PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data.

BACKGROUND: Detection of exotic plant pathogens and preventing their entry and establishment are critical for the protection of agricultural systems while securing the global trading of agricultural commodities. High-throughput sequencing (HTS) has been applied successfully for plant pathogen discovery, leading to its current application in routine pathogen detection. However, the analysis of massive amounts of HTS data has become one of the major challenges for the use of HTS more broadly as a rapid diagnostics tool. Several bioinformatics pipelines have been developed to handle HTS data with a focus on plant virus and viroid detection. However, there is a need for an integrative tool that can simultaneously detect a wider range of other plant pathogens in HTS data, such as bacteria (including phytoplasmas), fungi, and oomycetes, and this tool should also be capable of generating a comprehensive report on the phytosanitary status of the diagnosed specimen. RESULTS: We have developed an open-source bioinformatics pipeline called PhytoPipe (Phytosanitary Pipeline) to provide the plant pathology diagnostician community with a user-friendly tool that integrates analysis and visualization of HTS RNA-seq data. PhytoPipe includes quality control of reads, read classification, assembly-based annotation, and reference-based mapping. The final product of the analysis is a comprehensive report for easy interpretation of not only viruses and viroids but also bacteria (including phytoplasma), fungi, and oomycetes. PhytoPipe is implemented in Snakemake workflow with Python 3 and bash scripts in a Linux environment. The source code for PhytoPipe is freely available and distributed under a BSD-3 license. CONCLUSIONS: PhytoPipe provides an integrative bioinformatics pipeline that can be used for the analysis of HTS RNA-seq data. PhytoPipe is easily installed on a Linux or Mac system and can be conveniently used with a Docker image, which includes all dependent packages and software related to analyses. It is publicly available on GitHub at https://github.com/healthyPlant/PhytoPipe and on Docker Hub at https://hub.docker.com/r/healthyplant/phytopipe .

Translating virome analyses to support biosecurity, on-farm management, and crop breeding.

Virome analysis via high-throughput sequencing (HTS) allows rapid and massive virus identification and diagnoses, expanding our focus from individual samples to the ecological distribution of viruses in agroecological landscapes. Decreases in sequencing costs combined with technological advances, such as automation and robotics, allow for efficient processing and analysis of numerous samples in plant disease clinics, tissue culture laboratories, and breeding programs. There are many opportunities for translating virome analysis to support plant health. For example, virome analysis can be employed in the development of biosecurity strategies and policies, including the implementation of virome risk assessments to support regulation and reduce the movement of infected plant material. A challenge is to identify which new viruses discovered through HTS require regulation and which can be allowed to move in germplasm and trade. On-farm management strategies can incorporate information from high-throughput surveillance, monitoring for new and known viruses across scales, to rapidly identify important agricultural viruses and understand their abundance and spread. Virome indexing programs can be used to generate clean germplasm and seed, crucial for the maintenance of seed system production and health, particularly in vegetatively propagated crops such as roots, tubers, and bananas. Virome analysis in breeding programs can provide insight into virus expression levels by generating relative abundance data, aiding in breeding cultivars resistant, or at least tolerant, to viruses. The integration of network analysis and machine learning techniques can facilitate designing and implementing management strategies, using novel forms of information to provide a scalable, replicable, and practical approach to developing management strategies for viromes. In the long run, these management strategies will be designed by generating sequence databases and building on the foundation of pre-existing knowledge about virus taxonomy, distribution, and host range. In conclusion, virome analysis will support the early adoption and implementation of integrated control strategies, impacting global markets, reducing the risk of introducing novel viruses, and limiting virus spread. The effective translation of virome analysis depends on capacity building to make benefits available globally.

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated with potato ZC, and will be very useful for the potato quarantine programs and seed potato certification programs to ensure the availability of clean seed potato stocks and also for epidemiological studies on the disease.

Survival of Inoculum of Phytophthora capsici in Soil Through Time Under Different Soil Treatments.

From September 2001 until August 2002 and from September 2002 until August 2003, inoculum of Phytophthora capsici consisting of mycelium and oospores was buried in soil under three different soil treatments: soil solarization, fumigation with methyl bromide and chloropicrin, and white-on-black plastic mulch without fumigation or solarization. The effect of these soil treatments on the population and survival of P. capsici was evaluated through time after 28, 63, 119, 175, 245, and 343 days. Three techniques were used for detection of the localized inoculum in soil: soil dilution plating (SDP), a modified soil dilution plating technique with an overlay assay to allow for extra incubation (mSDPO), and lemon leaf baiting of soil (LLB). No viable inoculum was detected from any soil samples from the fumigated plots regardless of the soil detection technique used. By the last sampling date, viable oospore inoculum was still detected in both soil solarization and nontreated soils, but only using mSDPO and LLB. Overall, the mSDPO assay was the most sensitive assay, followed by LLB. Using mSDPO, populations in the last sampling date were 32.9 CFU/g soil for the untreated plots and 14.7 CFU/g soil for the solarized plots. Survival of P. capsici for a year would indicate that oospores have the potential to survive from year to year, and possibly much longer, in Florida and other locations.

Characterization of Phytophthora capsici Associated with Roots of Weeds on Florida Vegetable Farms.

Weeds were sampled in commercial vegetable fields in Palm Beach County, FL in August 2001, December 2001, and March 2002 for the presence of Phytophthora capsici. Fields sampled had a recent history of this plant pathogen. P. capsici was successfully isolated from the roots of six of 42 Carolina geranium (Geranium carolinianum) plants, four of 28 American black nightshade (Solanum americanum) plants, and two of 130 common purslane (Portulaca oleracea) plants. All but one of the 12 isolates were of the A1 mating type. All 12 isolates were resistant to mefenoxam, although at different levels. All but one isolate were strongly pathogenic on pepper seedlings. When two or three isolates recovered from each weed were inoculated onto the roots of their weed host of origin, P. capsici was recovered from the roots. Isolates of P. capsici were tested on four other solanaceous weeds of importance or potential importance to agricultural fields in Florida: Solanum nigrum, S. ptycanthum, S. carolinense, and S. capsicoides. Recovery of P. capsici from roots varied with weed species and isolate tested. P. capsici caused disease mortality on S. nigrum, and no reisolation of P. capsici was possible with S. capsicoides. This is the first report of S. americanum and G. carolinianum being alternative hosts for P. capsici under field conditions. This study also validated P. oleracea as an alternative host. In Florida, and perhaps elsewhere, weeds may contribute to pathogen survival in the absence of a host crop or when propagules may not readily survive in soil or plant debris.