Efficiency of the entomopathogenic fungus Verticillium lecanii in the ...

% of germinationBouhous and Larous 24371008060402001 2 3 4 5 6 8 10 11 12Time (hours)Figure 3. V. lecani spore germination at 24°C and H.R=100%.during 12 h (Drummond, 1987; Hall, 1976).Insect cultureTwo young bean plantations in the stage of the apparition of thetwo first leaves were infected by several aleurodes during 24 hin an insect cage (60x60x60 cm). Aleyrodes were eliminatedafter setting their eggs on the lower face of the leaves, so thatthe obtained population of eggs, larvae and adults will behomogenous.Eggs and stage II larvae treatmentLeaves were observed under a magnifying glass to localize theinfected zones. 7 mm diameter discs of leaves were cut, eachdisc contains at least 2 to 20 eggs or stage II larvae. Discs wereimmersed during 10 s in a freshly prepared fungus sporesuspension, and then 10 of these discs were placed on thesurface of the agar 1.5%. The Experiment was carried out intriplicate. Controls were provided for each experiment.Incubations were done in desiccators at 20°C under a relativehumidity of 100% with a photoperiod of 16 h per 24 h. Mortalitypercentage was determined from the 5th day of contact. LD50were calculated according to the method of Probit (Bliss, 1935;Drummond et al., 1987).Adults treatmentAdults of the same age were captured after cooling during 5min in the refrigerator. They were then recovered anddistributed in tubes to get 30 insects per tube. They werecooled again and put quickly in contact with a 7 days fungusculture. After 30 min of contact, spore infected adults werereleased in the desiccators each containing a host plant.Desiccators were adjusted to a relative humidity of 100% at20°C and exposed to light during 16 h per 24 h in a phytotronincubator (Drummond et al., 1987; Ekbom, 1979).Effect of relative humidity (RH)Eggs and Stage II larvae were treated with a dose of about1.5x10 7 spores/ml. The whole (eggs/ larvae/ spores) wereincubated at 20°C, first at 100% RH during 16 h and then at70% RH during 4, 8, 12, 20 and 96 h. It was finally put at a RHof 100% during 7 days compared to control. For each case, themortality rate was estimated after 7 days.RESULTSSpecies identificationSamples of infected leaves with different larval stagesof the aleurode were collected from two agriculturalregions (El Kennar and El Aouana) (Figure 2). Slideswere prepared and examined by the opticalmicroscope (Colles, 1958). Identification was doneaccording to Martin (1987). Leaves samples analysisallowed noting the total predominance of T.vaporariorum species. The species exists ongreenhouse cultures and even on other outsideadventitious plants.Spore germinationThe spore germination starts since the first hour andreaches a rate of 65% after 9 h and 92% after 12 h ofincubation (Figures 3 and 4).Effect of fungal spore concentration on differentdevelopment stages of T. vaporariorumEffect on eggsThe percentages of mortality after treatment showedan increase of about 6.9, 14 and 61.8% for the threedoses and contact times used. LD 50 were: 4.2x10 7spores/ml in day 5; 2.1x10 7 spores/ml in day 6 and0.59x10 7 spores /ml in day 7 (Table 1). Thus, LD 50 in

probitprobitprobit2438 Afr. J. Microbiol. Res.Figure 4. Germination of V. lecanii spores after 24 h of incubation at 24°C and at a RH of 100%. G 40X0.75.Table 1. Probit analysis of egg treatment.No. of insects (n) Slope ± SE LC (95%) Chi-square LD50788 0.69±0.10 (13.060-86.134)10 6 12.709 4.2x10 7788 0.73±0.09 (93.573-37.621)10 6 15.929 2.1x10 7788 1.02±0.10 (41.553-79.95)10 6 23.169 0.59x10 764a86b86c20-22 4 6 8 10Log dose of spores/ml420-22 4 6 8 10log dose of spores/ml420-22 4 6 8 10log dose of spores/mlFigure 5. Effect of V. lecanii on T. vaporariorum eggs. Day 5 (a), day 6 (b) and day 7 (c) after treatment.day 5 was 7 folds higher than that found in day 7(Figure 5a, b and c). Microscopic examination showedthat the infected T. vaporariorum egg was surroundedwith V. lecanii mycelium (Figure 6a and b).Effect on larvaeAs seen in Figure 7, the percentage of mortalityincreased according to contact time, whatever wasthe dose of spores used. LD 50 were: 11x10 3 , 3.4x10 3and 0.5x10 3 spores/ml for the three incubation times(Table 2). Figure 8 showed T. vaporariorum larvaewith an abnormal shape surrounded with V. lecaniimycelium.Effect on adultsAfter 7 days of incubation of 120 adult insects, only 81

