Phenotypic and Molecular Characterization of the Beetle Pathogens Paenibacillus popilliae and Paenibacillus lentimorbus
dc.contributor.author | Harrison, Helen A. | en |
dc.contributor.committeechair | Yousten, Allan A. | en |
dc.contributor.committeemember | Claus, George William | en |
dc.contributor.committeemember | Hilu, Khidir W. | en |
dc.contributor.department | Biology | en |
dc.date.accessioned | 2014-03-14T21:46:25Z | en |
dc.date.adate | 1999-09-23 | en |
dc.date.available | 2014-03-14T21:46:25Z | en |
dc.date.issued | 1999-09-16 | en |
dc.date.rdate | 2000-09-23 | en |
dc.date.sdate | 1999-09-20 | en |
dc.description.abstract | DNA similarity studies were used to determine the species of thirty-one strains of bacteria isolated from the hemolymph of infected larvae from Mexico and throughout Central and South America. Twenty-one of the strains were determined to be <I>Paenibacillus popilliae</I> and ten were found to be more closely related to <I>Paenibacillus lentimorbus</I>. Only one of the <I>P. popilliae</I> strains, an isolate from Mexico, was resistant to the antibiotic vancomycin, a trait characteristic of <I>P. popilliae</I> strains from other geographic areas. As expected, all <I>P. lentimorbus</I> strains were sensitive to vancomycin. The polymerase chain reaction (PCR) was used to amplify a portion of a ligase gene necessary for vancomycin resistance in the Mexican strain. Sequencing of the amplicon revealed a sequence identical to that obtained from a North American strain of <I>P. popilliae</I> previously described. The ability of <I>P. popilliae</I> and the inability of <I>P. lentimorbus</I> to grow on medium supplemented with 2% sodium chloride has been used as a phenotypic trait for differentiating between the two species. Approximately 86% of the <I>P. popilliae</I> strains were capable of growth on medium supplemented with 2% sodium chloride and 60% of the <I>P. lentimorbus</I> strains were not capable of growth on this medium. Microscopic examination revealed that all of the Mexican and Central and South American strains of <I>P. popilliae</I> and <I>P. lentimorbus</I> produced a parasporal body. PCR was used to amplify two different regions of the <I>cry18Aa1</I> gene encoding the paraspore in all of the isolates. One primer pair, CryBP2, detected the <I>cry18Aa1</I> gene in 17 of the 21 <I>P. popilliae</I> strains and in all ten of the <I>P. lentimorbus</I> strains. The second primer pair, CryBP4, detected the parasporal gene in 20 of the 21 <I>P. popilliae</I> strains and in all ten of the <I>P. lentimorbus</I> strains. Thirty of the thirty-one <I>P. popilliae</I> and <I>P. lentimorbus</I> strains produced amplicons of approximately 616 bp with the CryBP4 primers. The CryBP4 primers did not detect the paraspore gene in one of the <I>P. popilliae</I> strains. The CryBP2 primer pair produced amplicons of three different sizes, indicating possible variability in the parasporal proteins of <I>P. popilliae</I> and <I>P. lentimorbus</I>. Eleven of the <I>P. popilliae</I> strains produced CryBP2 amplicons approximately 660 bp in size and six of the <I>P. popilliae</I> strains produced CryBP2 amplicons approximately 1100 bp in size. The <I>cry</I> gene was not detected in four of the <I>P. popilliae</I> strains with the CryBP2 primers. The <I>P. lentimorbus</I> strains produced CryBP2 amplicons approximately 750 bp in size. Three PCR products representing the variable CryBP2 amplicon sizes were sequenced and compared to the published <I>cry18Aa1</I> gene sequence. Sequencing data revealed that the Central and South American CryBP2 amplicons are similar to the published <I>cry18Aa1</I> sequence, however, the 1100 bp amplicon has a 453 bp insert that is not found in the published <I>cry18Aa1</I> gene sequence. Paraspores are produced by <I>P. popilliae</I> and <I>P. lentimorbus</I> and is not a reliable phenotypic trait for differentiation between the two species. The ability of Mexican and Central and South American strains of <I>P. lentimorbus</I> to produce paraspores supports the previous findings of a North American group of <I>P. lentimorbus</I> strains that were also capable of producing paraspores. Except for one Mexican strain of <I>P. popilliae</I>, the Central and South American strains of <I>P. popilliae</I> are sensitive to vancomycin. This was unexpected since all North American strains of <I>P. popilliae</I> are vancomycin resistant. Vancomycin resistance could be useful in identifying strains of <I>P. popilliae</I> from North America but not for identifying strains of <I>P. popilliae</I> from Central and South America. So far, no vancomycin resistant strains of <I>P. lentimorbus</I> have been identified. There was variability in the ability of these organisms to grow on medium supplemented with 2% sodium chloride so the usefulness of this trait is debatable. However, the majority of <I>P. popilliae</I> strains from Mexico and Central and South America will grow on medium supplemented with 2% sodium chloride and the majority of the <I>P. lentimorbus</I> strains from these same areas will not grow on this medium. North American strains of <I>P. popilliae</I> and <I>P. lentimorbus</I> also showed variability of growth on medium supplemented with 2% sodium chloride. | en |
dc.description.degree | Master of Science | en |
dc.identifier.other | etd-092099-133341 | en |
dc.identifier.sourceurl | http://scholar.lib.vt.edu/theses/available/etd-092099-133341/ | en |
dc.identifier.uri | http://hdl.handle.net/10919/44890 | en |
dc.publisher | Virginia Tech | en |
dc.relation.haspart | chapter1.pdf | en |
dc.relation.haspart | chapter3.pdf | en |
dc.relation.haspart | summary.pdf | en |
dc.relation.haspart | introduction.pdf | en |
dc.relation.haspart | abstract.pdf | en |
dc.relation.haspart | resume.pdf | en |
dc.relation.haspart | title.pdf | en |
dc.relation.haspart | listoffigures.pdf | en |
dc.relation.haspart | acknowledgement.pdf | en |
dc.relation.haspart | tableofcontents.pdf | en |
dc.relation.haspart | conclusions.pdf | en |
dc.relation.haspart | chapter2.pdf | en |
dc.relation.haspart | chapter4a.pdf | en |
dc.relation.haspart | chapter4b.pdf | en |
dc.relation.haspart | listoftables.pdf | en |
dc.rights | In Copyright | en |
dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
dc.subject | milky disease | en |
dc.subject | Paenibacillus lentimorbus | en |
dc.subject | Paenibacillus popilliae | en |
dc.subject | vancomycin | en |
dc.subject | DNA similarity | en |
dc.subject | paraspore | en |
dc.subject | insect pathogens | en |
dc.title | Phenotypic and Molecular Characterization of the Beetle Pathogens Paenibacillus popilliae and Paenibacillus lentimorbus | en |
dc.type | Thesis | en |
thesis.degree.discipline | Biology | en |
thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
thesis.degree.level | masters | en |
thesis.degree.name | Master of Science | en |
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