International Research Journal of Microbiology (IRJM) (ISSN: 2141-5463) Vol. 2(8) pp.

285-291, September 2011 Available online http://www.interesjournals.org/IRJM Copyright 2011 International Research Journals

Full Length Research Paper

Recovery of Bacillus thuringiensis from forest soil after application for gypsy moth control using susceptible Insect larvae
Phyllis. A.W. Martin*, Elizabeth A. Mongeon, Dawn E. Gundersen-Rindal and Michael B. Blackburn
USDA/ARS Invasive Insect Biocontrol and Behavior Laboratory Beltsville, MD 20706
Accepted 08 August, 2011

Bacillus thuringiensis was recovered from forest soil sprayed two years previously with B. thuringiensis for gypsy moth control by amplifying the bacteria found in the soil on bacterial agar and feeding this mixed microbial population to tobacco hornworm larvae. These bacteria killed the larvae when fed on artificial diet. Most of the bacteria recovered from dead insects formed spores and belonged to the Bacillus cereus group (14) including three B. thuringiensis and one B. weihenstephanensis. All the B. thuringiensis strains and most of the B. cereus strains were closely related (<0.001 substitutions/site in the 16S rDNA) to the strain that was sprayed. Ten B. cereus sensu lato strains differed from the B. thuringiensis strain applied by five or fewer, of the 24 traits tested. The toxicity to gypsy moth larvae of the three B. thuringiensis strains isolated was similar to the applied B. thuringiensis strain. Thus, amplification of bacteria present in soil in combination with an insect susceptible to this strain can recover insect toxic strains that are related to an applied strain. Keywords: Gypsy INTRODUCTION Bacillus thuringiensis has been applied to many locations repeatedly over many years to control periodic outbreaks of gypsy moth, Lymantria dispar (Sharov et al., 2002, APHIS factsheet 2003). Although there are techniques that can recover B. thuringiensis from the environment (Ohba and Aizawa, 1986, Travers et al., 1987), they rely on isolating the bacteria first and then testing for toxicity. However, an alternative method can be developed around assay of mixed cultures grown directly from soil. When the bacteria occur at low levels, it is often necessary to have an enrichment step to detect the bacteria of interest. Traditionally this has Involved the use of selective media (Akiba and Katoh 1986, Bizzari and Bishop 2007, Andrzejczak and Lonc 2008). Alternatively, molecular techniques with specific probes for the toxin genes could be used, but these techniques have their own limitations when these bacteria exist as spores; molecular techniques often underestimate their abundance due to procedural difficultly in lysis of the spore stage (Ankolekar et al., 2009). In Maryland, at a site in the Frederick Municipal forest, B. thuringiensis has been sprayed repeatedly over several years for control of gypsy moth (R. Tichenor, Maryland Department of Agriculture pers. comm). In a previous study, we used toxicity to Manduca sexta L. as a model (Silva et al., 2002), to isolate lepidopteran pathogens from soil (Martin et al., 2008). In this study, we describe the use of toxicity to M. sexta as a means to determine the survival of a commercially applied toxic B. thuringiensis strain in a forest environment. moth, biological control, 16S rDNA gene sequences

*Corresponding author E-mail: Phyllis.martin@ars.usda.gov; Phone: 301-504-6331; Fax: 301-504-5725

