Emerging, re-emerging and endemic plant pathogens continue to challege our ability to safeguard plant health worldwide. Further, globalization, climate change, increased human mobility, and pathogen and vector evolution have combined to increase the spread of invasive plant pathogens. Early and accurate diagnoses and pathogen surveillance on local, regional, and global scales are necessary to predict outbreaks and allow time for development and application of mitigation strategies. Plant disease diagnostic networks have developed worldwide to address the problems of efficient and effective disease diagnosis and pathogen detection, engendering cooperation of institutions and experts within countries and across national borders. Networking maximizes impact in the face of shrinking government investments in agriculture and diminishing human resource capacity in diagnostics and applied pathology. New technologies promise to improve the speed and accuracy of disease diagnostics and pathogen detection. Widespread adoption of standard operating procedures and diagnostic laboratory accreditation serve to build trust and confidence among institutions. Case studies of national, regional, and international diagnostic networks are presented.

BackgroundPlant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer’s protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed.ResultsRecombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions.ConclusionsRPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0504-8) contains supplementary material, which is available to authorized users.

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Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.

Soybean rust (SBR) survival and host availability (kudzu, Pueraria spp.) were assessed from November 2006 through April 2007 at six sites from the panhandle to southwest Florida. Micro loggers recorded both temperature and relative humidity hourly at each location. Periods of drought and cumulative hours below 0°C correlated with kudzu defoliation. Inoculum potential from detached kudzu leaves was evaluated in vitro under various temperature and relative humidity levels. Kudzu leaves with SBR kept at 4°C produced viable urediniospores with the highest germination at all moisture levels over time. Freezing temperatures (–4 and –20°C) drastically reduced spore germination. However, when leaves were incubated at low (<35%) relative humidity, inoculum potential was prolonged. Results from this study demonstrate that both temperature and relative humidity impact P. pachyrhizi in the field and in vitro, and that detached kudzu leaves have the potential to serve as an inoculum source in kudzu stands.