J. Appl. Entomol.

Isolation of entomopathogenic fungi from the soil and their pathogenicity to two fruit y species (Diptera: Tephritidae)
P. Sookar1, S. Bhagwant2 & E. Awuor Ouna3
1 Ministry of Agro Industry & Fisheries, Entomology Division, Reduit, Mauritius 2 Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, Mauritius 3 International Centre of Insect Physiology and Ecology, Nairobi, Kenya

Keywords Bactrocera cucurbitae, Bactrocera zonata, Beauveria bassiana, Metarhizium anisopliae, biocontrol Correspondence Preeaduth Sookar (corresponding author), Ministry of Agro Industry & Fisheries, Entomology Division, Agricultural Services, Ministry of Agro Industry, Food Production and Security, Reduit, Republic of Mauritius. E-mail: psookar@mail.gov.mu Received: June 16, 2008; accepted: September 22, 2008. doi: 10.1111/j.1439-0418.2008.01348.x

Abstract The occurrence of deuteromycetous entomopathogenic fungi was determined by examining 224 soil samples from 19 locations in three climatic zones of Mauritius. Three sites were sampled per location: one site under vegetables cultivation, one site under sugar cane plantation and one natural site each within 1 km of each other. Soil samples were baited with the waxmoth larvae Galleria mellonella L. and incubated in the dark at 15, 20, 25 or 30C for 7, 14 and 21 days. Entomopathogenic fungi were isolated from 77 out of 224 (38.6%) soil samples. Metarhizium anisopliae was isolated from 42 (18.8%) samples, Beauveria bassiana from 24 (10.7%), Metarhizium spp. and Paecilomyces fumosoroseus from 5 (2.2%) each and Beauveria spp. from 1 (0.4%). It was observed that M. anisopliae was isolated more frequently from soils under vegetables as compared to soils under sugarcane or habitat with natural vegetation. Beauveria bassiana was isolated more frequently at the lowest incubation temperature (15C) while M. anisopliae isolates were recovered more frequently at higher temperatures (25 and 30C). The pathogenicity of seven isolates of M. anisopliae, ve isolates of B. bassiana and two isolates of P. fumosoroseus towards the adults of Bactrocera zonata and Bactrocera cucurbitae was tested by topical application of conidial suspension of 1 106 conidia/ml. All the isolates tested were pathogenic to the two fruit y species. Mortality of B. zonata varied between 12.0 and 98.0% and between 2.0 and 94.0% in B. cucurbitae at 5 days post-treatment. Our results suggest that entomopathogenic fungi present locally, could be integrated for the control of B. zonata and B. cucurbitae.

Introduction Tephritid fruit ies are among the major pests of fruits throughout the world and represent the most economically important group of phytophagous Diptera (White and Elson-Harris 1994). In Mauritius, peach fruit y Bactrocera zonata (Saunders) and the melon y Bactrocera cucurbitae (Coquillett) are important pests of fruits and cucurbits respectively. The Mauritian National Fruit Fly Control Programme, set
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up in 1994, aims at controlling fruit ies so that there is an increase in fruit production both in terms of quality and quantity (Permalloo et al. 1998). Bait sprays made up of a mixture of protein hydrolysate and insecticides have been used to attract and kill fruit ies. On the other hand, fruit growers make use of broad-spectrum insecticides to protect their crops from fruit y attack besides protein bait sprays (Sookar and Khayrattee 2000). The need for investigation into the biological control of fruit ies in

