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Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads
Journal of Invertebrate Pathology 93 (2006) 50–53 www.elsevier.com/locate/yjipa
Short communication
Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads R. Rajagopal a,¤, Sharad Mohan b, Raj K. Bhatnagar a a
International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 110 067, India b Indian Agricultural Research Institute (IARI), New Delhi 110 012, India Received 26 May 2005; accepted 17 May 2006 Available online 7 July 2006
Abstract Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 £ 107 Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch’s postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC50 dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h. © 2006 Elsevier Inc. All rights reserved. Keywords: Pathogenicity; Insect; Heterorhabditis; Photorhabdus; Nematode; Alginate beads
1. Introduction The motile Gram-negative bacteria Photorhabdus luminescens, potent insect pathogens, exist as symbionts of the entomopathogenic nematodes Heterorhabditis spp. (Boemare et al., 1993; Kaya and Gaugler, 1993). The inability to isolate these bacteria in a free-living form from the environment gave raise to doubts about their capacity to survive, multiply and infect insects in soil independent of their symbiont nematodes (Forst and Nealson, 1996). Studies by Gotz et al. (1981) showing that the nematode helps in overcoming the host’s (insect’s) defense by secreting an immune inhibitor served to reinforce these doubts. Nevertheless eVorts to establish the insect pathogenicity of P. luminescens in isolation from the nematode host continued. To check out the independent viability of the bacteria in soil, Poinar
*
Corresponding author. Fax: +91 11 26162316. E-mail address: [email protected] (R. Rajagopal).
0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2006.05.005
et al. (1980) investigated the persistence of Photorhabdus in sterile soil and were unable to recover any cells even on the day following the inoculation of the soil with the bacteria. However, Bleakley and Chen (1999) reported the successful survival of these bacteria for up to one month, in sterile (autoclaved) acidic soil augmented with calcium carbonate and gelatin or casamino acids, but they did not evaluate the insect pathogenicity of Photorhabdus under these conditions. All reports, until date, examining the direct virulence of P. luminescens to insect larvae employed artiWcial means, such as injecting live bacterial cells into the larvae (Bowen and Ensign, 1998; Gotz et al., 1981; Rajagopal and Bhatnagar, 2002). In an attempt to eVect a convergence of the directions charted out by all the earlier studies, we have focused on investigating the ability of P. luminescens in the soil environment to independently infect and kill an insect larvae. As such, we encapsulated P. luminescens akhurstii in sodium alginate beads for release in soil to assess their ability to kill Spodoptera litura 6th instar larvae upon feeding.
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53
2. Materials and methods 2.1. Culturing of bacteria and alginate beads preparation Photorhabdus luminescens sub sp. akhurstii strain K-1, isolated from Heterorhabditis indica, and Escherichia coli strain K-12 were grown overnight on Luria Bertani (LB) medium from starter cultures at 0.5% inoculum concentration. They were then processed as follows to result in six diVerent alginate bead preparations. T1 P. luminescens K-1; overnight cell suspension (50 ml) containing approximately 1 £ 109 cells/ml; medium was used without centrifugation to form beads in the ratio of 2.5 £ 107 P. luminescens cells per bead. T2 P. luminescens K-1; overnight cell suspension centrifuged at 15,000g at 4 °C, pellets resuspended in fresh LB medium (50 ml) containing approximately 1 £ 109 cells/ml; beads formed in the ratio of 2.5 £ 107 P. luminescens cells per bead. T3 P. luminescens K-1; supernatant from T2 passed through 0.22 m Wlter (50 ml); beads formed in the ratio of 0 P. luminescens cells per bead. T4 E. coli K-12; overnight cell suspension (50 ml) containing 1 £ 109 cells/ml; medium was used without centrifugation to form beads in the ratio of 2.5 £ 107 E. coli cells per bead. T5 Sterile water (50 ml); beads formed in the ratio of 0 P. luminescens cells per bead. T6 H. indica infective juveniles (IJ) in 50 ml sterile