Hypersensitive Reaction In Tobacco Research Articles

Flagellin, a plant-defense-activating protein identified from Xanthomonas axonopodis pv. Dieffenbachiae invokes defense response in tobacco

BackgroundSecretome analysis is a valuable tool to study host-pathogen protein interactions and to identify new proteins that are important for plant health. Microbial signatures elicit defense responses in plants, and by that, the plant immune system gets triggered prior to pathogen infection. Functional properties of secretory proteins from Xanthomonas axonopodis pv. dieffenbachiae (Xad1) involved in priming plant immunity was evaluated.ResultsIn this study, the secretome of Xad1 was analyzed under host plant extract-induced conditions, and mass spectroscopic analysis of differentially expressed protein was identified as plant-defense-activating protein viz., flagellin C (FliC). The flagellin and Flg22 peptides both elicited hypersensitive reaction (HR) in non-host tobacco, activated reactive oxygen species (ROS) scavenging enzymes, and increased pathogenesis-related (PR) gene expression viz., NPR1, PR1, and down-regulation of PR2 (β-1,3-glucanase). Protein docking studies revealed the Flg22 epitope of Xad1, a 22 amino acid peptide region in FliC that recognizes plant receptor FLS2 to initiate downstream defense signaling.ConclusionThe flagellin or the Flg22 peptide from Xad1 was efficient in eliciting an HR in tobacco via salicylic acid (SA)-mediated defense signaling that subsequently triggers systemic immune response epigenetically. The insights from this study can be used for the development of bio-based products (small PAMPs) for plant immunity and health.

First Report of Pseudomonas syringae Causing Bacterial Leaf Spot on Winterberry Holly (Ilex verticillata) in Ohio.

Winterberries (Ilex verticillata and hybrids) are deciduous species of holly whose branches bearing colorful fruit are cut in late Fall to be used for seasonal decorations. The annual wholesale value of the woody cuts is $1.5 million nationally (NASS, 2019). In June 2021, approximately 80% of the 45 Ilex verticillata 'Maryland Beauty' potted plants, which were maintained in a container yard at The Ohio State University research farm in Columbus, OH, presented leaves with irregular necrotic lesions surrounded by a chlorotic halo. No other symptoms were present on the plants. Bacterial streaming was observed from the lesions using a compound microscope and isolations were performed after surface disinfesting small sections of leaf tissue from the border of the lesions by soaking in 10% bleach for 30 sec, rinsing twice in sterile water, macerating in sterile water, and streaking the suspension on nutrient broth yeast extract agar. Creamy white, circular, smooth, and convex colonies were recovered after incubation at 28°C for 48 h. Bacterial identification of one representative isolate was initially pursued from single colonies of a purified culture using five discriminative phenotypic tests (i.e., LOPAT: "L", levan production; "O", oxidase activity; "P", pectinolytic activity; "A", arginine dehydrolase production; "T", tobacco hypersensitive reaction), which resulted in the L+ O- P- A- T+ profile consistent with the description of Pseudomonas syringae (Lelliott et al. 1996). Molecular identification was performed based on rpoD marker amplification and sequencing using primers PsrpoD FNP1/PsrpoDnprpcr1 (Parkison et al. 2011). NCBI GenBank BLASTn comparison of the rpoD sequence (GenBank Acc. No. OP221440) shared 99.12% identity to P. syringae pv. passiflorae (AB163366.1). Whole genome sequence analysis was conducted to strengthen the classification of the isolate species. To this extent, DNA was sequenced with an iSeq 100 Illumina benchtop sequencer using Illumina DNA Prep kit and iSeq 100 i1 Reagent v2 (Illumina, Inc, REF: 20060060 and 20031371). Illumina Local Run Manager software was used for base calling, demultiplexing, and trimming of the raw reads. Unicycler v0.5.0 was used for de novo assembly of the genome (Wick et al. 2017). The assembled genome size was 5.9 Mb with 959 contigs and 10× coverage (NCBI GenBank Biosample No. SAMN30281368; Acc. No. JANQCB010000000). Average nucleotide identity (ANI) analysis was performed on the server MiGA online (Rodriguez-R et al. 2018). Subgroup identification was inconclusive (p>0.05), positioning this isolate between P. syringae pv. actinidiae (96.45% ANI) and pv. viburni (96.65% ANI) (Rodriguez-R & Konstantinidis, 2016). Both these pathovars cause leaf spots on woody plants such as kiwi and viburnum (Donati et al. 2020; Garibaldi et al. 2005). To confirm pathogenicity, three separate branches on each of two I. verticillata 'Maryland Beauty' potted plants were selected, and 5-7 individual young leaves (>2 weeks from emergence) on each branch were infiltrated with a bacterial suspension (108 CFU/mL) in sterile water (SW) using a needleless syringe by delivering 30-50 µL of suspension per infiltration point. One additional branch per plant was infiltrated with SW to serve as control. Plants were covered with a plastic bag for two days post-inoculation (DPI) and maintained in the laboratory at an average of 23°C. All inoculated leaves showed necrotic lesions two DPI while control leaves remained asymptomatic. To fulfill Koch's postulates, the bacterium was re-isolated from the symptomatic leaves six DPI and confirmed to be identical to the original isolate based on rpoD gene sequencing. To the best of our knowledge, this report signifies the first instance of P. syringae causing bacterial leaf spot on winterberry worldwide. Ornamental plant sales are based primarily on visual appeal; therefore, identification and monitoring of emerging pathogens is essential to ensure the health of the industry.