LD50 (10 3 ) spores/mlBouhous and Larous 2439abFigure 6. A T. vaporariorum egg control (a) A T. vaporariorum egg surrounded with V. lecanii mycelium, (b) G 40X0.75.1510504 5 6 7 8Days after incubationFigure 7. Effect of V. lecanii on T. vaporariorum larvae after 5, 6, and 7 days of treatment.Table 2. Probit analysis of larvae treatment.No. of insects (n) Slope ± SE LC (95%) Chi-square LD50604 0.26± 0.08 17.357 10 3 -94.40 10 5 72.25 11X10 3604 0.30±0.08 42.156 10- 33.495 10 5 61.43 3.4X10 3604 0.28±0.09 25.64 -13.045 10 7 44.32 0.5X10 3were found to be completely invaded by means of thefungus mycelium (Figure 9).Influence of humidityThe incubation at 70% relative humidity did not showany inhibitory effect on the pathogenesis of thefungus, since the mortality rate of the larvae was100% and that of eggs was relatively high (Figure 10).Information about probit analysis (No. of insects(n=885), Slope ± S: 0.88± 0.10, LC (95%): 3.74-7.23,Chi-square: 1.44).DISCUSSIONExperiments on the efficiency of the fungus against T.vaporariorum investigated the influence of variousenvironmental factors. Experimental infections of

2440 Afr. J. Microbiol. Res.Figure 8. T. vaporariorum larvae with an abnormal shape surrounded with V. lecaniimycelium. G 40X0.75.Figure 9. An adult T. vaporariorum surrounded with V. lecanii mycelium G 40X0.75.larvae, adults and eggs showed that the observedmortality rates on the different stages treated byfungal spores are important and in particular in thecase of larvae (LD 50 : 0.5x10 3 spores/ml).Drummond et al. (1987) found an LD 50 of 1.5x10 5spores/ml in the same conditions of temperature andhumidity which confirmed the efficiency of the usedstrain against the development of T. vaporariorum. Incontrast to the results obtained with larvae, theefficiency of eggs treatment by fungal spores is lowerwith an LD 50 of 5.9x10 6 spores /ml. These results areconforming to those obtained by Hall (1982), whichobserved that, for adult insects, fungal sporetreatment was very efficient in small experimentalgreenhouses. In our experiments, the substitution ofexperimental greenhouses with desiccators confirmedthis efficiency. Therefore, 81 adults over 120 usedwere completely invaded by the fungal mycelium.Others died by drowning due to water condensationon desiccator’s walls.Eggs, larvae as well as adults treatment showedthat the used V. lecanii strain is very pathogenicagainst T. vaporariorum particularly in larval stage.The lower pathogenesis on eggs could be explainedby the presence of the rigid cuticle which hinders thepenetration of the mycelium. Given that insect

ProbitBouhous and Larous 2441Log time (hours)Development stages transfer of the insectsmaintained at an RH of 70% for 96 h did notinfluenced the mortality rates as compared with thecontrol maintained at 100% during the completeexperimentation period.Drummond et al. (1987) observed that more thefungus is pathogenic more it is independent on lowhumidity. However, Fargues et al. (2005) showed thatthe entomopathogenic Hyphomycetes have strongpotential for microbial control of whitefly larvaeinfesting tomato crops at moderate ambient humidityin Mediterranean greenhouses. The characteristic ofV. lecanii regarding the relative humidity could be ofgreat importance for its application in greenhouseswhere important humidity fluctuations are noted.Figure 10. Effect of relative humidity (70%) on T.vaporariorum eggs mortality after treatment with V. lecanii.Used dose =1.5x10 7 spores /ml.development inevitably passes by larval stages, whichlet us suppose that eggs that have escaped thetreatment will be attacked in the larval stage. Thepercentage of mortality of spore-treated eggs andlarvae increased throughout the incubation period inexperimental conditions, thus, fungus efficiencyrequires some contact time.Treatment efficiency depends closely on a highrelative humidity rate (95 to 100%) necessary to thefungal spore germination. A decrease in humidityattenuates the fungal infectious capacity (Milner andLuton, 1986). It is then recommended to maintain avery high relative humidity after treating the aleurodeby the fungus (Drummond et al., 1987). It is clear thathumidity in greenhouses is not constant, its recordingperformed in Jijel during May and June 2008, showeda decrease in the weekly mean from 57.34% in themorning to 96.22% in the night. The other importantfactor is the capacity of fungal spore germinationwhich should be higher than 90%, for the studiedstrain, spore germination reached 92% after 12 h ofincubation in the experimental conditions, which isconsidered as a good result.Germination capacity could be influenced byseveral factors such as spore age, temperature aswell as relative humidity; consequently, evaluation ofgermination capacity of the studied strain should becarried out permanently before each biological controltreatment. It was reported that one of the properties ofhighly pathogenic and virulent strains is their rapidgermination. According to Hall (1984), 50% of thespores of virulent strains germinate after 9 h ofincubation at 100% RH.Spore germination rate of the studied strain reached65% after 9 h of incubation at 100% RH. This fungalproperty is very important because a relative humidityhigher than 95% rarely persists in greenhouses duringmore than 12 h, as showed by the recordings carriedout in a greenhouse of Jijel. Because of the rapidgermination of the fungal strain, it depends a little onthe RH after an incubation period of 16 h at 100% RH.ConclusionBased on the realised tests, the entomopathogenicfungus V. lecanii is particularly infectious for larvaland adult stages, and slightly infectious for eggs. Inlarvae, LD 50 is relatively low (0.5x10 3 spores/ml) in theexperimentation conditions. The action time of thefungus reached its maximum in day 7. Concerning theeffect of humidity on the development of the fungusand on its virulence, results presented here allowedus to consider a future practical application of V.lecanii in the biological control of T. vaporariorum.Further applied research is required to determine thegreenhouse application conditions.REFERENCESBliss CI (1935). 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