J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

P. Sookar, S. Bhagwant and E. Awuor Ouna

Isolation and pathogenicity of entomopathogenic fungi from soil

Mauritius is ever more important as it becomes recognized that insecticides, although offering expedient and predictable results under certain conditions, are often inadequate and perceived as dangerous, if not physically dangerous to wildlife and humans alike. As an alternative to chemical control or as part of integrated pest management programmes, there is a resurgence of interest in the use of microbial insecticides for biological control of insect pests. Fungal agents are among the most promising group of biological control agents against insect pests as they infect their host through the cuticle. Entomopathogenic fungi have a global distribution (Zimmermann 1993) and wide insect host ranges (Mugnai et al. 1989). Their ability to regulate insect populations is well established in the tropics (Maniania 1991; Tigano-Milani et al. 1995) and temperate environments (Doane 1959). The development of resistance to chemical insecticides and concerns over the deleterious effects of chemicals on environmental and human safety have provided an impetus for exploring and developing microbial control agents for use in integrated control of insect pests (Inglis et al. 2001). Biopesticides based on entomopathogenic fungi show promise for insect pest management. About 750 species of such fungi have been reported (Babu 1992). Many of the entomopathogenic fungal species have many strains. Extensive collection of fungi and their characterization are needed. The genera most commonly encountered in nature include: Entomophthora, Beauveria, Metarhizium, or Aspergillus (Roberts and Yendol 1971). The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin has been isolated from 200 insects species including the orders of Lepidoptera, Coleoptera, Orthoptera, and Hemiptera. A large amount of genetic diversity has been reported in M. anisopliae and Beauveria bassiana (St Leger et al. 1992; Bidochka et al. 1994), and the potential existence of strains adapted to various hosts, environmental conditions, conidial survival and competitive saprophytic ability can profoundly inuence their virulence. Studies have shown that strains can be distinguished by their different levels of proteases, chitinases and lipases (Bridge et al. 1990; Varela and Morales 1996). De La Rosa et al. (1997, 2000) reported that the most effective strain of B. bassiana in the laboratory was less aggressive in the eld against the coffee berry borer, Hypothenermus hampei (Ferrari). Entomopathogenic fungi offer greater opportunity for biological control of adult fruit ies compared to bacteria and viruses, which must be ingested to be

effective. However, only a few studies have attempted to use them in fruit y management (Debouzie 1989; Strongman et al. 1997; Castillo et al. 2000; De La Rosa et al. 2002). The pathogenicity of locally isolated fungi to adults of B. zonata or B. cucurbitae, which are two important insect pests in Mauritius (Permalloo et al. 1998; Sookar and Khayrattee 2000), have not yet been studied. Moreover, Petch (1941) recorded several species of entomopathogenic fungi in Mauritius belonging to the subdivisions: Zygomycotina (Hypocrella spp., Myriangium spp., Torribiella spp.) and Deuteromycotina (Aschersonia spp., Aspergillus spp., Beauveria spp., Gibellula spp., Hirsutella spp., Hymenostilbe spp., M. anisopliae, Tetracrium spp.). Unfortunately, there is no collection of these fungi. The objectives of the study were therefore to search for locally present entomopathogenic fungi and to evaluate their pathogenicity to B. zonata and B. cucurbitae. Materials and Methods
Detection of entomopathogenic fungi from dead fruit ies in culture colonies

Dead ies of Bactrocera zonata (Saunders) and B. cucurbitae (Coquillett) were collected at weekly intervals from the fruit y colonies that are kept in the laboratory of the Entomology Division of the Agricultural Services of the Ministry of Agro Industry, Food Production and Security at Reduit. The dead ies were collected over a period of 6 months as from June 2003. The insects were placed in 70% alcohol for 35 s to facilitate wetting of the specimen, placed in 1% sodium hypochlorite for another 35 s, rinsed briey in three changes of sterile water and then blotted dry with sterile lter paper. They were placed in sealed glass Petri dishes on a humid sterile lter paper. The Petri dishes were kept in the dark at room temperature for a period of 14 days to allow fungal growth.
Detection of entomopathogenic fungi from fruit ies collected in pheromone baited traps

Entomology Division Ministry of Agriculture (EDMA) traps baited with male pheromones were placed at seven locations, namely: Labourdonnais, Plaisance and Curepipe in vegetable plantations, Fort William, Beau Bassin, Riviere Noire and Pamplemousses in mixed orchards (table 1 and g 1). At each location, there were three sets of traps baited with Cuelure, Methyl Eugenol or Trimedlure to
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J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

Isolation and pathogenicity of entomopathogenic fungi from soil

P. Sookar, S. Bhagwant and E. Awuor Ouna

Table 1 Coordinates and altitudes of the trapping and soil sample locations Location number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Altitude (m) 107 74 92 72 65 4 304 65 140 211 206 6 582 556 485 44 117 276 63 45 4 68 468

Location Belle Vue Labourdonnais Mapou Pamplemousses Bois Marchand Fort William Reduit Richelieu Roche Brunes Beau Bassin Barkly Riviere Noire Curepipe Plaine Sophie Bassin Blanc Bel Ombre Riviere des Anguilles Rose Belle Plaisance Bramsthan Belle Mare Flacq Nouvelle Decouverte