Screening and risk assessment of biological control agents against bacterial wilt of ginger

Bacterial wilt caused by Ralstonia pseudosolanacearum race 4 is a highly destructive disease in ginger. To screen for biological control agents of the disease, 118 bacteria were isolated from different cultivating soils. First, the suppressive activity of soil bacteria on lesion development by R. pseudosolanacearum was evaluated using ginger leaves. Subsequently, a tobacco hypersensitive reaction test was performed for risk assessment in plants. After the clonality test by repetitive element sequence-based PCR, 12 screened isolates were inoculated into silkworm larvae as a preliminary risk assessment in animals, and we observed that five isolates did not cause a lethal effect. Finally, based on the results of the disease suppression test in infested soil, Pseudomonas sp. 008 was selected as the most suitable isolate for biological control. To ascertain the taxonomic position of the isolate, biochemical and physiological tests, multilocus sequence analysis, and average nucleotide identity with related species analysis were performed. Our results suggest that Pseudomonas sp. 008 is a novel species belonging to the P. mandelii subgroup of the P. fluorescens group. Risk assessment and taxonomic approaches with robust resolution demonstrated in the present study are expected to contribute to the efficient screening of biological control agents.

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

First Report of Xanthomonas euvesicatoria Causing Bacterial Leaf Spot of Pepper (Capsicum annuum) in Montenegro.

Leaf spot of pepper was observed on different pepper cultivars in central Montenegro during summer and early autumn in three consecutive growing seasons (2017 - 2019). Necrotic spots were numerous, varying in size, irregular in shape, brown, and surrounded by a weak halo. The most intensive symptoms were observed on lower leaves. In conditions conducive or the infection, the lesions merged resulting in the leaf drop. Symptoms were not observed on pepper stems and fruits. A total of seventeen bacterial strains were isolated from infected pepper leaves collected in seven different localities in the seasons of 2017-19. They formed yellow, convex, and mucoid colonies on yeast extract-dextrose-CaCO3 (YDC) medium and induced hypersensitive reaction in tobacco leaves. They were Gram negative, strictly aerobic, oxidase negative, catalase-positive, hydrolyzed gelatine and esculin and did not reduce nitrate, nor grew on 0.1% TTC and at 37°C. Out of tested 17 strains, eight hydrolyzed starch and three showed pectolytic activity, thus differing in these biochemical traits from Xanthomonas euvesicatoria (Xe) the reference strain KFB 1 (Obradović et al., 2004) used in all tests as a positive control. PCR analysis, with primer pair XeF/XeR, produced a single characteristic band of 173 bp in all 17 strains (Koenraadt et al. 2009). Additionally, the BOX-PCR profile of all the strains produced with the BOX A1R primer (Schaad et al. 2001) showed 100% homology with KFB 1. Based on the locality and year of isolation, nine strains were selected for amplification and partial sequencing of the gyrB gene using sets of primers described by Parkinson et al. (2007). Obtained partial DNA sequences showed that all nine strains (GenBank nos. MZ569011, MZ574079, MZ574080, MZ574081, MZ574082, MZ574083, MZ574084, MZ574085, and MZ574086) share 99.86 to 100% identity of gyrB sequence with Xe type strain ICPM:109 as well as 98.71 to 100 % of gyrB sequence identity with Xe strain LMG930 isolated from pepper in The United States. Pathogenicity of all strains was confirmed by spraying young pepper plants (cv. Slonovo uvo) using a handheld sprayer with the bacterial suspension (108 CFU/ml of sterile tap water), in three replicates. Sterile distilled water and reference Xe strain (KFB 1) were used as negative and positive controls, respectively. The inoculated plants were incubated under plastic bags in the greenhouse providing high humidity conditions for 48h. Symptoms were monitored for two weeks after inoculation. Lesions surrounded by a halo appeared on leaves of all inoculated plants within 10 to 15 days after inoculation, while plants inoculated with SDW remained symptomless. Koch's postulates were confirmed by reisolation of the pathogen from necrotic tissue and identity check by PCR using primer set of Koenraadt et al. (2009). The pathogen race was determined according to the reaction of cv. Early Calwonder (ECW) and its isogenic lines (ECW-10R, ECW-20R, ECW-30R) (Stall et al. 2009). Obtained results indicated that all tested strains and reference strain Xe (KFB 1) belong to the pepper race P8. Based on pathogenic, biochemical, and molecular characteristics, the strains isolated from pepper leaves in Montenegro were identified as X. euvesicatoria. Pepper production is particularly significant for small farmers in Montenegro. Favorable climate, use of noncertified seed and lack of crop rotation contributes to the disease occurrence and severity. The disease has probably been around for years but the etiology was not confirmed so far. This is the first report of X. euvesicatoria affecting pepper in this country.

