Javier Tabima

Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction.

Many microbial, fungal, or oomcyete populations violate assumptions for population genetic analysis because these populations are clonal, admixed, partially clonal, and/or sexual. Furthermore, few tools exist that are specifically designed for analyzing data from clonal populations, making analysis difficult and haphazard. We developed the R package poppr providing unique tools for analysis of data from admixed, clonal, mixed, and/or sexual populations. Currently, poppr can be used for dominant/codominant and haploid/diploid genetic data. Data can be imported from several formats including GenAlEx formatted text files and can be analyzed on a user-defined hierarchy that includes unlimited levels of subpopulation structure and clone censoring. New functions include calculation of Bruvo’s distance for microsatellites, batch-analysis of the index of association with several indices of genotypic diversity, and graphing including dendrograms with bootstrap support and minimum spanning networks. While functions for genotypic diversity and clone censoring are specific for clonal populations, several functions found in poppr are also valuable to analysis of any populations. A manual with documentation and examples is provided. Poppr is open source and major releases are available on CRAN: http://cran.r-project.org/package=poppr. More supporting documentation and tutorials can be found under ‘resources’ at: http://grunwaldlab.cgrb.oregonstate.edu/.

The Irish potato famine pathogen Phytophthora infestans originated in central Mexico rather than the Andes

Significance The potato late blight pathogen was introduced to Europe in the 1840s and caused the devastating loss of a staple crop, resulting in the Irish potato famine and subsequent diaspora. Research on this disease has engendered much debate, which in recent years has focused on whether the geographic origin of the pathogen is South America or central Mexico. Different lines of evidence support each hypothesis. We sequenced four nuclear genes in representative samples from Mexico and the South American Andes. An Andean origin of P. infestans does not receive support from detailed analyses of Andean and Mexican populations. This is one of a few examples of a pathogen with a known origin that is secondary to its current major host. Phytophthora infestans is a destructive plant pathogen best known for causing the disease that triggered the Irish potato famine and remains the most costly potato pathogen to manage worldwide. Identification of P. infestan’s elusive center of origin is critical to understanding the mechanisms of repeated global emergence of this pathogen. There are two competing theories, placing the origin in either South America or in central Mexico, both of which are centers of diversity of Solanum host plants. To test these competing hypotheses, we conducted detailed phylogeographic and approximate Bayesian computation analyses, which are suitable approaches to unraveling complex demographic histories. Our analyses used microsatellite markers and sequences of four nuclear genes sampled from populations in the Andes, Mexico, and elsewhere. To infer the ancestral state, we included the closest known relatives Phytophthora phaseoli, Phytophthora mirabilis, and Phytophthora ipomoeae, as well as the interspecific hybrid Phytophthora andina. We did not find support for an Andean origin of P. infestans; rather, the sequence data suggest a Mexican origin. Our findings support the hypothesis that populations found in the Andes are descendants of the Mexican populations and reconcile previous findings of ancestral variation in the Andes. Although centers of origin are well documented as centers of evolution and diversity for numerous crop plants, the number of plant pathogens with a known geographic origin are limited. This work has important implications for our understanding of the coevolution of hosts and pathogens, as well as the harnessing of plant disease resistance to manage late blight.

Phylogeography and molecular epidemiology of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the most important virus affecting papaya and cucurbit plants in tropical and subtropical areas. PRSV isolates are divided into biotypes P and W: both the P and W types naturally infect plants in the family Cucurbitaceae, whereas the P type naturally infects papaya (Carica papaya). Understanding the origin and nature of the PRSV genetic diversity and evolution is critical for the implementation of control strategies based on cross-protection and the deployment of transgenic plants that show resistance to virus isolates highly similar to the transgene. The molecular epidemiology of PRSV was evaluated by analyzing the nucleotide sequence of the capsid protein (CP) and helper component-proteinase (HC-Pro) genes of isolates from around the world, including newly characterized ones from Colombia and Venezuela, using a relaxed molecular clock-based approach and a phylogeographic study. Our results confirm previous estimates on the origin of PRSV around 400 years ago and suggest distinct dispersion events from the Indian Peninsula to the rest of Asia, via Thailand, and subsequently to the Americas. A historical reconstruction of the P- and W-type characters in the phylogenetic study supports the need to revise the hypothesis that PRSV-P derives from PRSV-W since our results suggest that the ancestral state could be either of the two biotypes. Moreover, estimates of epidemic growth predict an increasing genetic diversity of the virus over time that has direct implications for control strategies of PRSV based on cross-protection and the use of transgenic plants.

Virulence Gene Expression in Malassezia spp from Individuals with Seborrheic Dermatitis

