while protective fungicides had a greater impact on the appearance and juiciness of the fruits. This study offers a potential novel solution for preserving fruits and vegetables which have been frequently infected by phytopathogens during storage, and consequently mitigate/reduce postharvest losses.

Biostimulant and antagonistic potential of endophytic fungi against fusarium wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici.

Endophytic fungal-based biopesticides are sustainable and ecologically-friendly biocontrol agents of several pests and diseases. However, their potential in managing tomato fusarium wilt disease (FWD) remains unexploited. This study therefore evaluated effectiveness of nine fungal isolates against tomato fusarium wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL) in vitro using dual culture and co-culture assays. The efficacy of three potent endophytes that inhibited the pathogen in vitro was assessed against FWD incidence, severity, and ability to enhance growth and yield of tomatoes in planta. The ability of endophytically-colonized tomato (Solanum lycopersicum L.) plants to systemically defend themselves upon exposure to FOL were also assessed through defence genes expression using qPCR. In vitro assays showed that endophytes inhibited and suppressed FOL mycelial growth better than entomopathogenic fungi (EPF). Endophytes Trichoderma asperellum M2RT4, Hypocrea lixii F3ST1, Trichoderma harzianum KF2R41, and Trichoderma atroviride ICIPE 710 had the highest (68.84-99.61%) suppression and FOL radial growth inhibition rates compared to EPF which exhibited lowest (27.05-40.63%) inhibition rates. Endophytes T. asperellum M2RT4, H. lixii F3ST1 and T. harzianum KF2R41 colonized all tomato plant parts. During the in planta experiment, endophytically-colonized and FOL-infected tomato plants showed significant reduction of FWD incidence and severity compared to non-inoculated plants. In addition, these endophytes contributed to improved growth promotion parameters and yield. Moreover, there was significantly higher expression of tomato defence genes in T. asperellum M2RT4 colonized than in un-inoculated tomato plants. These findings demonstrated that H. lixii F3ST1 and T. asperellum M2RT4 are effective biocontrol agents against FWD and could sustainably mitigate tomato yield losses associated with fusarium wilt.

Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta.

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

Vendors' handling practices of edible long-horned grasshoppers (Ruspolia differens) products and implications on microbial safety.

Edible grasshopper, Ruspolia ruspolia, has nutritional and cherished cultural and economic importance to people from diverse cultures, particularly in over 20 African countries. It is consumed at home or commercially traded as sautéed, deep-fried, or boiled products. However, there is limited information on the hygiene practices of the vendors and the implications on the microbial safety of the final product. This research aimed at assessing the food safety knowledge, handling practices and shelf life of edible long-horned grasshopper products among vendors and the microbial safety of ready-to-eat products sold in 12 different markets in Uganda. Samples of raw, deep-fried and boiled grasshoppers were randomly collected from 74 vendors (62% street and 38% market vendors) and subjected to microbial analysis. Over 85% of the vendors surveyed had no public health food handler's certificate and >95% had limited post-harvest handling knowledge. Total aerobic bacteria (7.30-10.49 Log10 cfu/g), Enterobacteriaceae (5.53-8.56 Log10 cfu/g), yeasts and molds (4.96-6.01 Log10 cfu/g) total counts were significantly high and above the acceptable Codex Alimentarius Commission and Food Safety Authority of Ireland (FSAI) limits for ready-to-eat food products. Eight key pathogenic bacteria responsible for foodborne diseases were detected and these isolates were characterized as Bacillus cereus, Hafnia alvei, Serratia marcescens, Staphylococcus aureus, S. xylosus, S. scuiri, S. haemolyticus, and Pseudomonas aeruginosa. Findings from this study highlight the urgent need to create local and national food safety policies for the edible grasshopper "nsenene" subsector to regulate and guide street and market vending along the value chain, to prevent the transmission of foodborne diseases to consumers.

Biochemistry and transcriptomic analyses of Phthorimaea absoluta (Lepidoptera: Gelechiidae) response to insecticides.

