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Insect Sci. Applic. Vol. 21, No. 2, 2001, pp. 133 -138

Effects of Formulated Imidacloprid on Two Mite Pathogens, Neozygites floridana (Zygomycotina: Zygomycetes) and Hirsutella thompsonii (Deuteromycotina: Hyphomycetes)

Surenda K. Dara and Fabien C. C. Hountondji

Plant Health Management Division, International Institute of Tropical Agriculture, 08 B.P. 0932 Cotonou, Rep. of Benin

Accepted 22 May 2001

Code Number: ti01016


Neozygites floridana (Weiser & Muma) Remaudière & Keller and Hirsutella thompsoniiFisher are important fungal pathogens of mites on cassava in Benin. Bioassays were conducted to evaluate the influence of four concentrations (50, 100, 200 and 500 ppm) of a water-dispersible formulation (Gaucho 70 WS) of imidacloprid on the conidial germination of these two fungi. Imidacloprid significantly reduced the germination of primary conidia and formation of infective capilliconidia in N. floridana. In contrast, imidacloprid concentrations of 100 ppm and above increased conidial germination in H. thompsonii. Another bioassay was conducted to evaluate possible synergism between imidacloprid and H. thompsonii in the mortality of the cassava green mite, Mononychellus tanajoa (Bondar). Hirsutella thompsonii alone and in combination with imidacloprid both killed around 95% of the mites after five days. In contrast, the mortality of mites treated with imidacloprid alone was close to that of untreated mites, which was around 30% on the fifth day after treatment.

Key Words: imidacloprid, Mononychellus tanajoa, Neozygites floridana, Hirsutella thompsonii, synergism


Neozygites floridana (Weiser & Muma) Remaudière & Keller et Hirsutella thompsonii Fisher sont des champignons pathogènes des acariens du manioc au Bénin. Des essais ont été conduits afin d’évaluer l’influence de quatre différentes concentrations (50, 100, 200 et 500 ppm) de la formulation en poudre hydrosoluble de l’insecticide imidacloprid sur la germination des conidies des deux champignons. Les résultats prouvent une action spécifique de l’imidacloprid sur chacun des deux champignons. La germination des conidies primaires et des capilloconidies de N. floridana est significativement affectée, alors que chez H. thompsonii, le pourcentage de germination est significativement plus élevé aux plus fortes concentrations. Vu ces résultats, de nouveaux essais ont été entrepris pour évaluer une éventuelle synergie de l’imidacloprid et de H. thompsonii pour lutter contre l’acarien vert du manioc, Mononychellus tanajoa Bondar. Comparativement au traitement à l’imidacloprid seul, la mortalité est significativement plus élevée chez les acariens traités avec H. thompsonii seul ou avec la combinaison imidacloprid-H. thompsonii. L’efficacité entre ces deux derniers traitements est comparable. 

Mots Clés: imidacloprid, Mononychellus tanajoaNeozygites floridanaHirsutella thompsonii, synergisme


The entomopathogenic fungi, Neozygites floridana (Weiser & Muma) Remaudière & Keller (Zygomycotina: Zygomycetes) and Hirsutella thompsonii Fisher (Deuteromycotina: Hyphomycetes) are two important pathogens of the cassava green mite, Mononychellus tanajoa (Bondar) (Acari: Tetranychidae), a foliage feeder on cassava (Manihot esculenta Crantz) in Benin (Dara et al., 1998). Imidacloprid is a chloroni-cotinyl insecticide that is effective against various sucking pests and some coleopterans, dipterans and lepidopterans, but non-toxic to phytophagous mites (Elbert et al., 1991; Leicht, 1993). Recent studies have demonstrated synergism of this insecticide with different entomopathogenic fungi and nematodes on various insect pests (Kaakeh et al., 1997; Koppenhöfer et al., 1997; McCoy et al., 1998; Quintela and McCoy, 1998a). Imidacloprid is also known to enhance the germination of some fungi as well as the egg production of predatory phytoseiid mites (James, 1997; Quintela and McCoy, 1997a). In light of these findings, we evaluated the potential of using sub-lethal doses of imidacloprid in the microbial control of the cassava green mite. Bioassays were conducted to evaluate the influence of the insecticide on the conidial germination of N. floridana and H. thompsonii and on the infection of M. tanajoa by H. thompsonii.

