Diagnostic activities for plant pests

A test was developed that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology.

In  this article it is reported that based on molecular analysis (ctshaplotype and BOX-polymerase chain reaction [PCR] electrophoretic pattern) of strains isolated from different regions of New Zealand two biovars could be distinguished. They have been called biovar 3 and biovar 4 to differentiate them from strains from Japan (biovar 1) or Korea (biovar 2), which have a different ctshaplotype or a different BOX-PCR pattern. Biovar 3, has also been present in Italy since 2008 and in France. 

Guignardia citricarpa Kiely (anamorph Phyllosticta citricarpa Van der Aa),  is subject to phytosanitary restrictions in the EU and USA, and consignments of citrus are rejected at import if citrus black spot is identified on inspection. As an aid to visual inspection of symptoms,  a method for detection ofG. citricarpa using loop-mediated isothermal amplification (LAMP) has been developed which can be used to confirm the presence of G. citricarpa in black spot lesions, including those lacking pycnidia.

Recent years have seen the description of a number of newly recognised species in the genus Phyllosticta that are associated with citrus. As new species emerge, and the taxonomy of the genus is resolved, it will be important to periodically re-evaluate the performance of DNA-based methods for detection of G. citricarpa, including the LAMP assay described here, such that the accuracy of diagnosis can be assured.

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium.

During the 2011/2012 potato growing season, root-knot nematode infected potato tubers were obtained from different potato growing regions in South Africa for identification of Meloidogyne spp. Using the intergenic region of the ribosomal DNA (IGS-rDNA) together with the region between the cytochrome oxidase small subunit II (COII) and the 16S rRNA gene in the mitochondrial DNA (mtDNA), five of the 78 composite samples received produced amplicon sizes of 705 bp for COII and 780 bp for IGS typical of M. enterolobii.  Nucleotide sequencing and phylogenetic analysis of the COII and IGS fragment showed that the five Meloidogyne populations were 100 % similar and they clustered closely with those of M. enterolobii in the GenBank database. The high damage potential of resistance-breaking populations of Meloidogyne species is a threat to profitable potato production and will require effective pest management programmes to be put in place.

Detection and identification of A. anomala is time consuming using conventional methods because the fungus can only be cultured from sporulating perithecia and the disease symptoms and signs only show 12 to 16 months after infection. In this study, a TaqMan real-time polymerase chain reaction (PCR) assay based on a ribosomal DNA internal transcribed spacer was developed for A. anomala. The assay was validated with multiple isolates of A. anomala, closely related species, common environmental microorganisms, and over 100 C. avellana samples. The real-time PCR assay detected as low as 0.12 pg of A. anomala genomic DNA, and positively diagnosed EFB on 82% of asymptomatic plants as early as 15 weeks from infection. The real-time PCR assay is more sensitive and faster than traditional diagnostic methods. It can facilitate hazelnut breeding and disease management by early and accurate diagnosis of EFB.

A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genusPospiviroid, family Pospiviroidae, reported in this ornamental previously.

Two new assays for the detection of Lso in New Zealand field samples were developed, and compared with previously available assays. Firstly, a single-tube semi-nested gel-based PCR assay was developed for the genus-specific detection of liberibacter species, and shown to provide increased sensitivity over standard and nested PCR. Secondly, a single-tube semi-nested SYBR Green real-time PCR (qPCR) assay was developed for the specific detection of Lso in field samples from New Zealand, with a limit of detection of five copies of the target gene per reaction. Semi-nested qPCR showed similar sensitivity compared with TaqMan qPCR with the primer-probe combination LsoF-HLBpr and was 10- to 50-fold more sensitive than the conventional PCR assays tested. Quantification of Lso in field samples of potato and tomato also revealed many samples with titres below the limit of detection of conventional PCR.

A new multiplex polymerase chain reaction (PCR) test was developed that allows to distinguish three different haplotypes: a Japanese/Korean group, a European group, and a Chinese group.The PCR test has a limit of detection of approximately 5 to 50 pg of purified DNA or of 5 × 102bacteria/PCR and was shown to work with both artificially and naturally infected plant tissues. Thus, the described method represents a suitable tool for detection of P. syringae pv. actinidiae and haplotype attribution, in particular, when testing a high number of samples during surveillance and prevention activities.