First Report of Xanthomonas campestris pv. campestris Causing Marginal Leaf Necrosis of Arugula (Eruca vesicaria subsp. sativa) in Serbia.

Arugula (Eruca vesicaria subsp. sativa (Miller) Thell., syn. Brassica eruca L.), is an annual cruciferous crop that is increasingly grown for fresh consumption in Serbia. In November 2018, a few detached leaves of cultivated arugula originating from a local producer, showing necrotic lesions, were observed in a fresh vegetable market in Belgrade, Serbia. Information about the disease incidence and severity was not available. Intensity of the observed symptoms was low, but it could be a consequence of the produce quality selection for the market. The leaves developed irregular chlorotic lesions starting from the leaf edge, and tissue within some of them turned dark brown and necrotic (Fig. 1a). From the lesions on different leaves, smooth, bright yellow pigmented, round and opalescent bacterial colonies were isolated on nutrient agar (NA) medium after 72 h of incubation at 26°C. Six bacterial isolates, obtained from three leaf subsamples which induced hypersensitive reaction in tobacco leaves (Nicotiana tabacum L. cv. Samsun), were selected for further studies. On yeast - dextrose - CaCO3 medium, the strains formed characteristic creamy yellow, mucoid, opaque and convex colonies. All isolates were Gram-negative, strictly aerobic, non-fluorescent and catalase positive, did not produce oxidase nor arginine dehydrolase, and did not show pectynolitic activity on potato tuber slices. They hydrolyzed starch, gelatine and esculin, used glucose and sucrose, but not arabinose as a carbon source, and did not reduce nitrates. They grew at 36°C, and tolerated 5% NaCl and 0.02% triphenyl-tetrazolium chloride (Lelliott and Stead, 1987). These growth characteristics were similar as for the reference Xanthomonas campestris pv. campestris (Xcc) strain KFB 105, used in all tests as a positive control (Obradović et al., 2000). The isolates were further characterized by polymerase chain reaction (PCR) using primers DLH120/DLH125, specific for the hrpF gene region of X. campestris according to Berg et al. (2005). Specific DNA fragment of 619 bp was amplified for all tested isolates. Amplification and partial sequencing of the gyrB gene of four isolates was performed using set of primers described by Parkinson et al. (2007). All obtained partial gyrB sequences were identical to each other. According to BLAST analysis (GenBank Acc. Nos. MW508894 - MW508897) they shared 100% of sequence identity with different Xcc strains and 99.5 % with the X.c. pv. raphani pathotype strain, deposited in the NCBI GenBank database. Pathogenicity of the isolates was tested by spraying leaves of 3-week old E. sativa seedlings grown in a commercial potting mix in a greenhouse, with a 24 h-old bacterial culture suspended in sterile distilled water (107 CFU/ml). Xcc strain KFB 105 was used as positive and sterile distilled water as negative control. Inoculated plants were incubated under plastic bags for 48 h and further maintained in a greenhouse at approx. 28°C. On inoculated plants, chlorotic lesions, spreading from the leaf margins, further coalescing into irregular, V-shaped tissue necrosis associated with blackening of veins, developed up to two weeks after inoculation (Fig. 1b, c). The colonies reisolated from symptomatic leaves were identified using PCR, as described above. Based on studied characteristics, all six isolates associated with arugula leaf lesions in Serbia belong to a clonal population. They were identified as X. campestris pv. campestris, the causal agent of black rot, a major disease affecting crucifers, including arugula worldwide (Romero et al., 2008; Rosenthal, et al., 2018). So far, it has been described on Brassica oleracea and B. napus in Serbia (Obradović et al., 2001; Popović et al., 2019). This is the first report of Xcc infecting arugula in this country. The severity of the symptoms developed on artificially inoculated plants indicated significant potential of the pathogen to affect arugula crop in conditions favoring infection. Being a minor crop, accurate information about severity of arugula diseases in Serbia is not available. Lack of crop rotation and close proximity of other Xcc host species on a farm could contribute to further spreading of this problem. Follow up of this arugula disease should reveal the distribution, population structure and genetic diversity of Xcc strains affecting this crop in Serbia.