water; contained approximately 300 IJ/ml; beads formed in the ratio of 7.5 H. indica IJ’s per bead. Each 50 ml treatment was mixed thoroughly with 50 ml of a solution containing 2% sodium alginate and 2% sucrose in sterile water and was then introduced dropwise into a 1.47% calcium chloride solution resulting in the formation of uniformly round 3–4 mm diameter beads. After 30 min, the calcium chloride was decanted and the beads were collected on a wire mesh Wlter. They were washed/ Xushed Wve times in succession with approximately 200 ml of sterile water each time, and were allowed to decant fully. The resulting beads were used immediately or were stored at 28 °C in polythene bags. Further, serial dilutions were made of the pelleted and resuspended P. luminescens preparation (T2) resulting in beads having a range of cells—from 2.5 £ 107 cells per bead to 2.5 cells per bead. 2.2. Insect bioassays Spodoptera litura were reared according to Rajagopal and Bhatnagar (2002). A 60 mm diameter Petri dish (Falcon) was half-Wlled with sterile soil and four alginate beads were distributed in it. One 6th instar S. litura larvae were released into each Petri dish and were observed until 48 h for mortality. For each of the above treatments there were 10 replications and each replication consisted of Wve Petri
51
plates having one insect each. All the bioassay experiments were conducted at room temperature (28 °C). The mortality data were recorded at 48 h post exposure of the insects to the diVerent treatments. 3. Results and discussion Alginate beads prepared from the six diVerent treatments were tested for insect pathogenicity on S. litura larvae. All the insects tested with alginate beads containing P. luminescens cells (T1, prepared from P. luminescens broth and T2, prepared by pelleting and resuspending the P. luminescens cells) resulted in 100% mortality within 48 h, while there was no mortality in the insects tested with alginate beads of E. coli and sterile water. The dead insects turned reddish in colour and luminesced strongly in dark. The bacteria were isolated from the body of the dead insect, the genomic DNA prepared and a PCR–RFLP analysis was conducted on the 16S rDNA gene according to Rajagopal and Bhatnagar (2002). The PCR–RFLP proWle of the reisolated bacteria and the initial bacteria (K-1) was identical thus satisfying all Koch’s postulates and establishing the Wdelity of infection and pathogenicity. Photorhabdus secretes insecticidal proteins into the culture medium, which have been puriWed (Bowen and Ensign, 1998; Rajagopal and Bhatnagar, 2002) and their encoding genes cloned (Bowen et al., 1998). Alginate beads prepared from the culture supernatant (T3) were also fed to the larvae to discriminate between the larval deaths due to the secreted toxin as against the direct infection of the insect by P. luminescens cells. Exposing the larvae to these alginate beads resulted in 20% mortality within 48 h, which pointed to retention of the secreted toxin protein complex’s insecticidal activity in the alginate beads. To understand the progress and mortality of infection, the larval behaviour every six hours after releasing them on alginate beads containing Photorhabdus cells (T2) was monitored. The insects could be seen moving around and nibbling at the beads at the 6th hour, and by the 12th hour approximately 40% of the beads were consumed and the larval mobility appeared signiWcantly reduced. By the 18th hour the larvae were immobile, and by the 24th hour they were dead. They turned reddish-black in colour and luminesced strongly in dark by the 48th hour. At each of the above four intervals, soil samples were taken and analysed for the presence of Photorhabdus colonies by serial dilution. No Photorhabdus CFU could be identiWed. Heterorhabditis indica IJ (carrying Photorhabdus in their gut) encapsulated in alginate beads (T6) did not result in the mortality of S. litura larvae until 48 h. (Mortality from the nematode treatment started to occur only after 72 h.) A comparison of the cadavers of the larvae killed by P. luminescens alone to those killed by H. indica (Fig. 1) revealed that while in the former there is complete degradation of the larval cadaver, in the latter the integrity of the cadaver was retained. A plausible explanation for this could be that as Heterorhabditis are bacterial feeders they need a constant supply of bacteria for completing their life cycle.
