African Journal of Microbiology Research Vol. 6(10), pp. 2435-2442, 16 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1502 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Efficiency of the entomopathogenic lecanii in the biological control of Trialeurodes vaporariorum, (Homoptera: Aleyrodidae), a greenhouse culture pest

Mostefa Bouhous1* and Larbi Larous2

1Laboratory of Molecular Toxicology, Faculty of Sciences, University of Jijel, Algeria. 2Laboratory of Microbiology, Faculty of Life and Natural Sciences, University of Ferhat Abbas, Sétif, Algeria.

Accepted 23 February, 2012

Our investigation in the region of Jijel revealed that are the predominant greenhouses pests; they are polyphagous, moreover, some species can transmit many plant viruses. The treatment method is based on the systematic use of that have side effects on both the consumer and the farmer. The objective of this study was to evaluate the use of biological control in situ and in vitro as an alternative method by using an Verticillium lecanii. In vitro experiments showed that the fungus was active during all stages of development of the , Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae): 7 3 Eggs (LD50 = 0.59. 10 / ml) larvae (LD50 = 0.5. 10 spores / ml) and adults. Our results showed the influence of concentration, contact time and relative humidity on the development of the parasite to reach an efficient anti-larval effect of 100%.

Key words: Verticillium lecanii, Trialeurodes vaporariorum, entomopathogenic, alleyrodidae, , biological control.

INTRODUCTION

The green house aleurode Trialeurodes vaporariorum (Faria and Wraight, 2001; Liu et al., 2011), in addition is a current polyphagous pest of leguminous cultures, to phytotoxicity problems and residual pesticides on for example, tomatoes, pepper, courgette, bean etc. legumes (Quinlan, 1988). Other control methods were (Vet et al., 1980; Van lenteren and Noldus, 1990). It proposed, including biological control by using was detected in 250 plants all around the world biopesticides based on ascomycetes and (Landa, 1994); it can multiply quickly and without hyphomycetes. interruption to many annual generations under Verticillium lecanii which was reclassified by Zare greenhouse conditions. In parallel to its multiplication, and Gams (2001) as lecanii is an sap puncture by different stages of development, important pathogen of , isolate from coccids, virus transmission and secretion of honeydew on the and whiteflies (Cortez-Madrigal et al., 2003; lower face of leaves promote sooty mold development Liu et al., 2011). (Coffin and Coutts, 1995; Guzman, 1997; Mckee, In the present work, we attempted to measure the 2007; Guevara-Coto et al., 2011). Thus, a efficiency of the microscopic fungus V. lecanii against depreciation of the product is seen, and a the multiplication of Trialeurodes vaporariorum. considerable decrease in photosynthesis making the leaves yellowing and falling (Dipietro, 1977). In the region of Jijel, the control method used today MATERIALS AND METHODS against the green house aleurode is chemical; however, spraying chemical pesticides causes an Sampling increase of resistance, environmental pollution and can be toxic to natural enemies Samples of infected leaves with different larval stages of the aleurode (Figure 1) were collected from two agricultural regions: El Kennar (36° 49’ 35” N/ 5° 57’ 17” E, latitude 12 meter) and El Aouana (36° 46’ 26” N/ 5° 37’ 09” E, latitude 20 m) (Figure 2). They were put in plastic bags and conserved in *Corresponding author. E-mail: [email protected]. Tel: the refrigerator. Larvae were used for species identification 00 213 34 502687. (Colles, 1958; Martin, 1987). 2436 Afr. J. Microbiol. Res.

agv advvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv

Figure 1. The whole white insect T. vaporariorum on the lower face of bean leaves in a greenhouse.

B é ni - B é laid

El - Kennar El Ouana

- ’ - – : : -

Figure 2. Regions where samples were collected.

Figure 2. Regions where samples were collected.

