Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora (amsC gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick TM ), the real-time PCR assays reliably detected at least 10 3 cells mL )l (c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.

The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

In May 2013, 20 plants in a production orchard of kiwifruit (Actinidia deliciosa) cv. Hayward in the seaside area of Primorska showed small, angular, coalescing necrotic leaf spots and cankers on green shoots. In the following 2 weeks, disease progressed to wilting and shoot dieback with exudates. Symptoms were consistent with Pseudomonas syringae pv. actinidiae. Circular, flat, granulated colonies with entire margins were isolated from leaf spots on King's medium B (KB) and on sucrose nutrient agar with boric acid, cephalexine, and cycloheximide. Strains were purified on KB and showed weak fluorescence upon a prolonged incubation (>10 days) and belonged to P. syringae LOPAT group Ia (+---+). DNA was extracted from strains and plant extracts with Chelex 100 resin and Bio-Nobile QuickPick Plant Kit (Turku, Finland), respectively. PCR products of expected sizes were generated by PCR assays (2,4) from all strains and plant extract, supporting the strains as being P. syringae pv. actinidiae. Two strains (NIB Z 1870 and 1871) were further identified by cytochrome C oxidase (negative), glucose metabolism (oxidative), aesculine (negative), and nitrate (negative). Their partial rpoD gene sequences (GenBank Accession Nos. KJ724117 and KJ724118) (3) were identical to the sequence of the P. syringae pv. actinidiae pathotype strain NCPPB 3739 (FN433222, 100% coverage) and to the sequence of P. syringae pv. theae at 96% coverage (FN433271). BOX-PCR fingerprinting and multilocus sequence analysis (MLSA) based on four housekeeping genes gapA (KJ733923 and KJ733924), gltA (KJ733925 and KJ733926), gyrB (KJ733927 and KJ733928), and rpoD identified both strains as biovar 3, a highly virulent biovar of P. syringae pv. actinidiae (5). The pathogenicity of the two strains was confirmed on four plants of A. deliciosa ‘Hayward’ for each strain. Six-month-old plants were sprayed on the abaxial sides of leaves with 30 ml cell suspension prepared from a 72-h-old culture of the appropriate strain (~8 × 106 CFU/ml in 0.01 M MgSO4), covered with plastic bags for 24 h, and incubated under high relative humidity (80%) with 14 h daylight and 24/21°C day/night temperature. Three positive and three negative control plants were inoculated with the Italian P. syringae pv. actinidiae virulent strain K9 (kindly provided by Dr. Gian Luca Bianchi of the Plant Health Service of Friuli Venezia Giulia region) and 0.01 M MgSO4, respectively. After 7 days, water-soaked brown spots with pale green halos were observed on all plants inoculated with bacteria. Re-isolated bacteria were identical to the original strains in their morphology, PCR products, and rpoD sequences. Negative control plants did not develop symptoms, and no growth was observed on media. This is the first laboratory confirmation of bacterial canker of kiwifruit in Slovenia. Visual inspections carried out by the plant health authorities in 2013 and laboratory analysis confirmed additional infection with P. syringae pv. actinidiae in a single, nearby orchard. The pest status of P. syringae pv. actinidiae in Slovenia is officially declared as present, subject to official control (1). References: (1) EPPO Reporting Service. Online publication: http://archives.eppo.int/EPPOReporting/2014/Rse-1402.pdf . No. 02 2014/026, 2014. (2) A. Gallelli et al. J. Plant Pathol 93:425, 2011. (3) N. Parkinson et al. Plant Pathol. 60:338, 2011. (4) J. Rees-George et al. Plant Pathol. 59:453, 2010. (5) J. L. Vanneste et al. Plant Dis. 97:708, 2013.

Foliar necrotic spots with narrow chlorotic halos were observed on different cultivars of Brazilian Jasmine (Mandevilla sanderi) during spring 2010 in several commercial greenhouses in Slovenia. Up to 70% were symptomatic and were unmarketable. No galls were observed on the stems of symptomatic plants. Circular, flat, granulated colonies with entire margins were isolated from symptomatic leaves of two plants from different greenhouses on King's B medium (KB). The isolates were negative for levan, oxidase, pectinolytic and arginine dihydrolase activity. They caused a hypersensitive reaction on tomato but not on tobacco cv. White Burley. Isolates were weakly fluorescent on KB under UV light. One isolate per sample (NIB Z 1413 and 1415) was further characterized. Partial sequences of 16S rDNA (1; GenBank KM603318 of 722 bp, KM603319 of 686 bp) grouped the isolates within genomospecies 2 of Pseudomonas. Repetitive polymerase chain reaction (PCR) assay using the BOXA1R primer (5) resulted in highly similar DNA fragment banding patterns of the two NIB Z isolates and other reference strains of genomospecies 2 (minimum 95.1% identity with Pearson's correlation). Partial sequences of rpoD (3) of the two Slovenian isolates (600 bp; GenBank KJ744202, KJ744201) were identical to the P. savastanoi isolate from Mandevilla B200 (W. Wohanka, Germany; GenBank KJ744203) and P. s. pv. nerii strain NCPPB 3334 (GenBank AB039513). The sequences differed in two nucleotides relative to the sequence of the pathotype strain of pv. nerii NCPPB 3278 (positions 487 and 510 relative to GenBank FN433279) and had an insertion of six nucleotides compared to available P. savastanoi pv. savastanoi rpoD sequences (NZ_JOJV01000073, CM001834). Pathogenicity of isolated bacteria (two isolates) was determined on M. sanderi cv. Pretty Rose inoculated by two different methods, spraying foliage and pricking stems. The abaxial and adaxial surfaces of leaves were sprayed with a 30-ml bacterial suspension (5 × 106 CFU/ml). Three plants were inoculated with each isolate: NIB Z 1413 and 1415 and the reference strain NCPPB 3278. Necrotic spots developed on leaves after 14 days of incubation, under >80% high relative humidity, with 16 h of daylight at 25°C and 8 h of dark at 21°C. One month after inoculation, necrosis also developed on stems and new growth. Inoculation of bacteria by pricking nodes of healthy M. sanderi cv. Pretty Rose with a needle dipped in the isolates grown on KB for 24 h (each of NIB Z 1413, 1415, and NCPPB 3278 for positive control) led to development of galls in 14 days at the inoculation points. The re-isolation was performed separately from necrotic spots on leaves, stems, new growth above the inoculation points, and galls. The BOX-PCR profiles of the bacteria isolated from symptomatic tissues were identical to the original profiles, thus confirming the systemic spread of the bacteria. None of the three negative control plants sprayed with 0.01M MgSO4 or pricked with a sterile needle developed symptoms. This is the first report of P. savastanoi on Mandevilla sanderi plants in greenhouse production in Slovenia. The galls caused by P. savastanoi have previously been reported from the United States (4) and Germany (2). This report broadens the geographical area where P. savastanoi, causing both galls on stems and necrotic spots on leaves, can be found in commercial production of Mandevilla spp. References: (1) U. Edwards et al. Nucleic Acids Res. 17:7843, 1989. (2) N. Eltlbany et al. Appl. Environ. Microbiol. 78:8492, 2012. (3) N. Parkinson et al. Plant Pathol. 60:338, 2011. (4) M. L. Putnam et al. Phytopathology 100:S104, 2010. (5) J. Versalovic et al. Methods Mol. Cell Biol. 5:25, 1994.