52
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53
Fig. 1. Spodoptera litura 6th instar larvae killed following exposure to Photorhabdus in alginate beads (A) and larvae killed due to exposure to Heterorhabditis indica in alginate beads (B). The cadavers are compared following four days of exposure to the respective beads.
Thus, the presence of H. indica exercises natural control on the multiplication of P. luminescens. Since alginate beads containing pelleted and resuspended P. luminescens cells resulted in 100% mortality, we studied the eVect of this treatment at varying doses on the insect larvae. Serially diluted doses of bacterial preparation revealed a sigmoid relationship between insect mortality and bacterial count at 48 h after exposure (Table 1). At 48 h, the LC50 dose was calculated by Probit analysis as 1010 (95% conWdence limits 552–1797) P. luminescens cells per larvae while the LC99 response was at 1,815,329 (95% conWdence limits 540,082–9,906,411) P. luminescens cells per larvae. The bacteria survived for two months following the encapsulation in alginate and retained their pathogenicity towards the larvae of S. litura (data not shown). Polymer-based formulations, like alginate, have been reported as successful carriers of microbes in the last 10–15 years. Encapsulation of the microbes by alginate protects them against harsh environmental extremes. It is well established that a gradual degradation of the alginate beads by Table 1 Mortality of Spodoptera litura larvae at diVerent concentrations of Photorhabdus luminescens impregnated in alginate beads Concentrations of bacteria per insect 8
6
1 £ 10 (§1.3 £ 10 ) 1 £ 107 (§3.6 £ 105) 1 £ 106 (§1.5 £ 104) 1 £ 105 (§4.0 £ 103) 1 £ 104 (§1.3 £ 102) 1 £ 103 (§4.3 £ 10) 1 £ 102 (§9.2) 10 (§0.57) 0 (§0)
% Mortality 100 (§0) 100 (§0) 100 (§0) 88 (§1.96) 76 (§1.76) 54 (§1.93) 26 (§1.35) 6 (§1.35) 0 (§0)
• Values in parentheses represent standard errors. • Mortality data at diVerent concentrations were recorded at 48 h and analysed by Probit analysis to calculate the LC50 dose of 1010 cells (95% conWdence limits 552–1797). • Data represent an average of 50 insects (grouped into 10 replications of 5 insects each) for each treatment concentration. • Each larva was exposed to four alginate beads, each bead containing one fourth of the targeted concentration of Photorhabdus luminescens cells in a treatment.
the soil microbes releases the encapsulated microbes into the environment in a biologically competent state (Bashan, 1998). VAM fungi (Ganry et al., 1982), mycoherbicidal fungi Fusarium oxysporum (Amsellem et al., 1999), growth promoting bacteria of plants (PGPB) like Rhizobium, Azospirillum and Pseudomonas Xuorescens (Bashan, 1986) and plant disease controlling bacteria (Aino et al., 1997) are a few successful examples of encapsulation and release of viable microbes in alginate beads. In conclusion, these results suggest that the P. luminescens bacteria, protected by encapsulation within alginate beads, maintain their viability in a form that is directly pathogenic to insects. This is the Wrst such report describing the ability of P. luminescens bacteria to kill insects when applied in soil independent of their nematode host. References Aino, M., Maekawa, Y., Myama, S., Kato, H., 1997. Biocontrol of bacterial wilt of tomato by producing seedlings colonised with endophytic antagonistic pseudomonads. In: Ogoshi, A. (Ed.), Plant Growth-promoting Rhizobacteria—Present Status and Future Prospects. Faculty of Agriculture, Hokkaido University, Sapporo, Japan. pp. 120–123. Amsellem, Z., Zidack, N.K., Quimby, P.C., Gressel, J., 1999. Longterm dry preservation of viable mycelia of two mycoherbicidal organisms. Crop Prot. 18, 643–649. Bashan, Y., 1986. Alginate beads as synthetic inoculant carriers for the slow release of bacteria that eVect plant growth. Appl. Environ. Microbiol. 51, 1089–1098. Bashan, Y., 1998. Inoculants for plant growth-promoting bacteria in agriculture. Biotechnol. Adv. 16, 729–770. Bleakley, B.H., Chen, X., 1999. Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil. Can. J. Microbiol. 45, 273–278. Boemare, N.E., Akhurst, R.J., Mourant, R.G., 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int. J. Syst. Bacteriol. 43, 249–255. Bowen, D.J., Ensign, J.C., 1998. PuriWcation and characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64, 3029–3035. Bowen, D.J., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., Vrench-Constant, R.H., 1998. Novel insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132.
