Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny

Available online at www.sciencedirect.com Journal of

INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 97 (2008) 171–181 www.elsevier.com/locate/yjipa

Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny Douglas W. Dingman

*

Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, 123 Huntington Street, P.O. Box 1106, New Haven, CT 06504, USA Received 5 July 2007; accepted 6 September 2007 Available online 14 September 2007

Abstract A phylogenetic grouping of 48 different isolates of milky disease bacteria isolated in the United States was determined using genomic RFLP analysis and 16S rDNA sequence comparison. A clear distinction between Paenibacillus popilliae isolates and Paenibacillus lentimorbus isolates was evident from the results of each procedure. The P. popilliae isolates segregated into two phylogenetic groups and the P. lentimorbus isolates segregated into three phylogenetic groups. In the United States, P. popilliae group 1 was generally isolated from insects collected west of the Appalachian Mountains. P. popilliae group 2 was only isolated from insects collected east of the Appalachian Mountains. P. lentimorbus groups 1 and 2 were obtained from insects collected west and south of the Appalachians. P. lentimorbus group 3 was identified in insects collected east of the mountains. From five different locations in Connecticut, 12 milky disease bacterial isolates were classified as P. popilliae and three were classified as P. lentimorbus. Except for one isolate, all P. popilliae isolates were of phylogenetic group 2. The three P. lentimorbus strains were isolated from diseased insects that had been collected from a localized area in the state. These three strains formed a separate phylogenetic grouping (i.e., group 3) of P. lentimorbus and, based on 16S rDNA sequence comparisons, were most similar to the newly identified P. lentimorbus Semadara strain recently isolated in Japan. All milky disease bacteria that had been isolated from commercially available insecticide preparations were identified as P. popilliae group 1.  2007 Elsevier Inc. All rights reserved. Keywords: Paenibacillus; Milky spore disease; Scarab beetle larvae; Bacterial phylogeny; Bacterial ecology

1. Introduction Paenibacillus popilliae and Paenibacillus lentimorbus are pathogens of Japanese beetle (Popillia japonica Newman) larvae, and larvae of several other related scarab beetles (i.e., ‘‘white grubs’’), causing ‘‘Milky Spore Disease’’ (Dutky, 1963). Paenibacillus popilliae and P. lentimorbus are very closely related microorganisms and considerable debate on taxonomic separation has occurred for decades. The initial work on milky disease bacteria (then called Bacillus popilliae and Bacillus lentimorbus) performed by S. R. Dutky led him to define two separate species based on characteristics of the diseased insect and bacterial cell

*

Fax: +1 203 974 8502. E-mail address: [email protected]

0022-2011/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.09.002

morphology (Dutky, 1940). The production of a parasporal body by P. popilliae, but not by P. lentimorbus, prompted Gordon et al. (1973) and Krieg (1961) to distinguish these bacteria as separate species. However, serological studies by Krywienczyk and Lu¨thy (1974), physiological and morphological characterizations by Wyss (1971), and studies by Milner (1981) on the spore/parasporal body size and the cellular position in the sporangium have led them to purpose a single species with two varieties. Rippere et al. (1998) reported P. popilliae and P. lentimorbus to be separate species based on DNA–DNA similarity tests and randomly amplified polymorphic DNA (RAPD) analysis of genomic DNA. Harrison et al. (2000), using milky disease bacteria isolated in Central and South America, also report segregation between P. popilliae and P. lentimorbus isolates. These RAPD analyses have resulted in the construction of phylogenetic trees for

172

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

milky disease bacteria. Currently, two sub-groupings within each of the two species are reported. Neither of the two investigations identified a correlation between host and pathogen nor geographic distribution of the pathogen based on phylogeny. However, Central and South American isolates were identified as being phylogenically distinct from isolates obtained in North America. MacDonald and Kalmakoff (1995), using pulsed field gel electrophoresis (PFGE) for comparative analysis of genomic DNA fingerprints, have demonstrated relatedness among six P. popilliae strains based on geographical origins in New Zealand. Correa and Yousten (2001) have used PFGE for DNA fingerprinting of milky disease bacteria to investigate strain relatedness and the usefulness of PFGE for ecological and specificity studies. No investigation correlating phylogenic groupings of milky disease bacteria and geographic distribution has been reported. An understanding of milky disease bacterial phylogeny in relation to the ecology (i.e., host-specificity and environmental segregation) of milky disease bacteria will help efforts to utilize these bacteria as effective biocontrol agents for white grubs. I present here further information on phylogenic relationships between P. popilliae and P. lentimorbus and identify a third subgroup for P. lentimorbus. Additionally, I report an association between the phylogenic grouping and geographic origin of milky disease bacteria within the United States (USA). From these findings, a factor relating to efficacy problems encountered with commercial insecticides containing milky disease bacteria is suggested. 2. Materials and methods 2.1. Bacterial strains and growth media Paenibacillus popilliae and P. lentimorbus strains used in this study, and informational profiles specific to each strain, are listed in Table 1. Bacteria were grown on MYPGP agar plates or in MYPGP broth (Dingman and Stahly, 1983) at 30 C with aeration. Chemical supplies were obtained from Fisher Scientific (Pittsburgh, PA) and Sigma Chemical Co. (St. Louis, MO).