TO THE EDITOR Seborrheic dermatitis (SD) is a chronic inflammatory disease that compromises skin areas rich in sebaceous glands, such as the face, scalp, and upper trunk. The condition is frequently observed in acquired immune deficiency syndrome patients (30–85% compared with 3–5% of immunocompetent adults). Its appearance is considered to be an early marker of the evolutionary trend of HIV infection (Gupta et al., 2001; Gupta and Bluhm, 2004; Ashbee, 2007; Naldi and Rebora, 2009). The cause or causes of SD and the pathogenic role of Malassezia spp are, at present, not completely understood (Gupta et al., 2000; DeAngelis et al., 2005; Tajima et al., 2008). At this time, we can only rely on hypotheses of the pathogenicity mechanisms and the pathogenic determinants (Ran et al., 1993; Brunke and Hube, 2006; Xu et al., 2007). Analyses of the complete genome of Malassezia globosa and the partial genome of M. restricta (Xu et al., 2007) have presented gene-encoding enzymes of the lipase and phospholipase families that could explain the lipid dependency of the genus. The secretion of enzymes by human pathogenic fungi has been considered an important factor in the invasion and dissemination in the host (Staib et al., 1999; Descamps et al., 2002) and, thus, it is suggested that lipases and phospholipases are involved in the mechanisms of pathogenicity of Malassezia spp. However, except for an early approximation of M. globosa lipase gene expression on human scalp, the expression of these genes has never been tested during disease development (Xu et al., 2007). In order to assess the induction of lipases and phospholipases in SD, we analyzed the expression profiles of candidate virulence genes of Malassezia spp during host infection. A total of 40 Malassezia isolates were obtained from four groups of individuals (non-Malassezia lesion (NML), SD, NMLþHIV, and SDþHIV) (Table 1). The selection of SD and SDþHIV patients was based on dermatologist evaluation of the presence of erythema and peeling in areas such as the face and scalp. We assessed isolates for morphological and physiological features, and we confirmed their identification by amplification and sequencing of 5.8S rDNA-ITS2 regions (Supplementary Materials and Methods online). Six Malassezia spp strains were used as a reference (M. furfur CBS 1878, M. sympodialis CBS 7222, M. globosa CBS 7966, M. restricta CBS 7877, M. slooffiae CBS 7956, and M. pachydematis CBS 1879). All of the Malassezia species isolated from NML, SD, NMLþHIV, and SDþHIV individuals exhibited typical morphological, physiological, and molecular features (Supplementary Table S1 online), with the exception of one: M. Furfur, which presented atypical Tween assimilation pattern (Gonzalez et al., 2009). We found M. restricta to be the predominant species in all groups of individuals, and M. furfur was found in all groups except the SD group. M. furfur with an atypical Tween assimilation pattern was present only in SDþHIV individuals, and M. globosa was present in SD and SDþHIV individuals. These results coincide with previous reports (Tajima et al., 2008; Oh et al., 2010). For the expression assays, four genes, Mgl0797/M. globosa LIP1, Mgl0798/M. globosa hypothetical secretory lipase, Mgl3326/M. globosa hypothetical phospholipase, and Mflip 1/M. furfur MfLIP1 were used for a quantitative analysis using real-time PCR (Supplementary Table S2 online). All the genes were detected in all samples.

Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales.

Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates.

Speciation in Fungal and Oomycete Plant Pathogens

The process of speciation, by definition, involves evolution of one or more reproductive isolating mechanisms that split a single species into two that can no longer interbreed. Determination of which processes are responsible for speciation is important yet challenging. Several studies have proposed that speciation in pathogens is heavily influenced by host-pathogen dynamics and that traits that mediate such interactions (e.g., host mobility, reproductive mode of the pathogen, complexity of the life cycle, and host specificity) must lead to reproductive isolation and ultimately affect speciation rates. In this review, we summarize the main evolutionary processes that lead to speciation of fungal and oomycete plant pathogens and provide an outline of how speciation can be studied rigorously, including novel genetic/genomic developments.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

To design and validate a colorimetric loop‐mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA.

Gall-ID: tools for genotyping gall-causing phytopathogenic bacteria

Understanding the population structure and genetic diversity of plant pathogens, as well as the effect of agricultural practices on pathogen evolution, is important for disease management. Developments in molecular methods have contributed to increase the resolution for accurate pathogen identification, but those based on analysis of DNA sequences can be less straightforward to use. To address this, we developed Gall-ID, a web-based platform that uses DNA sequence information from 16S rDNA, multilocus sequence analysis and whole genome sequences to group disease-associated bacteria to their taxonomic units. Gall-ID was developed with a particular focus on gall-forming bacteria belonging to Agrobacterium, Pseudomonas savastanoi, Pantoea agglomerans, and Rhodococcus. Members of these groups of bacteria cause growth deformation of plants, and some are capable of infecting many species of field, orchard, and nursery crops. Gall-ID also enables the use of high-throughput sequencing reads to search for evidence for homologs of characterized virulence genes, and provides downloadable software pipelines for automating multilocus sequence analysis, analyzing genome sequences for average nucleotide identity, and constructing core genome phylogenies. Lastly, additional databases were included in Gall-ID to help determine the identity of other plant pathogenic bacteria that may be in microbial communities associated with galls or causative agents in other diseased tissues of plants. The URL for Gall-ID is http://gall-id.cgrb.oregonstate.edu/.

Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species complex (Cordyceps sensu stricto)

In the Amazon the only described species of Cordyceps sensu stricto (Hypocreales, Cordycipitaceae) that parasitize insects of Orthopterida (orders Orthoptera and Phasmida) are Cordyceps locustiphila and C. uleana. However, the type specimens for both taxa have been lost and the concepts of these species are uncertain. To achieve a more comprehensive understanding of the systematics of these species, collections of Cordyceps from the Amazon regions of Colombia, Ecuador and Guyana were subjected to morphological, ecological and molecular phylogenetic studies. Phylogenetic analyses were conducted on partial sequences of SSU, LSU, TEF, RPB1 and RPB2 nuclear loci. Two new species are proposed including C. diapheromeriphila, a parasite of Phasmida, and C. acridophila, a parasite of the superfamily Acridomorpha (Orthoptera), which is broadly distributed across the Amazon. For C. locustiphila a lectotypification and an epitypification are made. Cordyceps locustiphila is host specific with Colpolopha (Acridomorpha: Romaleidae), and its distribution coincides with that of its host. The phylogenetic placement of these three species was resolved with strong support in the Beauveria clade of Cordyceps s. str. (Cordycipitaceae). This relationship and the morphological similarity of their yellow stromata with known teleomorphs of the clade, suggest that the holomorphs of these species may include Beauveria or Beauveria-like anamorphs. The varying host specificity of the beauverioid Cordyceps species suggest the potential importance of identifying the natural host taxon before future consideration of strains for use in biological control of pest locusts.