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53 Forst, S., Nealson, K., 1996. Molecular biology of the symbiotic pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21–43. Ganry, F., Diem, H.G., Dommergues, Y.R., 1982. EVect of inoculation with Glomus mosseae on nitrogen Wxation by Weld grown soybeans. Plant Soil 68, 321–329. Gotz, P., Boman, A., Boman, H.G., 1981. Interactions between insect immunity and an insect pathogenic nematode with symbiotic bacteria. Proc. R. Soc. Lond. B 212, 333–350.
53
Kaya, H.K., Gaugler, R., 1993. Entomopathogenic nematodes. Annu. Rev. Entomol. 38, 181–206. Poinar Jr., G.O., Thomas, G., Haygood, H., Nealson, K.H., 1980. Growth and luminescence of the symbiotic bacteria associated with the nematode Heterorharbditis bacteriophora. Soil Biol. Biochem. 12, 5–10. Rajagopal, R., Bhatnagar, R.K., 2002. Insecticidal toxic proteins produced by P. luminescens akhurstii the symbiont of Heterorhabditis indica. J. Nematol. 34, 23–27.
Short communication
Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads R. Rajagopal a,¤, Sharad Mohan b, Raj K. Bhatnagar a a
International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 110 067, India b Indian Agricultural Research Institute (IARI), New Delhi 110 012, India Received 26 May 2005; accepted 17 May 2006 Available online 7 July 2006
Abstract Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 £ 107 Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch’s postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC50 dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h. © 2006 Elsevier Inc. All rights reserved. Keywords: Pathogenicity; Insect; Heterorhabditis; Photorhabdus; Nematode; Alginate beads
1. Introduction The motile Gram-negative bacteria Photorhabdus luminescens, potent insect pathogens, exist as symbionts of the entomopathogenic nematodes Heterorhabditis spp. (Boemare et al., 1993; Kaya and Gaugler, 1993). The inability to isolate these bacteria in a free-living form from the environment gave raise to doubts about their capacity to survive, multiply and infect insects in soil independent of their symbiont nematodes (Forst and Nealson, 1996). Studies by Gotz et al. (1981) showing that the nematode helps in overcoming the host’s (insect’s) defense by secreting an immune inhibitor served to reinforce these doubts. Nevertheless eVorts to establish the insect pathogenicity of P. luminescens in isolation from the nematode host continued. To check out the independent viability of the bacteria in soil, Poinar
*
Corresponding author. Fax: +91 11 26162316. E-mail address: [email protected] (R. Rajagopal).
0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2006.05.005
et al. (1980) investigated the persistence of Photorhabdus in sterile soil and were unable to recover any cells even on the day following the inoculation of the soil with the bacteria. However, Bleakley and Chen (1999) reported the successful survival of these bacteria for up to one month, in sterile (autoclaved) acidic soil augmented with calcium carbonate and gelatin or casamino acids, but they did not evaluate the insect pathogenicity of Photorhabdus under these conditions. All reports, until date, examining the direct virulence of P. luminescens to insect larvae employed artiWcial means, such as injecting live bacterial cells into the larvae (Bowen and Ensign, 1998; Gotz et al., 1981; Rajagopal and Bhatnagar, 2002). In an attempt to eVect a convergence of the directions charted out by all the earlier studies, we have focused on investigating the ability of P. luminescens in the soil environment to independently infect and kill an insect larvae. As such, we encapsulated P. luminescens akhurstii in sodium alginate beads for release in soil to assess their ability to kill Spodoptera litura 6th instar larvae upon feeding.
