Pathogenicity of multiple Providencia species (Enterobacteriales: Morganellaceae) to the mass-reared Mexican fruit fly (Diptera: Tephritidae).

Mexican fruit fly (Anastrepha ludens (Loew)) (Diptera: Tephritidae) represents a major threat to fruit production in the Western Hemisphere. Sterile insect technique is used to suppress and eradicate wild populations. Success of this control method necessitates weekly production of hundreds of millions of flies, their sterilization by irradiation, and their aerial release. Diet needed to produce large fly numbers are conducive to the spread of bacteria. Pathogenic bacteria were isolated from 3 rearing facilities and from multiple sources: eggs, larvae, pupae and spent diet, and were found to include some isolates identified to the genus Providencia (Enterobacteriales: Morganellaceae). We identified 41 Providencia isolates and tested their pathogenicity to A. ludens. Based on 16s rRNA sequences, 3 groups were clustered into several species of Providencia with varying capacities to affect the Mexican fruit fly production. Isolates putatively identified as P. alcalifaciens/P. rustigianii were all pathogenic causing larval and pupal yield reduction of 46-64% and 37-57%, respectively. Among them, Providencia isolate 3006 was the most pathogenic reducing larval and pupae yield by 73 and 81%, respectively. Isolates identified as P. sneebia were not pathogenic. The final cluster, P. rettgeri/P. vermicola, were variable in pathogenicity with 3 isolates yielding like the control and the rest causing larval and pupal yield reduction of 26-53% and 23-51%, respectively. Isolates putatively identified as P. alcalifaciens/P. rustigianii were more virulent than P. rettgeri/P. vermicola. Accurate identification of species is needed to diagnose and monitor pathogenic versus nonpathogenic Providencia strains.

Large-scale intersubspecific recombination in the plant-pathogenic bacterium Xylella fastidiosa is associated with the host shift to mulberry.

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.

PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes.

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.

Multilocus sequence typing reveals two evolutionary lineages of Acidovorax avenae subsp. citrulli.

Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, has caused considerable damage to the watermelon and melon industry in China and the United States. Understanding the emergence and spread of this pathogen is important for controlling the disease. To build a fingerprinting database for reliable identification and tracking of strains of A. avenae subsp. citrulli, a multilocus sequence typing (MLST) scheme was developed using seven conserved loci. The study included 8 original strains from the 1978 description of A. avenae subsp. citrulli, 51 from China, and 34 from worldwide collections. Two major clonal complexes (CCs), CC1 and CC2, were identified within A. avenae subsp. citrulli; 48 strains typed as CC1 and 45 as CC2. All eight original 1978 strains isolated from watermelon and melon grouped in CC1. CC2 strains were predominant in the worldwide collection and all but five were isolated from watermelon. In China, a major seed producer for melon and watermelon, the predominant strains were CC1 and were found nearly equally on melon and watermelon.

Detecting natural selection at the molecular level: a reexamination of some "classic" examples of adaptive evolution.

An important criterion used to detect adaptive evolution in DNA sequence data is omega(i) > 1, where omega(i) is the ratio of nonsynonymous to synonymous substitution rates in lineage i. However, the evaluation of multiple omega(i) within a phylogenetic tree can easily inflate the statistical type I error rate. We developed two rigorous methods of analysis that avoid this and other potential pitfalls. We applied these methods to four published examples of adaptive evolution. One case was strongly supported by our reanalysis (abalone sperm lysin), and one was weakly supported (baboon alpha-globin), but two examples (primate lysozyme and Antarctic fish beta-globin) did not show significant evidence of adaptive evolution. Our first method is a "bottom-up" hierarchical maximum likelihood approach, which (1) tests for significant heterogeneity in omega across the phylogeny, (2) locates its source using a sequence of planned comparisons, and (3) tests homogeneous groups of omega for omega > 1, using a modified level of significance that incorporates the pretesting. The second method is a "top-down" log-linear analysis based on estimates of nonsynonymous and synonymous substitutions in pairs of lineages. The log-linear test is applied to pairs of lineages joined at progressively deeper nodes. For each pair, the analysis simultaneously tests for adaptive evolution (omega > 1), a shift in natural selection (omega1 does not = omega2), and unequal evolution rate (the relative rate test). In both tests, we emphasized that the criterion omega1 not equal omega2 is an important additional indicator of a phylogenetic shift in the balance between natural selection and genetic drift between two related lineages.

Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity.

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.

A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a pathogen that causes leaf scorch and related diseases in over 100 plant species, including Pierce's disease in grapevines (PD), phony peach disease (PP), plum leaf scald (PLS), and leaf scorch in almond (ALS), oak (OAK), and oleander (OLS). We used a high-resolution DNA sequence approach to investigate the evolutionary relationships, geographic variation, and divergence times among the X. fastidiosa isolates causing these diseases in North America. Using a large data set of 10 coding loci and 26 isolates, the phylogeny of X. fastidiosa defined three major clades. Two of these clades correspond to the recently identified X. fastidiosa subspecies piercei (PD and some ALS isolates) and X. fastidiosa subsp. multiplex (OAK, PP, PLS, and some ALS isolates). The third clade grouped all of the OLS isolates into a genetically distinct group, named X. fastidiosa subsp. sandyi. These well-differentiated clades indicate that, historically, X. fastidiosa has been a clonal organism. Based on their synonymous-site divergence ( approximately 3%), these three clades probably originated more than 15,000 years ago, long before the introduction of the nonnative plants that characterize most infections. The sister clades of X. fastidiosa subsp. sandyi and X. fastidiosa subsp. piercei have synonymous-site evolutionary rates 2.9 times faster than X. fastidiosa subsp. multiplex, possibly due to generation time differences. Within X. fastidiosa subsp. multiplex, a low level ( approximately 0.1%) of genetic differentiation indicates the recent divergence of ALS isolates from the PP, PLS, and OAK isolates due to host plant adaptation and/or allopatry. The low level of variation within the X. fastidiosa subsp. piercei and X. fastidiosa subsp. sandyi clades, despite their antiquity, suggests strong selection, possibly driven by host plant adaptation.