phoresis was conducted at 5 V/cm, and DNA bands were visualized by UV illumination at 365 nm. 2.3. PCR amplification methods Paenibacillus popilliae and P. lentimorbus 16S rDNA was PCR amplified from purified genomic DNA using the oligonucleotide primers RNA31F (5 0 -GCGCAAGCTTAGA GTTTGATCCTGGCTCAGGACG-3 0 ) and RNA1484R (5 0 -GCGGATCCTACCTTGTTACGACTTCACCCCA-3 0 ) (Lane et al., 1985; Pettersson et al., 1999). TITANIUM Taq DNA polymerase kit (BD Biosciences/Clontech, Palo Alto, CA) was used for PCR amplification. Each PCR mixture (50 ll), made according to the manufacturer’s instructions, contained 100 lM (each) deoxynucleotide triphosphate plus 0.8 lM of each primer. The PCR amplification cycle protocol was as follows: 5 min at 94 C, then 25 cycles of 1 min denaturation at 94 C, 1.5 min of annealing at 58 C, and 1.5 min elongation at 72 C, followed by a final elongation of 7 min at 72 C. Agarose gel electrophoresis of PCR products was done in the presence of a molecular size ladder standard and a low mass ladder standard (Invitrogen, Carlsbad, CA) to ensure proper amplification and to determine DNA concentration. Amplified PCR samples were cleaned using the Qiaquick PCR purification kit (Qiagen Inc., Santa Clarita, CA) prior to sequence analysis. 2.4. DNA sequencing PCR amplified 16S rDNA was custom sequenced by the DNA sequencing group of the Keck Biotechnology/HHMI Biopolymer Laboratory at Yale University (New Haven, CT) using Applied Biosystems 377 gel DNA Sequencers and a capillary ABI 3700 DNA Analyzer. Primer DNA (2 ll of a 4 lM solution) and template DNA (20 ng template for every 200 bases of PCR fragment length) were mixed to an 18 ll total volume in premixed reactions for sequencing. The oligonucleotide primers RNA31F and RNA1484R were used for bi-directional determinations of all DNA sequences. Raw sequence data were aligned using ChromasPro version 1.33 software (Technelysium Pty Ltd., Tewantin, QLD 4565, Australia) for production of corrected 16S rDNA sequences.

2.2. DNA extraction and agarose gel electrophoresis procedures

2.5. Restriction fragment length polymorphism (RFLP) analysis of bacterial genomic DNA

Genomic DNA was isolated from bacteria that had been grown overnight in broth culture (1.5 ml). The QIAamp Tissue kit (Qiagen Inc., Santa Clarita, CA) was used according to the manufacturer’s protocol to extract DNA. Final suspension of genomic DNA was in 400 ll of the QIAamp AE buffer. Agarose gel electrophoresis was performed as described by Sambrook et al. (1989) using agarose gels (1%) prepared in TAE electrophoresis buffer (40 mM Tris–acetate, pH 7.5–7.8; 1 mM EDTA, pH 8.0) containing ethidium bromide (1 lg/ml). Electro-

Pulsed field gel electrophoresis (PFGE) of I-CeuI digested genomic DNA was used to produce RFLP fingerprint patterns for all bacterial strains investigated. PFGE was performed according to the procedure described by Gautom (1997). SeaKem Gold agarose (Cambrex BioScience Rockland Inc., Rockland, ME) was used to imbed bacterial cells and mutanolysin (20 U; Sigma Chemical Co., St. Louis, MO) was used for disruption of the bacterial cells for production of imbedded genomic DNA. A 1 mm slice of agarose containing imbedded genomic

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

173

Table 1 P. popilliae and P. lentimorbus strains studied Isolate

Host insecta

Geographic origin (Date)

Isolation/source (reference)d

P. popilliae Group 1 NRRL B-2309 NRRL B-3391 NRRL B-2519 NRRL B-2527 NRRL B-2529 NRRL B-4223 Dutky 1S BL Pj1 KLN 1 Pj3 Pj4 DNG 12 Pj1 Pj2-1 Pj2-2 Pj2-3 Pj5

European chaferb Japanese Beetle Japanese Beetle Japanese Beetle Japanese Beetle Unknown Unknown Japanese Beetle Japanese Beetle Japanese Beetle Japanese Beetle Oriental Beetle Insecticide Insecticide Insecticide Insecticide Insecticide

Newark, NY (1951); DeBryne derivative of NRRL B-2309 S. Dutky slide III-2 (circa 1940) S. Dutky slide III-6 (circa 1940) S. Dutky slide III-4 (circa 1940) Ohio Unknown (1979) Akron, OH. (1975) Wooster, OH (1987) Wooster, OH (1990) Moorestown, NJ (1944) Groton, CT (1989) Milky Spore(Reuter Lab., 1980) Grub Attack (Reuter Lab., 1988) Grub Attack (Reuter Lab., 1988) Grub Attack (Reuter Lab., 1988) Doom (Fairfax Biol. Lab., 1983)

H. Tashiro/D. Stahly W. Haynes/L. Nakamura W. Haynes/L. Nakamura W. Haynes/L. Nakamura W. Haynes/L. Nakamura G. Sharpe/A. Rooney C. Fields/D. Stahly M. Klein and D. Stahly D. Dingman/M. Klein M. Klein and D. Stahly M. Klein/D. Stahly D. Dingman/J. Hanula D. Stahly M. Klein and D. Stahly M. Klein and D. Stahly M. Klein and D. Stahly M. Klein and D. Stahly

P. popilliae Group 2 NRRL B-2524 NRRL B-4265 DNG 1 DNG 8 DNG 18 DNG 4 DNG 5 DNG 11 AGB 1 DNG 9 DNG 2 DNG 19 DNG 20 KLN 3 KLN 4 BpPa Vmr

Japanese Beetle Chemostat isolate Oriental Beetle Japanese Beetle Japanese Beetle Oriental Beetle Japanese Beetle Oriental Beetle Asiatic Garden Beetle Japanese Beetle Japanese Beetle Japanese Beetle Japanese Beetle Japanese Beetle Anomala flavipennis June Beetle

H. Tashiro feeding expt. (1955) derivative of NRRL B-2309 Norwalk, CT (1987) Norwalk, CT (1987) Norwalk, CT (1991) Groton, CT (1987) Groton, CT (1987) Groton, CT Groton, CT (1990) Danbury, CT (1989) Windsor, CT (1987) Windsor, CT (1987) Windsor, CT (1987) Moorestown, NJ (1945) Wilmington, NC (1986) Todd, NC (1984)