First Report of Leaf Blight of Adenophora triphylla var. japonica caused by Pseudomonas viridiflava in Korea.

Severe disease with leaf spots and necrotic symptoms were observed in Adenophora triphylla var. japonica (Regel) Hara (A. triphylla) during the survey in July 2020 on a field in Andong, Gyeongbuk province, Korea. It is a highly valued medicinal plant used to treat various diseases, including cough, cancer, and obesity. The infected plants initially showed spots with halo lesions, at later stages, enlarged and spread to the leaves, which the lesions becoming yellowing and chlorotic (Fig. 1). In some areas, disease incidence was up to 15% of the plants. The symptomatic samples were collected from A. triphylla and cut into 4 to 5 mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated in sterile distilled water (SDW). They were spread onto nutrient agar (NA) plates and incubated at 28°C for 3 days. The representative bacterial strains selected for identification showed fluorescent colonies on King's medium B (KB). Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. The isolates induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: - - + - +. The Biolog GN2 microplate and the Release 4.20 system putatively found the isolate to exhibit 93% similarity with the bacterium, Pseudomonas viridiflava. Likewise, analysis of FAME profiles using the Microbial identification system (Sherlock version 3.1) also characterized the representative bacterial strain as P. viridiflava with 87% similarity. The genomic DNA of the isolate was extracted, and the 16S rDNA sequence was amplified with a universal bacterial primer set (27F and 1492R). The sequence was submitted to GenBank under the accession number MT975233. BLASTn analysis yielded 99.79% identity with P. viridiflava strain RT228.1b (accession no. AY604846.1) and 99.72% similarity with P. viridiflava KNOX249.1b strain (accession no. AY604848.1). Phylogenetic dendrogram constructed from the comparative analysis of 16S rDNA gene sequences showing the relationship between P. viridiflava GYUN274 and related Pseudomonas species (Fig. 2). Pathogenicity tests were conducted three times on seedling of A. triphylla by spraying 50 ml of bacterial suspensions of a 24-h culture in KB medium (108 CFU/ml). The leaves inoculated with SDW alone did not develop symptoms; however, the plants treated with isolated bacterial suspensions developed halo and blight symptoms similar to those observed in the field 7 days post-inoculation. Finally, Koch's postulates were verified by re-isolating P. viridiflava from all symptomatic tissues and determined to be morphologically identical to the original isolates. To our knowledge, this is the first report of leaf blight disease of A. triphylla caused by P. viridiflava in Korea. Based on the observed symptoms, and identification by morphological characteristics, molecular data, and pathogenicity against the host plant, the proper control measures can be identified in future studies.

The first report of bacterial streak and rot of onion caused by Pseudomonas viridiflava in Japan.