Longitude 200358.27S 20415.47S 20447.67S 200614.09S 200653.05S 200855.81S 201405.13S 201130.99S 201446.51S 201316.85S 201333.84S 202110.74S 202004.33S 202133.06S 202712.36S 203038.25S 202923.18S 202406.71S 202544.72S 201236.66S 201159.49S 201213.91S 201102.27S

Latitude 573631.07E 573658.88E 573630.69E 573447.70E 572734.61E 572937.79E 572936.69E 572734.61E 572518.03E 572802.81E 572742.66E 572153.87E 573057.28E 572924.37E 572839.77E 573207.44E 573329.80E 573549.75E 573957.05E 574356.38E 574651.56E 574304.26E 573538.41E

The locations are shown in g. 1.

attract B. cucurbitae, B. zonata and Ceratitis rosa Karsch, Ceratitis capitata (Wiedemann) respectively. The traps were placed in the eld at the beginning of June 2003 for a period of 6 months. They were checked and the pheromones renewed at fortnightly intervals. Fruit ies collected from the different traps were brought to the laboratory in separate containers. They were surface sterilized and incubated as described above.
Soil baiting with the waxmoth larvae, Galleria melonella L.

Soil samples were collected at 19 locations in three climatic zones of the island: Belle Mare, Belle Vue, Bel Ombre, Bois Marchand, Bramsthan, Labourdonnais, Mapou, Plaisance, Richelieu and Roche Brunes in the sub humid zone with an annual rainfall below 1200 mm; Barkly, Bassin Blanc, Flacq, Nouvelle Decouverte, Reduit and Riviere des Anguilles in the humid zone with an annual rainfall of 1200 2400 mm; Curepipe, Plaine Sophie and Rose Belle in the super humid zone with an annual rainfall of
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more than 2400 mm (table 1 and g. 1). Three sites were sampled per location: one site under vegetables, one site under sugarcane and one site under natural vegetation each within 1 km of each other. At Barkly there was no site under sugarcane plantation. A total of 224 soil samples were collected from July to November 2003. Approximately 1 kg of soil was taken from the top 10 cm from each site. The samples were placed in plastic bags. Each soil sample was sifted through 0.5-cm mesh and then divided into four portions. Each portion was placed into a 250-ml plastic cup with a lid. Samples were processed within 24 h. Waxmoth larvae Galleria mellonella L. are susceptible to infection by entomopathogenic fungi (Zimmermann 1986; Vanninen 1996; Bidochka et al. 1998). The larvae were used as bait insects. Honey combs infected with the waxworm larvae were collected from beekeepers and brought to the laboratory to set up a culture. Adults laid eggs on a dry honey comb, which was renewed every 45 days. The honey combs with eggs were kept in a separate cage. Larvae were fed with dry honey comb and those that were in their pupal case were used as the bait insects. Several larvae were placed in sterilized Petri dishes on a humid lter paper that were sealed with a cling lm prior to the baiting experiments to ensure that they were disease free. The collected soil samples were moistened with sterile distilled water and then baited with the waxmoth larvae. Three larvae (L4) were placed into the soil with sterile forceps. One container from each group of four was incubated in the dark at 15, 20, 25 or 30C. These temperatures were chosen to investigate dependence of infection on temperature. Larvae were checked after 7, 14 and 21 days. Dead larvae were removed from the soil with sterile forceps and placed in a 1% sodium hypochlorite solution for 3 min to surface sterilize the larvae, after which they were washed in sterile distilled water for 3 min. Excess water was removed by dabbing with a piece of sterilized paper, and the dead larvae were placed individually in sterilized 9-cm glass Petri dishes on humid lter paper. The Petri dishes were sealed with Para lm, incubated at 25C and checked for fungal outgrowth every 2 days. When the presence of a fungus was observed, conidia were removed from the insect surface with a sterile loop and streaked onto potato dextrose agar plates containing 0.05 g/l chloramphenicol previously dissolved in 10 ml absolute alcohol. The fungi were identied by microscopical examination of the sporulating structures. A fungus recovered from an

J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

P. Sookar, S. Bhagwant and E. Awuor Ouna

Isolation and pathogenicity of entomopathogenic fungi from soil

1 2 3 4 5 6 8 9 7 10 11 21 20 13 14 12 15 16 17 18 19 23 22

10 km

Fig. 1 Map of Mauritius showing trapping () and soil sample (s) locations.