W. Haynes/D. Stahly E. Sharpe/A. Rooney D. Dingman/J. Hanula D. Dingman/J. Hanula D. Dingman (this study) D. Dingman/J. Hanula D. Dingman/J. Hanula D. Dingman/J. Hanula D. Dingman (this study) D. Dingman/J. Hanula D. Dingman/J. Hanula D. Dingman (this study) D. Dingman (this study) D. Dingman/M. Klein D. Dingman/M. Klein M. Klein and D. Stahly

P. lentimorbus Group 1 NRRL B-2522 NRRL B-2521 KLN 2

Japanese Beetle Japanese Beetle Japanese Beetle

S. Dutky slide II-15 (circa 1940) S. Dutky slide II-9 (circa 1940) Akron, OH (1975)

W. Haynes/L. Nakamura W. Haynes/L. Nakamura D. Dingman/M. Klein

P. lentimorbus Group 2 Pa1 Cp1 DGB 1 Cb1 Cb2 DNG 14 DNG 15 DNG16 DNG 17

June Beetle Cyclocephala parallela Cyclocephala parallela N. Masked Chafer N. Masked Chafer N&S masked Chafer N&S masked Chafer Green June Beetle Phyllophaga species

Todd, NC (1984) Belle Glade, FL (1990) South Florida (1986) Ohio (1983) Ohio (1985) Columbia, MO (2002) Columbia, MO (2003) Columbia, MO (2003) Brunswick, MO (2004)

M. Klein and D. Stahly M. Klein and D. Stahly D. Dingman/D. Boucias M. Klein and D. Stahly M. Klein and D. Stahly D. Dingman/B. Puttler (this D. Dingman/B. Puttler (this D. Dingman/B. Puttler (this D. Dingman/B. Puttler (this

feeding expt., CT (1991) Hamden, CT (1990) Hamden, CT (1994)

D. Dingman D. Dingman D. Dingman (this study)

P. lentimorbus Group 3 (Semadara-like) E-10 Oriental Beetlec DNG10 Japanese Beetle DNG 21 Oriental Beetle a

study) study) study) study)

European chafer, Rhizotrogus majalis; Japanese beetle, Popillia japonica; Oriental beetle, Anomala orientalis; Asiatic garden beetle, Maladerea castanea; June beetle, Phyllophaga anxia; Northern masked chafer, Cyclocephala borealis; Southern masked chafer, Cyclocephala lurida. b The host origin of this strain is uncertain. The DeBryne strain was reportedly obtained from a European chafer exposed to spores produced from type A regular and anxia strains. See references; Tashiro and White (1954), Haynes et al. (1961) and Tashiro and Steinkraus (1966). c Spore feeding experiment using Oriental beetle larvae and Brevibacillus laterosporus DNG 6. d The original strain citations occur in the following references; Tashiro and White (1954), Tashiro (1957), Haynes et al. (1961), Gordon et al. (1973), Sharpe and Bulla (1978), Stahly et al. (1992), and Dingman (1994b).

174

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

DNA was digested with 2.5 U of I-CeuI for 3.5 h at 37 C in 100 ll of restriction digestion buffer. The slice was then added to a 1% agarose gel (SeaKem Gold Agarose) in 0.5· TBE buffer (45 mM Tris–base, 45 mM boric acid, 1.0 mM EDTA, pH 8.3; circulated at 14 C) and subjected to electrophoresis using a CHEF-DR II pulsed field electrophoresis system (Bio-Rad Laboratories, Hercules, CA). Electrophoresis parameters (run time of 18 h at 6 V/cm with a 2.5–90 s switch time ramp at an included angle of 120) for separation of DNA fragments less than 900 kb in size were used. Agarose imbedded bacteriophage lambda DNA and Saccharomyces cerevisiae DNA size standards (Bio-Rad Laboratories) were included in all electrophoresis procedures. Following electrophoresis, RFLP DNA banding patterns were visualized using SYBR Green (Molecular Probes Inc., Eugene, OR) staining as described by the manufacturer’s protocol. A digital image was captured for each PFGE gel and molecular sizes of the DNA restriction bands were determined using Image J (version 1.32j) and Microsoft Excel (version 10.0.2614.0). 2.6. Construction of the phylogenetic tree Assembly of a binary matrix (presence/absence characteristic) for 38 different RFLP DNA band sizes was done using DNA restriction band sizes measured following PFGE. Using the RFLP binary matrix and the software program FreeTree (version 0.9.1.50; Pavlicek et al., 1999), a distance/similarity matrix was produced via Nei and Li/ Dice analysis with tree construction by the unweighted pair group method with arithmetic averages (UPGMA). Bootstrap resampling for 1000 datasets was performed to test tree robustness. The software program TreeView (version 1.0.0.0; Page, 1996) was used for visualization of phylogenetic trees. 2.7. GenBank accession numbers Previously unreported partial 16S rDNA sequences determined for the various strains of P. popilliae and P. lentimorbus tested were deposited in the GenBank database. All accession numbers for 16S rDNA sequences cited in this report are listed in Fig. 3. 3. Results 3.1. Phylogenic tree construction using RFLP comparisons of genomic DNA Forty-eight isolates of milky disease bacteria (comprising P. popilliae and P. lentimorbus) that had been collected from various locations in the eastern half of the USA and from several commercially produced insecticides (Table 1) were phylogenically grouped using RFLP DNA analysis of complete bacterial genomes. Following PFGE of I-CeuI digested genomic DNA taken from milky disease bacteria (Fig. 1), DNA restriction fragment sizes (9–900 kb) within

