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Laboratory evaluation of virulence of Chinese Beauveria bassiana and Metarhizium anisopliae isolates to engorged female Rhipicephalus (Boophilus) microplus ticks
Biological Control 63 (2012) 98–101
Contents lists available at SciVerse ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Laboratory evaluation of virulence of Chinese Beauveria bassiana and Metarhizium anisopliae isolates to engorged female Rhipicephalus (Boophilus) microplus ticks Qiaoyun Ren, Zhijie Liu, Guiquan Guan, Ming Sun, Miling Ma, Qingli Niu, Youquan Li, Aihong Liu, Junlong Liu, Jifei Yang, Hong Yin ⇑, Jianxun Luo ⇑ State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory of Grazing Animal Diseases MOA, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" We evaluated the virulence of 20
isolates against Rhipicephalus (Boophilus) microplus. " Four isolates of entomopathogenic fungi were highly virulent against this tick. " Four isolates have potential for applications to control this tick.
a r t i c l e
i n f o
Article history: Received 8 July 2011 Accepted 2 July 2012 Available online 25 July 2012 Keywords: Rhipicephalus (Boophilus) microplus Beauveria bassiana Metarhizium anisopliae Biological control Entomopathogenic fungi
a b s t r a c t Rhipicephalus (Boophilus) microplus, an important ectoparasite that can transmit Babesia and Anaplasma, has caused inestimable economic losses around the world. Traditionally, acaricides are used for the control of ticks. However, drawbacks of chemical control, such as resistance, environmental pollution, and traces in food promote alternative strategies to pesticides. Microbial control is one option to reduce tick populations. In this study, we investigated the pathogenicity of thirteen Beauveria bassiana isolates and seven Metarhizium anisopliae isolates to the engorged female R. (B.) microplus ticks using different conidial concentrations of 107, 108 and 109 conidia mL–1. Fourteen days after treatment, three B. bassiana isolates (B.bAT01, B.bAT03, B.bAT13) and one M. anisopliae isolate (M.aAT04) resulted in 100% mortality of engorged female ticks with conidial concentrations of 108 and 109 conidia mL–1. Isolates of B.bAT01, B.bAT03, B.bAT13 and M.aAT04 at all conidial concentrations could reduce the reproductive efficiency index (REI) of R. (B.) microplus ticks. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Ticks, obligatory ectoparasites of terrestrial vertebrates, transmit various pathogenic microorganisms, such as protozoa, rickettsiae, spirochaetes and viruses (Jongejan and Uilenberg, 2004). The cattle tick Rhipicephalus (Boophilus) microplus (Acari, Ixodidae) is an obligatory haematophagous ectoparasite and a single host species in almost all tropical and subtropical countries. Apart from ⇑ Corresponding authors. Fax: +86 931 8340977 (J. Luo), +86 931 8340977 (H. Yin). E-mail addresses: [email protected] (H. Yin), [email protected] (J. Luo). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.07.002
physical damage to their host and blood spoliation, R. (B.) microplus is a major vector for bovine babesiosis and anaplasmosis (Monteiro et al., 2010; Jittapalapong et al., 2010). In China, R. (B.) microplus results in severe economic losses (Luo et al., 2003). Though various techniques have been developed, tick population control still relies on chemical acaricides during their parasitic phase. Presently, chemical acaricides (that include organochlorines, organophosphates, carbamates, amidines and pyrethroids) are most effective reagents in tick control (Tuininga et al., 2009). The unlimited and indiscriminate use of these acaricides has resulted in many problems, such as heritable resistance of ticks, risk to non-target organisms, environmental pollution, trace contamination in food
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Q. Ren et al. / Biological Control 63 (2012) 98–101
products, and increasing cost of tick control (Pourseyed et al., 2010; Angel-Sahagún et al., 2010). Therefore, alternative approaches are needed to reduce or replace the application of acaricides. During recent years, biological control of ticks with entomopathogenic fungi has received considerable attention (Ment et al., 2010). Because of their wide distribution, low risk for humans and animals, environmental safety, high virulence against ticks and potential low cost, entomopathogenic fungi have been investigated as potential agents for biological control of ticks (Samish and Rehacek, 1999; Pirali-Kheirabadi et al., 2007). Among the entomopathogenic fungi examined for pathogenicity against ticks, two species commonly investigated are Beauveria bassiana and Metarhizium anisopliae (Polar et al., 2008). B. bassiana and M. anisopliae show high virulence to various life stages of ticks, and several studies substantiate the effect of these fungi on R. (B.) microplus. OjedaChi et al. (2010) reported that isolates of M. anisopliae killed 100% of engorged female R. (B.) microplus ticks at 1 106 conidia mL–1 dose. Some strains of M. anisopliae caused up to 100% mortality in R. (B.) microplus in vitro within 2 days (Leemon and Jonsson, 2008). Fernandes et al. (2003) observed a high percentage of larval mortality and a low percentage of egg hatching in ticks treated with B. bassiana. The entomopathogenic fungi B. bassiana and M. anisopliae have been used widely for biological control of agricultural and forest pests (Quesada-Moraga et al., 2007; Wraight et al., 2010), but few studies have been reported regarding their virulence against R. (B.) microplus in China. The aim of the present study was to evaluate the susceptibility of engorged female R. (B.) microplus ticks to thirteen strains of B. bassiana and seven strains of M. anisopliae, to discover potential strains for controlling R. (B.) microplus ticks in China.