Phthorimaea absoluta is an invasive solanaceous plant pest with highly devastating effects on tomato plant. Heavy reliance on insecticide use to tackle the pest has been linked to insecticide resistance selection in P. absoluta populations. To underline insights on P. absoluta insecticide resistance mechanisms to diamides and avermectins, we evaluated the transcriptomic profile of parental (field-collected) and F8 (lab-reared) populations. Furthermore, to screen for the presence of organophosphate and pyrethroid resistance, we assessed the gene expression levels of acetylcholinesterase (ace1) and para-type voltage-gated sodium channel (VGSG) genes in the F1 to F8 lab-reared progeny of diamide and avermectin exposed P. absoluta field-collected populations. The VGSG gene showed up-regulation in 12.5% and down-regulation in 87.5% of the screened populations, while ace1 gene showed up-regulation in 37.5% and down-regulation in 62.5% of the screened populations. Gene ontology of the differentially expressed genes from both parental and eighth generations of diamide-sprayed P. absoluta populations revealed three genes involved in the metabolic detoxification of diamides in P. absoluta. Therefore, our study showed that the detoxification enzymes found could be responsible for P. absoluta diamide-based resistance, while behavioural resistance, which is stimulus-dependent, could be attributed to P. absoluta avermectin resistance.

One stone for two birds: Endophytic fungi promote maize seedlings growth and negatively impact the life history parameters of the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW) Spodoptera frugiperda, is a voracious pest of cereals native to the Americas and which invaded Africa in 2016. Chemical control is the main management option, which however remains ineffective and unsustainable. Fungal endophytes are increasingly used as alternative for the management of insect pests of economic importance. This study assessed the potential of eight endophytic fungal isolates to colonize maize plant and their ability to promote seedlings growth through seed and foliar inoculations, as well as their suppressive effects on FAW. Fungal colonization rates of different plant parts by the endophytes varied as per the inoculation methods. Beauveria bassiana ICIPE 279 colonized more than 60% of all the seedling parts while B. bassiana G1LU3 only colonized stem (25%) and leaf (5%) tissues through foliar inoculation. Trichoderma atroviride F2S21, T. asperellum M2RT4, T. harzianum F2R41, Trichoderma sp. F2L41, Hypocrea lixii F3ST1 and Fusarium proliferatum F2S51 successfully colonized all the plant parts and therefore were selected and further evaluated through seed inoculation for their endophytic persistence, effect on plant growth, and pathogenicity to Spodoptera frugiperda immature and adult stages. Weekly assessment showed varied effect of the endophytes on maize plant growth parameters compared to the control. During the first week, percentage colonization of the plant parts ranges between 90%-100%, 65%-100%, and 60%-100%, in the roots, stems, and leaves, respectively for all the five tested isolates. However, the colonization pattern/rates significantly decreased over time for H. lixii F3ST1 in the stems and leaves, and for T. harzianum F2R41 in the leaves and for T. asperellum M2RT4 in the roots. In addition, T. harzianum F2R41 outperformed all the other isolates in boosting the plant height, whereas H. lixii F3ST1 and T. asperellum M2RT4 outperformed all the other isolates in increasing the wet and dry shoots weight. Furthermore, the number of egg masses laid on endophytically-colonized maize plants varied among the treatments. Trichoderma asperellum M2RT4 and H. lixii F3ST1 endophytically-colonized maize plants significantly reduced the number of egg masses and the defoliation/feeding rates of the pest compared to the control. Additionally, T. harzianum F2R41 had the highest negative impact on the pupation and adult emergence of S. frugiperda with a female-biased sex ratio. Our findings indicate that T. asperellum M2RT4, T. harzianum F2R41, and H. lixii F3ST1 hold a potential to be developed as endophytic-fungal-based biopesticides for sustainable management of S. frugiperda and as plant growth promoters.

The Endophyte Trichoderma asperellum M2RT4 Induces the Systemic Release of Methyl Salicylate and (Z)-jasmone in Tomato Plant Affecting Host Location and Herbivory of Tuta absoluta.