infected waxmoth larva is considered to be an isolate. The fungal isolates were sent to International Centre of Insect Physiology & Ecology (ICIPE), Nairobi, Kenya for identication. Pathogenicity Test
Insects

2 ml for 2.5 kg of larval diet. Adult ies were reared in 1 m3 nylon netting insect cage at room temperature and were provided with tap water and a diet of enzymatic yeast hydrolysate (USB, Corporation, Cleveland, OH, USA) and sugar in a ratio 3 : 1. In all experiments, 5 to 10-day-old adult ies were used for the bioassays.
Conidial viability tests of the fungi

Colonies of two species, B. zonata or B. cucurbitae, were mass reared in the laboratory of the Entomology Division of the Ministry of Agro Industry & Fish eries, Reduit. The initial colony of B. zonata was derived from guava Psidium guajava L., mango Mangifera indica L. and Indian almond Terminalia catappa L. fruits collected from different parts of the island. The colony of B. cucurbitae was derived from cucurbit fruits collected from farmers elds. The larvae of the two species were fed on an articial diet comprising of sugarcane bagasse (6%), ground maize (6%), cane sugar (11%), waste brewers yeast (6%), wheat bran (6%), sodium benzoate (0.1%), nipagen (0.1%) and water (64.8%) by weight. The egg seeding rate was

Conidia were harvested by scraping the surface of 3-week-old culture. Spores were suspended in 20-ml sterile distilled water, 0.05% Triton X-100 in glass bottles containing 3 mm glass beads. Bottles were stoppered and vortexed for 5 min to produce a homogeneous conidial suspension. Conidia were then quantied with a haemocytometer following serial dilution in sterile distilled water. The viability of conidia was determined by spread-plating 0.1 ml of conidial suspension (titrated to 3 106 conidia ml)1) on four Sabouraud Dextrose Agar plates. Sterile microscope cover slips were placed on each plate. The plates were incubated at 2429C and examined
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J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

Isolation and pathogenicity of entomopathogenic fungi from soil

P. Sookar, S. Bhagwant and E. Awuor Ouna

after 20 h. Percentage germination was determined by counting approximately 100 spores for each plate at 200 magnication. Each plate served as a replicate with four replications per isolate.
Bioassay

probit analysis method for correlation data (Throne et al. 1995). All analyses were performed using the sas package (SAS Institute, 2001). Results

Conidial suspension of 1 106 conidia ml)1 was prepared as described above. One microlitre of the conidial suspension was applied with a micropipette to the ventral surface of the abdomen of 2-day-old adult fruit ies (ve males and ve females), which had previously been anaesthetized with ice. There were ve replicates. Control ies were treated with 1 ll of 0.05% Tween 80. Treated ies were placed in a cage (150 150 200 mm). The insects were fed on a 3 : 1 mixture of sugar and yeast hydrolysate. A sponge soaked in water was also provided as a water source. Mortality was recorded daily for a period of 10 days. Dead insects were surface-sterilized in 70% alcohol followed by three rinses in sterile distilled water and transferred to Petri dishes lined with damp sterilized lter paper to allow fungal growth on the surface of the cadaver. Mycosis was conrmed by microscopic examination. Statistical Analysis The effect of geographical location (three zones), habitat type (vegetables, sugarcane or natural), soil humidity and incubation temperature (15, 20, 25, or 30C) on the occurrence of entomopathogenic fungi obtained from waxworm larvae baited in soil samples was examined using a full analysis of variance followed by a factorial analysis. The Tukey honesty signicant difference (Tukey HSD) multiple comparison test (post hoc) was then employed to determine signicant differences between means in the event of a signicant F value. Percentage germination of conidia for the isolates was arcsine transformed before analysis of variance. Means were separated by Student-Newman-Keuls (SNK) test. Fly mortalities were adjusted for natural mortality in the control insects using Abbotts formula (Abbott 1925), where the proportion of insects killed by the entomopathogen alone (P) was [(C-T)/C)] x 100, where C is the percentage of the control insects that are living and T is the proportion of treated insects that are living after the experimental period. anova was performed on the percentage mortality data after transformation to arcsine (%) scale) to homogenize the variance. Mean values were separated by the SNK test. LT90 values were determined for each replicate using the
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Detection of entomopathogenic fungi from dead fruit ies in culture colonies and in trapped fruit ies

Both laboratory reared and trapped fruit ies were surface-sterilized and then incubated to allow fungal growth. The laboratory-reared fruit ies comprised 3486 B. zonata and 3990 B. cucurbitae while the trapped fruit ies included 14 331 B. zonata, 15 715 B. cucurbitae, 1561 C. capitata and 3930 C. rosa. No entomopathogenic fungi were recovered from the incubated insects.
Soil baiting with the waxmoth larvae, Galleria melonella L.