Table 1 Details of fungal strains used in this study. Strain
Fungus
Original host
Geographical origin
B.bAT01 B.bAT02 B.bAT03 B.bAT04 B.bAT05 B.bAT06 B.bAT07 B.bAT08 B.bAT09 B.bAT10 B.bAT11 B.bAT12 B.bAT13 M.aAT01 M.aAT02 M.aAT03 M.aAT04 M.aAT05 M.aAT06 M.aAT07
B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae
Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil B. microplus Soil Soil Soil Soil Soil Soil Soil
Anhui, China Anhui, China Guizhou, China Zhejiang, China Zhejiang, China Jiangsu, China Fujian, China Hunan, China Heilongjiang, China Guizhou, China Guangxi, China Hubei, China Guizhou, China Sichuan, China Shanxi, China Anhui, China Henan, China Gansu, China Guangdong, China Guizhou, China
approximately 30 s and then placing them on paper towel to soak up the excess. Control groups (with 10 engorged R. (B.) microplus females per group) were treated with sterile water containing 0.05% Tween 80 instead of conidial suspension. Each trial was repeated three times. After treatment, ticks were individually placed in glass tubes sealed with hydrophilic cotton and kept in an incubator at 27 ± 1° C and 80 ± 5% RH for oviposition. Ticks were observed daily to evaluate biological parameters. 2.4. Data analysis
2. Materials and methods 2.1. Ticks R. (B.) microplus ticks were originally obtained from naturally infested cattle in Sichuan Province in western China. The ticks were maintained in an incubator at 27 ± 1° C and 80 ± 5% RH (relative humidity) in our laboratory. For this study, the larval R. (B.) microplus ticks were put onto two calves, and the engorged female ticks were collected, which were randomly divided into different groups. 2.2. Fungal growth and preparation of conidial suspensions Thirteen isolates of B. bassiana and seven isolates of M. anisopliae were originally obtained from soil and a tick collected from 14 provinces of China (Table 1). These fungal isolates were maintained on potato dextrose agar (PDA) and kept at 4° C. All isolates were cultured with PDA on petri plates in an incubator at 26–28° C and RH > 80% for 12 days. Conidia were harvested by scraping the surface of the plate and placed into sterilized aqueous solution containing 0.05% Tween 80. After homogenization, concentrations of conidia were determined with a haemocytometer and adjusted to final concentrations of 107, 108 and 109 conidia mL–1 with 0.05% Tween 80 in distilled water.
Treatment data were evaluated using a formula described by Ojeda-Chi et al. (2010). The reproductive efficiency index (REI = egg mass weight/engorged female weight) was determined for each female tick. Dead ticks were counted and mortality rates were calculated. Analyses of variance (ANOVA) were used to evaluate levels of significance of the virulence of B. bassiana and M. anisopliae against R. (B.) microplus female ticks. 3. Results All isolates of B. bassiana and M. anisopliae were pathogenic to R. (B.) microplus at concentration of 107, 108 and 109 conidia mL–1 in the laboratory. The lethal activity of all treatment and control groups to engorged females are presented in Fig. 1. Fourteen days
2.3. Laboratory bioassays Evaluation of fungal virulence was carried out according to the method described in Frazzon et al. (2000). Bioassays of B. bassiana and M. anisopliae suspensions (107, 108 and 109 conidia mL–1 of each isolate) were conducted for pathogenicity by immersing 10 engorged R. (B.) microplus females in each of the suspensions for
Fig. 1. Mortality of R. (B.) microplus female caused by M. anisopliae and B. bassiana (concentrations of fungal strains: (A) 107 conidia mL–1, (B) 108 conidia mL–1, (C) 109 conidia mL–1).
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Fig. 2. Rhipicephalus (Boophilus) microplus infected with Metarhizium anisopliae and Beauveria bassiana. (A) M.aAT04 (108 conidia mL–1); (B) B.bAT01 (108 conidia mL–1); (C) B.bAT03 (108 conidia mL–1); (D) B.bAT13 (108 conidia mL–1).
after treatment, mortality ranges of treated females were 0–96.7%, 10–100%, and 20–100% for concentrations of 107, 108 and 109 conidia mL–1 of M. anisopliae and B. bassiana strains, respectively. Mortality in the control group was zero. Isolates of B.bAT03 and M.aAT04 caused over 90% mortality at the 107 conidia mL–1 concentration. In contrast, the isolates M.aAT02 and M.aAT05 had little effect on the survival of ticks at a concentration of 107 conidia mL–1. Infected females of R. (B.) microplus were completely covered with fungal hyphae (Fig. 2). Results on the LT50 are shown in Table3. The LT50 varied from 6.4884 to 7.3759 days on the four selected fungal isolates at the 107 conidia mL–1 concentration (Table 3).
