Two isolates of a novel bipartite begomovirus, tentatively named malvastrum bright yellow mosaic virus (MaBYMV), were molecularly characterized from naturally infected plants of the genus Malvastrum showing bright yellow mosaic disease symptoms in South Texas. Six complete DNA-A and five DNA-B genome sequences of MaBYMV obtained from the isolates ranged in length from 2,608 to 2,609 nucleotides (nt) and 2,578 to 2,605 nt, respectively. Both genome segments shared a 178- to 180-nt common region. In pairwise comparisons, the complete DNA-A and DNA-B sequences of MaBYMV were most similar (87-88 % and 79-81 % identity, respectively) and phylogenetically related to the corresponding sequences of sida mosaic Sinaloa virus-[MX-Gua-06]. Further analysis revealed that MaBYMV is a putative recombinant virus, thus supporting the notion that malvaceous hosts may be influencing the evolution of several begomoviruses. The design of new diagnostic primers enabled the detection of MaBYMV in cohorts of Bemisia tabaci collected from symptomatic Malvastrum sp. plants, thus implicating whiteflies as potential vectors of the virus.

Isaria poprawskii is described as a new entomopathogenic species similar to Isaria javanica (=Paecilomyces javanicus). It was discovered on the sweet potato whitefly, Bemisia tabaci biotype B in the Lower Rio Grande Valley of Texas (LRGV), USA. Morphological and DNA examinations indicated the distinctness of I. poprawskii from the ex-type isolate of I. javanica. I. poprawskii produced light yellow young colonies to darker yellow with a grayish-violet center to a taupe or a brownish-gray mature conidial mass; conidia hyaline, one-celled, 3.9 (2.9-4.6) mu m long x 1.6 (1.4-2.1) mu m wide; colored synnemata, but I. javanica ex-type produced white colony, hyaline conidia and no synnemata. A phylogenetic position of I. poprawskii was inferred by a nucleotide sequence analysis of beta-tubulin along with standard beta-tubulin sequences from GenBank. Fifteen unsequenced isolates, including eight from the LRGV, were investigated. The analysis confirmed that I. poprawskii could be recovered from LRGV fields, and that both I. javanica and I. poprawskii are present in the LRGV in sympatry. I. poprawskii was shown to be closely related to I. javanica; however, it formed its own unique clade, thus confirming its status as a new fungal species. (C) 2012 The Mycological Society of Japan. Published by Elsevier B. V. All rights reserved.

American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and "'omics" and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen.

Background We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis , an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture. Results We compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website. Conclusions This comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management.

In this study we developed an electrotransformation method for use with the Gram-positive bacterium Paenibacillus larvae-a deadly pathogen of honey bees. Combining multiple Bacillus electrotransformation methods to generate an initial protocol, we then optimized the following parameters for use with P larvae: cell density of culture at harvest time, contents of the washing/electroporation solution, field strength of the electrical pulse, recovery growth medium, and recovery time period. With the optimized method, we achieved an average transformation efficiency of 1.9 x 10(5) transformants/mu g DNA. The method is substantially different from the only other electrotransformation method for a Paenibacillus species found in the literature. This work should facilitate the study of the several previously discovered natural plasmids of P. larvae, and is a step toward developing a genetic system for this species. Published by Elsevier B.V.

This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae--a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling-circle mechanism, and sequence comparisons place it in the pMV158 family of rolling-circle replicons. The plasmid contains predicted rep, cop, and rnaII genes for control of replication initiating at a predicted double-strand origin. The plasmid has an ssoT single-strand origin, which is efficient enough to allow only very small amounts of the single-stranded DNA intermediate to accumulate. The overall efficiency of replication is sufficient to render the plasmid segregationally stable without selection in P. larvae and in Bacillus megaterium, but not in Escherichia coli. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements.

The gram-positive bacterium, Paenibacillus larvae, causes a serious honey bee disease, American Foulbrood. For several decades, commercial and hobbyist beekeepers have controlled this disease with the antibiotic oxytetracycline. However, in recent years there have been reports of oxytetracycline-resistant P larvae. In this study, we report that the reason for the oxytetracycline resistance in P larvae is the presence of a novel plasmid carrying a tetracycline resistance gene-tetL. We tested 36 strains of P. larvae from the USA and Canada and found this plasmid in all 21 oxytetracycline-resistant strains and in none of the 15 oxytetracycline-sensitive strains. We cloned and expressed the P larvae tetL gene in Escherichia coli and showed it was functional. Sequencing of the entire plasmid, which we named pMA67, revealed that it is likely a mobilizable rolling-circle replication plasmid. This work provides the first sequence information for any P. larvae plasmid, a new tetL ortholog with significant sequence divergence from tetL genes found in other species, and the first tetracycline-resistance gene found in the entire Paenibacillus genus.

Ascosphaera spp. fungi are associated with social and solitary bees, in some cases as pathogens causing chalkbrood disease. As a supplement to morphological identification, we developed a simple PCR-based method for selected Ascosphaera species. We exploited sequence differences in the internal transcribed spacer regions of rDNA to design species-specific primers. Analysis involves simply scoring the presence or absence of a single band for a given pair of primers. The method can distinguish the four Ascosphaera species known to be associated with honey bees. It also distinguishes Ascosphaera aggregata, the chalkbrood pathogen of the alfalfa leafcutting bee, from other Ascosphaera species associated with this bee. We expect the method will be useful for determining purity of Ascosphaera cultures, and may be a first step toward development of an early detection method of chalkbrood infection in honey bees and leafcutting bees. We also present a new, quick and reliable method for preparing fungal DNA suitable for PCR amplification from mycelia grown in liquid or on solid media.