Spore germination and preparation of V. lecanii doses surface of the mycelium. The plate was slowly agitated and spore suspension was recovered, vigorously agitated by vortex V. lecanii strain provided by the University of Kiev (Ukraine) and filtrated, spore count was carried out using a Malassez was plated on Czapek–dox (CD) medium pooled in Petri hematimetric cell. For every test, different concentrations were dishes. After incubation at 25°C for 7 days, cultures were studied: 105, 106 and 107 spores/ml. 0.1 ml of spore suspension conserved in a freezer (-10°C). Frozen fungus spores were (107 spore /ml) was plated on agar (2%). The tests were then collected and used to inoculate a CD agar plate and realised in triplicate and the whole material was maintained at incubated at 23°C for 7 days. A volume of 10 ml distilled water 23°C at a relative humidity (RH) of 100%. Spores of the supplemented with Tween 80 at 0.5% was pooled on the germing fungus were counted by optical microscope every hour Bouhous and Larous 2437

100

80

60

40 % of germination 20

0 1 2 3 4 5 6 8 10 11 12 Time (hours)

Figure 3. V. lecani spore germination at 24°C and H.R=100%.

during 12 h (Drummond, 1987; Hall, 1976). 1.5x107 spores/ml. The whole (eggs/ larvae/ spores) were incubated at 20°C, first at 100% RH during 16 h and then at 70% RH during 4, 8, 12, 20 and 96 h. It was finally put at a RH Insect culture of 100% during 7 days compared to control. For each case, the mortality rate was estimated after 7 days. Two young bean plantations in the stage of the apparition of the two first leaves were infected by several aleurodes during 24 h in an insect cage (60x60x60 cm). Aleyrodes were eliminated RESULTS after setting their eggs on the lower face of the leaves, so that the obtained population of eggs, larvae and adults will be homogenous. Species identification

Samples of infected leaves with different larval stages Eggs and stage II larvae treatment of the aleurode were collected from two agricultural regions (El Kennar and El Aouana) (Figure 2). Slides Leaves were observed under a magnifying glass to localize the were prepared and examined by the optical infected zones. 7 mm diameter discs of leaves were cut, each microscope (Colles, 1958). Identification was done disc contains at least 2 to 20 eggs or stage II larvae. Discs were immersed during 10 s in a freshly prepared fungus spore according to Martin (1987). Leaves samples analysis suspension, and then 10 of these discs were placed on the allowed noting the total predominance of T. surface of the agar 1.5%. The Experiment was carried out in vaporariorum species. The species exists on triplicate. Controls were provided for each experiment. greenhouse cultures and even on other outside Incubations were done in desiccators at 20°C under a relative adventitious plants. humidity of 100% with a photoperiod of 16 h per 24 h. Mortality percentage was determined from the 5th day of contact. LD50 were calculated according to the method of Probit (Bliss, 1935; Spore germination Drummond et al., 1987).

The spore germination starts since the first hour and Adults treatment reaches a rate of 65% after 9 h and 92% after 12 h of incubation (Figures 3 and 4). Adults of the same age were captured after cooling during 5 min in the refrigerator. They were then recovered and distributed in tubes to get 30 insects per tube. They were Effect of fungal spore concentration on different cooled again and put quickly in contact with a 7 days fungus culture. After 30 min of contact, spore infected adults were development stages of T. vaporariorum released in the desiccators each containing a host plant. Desiccators were adjusted to a relative humidity of 100% at Effect on eggs 20°C and exposed to light during 16 h per 24 h in a phytotron incubator (Drummond et al., 1987; Ekbom, 1979). The percentages of mortality after treatment showed an increase of about 6.9, 14 and 61.8% for the three 7 Effect of relative humidity (RH) doses and contact times used. LD50 were: 4.2x10 7 spores/ml in day 5; 2.1x10 spores/ml in day 6 and 7 Eggs and Stage II larvae were treated with a dose of about 0.59x10 spores /ml in day 7 (Table 1). Thus, LD50 in 2438 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 LD50 788 0.69±0.10 (13.060-86.134)106 12.709 4.2x107 788 0.73±0.09 (93.573-37.621)106 15.929 2.1x107 788 1.02±0.10 (41.553-79.95)106 23.169 0.59x107

6 8

a 8 probit

probit b 6 4 probit 6 c 4 4 2 2 2 0 2 4 6 8 10 0 0 2 4 6 8 10 2 4 6 8 10 Log dose of spores/ml -2 -2 log dose of spores/ml -2 log dose of spores/ml

Figure 5. Effect of V. lecanii on T. vaporariorum eggs. Day 5 (a), day 6 (b) and day 7 (c) after treatment.