The use of endophytic fungi has dramatically increased plant performance through the enhancement of plant protection against abiotic and biotic stressors. We previously demonstrated that the endophytic fungus Trichoderma asperellum M2RT4 improves tomato defenses against the tomato leafminer Tuta absoluta through the reduction of oviposition, leafmining, pupation, and adult emergence. However, the underlying mechanism by which the presence of this endophytic fungus within tomato host plant affects T. absoluta host selection and life-history traits is unknown. We tested the behavioral responses of T. absoluta in Y-tube olfactometer bioassays and found that females preferred non-inoculated tomato plants against those inoculated by endophytes. Additionally, T. absoluta females were not attracted to non-inoculated infested nor to inoculated-infested tomato plants. Chemical analysis revealed the emission of methyl salicylate in inoculated tomato plant and an increase in the amounts of monoterpenes emitted from non-inoculated infested plants. Additionally, we found that upon herbivory, T. asperellum M2RT4 modulates tomato plant chemistry through the production of (Z)-jasmone thus activating both salicylic and jasmonic acid defense pathways. Further, T. absoluta females were attracted to monoterpernes including α-pinene, 2-carene, and ß-phellandrene but repelled by methyl salicylate. Methyl salicylate could therefore be considered as a good semiochemical-based candidate for sustainable T. absoluta management using a "push-pull" approach. However, in dose-response bioassays, females of T. absoluta did not show any preference to the four component-blend (α-pinene, 2-carene, ß-phellandrene, and methyl salicylate). (Z)-jasmone-treated tomato leaflets significantly reduced the leafmining activity of the pest at the concentration of 10 ng/µL and causing the highest larval mortality rate (83%) with the shortest LT50 (1.73 days) 7 days post-treatment. T. asperellum M2RT4 effect on herbivore performance was then (Z)-jasmone-mediated. These findings expand our understanding of how the endophytic fungus T. asperellum M2RT4 could mediate chemical interactions between T. absoluta and its host plant which are potentially important for development of environmentally friendly T. absoluta management programs.

Household perception and infestation dynamics of bedbugs among residential communities and its potential distribution in Africa.

Bedbugs have experienced an extraordinary upsurge in the recent past across the world. This cross-sectional study aimed to explore the community perception of the pest outbreaks, the population dynamics, and dispersal patterns under different habitat systems. A survey was conducted within communities in nine counties in Kenya, where geographical coordinates of the sites of bedbug presence were recorded and maximum entropy distribution modelling (MaxEnt) was used to map and predict the potentially suitable habitat, while system thinking and system dynamics approach with Vensim PLE 8.0.9 software was applied to implement bedbug infestation dynamics. Our results indicated that majority of the respondents had ample knowledge on bedbugs and were concerned about the physico-psychologic and socio-economic health effects. Spatial distribution analysis showed regions in Kenya with optimal to suitable for bedbug occurrence in the whole country, and similar results were found at continental level across Africa. Furthermore, infestation dynamics results showed a rapid mobility of bedbug from one house to another. In terms of management strategies, the models showed that the combination of chemical with other control methods was considerably much more effective compared to the use of chemical approach only, appointing integrated pest management strategy as a better intervention approach in controlling the pest.

The Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana for Management of the Melon Fly Zeugodacus cucurbitae: Pathogenicity, Horizontal Transmission, and Compatability with Cuelure.

In the laboratory, the pathogenicity of thirteen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin against the melon fly Zeugodacus cucurbitae (Coquillett) were assessed by exposing adults to 0.3 g of dry conidia (~3 × 109 conidia) of each isolate for 5 min and monitoring mortality for up to 5 days. Compatibility with a male pheromone, cuelure, (4-(p-acetoxyphenyl)-2-butanone), was determined by testing conidial germination and germ tube growth of the most promising isolate, M. anisopliae ICIPE 69, in the presence of cuelure at different temperatures. For horizontal transmission, the flies were separated by sex, separately exposed to M. anisopliae ICIPE 69, and subsequently mixed with non-exposed flies from the other sex. The most pathogenic isolates were M. anisopliae ICIPE 69, 18, and 30, causing mortalities of 94, 87, and 81%, with 5 days post-exposure, respectively. Metarhizium anisopliae ICIPE 69 caused the highest pupal mortality of 74%, with 15 days post-exposure. Horizontal transmission of M. anisopliae ICIPE 69 among male and female Z. cucurbitae was confirmed by 59 and 67% mortality after exposure to infected donor males and females, respectively. Metarhizium anisopliae ICIPE 69 affected the oviposition, but not hatchability, of infected Z. cucurbitae females. Metarhizium anisopliae ICIPE 69 is, therefore, a potential isolate for biopesticide development for Z. cucurbitae management in cucurbit production systems.