286 Int. Res. J. Microbiol.

MATERIALS AND METHODS Bacterial strains and media Three B. thuringiensis strains isolated from commercial preparations were used as controls for toxicity and phenotypic properties. B. thuringiensis subsp. kurstaki, strain IBL 455, was originally isolated from a 1980 preparation of Dipel (Abbott Laboratories, Chicago, IL). Strain IBL 451 was isolated from a 2001 preparation of Foray (Novo Nordisk, Danbury, CT) which was applied to the Fishing Creek site in 2001. IBL 451 was used as a control for toxicity testing and for comparison to the bacteria recovered from the environment. Strain IBL 1410, B. thuringiensis subsp. tenebrionis, was isolated from a 1991 preparation of Novodur (Mycogen, San Diego, CA) and was used along with IBL 455 as a controls for phenotypic tests. IBL 10003, which was IPS 82, the B. thuringiensis subsp. israelensis international standard fermented in 1982 was used for comparisons of 16S rDNA gene sequences. Bacteria were grown on L-agar (Atlas, 2003) or RM ( L) for enumeration and recovery. If crystals were detected, bacteria were grown on T3 (Travers et al., 1987) for maximum crystal production for insect assays. Biochemical media were as described for Bacillus and modified for B. thuringiensis (Martin et al., 1985; Martin et al., 2010). Fourteen different media were used to test for various phenotypic traits including acid production from glucose, arabinose, xylose, mannitol, mannose, salicin and sucrose, utilization of citrate, hydrolysis of esculin, production of protease, amylase, urease, phosopholipase C, and hemolysin. Only those traits that differed among isolated strains are discussed. Additionally, eight antibiotic susceptibilities which we anticipated might be more variable than other phenotypic properties (ampicillin, vancomycin, chloramphenicol, triple sulfa, neomycin, tetracycline and kanamycin) were tested using disk diffusion (Remel, Lenexa, KS) on Lagar. Zones were measured after incubation at 30C for 24h and levels of sensitivity: sensitive, intermediate and resistant determined by the information supplied for each antibiotic. Soil samples Twenty soil samples were collected along Fishing Creek in the City of Frederick(MD) Municipal Forest in July 2004 and stored at ambient temperature in sterile plastic bags. Soil was suspended 1:10 wt: vol in sterile distilled water, shaken on a vortex mixer for 30 minutes, and plated for initial enumeration of bacteria. An undiluted sample of this soil suspension was spread on RM and incubated at 25C for 48 h, increasing the number of culturable bacteria available from each sample (Martin et al.,

2008). The bacteria harvested from these plates were fed to M. sexta larvae. Bacteria recovered from these soil suspensions harvested after growth were enumerated on RM. For comparison to this technique, bacteria isolated from other soil samples (614) that were previously taken in Maryland were compared to the samples taken along Fishing Creek. B. thuringiensis and other sporeformers were isolated from these samples using acetate selection (Travers et al., 1987). Phenotypes were tested as described (Martin et al., 2010). In 2009, 75 additional soil samples were taken from the same area for comparison of techniques. Bacteria from these soil samples were suspended in water, a 100 l sample heated for 3 min at 80C and plated on L-agar. Colonies were grown overnight, transferred to T3, incubated for 48-72 h at 30C and checked for spores and crystals using Nomarski optics. Insects and bioassays For bioassays, tobacco hornworm diet (THW diet) was used as re-hydrated freeze dried pellets (Martin 2004, Martin and Blackburn 2007). Manduca sexta L. eggs were received from J. Pennington (U. Arizona) and reared on strips of THW diet at 24 C, 46% RH, and 16:8 light: dark cycle until the 2nd instar. Diet was changed every 2-4 days. Sixteen diet pellets were used for each treatment in bioassays with one diet pellet/well (1.6 cm diameter x 1.6 cm deep) in white plastic bioassay trays (C-D International, Ocean City, NJ). Pellets were re-hydrated with 0.3 ml of water (controls) or suspensions containing extracts of soil or dilutions of bacteria. One tobacco hornworm, 2nd instar larva, was added to each pellet in individual wells. Wells were sealed with a clear plastic film and holes made in the film with insect pins for oxygen transfer. Larvae were incubated as for rearing. Mortality was recorded as early as 16, 24 and 48 h. Gypsy moths were received as egg masses from USDA/ARS, Otis Air National Guard Base (MA). Eggs were hatched and larvae reared to 2nd instar on a wheat germ-based diet (Bell et al. 1981) at 25 on a 16:8 h L:D C cycle without humidity control. For initial toxicity testing 0.3 ml of each dilution of each B. thuringiensis strain was added to gypsy moth diet on 16 freeze dried pellets. Mortality was recorded daily. LC50s were done on crystal forming strains against gypsy moths using the lepidopteran active IBL 455 and IBL 451 as standard strains for comparison. Recovery of bacteria from insects From each treatment with larval mortality, one dead larva

