Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence.

The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains unclear, especially in a field-based production such as for leek plants in Flanders. Therefore, more insight in bacteria-phage interaction is required, along with the evaluation of different application strategies. In this study, we further characterized the infection strategy of two Pspo phages, KIL3b and KIL5. We found that both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium. LPS is an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests showed that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. However, the impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. Our data suggest that the phage cocktail has promising potential to lower the prevalence of Pspo and to be integrated in a pest management strategy. Targeted research is needed to further explore the applicability of the phages in combination with other disease control strategies.

Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.

Four bacterial strains, designated M89, M92, M97(T), and M106, were isolated in a previous study from surface-sterilized leaves of rice (Oryza sativa) or murainagrass (Ischaemum rugosum) at three sites in Mali, Africa. Here they were examined by a polyphasic taxonomic approach and analysis of a whole-genome sequence. Phylogenetic analyses based on 16S rRNA sequence and multilocus sequence analysis of seven genes showed that these four strains formed a distinct lineage representing a novel species within the genus Xanthomonas. This was supported by whole-genome average nucleotide identity values calculated from comparisons of strain M97(T) with established Xanthomonas species. The strains can be differentiated from the known Xanthomonas species on the basis of their fatty acid and carbohydrate utilization profiles. Population growth studies on rice confirmed that these bacteria multiply in rice leaves without causing symptoms. Identification of this novel species can be accomplished by using diagnostic primer sets or by gyrB gene sequence analysis. We propose to classify these rice- and grass-associated bacteria as Xanthomonas maliensis sp. nov. with strain M97(T) = CFBP7942(T) = LMG27592(T) as the type strain.

Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle.

BACKGROUND: The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health. RESULTS: To get more insight in the genetic features linked to the lifestyle of these bacteria, a whole-genome sequence of the tomato seed-borne non-pathogenic Clavibacter LMG 26808 was determined. To gain a better understanding of the molecular determinants of pathogenicity, the genome sequence of LMG 26808 was compared with that of the pathogenic Cmm strain (NCPPB 382). The comparative analysis revealed that LMG 26808 does not contain plasmids pCM1 and pCM2 and also lacks the majority of important virulence factors described so far for pathogenic Cmm. This explains its apparent non-pathogenic nature in tomato plants. Moreover, the genome analysis of LMG 26808 detected sequences from a plasmid originating from a member of Enterobacteriaceae/Klebsiella relative. Genes received that way and coding for antibiotic resistance may provide a competitive advantage for survival of LMG 26808 in its ecological niche. Genetically, LMG 26808 was the most similar to the pathogenic Cmm NCPPB 382 but contained more mobile genetic elements. The genome of this non-pathogenic Clavibacter strain contained also a high number of transporters and regulatory genes. CONCLUSIONS: The genome sequence of the non-pathogenic Clavibacter strain LMG 26808 and the comparative analyses with other pathogenic Clavibacter strains provided a better understanding of the genetic bases of virulence and adaptation mechanisms present in the genus Clavibacter.

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.

Temperature-dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii.

The broad mite, Polyphagotarsonemus latus (Banks), is one of the major pests causing severe economic damage in Rhododendron simsii Planch hybrid production in Belgium. In order to optimize biological control programs and to parameterize warning programs, we studied the effect of environmental temperature on the development of P. latus on R. simsii leaves. In combination with a photoperiod of 16:8 h (L:D) and a relative humidity of 80 ± 5 %, six constant temperatures (15, 17, 20, 25, 30 and 33 ± 1 °C), were studied. Total developmental times of 13.3, 10.5, 6.6, 4.2, 3.5 and 4.0 days were measured, respective to each of the aforementioned temperatures. Development of females took significantly longer than that of males at 15, 17, 20 and 30 °C. Survival rates observed between 17 and 30 °C varied between 43.5 and 96.9 %. Lower survival rates were found at 15 and 33 °C, i.e. 31.8 and 23.6 %, respectively. The lower, optimal and upper developmental threshold (t min , t opt and t max , respectively) and thermal constant (K) of the pest were estimated for each life stage by a linear and two non-linear models. Based on measurements of total development of P. latus thermal thresholds of 10.0, 30.1 and 36.0 °C were calculated for t min , t opt and t max , respectively. The number of degree-days needed to complete immature development when feeding on R. simsii was 66.7.

Draft genome sequence of Xanthomonas fragariae reveals reductive evolution and distinct virulence-related gene content.

BACKGROUND: Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content. RESULTS: The Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cell-wall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ß-ketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones. CONCLUSIONS: The draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant.

Multilocus variable-number-tandem-repeats analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium.

BACKGROUND: Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains. RESULTS: A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010-2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons. CONCLUSIONS: We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm.

Survival of enteric pathogens during butterhead lettuce growth: crop stage, leaf age, and irrigation.