3 3 day 5 was 7 folds higher than that found in day 7 the dose of spores used. LD50 were: 11x10 , 3.4x10 (Figure 5a, b and c). Microscopic examination showed and 0.5x103 spores/ml for the three incubation times that the infected T. vaporariorum egg was surrounded (Table 2). Figure 8 showed T. vaporariorum larvae with V. lecanii mycelium (Figure 6a and b). with an abnormal shape surrounded with V. lecanii mycelium.

Effect on larvae Effect on adults As seen in Figure 7, the percentage of mortality increased according to contact time, whatever was After 7 days of incubation of 120 adult insects, only 81 Bouhous and Larous 2439

b a

Figure 6. A T. vaporariorum egg control (a) A T. vaporariorum egg surrounded with V. lecanii mycelium, (b) G 40X0.75.

15

10

) spores/ml 3

5 LD50 (10

0 4 5 6 7 8

Days after incubation

Figure 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 LD50 604 0.26± 0.08 17.357 103-94.40 105 72.25 11X103 604 0.30±0.08 42.156 10- 33.495 105 61.43 3.4X103 604 0.28±0.09 25.64 -13.045 107 44.32 0.5X103

were found to be completely invaded by means of the Information about probit analysis (No. of insects fungus mycelium (Figure 9). (n=885), Slope ± S: 0.88± 0.10, LC (95%): 3.74-7.23, Chi-square: 1.44).

Influence of humidity DISCUSSION The incubation at 70% relative humidity did not show any inhibitory effect on the pathogenesis of the Experiments on the efficiency of the fungus against T. fungus, since the mortality rate of the larvae was vaporariorum investigated the influence of various 100% and that of eggs was relatively high (Figure 10). environmental factors. Experimental of 2440 Afr. J. Microbiol. Res.

Figure 8. T. vaporariorum larvae with an abnormal shape surrounded with V. lecanii mycelium. G 40X0.75.

Figure 9. An adult T. vaporariorum surrounded with V. lecanii mycelium G 40X0.75.

larvae, adults and eggs showed that the observed treatment was very efficient in small experimental mortality rates on the different stages treated by greenhouses. In our experiments, the substitution of fungal spores are important and in particular in the experimental greenhouses with desiccators confirmed 3 case of larvae (LD50: 0.5x10 spores/ml). this efficiency. Therefore, 81 adults over 120 used 5 Drummond et al. (1987) found an LD50 of 1.5x10 were completely invaded by the fungal mycelium. spores/ml in the same conditions of temperature and Others died by drowning due to water condensation humidity which confirmed the efficiency of the used on desiccator’s walls. strain against the development of T. vaporariorum. In Eggs, larvae as well as adults treatment showed contrast to the results obtained with larvae, the that the used V. lecanii strain is very pathogenic efficiency of eggs treatment by fungal spores is lower against T. vaporariorum particularly in larval stage. 6 with an LD50 of 5.9x10 spores /ml. These results are The lower pathogenesis on eggs could be explained conforming to those obtained by Hall (1982), which by the presence of the rigid cuticle which hinders the observed that, for adult insects, fungal spore penetration of the mycelium. Given that insect Bouhous and Larous 2441

Development stages transfer of the insects maintained at an RH of 70% for 96 h did not influenced the mortality rates as compared with the control maintained at 100% during the complete experimentation period. Drummond et al. (1987) observed that more the

Probit fungus is pathogenic more it is independent on low humidity. However, Fargues et al. (2005) showed that the entomopathogenic Hyphomycetes have strong potential for microbial control of whitefly larvae infesting tomato crops at moderate ambient humidity in Mediterranean greenhouses. The characteristic of V. lecanii regarding the relative humidity could be of great importance for its application in greenhouses where important humidity fluctuations are noted. Log time (hours)