Martin et al. 287

was used to recover bacteria. Each dead larva was surface sterilized by dipping for 3 s in 10% bleach, rinsed in sterile distilled water, placed in a sterile plastic bag with five ml sterile distilled water, and ground in a stomacher blender (Techmar, Cincinnati, OH) for 60 s on high. The bacteria isolated from the larval extract were enumerated on RM. Plates were incubated at 25 for 48 h. C Colonies were enumerated and the predominant colony type was isolated and checked for the formation of spores and crystals by phase-contrast microscopy. These colonies were then characterized by their substrate utilization profiles and sensitivity to antibiotics. Statistical analysis Using protein concentration as dose, the LC50 was determined using probit analysis (SAS Inc. 2008) for each strain. Bacterial Identification and cry analysis In addition to characterization of spores and crystals by phase contrast microscopy and strains by substrate utilization, individual isolates were identified by PCR amplification and sequencing of conserved 16S rDNA genes. For each isolate, DNA was purified from a 2 ml culture grown only 8 hours. DNA was isolated using the Quantum Prep miniprep kit (BioRad, Hercules, CA) as specified by the manufacturer for use as a template in polymerase chain reaction (PCR). Nearly full length 16S rDNA was amplified for each isolate using primers universal to prokaryotes, R16F0 and R16R0 (Lee et al. 1993). Thirty-five PCR cycles were conducted in a model 9700 thermocycler (Applied Biosystems, Foster City, CA). The amplification primers and a nested universal primer 533F (5'-GTGCCAGCMGCCGCGGTAA-3') using 30 sec denaturation at 94C, 1.5-min annealing at 55C, and 2min primer extension (10-min in final cycle) at 72C. Bacterial 16S rRNA gene amplicons were sequenced directly. Products were separated on 1.5% NuSieve agarose gel (FMC, Rockland, ME) in modified-1X TAE (0.04 M Tris-acetate and 0.1 mM EDTA), and excised for sequencing using ABI BigDye V1.1 Automatic sequencing was carried out on an ABI Prism Model 3100 (Applied Biosystems, Foster City CA). Sequences were edited and assembled (DNASTAR, SeqMan component); BLAST (Altschul et al., 1990) searches were conducted to identify bacterial isolates based on rDNA sequence homology to known bacteria. Sequences thus obtained for 16S rDNA were deposited in GenBank with accession numbers EU168402- EU168418. Strains were also tested for cry1 gene using degenerate cry1 primers (Juarez-Perez et al., 1997).

RESULTS Bacteria grown from various soil samples and fed to M. sexta killed the larvae within 48 h. Larval mortality was 100% for 17 of 20 samples. While the initial bacterial concentration applied to diet pellets was high, the larvae did not noticeably consume any of the diet pellets before they died. The bacterial concentration averaged 1.11+/0.82 x108cfu (colony forming units)/ diet pellet and ranged from 9.9 x 106 to 3.0 x 108cfu/ diet pellet. The number of bacteria recovered from various larvae averaged 3.02 +/2.6 x 107 cfu/larva and ranged from 5.0 x105 to 8.2 x107cfu/larva. For most larvae only a single colony type was obtained. For others, one colony type predominated. The major colony type was isolated, characterized by phenotypic tests and fed to other M. sexta larvae. One sample (10) had two colony types of approximately the same numbers. One type made round spores that swelled the sporangia and the other type made oval spores and a bipyramidal crystal. Only the bacteria which had 100% mortality when refed made crystals. Bacteria (15 of 20) recovered from dead larvae from the second round were found to be identical in phenotype to the bacteria isolated initially. Once a pure culture was isolated it was assigned a strain designation. These strains were identified to species by 16S rDNA sequencing. Relatedness based on 16S rDNA genes Putative identities based on nearly full 16S rRNA genes showed that 14 strains belonged to the Bacillus cereus species complex (Figure 1). Three strains (IBL 1056, IBL 1067, IBL 1445) also formed bipyramidal crystals upon sporulation (from three different samples 10, 8, and 18) and were identified as B. thuringiensis. Another spore former, IBL 1077 (from sample 5), was identified as B. weihenstephanensis and grew at 4 C. A third spore forming strain (IBL10B1445), which was co- isolated with crystal forming IBL 1445, formed spherical to oval spores that swelled the sporangia and was identified as Lysinibacillus fusiformis (Ahmed et al., 2007). The relatedness of these strains based on their 16S rRNA genes are shown in Figure 1. The B. thuringiensis and the B. cereus strains clustered closely together differing by fewer than 5 bases in approximately a 1400 base sequence. Therefore a method for more precisely differentiating stains was needed. Relatedness based on phenotypes Phenotypes (substrate utilization and antibiotic resistance profiles) were chosen to distinguish the strains in the B.