Efficacy of Metarhizium anisopliae and (E)-2-hexenal combination using autodissemination technology for the management of the adult greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae).

The efficiency of an autodissemination technique in controlling adult whiteflies, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) on tomato, Solunum lycopersicum was investigated with previously identified potent fungal isolates of Metarhizium anisopliae ICIPE 18, ICIPE 62 and ICIPE 69 under screenhouse or semi-field conditions. The autodissemination device was inoculated with dry conidia of the M. anisopliae isolates, while control insects were exposed to a fungus-free device. Sampling for conidia uptake, conidial viability and persistence, and insect mortality was done at 1, 2, 3, 5 and 8 days post-exposure, and collected insects were monitored for mortality over ten days. Overall, mortality was higher in insects exposed to ICIPE 18 (62.8%) and ICIPE 69 (61.8%) than in those exposed to ICIPE 62 (42.6%), with median lethal times, (LT50) ranging between 6.73-8.54 days. The control group recorded the lowest mortality rates (18.9%). A general linear reduction in conidial viability with exposure time was observed, although this was more pronounced with M. anisopliae ICIPE 62. Insects exposed to M. anisopliae ICIPE 69 also recorded the highest conidia uptake, hence selected for further evaluation with a T. vaporariorum attractant volatile organic compound, (E)-2-hexenal. The volatile inhibited fungal germination in laboratory compatibility tests, therefore, spatial separation of M. anisopliae ICIPE 69 and (E)-2-hexenal in the autodissemination device was conducted. The inhibitory effects of the volatile were significantly reduced by spatial separation at a distance of 5 cm between the fungus and the volatile, which was found to be more suitable and chosen for the subsequent experiments. Results showed that (E)-2-hexenal did not influence conidia uptake by the insects, while fungal viability and the subsequent mortality variations were more related to duration of exposure. The fungus-volatile compatibility demonstrated with spatial separation provides a basis for the optimisation of the volatile formulation to achieve better T. vaporariorum suppression with an excellent autodissemination efficiency when used in the management of whiteflies under screenhouse conditions.

Evaluating the efficacy of Mazao Tickoff (Metarhizium anisopliae ICIPE 7) in controlling natural tick infestations on cattle in coastal Kenya: Study protocol for a randomized controlled trial.

Ticks and tick-borne diseases cause substantial economic losses to the livestock industry in sub-Saharan Africa. Mazao Tickoff is a novel bioacaricide developed for tick control and is based on the entomopathogenic fungus Metarhizium anisopliae sensu lato (s.l.) isolate ICIPE 7. To date, no randomized controlled study has been undertaken to demonstrate the efficacy of this bioacaricide in reducing natural tick infestation on cattle. To this end, this field trial is designed to evaluate the anti-tick efficacy of Mazao Tickoff on cattle in coastal Kenya compared to a standard chemical tick control protocol. In this prospective, multi-center randomized controlled trial, eligible herds will be randomized by the herd size to the intervention arm in a 1:1:1 ratio to either Triatix® (active ingredient: amitraz); Mazao Tickoff (active ingredient: M. anisopliae ICIPE 7); or placebo (excipients of the Mazao Tickoff), with a total enrollment target of 1,077 cattle. Treatments will be dispensed on Day 0 (defined individually as the day each animal receives the first treatment) and thereafter every two weeks until Day 182. Ticks will be counted on every animal in each herd (herds to be included have at least one animal bearing at least one tick on Day 0), and thereafter on bi-weekly intervals until Day 182. The primary efficacy assessments of Mazao Tickoff will be based on the mean percentage reduction in tick counts at each post-treatment follow-up visit compared to the placebo group and the Triatix® arm. Further, the effect of Mazao Tickoff on the prevalence of common cattle pathogens, Anaplasma marginale and Theileria parva, will be determined by assessing incidence and seroprevalence at four different time points. This protocol describes the first rigorous evaluation of the efficacy of Mazao Tickoff and its potential as a viable alternative non-chemical acaricide tool for tick control in Kenya and elsewhere.

