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International Research Journal of Microbiology (IRJM) (ISSN: 2141-5463) Vol. 2(8) pp. 285-291, September 2011Available online http://www.interesjournals.org/IRJMCopyright 2011 International Research Journals

Full Length Research Paper

Recovery of Bacillus thuringiensisfrom forest soil afterapplication 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.thuringiensisfor gypsy moth control by amplifying the bacteria found in the soil on bacterial agar andfeeding this mixed microbial population to tobacco hornworm larvae. These bacteria killed the larvaewhen fed on artificial diet. Most of the bacteria recovered from dead insects formed spores andbelonged to the Bacillus cereus group (14) including three B. thuringiensis and one B.weihenstephanensis. All the B. thuringiensisstrains and most of the B. cereus strains were closelyrelated (


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286 Int. Res. J. Microbiol.

MATERIALS AND METHODS

Bacterial strains and media

Three B. thuringiensis strains isolated from commercialpreparations were used as controls for toxicity andphenotypic properties. B. thuringiensis subsp. kurstaki,strain IBL 455, was originally isolated from a 1980preparation of Dipel (Abbott Laboratories, Chicago, IL).Strain IBL 451 was isolated from a 2001 preparation ofForay (Novo Nordisk, Danbury, CT) which was appliedto the Fishing Creek site in 2001. IBL 451 was used as acontrol for toxicity testing and for comparison to thebacteria recovered from the environment. Strain IBL1410, B. thuringiensis subsp. tenebrionis, was isolatedfrom a 1991 preparation of Novodur (Mycogen, SanDiego, CA) and was used along with IBL 455 as acontrols for phenotypic tests. IBL 10003, which was IPS

82, the B. thuringiensis subsp. israelensis internationalstandard fermented in 1982 was used for comparisons of16S rDNA gene sequences.

Bacteria were grown on L-agar (Atlas, 2003) or RM (L) for enumeration and recovery. If crystals weredetected, bacteria were grown on T3 (Travers et al.,1987) for maximum crystal production for insect assays.Biochemical media were as described for Bacillus andmodified for B. thuringiensis (Martin et al., 1985; Martin etal., 2010). Fourteen different media were used to test forvarious phenotypic traits including acid production fromglucose, arabinose, xylose, mannitol, mannose, salicinand sucrose, utilization of citrate, hydrolysis of esculin,

production of protease, amylase, urease, phosopholipaseC, and hemolysin. Only those traits that differed amongisolated strains are discussed.

Additionally, eight antibiotic susceptibilities which weanticipated might be more variable than other phenotypicproperties (ampicillin, vancomycin, chloramphenicol,triple sulfa, neomycin, tetracycline and kanamycin) weretested using disk diffusion (Remel, Lenexa, KS) on L-agar. Zones were measured after incubation at 30C for24h and levels of sensitivity: sensitive, intermediate andresistant determined by the information supplied for eachantibiotic.

Soil samples

Twenty soil samples were collected along Fishing Creekin the City of Frederick(MD) Municipal Forest in July 2004and 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 forinitial enumeration of bacteria. An undiluted sample ofthis soil suspension was spread on RM and incubated at25C for 48 h, increasing the number of culturablebacteria available from each sample (Martin et al.,

2008). The bacteria harvested from these plates were fedto M. sexta larvae. Bacteria recovered from these soisuspensions harvested after growth were enumerated onRM.

For comparison to this technique, bacteria isolated fromother soil samples (614) that were previously taken inMaryland were compared to the samples taken alongFishing Creek. B. thuringiensis and other sporeformerswere isolated from these samples using acetate selection(Travers et al., 1987). Phenotypes were tested asdescribed (Martin et al., 2010). In 2009, 75 additional soisamples were taken from the same area for comparisonof techniques. Bacteria from these soil samples weresuspended in water, a 100 l sample heated for 3 min at80C and plated on L-agar. Colonies were grownovernight, transferred to T3, incubated for 48-72 h at30C and checked for spores and crystals usingNomarski optics.