Figure 10. Effect of relative humidity (70%) on T. vaporariorum eggs mortality after treatment with V. lecanii. Conclusion Used dose =1.5x107 spores /ml. Based on the realised tests, the entomopathogenic fungus V. lecanii is particularly infectious for larval and adult stages, and slightly infectious for eggs. In 3 development inevitably passes by larval stages, which larvae, LD50 is relatively low (0.5x10 spores/ml) in the let us suppose that eggs that have escaped the experimentation conditions. The action time of the treatment will be attacked in the larval stage. The fungus reached its maximum in day 7. Concerning the percentage of mortality of spore-treated eggs and effect of humidity on the development of the fungus larvae increased throughout the incubation period in and on its virulence, results presented here allowed experimental conditions, thus, fungus efficiency us to consider a future practical application of V. requires some contact time. lecanii in the biological control of T. vaporariorum. Treatment efficiency depends closely on a high Further applied research is required to determine the relative humidity rate (95 to 100%) necessary to the greenhouse application conditions. fungal spore germination. A decrease in humidity attenuates the fungal infectious capacity (Milner and REFERENCES Luton, 1986). It is then recommended to maintain a very high relative humidity after treating the aleurode Bliss CI (1935). The calculation of the dosage-mortality curve. Ann. by the fungus (Drummond et al., 1987). It is clear that Appl. Biol., 22: 134-167. humidity in greenhouses is not constant, its recording Colles DH (1958). An improved technique for permanent mouts of smale insects and nematodes. Bull. Entomol. Res., 49(1): 45-47. performed in Jijel during May and June 2008, showed Coffin RS, Coutts RHA (1995). Relationship among Trialeurodes a decrease in the weekly mean from 57.34% in the vaporariorum –transmitted yellowing viruses from Europe and morning to 96.22% in the night. The other important North America. J. Phytopathol., 143: 375-380. factor is the capacity of fungal spore germination Cortez-Madrigal H, Allatorre-Rosas R, Mora-Aguilera G, Bravo- Mojica H, Ortiz-Garcia CF, Aceves-Navarro LA (2003). which should be higher than 90%, for the studied Characterization of multisporic and monosporic isolates of strain, spore germination reached 92% after 12 h of Lecanicillium(=Verticillium)lecanii for the management of incubation in the experimental conditions, which is Toxoptera aurantii in cocoa. BioControl, 48: 321-334. considered as a good result. Dipietro JP (1977). Contribution à l’étude d’une méthodologie de lutte biologique contre l’aleurode des serres Trialeurodes Germination capacity could be influenced by vaporariorum West. (Homopt., Aleurodidae). Thèse Doct. several factors such as spore age, temperature as Toulouse, 112 p. well as relative humidity; consequently, evaluation of Drummond I, Heale JB, Gillespie AT (1987). Germination and effect germination capacity of the studied strain should be of reduced humidity on expression of pathogenicity in Verticillium lecanii against the glasshouse whitefly Trialeurode carried out permanently before each biological control vaporariorum. Appl. Biol., 11(1): 123-201. treatment. It was reported that one of the properties of Ekbom BS (1979). Investigations on the potential of the fungus highly pathogenic and virulent strains is their rapid Verticillium lecanii for biological control of the greenhouse germination. According to Hall (1984), 50% of the whitefly Trialeurode vaporariorum. Swedish J. Agric. Res., 2: 125-138. spores of virulent strains germinate after 9 h of Fargues J, Smits N, Rougier M, Boulard T, Ridray G, Lagier J, incubation at 100% RH. Jeannequin B, Fatnassi H, Mermier M (2005). Effect of Spore germination rate of the studied strain reached microclimate heterogeneity and ventilation system on 65% after 9 h of incubation at 100% RH. This fungal entomopathogenic hyphomycete of Trialeurodes vaporariorum (Hmoptera:Aleyrodidae) in Mediterranean property is very important because a relative humidity greenhouse tomato. Biol. Control, 32: 461-472. higher than 95% rarely persists in greenhouses during Faria M, Wraight SP (2001). Biological control of Bemisia tabaci more than 12 h, as showed by the recordings carried with fungi. Crop Prot., 20: 767-778. out in a greenhouse of Jijel. Because of the rapid Guevara-Coto JA, Barboza-Vargas N, Hernandez-Jimenez E, Hammond RW, Ramirez-Fonseca P (2011). Bemisia tabaci germination of the fungal strain, it depends a little on biotype Q is present in Costa Rica. Eur. J. Plant Pathol., 131: the RH after an incubation period of 16 h at 100% RH. 2167-170. 2442 Afr. J. Microbiol. Res.

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