Susceptibility of the Western Honey Bee Apis mellifera and the African Stingless Bee Meliponula ferruginea (Hymenoptera: Apidae) to the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana.

This study assessed the nontarget effect of entomopathogenic fungi on the Western honey bee Apis mellifera L. and the African stingless bee Meliponula ferruginea Cockrell (Hymenoptera: Apidae). Pathogenicity of five Metarhizium anisopliae (ICIPE 7, ICIPE 20, ICIPE 62, ICIPE 69, and ICIPE 78) (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) and one of Beauveria bassiana (ICIPE 284) (Balsamo) Vuillemin (Hypocreales: Cordicipitaceae) isolates were evaluated on bees at 108 conidia/ml. Conidial acquisition was evaluated immediately after exposure. Apis mellifera acquired more conidia (2.8 × 104-1.3 × 105 conidia per bee) compared to M. ferruginea (1.1 × 104-2.3 × 104 conidia per bee). In the bioassay with A. mellifera, ICIPE 7, ICIPE 20, and ICIPE 69 moderately reduced the survival by 16.9, 17.4, 15.3%, with lethal times LT10 = 7.4, 7.6, 8.1 d and LT25 = 8.7, 10.0, 9.9 d, respectively. The three isolates caused A. mellifera mycosis of 11.6-18.5%. None of the isolates had a significant effect on M. ferruginea. The tested isolates are nontoxic to bees according to the International Organization of Biological Control (IOBC) classification. However, the effect of ICIPE 7, ICIPE 20, and ICIPE 69 merits further studies on bee colonies, especially those of A. mellifera, under field conditions.

Temperature-dependent modelling and spatial prediction reveal suitable geographical areas for deployment of two Metarhizium anisopliae isolates for Tuta absoluta management.

Tuta absoluta is one of the most devastating pests of Solanaceae crops in Africa. We previously demonstrated the efficacy of Metarhizium anisopliae isolates ICIPE 18, ICIPE 20 and ICIPE 665 against adult T. absoluta. However, adequate strain selection and accurate spatial prediction are fundamental to optimize their efficacy and formulations before field deployment. This study therefore assessed the thermotolerance, conidial yield and virulence (between 15 and 35 °C) of these potent isolates. Over 90% of conidia germinated at 20, 25 and 30 °C while no germination occurred at 15 °C. Growth of the three isolates occurred at all temperatures, but was slower at 15, 33 and 35 °C as compared to 20, 25 and 30 °C. Optimum temperatures for mycelial growth and spore production were 30 and 25 °C, respectively. Furthermore, ICIPE 18 produced higher amount of spores than ICIPE 20 and ICIPE 665. The highest mortality occurred at 30 °C for all the three isolates, while the LT50 values of ICIPE 18 and ICIPE 20 were significantly lower at 25 and 30 °C compared to those of ICIPE 665. Subsequently, several nonlinear equations were fitted to the mortality data to model the virulence of ICIPE 18 and ICIPE 20 against adult T. absoluta using the Entomopathogenic Fungi Application (EPFA) software. Spatial prediction revealed suitable locations for ICIPE 18 and ICIPE 20 deployment against T. absoluta in Kenya, Tanzania and Uganda. Our findings suggest that ICIPE 18 and ICIPE 20 could be considered as effective candidate biopesticides for an improved T. absoluta management based on temperature and location-specific approach.

Virulence and horizontal transmission of Metarhizium anisopliae by the adults of the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) and the efficacy of oil formulations against its nymphs.