Insects and bioassays

For bioassays, tobacco hornworm diet (THW diet) wasused as re-hydrated freeze dried pellets (Martin 2004Martin and Blackburn 2007). Manduca sexta L. eggswere received from J. Pennington (U. Arizona) andreared on strips of THW diet at 24 C, 46% RH, and 16:8light: dark cycle until the 2

nd 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-DInternational, Ocean City, NJ). Pellets were re-hydratedwith 0.3 ml of water (controls) or suspensions containingextracts of soil or dilutions of bacteria. One tobaccohornworm, 2

nd instar larva, was added to each pellet in

individual wells. Wells were sealed with a clear plasticfilm and holes made in the film with insect pins for oxygentransfer. Larvae were incubated as for rearing. Mortalitywas recorded as early as 16, 24 and 48 h.

Gypsy moths were received as egg masses fromUSDA/ARS, Otis Air National Guard Base (MA). Eggswere hatched and larvae reared to 2

nd instar on a whea

germ-based diet (Bell et al.1981) at 25C on a 16:8 h L:D

cycle without humidity control.For initial toxicity testing 0.3 ml of each dilution of eachB. thuringiensis strain was added to gypsy moth diet on16 freeze dried pellets. Mortality was recorded dailyLC50s were done on crystal forming strains against gypsymoths using the lepidopteran active IBL 455 and IBL 451as standard strains for comparison.

Recovery of bacteria from insects

From each treatment with larval mortality, one dead larva


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was used to recover bacteria. Each dead larva wassurface sterilized by dipping for 3 s in 10% bleach, rinsedin sterile distilled water, placed in a sterile plastic bag withfive ml sterile distilled water, and ground in a stomacher

blender (Techmar, Cincinnati, OH) for 60 s on high. Thebacteria isolated from the larval extract were enumeratedon RM. Plates were incubated at 25C for 48 h.Colonies were enumerated and the predominant colonytype was isolated and checked for the formation ofspores and crystals by phase-contrast microscopy. Thesecolonies were then characterized by their substrateutilization profiles and sensitivity to antibiotics.

Statistical analysis

Using protein concentration as dose, the LC50 wasdetermined using probit analysis (SAS Inc. 2008) for

each strain.

Bacterial Identification and cry analysis

In addition to characterization of spores and crystals byphase contrast microscopy and strains by substrateutilization, individual isolates were identified by PCRamplification and sequencing of conserved 16S rDNAgenes. For each isolate, DNA was purified from a 2 mlculture grown only 8 hours. DNA was isolated using theQuantum Prep miniprep kit (BioRad, Hercules, CA) asspecified by the manufacturer for use as a template in

polymerase chain reaction (PCR). Nearly full length 16SrDNA was amplified for each isolate using primersuniversal to prokaryotes, R16F0 and R16R0 (Lee et al.1993). Thirty-five PCR cycles were conducted in a model9700 thermocycler (Applied Biosystems, Foster City, CA).The amplification primers and a nested universal primer533F (5'-GTGCCAGCMGCCGCGGTAA-3') using 30 secdenaturation at 94C, 1.5-min annealing at 55C, and 2-min primer extension (10-min in final cycle) at 72C.Bacterial 16S rRNA gene amplicons were sequenceddirectly. Products were separated on 1.5% NuSieveagarose 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 Automaticsequencing was carried out on an ABI Prism Model 3100(Applied Biosystems, Foster City CA). Sequences wereedited and assembled (DNASTAR, SeqMan component);BLAST (Altschul et al., 1990) searches were conductedto identify bacterial isolates based on rDNA sequencehomology to known bacteria. Sequences thus obtainedfor 16S rDNA were deposited in GenBank withaccession numbers EU168402- EU168418. Strains werealso tested for cry1gene using degenerate cry1 primers(Juarez-Perez et al., 1997).

Martin et al. 287

RESULTS

Bacteria grown from various soil samples and fed to Msexta killed the larvae within 48 h. Larval mortality was

100% for 17 of 20 samples. While the initial bacteriaconcentration applied to diet pellets was high, the larvaedid not noticeably consume any of the diet pellets beforethey died. The bacterial concentration averaged 1.11+/0.82 x10

8cfu (colony forming units)/ diet pellet and ranged

from 9.9 x 106to 3.0 x 10

8cfu/ diet pellet. The number o

bacteria recovered from various larvae averaged 3.02 +/2.6 x 10

7cfu/larva and ranged from 5.0 x10

5 to 8.2

x107cfu/larva. For most larvae only a single colony type

was obtained. For others, one colony type predominatedThe major colony type was isolated, characterized byphenotypic tests and fed to other M. sexta larvae. Onesample (10) had two colony types of approximately thesame numbers. One type made round spores tha

swelled the sporangia and the other type made ovaspores and a bipyramidal crystal. Only the bacteriawhich had 100% mortality when refed made crystalsBacteria (15 of 20) recovered from dead larvae from thesecond round were found to be identical in phenotype tothe bacteria isolated initially. Once a pure culture wasisolated it was assigned a strain designation. Thesestrains were identified to species by 16S rDNAsequencing.