The Entomopathology Unit of ICIPE identied M. anisopliae (Metchnikoff), Metarhizium spp., B. bassiana (Balsamo) Vuillemin, Beauveria spp. and Paecilomyces fumosoroseus from the fungal isolates. Fungi were isolated from 77 of 224 (34.4%) soil samples. Metarhizium anisopliae was isolated from a total of 42 (18.8%) samples, B. bassiana from 24 (10.7%), Metarhizium spp. and P. fumosoroseus from 5 (2.2%) each and Beauveria spp. from one sample (0.4%). The number of fungal isolates by location was as follows: M. anisopliae 15 out of 19 (78.9%), Metarhizium spp. 3 (15.8%), B. bassiana 11 (57.9%), Beauveria spp. 1 (5.3%) and P. fumosoroseus 4 (21.1%). At least one entomopathogenic fungus was isolated from any one of the locations. Location showed a signicant effect on occurrence of M. anisopliae (anova: F18, 671 = 2.85, P < 0.0001), B. bassiana (anova: F18, 671 = 4.10, P < 0.0001) and P. fumosoroseus (anova: F18, 671 = 2.65, P = 0.0002). Zone had no signicant effect on the occurrence of either M. anisopliae (anova: F2, 671 = 0.20, P = 0.8173) or P. fumosoroseus (anova: F2, 671 = 2.55, P = 0.0786) (table 2). However, the occurrence of B. bassiana was signicantly affected by zone (anova: F2, 671 = 6.54, P = 0.0015). Signicantly more B. bassiana isolates were recovered in the super humid zone as compared to either the humid or sub humid zones (P < 0.05) while there was no signicant difference on the number of isolates obtained between the humid and sub humid zones (P > 0.05). There was a signicant difference on the occurrence of M. anisopliae (anova: F2, 671 = 8.45,

J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

P. Sookar, S. Bhagwant and E. Awuor Ouna

Isolation and pathogenicity of entomopathogenic fungi from soil

Table 2 Occurrence of entomopathogenic fungi by zone Paecilomyces fumosoroseus nf 4 0 1 %f 3.3 0.0 2.8

Zone Subhumid Humid Superhumid

1 2

Total no. of soil samples 120 68 36

Metarhizium anisopliae nf1 24 16 2 %f2 20.0 23.5 5.6

Metarhizium spp. nf 2 3 0 %f 1.7 4.4 0.0

Beauveria bassiana nf 10 6 8 %f 8.3 8.8 22.2

Beauveria spp. nf 1 0 0 %f 0.8 0.0 0.0

Total nf 41 25 11 %f 34.2 36.8 30.6

No. of soil samples with fungi. Percentage no. of waxmoth larvae with fungi

P = 0.0002) and B. bassiana (anova: F2, 671 = 9.89, P < 0.0001) by habitat type (table 3). Signicantly more M. anisopliae isolates were recovered in vegetable plantations than either sugarcane plantations or habitat with natural vegetation (P < 0.05). However, there was no signicant difference in the number of M. anisopliae isolates obtained between sugar cane plantations and habitat with natural vegetation (P > 0.05). Signicantly more B. bassiana isolates were obtained in habitat with natural vegetation as compared to either vegetables or sugar cane plantations (P < 0.05). On the other hand, occurrence of P. fumosoroseus was not affected by the habitat (anova: F2, 671 = 2.92, P = 0.0547). The humidity of soil samples did not affect the occurrence of M. anisopliae (anova: F1, 671 = 0.24, P = 0.6242), B. bassiana (anova: F1, 671 = 0.26, P = 0.6101) or P. fumosoroseus (anova: F1, 671 = 2.09, P = 0.1489). The recovery of M. anisopliae (anova: F3, 671 = 14.66, P < 0.0001), B. bassiana (anova: F3, 671 = 29.62, P < 0.0001) or P. fumosoroseus (anova: F3, 671 = 5.36, P < 0.0012) was affected by the temperatures at which the soil samples were incubated (table 4). Higher number of M. anisopliae isolates were recovered from soil samples incubated at either 25 or 30C than at 15C or 20C (P < 0.05). The