The pathogenicity of dry conidia and fungal suspensions of 16 entomopathogenic fungal isolates (10 Metarhizium anisopliae and six Beauveria bassiana) was evaluated against adults and second instar nymphs of the greenhouse whitefly, Trialeurodes vaporariorum respectively. All the tested isolates were pathogenic to T. vaporariorum and caused mortality of 45-93% against the adults and 24-89% against the nymphs. However, M. anisopliae strains showed higher virulence to both developmental stages as compared to B. bassiana strains. The three most virulent isolates that caused high mortalities in adults were M. anisopliae ICIPE 18, ICIPE 62 and ICIPE 69, with cumulative mortalities of 82, 91 and 93%, and median lethal times (LT50) of 5.20, 5.05 and 4.78 days, respectively. These isolates were further assessed for spore acquisition and retention by the adult insects at 0, 24, 48 and 72 h after exposure to dry conidia spores. There was no significant difference among isolates on their acquisition by the insects, although the effect of time on the number of spores retained by each insect was significant. For M. anisopliae ICIPE 62 and ICIPE 69, spore number was significantly higher immediately after exposure at 0 h than at 24, 48 and 72 h, whereas for M. anisopliae ICIPE 18, the spore number remained constant for all the days. The infected "donor" insects were able to horizontally transmit the acquired spores to uninfected "recipient" insects causing high mortality rates in both donor and recipient groups. Metarhizium anisopliae ICIPE 7, ICIPE 18 and ICIPE 62 were the most virulent isolates against the nymphs in aqueous formulation during the first screening with >80% mortality. However, in 2% (v/v) oil formulations at 1 × 108 conidia/ml, canola formulated ICIPE 62, ICIPE 18 and olive formulated ICIPE 18 were the most effective, resulting in 87.8, 88.1 and 99.4% nymphal mortalities respectively and with lower LT50. Oil formulations significantly enhanced the efficacy and virulence of the isolates against the nymphs compared to aqueous formulations.

Infection of the Stable Fly, Stomoxys calcitrans, L. 1758 (Diptera: Muscidae) by the Entomopathogenic Fungi Metarhizium anisopliae (Hypocreales: Clavicipitaceae) Negatively Affects Its Survival, Feeding Propensity, Fecundity, Fertility, and Fitness Parameters.

Entomopathogenic fungi can cause substantial mortality in harmful insects. Before killing the insect, these pathogens start by negatively affecting the biological parameters of the host. Prior to our study, the information about how fungal exposure affects the biological parameters of the stable fly, Stomoxys calcitrans was still elusive. Therefore, we aimed to assess the infection of S. calcitrans with some Metarhizium anisopliae strains, and their impact on feeding, fecundity, fertility and other life-history traits of this fly. Among the 11 M. anisopliae strains screened, we identified ICIPE 30 as the most virulent strain against S. calcitrans. We observed that the infectivity of this strain was sex and age-dependent. Infected male S. calcitrans died earlier than their counterpart females. Older infected S. calcitrans died faster than infected young ones. Also, male and female S. calcitrans successfully transmitted ICIPE 30 conidia to their mates. We demonstrated that infection by ICIPE 30 extended the feeding time of S. calcitrans and consequently reduced the feeding probability of the fly and the amount of blood taken. Using a dual test oviposition bioassay, we determined that uninfected gravid female S. calcitrans avoided laying eggs on substrates amended with ICIPE 30 conidia. We showed that these conidia could lower the hatchability of the eggs deposited by gravid females. Using, a no-choice test, we showed that gravid female S. calcitrans infected with ICIPE 30 laid fewer eggs than uninfected females and those eggs hatched less. Using 11 strains of M. anisopliae and four high concentrations of ICIPE 30 conidia, we verified that S. calcitrans larvae were not susceptible to fungal infection. Further, we showed that though these larvae were tolerant to fungal infection, there was a significant effect on their fitness, with contaminated larvae having a small bodyweight coupled with longer developmental time as compared to uncontaminated larvae. Our study provides detailed information on how fungal infection affects the biology of S. calcitrans and the potential of using M. anisopliae ICIPE 30 as a biopesticide to reduce the fly population. Such knowledge can assist in developing fungal-based control strategies against this harmful fly.