In March 2020, a bacterial streak and rot symptom was observed on the onion (Allium cepa L.) leaves in Akita Prefecture of Japan. On the beginning, oval and dark-greenish water-soaked lesions with grayish-white necrotic center, 2-3 mm in diameter, appeared on the middle or the tip of upper leaves. Lesions, frequently surrounded by light yellow halo, expanded along veins and overlapped together. As lesions grew, the center of the lesions turned to light brown necrosis. The basal areas of diseased leaves often rotted, causing the withering of a whole leaf at last. From the water-soaked tissues of young lesions, a bacterium forming cream white colonies and producing fluorescent pigment on King's medium B was consistently isolated, and suggested to be a member of genus Pseudomonas. The isolates were positive for potato soft rot and tobacco hypersensitive reaction, and negative for levan production, oxidase and arginine dihydrolase activity, indicating that they belonged to LOPAT group II, Pseudomonas viridiflava, defined by Lelliott et al. (1966). P. viridiflava is known as a pathogen of bacterial streak and bulb rot of onion in United States and Uruguay, though it has not been reported in Japan. Four isolates were chosen for further examinations. Pathogenicity tests on onion leaves stubbed with selected isolates reproduced similar symptoms as observed in field's samples. After 2 days of inoculation, isolates produced water-soaking around the stabbed holes, developed into grayish-white necrosis on 4 days after inoculation. After 10 days of inoculation, lesions grew to necrotic streaks with light blown center surrounded by water-soaking and light yellow halo. Additionally, on onion scales, isolates caused water-soaked rot, yellow- to brown-colored, in 1-2 days after inoculation. From water-soaked areas of both of leaves and scales, inoculum was reisolated and fulfilled Koch´s postulates. In phenotypic properties, isolates showed consistent results as P. viridiflava strains identified in former studies. They were gram negative and aerobic, positive for hydrolysis of esculin, casein and gelatin, and negative for nitrate reduction and indole production. Regarding the utilization of carbohydrates, positive for 2-keto-gluconate, glucose, fructose, D-galactose, mannose, L(+)arabinose, glycerol, mannitol, sorbitol, myo-inositol, meso-erythritol, meso-tartrate, D(-)-tartrate, gluconate, n-caprate, dl-malate, citrate, L-arginine, L-aspartate, L-glutamate, and negative for D-arabinose, maltose, sucrose, raffinose, adonitol, trehalose, L-tartrate, L-rhamnose, acetate. The 16S rRNA gene sequences (Frank et al 2008) of four isolates (1,377 bp) showed 100% similarity as that of P. viridiflava type strain LMG 2352T (GenBank Accession No. Z76671) in BLASTN search. In phylogenetic analysis using gyrB (910 bp) and rpoD (801 bp) genes (Maeda et al 2006), isolates formed a cluster with P. viridiflava strains deposited in public databases, independent from other closely related Pseudomonas species. Sequences of 16S rRNA (GenBank Accession Nos. LC597475-LC597478), gyrB (LC597479-LC597482) and rpoD (LC597483-LC597486) genes were deposited in DNA Data Bank of Japan. According to these results, the isolates were identified as P. viridiflava (Burkholder 1930) Dowson 1939. This is the first report of the occurrence of bacterial streak and rot of onion caused by P. viridiflava in Japan, causing severe damage on onion growth.

First Report of Xanthomonas arboricola pv. pruni Causing Leaf and Fruit Spot on Apricot (Prunus armeniaca L.) in Montenegro.

In July 2020, symptoms of leaf and fruit spot were observed on two-year old apricot plants (Prunus armeniaca L.), cultivar Rubista in plantation covering approximately 0,5 ha near Podgorica, central Montenegro. The intensity of infection on leaves was more than 70%. Initially, leaf spots were mainly circular, 2 to 5 mm in diameter, water-soaked, surrounded by a weak chlorotic halo, but later became light to dark brown and necrotic. Eventually, the spots merged and necrotic tissue dropped out, leaving a "shot-hole" leaf appearance. On apricot fruits small, dark brown, mainly circular superficial lesions were observed. The lesions merged and formed large necrotic areas reducing the quality of fruits. Symptoms were not observed on woody parts, such as twigs or stem. A total of 10 bacterial strains, forming yellow, convex, and mucoid colonies on yeast extract-dextrose-CaCO3 (YDC) medium, were isolated from symptomatic leaf and fruit tissue. All strains induced hypersensitive reaction in tobacco leaves. They were Gram-negative, strictly aerobic, oxidase negative, catalase positive, hydrolyzed gelatine and esculin but not starch, and did not grow at 37°C, showing similar biochemical properties as a reference strain Xanthomonas arboricola pv. pruni (Xap) (NCPPB 416) used in all tests as a positive control. Strains were further identified by PCR analysis, using primer pair XapY17-F/XapY17-R (Pagani 2004; Pothier et al. 2011), resulting in a single band of 943 bp, characteristic for Xap. Additionally, BOX-PCR with the BOX A1R primer (Schaad et al. 2001) showed 100% homology in genetic profiles of all tested strains and control strain. Amplification and partial sequencing of the gyrB gene of four representative strains was performed using set of primers described by Parkinson et al. (2007). Obtained DNA sequences showed that analysed strains (GenBank nos. MW473770, MW473771, MW473772, and MW473773) share 99.44 to 99.57% of gyrB sequence identity with Xap pathotype strain ICMP51. Pathogenicity of all strains was confirmed by spraying young apricot shoots using a hand-held sprayer, and by infiltration of apricot leaves (cv. Roksana) from the abaxial surface using a syringe without needle, with the bacterial suspension (107 CFU/ml in sterile distilled water), in three replicates. Sterile distilled water and reference Xap strain (NCPPB 416), were used as negative and positive controls, respectively. The inoculated shoots and leaves were maintained at approx. 25°C and high humidity conditions. Tissue necrosis appeared on all inoculated shoots 5 to 11 days and leaves 5 to 9 days after inoculation. Koch's postulates were completed by re-isolation of the pathogen from inoculated tissue and identification by PCR using XapY17-F/XapY17-R primers. Based on pathogenic, biochemical and molecular characteristics, the strains isolated from apricot leaves and fruits in Montenegro were identified as Xap - causal agent of bacterial leaf spot and canker of stone fruits. This quarantine pathogen was previously reported on almond (Panić et al. 1998) and on peach (Popović et al. 2020) in Montenegro. This is the first report of Xap affecting apricot in this country. Therefore, strict phytosanitary measures have to be implemented to prevent spread of the pathogen in other areas and other susceptible hosts.