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53
2. Materials and methods 2.1. Culturing of bacteria and alginate beads preparation Photorhabdus luminescens sub sp. akhurstii strain K-1, isolated from Heterorhabditis indica, and Escherichia coli strain K-12 were grown overnight on Luria Bertani (LB) medium from starter cultures at 0.5% inoculum concentration. They were then processed as follows to result in six diVerent alginate bead preparations. T1 P. luminescens K-1; overnight cell suspension (50 ml) containing approximately 1 £ 109 cells/ml; medium was used without centrifugation to form beads in the ratio of 2.5 £ 107 P. luminescens cells per bead. T2 P. luminescens K-1; overnight cell suspension centrifuged at 15,000g at 4 °C, pellets resuspended in fresh LB medium (50 ml) containing approximately 1 £ 109 cells/ml; beads formed in the ratio of 2.5 £ 107 P. luminescens cells per bead. T3 P. luminescens K-1; supernatant from T2 passed through 0.22 m Wlter (50 ml); beads formed in the ratio of 0 P. luminescens cells per bead. T4 E. coli K-12; overnight cell suspension (50 ml) containing 1 £ 109 cells/ml; medium was used without centrifugation to form beads in the ratio of 2.5 £ 107 E. coli cells per bead. T5 Sterile water (50 ml); beads formed in the ratio of 0 P. luminescens cells per bead. T6 H. indica infective juveniles (IJ) in 50 ml sterile water; contained approximately 300 IJ/ml; beads formed in the ratio of 7.5 H. indica IJ’s per bead. Each 50 ml treatment was mixed thoroughly with 50 ml of a solution containing 2% sodium alginate and 2% sucrose in sterile water and was then introduced dropwise into a 1.47% calcium chloride solution resulting in the formation of uniformly round 3–4 mm diameter beads. After 30 min, the calcium chloride was decanted and the beads were collected on a wire mesh Wlter. They were washed/ Xushed Wve times in succession with approximately 200 ml of sterile water each time, and were allowed to decant fully. The resulting beads were used immediately or were stored at 28 °C in polythene bags. Further, serial dilutions were made of the pelleted and resuspended P. luminescens preparation (T2) resulting in beads having a range of cells—from 2.5 £ 107 cells per bead to 2.5 cells per bead. 2.2. Insect bioassays Spodoptera litura were reared according to Rajagopal and Bhatnagar (2002). A 60 mm diameter Petri dish (Falcon) was half-Wlled with sterile soil and four alginate beads were distributed in it. One 6th instar S. litura larvae were released into each Petri dish and were observed until 48 h for mortality. For each of the above treatments there were 10 replications and each replication consisted of Wve Petri
51
plates having one insect each. All the bioassay experiments were conducted at room temperature (28 °C). The mortality data were recorded at 48 h post exposure of the insects to the diVerent treatments. 3. Results and discussion Alginate beads prepared from the six diVerent treatments were tested for insect pathogenicity on S. litura larvae. All the insects tested with alginate beads containing P. luminescens cells (T1, prepared from P. luminescens broth and T2, prepared by pelleting and resuspending the P. luminescens cells) resulted in 100% mortality within 48 h, while there was no mortality in the insects tested with alginate beads of E. coli and sterile water. The dead insects turned reddish in colour and luminesced strongly in dark. The bacteria were isolated from the body of the dead insect, the genomic DNA prepared and a PCR–RFLP analysis was conducted on the 16S rDNA gene according to Rajagopal and Bhatnagar (2002). The PCR–RFLP proWle of the reisolated bacteria and the initial bacteria (K-1) was identical thus satisfying all Koch’s postulates and establishing the Wdelity of infection and pathogenicity. Photorhabdus secretes insecticidal proteins into the culture medium, which have been puriWed (Bowen and Ensign, 1998; Rajagopal and Bhatnagar, 2002) and their encoding genes cloned (Bowen et al., 1998). Alginate beads prepared from the culture supernatant (T3) were also fed to the larvae to