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Neotropical Entomology

Print version ISSN 1519-566X

Neotrop. entomol. vol.40 no.2 Londrina Mar./Apr. 2011 



Laboratory and field evaluation of Metarhizium anisopliae var. anisopliae for controlling subterranean termites



A HussainI; S AhmedII; M ShahidIII

ICollege of Natural Resources and Environment, South China Agricultural Univ, Guangzhou, China
IIDept of Agricultural Entomology
IIIDept of Chemistry and Biochemistry, Univ of Agriculture, Faisalabad, Pakistan





The efficacy of the Metarhizium anisopliae strain ARSEF 6911 was determined in the laboratory and field against two sugarcane pests, Microtermes obesi Holmgren and Odontotermes obesus Rambur (Termitidae: Isoptera). The susceptibility of both termite species to different conidial suspensions (1 × 1010, 1 × 108, 1 × 106 and 1 × 104 conidia/ml) was determined in laboratory. All conidial suspensions were able to induce mortality. Termite mortality caused by the fungal suspensions was dose dependent. There were no significant differences in the LT50 values between species. Field evaluation of M. anisopliae alone or in combination with diesel oil and thiamethoxam was carried out in two growing seasons (autumn 2005 and spring 2006) at two sites located in Punjab, Pakistan. Dipping the sugarcane setts in these suspensions was tried to determine their effects on germination and percentage of bud damage to sugarcane setts. All treatments significantly reduced termite infestation compared to the untreated control. The combined treatment of M. anisopliae and diesel oil significantly reduced insect damage by attaining higher germination > 55% and lower bud damage < 5.50% at both sites in both seasons. The results suggest that the application of M. anisopliae and diesel oil in combination might be a useful treatment option for the management of termites in sugarcane.

Keywords: Microtermes obesi, Odontotermes obesus, field application, sugarcane, diesel oil, thiamethoxam, repellency




The termite fauna of Pakistan consists of 50 species, and nine of them belong to Termitidae and are recorded as pests of agricultural crops (Akhtar & Shahid 1993). The most important termite species causing damage to sugarcane in Pakistan are Microtermes obesi Holmgren, M. mycophagus Desneux and Odontotermes obesus Rambur (Sattar & Salihah 2001, Ahmed et al 2006).

Termites attack sugarcane when setts are placed in furrows and are covered with soil. Termite's infestation at this time results in low or no germination of seedlings at all. After cane formation, attack by termites may cause lodging or production of a cane with poor quality for milling. Thus, insecticides are applied to setts to protect them from termite's infestation in three ways: (i) application to setts at the time of placing in furrows, (ii) dipping setts in insecticide solutions and then placing in furrows, or (iii) applying insecticides at the time of first irrigation, but this is the least preferred (Ahmed et al 2006).

Management of subterranean termites has relied heavily on chemicals such as soil treatment, baiting and dusting. Soil termiticide treatment creates a chemical barrier for exclusion of subterranean termites (Bläske et al 2003), and has been a popular termite management tool over the last 50 years (Su et al 1997). In the past, persistent organo chlorine insecticides were available for termite control, but were later replaced by many of the current available insecticides. Chlorpyrifos, bifenthrin, imidacloprid and thiamethoxam have proven to be effective against many species of termites. But the shortcomings associated with the use of insecticides in recent years have prompted policy makers and scientists to evaluate the potential of biological control strategies to suppress subterranean termite populations (Culliney & Grace 2000).

Biological management has been investigated as an alternative to conventional insecticide use for a number of termite species, and laboratory data on the efficacy of fungal pathogens are available against Reticulitermes spp. (Ramakrishnan et al 1999), Coptotermes spp. (Ahmed et al 2009, Hussain et al 2010a), Nasutitermes spp. (Milner 2003), damp wood termites (Rosengaus & Traniello 1997), Heterotermes tenuis Hagen (Moino et al 2002) and Cornitermes cumulans Kollar (Neves & Alves 1999).

The studies carried out in a number of laboratories have focused on the possible avoidance behavior of termites against repellent entomopathogenic fungi. Recent studies by Mburu et al (2009) on M. anisopliae and B. bassiana suggested that termite response towards entomopathogenic fungi is directly related to the potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided. The repellent action of conidia has also been utilized to protect maize crops from termites, which significantly reduce maize plant logging and increased grain yield in Kenya (Maniania et al 2002). There are also some reports on the successful control of termites by direct blowing of conidia of less virulent strains into termite galleries, resulting in a successful control of only those termites which were directly hit by the conidia (Milner 2000). However, no attempt has been made to test their susceptibility to fungal pathogens against many species of Odontotermes, Microtermes and Macrotermes, prevalent in agricultural lands in Pakistan, which have been found attacking sugarcane, wheat, maize and gram.