Table 2 Reproductive efficiency index (REI) of R. (B.) microplus females treated with M. anisopliae and B. bassiana. Isolates
Treatment group REI 107 Conidial mL
B.bAT01 B.bAT02 B.bAT03 B.bAT04 B.bAT05 B.bAT06 B.bAT07 B.bAT08 B.bAT09 B.bAT10 B.bAT11 B.bAT12 B.bAT13 M.aAT01 M.aAT02 M.aAT03 M.aAT04 M.aAT05 M.aAT06 M.aAT07 Control
0.15 ± 0.01 0.48 ± 0.01 0.15 ± 0.01 0.37 ± 0.03 0.45 ± 0.03 0.4 ± 0.02 0.4 ± 0.00 0.48 ± 0.03 0.54 ± 0.02 0.53 ± 0.01 0.47 ± 0.01 0.48 ± 0.04 0.07 ± 0.00 0.47 ± 0.04 0.55 ± 0.03 0.46 ± 0.00 0.17 ± 0.01 0.55 ± 0.03 0.29 ± 0.02 0.29 ± 0.01 0.57 ± 0.00
1
108 Conidial mL 0.12 ± 0.01 0.45 ± 0.02 0.09 ± 0.02 0.36 ± 0.01 0.43 ± 0.01 0.43 ± 0.03 0.45 ± 0.02 0.44 ± 0.01 0.53 ± 0.00 0.53 ± 0.03 0.43 ± 0.01 0.44 ± 0.03 0.03 ± 0.00 0.42 ± 0.04 0.53 ± 0.02 0.42 ± 0.02 0.01 ± 0 0.55 ± 0.02 0.28 ± 0.02 0.17 ± 0.01
1
109 Conidial mL
1
0.09 ± 0.01 0.41 ± 0.03 0.07 ± 0.01 0.32 ± 0.02 0.40 ± 0.02 0.40 ± 0.00 0.45 ± 0.03 0.41 ± 0.01 0.53 ± 0.01 0.52 ± 0.01 0.44 ± 0.01 0.43 ± 0.01 0.00 ± 0.00 0.44 ± 0.03 0.51 ± 0.03 0.42 ± 0.01 0.03±.00 0.54 ± 0.02 0.26 ± 0.01 0.12 ± 0.01
Means followed by the same lowercase letter within the same column do not differ statistically (p < 0.05).
Egg production of R. (B.) microplus females was reduced after treatment with isolates of B. bassiana and M. anisoplia (Table 2). Specifically, 0.15, 0.15, 0.07 and 0.17 of the REI of R. (B.) microplus infected with isolates of B.bAT01, B.bAT03, B.bAT13 and M.aAT04 at 107 conidia mL–1, compared with 0.57 in the control group. 4. Discussion Entomopathogenic fungi are wide distributed, low risk to nontarget organisms, environmental safety, and high virulence against ticks, thus they have been rapidly developed for biological control of pests. Studies showed that tick death resulting from fungi is usually due to a combination of actions that include depletion of nutrients, physical obstruction or invasion of organs, or toxicosis (Fernandes et al., 2012.). Entomopathogenic fungi commonly investigated for pathogenicity against ticks are the mycoinsecticidal B. bassiana, M. anisopliae and Paecilomyces fumosoroseus (Campos et al., 2005; Zabalgogeazcoa et al., 2008). Previous studies have reported effectiveness of B. bassiana and M. anisopliae against all stages of R. (B.) microplus. In this study, three B. bassiana isolates (B.bAT01, B.bAT03, B.bAT13) and one M. anisopliae isolate (M.aAT04) demonstrated more virulence against female ticks, which caused up to 100% mortality for the ticks at concentrations of 108 and 109 conidia mL–1. Gindin et al. (2001) reported that the Ma-7 strain of M. anisopliae was virulent to females and caused 100% mortality at a concentration of 108 spores mL–1. Our results were in agreement with this result. In addition, another four isolates of B. bassiana and two isolates of M. anisopliae were also pathogenic against R. (B.) microplus, causing mortality greater than 80%. All the treatments caused a reduction of the REI, while the control group did not. We observed that the reproductive efficiency of R. (B.) microplus was reduced from 0.40 to 0.50 at concentration of 107 spores mL–1when treated. Similarly, Kaaya et al. (1996) observed that 5% of ticks infected with B. bassiana failed to lay eggs. In our study, the LT50 of most virulent fungi isolates ranged from 6.4884–7.3759 days at the 107 conidia mL–1 concentration. The estimated LT50 for ticks immersed in M.aAT04 and B.bAT13
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Q. Ren et al. / Biological Control 63 (2012) 98–101 Table 3 Lethal time (LT50) of M. anisopliae e and B. bassiana against R. (B.) microplus engorged females. Isolates
LT50 (days) 107 Conidial mL
B.bAT01 B.bAT03 B.bAT13 M.aAT04
7.0992 7.3759 6.6173 6.4884
1
(6.4830–7.7258) (6.7958–7.9709) (5.9683–7.2834) (5.8599–7.1306)