The survival of Salmonella enterica serovar Thompson and Escherichia coli O157 was investigated on growing butterhead lettuce plants in the plant-growth chamber and greenhouse. All inoculation tests were made under conditions that approximate the greenhouse conditions for butterhead lettuce cultivation in Flanders (Belgium). The survival and proliferation of the pathogens on the leaves was determined at days 0, 4, and 8 after inoculation using standard plating techniques on selective medium. In the growth chamber, the extent to which both pathogens were able to multiply on the lettuce leaves was influenced by crop stage and leaf age. On young plants, the older leaves supported pathogen survival better. On nearly mature plants, pathogen population sizes were significantly higher on the old and young leaves compared with middle-aged leaves (p<0.001). In the greenhouse, the environmental regimen with high fluctuations in temperature and relative humidity was less conducive to the survival of E. coli O157, though its survival on nearly mature lettuce was enhanced by overhead irrigation. The moist conditions between the folded inner leaves are likely contributing to the survival of enteric pathogens in the lettuce head. Butterhead lettuce grown in greenhouses with a sprinkle irrigation system may present a potential health hazard when contaminated near harvest. Experimental design (growth chamber versus greenhouse) largely influences enteric pathogen behavior on growing lettuce plants.

A suggested new bacteriophage genus: "Viunalikevirus".

We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.

The potential of bacteriophages to control Xanthomonas campestris pv. campestris at different stages of disease development.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that invades the xylem of Brassica crops. Current chemical and antibiotics-based control measures for this bacterium are unsustainable and inefficient. After establishing a representative collection of Xcc strains, we isolated and characterized bacteriophages from two clades of phages to assess their potential in phage-based biocontrol. The most promising phages, FoX2 and FoX6, specifically recognize (lipo) polysaccharides, associated with the wxc gene cluster, on the surface of the bacterial cell wall. Next, we determined and optimized the applicability of FoX2 and FoX6 in an array of complementary bioassays, ranging from seed decontamination to irrigation- and spray-based applications. Here, an irrigation-based application showed promising results. In a final proof-of-concept, a CaCl2 -formulated phage cocktail was shown to control the outbreak of Xcc in the open field. This comprehensive approach illustrates the potential of phage biocontrol of black rot disease in Brassica and serves as a reference for the broader implementation of phage biocontrol in integrated pest management strategies.

Aberrant genome size and instability of Phytophthora ramorum oospore progenies.

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen's ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses".

Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."

Identification of new polymorphic microsatellite markers in the NA1 and NA2 lineages of Phytophthora ramorum.

Phytophthora ramorum is a recently introduced pathogen in Europe and North America consisting of three clonal lineages. Due to the limited intralineage genetic variation, only a few polymorphic markers are available for use in studies involving the epidemiology and evolution of P. ramorum. A total of 159 primer pairs for candidate polymorphic SSR loci were tested with universal labeling. Four polymorphic microsatellite loci were identified within the NA1 lineage and one within the NA2 lineage, demonstrating the power and flexibility of the screening technique. The markers may significantly increase the number of genotypes that can be identified and as such can help better characterize the North American lineages of P. ramorum.

Willow wood sap promotes the density-dependent pathogenesis of Brenneria salicis.

Brenneria salicis resides in symptomless willow (Salix spp.) and other tree species, but only willow trees develop watermark disease. To understand the conversion of B. salicis into a pathogen, its pathogenicity and differential growth in the various tree species are studied. Brenneria salicis was detected by plating and polymerase chain reaction-based techniques. Cell wall degradation and quorum sensing (QS) were assayed as possible pathogenicity mechanisms in wood. Differences in B. salicis growth capacities were tested in wood sap of the trees. Watermark diseased willow wood contained high concentrations of B. salicis with QS-induced cellulase activity. In the fall, wood sap of willow, and not of poplar and alder, promoted high density growth of B. salicis. In situ, B. salicis was the dominant bacterial type in willow wood during the fall and winter period. Willow sustains high densities of B. salicis at the time of leaf shedding. The cellulase in the immobilized wood sap has then a long-lasting contact with the xylem cell wall. Timing of dormancy and subsequent winter conditions might interfere with sap composition, B. salicis density, activity and survival, and be the reason, at least partly, for the variable occurrence of the disease.

Brenneria salicis, the bacterium causing watermark disease in willow, resides as an endophyte in wood.

Brenneria salicis has been studied in willow wood only in relation to watermark disease. In this pathogenic condition, the bacterium occurs at high concentrations. Pathogenicity of B. salicis is still uncontrollable and the disease unpredictable because the plant-bacteria interaction is not understood. Thanks to molecular techniques B. salicis can be detected at low concentrations, which are found in most non-pathogenic interactions. Brenneria salicis was identified and traced with a new specific three-primer polymerase chain reaction and its identity and relative concentration in biological samples confirmed through denaturing gradient gel electrophoresis profiling. Brenneria salicis was found in symptomless willows sampled randomly in Flanders agricultural areas, in young nursery willows, and also in poplar (Populus) and alder (Alnus). It harboured the nitrogenase reductase gene NifH and promoted growth and chlorophyll in willow. Inoculated luminescence-marked B. salicis circulated through the whole plant without inducing disease and exuded at the leaf margins. Other willow endophytes identified were Rahnella, Sphingomonas and Methylobacterium. In conclusion, because endophytic B. salicis is generally observed in willow, disease must not be dependent on infection. Leaf-to-leaf spread is proposed as an important mechanism for spread of B. salicis.

Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.

Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.

Pseudomonas cichorii as the causal agent of midrib rot, an emerging disease of greenhouse-grown butterhead lettuce in Flanders.

Bacterial midrib rot of greenhouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) is an emerging disease in Flanders (Belgium) and fluorescent pseudomonads are suspected to play an important role in the disease. Isolations from infected lettuces, collected from 14 commercial greenhouses in Flanders, yielded 149 isolates that were characterized polyphasically, which included morphological characteristics, pigmentation, pathogenicity tests by both injection and spraying of lettuce, LOPAT characteristics, FAME analysis, BOX-PCR fingerprinting, 16S rRNA and rpoB gene sequencing, as well as DNA-DNA hybridization. Ninety-eight isolates (66%) exhibited a fluorescent pigmentation and were associated with the genus Pseudomonas. Fifty-five of them induced an HR+ (hypersensitive reaction in tobacco leaves) response. The other 43 fluorescent isolates were most probably saprophytic bacteria and about half of them were able to cause rot on potato tuber slices. BOX-PCR genomic fingerprinting was used to assess the genetic diversity of the Pseudomonas midrib rot isolates. The delineated BOX-PCR patterns matched quite well with Pseudomonas morphotypes defined on the basis of colony appearance and variation in fluorescent pigmentation. 16S rRNA and rpoB gene sequence analyses allowed most of the fluorescent isolates to be allocated to Pseudomonas, and they belonged to either the Pseudomonas fluorescens group, Pseudomonas putida group, or the Pseudomonas cichorii/syringae group. In particular, the isolates allocated to this latter group constituted the vast majority of HR+ isolates and were identified as P. cichorii by DNA-DNA hybridization. They were demonstrated by spray-inoculation tests on greenhouse-grown lettuce to induce the midrib rot disease and could be re-isolated from lesions of inoculated plants. Four HR+ non-fluorescent isolates associated with one sample that showed an atypical midrib rot were identified as Dickeya sp.

Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum x morifolium based on rDNA ITS sequence analysis.

Isolates of the most important Puccinia species that have been reported on Chrysanthemum x morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.

Identification of quorum sensing signal molecules and oligolignols associated with watermark disease in willow (Salix sp.).

The bacterium Brenneria salicis is the causal agent of watermark disease in willow. This work shows the importance of in situ studies and high-resolution separation of biological samples with ultrahigh performance liquid chromatography combined with ion trap mass spectrometry to unambiguously identify molecular compounds associated with this disease. Approximately 40 oligolignols accumulated in wood sap of watermark diseased willow, and are indicative for degradation of the xylem cell wall, of which 15 were structurally assigned based on an earlier study. Many bacteria are known to produce and release quorum sensing signal molecules that switch on the expression of specific, sometimes pathogenic functions. Two quorum sensing signal molecules, N-(3-oxohexanoyl)-l-homoserine lactone and N-(hexanoyl)-l-homoserine lactone, were present in 4/1 ratios in diseased wood and in high-density in vitro cultures of B. salicis at 0.13-1.2 microM concentrations, and absent in healthy wood and in low-density in vitro cultures of B. salicis. Although it is not a proof, it can be an indication for involvement of quorum sensing in B. salicis pathogenesis. Cyclic dipeptides were present at high concentrations in high-density in vitro cultures of B. salicis, but not in situ, and were found not to be involved in quorum sensing signaling, therefore, the attribution of quorum signal properties to cyclic dipeptides isolated from in vitro cultures of pathogenic bacteria should be reconsidered.

The Use of High-Throughput Sequencing for the Study and Diagnosis of Plant Viruses and Viroids in Pollen.

This protocol details the wet lab preparation, extraction of fruit pollen samples, and analysis of the sequencing data following Illumina NextSeq small and total RNA sequencing. The protocol was developed for virus and viroid detection using NGS sequencing and was based on the results of a comparison between different extraction methods followed by yield, RNA purity, and integrity assessment. Moreover, the advantage of an additional ribosomal (r)RNA depletion step to the total RNA extraction protocol was evaluated. The smallRNA procedure is the preferred method of choice. If the total RNA protocol is chosen, the use of the mirVana kit followed by an rRNA depletion step is the best option. The library preparation and sequencing steps were outsourced. As a final step in the data analysis, the VirusDetect software was used to detect the viruses and viroids in the pollen samples.