qPCR Assays for the Detection of Cylindrocladium buxicola in Plant, Water, and Air Samples.

Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the ß-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.

First Report of Xanthomonas arboricola pv. pruni in Ornamental Prunus laurocerasus in the Netherlands.

In 2008, Dutch ornamental plant growers observed a leaf spot of cherry laurel (Prunus laurocerasus) at a greater incidence (5 to 50%) than the usual sporadic level (<1%). For advice on disease control, ~5 to 10% of these growers contacted Dutch regulatory officials. In November and December 2008, six symptomatic samples from northern and southern parts of the Netherlands were submitted for diagnosis. Leaf spots were chlorotic, most had a necrotic brown center with a distinct margin, and the spots readily abscised, resulting in a "shot-hole" appearance. Leaf spots from the samples were surface sterilized (2 s in 70% vol/vol alcohol), blotted dry on tissue paper, chopped into pieces (1 to 2 mm in diameter), and incubated for 30 min in 10 mM phosphate-buffered saline (PBS) (1). A 20-µl aliquot of extract per sample was streaked by dilution plating on four plates of yeast peptone glucose agar medium (1), and the plates were incubated for 2 to 3 days at 28°C. Isolations from all six samples yielded Xanthomonas-like colonies. After purification, characterization of all six isolates revealed oxidative, nonfermentative metabolism of glucose by rod-shaped, gram-negative bacterial cells. All six isolates were identified as Xanthomonas arboricola pv. pruni based on biochemical tests (1), fatty acid analysis (4), and immunofluorescence (IF) using polyclonal antibodies (Plant Research International, the Netherlands). Pathogenicity was tested on potted peach plants (cvs. Peregrine and Vaes Oogst) and on detached leaves of P. laurocerasus (cv. Novita) (1). The six field isolates from 2008 were each inoculated (108 CFU/ml) onto four leaves per plant of each of two peach plants (replicates). As positive control treatments, two reference strains (ATCC 19312 and PD740) were each inoculated onto the same number of leaves and plants, and as a noninoculated negative control treatment, leaves of two peach plants were treated with sterile 10 mM PBS buffer (1). All leaves inoculated with the six field isolates and the two reference strains developed typical bacterial spot symptoms in 3 to 4 weeks. Negative control plants showed no symptoms. The detached leaf assay performed with the same treatments on each of two leaves (replicates) showed identical results. The bacterium was reisolated from leaf spots associated with each of the eight symptomatic treatments and identity of the reisolates was confirmed by IF. Additionally, genotypic variation of 35 Dutch isolates of X. arboricola pv. pruni was assessed by BOX-PCR assay with the BOX A1R primer set (3), and Gyrase B gene sequencing (2). Both methods revealed 100% homology among the 35 isolates, suggesting a single, recent introduction of X. arboricola pv. pruni into the Netherlands. In a 2009 survey to assess distribution of the disease in the Netherlands, X. arboricola pv. pruni was found in 41 fields. Infected hosts included P. laurocerasus cvs. Otto Luyken, Rotundifolia, Novita, Etna, Anbri, Herbergii, Mischeana, and Caucasia. X. arboricola pv. pruni is a quarantine organism in countries affiliated under the EPPO (European and Mediterranean Plant Protection Organization). Phytosanitary measures were taken to prevent movement of infested plants from nurseries where X. arboricola pv. pruni was detected. References: (1) Anonymous. EPPO Bull. 36:129, 2006. (2) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009. (3) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994. (4) S. A. Weller et al. EPPO Bull. 30:375, 2000.

First Report of Tomato yellow leaf curl virus in Tomato in the Netherlands.

Tomato yellow leaf curl virus (TYLCV) is an economically important virus with tomato (Solanum lycopersicum L.) as its main host. The virus is widely distributed in subtropical areas and is transmitted by the tobacco whitefly (Bemisia tabaci) in a persistent manner. TYLCV has a quarantine status (IIAII) in the European Union (EU directive 2000/29/EC). It was not previously recorded in the Netherlands. In September 2007, symptoms were observed in tomato crops in a few greenhouses located in close proximity from each other in the western part of the Netherlands. Infected plants showed TYLCV-like symptoms, i.e., stunting, leaf curl, and marginal and interveinal chlorosis. Similar symptoms were evoked after grafting symptomatic tips onto healthy tomato seedlings, whereas no viruses were transmitted by mechanical inoculation to herbaceous test plants. Extracted DNA from symptomatic leaves was used in PCR with two sets of primers for universal detection of begomoviruses (1,2). Analysis of the overlapping amplified products revealed the highest identity to isolate TYLCV-Alm (GenBank Accession No. AJ489258) from Almeria, Spain. To amplify the remaining 60% of the virus genome, three additional primer sets were designed: TYLCV965F 5'-GGCAGCCAAGTACGAGAACC-3' and TYLCV1736R 5'-CCACTATCTTCCTCTGCAATCC-3'; TYLCV1598F 5'-TACTTGCGAACAGTGGCTCG-3' and TYLCV2282R 5'-TCCAAATCGATGGCAGATCAG-3'; TYLCV2229F 5'-ATGCGTCGTTGGCAGATTG-3' and TYLCV68R 5'-CAGTGACGTCTGTGGAACCCT-3'. Analysis of the five overlapping PCR products of one isolate revealed a total virus genome of 2,781 nucleotides. The complete sequence of the Netherlands Isolate (GenBank Accession No. FJ439569) showed 99.3% nucleotide identity to isolate TYLCV-Alm (AJ489258), and therefore, the virus was identified as TYLCV-Alm. After the initial identification, a survey was conducted in all tomato crops in a surrounding area of approximately 40 km2. TYLCV was found in 19 of 27 cultivations. The identity of one isolate per cultivation was confirmed by sequence analysis of the products obtained with the Wyatt and Brown primers (2) occasionally in combination with the Deng primers (with 99.1 to 100% and 99.2 to 100% nucleotide identity to the Netherlands isolate [FJ439569], respectively) (1). As many as 25 symptomatic plants were recorded per greenhouse. A subsequent survey of 34 randomly selected tomato growers in other areas of the country revealed no further infections. Results of the sequence analyses and surveys suggested that the outbreak resulted from a single introduction of the virus, whereas the insect vector B. tabaci accounted for local spread. Measures taken to eliminate the virus included the removal and subsequent destruction of infected tomato plants as well as eradication of B. tabaci. No TYLCV infections were found during surveys in 2008, and therefore, it is believed that the virus was eradicated effectively. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.