is lower than in B.bAT01 and B.bAT03. These results demonstrated the virulence of B. bassiana and M. anisopliae to female, which indicates a potential use of these species for biocontrol of R. (B.) microplus ticks in China. Acknowledgments This study was financially supported by the Supporting Plan (2007BAD40B06), ‘‘863’’ Project (2006AA10A207), 973 Program (2010CB530206), ‘‘948’’(2010-S04), Key Project of Gansu Province (1002NKDA035 and 0801NKDA033), Beef and Yak Production System Programme, MOA, Specific Fund for Sino-Europe Cooperation, MOST, China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019); The research was also facilitated by EPIZONE (FOOD-CT-2006-016236), ASFRISK(No: 211691), ARBOZOONET(No: 211757) and PIROVAC (KBBE-3-245145) of European Commission, Brussels, Belgium. References Angel-Sahagún, C.A., Lezama-Gutiérrez, R., Molina-Ochoa, J., Pescador-Rubio, A., Skoda, S.R., Cruz-Vázquez, C., Lorenzoni, A.G., Galindo-Velasco, E., FragosoSánchez, H., Foster, J.E., 2010. Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus = B. microplus (Acari: Ixodidae) larvae and the efficacy of conidia formulations to reduce larval tick density under field conditions. Vet. Parasitol. 170, 278–286. Campos, R.A., Arruda, W., Boldo, J.T., da Silva, M.V., de Barros, N.M., de Azevedo, J.L., Schrank, A., Vainstein, M.H., 2005. Boophilus microplus infection by Beauveria amorpha and Beauveria bassiana: SEM analysis and regulation of subtilisin-like proteases and chitinases. Curr. Microbiol. 50, 257–261. Fernandes, É.K., Bittencourt, V.R., Roberts, D.W., 2012. Perspectives on the potential of entomopathogenic fungi in biological control of ticks. Exp. Parasitol. 130, 300–305. Fernandes, E.K., da Costa, G.L., de Souza, E.J., de Moraes, A.M., Bittencourt, V.R., 2003. Beauveria bassiana isolated from engorged females and tested against eggs and larvae of Boophilus microplus (Acari: Ixodidae). J. Basic Microbiol. 43, 393–398. Frazzon, A.P., da Silva Vaz Jr., I., Masuda, A., Schrank, A., Vainstein, M.H., 2000. In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet. Parasitol. 94, 117–125. Gindin, G., Samish, M., Alekseev, E., Glazer, I., 2001. The susceptibility of Boophilus annulatus (Ixodidae) ticks to entomopathogenic fungi. Biocontrol. Sci. Technol. 11, 111–118. Jittapalapong, S., Kaewhom, P., Pumhom, P., Canales, M., de la Fuente, J., Stich, R.W., 2010. Immunization of rabbits with recombinant serine protease inhibitor
108 Conidial mL 6.5242 6.2565 6.2172 6.1154
1
(5.9544–7.0912) (5.7010–6.8067) (5.6925–6.7323) (5.5614–6.6648)
109 Conidial mL 5.7315 6.0025 5.7313 5.4579
1
(5.2245–6.2213) (5.5141–6.5200) (5.2499–6.2105) (4.9323–5.9671)
reduces the performance of adult female Rhipicephalus microplus. Transbound. Emerg Dis. 57, 103–106. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129, S3–S14. Kaaya, G.P., Mwangi, E.N., Ouna, E.A., 1996. Prospects for biological control of livestock ticks, Rhipicephalus appendiculatus and Amblyomma variegatum, using the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. J. Invertebr. Pathol. 67, 15–20. Leemon, D.M., Jonsson, N.N., 2008. Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus. J. Invertebr. Pathol. 97, 40-49. Luo, J., Chen, F., Lu, W., Guan, G., Ma, M., Yin, H., 2003. Experimental transmission of an unnamed bovine Babesia by Hyalomma spp., Haemaphysalis longicornis and Boophilus microplus. Vet. Parasitol. 116, 115–124. Ment, D., Gindin, G., Soroker, V., Glazer, I., Rot, A., Samish, M., 2010. Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface. J. Invertebr. Pathol. 103 (2), 132–139. Monteiro, C.M., Prata, M.C., Furlong, J., Faza, A.P., Mendes, A.S., Andaló, V., MoinoJunior, A., 2010. Heterorhabditis amazonensis (Rhabditidae: Heterorhabditidae), strain RSC-5, for biological control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitol. Res. 106, 821–826. Ojeda-Chi, M.M., Rodriguez-Vivas, R.I., Galindo-Velasco, E., Lezama-Gutiérrrez, R., 2010. Laboratory and field evaluation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) for the control of Rhipicephalus microplus (Acari: Ixodidae) in the Mexican tropics. Vet. Parasitol. 170, 348–354. Pirali-Kheirabadi, K., Haddadzadeh, H., Razzaghi-Abyaneh, M., Bokaie, S., Zare, R., Ghazavi, M., Shams-Ghahfarokhi, M., 2007. Biological control of Rhipicephalus (Boophilus) annulatus by different strains of Metarhizium anisopliae, Beauveria bassiana and Lecanicillium psalliotae fungi. Parasitol. Res. 100, 1297–1302. Polar, P., Moore, D., Kairo, M.T., Ramsubhag, A., 2008. Topically applied mycoacaricides for the control of cattle ticks: overcoming the challenges. Exp. Appl. Acarol. 46, 119–148. Pourseyed, S.H., Tavassoli, M., Bernousi, I., Mardani, K., 2010. Metarhizium anisopliae (Ascomycota: Hypocreales): an effective alternative to chemical acaricides against different developmental stages of fowl tick Argas persicus (Acari: Argasidae). Vet. Parasitol. 172, 305–310. Quesada-Moraga, E., Navas-Cortés, J.A., Maranhao, E.A., Ortiz-Urquiza, A., SantiagoAlvarez, C., 2007. Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils. Mycol. Res. 111, 947– 966. Samish, M., Rehacek, J., 1999. Pathogens and predators of ticks and their potential in biological control. Annu. Rev. Entomol. 44, 159–182. Tuininga, A.R., Miller, J.L., Morath, S.U., Daniels, T.J., Falco, R.C., Marchese, M., Sahabi, S., Rosa, D., Stafford 3rd., K.C., 2009. Isolation of entomopathogenic fungi from soils and Ixodes scapularis (Acari: Ixodidae) ticks: prevalence and methods. J. Med. Entomol. 46, 557–565. Wraight, S.P., Ramos, M.E., Avery, P.B., Jaronski, S.T., Vandenberg, J.D., 2010. Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops. J. Invertebr. Pathol. 103, 186–199. Zabalgogeazcoa, I., Oleaga, A., Pérez-Sánchez, R., 2008. Pathogenicity of endophytic entomopathogenic fungi to Ornithodoros erraticus and Ornithodoros moubata (Acari: Argasidae). Vet. Parasitol. 158, 336–343.
Contents lists available at SciVerse ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Laboratory evaluation of virulence of Chinese Beauveria bassiana and Metarhizium anisopliae isolates to engorged female Rhipicephalus (Boophilus) microplus ticks Qiaoyun Ren, Zhijie Liu, Guiquan Guan, Ming Sun, Miling Ma, Qingli Niu, Youquan Li, Aihong Liu, Junlong Liu, Jifei Yang, Hong Yin ⇑, Jianxun Luo ⇑ State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory of Grazing Animal Diseases MOA, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou, Gansu 730046, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" We evaluated the virulence of 20
isolates against Rhipicephalus (Boophilus) microplus. " Four isolates of entomopathogenic fungi were highly virulent against this tick. " Four isolates have potential for applications to control this tick.
a r t i c l e
i n f o
Article history: Received 8 July 2011 Accepted 2 July 2012 Available online 25 July 2012 Keywords: Rhipicephalus (Boophilus) microplus Beauveria bassiana Metarhizium anisopliae Biological control Entomopathogenic fungi
a b s t r a c t Rhipicephalus (Boophilus) microplus, an important ectoparasite that can transmit Babesia and Anaplasma, has caused inestimable economic losses around the world. Traditionally, acaricides are used for the control of ticks. However, drawbacks of chemical control, such as resistance, environmental pollution, and traces in food promote alternative strategies to pesticides. Microbial control is one option to reduce tick populations. In this study, we investigated the pathogenicity of thirteen Beauveria bassiana isolates and seven Metarhizium anisopliae isolates to the engorged female R. (B.) microplus ticks using different conidial concentrations of 107, 108 and 109 conidia mL–1. Fourteen days after treatment, three B. bassiana isolates (B.bAT01, B.bAT03, B.bAT13) and one M. anisopliae isolate (M.aAT04) resulted in 100% mortality of engorged female ticks with conidial concentrations of 108 and 109 conidia mL–1. Isolates of B.bAT01, B.bAT03, B.bAT13 and M.aAT04 at all conidial concentrations could reduce the reproductive efficiency index (REI) of R. (B.) microplus ticks. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Ticks, obligatory ectoparasites of terrestrial vertebrates, transmit various pathogenic microorganisms, such as protozoa, rickettsiae, spirochaetes and viruses (Jongejan and Uilenberg, 2004). The cattle tick Rhipicephalus (Boophilus) microplus (Acari, Ixodidae) is an obligatory haematophagous ectoparasite and a single host species in almost all tropical and subtropical countries. Apart from ⇑ Corresponding authors. Fax: +86 931 8340977 (J. Luo), +86 931 8340977 (H. Yin). E-mail addresses: [email protected] (H. Yin), [email protected] (J. Luo). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.07.002
physical damage to their host and blood spoliation, R. (B.) microplus is a major vector for bovine babesiosis and anaplasmosis (Monteiro et al., 2010; Jittapalapong et al., 2010). In China, R. (B.) microplus results in severe economic losses (Luo et al., 2003). Though various techniques have been developed, tick population control still relies on chemical acaricides during their parasitic phase. Presently, chemical acaricides (that include organochlorines, organophosphates, carbamates, amidines and pyrethroids) are most effective reagents in tick control (Tuininga et al., 2009). The unlimited and indiscriminate use of these acaricides has resulted in many problems, such as heritable resistance of ticks, risk to non-target organisms, environmental pollution, trace contamination in food
99