Materials and Methods

Influence of imidacloprid on conidial germination of N. floridana and H. thompsonii

Neozygites floridana and H. thompsoniiwere isolated from M. tanajoa in Benin. Since members of the genus Neozygites are difficult to grow on artificial media (Gustafsson, 1965; Le Rü et al.,1985; Saito et al., 1989), N. floridana was cultured in vivo in M. tanajoa, while H. thompsonii was grown on potato dextrose agar (Bacto®, Difco laboratories, Detroit MI, USA). Neozygites floridana was produced by exposing healthy females of M. tanajoa to sporulating mite cadavers infected with the fungus (sporulation was obtained after two-day incubation at ca. 20 oC, near 100% RH and in the dark), on 2 cm diam. cassava leaf discs deposited on moist cotton inside Petri dishes (15 cm diam.). Exposed mites were maintained at ca. 28 oC and under the natural light-dark regime and mummified infected mites collected 4 to 7 days later and used in the experiment. Field-collected M. tanajoa infected with H. thompsonii were surface-disinfected using 5% bleach for 1 minute and deposited on potato dextrose agar medium at ca. 28 oC. Mature mycelia mat with spores formed 14 days later and were used immediately. 

Four concentrations—50, 100, 200, and 500 ppm— of a water-dispersible powder formulation of imidacloprid (Gaucho 70 WS) were tested, along with distilled water as control, for their effect on the conidial germination of the two fungi. Glass coverslips were immersed in respective concentrations of treatment solutions for 15 seconds and dried at room temperature for two hours. The coverslips were then separately inoculated with conidia from one or the other of the two fungi. Hirsutella thompsonii mycelia with spores were scrapped from the surface of the medium and mixed with sterile distilled water using a glass rod until a high spore concentration was obtained. Conidial suspension was separated from mycelia using sterile syringes and diluted to 105 conidia/ml; 0.1 ml of this suspension was applied onto the surface of the coverslip and allowed to dry.

For N. floridana, a coverslip was placed on moist cotton padding inside a Petri dish (60 mm dia. by 15 mm) and exposed to the conidial showers from a N. floridana-killed mite glued to the inside of its lid with a double-sided sticky tape, at 20 °C in the dark for 12 h. For each of the fungi, 10 coverslips were used for each concentration of imidacloprid along with the control. Coverslips, laid on moist cotton padding inside Petri dishes, were incubated at 25 oC for 24 h in the dark and the germination of conidia was observed using a compound microscope. The number of germinated and ungerminated conidia in four randomly selected locations on each coverslip was recorded and the percent germination determined. In the case of N. floridana, total conidial germination included conidia that were in the process of forming and had already formed secondary conidia. This bioassay was repeated three times.

Effect of imidacloprid on infection of M. tanajoa by H. thompsonii

Since imidacloprid is not toxic to phytophagous mites, the concentration 500 ppm was used in this trial because it gave the higher proportion of germinated H. thompsonii spores. The effectson M. tanajoa mortality of imidacloprid (500 ppm) and H. thompsonii (2.9 x 106 conidia/ml), alone or in combination, were therefore compared, with distilled water serving as a control. Healthy leaves were collected from greenhouse cassava plants, washed with distilled water and discs of 20 mm diameter were cut from them. Leaf discs were checked under a dissecting microscope to ensure that they were clean and particularly free of M. tanajoa eggs. Twenty leaf discs were respectively immersed in each treatment solution for thirty seconds and allowed to dry at room temperature. After drying, leaf discs were placed on moist cotton inside a plastic box (190 x 150 x 50 mm) with mite-proof screen attached to the lid. Twenty 12-day-old adult female mites, reared on potted cassava plants in the laboratory, were transferred to each leaf disc and incubated at 25 oC and 12:12 (L : D) h photoperiod. Mites were observed every day and mortality was recorded for 10 days after treatment (DAT). Dead mites without H. thompsonii symptoms were incubated overnight and checked under the dissecting microscope for the presence of mycelia of the fungus. The experiment was repeated three times.

Statistical analyses

Data were arcsine-transformed to stabilise variances and analyses were conducted using PROC GLM of SAS software (SAS Institute, 1996). CONTRAST statement was used to separate significant means. 


Influence of imidacloprid on conidial germination of N. floridana and H. thompsonii

The germination of the primary conidia and formation of the capilliconidia of N. floridana as well as the germination of conidia of H. thompsonii showed different responses to imidacloprid treatment (Fig. 1). Both the conidial germination (F = 33.90; df = 4, 153; P < 0.001) and capilliconidial formation (F = 32.98; df = 4, 153; P = 0.001) of N. floridana were negatively correlated to imidacloprid concentrations and at 500 ppm imidaclaloprid virtually prevented spore germination (Fig. 1A). In contrast, imidacloprid had a slightly positive influence on the conidial germination of H. thompsonii (Fig. 1B). Significantly higher (F = 2.8; df = 4, 173; P < 0.05) proportions of conidia germinated at 100, 200, and 500 ppm of imidacloprid compared with the control (Fig. 1B).

