Unrestricted Access to T-BAS Phylogeny for Emerging Phytophthora Species
by Jorge Luis Alonso with ChatGPT
North Carolina State University conducted a study published in PLOS ONE with three goals: to create an open T-BAS phylogeny by collecting information from published sources, to make it easy to update as new Phytophthora species are discovered, and to develop a search engine to identify different genotypes of P. infestans SSR data. This is a summary of its contents.
Phytophthora is a destructive genus of oomycete plant pathogens that cause severe plant diseases in food crops, ornamentals, forests, and riparian ecosystems. Within the genus, Phytophthora infestans is the first described species and was responsible for the Irish potato famine in the 1840s. It is a species that continues to pose a significant threat to crop production worldwide. For example, P. ramorum and P. cinnamomi are threaten agricultural production and natural ecosystems; P. megakarya is responsible for the black pod disease of cocoa in West Africa, and P. palmivora threatens cocoa production worldwide.
Researchers have used morphological features to identify Phytophthora species based on observed characteristics, and matrix-based Lucid keys have been developed to aid in species identification. However, the number of newly discovered species has increased greatly (Fig 1), resulting in many recently described species that are not included in the Lucid key resource.
It should be noted that molecular identification methods such as single gene or multilocus sequencing and whole genome sequencing have become essential in species identification. Multilocus genotyping is used to differentiate species and a robust phylogeny has been developed for the genus, including many newly described species.
The IDphy tool includes a detailed Lucid key for species identification based on the morphology of over 160 species, as well as fact sheets with images and tools for performing Basic Local Alignment Search Tool (BLAST) searches to identify species based on multilocus sequences. However, there is still a need to integrate the available Phytophthora multilocus sequence data into a centralized maximum likelihood tree with metadata to facilitate species identification and to study the evolution and emergence of species within the genus.
In this context, the research team has used the Tree-Based Alignment Selector (T-BAS) software toolkit to integrate phylogenetic alignment and visualization of biological metadata. A summary of their methods, results, discussion and future directions is provided below.
Methods
To collect data on different Phytophthora species, the research team decided to download publicly available data from a database called GenBank. However, they encountered some problems with the accuracy of the identification and naming of some isolates. To overcome this problem, they used the ITS (Internal Transcript Spacer) and CoxI datasets from known collections and species descriptions for new species. They also double-checked isolates with known identification problems to ensure accuracy.
They also amplified and sequenced seven loci for two newly described Phytophthora species and tested the placement of other species in the evolutionary tree using multiple sequence datasets. They collected isolates for each species, extracted DNA using the cetyltrimethylammonium bromide (CTAB) method, and used primers from previous studies to amplify each locus. They then sequenced the amplified regions using Sanger sequencing, resulting in sequence data for 194 Phytophthora species, 30 informal Phytophthora taxa, and 3 outgroups of related oomycete genera.
The researchers used this data set to construct phylogenetic trees using Randomized Axelerated Maximum Likelihood (RAxML) with 1000 bootstrap replicates under the GTRGAMMA model. They compared the resulting trees to the most recent phylogeny by adding 50 new taxa using this larger set of loci.
Results
As mentioned above, the study examined a total of 194 Phytophthora species and 33 related species using a method called phylogeny. To create the phylogeny, they looked at eight different parts of the genetic code and used RAxML to find the most likely relationships between the different species. The researchers also compared the Phytophthora species with other similar species to see if they belonged to the same group. They found that the Phytophthora species were all closely related to each other, forming a single group called a monophyletic clade.
The results of this study were consistent with previous research, but some new information was discovered. For example, some species previously thought to be related were found to be more distantly related, while others were found to be more closely related than previously thought. In addition, the researchers found that some Phytophthora species are more likely to live in the soil, while others are more likely to live in the air. They also found that the degree of host specificity varied widely among the different species. Overall, this study provides new insights into the relationships and characteristics of the genus Phytophthora.