288 Int. Res. J. Microbiol.

Figure1: Dendrogram showing the relationship of Bacillus strains isolated from dead larvae to known B. thuringiensis strains

cereus group because previous as well as ongoing research has shown that the closely related strains of B. thuringiensis can be differentiated using these characteristics (Martin and Travers 1989, Martin et al. 2010). The three B. thuringiensis strains recovered from M. sexta larvae differed from the strain that had been sprayed (IBL 451) by two (IBL 1445 - kanamycin and tetracycline sensitivity), four (IBL 1056 urease production, esculin hydrolysis; triple sulfa and kanamycin sensitivity;) or five (IBL 1067- acid production from salicin; vancomycin, triple sulfa, neomycin and tetracycline sensitivity) phenotypic traits (Table 1). The four most closely related B. cereus strains differed from the B. thuringiensis strain that had been sprayed by three (IBL 1050, 1058, 1075 and 1078) phenotypic traits other than crystal production (Table 1).

Relatedness based on cry genes and toxicity The initial mixture of B. thuringiensis strain IBL 1445 and L. fusiformis strain IBL 10B1445 was toxic when fed to M. sexta larvae. The L. fusiformis strain, however, was not toxic when fed separately, but the B. thuringiensis strain was toxic when fed separately. All the crystal forming strains had cry1 genes by PCR. One strain that did not form crystals, IBL 1050, also had a cry1 gene. The toxicity of recovered B. thuringiensis strains to gypsy moth was not significantly different than the strain that was applied (IBL 451). The LC50 for the applied strain (IBL 451) in pure culture was 40ng protein/diet pellet, 12ng protein/diet pellet for IBL 1067 and 38ng/diet pellet for IBL 1056. IBL 1050 which did not make a crystal killed 60% of the M. sexta larvae at a screening concentration

Martin et al. 289

Table 1: Phenotypes of Bacillus cereus group strains recovered (bold) applied (highlighted) and control strains

Soil no. 10 8 18 12 21 11 19 16 17 14 7 1 9 5 control control control

IBL no 451 1445 1067 1056 1050 1439 1075 1058 1078 1080 1054 1065 1048 1068 1077 10003 1410 455

T + + + + + + + + + + + + + + + + + +

L + + + + + + + + + + + + + + +

U + + + + + + + +

S + +

A + + + + + + + + + + +

E Am van cam ery 3S neo + R S S S S S + R S S S S S + R I S S I I - R S S S R S + R S S S S S - R S S I I I + R S S S S S + R S S S R S + R S S S I S + R S I S R I + R S S S I R - R S S S I I - R S S S I S + R S S S I I - R S S S S I R S R S S S S I S S S S S S I S S S

tet S I I S I S I S S S I S S R S S S R

kan Species R Bt I Bt R Bt S Bt I Bc S Bc I Bc S Bc S Bc I Bc I Bc I Bc S Bc S Bc I Bw I I I Bt Bt Bt

+ - - - - - + - + + - + +

Abbreviations used, T - amylase production on starch, L- Lecithinase or phospholipase C production, U- production of urease, S acid production from sucrose, A- acid production from salicin, E- hydrolysis of esculin. Antibiotics amp- 10 ug ampicillin, van, 30 ug vancomycin, cam 30 ug chloramphenicol, ery, 15 ug erythromycin, 3S 1 mg triple sulfa, neo 30 ug neomycin, tet, 30 ug tetracycline kan 30 ug kanamycin. Acid production from mannitol, arabinose and xylose was negative for all strains as was utilization of citrate. All strains were hemolytic, produced proteases and produced acid from glucose.