Relatedness based on 16S rDNA genes

Putative identities based on nearly full 16S rRNA genesshowed that 14 strains belonged to the Bacillus cereusspecies complex (Figure 1). Three strains (IBL 1056, IBL1067, IBL 1445) also formed bipyramidal crystals uponsporulation (from three different samples 10, 8, and 18)and were identified as B. thuringiensis. Another sporeformer, IBL 1077 (from sample 5), was identified as Bweihenstephanensis and grew at 4 C. A third sporeforming strain (IBL10B1445), which was co- isolated withcrystal forming IBL 1445, formed spherical to oval sporesthat swelled the sporangia and was identified asLysinibacillus fusiformis (Ahmed et al., 2007).

The relatedness of these strains based on their 16S

rRNA genes are shown in Figure 1. The B. thuringiensisand the B. cereus strains clustered closely togethediffering by fewer than 5 bases in approximately a 1400base sequence. Therefore a method for more preciselydifferentiating stains was needed.

Relatedness based on phenotypes

Phenotypes (substrate utilization and antibiotic resistanceprofiles) were chosen to distinguish the strains in the B.


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

cereus group because previous as well as ongoingresearch has shown that the closely related strains of B.thuringiensis can be differentiated using thesecharacteristics (Martin and Travers 1989, Martin et al.

2010). The three B. thuringiensisstrains recovered fromM. sexta larvae differed from the strain that had beensprayed (IBL 451) by two (IBL 1445 - kanamycin andtetracycline sensitivity), four (IBL 1056 ureaseproduction, esculin hydrolysis; triple sulfa and kanamycinsensitivity;) or five (IBL 1067- acid production fromsalicin; vancomycin, triple sulfa, neomycin andtetracycline sensitivity) phenotypic traits (Table 1). Thefour most closely related B. cereusstrains differed fromthe B. thuringiensisstrain that had been sprayed by three(IBL 1050, 1058, 1075 and 1078) phenotypic traits otherthan crystal production (Table 1).

Relatedness based on cry genes and toxicity

The initial mixture of B. thuringiensisstrain IBL 1445 andL. fusiformisstrain IBL 10B1445 was toxic when fed to M

sexta larvae. The L. fusiformis strain, however, was notoxic when fed separately, but the B. thuringiensis strainwas toxic when fed separately. All the crystal formingstrains had cry1 genes by PCR. One strain that did noform crystals, IBL 1050, also had acry1gene.

The toxicity of recovered B. thuringiensis strains togypsy moth was not significantly different than the strainthat was applied (IBL 451). The LC50for the applied strain(IBL 451) in pure culture was 40ng protein/diet pellet12ng protein/diet pellet for IBL 1067 and 38ng/diet pelletfor IBL 1056. IBL 1050 which did not make a crystal killed60% of the M. sextalarvae at a screening concentration


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Martin et al. 289

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

Soil no. IBL no T L U S A E Am van cam ery 3S neo tet kan Species

451 + + + - + + R S S S S S S R Bt

10 1445 + + + - + + R S S S S S I I Bt8 1067 + + + - - + R I S S I I I R Bt

18 1056 + + - - + - R S S S R S S S Bt

12 1050 + + - - + + R S S S S S I I Bc

21 1439 + + + - - - R S S I I I S S Bc

11 1075 + + - - + + R S S S S S I I Bc

19 1058 + + - - + + R S S S R S S S Bc

16 1078 + + - - + + R S S S I S S S Bc

17 1080 + + - - + + R S I S R I S I Bc

14 1054 + + + - + + R S S S I R I I Bc

7 1065 + + - + + - R S S S I I S I Bc

1 1048 + + + - - - R S S S I S S S Bc

9 1068 + + + - - + R S S S I I R S Bc

5 1077 + + + + + - R S S S S I S I Bw

control 10003 + + - - - - R S S S S S S I Bt

control 1410 + - - + - - S S I S S I S I Bt

control 455 + + + - + + R S S S S S R I Bt

Abbreviations used, T - amylase production on starch, L- Lecithinase or phospholipase C production, U- productionof urease, S acid production from sucrose, A- acid production from salicin, E- hydrolysis of esculin. Antibioticsamp- 10 ug ampicillin, van, 30 ug vancomycin, cam 30 ug chloramphenicol, ery, 15 ug erythromycin, 3S 1 mg triplesulfa, neo 30 ug neomycin, tet, 30 ug tetracycline kan 30 ug kanamycin. Acid production from mannitol, arabinoseand 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