recovery of B. bassiana was highest at 15C than at 20, 25 or 30C (P < 0.05). The incubation period of the soil samples baited with the waxmoth larvae signicantly affected recovery rates of M. anisopliae (anova: F2, 668 = 84.45, P < 0.0001), B. bassiana (anova: F2, 668 = 11.78, P < 0.0001) or P. fumosoroseus (anova: F2, 668 = 5.80, P < 0.0032). Signicantly more waxmoth larvae were infected with M. anisopliae after either 7 or 14 days post-incubation compared to 21 days (P < 0.05).
Conidial viability tests of the fungi

In viability tests, germination of conidia ranged from 40.5 to 100% after 20 h. Seven M. anisopliae isolates, two P. fumosoroseus and ve B. bassiana isolates, which had germination above 94% were selected for the pathogenicity tests against B. zonata and B. cucurbitae. Fungal isolates with irregular germination pattern were excluded.
Pathogenicity tests

Mortality in the controls was 4.0% in B. zonata and 2.0% in B. cucurbitae, 8 days after treatment (tables 5 and 6 respectively). The three fungal spe-

Table 3 Occurrence of entomopathogenic fungi by habitat Paecilomyces fumosoroseus

2

Habitat Natural Sugarcane Vegetables

1 2

No. of soil samples 76 72 76

Metarhizium anisopliae nf
1

Metarhizium spp. nf 1 2 2 %f 1.3 2.8 2.6

Beauveria bassiana nf 8 6 10 %f 10.5 8.3 13.2

Beauveria spp. nf 0 1 0
1

Total nf 27 17 38 %f 35.5 23.6 50.0

%f

%f

nf 4 1 5

%f 5.3 1.4 6.6

14 7 21

18.4 9.7 27.6

0.0 1.4 0.0

No. of soil samples with fungi. Percentage no. of waxmoth larvae with fungi.

J. Appl. Entomol. 132 (2008) 778788 2008 Ministry of Agro Industry, Food Production and Security Journal compilation 2008 Blackwell Verlag, Berlin

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Table 4 Occurrence of entomopathogenic fungi in soil samples incubated at different temperatures Paecilomyces fumosoroseus %f2 0.0 0.0 0.6 0.6 nf 8 3 0 0 %f 4.8 1.8 0.0 0.0

Incubated temperature C 15 20 25 30

M. anisopliae nf1 25 41 74 64 %f2 14.9 24.4 44.0 38.1

Metarhizium spp. nf 3 4 2 2 %f 1.8 2.4 1.2 1.2

B. bassiana nf 60 27 15 4 %f 35.7 16.1 8.9 2.4

Beauveria spp. nf1 0 0 1 1

Total nf 96 75 92 71 %f 57.1 44.6 54.8 42.3

At each temperature there were 56 soil samples and 168 waxmoth larvae. No. of soil samples with fungi. 2 Percentage no. of waxmoth larvae with fungi.
1

Table 5 Pathogenicity of Metarhizium anisopliae, Paecilomyces fumosoroseus and Beauveria bassiana isolates against Bactrocera zonata % Mortality (% SE) 95.8 32.7 34.7 98.0 16.0 46.7 71.3 89.8 12.0 14.2 20.2 67.6 98.0 36.4 2.0 2.6 1.9 2.3 2.0 5.1 4.6 3.9 3.2 3.7 2.4 4.4 3.4 2.0 4.8 2.0 LT90 (95% CI) a 5.2 (5.15.3) cd 9 (8.79.2) cd 14.4 (13.515.4) a 4.6 (4.54.8) e 9.3 (9.19.6) c 11.9 (11.312.5) b 7.4 (7.27.6) a 5.2 (5.15.4) e 10.4 (10.110.8) e 28 (23.934.2) de 9.2 (8.99.4) b 7.4 (7.37.6) a 4.2 (4.14.3) cd 9.2 (8.99.6) f Slope (SE) 0.73 0.42 0.19 0.84 0.38 0.24 0.41 0.36 0.41 0.10 0.42 0.54 0.60 0.28 0.02 0.01 0.01 0.03 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01