First Report of Pseudomonas viridiflava Causing Bacterial Blight on Globe Artichoke in Turkey

The production of globe artichoke (Cynara scolymus L.), which is a major part of Mediterranean cuisine and packs an abundance of nutritional value, has been increasing over recent decades in Turkey. In the spring of 2018, globe artichoke (cv. Sakiz) plants with blight symptoms on outer bracts, dark brown sunken lesion on inner sides, red-brown areas on margins, and small and malformed buds were found in artichoke-growing areas in the Karaburun district of Izmir, Turkey. In some areas, disease incidence was up to 5% of the plants. Several buds in harvest stage exhibiting symptoms were collected from 30-ha artichoke production fields that have heavy soil texture and humid weather conditions and processed to identify the causal agent on 15 artichoke samples. Symptomatic outer bracts showing characteristics of bacterial blight symptoms as well as spines were surface sterilized with 1% NaOCl, rinsed in sterilized distilled water (SDW), and cultured on King’s medium B (KB) for 24 h at 28°C. After incubation, a predominant bacterium was isolated from all samples. Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. Round, flat, and blue-green translucent colonies fluorescing under 360-nm UV light when grown on KB medium were observed in all samples taken. The isolates caused soft rot of potato tubers, induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. Acid was produced from mannitol, erythritol glycerol, D-sorbitol, D-mannose, and inositol but not from L-tyrosine, D-cellobiose, D-arabinose, and D-trehalose, all of which are typical characteristics of Pseudomonas viridiflava (Lelliott et al. 1966; Schaad et al. 2001). The bacterial identity was further confirmed by amplifying the 16S rRNA gene using the universal bacterial primers 8F and 1492R (Galkiewicz and Kellogg 2008) and gyrase subunit B (gyrB) gene using primers gyrB-Rps/gyrB-Fps (Sawada et al. 1999). The sequences were submitted to GenBank under accession numbers MH763694 for 16S and MK546431 for gyrB. BLASTn analysis of the 1,042-bp amplicon for 16S and 545-bp amplicon for gyrB indicated that it had 99% similarity with P. viridiflava strain 4G1113 (accession no. MG972916) and 100% similarity with P. viridiflava ATCC 13223 strain (accession no. KY612187), respectively. Pathogenicity tests were conducted twice on outer bracts and main stems of artichoke cultivar Sakiz by inoculating 20 µl of bacterial suspension of a 24-h culture in KB medium (1.9 × 10⁹ CFU/ml in SDW). The inoculated plants were placed in a transparent box for 7 days and kept at 25°C and 70% relative humidity. The bract leaves inoculated with only SDW did not develop symptoms; however, the bracts treated with isolated bacterial suspension developed blight symptoms similar to those observed in the field, 7 days postinoculation. The stems inoculated with the isolate also exhibited soft rot symptoms, and the same bacterium was consistently reisolated from both bracts and stems but not from parts of negative control plants. Reisolated bacteria exhibited the same biochemical profile with our isolate, thereby fulfilling Koch’s postulates. P. viridiflava causing a bacterial blight of artichoke bracts was previously reported in Greece (Sarris et al. 2012). To our knowledge, it is the first report of this bacterium causing bacterial blight on leaves and soft rot on stems of globe artichoke in Turkey. Given the availability of climatic conditions, the potential of disease spreading can cause a serious reduction in yield and marketability of this vegetable, which has increasing popularity owing to having high nutritional value, leading to substantial economic losses.