discriminate between the larval deaths due to the secreted toxin as against the direct infection of the insect by P. luminescens cells. Exposing the larvae to these alginate beads resulted in 20% mortality within 48 h, which pointed to retention of the secreted toxin protein complex’s insecticidal activity in the alginate beads. To understand the progress and mortality of infection, the larval behaviour every six hours after releasing them on alginate beads containing Photorhabdus cells (T2) was monitored. The insects could be seen moving around and nibbling at the beads at the 6th hour, and by the 12th hour approximately 40% of the beads were consumed and the larval mobility appeared signiWcantly reduced. By the 18th hour the larvae were immobile, and by the 24th hour they were dead. They turned reddish-black in colour and luminesced strongly in dark by the 48th hour. At each of the above four intervals, soil samples were taken and analysed for the presence of Photorhabdus colonies by serial dilution. No Photorhabdus CFU could be identiWed. Heterorhabditis indica IJ (carrying Photorhabdus in their gut) encapsulated in alginate beads (T6) did not result in the mortality of S. litura larvae until 48 h. (Mortality from the nematode treatment started to occur only after 72 h.) A comparison of the cadavers of the larvae killed by P. luminescens alone to those killed by H. indica (Fig. 1) revealed that while in the former there is complete degradation of the larval cadaver, in the latter the integrity of the cadaver was retained. A plausible explanation for this could be that as Heterorhabditis are bacterial feeders they need a constant supply of bacteria for completing their life cycle.
52
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53
Fig. 1. Spodoptera litura 6th instar larvae killed following exposure to Photorhabdus in alginate beads (A) and larvae killed due to exposure to Heterorhabditis indica in alginate beads (B). The cadavers are compared following four days of exposure to the respective beads.
Thus, the presence of H. indica exercises natural control on the multiplication of P. luminescens. Since alginate beads containing pelleted and resuspended P. luminescens cells resulted in 100% mortality, we studied the eVect of this treatment at varying doses on the insect larvae. Serially diluted doses of bacterial preparation revealed a sigmoid relationship between insect mortality and bacterial count at 48 h after exposure (Table 1). At 48 h, the LC50 dose was calculated by Probit analysis as 1010 (95% conWdence limits 552–1797) P. luminescens cells per larvae while the LC99 response was at 1,815,329 (95% conWdence limits 540,082–9,906,411) P. luminescens cells per larvae. The bacteria survived for two months following the encapsulation in alginate and retained their pathogenicity towards the larvae of S. litura (data not shown). Polymer-based formulations, like alginate, have been reported as successful carriers of microbes in the last 10–15 years. Encapsulation of the microbes by alginate protects them against harsh environmental extremes. It is well established that a gradual degradation of the alginate beads by Table 1 Mortality of Spodoptera litura larvae at diVerent concentrations of Photorhabdus luminescens impregnated in alginate beads Concentrations of bacteria per insect 8
6
1 £ 10 (§1.3 £ 10 ) 1 £ 107 (§3.6 £ 105) 1 £ 106 (§1.5 £ 104) 1 £ 105 (§4.0 £ 103) 1 £ 104 (§1.3 £ 102) 1 £ 103 (§4.3 £ 10) 1 £ 102 (§9.2) 10 (§0.57) 0 (§0)
% Mortality 100 (§0) 100 (§0) 100 (§0) 88 (§1.96) 76 (§1.76) 54 (§1.93) 26 (§1.35) 6 (§1.35) 0 (§0)
• Values in parentheses represent standard errors. • Mortality data at diVerent concentrations were recorded at 48 h and analysed by Probit analysis to calculate the LC50 dose of 1010 cells (95% conWdence limits 552–1797). • Data represent an average of 50 insects (grouped into 10 replications of 5 insects each) for each treatment concentration. • Each larva was exposed to four alginate beads, each bead containing one fourth of the targeted concentration of Photorhabdus luminescens cells in a treatment.