the RFLP profiles were measured to identify the various fragment sizes and a binary matrix was assembled based on the presence/absence of the various restriction fragment sizes. I-CeuI is a homing endonuclease that specifically recognizes a 26 bp DNA sequence only found within the highly conserved 23S rRNA gene (Liu et al., 1993). Changes in RFLP patterns due to a point mutation at the restriction recognition site are not likely to occur and only significant genomic changes (i.e., additions or deletions) will be exhibited in the banding patterns. The unrooted phylogenetic tree (Fig. 2), constructed using the binary matrix and phylogenic-based computer software, showed separation of P. popilliae isolates from P. lentimorbus isolates. The tree also demonstrated two groups for the P. popilliae isolates and three groups for the P. lentimorbus isolates. Milky disease isolates Pa1, Cp1, Cb1, Cb2, DGB 1, and DNG 10 (identified as P. popilliae; Stahly et al., 1992) were reclassified as P. lentimorbus and isolate BlPj1 (identified as P. lentimorbus) was identified as P. popilliae. Except for isolates DNG 10 and NRRL B-2522, these reclassification findings agreed with those previously reported by Rippere et al. (1998). No significant correlation between phylogenic groups of milky disease bacteria and host insect species was observed. It was noted that 8 of 10 P. popilliae group 1 isolates having a known insect host origin originated from Japanese beetle larvae (Table 1). In addition, most bacterial isolates obtained from Cyclocephala spp. were classified into P. lentimorbus group 2 and none of the isolates within this phylogenic group was from Japanese beetle or oriental beetle larvae. A strain of P. popilliae (i.e., Ch 1) has been isolated from spore powder produced using Cyclocephala hirta larvae (Stahly et al., 1992). Further research, utilizing many new isolates of milky disease bacteria, is required to determine if a correlation exists between phylogenic groups of these bacteria and host insect species. For the five different samples of commercial insecticides obtained from 1980–1988, all milky disease isolates were identified as being within P. popilliae group 1. 3.2. Comparative alignment of 16S rDNA sequences for phylogenetic separation DNA sequence comparison using 16S rRNA gene segments (i.e., approximately 1450 bp fragments that had been PCR amplified from each of the strains) was performed in support of the phylogenic results obtained via RFLP analysis. Sequence data for P. popilliae and P. lentimorbus 16S rDNA previously listed in the GenBank database were included in the comparisons. Sequence alignments of the 16S rDNA fragments for 11 variable regions (Fig. 3) also exhibited a separation between P. popilliae isolates and P. lentimorbus isolates. Nucleotide differences within the sequence ranges of 70– 98 and 212–221 were significantly characteristic of a separation between the two milky disease bacteria. Division of milky disease isolates, based on sequence similarities/dif-

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

175

Fig. 1. RFLP patterns of milky disease isolates following I-CeuI digestion of genomic DNA and PFGE. Photographs of the electrophoresis patterns were digitally manipulated using Adobe Photoshop CS to better demonstrate banding patterns.

ferences, was in agreement with the phylogenic tree. Within P. popilliae, 16S rDNA sequences did not show distinctive differences with which to identify different groupings. However, comparative analysis of the 16S rDNA sequences in P. lentimorbus demonstrated the three phylogenetic groupings as previously determined via the RFLP results. DNA sequence comparisons also classified milky disease isolates

Pa1, Cp1, Cb1, Cb2, DGB 1, and DNG 10 within P. lentimorbus and BlPj1 within P. popilliae (data not shown). Alignment of 16S rDNA sequences of the P. lentimorbus group 3 isolates to sequence data for P. lentimorbus 16S rDNA present in the GenBank database identified a match with P. lentimorbus strain Semadara (Fig. 3). Previously unknown, strain Semadara was recently isolated from

176

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

Fig. 2. Unrooted phylogenetic tree for 33 P. popilliae and 15 P. lentimorbus strains. The tree was constructed based on UPGMA analysis of a RFLP binary matrix. Bootstrap values for branch points are given and branch lengths reflect genetic distances. Isolates obtained from Connecticut ( ) and from commercial bioinsecticide (m) are shown.

oriental beetle larvae collected from turf in Japan (Yokoyama et al., 2003). This is the first report for P. lentimorbus Semadara-like strains within the USA and outside of Japan.

morbus (i.e., group 3) was the only type of isolate for this milky disease bacterium collected in Connecticut.

3.3. Phylogenic diversity of milky disease bacteria in Connecticut

Using source records available for those isolates examined, geographic collection points were mapped for the phylogenic groups of P. popilliae and P. lentimorbus in the eastern half of the USA (Fig. 4). For 14 P. popilliae group 2 isolates of known geographic origin, all originated east of the Appalachian Mountain range. For P. popilliae group 1, 5 of the 7 isolates with known geographic origins were obtained from insects collected west of the Appalachian Mountains. A single P. popilliae isolate obtained in California (strain Ch1) was also identified as group 1 (data not presented). Generally, P. lentimorbus isolates in groups 1 and 2 were obtained from insect larvae that had been collected west of the Appalachian mountain range. In the southern half of the United States, P. lentimorbus group 2 isolates were found east of the Appalachian Mountains in Florida and North Carolina. The majority (9 of the 15 isolates) of P. lentimorbus strains were classified into group 2. Except for the three P. lentimorbus group 3 strains (i.e., Semadara-like isolates) identified in Connecticut, no P. lentimorbus isolates examined in this study were obtained

Geographic locations in the state of Connecticut for specific P. popilliae or P. lentimorbus isolates, based on the collection points for host insect larvae, are shown in Fig. 4. For 15 milky disease isolates obtained from 5 regions over a 7 year period, 12 of the isolates were identified as P. popilliae and 11 were classified into phylogenic group 2 (Fig. 2). P. popilliae DNG 12 was classified into group 1. During this time, only three P. lentimorbus isolates were collected within Connecticut and two of the isolates were limited to the New Haven area. The third P. lentimorbus isolate (i.e., E-10) was cultured from a white grub that had been collected from an uncertain location within south central Connecticut. Forming group 3 on the phylogenic tree, these three P. lentimorbus isolates segregated from the other P. lentimorbus strains (Fig. 2). In Connecticut, P. popilliae was the predominate cause of milky disease and these bacterial isolates were largely restricted to group 2. The Semadara-like strain of P. lenti-