Q. Ren et al. / Biological Control 63 (2012) 98–101
products, and increasing cost of tick control (Pourseyed et al., 2010; Angel-Sahagún et al., 2010). Therefore, alternative approaches are needed to reduce or replace the application of acaricides. During recent years, biological control of ticks with entomopathogenic fungi has received considerable attention (Ment et al., 2010). Because of their wide distribution, low risk for humans and animals, environmental safety, high virulence against ticks and potential low cost, entomopathogenic fungi have been investigated as potential agents for biological control of ticks (Samish and Rehacek, 1999; Pirali-Kheirabadi et al., 2007). Among the entomopathogenic fungi examined for pathogenicity against ticks, two species commonly investigated are Beauveria bassiana and Metarhizium anisopliae (Polar et al., 2008). B. bassiana and M. anisopliae show high virulence to various life stages of ticks, and several studies substantiate the effect of these fungi on R. (B.) microplus. OjedaChi et al. (2010) reported that isolates of M. anisopliae killed 100% of engorged female R. (B.) microplus ticks at 1 106 conidia mL–1 dose. Some strains of M. anisopliae caused up to 100% mortality in R. (B.) microplus in vitro within 2 days (Leemon and Jonsson, 2008). Fernandes et al. (2003) observed a high percentage of larval mortality and a low percentage of egg hatching in ticks treated with B. bassiana. The entomopathogenic fungi B. bassiana and M. anisopliae have been used widely for biological control of agricultural and forest pests (Quesada-Moraga et al., 2007; Wraight et al., 2010), but few studies have been reported regarding their virulence against R. (B.) microplus in China. The aim of the present study was to evaluate the susceptibility of engorged female R. (B.) microplus ticks to thirteen strains of B. bassiana and seven strains of M. anisopliae, to discover potential strains for controlling R. (B.) microplus ticks in China.
Table 1 Details of fungal strains used in this study. Strain
Fungus
Original host
Geographical origin
B.bAT01 B.bAT02 B.bAT03 B.bAT04 B.bAT05 B.bAT06 B.bAT07 B.bAT08 B.bAT09 B.bAT10 B.bAT11 B.bAT12 B.bAT13 M.aAT01 M.aAT02 M.aAT03 M.aAT04 M.aAT05 M.aAT06 M.aAT07
B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana B. bassiana M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae M. anisopliae
Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil Soil B. microplus Soil Soil Soil Soil Soil Soil Soil
Anhui, China Anhui, China Guizhou, China Zhejiang, China Zhejiang, China Jiangsu, China Fujian, China Hunan, China Heilongjiang, China Guizhou, China Guangxi, China Hubei, China Guizhou, China Sichuan, China Shanxi, China Anhui, China Henan, China Gansu, China Guangdong, China Guizhou, China
approximately 30 s and then placing them on paper towel to soak up the excess. Control groups (with 10 engorged R. (B.) microplus females per group) were treated with sterile water containing 0.05% Tween 80 instead of conidial suspension. Each trial was repeated three times. After treatment, ticks were individually placed in glass tubes sealed with hydrophilic cotton and kept in an incubator at 27 ± 1° C and 80 ± 5% RH for oviposition. Ticks were observed daily to evaluate biological parameters. 2.4. Data analysis
2. Materials and methods 2.1. Ticks R. (B.) microplus ticks were originally obtained from naturally infested cattle in Sichuan Province in western China. The ticks were maintained in an incubator at 27 ± 1° C and 80 ± 5% RH (relative humidity) in our laboratory. For this study, the larval R. (B.) microplus ticks were put onto two calves, and the engorged female ticks were collected, which were randomly divided into different groups. 2.2. Fungal growth and preparation of conidial suspensions Thirteen isolates of B. bassiana and seven isolates of M. anisopliae were originally obtained from soil and a tick collected from 14 provinces of China (Table 1). These fungal isolates were maintained on potato dextrose agar (PDA) and kept at 4° C. All isolates were cultured with PDA on petri plates in an incubator at 26–28° C and RH > 80% for 12 days. Conidia were harvested by scraping the surface of the plate and placed into sterilized aqueous solution containing 0.05% Tween 80. After homogenization, concentrations of conidia were determined with a haemocytometer and adjusted to final concentrations of 107, 108 and 109 conidia mL–1 with 0.05% Tween 80 in distilled water.