the soil microbes releases the encapsulated microbes into the environment in a biologically competent state (Bashan, 1998). VAM fungi (Ganry et al., 1982), mycoherbicidal fungi Fusarium oxysporum (Amsellem et al., 1999), growth promoting bacteria of plants (PGPB) like Rhizobium, Azospirillum and Pseudomonas Xuorescens (Bashan, 1986) and plant disease controlling bacteria (Aino et al., 1997) are a few successful examples of encapsulation and release of viable microbes in alginate beads. In conclusion, these results suggest that the P. luminescens bacteria, protected by encapsulation within alginate beads, maintain their viability in a form that is directly pathogenic to insects. This is the Wrst such report describing the ability of P. luminescens bacteria to kill insects when applied in soil independent of their nematode host. References Aino, M., Maekawa, Y., Myama, S., Kato, H., 1997. Biocontrol of bacterial wilt of tomato by producing seedlings colonised with endophytic antagonistic pseudomonads. In: Ogoshi, A. (Ed.), Plant Growth-promoting Rhizobacteria—Present Status and Future Prospects. Faculty of Agriculture, Hokkaido University, Sapporo, Japan. pp. 120–123. Amsellem, Z., Zidack, N.K., Quimby, P.C., Gressel, J., 1999. Longterm dry preservation of viable mycelia of two mycoherbicidal organisms. Crop Prot. 18, 643–649. Bashan, Y., 1986. Alginate beads as synthetic inoculant carriers for the slow release of bacteria that eVect plant growth. Appl. Environ. Microbiol. 51, 1089–1098. Bashan, Y., 1998. Inoculants for plant growth-promoting bacteria in agriculture. Biotechnol. Adv. 16, 729–770. Bleakley, B.H., Chen, X., 1999. Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil. Can. J. Microbiol. 45, 273–278. Boemare, N.E., Akhurst, R.J., Mourant, R.G., 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int. J. Syst. Bacteriol. 43, 249–255. Bowen, D.J., Ensign, J.C., 1998. PuriWcation and characterization of a high molecular weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ. Microbiol. 64, 3029–3035. Bowen, D.J., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., Vrench-Constant, R.H., 1998. Novel insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129–2132.
R. Rajagopal et al. / Journal of Invertebrate Pathology 93 (2006) 50–53 Forst, S., Nealson, K., 1996. Molecular biology of the symbiotic pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21–43. Ganry, F., Diem, H.G., Dommergues, Y.R., 1982. EVect of inoculation with Glomus mosseae on nitrogen Wxation by Weld grown soybeans. Plant Soil 68, 321–329. Gotz, P., Boman, A., Boman, H.G., 1981. Interactions between insect immunity and an insect pathogenic nematode with symbiotic bacteria. Proc. R. Soc. Lond. B 212, 333–350.
53
Kaya, H.K., Gaugler, R., 1993. Entomopathogenic nematodes. Annu. Rev. Entomol. 38, 181–206. Poinar Jr., G.O., Thomas, G., Haygood, H., Nealson, K.H., 1980. Growth and luminescence of the symbiotic bacteria associated with the nematode Heterorharbditis bacteriophora. Soil Biol. Biochem. 12, 5–10. Rajagopal, R., Bhatnagar, R.K., 2002. Insecticidal toxic proteins produced by P. luminescens akhurstii the symbiont of Heterorhabditis indica. J. Nematol. 34, 23–27.