Presence of Ralstonia pseudosolanacearum (Phylotype I) in Aquatic Environments in the Netherlands.

Ralstonia pseudosolanacearum, a European Union quarantine organism, was until recently absent in the aquatic environments and outdoor cultivation systems of the region. This bacterium was only sporadically reported in restricted greenhouse cultivation systems in some EU countries. In this paper, we report the first findings of R. pseudosolanacearum (phylotype I) in surface water in two distinct geographic locations in the Netherlands in 2020. In 2021, the population of R. pseudosolanacearum in surface water ranged from 104 to 106 CFU/liter. An inoculum reservoir for R. pseudosolanacearum in these aquatic environments was the wild bittersweet plant where population densities ranged from 105 to 107 CFU/ml concentrated bittersweet extract. The virulence of the R. pseudosolanacearum isolates from surface water and bittersweet was confirmed by a pathogenicity test on Solanum lycopersicum cv. Moneymaker plants, resulting in wilting and necrosis of the plants. Sequence analysis of the egl locus of R. pseudosolanacearum isolates from surface water and bittersweet revealed that these isolates are closely related to R. pseudosolanacearum (phylotype I) isolates found previously in the Netherlands on rose. R. pseudosolanacearum (phylotype I) has a very broad host plant range, including potato, many ornamentals, and other economically important crops. This highlights the risk for various host plants grown in the vicinity of the geographic locations where R. pseudosolanacearum has been found and shows the importance of unraveling the epidemiological parameters of the survival, establishment, and spread of R. pseudosolanacearum in temperate climates.

Phylogenetic Assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) Isolated from Rosa spp.

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

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.

Assessment of genotypic diversity of antibiotic-producing pseudomonas species in the rhizosphere by denaturing gradient gel electrophoresis.

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD(+) reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD(+) genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD(+) genotypes directly in rhizosphere samples with a detection limit of approximately 5 x 10(3) CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD(+) genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD(+) isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD(+) genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD(+) genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.