Treatment data were evaluated using a formula described by Ojeda-Chi et al. (2010). The reproductive efficiency index (REI = egg mass weight/engorged female weight) was determined for each female tick. Dead ticks were counted and mortality rates were calculated. Analyses of variance (ANOVA) were used to evaluate levels of significance of the virulence of B. bassiana and M. anisopliae against R. (B.) microplus female ticks. 3. Results All isolates of B. bassiana and M. anisopliae were pathogenic to R. (B.) microplus at concentration of 107, 108 and 109 conidia mL–1 in the laboratory. The lethal activity of all treatment and control groups to engorged females are presented in Fig. 1. Fourteen days
2.3. Laboratory bioassays Evaluation of fungal virulence was carried out according to the method described in Frazzon et al. (2000). Bioassays of B. bassiana and M. anisopliae suspensions (107, 108 and 109 conidia mL–1 of each isolate) were conducted for pathogenicity by immersing 10 engorged R. (B.) microplus females in each of the suspensions for
Fig. 1. Mortality of R. (B.) microplus female caused by M. anisopliae and B. bassiana (concentrations of fungal strains: (A) 107 conidia mL–1, (B) 108 conidia mL–1, (C) 109 conidia mL–1).
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Q. Ren et al. / Biological Control 63 (2012) 98–101
Fig. 2. Rhipicephalus (Boophilus) microplus infected with Metarhizium anisopliae and Beauveria bassiana. (A) M.aAT04 (108 conidia mL–1); (B) B.bAT01 (108 conidia mL–1); (C) B.bAT03 (108 conidia mL–1); (D) B.bAT13 (108 conidia mL–1).
after treatment, mortality ranges of treated females were 0–96.7%, 10–100%, and 20–100% for concentrations of 107, 108 and 109 conidia mL–1 of M. anisopliae and B. bassiana strains, respectively. Mortality in the control group was zero. Isolates of B.bAT03 and M.aAT04 caused over 90% mortality at the 107 conidia mL–1 concentration. In contrast, the isolates M.aAT02 and M.aAT05 had little effect on the survival of ticks at a concentration of 107 conidia mL–1. Infected females of R. (B.) microplus were completely covered with fungal hyphae (Fig. 2). Results on the LT50 are shown in Table3. The LT50 varied from 6.4884 to 7.3759 days on the four selected fungal isolates at the 107 conidia mL–1 concentration (Table 3).
Table 2 Reproductive efficiency index (REI) of R. (B.) microplus females treated with M. anisopliae and B. bassiana. Isolates
Treatment group REI 107 Conidial mL
B.bAT01 B.bAT02 B.bAT03 B.bAT04 B.bAT05 B.bAT06 B.bAT07 B.bAT08 B.bAT09 B.bAT10 B.bAT11 B.bAT12 B.bAT13 M.aAT01 M.aAT02 M.aAT03 M.aAT04 M.aAT05 M.aAT06 M.aAT07 Control
0.15 ± 0.01 0.48 ± 0.01 0.15 ± 0.01 0.37 ± 0.03 0.45 ± 0.03 0.4 ± 0.02 0.4 ± 0.00 0.48 ± 0.03 0.54 ± 0.02 0.53 ± 0.01 0.47 ± 0.01 0.48 ± 0.04 0.07 ± 0.00 0.47 ± 0.04 0.55 ± 0.03 0.46 ± 0.00 0.17 ± 0.01 0.55 ± 0.03 0.29 ± 0.02 0.29 ± 0.01 0.57 ± 0.00
1
108 Conidial mL 0.12 ± 0.01 0.45 ± 0.02 0.09 ± 0.02 0.36 ± 0.01 0.43 ± 0.01 0.43 ± 0.03 0.45 ± 0.02 0.44 ± 0.01 0.53 ± 0.00 0.53 ± 0.03 0.43 ± 0.01 0.44 ± 0.03 0.03 ± 0.00 0.42 ± 0.04 0.53 ± 0.02 0.42 ± 0.02 0.01 ± 0 0.55 ± 0.02 0.28 ± 0.02 0.17 ± 0.01
1
109 Conidial mL
1
0.09 ± 0.01 0.41 ± 0.03 0.07 ± 0.01 0.32 ± 0.02 0.40 ± 0.02 0.40 ± 0.00 0.45 ± 0.03 0.41 ± 0.01 0.53 ± 0.01 0.52 ± 0.01 0.44 ± 0.01 0.43 ± 0.01 0.00 ± 0.00 0.44 ± 0.03 0.51 ± 0.03 0.42 ± 0.01 0.03±.00 0.54 ± 0.02 0.26 ± 0.01 0.12 ± 0.01
Means followed by the same lowercase letter within the same column do not differ statistically (p < 0.05).