Influence of imidacloprid on infection of M. tanajoa by H. thompsonii

The mortality of mites was significantly higher (< 0.05) from H. thompsonii, when used alone and together with imidacloprid, than from imidacloprid alone and the control (Fig. 2a). No synergism was found between the fungus and the insecticide and their combination was as effective as the fungus alone on the mortality of mites (> 0.05). Imidacloprid appeared to be slightly toxic to M. tanajoa compared with the control (Fig. 2A). Mortality was not significantly different on 1 and 2 DAT for all treatments (p > 0.05) except for the control, which showed a significantly lower mortality on 2 DAT (P < 0.05). From 3 DAT to the end of the experiment, significantly higher mortalities were observed in H. thompsonii alone and in combination with imidacloprid compared with imidacloprid alone.

Significantly lower mortalities were found in the control throughout the experiment. Mortality reached 99.8, 99.7, 90.5 and 82.5% for H. thompsonii, imidacloprid + H. thompsonii, imidacloprid alone and the control respectively on 10 DAT. Hirsutella thompsonii infection was found from 2 DAT in mites treated with H. thompsonii alone, while in the treatment where the fungus was applied with imidacloprid, infection occurred 1 DAT. The only infected mite collected 1 DAT in this treatment was found infected following overnight incubation of dead mites. However, the peak of infection was reached on 8 DAT for both treatments (Fig. 2b). The level of infection between these treatments was similar (> 0.05) throughout the observation period.


Most of the studies conducted on the interaction between imidacloprid and entomopathogenic fungi have concentrated on the effect of this insecticide on the infectivity of either or both of the two deuteromycetous fungi Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin (Boucias et al., 1996; Kaakeh et al., 1997; Quintela and McCoy, 1997b, 1998a, 1998b). However, little attention has been paid to the influence of imidacloprid on spore germination.Quintela and McCoy (1997a) found a positive correlation between M. anisopliae conidial germination on water agar and imidacloprid concentration and attributed this to both the inert carrier and the active ingredient of the formulation. Imidacloprid formulation probably favoured the germination of H. thompsonii spores as did the presence of potassium for H. rhossiliensis spores (Eayre et al., 1990), or induced more rapid germination as was the case of Fusarium oxysporum f. sp. Vasinfectum and Verticillium albo-atrum on agar adjusted to - 10 or - 20 bars with salt or sucrose (Manandhar and Bruehl, 1973). In this study, germination was checked only 24 h after installation of spores, and we cannot conclude on the effect of imidacloprid on the germination speed.

Inhibition of conidial germination of N. floridana by another pesticide, benomyl, was reported on Tetranychus urticae, and the incidence of the fungus in the field was found to be affected by it (Brandenberg and Kennedy, 1983). By inhibiting the formation of capilliconidia (the infective spores) of N. floridana, imidacloprid significantly reduces its infectivity on mites, and thus renders it incompatible with N. floridana for mite control.

According to previous studies, the synergistic action of sub-lethal dosages of insecticides with entomopathogens may occur through the weakening of the pest, making it more susceptible to the pathogen (Boucias et al., 1996; Quintela, 1996; Quintela and McCoy, 1997b, c; Hiromori and Nishigaki, 1998). The slight toxicity of imidacloprid observed in this study suggests a certain stressful effect of the insecticide on the mites. However, no additive effect of imidacloprid was observed in H. thompsonii infectivity despite its positive effect on spore germination as observed in M. anisopliae and B. bassiana (Quintela and McCoy, 1997a).

The infection observed 1 DAT could have been initiated by early-germinating spores possibly due to imidacloprid’s effect, but it did not completely invade the host. The mite most probably died from the effect of imidacloprid rather than from fungus infection: more mites died on 1 DAT among imidacloprid-treated mites. Other mites of the same treatment might have been infected on 1 DAT but were able to survive until the 2nd or 3rd day.

The present results did not show a synergistic effect of imidacloprid with H. thompsonii on the cassava green mite. However, the enhancing effect of the insecticide on germination of spores of this fungus could be beneficial under field conditions, where multiple factors are involved. Further studies should consider assessing germination in time and evaluating the interactions between imidacloprid and H. thompsonii in the field. 


We thank Honoré Dossounon, Roland Bocco, Jacques Kuinsou and Hugues Baimey for their technical assistance.


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© 2001 ICIPE

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