3.4. National distribution of milky disease bacteria based on phylogeny

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

P. popilliae Group 1: ATCC14706(T); NRRL B-2309 Pj1 Dutky 1S Group 2: NRRL B-2524 KLN 3 DNG 9

177

GenBank# AF071859 EF190487 EF190488 EF190490

70 95 | | -GACTCAACTGTTTCCTTCGGGAAACCGTTAGGTT---CTC---G---T--------A---------G-----CTC---G---T--------A---------G-----CTC---G---T--------A---------G---

125 140 | | -GTAACCTGCCCTTAAGACYG-------------------Y--------------------Y--------------------T--

185 | -TTTGCTCGCATGAGGGAAT----G-------------------G-------------------G----------------

EF190489 EF190491 EF190493

---CTC---G---T--------A---------G-----CTC---G---T--------A---------G-----CTC---G---T--------A--Y------G---

-------------------T--------------------T--------------------Y--

----G-------------------G-------------------G----------------

AF071861 EF190496 EF190497 EF190498

-GAGCGA:CGGTTCCCTTCGGGGAACCGTTAGCTT---GCG-:YG---C--------G---------C-----GCG-:-G---C--------G---------C-----GCG-:-G---C--------G---------C---

-GTAACCTGCCCTTAAGACCG-------------------C--------------------C--------------------C--

-TTTCCTCGCATGAGGGAAT----C-------------------C-------------------C----------------

EF190504 EF190505 EF190501 EF190506

---GCG-T-:---T--------G---GA--G-C-----GCG-T-:---T--------G---GA--G-C-----GCG-T-:---T--------G---GA--G-C-----GCG-T-:---T--------G---GA--G-C---

--------------G----C---------------G----C---------------G----C---------------G----C--

----G-------------------G-------------------G-------------------G----------------

AB110988 EF190499 EF190500

---GC:-A-G---C----A---G----A----C-----GC:-A-G---C----A---G----A----C-----NC:-A-G---C----N---G----A----C---

--C----------------C---C----------------C---C------N---------C--

----C-------------G-----C-------------G-----C-------------G--

P. lentimorbus Group 1: ATCC14707(T); NRRL B-2522 KLN 2 NRRL B-2521 Group 2: Pa1 Cb2 DNG14 Cb1 Group 3: Semadara; DNG 10 DNG 21

P. popilliae Group 1: ATCC14706(T); NRRL B-2309 Pj1 Dutky 1S Group 2: NRRL B-2524 KLN 3 DNG 9

GenBank# AF071859 EF190487 EF190488 EF190490

210 225 | | -CGG:::::AGCAATC:::::TGCCAC----:::::-GC----:::::----------:::::-GC----:::::----------:::::-GC----:::::----G--

265 290 | | -GRGGTAACGGCTCACCAAGGCGACGATGCGTAG--R-----Y---------------------------R-----Y---------------------------G--------------------------------

475 | -TGTTCCAT---T--C-----T--C-----T--C---

EF190489 EF190491 EF190493

----:::::-GC----:::::----------:::::-GC----:::::----------:::::-GC----:::::-------

--R-----Y---------------------------G---------------------------------R-----Y--------------------------

---T--C-----T--C-----T--C---

AF071861 EF190496 EF190497 EF190498

-CGGTTCCGAWTAATCGGGGCTGCCAC----TTCCG-WT----GGGGC----------TTCCG-TT----GGGGC----------TTCCG-TT----GGGGC-------

-GGGGTAACGGCTCACCAAGGCGACGATGCGTAG--G---R-M------M---------R----SW----G---G-----------------------------G---R-M----------------R----SW---

-TGCTCTAT---C--T-----C--T-----C--T---

EF190504 EF190505 EF190501 EF190506

----TTTCG-TTT---GGGGC----------TTTCG-TTT---GGGGC----------TTTCG-TTT---GGGGC----------TTTCG-TTT---GGGGC-------

--G---------------------------------G---------------------------------G---------------------------------G--------------------------------

---C--T-----C--T-----C--T-----C--T---

AB110988 EF190499 EF190500

----TTCCG-TTT---GGGGC----------TTCCG-TTT---GGGGC---T------TTCCG-TTT---GGGGC---T---

--G-----G---------------------------G---------------------------------G--------------------------------

---C--T-----C--T-----C--T---

GenBank# AF071859 EF190487 EF190488 EF190490

710 | -AGAT----------------

1005 1025 | | -CCCTCTGACCGCGCTAGAGATAGGG----T--------G----------G-----T--------G----------G-----T--------G----------G--

1140 | -TTGAG-------------------

1260 1270 | | -GGAAGCGAAGCCGCG----A---------------A---------------A------------

1425 | -TTTACAA-------------------------

EF190489 EF190491 EF190493

----------------

----T--------G----------G-----T--------G----------G-----T--------G----------G--

-------------------

----A---------------A---------------A------------

-------------------------

AF071861 EF190496 EF190497 EF190498

-AGAT----------------

-CCCACTGACCGCTCTAGAGATAGAG----A--------T----------A-----A--------T----------A-----A--------T----------A--

-TTGAG-------------------

-GGAGGCGAAGCCGCG----G---------------G---------------G--------C---

-TTTACAA-------------------------

EF190504 EF190505 EF190501 EF190506

---------------------

----T--------TT---------A-----T--------TT---------A-----T--------TT---------A-----T--------TT---------A--

---R-----A-----A-----R---

----G---------------G---------------G---------------G------------

---------------------------------

AB110988 EF190499 EF190500

--T----T----T---

----G--------T----------A-----G--------T----------A-----G--------T----------A--

-------------------

----G---------------G---------------G------------

----G-------G-----N-G----

P. lentimorbus Group 1: ATCC14707(T); NRRL B-2522 KLN 2 NRRL B-2521 Group 2: Pa1 Cb2 DNG14 Cb1 Group 3: Semadara; DNG 10 DNG 21