Table 6 Pathogenicity of Metarhizium anisopliae, Paecilomyces fumosoroseus and Beauveria bassiana isolates against Bactrocera cucurbitae % Mortality (% SE) 42.0 90.0 56.0 94.0 46.0 48.0 80.0 48.0 46.0 2.0 84.0 34.0 28.0 16.0 LT90 (95% CI) Slope ( SE) 0.45 0.39 0.53 0.45 0.39 0.34 0.51 0.54 0.38 0.18 0.53 0.37 0.44 0.29 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.01 0.01 0.01 0.01

Treatment M65 M103 M196 M235 M394 M471 M499 P. fumosoroseus M603 M637 B. bassiana M257 M265 M397 M421 M433 Control M. anisopliae

Treatment M65 M103 M196 M235 M394 M471 M499 P. fumosoroseus M603 M637 B. bassiana M257 M265 M397 M421 M433 Control 0d M. anisopliae

3.7 b 7.6 (7.47.8) 7.8 a 5.7 (5.55.8) 5.1 b 7.4 (7.27.5) 4.0 a 5.2 (5.15.4) 2.4 b 8.1 (7.98.3) 2.0 b 9 (8.89.4) 8.9 a 6.1 (5.96.2) 3.7 b 6.5 (6.36.6) 11.7 b 9.1 (8.89.4) 2.0 d 23.6 (19.730.5) 4.0 a 6.4 (6.26.5) 2.4 bc 10.1 (9.810.4) 3.7 bc 8.5 (8.38.7) 2.4 c 11.5 (11.012.0)

Percent mortality at 5 days post-treatment and LT90 values with 95% condence interval. Within column, mean values followed by the same letters are not signicantly different (Student-Newman-Keuls SNK, P = 0.05). Data were arcsine transformed before analysis.

Percent mortality at 5 days post-treatment and LT90 values with 95% condence interval. Within column, mean values followed by the same letters are not signicantly different (Student-Newman-Keuls SNK, P = 0.05). Data were arcsine transformed before analysis.

cies were pathogenic to both B. zonata and B. cucurbitae, but mortality was signicantly different among the isolates at 18 days after treatment. Mortality of B. zonata varied between 12.0 and 98.0% (F = 49.5; d.f. = 14, 74; P < 0.0001) (table 5) and between 2.0 and 94.0% in B. cucurbitae (F = 57.8; d.f. = 14, 74; P < 0.0001) (table 6) at 5 days post-treatment. The lethal time to 90% mortality (LT90) varied from 4.2 to 27.8 days in B. zonata and from 5.2 to 23.6 days in B. cucurbitae. Beauveria bassiana isolate M421 had the shortest LT90 value (4.2 days) for
784

B. zonata (table 5), while M. anisopliae isolate M235 had the shortest LT90 value (5.2 days) for B. cucurbitae (table 6). Slopes of regression equation ranged from 0.10 to 0.84 in B. zonata (table 5) and 0.18 to 0.54 in B. cucurbitae (table 6). Discussion and Conclusion No entomopathogenic fungus was recovered from either the laboratory reared or the trapped fruit ies. This shows that the fruit y colony in the laboratory was free from entomopathogenic fungi. Likewise,

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the absence of entomopathogenic fungi from trapped fruit ies suggests that either natural contamination is rare or that infected adult fruit ies may be too sick to y to traps. These data conrmed the presence of M. anisopliae, B. bassiana and P. fumosoroseus in the soils of Mauritius as reported by Petch (1941). Metarhizium anisopliae and B. bassiana were more frequent widespread than P. fumosoroseus. The occurrence of these fungi in different localities of Mauritius conrms their cosmopolitan distribution, M. anisopliae and B. bassiana have been isolated from tropical South America (Tigano-Milani et al. 1995), Asia and Africa (Humber 1992), to the Arctic Circle in Finland (Vanninen 1996), Canada (Bidochka et al. 1998) and Russia (Arkhipova 1965). Metarhizium anisopliae was isolated more frequently in the sub humid and humid zones, compared to the super humid zone. On the other hand, B. bassiana was recovered more frequently in the super humid zone compared to the sub humid or the humid zones. Beauveria bassiana isolates were recovered in higher numbers in wet soil samples compared to dry ones. Recovery of M. anisopliae was not inuenced by the humidity of the soil samples. Laboratory trials demonstrated the possibility of infecting insects with fungal entomopathogens at relatively low ambient humidities (4570%) Fargues et al. 1997b; Milner et al. 1997; James et al. 1998; Arthurs and Thomas 2001). However, the level of humidity inuences the transmission of insect pathogens. High environmental moisture has been found to be important for fungal entomopathogens to sporulate (that is to produce infective asexual conidia on the host cadavers during the necrophytic phase of the fungus) (Hall and Papierok 1982; Benz 1987; Carruthers and Soper 1987; Hajek and St Leger 1994). Signicantly more B. bassiana isolates were obtained in habitat with natural vegetation as compared to either vegetables or sugarcane plantations. Metarhizium anisopliae preferred soils under vegetables cultivation as compared to soils under sugarcane cultivation or habitat natural with natural vegetation. Similar ndings have been reported by Rath et al. 1992; Vanninen 1996 and Bidochka et al. 1998;. Several factors inuence the survival of fungal entomopathogens in soils (Fargues and Robert 1985; Daoust and Pereira 1986), and the general nding is for M. anisopliae to persist for extended periods in soils, while B. bassiana is sensitive to competition from soil microbial populations (Clerk 1969; Lingg and Donaldson 1981; Shield et al. 1981).

