Abstract: The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses.
Abstract: In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.
Abstract: Bemisia tabaci biotype B is considered to be the primary vector of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus). However, Trialeurodes abutiloneus also has been shown to transmit SPCSV in a semipersistent manner. Mixed infection of SPCSV with the aphid-transmitted Sweet potato feathery mottle virus (SPFMV, Potyvirus) causes sweetpotato (Ipomoea batatas) virus disease (SPVD), the major virus disease affecting this crop. High populations of B. afer sensu lato are seasonally associated with sweetpotato in Peru during times of low B. tabaci incidence. The transmission of SPCSV (in single and double infection with SPFMV) by laboratory-reared B. afer sensu lato and B. tabaci biotype B was investigated. For SPCSV transmission efficiency, individual adult insects were allowed 48 h for acquisition and inoculation access periods at both 20 and 25°C. SPCSV was transmitted by both whiteflies, with similar transmission efficiency when the virus was acquired from plants singly infected by SPCSV or doubly infected with SPCSV and SPFMV, at 20 and 25°C. We conclude that B. afer sensu lato is a newly identified vector of SPCSV. This finding may have important epidemiological significance for the spread of SPCSV and SPVD.
Abstract: Identification of the juveniles of economically important thrips species on imports by morphology alone can be challenging and culturing is usually required. In the case of EU quarantine species such as Thrips palmi, rapid and accurate identification is essential. DNA barcoding using the Cytochrome oxidase I (COI) gene has become a popular technique for species identification; however, in some invertebrate genera COI has been shown to provide insufficient variability for species discrimination. This study presents a comparison of five different loci to investigate their ability to discriminate a small number of Thrips species. All five loci discriminated the species by neighbour-joining tree and varying degrees of discrimination were determined upon further investigation of the intraspecific and interspecific distances. Two distinct COI clades were observed for T. palmi and judged to be COI haplotypes when data from the other four additional loci and geographical collection data were taken into consideration. COI was shown to provide sufficient variation to be used in future DNA barcoding efforts within the genus Thrips.
Abstract: A novel, unbiased approach to plant viral disease diagnosis has been developed which requires no a priori knowledge of the host or pathogen. Next-generation sequencing coupled with metagenomic analysis was used to produce large quantities of cDNA sequence in a model system of tomato infected with Pepino mosaic virus. The method was then applied to a sample of Gomphrena globosa infected with an unknown pathogen originally isolated from the flowering plant Liatris spicata. This plant was found to contain a new cucumovirus, for which we suggest the name 'Gayfeather mild mottle virus'. In both cases, the full viral genome was sequenced. This method expedites the entire process of novel virus discovery, identification, viral genome sequencing and, subsequently, the development of more routine assays for new viral pathogens.
Abstract: The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.
Abstract: The whiteflies Trialeurodes lauri and T. ricini have been found to be moving in international plant trade. The taxonomic validity and separation of these species is relevant to the plant health quarantine services of the European Union as T. lauri is oligophagous, nor recorded as a virus vector and present in the EU, whereas T. ricini is polyphagous, reported to be a virus vector, and absent from the EU (except for the Canary Islands). Yet doubt has been cast on the validity of the two species, with the suggestion that T. lauri is merely a variant of T. ricini. The taxonomic relationship was therefore investigated using morphological and molecular data. One morphological character traditionally used for the separation of these two species, the arrangement of the submarginal papillae, was found to be unreliable but morphological differences between the two species were found in the cephalic setal state, body outline and dorsal pigmentation. However, the differences were subtle and not always reliable. The molecular data, based on the sequence of a fragment of the COI gene, support the hypothesis that T. ricini and T. lauri are distinct valid species.