Egg production of R. (B.) microplus females was reduced after treatment with isolates of B. bassiana and M. anisoplia (Table 2). Specifically, 0.15, 0.15, 0.07 and 0.17 of the REI of R. (B.) microplus infected with isolates of B.bAT01, B.bAT03, B.bAT13 and M.aAT04 at 107 conidia mL–1, compared with 0.57 in the control group. 4. Discussion Entomopathogenic fungi are wide distributed, low risk to nontarget organisms, environmental safety, and high virulence against ticks, thus they have been rapidly developed for biological control of pests. Studies showed that tick death resulting from fungi is usually due to a combination of actions that include depletion of nutrients, physical obstruction or invasion of organs, or toxicosis (Fernandes et al., 2012.). Entomopathogenic fungi commonly investigated for pathogenicity against ticks are the mycoinsecticidal B. bassiana, M. anisopliae and Paecilomyces fumosoroseus (Campos et al., 2005; Zabalgogeazcoa et al., 2008). Previous studies have reported effectiveness of B. bassiana and M. anisopliae against all stages of R. (B.) microplus. In this study, three B. bassiana isolates (B.bAT01, B.bAT03, B.bAT13) and one M. anisopliae isolate (M.aAT04) demonstrated more virulence against female ticks, which caused up to 100% mortality for the ticks at concentrations of 108 and 109 conidia mL–1. Gindin et al. (2001) reported that the Ma-7 strain of M. anisopliae was virulent to females and caused 100% mortality at a concentration of 108 spores mL–1. Our results were in agreement with this result. In addition, another four isolates of B. bassiana and two isolates of M. anisopliae were also pathogenic against R. (B.) microplus, causing mortality greater than 80%. All the treatments caused a reduction of the REI, while the control group did not. We observed that the reproductive efficiency of R. (B.) microplus was reduced from 0.40 to 0.50 at concentration of 107 spores mL–1when treated. Similarly, Kaaya et al. (1996) observed that 5% of ticks infected with B. bassiana failed to lay eggs. In our study, the LT50 of most virulent fungi isolates ranged from 6.4884–7.3759 days at the 107 conidia mL–1 concentration. The estimated LT50 for ticks immersed in M.aAT04 and B.bAT13
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Q. Ren et al. / Biological Control 63 (2012) 98–101 Table 3 Lethal time (LT50) of M. anisopliae e and B. bassiana against R. (B.) microplus engorged females. Isolates
LT50 (days) 107 Conidial mL
B.bAT01 B.bAT03 B.bAT13 M.aAT04
7.0992 7.3759 6.6173 6.4884
1
(6.4830–7.7258) (6.7958–7.9709) (5.9683–7.2834) (5.8599–7.1306)
is lower than in B.bAT01 and B.bAT03. These results demonstrated the virulence of B. bassiana and M. anisopliae to female, which indicates a potential use of these species for biocontrol of R. (B.) microplus ticks in China. Acknowledgments This study was financially supported by the Supporting Plan (2007BAD40B06), ‘‘863’’ Project (2006AA10A207), 973 Program (2010CB530206), ‘‘948’’(2010-S04), Key Project of Gansu Province (1002NKDA035 and 0801NKDA033), Beef and Yak Production System Programme, MOA, Specific Fund for Sino-Europe Cooperation, MOST, China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019); The research was also facilitated by EPIZONE (FOOD-CT-2006-016236), ASFRISK(No: 211691), ARBOZOONET(No: 211757) and PIROVAC (KBBE-3-245145) of European Commission, Brussels, Belgium. References Angel-Sahagún, C.A., Lezama-Gutiérrez, R., Molina-Ochoa, J., Pescador-Rubio, A., Skoda, S.R., Cruz-Vázquez, C., Lorenzoni, A.G., Galindo-Velasco, E., FragosoSánchez, H., Foster, J.E., 2010. Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus = B. microplus (Acari: Ixodidae) larvae and the efficacy of conidia formulations to reduce larval tick density under field conditions. Vet. Parasitol. 170, 278–286. Campos, R.A., Arruda, W., Boldo, J.T., da Silva, M.V., de Barros, N.M., de Azevedo, J.L., Schrank, A., Vainstein, M.H., 2005. Boophilus microplus infection by Beauveria amorpha and Beauveria bassiana: SEM analysis and regulation of subtilisin-like proteases and chitinases. Curr. Microbiol. 50, 257–261. Fernandes, É.K., Bittencourt, V.R., Roberts, D.W., 2012. Perspectives on the potential of entomopathogenic fungi in biological control of ticks. Exp. Parasitol. 130, 300–305. Fernandes, E.K., da Costa, G.L., de Souza, E.J., de Moraes, A.M., Bittencourt, V.R., 2003. Beauveria bassiana isolated from engorged females and tested against eggs and larvae of Boophilus microplus (Acari: Ixodidae). J. Basic Microbiol. 43, 393–398. Frazzon, A.P., da Silva Vaz Jr., I., Masuda, A., Schrank, A., Vainstein, M.H., 2000. In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet. Parasitol. 94, 117–125. Gindin, G., Samish, M., Alekseev, E., Glazer, I., 2001. The susceptibility of Boophilus annulatus (Ixodidae) ticks to entomopathogenic fungi. Biocontrol. Sci. Technol. 11, 111–118. Jittapalapong, S., Kaewhom, P., Pumhom, P., Canales, M., de la Fuente, J., Stich, R.W., 2010. Immunization of rabbits with recombinant serine protease inhibitor
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1
(5.9544–7.0912) (5.7010–6.8067) (5.6925–6.7323) (5.5614–6.6648)
109 Conidial mL 5.7315 6.0025 5.7313 5.4579
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