From the retrospective samples, taken from Marylandsoils by acetate selection, 165 samples contained morethan 10 B. thuringiensisisolates and 189 contained no B.thuringiensis isolates. Eleven isolates had the samephenotype as the strain applied (without antibiotic tests).From 75 soil samples collected in 2009, B. thuringiensiswas recovered from 41 samples. Only 22 samples hadspore forming bacteria which made bipyramidal crystalsand only 13 of these from 4 samples had the samephenotypic profile (Martin et al. 2010) as the B.thuringiensisstrain applied.

Comparing all the isolates, 725 B. thuringiensis wereisolated along with 304 spore forming bacteria that didnot make crystals. The most common phenotypes areshown in Table 2. All but 3 of the 67 TLUAE (starchutilization, lecithinase and urease production, acid

production from salicin and esculin hydrolyisisphenotypes were recovered from areas previously knownto be sprayed with B. thuringiensis.

DISCUSSION

Using a pest insect sensitive to microbial insecticides, thetobacco hornworm, three distinct crystal forming Bthuringiensisstrains and 12 other strains in the B. cereusgroup were recovered from 15 different sample sitesThese sites had been sprayed with a commerciaformulation of B. thuringiensis five different times in the22 years prior to our sampling (1983, 1986, 1989, 1995and 2001; R. Tichenor, Maryland Department oAgriculture, pers. comm.). Two of the three crystaformers recovered from the application sites (IBL 1445and IBL 1067) were highly similar to the applied Bthuringiensisvar. kurstaki (Foray; IBL 451), by patterns osubstrate utilization, toxicity, antibiotic susceptibility, and16S rRNA gene sequence. The third, IBL 1056, differedin its antibiotic resistance profile and did no


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

Phenotype Bt % total Bt Other spore formers % other spore formers

TL 176 24.3 111 36.0

TLU 82 11.3 31 10.2

TLAE 50 6.9 16 5.3

TLUAE* 23 3.2 7 2.3

Null (0) 7 1.0 69 22.6

Total of all phenotypes** 725 304

*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 recoverapplied strains.Interestingly, a number of toxic B. cereus (non-crystal

forming isolates) were isolated using our technique, andexcept for the lack of a parasporal crystal, a number ofthese had phenotypic profiles resembling the appliedstrain. Some of these toxic non-crystal forming isolateshad 16S rRNA gene sequences identical to the appliedstrain and may represent descendants of the appliedstrain that have lost the ability to form a crystal. However,other strains had 16S rRNA gene sequence that differedslightly from Foray, indicating that they are not derivedfrom the applied strain. Although these may haveacquired their pathogenicity from the applied strain via

conjugation (Grohmann et al., 2003), it seems moreprobable that they, and the other non-crystal formingisolates represent naturally occurring varieties ofentomopathogenic B. cereus that have previouslyescaped detection with isolation methods based onselection of crystal forming isolates.

We have recently demonstrated that urease productionis an important trait that appears to improve replication ofB. thuringiensisin gypsy moth larvae (Martin et al., 2009).The relatively high numbers of urease producers amongour isolates (50%), compared with urease producers inour general collection (ca. 20%) may reflect differences inpathogenicity based selection used in this study and

selection based on crystal formation. We plan toinvestigate these possibilities using multilocus sequencetyping (Priest et al., 2004) to determine the phylogeneticrelationships between these isolates.

ACKNOWLEDGMENTS

Thanks to L. Liska for rearing insects, and A. Mitchell andR. Farrar for general technical assistance. Mention of atrademark or proprietary product does not constitute aguarantee or warranty of the product by the United States

Department of Agriculture and does not imply its approvalto the exclusion of other products that may also be

suitable.

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