P. popilliae Group 1: ATCC14706(T); NRRL B-2309 Pj1 Dutky 1S Group 2: NRRL B-2524 KLN 3 DNG 9

P. lentimorbus Group 1: ATCC14707(T); NRRL B-2522 KLN 2 NRRL B-2521 Group 2: Pa1 Cb2 DNG14 Cb1 Group 3: Semadara; DNG 10 DNG 21

Fig. 3. Nucleotide sequence alignments of variable regions present in the 16S rDNA sequences of select P. popilliae and P. lentimorbus isolates based on phylogenic grouping; (-) identical base; (:) missing base. Nucleotides are presented via the IUPAC-IUB single-letter base code and highlights are used to demonstrate differences within P. popilliae and P. lentimorbus classifications. Total sequence length compared corresponded to nucleotides 31–1488 of the 16S rRNA gene. Base pair numbering is based on Bacillus subtilis rRNA numbering (Van de Peer et al., 1996).

178

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

Fig. 4. Maps of the eastern half of the United States and the state of Connecticut showing geographical origins of P. popilliae group 1 (s) and group 2 (d) plus P. lentimorbus group 1 (m), group 2 (4), and group 3 ( ) isolates.

from insects collected east of the mountain range in the northern regions of the USA. 4. Discussion The phylogenic separation of milky disease bacteria into either species (P. popilliae and P. lentimorbus) or subspecies (P. popilliae var. popilliae and P. popilliae var. lentimorbus) designation has been in question for decades. The current classification of milky disease bacteria, based on DNA– DNA homology (DDH) and RAPD analysis experiments (Rippere et al., 1998; Harrison et al., 2000), is as two species. This investigation, using RFLP and 16S rDNA sequence comparisons (Figs. 2 and 3), has also demonstrated a clear distinction between P. popilliae and P. lentimorbus isolates. No consensus has been reached to the general question on what defines a bacterial species (Cohan, 2002). Despite the clear demarcation between isolates of milky disease bacteria in this and previous studies, species/subspecies classification of these bacteria is not clear. A 70% level of genomic homology is reported as the ‘‘gold standard’’ for determining species differentiation by DDH (Cohan,

2002). Harrison et al. (2000) report some P. popilliae and all P. lentimorbus to have <70% DNA similarity within species when comparing North American and Central American isolates by DDH. Although a separate variety within species is suggested, by the gold standard, these results show Central American and North American milky disease isolates of P. lentimorbus to be different species. In addition, 16S rDNA sequences of the P. lentimorbus isolates differed by 27–31 bases (approximately 1.8–2.1%) when compared to the sequences of P. popilliae isolates over a length of approximately 1480 bp (Fig. 3). Based only on this finding and that a >2–3% 16S rRNA difference has been a ‘‘gold standard’’ for species separation (Ludwig and Schleifer, 1999; Ward, 2006), a classification of the two species into one species with subspecies designation would be the result. Prokaryotic systematics are expanding beyond simple standards (e.g., <70% DNA–DNA homology match, >2– 3% 16S rRNA difference). Signature sequences associated with several specific proteins (Gupta, 2002), multilocus sequence analysis of house keeping genes (Gevers et al., 2005), and use of a macrobiological perspective (Ward, 2006) are proposed as newer ways to define species/subspe-

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

cies differentiation of bacteria. Analyses going beyond single standards are needed to test the validity of the current classification of milky disease bacteria into two species. However, for clarity, continuity, and adherence to current speciation standards, all further discussion in this manuscript will refer to a separate designation (i.e., species) for P. popilliae and P. lentimorbus isolates. Milky disease bacteria are fastidious microorganisms that readily replicate and sporulate within the hemocoel of the insect host (Dutky, 1963). Whether these bacteria naturally exist as vegetative cells outside of the host insect is unknown. Spore release into the soil following death and decomposition of a diseased insect, disperses the contagion (i.e., the spores). The effective use of spore powder to induce milky disease (Schread, 1945; Fleming, 1968) and a peroral injection study using P. popilliae (Dingman, 1996) support the endospore as the natural contagion. Because the contagion remains dormant within the soil until consumed by scarab beetle larvae, the rate of disease spread into a large geographic region from the point of spore deposit will be low. This slow dispersal of contagion has likely resulted in the development of phylogenic groupings of milky disease bacteria based on geographic localization of the disease (i.e., geotypes) at international, national, and regional levels. PFGE fingerprinting of P. popilliae field isolates by MacDonald and Kalmakoff (1995) in New Zealand, demonstrated bacterial relatedness based on geographic origins. In addition, Harrison et al. (2000) have noted a geographic distribution of resistance to vancomycin between P. popilliae isolates of North and Central America. I report the examination of 48 different milky disease bacterial isolates obtained from diseased insects collected in the eastern half of the USA. Thirty-three isolates had knowledge on geographic origin. Phylogenic groups of milky disease bacteria identified in the USA comprised two groupings within P. popilliae and three groupings within P. lentimorbus (Fig. 2). These findings matched well with those reported by Rippere et al. (1998) and Harrison et al. (2000) and identified a new group of P. lentimorbus previously reported only in Japan (Yokoyama et al., 2003). The mapping of milky disease bacteria, using the collection location for the diseased insect host and the phylogenic grouping of the isolate, demonstrated a geographic distribution in the eastern half of the USA (Fig. 4). Out of 12 P. popilliae isolations made from insects collected in Connecticut, only one group 1 P. popilliae isolate (strain DNG 12) was identified. Further, only one other group 1 isolate (strain Pj4 in New Jersey) was obtained from insects collected in northern states east of the Appalachian Mountains. The predominant P. popilliae group identified on the northeastern seaboard (specifically within Connecticut) was group 2. No group 2 P. popilliae were isolated from insects collected west of the Appalachian Mountains. Excluding the isolates of group 3 identified only in Connecticut, P. lentimorbus isolates were only obtained west and south of the Appalachian Mountain range. In Mis-