The present study was initiated in order to evaluate the virulence of a strain (ARSEF 6911) of M. anisopliae to M. obesi and O. obesus workers. Further, chemicals were added to enhance the biocontrol potential of this entomopathogen. Although the idea is not novel, there is limited data on this tactic for pest control, especially in field conditions. Hence, mass produced spores of M. anisopliae ARSEF 6911 alone or in combination with different chemicals were evaluated by dipping sugarcane setts in their solutions in order to implement a practical approach to control termites in sugarcane; tests were performed in two successive growing seasons (autumn 2005 and spring 2006) at two different sites. The overall objective was to demonstrate the potential of the entomopathogenic fungus M. anisopliae ARSEF 6911 in order to develop an inexpensive IPM strategy for termites control in sugarcane.


Material and Methods


Subterranean termites such as O. obesus and M. obesi were collected from the sugarcane fields within damaged canes and from the corrugated cardboard in PVC traps installed at different places at the experimental areas of the University of Agriculture, Faisalabad, Pakistan.

Fungal isolate

Metarhizium anisopliae var. anisopliae strain ARSEF 6911 was obtained from the USDA, the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF). Strain ARSEF 6911 was originally isolated from Coptotermes formosanus Shiraki in Lake Charles, Louisiana, USA and was cultured on Potato Dextrose Agar (PDA, Oxoid, Hampshire, UK) at 27 ± 2 °C in complete darkness. The isolate selection was based on its pathogenicity to O. obesus and M. obesi workers found in preliminary studies (data not given).

Laboratory bioassay

Conidial suspensions. Conidia of M. anisopliae were harvested from 28-day-old sporulating culture into 0.03% Tween 80 (Sigma-Aldrich). The conidial suspension was sieved through a piece of cheese cloth (Mesh: 36 × 36 threads/cm2) to remove clumps of mycelia and the suspension was adjusted to concentrations of 1 × 1010, 1 × 108, 1 × 106 and 1 × 104 conidia/ml. The viability of conidia was determined by inoculating PDA plates (90 × 15 mm) with 10 μl of 1 × 106 conidia/ml at 25°C for 24h. Plates were then observed for germination of 100 conidia randomly selected in three separate fields of the Petri dishes using a compound microscope (×400). The viability was 94.3 ± 1.5%.

Laboratory experimental set up. The infectivity of the isolate was evaluated by directly applying conidial suspensions at different concentrations to individual termite workers. A Precision (Microprocessor Controller) PAX100-3 Microapplicator (Burkard, UK) was used to dispense from 1 μl to 10 μl per individual termite. Workers of M. obesi and O. obesus were placed separately in Petri dishes containing sterilized moistened Whatman No. 1 filter paper. The Petri dishes were covered and then placed over ice to lower termite activity. Individual workers were held from their heads and 5 μl were applied to the ventral side of their abdomen. After drying, forty workers/treatment were transferred to Petri dishes (90 × 15 mm) that contained 10 g sifted sterilized soil and a strip of sugarcane (10 × 60 mm), dampened with sterile distilled water. The dishes were sealed with laboratory film (Parafilm® Pechiney Plastic Packaging; Menasha. WI. US) and incubated at 27 ± 2°C in complete darkness. The mortality was recorded daily until all termites were dead. At each observation, dead termites were counted and removed from the Petri dishes. At each observation, dead termites were removed, counted and placed in dishes (90 × 15 mm) lined with wet filter paper and maintained at 25°C in growth chamber for mummification and sporulation to prove that the insects died because of the fungi used.

Data analysis. The Abbott's formula (Abbott 1925) was applied to correct for the percent mortality in the control. The data on dose-mortality were subjected to probit analysis to estimate the LT50. All the values obtained were subjected to one way analysis of variance (ANOVA) and means were compared by using the Student Newman-Keuls test (SAS Institute 2000). The comparisons between termite species at all the concentrations were performed on LT50 values using the t-test.

