Phenotypic and Molecular Characterization of the Beetle Pathogens Paenibacillus popilliae and Paenibacillus lentimorbus

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.