First Report of Pseudomonas syringae pv. syringae Associated with Bacterial Blossom Blast on Apple (Malus pumila) in the United States

In May 2017, dead buds at pink and tight cluster growth stages were observed on young apple trees of cultivar SnapDragon at two orchard sites in Marlboro, NY. Disease symptoms included necrosis of lateral and terminal flower buds that were wilted and drooping. Bark below the dead buds on severely affected trees showed black streaking with or without interspersing white streaks. On the cross-section of branch wood, just below the buds, light brown xylem and glossy water-soaked sections resembling frost damage were observed. After bark shaving, dark xylem tissue was visible along the branch length, in contrast to the light or pale color of the healthy tissue. The incidence of the disease on trees was 3 to 5%, and 0.5% of trees died. There were five near-frost events recorded on 4, 8, 9, 10, and 11 May 2017, leading us to suspect that the observed symptoms were blossom blast caused by the ice-nucleating strains of Pseudomonas syringae. Isolation from the affected tree samples was performed on King’s B (KB) and nutrient sucrose agar (NSA) media. After 3 days at 25°C, most of bacterial isolates formed round, shiny, gray-white colonies producing green fluorescent pigment on KB medium and typical levan-positive colonies on NSA. Eight isolates, inducing a hypersensitive reaction on tobacco plants, were further characterized by conventional bacteriological and molecular tests (Braun-Kiewnick and Sands 2001). The analyzed bacterial isolates were Gram-negative, aerobic, hydrolyzed gelatin and aesculin, but not pectate, and were negative for oxidase and arginine-dihydrolase activity. All strains were ice-nucleation active and utilized sucrose, sorbitol, and inositol as carbon sources, but not tyrosine and tartaric acid. Results of levan, cytochrome c oxidase, potato soft rot, arginine dihydrolase, and tobacco hypersensitive reaction (LOPAT) tests indicated that the isolates belong to P. syringae group Ia, and gelatin hydrolysis, aesculin hydrolysis, tyrosinase activity, and tartrate utilization (GATTa) tests designated them as pathovar (pv.) syringae (Kaluzna et al. 2012). Polymerase chain reaction (PCR) with specific primers B1/B2 (Sorensen et al. 1998) and SyD1/SyD2 (Bultreys and Gheysen 1999) showed that isolates possessed genes responsible for synthesis (syrB) and secretion (syrD) of syringomycin, respectively. Partial sequencing of the 16S rDNA gene of three isolates (GenBank MG788015 to MG788017) showed 99 to 100% identity with sequences of P. syringae pv. syringae previously deposited in the NCBI GenBank database. To complete Koch’s postulates, pathogenicity of the four isolates was confirmed by inoculation of 1-month-old potted apple plantlets grown from seeds collected from mature apple fruit of cultivar SnapDragon. One plant per isolate was inoculated multiple times as follows: using a needleless syringe, two leaves per plant were spot-infiltrated with bacterial suspension (10⁶ CFU/ml); on the same plants, stem was also inoculated by inserting a sterile toothpick previously dipped into a bacterial colony. Sterile distilled water was used as a negative control. Inoculated plantlets were sealed within plastic bags for 48 h and further incubated at 22°C. Inoculation test was repeated in two independent experiments. Necrosis of the leaf and stem tissue developed at the point of inoculation, and its progression was observed daily for 2 weeks. No symptoms developed on the control plants. Bacteria were reisolated from inoculated tissues and confirmed as P. syringae pv. syringae by PCR for syrB and syrD genes. This is the first report of P. syringae pv. syringae causing blossom blast symptoms on developing apple buds in commercial orchards in the United States. Even though it rarely occurs on apples, with most recent frosts significantly lowering apple yields in Michigan (2012) and New York (2016), blossom blast might become a more common problem in U.S. orchards. Previously, P. syringae pv. syringae was isolated from dormant apple buds without and with internal necrosis in the United States (Burr and Katz 1984).