Beauveria bassiana was isolated more frequently at the lowest incubation temperature (15C), while M. anisopliae isolates were recovered more frequently at the higher temperatures (25 and 30C). These ndings concur with temperature effects on germination of these fungi in nutrient media and insect pathogenicity tests reported elsewhere (Schaerffenberg 1964; Walstad et al. 1970; Hywel-Jones and Gillespie 1990). Temperature also affects the survival rate of entomopathogenic fungi (Roberts and Campbell 1977; Benz 1987). Optimal temperatures for entomopathogenic fungal growth, sporulation and infection often range between 20 and 30C, but variation in temperature tolerance within a strain can be signicant (Hall and Bell 1960, 1961; Stimmann 1968; Walstad et al. 1970; Roberts and Campbell 1977; Carruthers and Haynes 1986; Fargues et al. 1997a). All the isolates of M. anisopliae, P. fumosoroseus and B. bassiana were pathogenic to B. zonata and B. cucurbitae (tables 5 and 6). This nding conrms the susceptibility of fruit ies to entomopathogenic fungi reported previously (Garcia et al. 1984; Castillo et al. 2000; Lezama-Cutierrez et al. 2000; De La Rosa et al. 2002; Ekesi et al. 2002; Dimbi et al. 2003a,b; Uziel et al. 2003; Mochi et al. 2006; Quesada-Moraga et al. 2006). The pathogenicity of entomopathogenic fungi to B. zonata has never been reported before, while the pathogenicity of M. anisopliae and B. bassiana has been reported to other fruit y species, such as Ceratitis capitata (Wiedemann) (Mochi et al. 2006; Quesada-Moraga et al. 2006), Anastrepha ludens (Lezama-Cutierrez et al. 2000; De La Rosa et al. 2002), Ceratits cosyra (Walker) and C. fasciventris (Bezzi) (Ekesi et al. 2002; Dimbi et al. 2003a,b). Although all fungal isolates were pathogenic to the two adult fruit y species, there were considerable variations in their pathogenic activity. The possibility of contaminating adults by using fungus-contamination devices that attract insects to baited stations where they are contaminated with the pathogen and then escape to potentially transmit disease to noninfected individuals could be explored (Maniania 2002). Furthermore, the effect of entomopathogenic fungi against prepupating larvae and puparia in the soil, which provides a favourable environment for fungal growth, should also be explored (Ekesi et al. 2002). This study has demonstrated the virulence under controlled conditions of several isolates of M. anisopliae, B. bassiana and P. fumosoroseus from the soils of Mauritius against B. cucurbitae and for the rst time, against B. zonata too. This is an essential rst step in
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the development of a management strategy which integrates entomopathogenic fungi. Acknowledgements We would like to express our thanks to Dr Noel Govinden, Deputy Director of the Mauritius Sugar Industry Research Institute for his valuable comments on the manuscript and the International Centre of Insect Physiology & Ecology, Nairobi, Kenya for the identication of the fungi. This study has been nanced by the Tertiary Education Commission, University of Mauritius and the Ministry of Agro Industry & Fisheries, Republic of Mauritius. The participation of Mr Sookar in the TEAM meeting in Mallorca, Spain in 2008 was nanced by the Programme Regional de Protection des Vegetaux, Indian Ocean Commission, European Union. References
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