Discussion
The phylogeny presented in this text resolves some questions regarding the placement of certain species and extends currently published phylogenies. For example, the placement of P. quercina has changed since its inclusion in the first molecular phylogeny of the genus Phytophthora. On the other hand, the phylogeny supports the conclusion that P. quercina, P. versiformis, P. tubulina, and P. castanetorum form a unique monophyletic clade referred to as clade 12. Besides, the addition of new species in this work supports the subdivision of clade 9 into clades 9a and 9b, where 9b is monophyletic and 9a consists of three monophyletic subclades, 9a1, 9a2, 9a3. The clade placement of P. acaciae and P. betacei was also confirmed.
Future directions
To expand the Phytophthora genus phylogeny, researchers can use the T-BAS system to add metadata, sequence data, and new taxa as new species are discovered. They can also add taxonomic characters and phenotypic data to the tree if it’s relevant to the larger community. It should be noted that researchers must submit metadata and sequence data for curation via the T-BAS website before uploading.
By regularly updating the phylogeny, scientists can keep pace with new discoveries of species and characters. Adding metadata to the phylogeny could also help Phytophthora researchers identify where new outbreaks are occurring. As a result, the T-BAS tool will expand its mapping capabilities, and a community of researchers involved in Phytophthora research will need to validate the genus-level phylogeny and P. infestans classifier.
To identify a species, researchers must use morphology and sequence the loci used in this study for phylogenetic placement in the T-BAS. For new species descriptions, researchers should study them more thoroughly and sequence at least three nuclear loci in addition to the morphological requirements. Placement in both the nuclear and mitochondrial trees is also recommended for species identification. The T-BAS system makes it easy to add new information to the phylogeny and make it available to the community without having to publish an updated description.
Newer Phytophthora species are often found in natural ecosystems, such as riparian buffers, and aquatic and forested areas, rather than in agricultural systems. Researchers should explore these areas to discover more Phytophthora species.
The usefulness of the T-BAS system for the plant-pathogenic genus Phytophthora could serve as a model for other pathogen phylogenies. For example, the creation of a separate living phylogeny for downy mildew fungi could be an excellent additional tool for the oomycete research community. In addition, enhancing pathogen phylogenies with metadata and live taxon placement will facilitate research on a variety of pathogen species. Researchers should share and standardize data, including the phylogeny, multiple sequence alignments and sequence data, biological sample data, specimen vouchers, and other associated metadata.
The live phylogeny presented here relies on the open sharing of data, including cultures of newly described species, among researchers and their deposit in respected culture collections by the global Phytophthora research community. Many, but not all, of the species described here, are available to researchers from the Ristaino Phytophthora Collection at NC State.
Conclusion
The team has accomplished significant milestones in developing a live phylogeny for the Phytophthora genus and creating a microsatellite-based classifier for P. infestans genotypes. Here are the details in bullet points:
Live Phylogeny for Phytophthora Genus:
- Developed a live phylogeny for the Phytophthora genus
- Updated by the community, making it an important resource for future research
- Successfully integrated newly discovered species to the phylogeny (Table 1)
- Provides an innovative live format for tracking the evolution of the Phytophthora genus
- Can be extended to other pathogenic microbes, making it a valuable tool for the broader scientific community
Microsatellite-Based Classifier for P. Infestans Genotypes:
- Created a microsatellite-based classifier for P. infestans genotypes
- Allows for faster identification of P. infestans genotypes
- Essential to update the classifier regularly to keep up with the increasing number of genotypes
- Provides a reliable method for identifying P. infestans genotypes for disease management and control
Overall, these developments are a significant breakthrough in pathogenic microbiology research and provide researchers with valuable tools to further advance the field.
Source: Coomber, A., Saville, A., Carbone, I., & Ristaino, J. B. (2023). An open-access T-BAS phylogeny for emerging Phytophthora species. PLOS ONE, 18(4), e0283540.
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