179

souri and Florida, the finding of only P. lentimorbus isolates obtained from different sites over several years suggested this as the predominant milky disease bacterium in these regions. Group 3 P. lentimorbus isolates (i.e., Semadara-like strains) were identified only within a limited area of Connecticut. More isolates of milky disease bacteria belonging to P. lentimorbus group 3 are needed to determine the geodistribution. One explanation for the phylogenic distribution observed in Connecticut is that all phylogenic groupings of milky disease bacteria are naturally present, but group 1 P. popilliae and all the P. lentimorbus groupings are present in proportions much lower than group 2 P. popilliae. Alternatively, group 1 P. popilliae and P. lentimorbus (excluding the group 3 isolates, which may be a regionally localized grouping) may not be naturally present in Connecticut (or east of the Appalachian Mountains). The two P. popilliae group 1 isolations found in the northeast could be reflective of commercial bioinsecticide use. Commercial bioinsecticides using endospores of milky disease bacteria have been sold and used extensively throughout the eastern United States since the 1950 s (Stahly and Klein, 1992). For the three different commercially produced bioinsecticide samples examined in this investigation, all isolated milky disease bacteria were P. popilliae group 1 (Table 1). No P. lentimorbus isolates were obtained from commercial bioinsecticides. Because these insecticide samples had been collected over 8 years, it is probable that the bioinsecticides have always contained group 1 P. popilliae. Application of these commercial insecticides over decades should have distributed group 1 P. popilliae into Connecticut (and other eastern seaboard states). Therefore, why was group 1 P. popilliae not isolated more frequently from insects collected in Connecticut? A low onset of disease due to inhibition of the bacterium and/or use of ineffective bioinsecticides, combined with the slow progress at which the disease spreads, would hinder establishment of group 1 P. popilliae over large areas of Connecticut soil. Factors such as low soil temperature (Milner et al., 1980) and susceptibility to synthetic pesticides that are applied to turf (Dingman, 1994a) are inhibitory to milky disease bacteria. Additionally, multi-year field trials of commercial bioinsecticides against populations of Japanese beetle larvae in Kentucky showed the products to be ineffective in inducing higher levels of milky disease or in reducing larvae populations (Redmond and Potter, 1995). Another factor for consideration is that phylogenic groups of milky disease bacteria, due to geographic confinement, are environmentally adapted. This adaptation might influence the ability of the bacterium to cause disease and establish itself into a new region. For example, Beard (1945) has reported that soil pH affects the potency of milky disease spores. Based on USDA-NRCS soil geochemistry data (http://soils.usda.gov/survey/geochemistry/index.html ‘‘Access to data’’), soil pH for 17 out of 23 sites in Connecticut (group 2 P. popilliae and group 3

180

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181

P. lentimorbus) was less than 4.5. The soil pH for one site in central Ohio (group 1 P. popilliae and P. lentimorbus groups 1 and 2) was approximately 5.3 and for 7 out of 9 sites in Missouri (group 2 P. lentimorbus), the pH was greater than 6.4. Use of milky disease bacteria adapted to a specific pH range (i.e., group 1 bacteria from west of the Appalachian Mountains) in an environment with a more acidic soil pH (i.e., northeast coast) may cause poor performance of the bacteria and hinder establishment of a new milky disease group into a new area. In the government-sponsored program from 1939 to 1951, endospores of milky disease bacteria (the contagion) were effectively used to suppress Japanese beetle infestations in the eastern United States (Schread, 1945; Fleming, 1968). Interestingly, the contagion was produced using milky disease spores and insects that had been collected from the areas the contagion was used to treat. This investigation has shown a geographic placement, based on phylogenic grouping, of milky disease bacteria in the eastern half of the United States (Fig. 4). However, these findings represent an initial mapping. Continued isolations and phylogenic analysis, referencing the geographic isolation point, are needed to improve mapping of the distribution of milky disease bacteria within the USA and worldwide. Understanding the geographic distribution of milky disease bacteria at the phylogenic level, and identifying characteristics that influence environmental adaptation of these bacteria, will help to enhance utilization of these organisms as bioinsecticides. Acknowledgments I thank S.M. Douglas for helpful suggestions in the preparation of this manuscript and Cindy Musante for technical assistance. This research was supported by Federal Hatch fund USDA CONH00243. References Beard, R.L., 1945. Studies on the milky disease of Japanese beetle larvae. Conn. Agric. Exp. Station Bull., 491. Cohan, F.M., 2002. What are bacterial species? Annu. Rev. Microbiol. 56, 457–487. Correa, M.M., Yousten, A.A., 2001. Pulsed-field gel electrophoresis for the identification of bacteria causing milky disease in scarab larvae. J. Invertebr. Pathol. 78, 278–279. Dingman, D.W., 1994a. Inhibitory effects of turf pesticides on Bacillus popilliae and the prevalence of milky disease. Appl. Environ. Microbiol. 60, 2343–2349. Dingman, D.W., 1994b. Physical properties of three plasmids and the presence of interrelated plasmids in Bacillus popilliae and Bacillus lentimorbus. J. Invertebr. Pathol. 63, 235–243. Dingman, D.W., 1996. Description and use of a peroral injection technique for studying milky disease. J. Invertebr. Pathol. 67, 102–104. Dingman, D.W., Stahly, D.P., 1983. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol. 46, 860–869. Dutky, S.R., 1940. Two new spore-forming bacteria causing milky diseases of Japanese beetle larvae. J. Agric. Res. 61, 57–68.