of 107cfu/ diet pellet. Comparison with the standard method

production from salicin and esculin hydrolyisis) phenotypes were recovered from areas previously known to be sprayed with B. thuringiensis. DISCUSSION

From the retrospective samples, taken from Maryland soils by acetate selection, 165 samples contained more than 10 B. thuringiensis isolates and 189 contained no B. thuringiensis isolates. Eleven isolates had the same phenotype as the strain applied (without antibiotic tests). From 75 soil samples collected in 2009, B. thuringiensis was recovered from 41 samples. Only 22 samples had spore forming bacteria which made bipyramidal crystals and only 13 of these from 4 samples had the same phenotypic profile (Martin et al. 2010) as the B. thuringiensis strain applied. Comparing all the isolates, 725 B. thuringiensis were isolated along with 304 spore forming bacteria that did not make crystals. The most common phenotypes are shown in Table 2. All but 3 of the 67 TLUAE (starch utilization, lecithinase and urease production, acid

Using a pest insect sensitive to microbial insecticides, the tobacco hornworm, three distinct crystal forming B. thuringiensis strains and 12 other strains in the B. cereus group were recovered from 15 different sample sites. These sites had been sprayed with a commercial formulation of B. thuringiensis five different times in the 22 years prior to our sampling (1983, 1986, 1989, 1995 and 2001; R. Tichenor, Maryland Department of Agriculture, pers. comm.). Two of the three crystal formers recovered from the application sites (IBL 1445 and IBL 1067) were highly similar to the applied B. thuringiensis var. kurstaki (Foray; IBL 451), by patterns of substrate utilization, toxicity, antibiotic susceptibility, and 16S rRNA gene sequence. The third, IBL 1056, differed in its antibiotic resistance profile and did not

290 Int. Res. J. Microbiol.

Table 2: Common phenotypes of spore forming bacteria isolated in Maryland

Phenotype TL TLU TLAE TLUAE* Null (0) Total of all phenotypes**

Bt 176 82 50 23 7 725

% total Bt 24.3 11.3 6.9 3.2 1.0

Other spore formers 111 31 16 7 69 304

% other spore formers 36.0 10.2 5.3 2.3 22.6

*Phenotype of the strain that was applied ** including rare phenotypes not shown

produce urease. These results indicate that an insect, such as the tobacco hornworm, can be used to select B. thuringiensis from mixtures of soil bacteria, and recover applied strains. Interestingly, a number of toxic B. cereus (non-crystal forming isolates) were isolated using our technique, and except for the lack of a parasporal crystal, a number of these had phenotypic profiles resembling the applied strain. Some of these toxic non-crystal forming isolates had 16S rRNA gene sequences identical to the applied strain and may represent descendants of the applied strain that have lost the ability to form a crystal. However, other strains had 16S rRNA gene sequence that differed slightly from Foray, indicating that they are not derived from the applied strain. Although these may have acquired their pathogenicity from the applied strain via conjugation (Grohmann et al., 2003), it seems more probable that they, and the other non-crystal forming isolates represent naturally occurring varieties of entomopathogenic B. cereus that have previously escaped detection with isolation methods based on selection of crystal forming isolates. We have recently demonstrated that urease production is an important trait that appears to improve replication of B. thuringiensis in gypsy moth larvae (Martin et al., 2009). The relatively high numbers of urease producers among our isolates (50%), compared with urease producers in our general collection (ca. 20%) may reflect differences in pathogenicity based selection used in this study and selection based on crystal formation. We plan to investigate these possibilities using multilocus sequence typing (Priest et al., 2004) to determine the phylogenetic relationships between these isolates. ACKNOWLEDGMENTS Thanks to L. Liska for rearing insects, and A. Mitchell and R. Farrar for general technical assistance. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States

Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

REFERENCES Akiba Y, Katoh K (1986). Microbial ecology of Bacillus thuringiensis V. Selective medium for Bacillus thuringiensis vegetative cells. Appl. Entomol. Zool. 21: 210215. Ahmed I, Yokota A, Yamazoe A, Fujiwara T (2007). Proposal of Lysinibacillus boroitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Intern. J. Syst. Evol. Microbiol. 57:117-1125. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic local alignment search tool. J. Mol. Biol. 215: 403-410. Andrzejczak S, Lonc E (2008). Selective isolation of Bacillus thuringiensis from soil by the use of L-serine as a minimal media supplement. Pol. J. Microbiol. 57:333-335. APHIS factsheet (2003). Gypsy moth: slow the spread. http://www.docstoc.com/docs/5427202/APHIS-Plant-Protection-andQuarantine-Factsheet-April Gypsy-Moth. Accessed February 4, 2010. Atlas RM (2004). Handbook of microbiological media. Third edition. CRC Press, Inc. Boca Raton, FL. Pp 904. Bell RA, Owens CD, Shapiro M, Tardif JGR (1981). Development of mass rearing technology, in The gypsy moth: Research toward integrated pest management (Doane CC, McManus ML, Eds.) U.S. Department of Agriculture Technical Bulletin 1584, Washington, pp 599-633. Bizzarri MF, AH Bishop (2007). Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season. J. Invertebr. Pathol. 94: 38-47. Grohmann E, Muth G, Espinosa M (2003). Conjugative plasmid transfer in Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67: 277-301. Juarez-Perez VM, Ferrandis MD, Frutos R (1997). PCR-based approach for detection of novel Bacillus thuringiensis cry genes. Appl. Environ. Microbiol. 63:29973002. Lee IM, Hammond RW, Davis RE, Gundersen DE (1993). Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma like organisms. Mol. Plant Pathol. 83: 834-842. Martin PAW, Haransky EB, Travers RS, Reichelderfer CF (1985). Rapid biochemical testing of large numbers of Bacillus thuringiensis isolates using agar dots. BioTechniques 3:386-392. Martin PAW, Travers RS (1989). Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55: 24372442. Martin PAW (2004). A freeze-dried diet to test pathogens of Colorado

Martin et al. 291

potato beetle. Biol. Cont. 29: 09-14. Martin PAW, Blackburn MB (2007). Using combinatorics to screen Bacillus thuringiensis isolates for toxicity against Manduca sexta and Plutella xylostella. Biol. Cont. 42:226-232. Martin PAW, Mongeon EA, Gundersen-Rindal DE (2008). Microbial combinatorics: a simplified approach to isolating insecticidal bacteria. Biocont. Sci. Technol. 18: 291-305. Martin PAW, Farrar Jr RR, Blackburn MB (2009). Survival of diverse Bacillus thuringiensis strains in gypsy moth (Lepidoptera: Lymantriidae) is correlated with urease production. Biol. Cont. 51:147-151. Martin PAW, Gundersen-Rindal DE, Blackburn MB (2010). Distribution of phenotypes among Bacillus thuringiensis strains. Syst. Appl. Microbiol. 33:204-208. Ohba M, Aizawa K (1986). Insect toxicity of Bacillus thuringiensis isolated from soils of Japan. J. Invertebr. Pathol. 47:12-20.

Priest FG, Barker M, Baillie LWJ, Holmes EC, Maiden MCJ (2004). Population structure and evolution of the Bacillus cereus group J. Bacteriol. 186: 79597970. SAS Institute Inc. (2008). SAS OnlineDoc7. Version 9.1. SAS Institute Inc. Cary, NC. Sharov AA, Leonard D, Liebhold AM, Clemens NS (2002). Evaluation of preventive treatments in low-density gypsy moth populations using pheromone traps. J. Econ. Entomol. 95: 1205-1215. Silva CP, Waterfield NR, J Daborn P, Dean P, Chilver T, Au CPY, Sharma S, Potter U, Reynolds SE, ffrench-Constant RH (2002). Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cell Microbiol. 4: 329-339. Travers RS, Martin PAW, Reichelderfer CF (1987). Selective process for efficient isolation of soil Bacillus spp. Appl. Environ. Microbiol. 53:1263-1266