Field experiments

Production of conidia on rice. One milliliter of a suspension of M. anisopliae ARSEF 6911 at a concentration of 1 × 106 conidia/ml was poured into a 250 ml flask containing 100 ml of the growth medium described in Table 1. Flasks were then put on a rotary shaker at 150 rpm for four days at 27 ± 2°C in complete darkness. Content of flasks were then inoculated at a rate of 150 ml per kg of autoclaved parboiled rice with the help of a syringe. The bags of inoculated rice were then incubated for 18 days at room conditions (24 ± 2°C, 75-85% RH). Rice bags with conidia were then allowed to dry for seven days at 30°C, before conidia were separated from the rice by sieving through a 300 μm mesh. Plastic sheeting was taped around both the top and bottom edges of the sieve and sealed at the top. A collecting vessel, such as a bucket was fitted to the plastic sheeting at the bottom of the sieve to create a funnel into the collecting vessel. The sieve was shaken until all the loose conidial powder had been removed from the rice and had been collected in the vessel below. The conidial powder was then further sieved using a 106 μm sieve to separate the larger rice dust particles from the conidial powder. Dry conidia were then stored at 4°C until used in field experiments (35-day-old conidia were used, with >94.5% germination after 24h on PDA). The compatibility of diesel oil and thiamethoxam was checked in preliminary studies. The viability of conidia in diesel oil and thiamethoxam showed >89% after 24h on PDA.

Field experimental set up. Sugarcane (Saccharum officinarum) variety HoSG 559 was planted in 3.35 × 7.32 m plots with a distance between rows of 1.22 m at the Ashabah Farm Shakarganj Sugar Mills Jhang, Punjab, Pakistan (site 1, 31° 09' 38" N, 72° 21' 55" E, 154 m elevation) and the Postgraduate Agricultural Research Station (PARS), University of Agriculture Faisalabad, Pakistan (site 2, 31° 23' 3" N, 73° 01' 4" E, 177 m elevation), in the autumn 2005 and in spring 2006. Sugarcane setts were inoculated by dipping them in the solutions presented in Table 2. The compatibility of chemicals with the fungal isolate was evaluated on PDA. The chemicals used had no negative effect on conidial germination of the studied strain of M. anisopliae. After setts had been treated, they were planted and each plot was irrigated separately when needed. Treatments were arranged in a randomized complete block design with three replicates.

The number of buds were counted before and two months after planting to determine the percentage of germination of sugarcane setts for each treatment. The presence of bud damage was also assessed for two months after planting by randomly choosing five 1m2 areas from each plot. Sugarcane setts were carefully removed from the soil and the number of buds damaged by termites were counted.

Data analysis. The data on germination and bud damage percentage were angularly transformed before analysis. A two way factorial analysis consisting of two seasons and six treatments was conducted for germination and bud damage percentage in order to investigate the main effect of these two factors. Significant differences among means were separated by the Tukey's Honestly Significant Difference Test (SAS Institute 2000).



Laboratory bioassay

Infectivity of M. anisopliae differed significantly when M. obesi (F = 54.71; df = 3; P < 0.05) and O. obesus (F = 133.37; df = 3; P < 0.05) workers were directly exposed with different conidial concentrations in the laboratory. Both termite species were susceptible to M. anisopliae in a dose-dependent manner. With decreasing conidial concentrations, LT50 values tended to be longer for both termite species (Table 3). At 1 × 1010 conidia/ml, the LT50 values were 2.82 and 3.08 days for M. obesi and O. obesus, respectively. No significant differences were observed between M. obesi and O. obesus workers exposed to M. anisopliae suspension at the concentrations of 1 × 1010, 1 × 108 and 1 × 106 conidia/ml (P > 0.05). The variation in LT50 values was statistically significant (P < 0.02), when workers of both species were exposed to the lowest concentration (1 × 104 conidia/ml) (Table 3).



Field experiment

The percent germination of sugarcane recorded after two months post-treatment differed significantly among treatments at site 1 (Ashabah Farm Shakarganj Sugar Mills Jhang, Punjab, Pakistan) (F = 28.08; df = 5, 22; P < 0.01) and site 2 (Postgraduate Agricultural Research Station, University of Agriculture Faisalabad, Pakistan) (F = 30.02; df = 5, 22; P < 0.01). Mean percent germination of sugarcane setts in controls were low, ranging from 23.7-24.6% (site 1) and 22.0-26.4% (site 2). All treatments except diesel oil had significantly higher germination than the control in both cropping seasons and at both sites (Fig 1). The combined effect of M. anisopliae conidia with diesel oil during autumn 2005 and spring 2006 at both sites resulted in significantly higher percent germination over that caused by diesel oil alone, but it was similar to M. anisopliae conidia alone. When thiamethoxam was applied with M. anisopliae conidia, germination of sugarcane was not improved if compared with these treatments alone. There was no significant difference in the germination of sugarcane setts between autumn 2005 and spring 2006 at site 1 (F = 2.80; df = 1, 22; P > 0.10) and site 2 (F = 2.49; df = 1, 22; P > 0.12).