Dutky, S.R., 1963. The milky diseases. In: Steinhaus, E.A. (Ed.), Insect Pathology: An Advanced Treatise, vol. 2. Academic Press, New York, pp. 75–115. Fleming, W.E., 1968. Biological control of the Japanese beetle. U.S. Dep. Agric. Tech. Bull. 1383, 1–78. Gautom, R.K., 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other Gram-negative organisms in 1 day. J. Clin. Microbiol. 35, 2977–2980. Gevers, D., Cohan, F.M., Lawrence, J.G., Spratt, B.G., Coenye, T., Feil, E.J., Stackebrandt, E., Van de Peer, Y., Vandamme, P., Thompson, F.L., Swings, J., 2005. Re-evaluating prokaryotic species. Nat. Rev. 3, 733–739. Gordon, R.E., Haynes, W.C., Pang, C.H. 1973. The genus Bacillus, In: Agriculture handbook no. 427, U.S. Dept. Agriculture, Washington, DC. Gupta, R.S., 2002. Phylogeny of bacteria: are we now close to understanding it? ASM News 68, 284–291. Harrison, H., Patel, R., Yousten, A.A., 2000. Paenibacillus associated with milky disease in Central and South American scarabs. J. Invertebr. Pathol. 76, 169–175. Haynes, W.C., St. Julian Jr., G., Shekleton, M.C., Hall, H.H., Tashiro, H., 1961. Preservation of infectious milky disease bacteria by lyophilization. J. Insect Pathol. 3, 55–61. Krieg, A., 1961. Grundlagen der insektenpathologie. Darmstadt, Steinkopff. Krywienczyk, J., Lu¨thy, P., 1974. Serological relationship between three varieties of Bacillus popilliae. J. Invertebr. Pathol. 23, 275–279. Lane, D.A., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., Pace, N.R., 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82, 6955– 6959. Liu, S., Hessel, A., Sanderson, K.E., 1993. Genomic mapping with I-Ceu I, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli, and other bacteria. Proc. Natl. Acad. Sci. USA 90, 6874–6878. Ludwig, W., Schleifer, K., 1999. Phylogeny of Bacteria beyond the 16S rRNA standard. ASM News 65, 752–757. MacDonald, R., Kalmakoff, J., 1995. Comparison of pulse-field gel electrophoresis DNA fingerprints of field isolates of the entomopathogen Bacillus popilliae. Appl. Environ. Microbiol. 61, 2446– 2449. Milner, R.J., 1981. Identification of the Bacillus popilliae group of insect pathogens. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Diseases 1970–1980. Academic Press, London, pp. 45–59. Milner, R.J., Wood, J.T., Williams, E.R., 1980. The development of milky disease under laboratory and field temperature regimes. J. Invertebr. Pathol. 36, 203–210. Page, R.D.M., 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comp. Appl. Biosci. 12, 357–358. Pavlicek, A., Hrda, S., Flegr, J., 1999. FreeTree-Freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of the genus Frenkelia. Folia Biol. (Praha) 45, 97–99. Pettersson, B., Rippere, K.E., Yousten, A.A., Priest, F.G., 1999. Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov. Int. J. Syst. Bacteriol. 49, 531–540. Redmond, C.T., Potter, D.A., 1995. Lack of efficacy of in vivo- and putatively in vitro-produced Bacillus popilliae against field populations of Japanese beetle (Coleoptera: Scarabaeidae) grubs in Kentucky. J. Econ. Entomol. 88, 846–854. Rippere, K.E., Tran, M.T., Yousten, A.A., Hilu, K.H., Klein, M.G., 1998. Bacillus popilliae and Bacillus lentimorbus, bacteria causing milky disease in Japanese beetles and related scarab larvae. Int. J. Syst. Bacteriol. 48, 395–402.

D.W. Dingman / Journal of Invertebrate Pathology 97 (2008) 171–181 Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schread, J.C., 1945. Status of the natural enemies of the Japanese beetle in Connecticut. Conn. Agric. Exp. Station Bull. 488, 331–339. Sharpe, E.S., Bulla Jr., L.A., 1978. Characteristics of the constituent substrains of Bacillus popilliae growing in batch and continuous cultures. Appl. Environ. Microbiol. 35, 601–609. Stahly, D.P., Klein, M.G., 1992. Problems with in vitro production of spores of Bacillus popilliae for use in biological control of the Japanese beetle. J. Invertebr. Pathol. 60, 283–291. Stahly, D.P., Takefman, D.M., Livasy, C.A., Dingman, D.W., 1992. Selective medium for quantitation of Bacillus popilliae in soil and in commercial spore powders. Appl. Environ. Microbiol. 58, 740–743. Tashiro, H., 1957. Susceptibility of European chafer and Japanese beetle larvae to different strains of milky disease organisms. J. Econ. Entomol. 50, 350–352.

181

Tashiro, H., Steinkraus, K.H., 1966. Virulence of species and strains of milky disease bacteria in the European chafer, Amphimallon majalis (Razoumowsky). J. Invertebr. Pathol. 8, 382–389. Tashiro, H., White, R.T., 1954. Milky diseases of European chafer larvae. J. Econ. Entomol. 47, 1087–1092. Van de Peer, Y., Nicolaı¨, S., De Rijk, P., De Wachter, R., 1996. Database on the structure of small ribosomal subunit RNA. Nucleic Acids Res. 24, 86–91. Ward, D.M., 2006. A macrobiological perspective on microbial species. Microbe 1, 269–278. Wyss, C., 1971. Sporulationsversuche mit drei varieta¨ten von Bacillus popilliae Dutky. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 2 126, 461–492. Yokoyama, T., Tanaka, M., Fujiie, A., Hasegawa, M., 2003. A new strain of Paenibacillus lentimorbus isolated from larvae of the oriental beetle, Blitopertha orientalis (Coleoptera: Scarabaeidae), in Chiba Prefecture, Japan. Appl. Entomol. Zool. 38, 523–528.