There was no significant difference in percentage of bud damage of the sugarcane setts between autumn 2005 and spring 2006 at site 1 (F = 0.89; df = 1, 22; P > 0.05) and site 2 (F = 1.134; df = 1, 22; P = 0.297), but there was a significant difference among treatments in comparison with the control at site 1 (F = 54.87; df = 5, 22; P < 0.01) and site 2 (F = 66.28; df = 5, 22; P < 0.01). The lowest bud damage < 5.5% was observed following exposure with conidia + diesel oil at both sites in autumn 2005 and spring 2006, except that this treatment was not statistically different from M. anisopliae + thiamethoxam and thiamethoxam alone in autumn 2005 and M. anisopliae + thiamethoxam in spring 2006 (Fig 2). Higher percentages of bud damage occurred in untreated control plots (> 35%). Treatment efficacy to prevent termite damage in sugarcane increased upon combination of conidia and diesel oil as compared to the each one of these treatments alone. This effect was not observed for thiamethoxam.




The LT50 values obtained demonstrated that M anisopliae is highly pathogenic to M. obesi and O. obesus. Significant differences in LT50 values between both studied termite species at the lowest concentration (1 ×104 conidia ml-1) were observed, which could reflect different host susceptibility at this concentration. However, there were no significant differences in the susceptibility of both species at higher concentrations.

Application of mass-produced conidia on sugarcane setts in combination with diesel oil and thiamethoxam yielded encouraging results as compared to the other treatments. The germination and bud damage to sugarcane setts at our study did not vary considerably between seasons at both sites. The application of M. anisopliae with diesel oil proved to be a promising alternative to insecticides against termites in sugarcane by producing higher germination with the lowest bud damage < 5.5%. The effectiveness of entomopathogenic fungi in oil formulations is consistent with previous work reported by Bateman et al (1993) against locusts. Furthermore, the findings of Prior et al (1988) also suggested that Hypocreales fungi are more infectious when applied in oil formulations. Entomopathogenic fungi in oil formulations maintain their viability for longer periods than those in aqueous formulations (Daoust et al 1983).

Diesel oil + M. anisopliae conidia had the most pronounced effect in reducing bud damage when compared to treatments where only M. anisopliae conidia (13.99-16.40%) or diesel oil (18.13-25.45%) were used, suggesting that the synergistic effect of M. anisopliae + diesel oil may create a barrier that could offer more effective protection to the sugarcane setts than causing disease to the subterranean termites at the time of planting the sugarcane crop. The nature of fungal repellency by the species within the order Isoptera has been widely assessed in the laboratory (Staples & Milner 2000, Hussain et al 2010b). This type of avoidance behavior is very important for the management of insect pests like termites residing in difficult-to-reach locations, such as in underground nests. Similarly, Sun et al (2008) found that organic mulches supplemented with M. anisopliae significantly repelled foraging C. formosanus and reduced mulch consumption by up to 71%.

Our work has also provided evidence that suggests that the variation in effectiveness among different treatments is due to the alteration in sensory perception of termites. The impregnation of sugarcane setts in oil-fungal formulation is helpful tool for the management of insects living in concealed habitats with millions of individuals by changing the volatile gaseous profile of the host. Otherwise, this habitat makes it impractical to inoculate fungal spores directly onto most colony members whose galleries may stretch more than 100 m. The repellent action of conidia has also been utilized to protect other crops such as maize from termites. Maniania et al (2002) concluded that the repellency of entomopathogenic fungi significantly reduced the maize plant logging and ultimately increased the grain yield in Kenya.

In our study, the neonicotinoid thiamethoxam tested alone was not effective against termites, resulting in 41.08-46.20% germination. However, the combined formulation or application of M. anisopliae + thiamethoxam was moderately effective and showed 47.43-53.40% germination of sugarcane setts and gave better results in lowering bud damage by 5.49-7.06% as compared to the untreated control. Studies on the compatibility of thiamethoxam with M. anisopliae showed that it did not affect the inoculum of M. anisopliae when applied in the field (Filho et al 2001). Similarly, the findings of Neves et al (2001) also explained the integration of insecticides acetamiprid, imidacloprid and thiamethoxam with fungal conidia on the basis of compatibility in order to enhance the biocontrol potential of entomopathogenic fungi.

In summary, our study indicates that the addition of diesel oil to M. anisopliae has pronounced positive effect in the control of termites at the time of sugarcane planting by increasing germination and reducing sugarcane bud damage.



We thank Dr Richard A Humber (USDA-ARS) for providing Metarhizium anisopliae var. anisopliae strain ARSEF 6911. Special thanks to Prof. Tian MY (South China Agricultural University, Guangzhou, P. R. China) for reading and providing useful comments on the earlier draft of the manuscript.



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College of Natural Resources and Environment
South China Agricultural University
Guangzhou, 510642, P.R. China

Received 01 August 2009 and accepted 11 December 2010



Edited by Italo Delalibera Jr - ESALQ/USP

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