Characterisation of Pseudomonas syringae isolates from apricot orchards in north-eastern Italy

A set of 31 isolates were obtained from apricot (Prunus armeniaca L.) orchards showing symptoms of blast and gummosis in orchards located in north-eastern Italy, mostly during three subsequent growing seasons (2014–2016). These isolates were identified by means of their colony morphology, pathogenicity (hypersensitive reaction in tobacco leaves and the detection of both syringomycin and coronatine coding genes), genetic features (BLASTn analysis of the rpoD gene) and physiological tests (GATTa) as Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum race 1 and race 2 strains, respectively. Phylogenetic analysis based on the rpoD gene sequence showed that the two pathovars and two races are well separated and highlighted high intrapathovar/race variation for both P. syringae pv. syringae and P. syringae pv. morsprunorum race 2. Pseudomonas syringae pv. morsprunorum race 1 formed a unique clade that had high sequence homology. Repetitive element-based polymerase chain reaction fingerprinting analysis using the ERIC, REP and BOX primers again showed both high intra-pathovar variation within the P. syringae pv. syringae isolates, which grouped into six distinct clusters, and notable intra-race genetic diversity within both P. syringae pv. morsprunorum races. All 31 strains showed tolerance to copper at 200 ppm in vitro. Among these isolates, 13 were highly resistant (up to 500 ppm) and revealed the presence of the cusCBA gene cluster. All P. syringae pv. syringae strains showed ice nucleation activity, and nine were markedly active (up −3 °C) and characterised by the presence of the INAz gene. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 2 strains from apricot in Italy.

Characterization and population diversity of Erwinia amylovora strains originating from pome fruits in Serbia

The diversity of 30 Erwinia amylovora strains, isolated from quince, pear and apple trees on 14 localities in Serbia, was studied using bacteriological and molecular methods. In pathogenicity tests, all strains caused necrosis and oozing of bacterial exudate on inoculated immature pear, cherry and plum fruits, and induced hypersensitive reaction in tobacco leaves. The studied strains were Gram and oxidase negative, non-fluorescent, levan and catalase positive and facultatively anaerobic. The strains did not reduce nitrates, but utilized citrate and produced acid from sorbitol, hydrolyzed gelatine, produced reducing substances from sucrose and grew in the presence of 5% NaCl, but not at 36oC. Identity of the strains was confirmed by conventional and nested PCR methods. Rep-PCR with REP, ERIC and BOX primers resulted in amplification of several DNA fragments respectively, but showed no variation within the strains. However, different genetic profiles were obtained with RAPD-PCR by using six primers which enabled differentiation of the strains into four groups. Genetic differences between the studied strains did not correlate with the host plants, geographical origin or year of isolation.

First Report of Pseudomonas cichorii Causing Bacterial Leaf Blight of Plumeria pudica in Hawaii

Bridal Bouquet Plumeria (Plumeria pudica) is a commonly grown ornamental in Hawaii. In November 2014, a nursery grower located in Panaewa on the eastern side of Hawaii Island noticed water-soaked, irregular greyish-black leaf spots and leaf blight followed by defoliation on potted plants of P. pudica. Two leaf samples were submitted to the University of Hawaii Agricultural Diagnostic Service Center located at the Komohana Research and Extension Center in Hilo, HI for diagnosis. Isolations were made on King’s Medium B agar (KMB) and bacteria were consistently isolated from diseased tissue. Culture plates were forwarded to the USDA-ARS facility in Hilo, HI for further purification and molecular identification. Isolations on KMB resulted in essentially pure cultures that were non-mucoid, cream and fluorescent under 365 nm UV light. Four representative bacterial isolates were examined and characterized as gram-negative, oxidase positive, and produced a hypersensitive reaction in tobacco. Colonies were identif...

First Report of Bacterial Blight of Crucifers Caused by Pseudomonas cannabina pv. alisalensis in Minnesota on Arugula (Eruca vesicaria subsp. sativa).

In 2011, bacterial blight of arugula (Eruca vesicaria subsp. sativa; cv. Roquette) was observed in organically grown plants under overhead irrigation in a field near Delano, MN. Approximately 80 to 100% of each planting was affected, with greater rates of infection occurring after periods of high humidity. Small, water-soaked, angular spots apparent on both sides of the leaves comprised the initial symptoms, which sometimes expanded and coalesced. Lesions maintained a dark water-soaked appearance or dried and turned a brown/tan color. Additionally, some lesions were outlined by a purple margin. Blue-green fluorescent pseudomonads were isolated consistently on King's Medium B agar (KMB) from symptomatic leaf tissue surface-disinfested with sodium hypochlorite (0.525%). The isolates nucleated ice and produced levan. Isolates were oxidase and arginine dihydrolase negative. They did not rot potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliott's LOPAT group 1 (2). DNA fragment banding patterns generated by amplifying DNA of the arugula isolates using repetitive extragenic palindromic sequence-polymerase chain reaction (rep-PCR) and the BOX A1R primer were identical and nearly identical to the banding patterns of the Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis) (1) strain (CFBP1637) and the pathotype strain (CFBP 6866PT), respectively. Pathogenicity was confirmed on the arugula cv. My Way in two independent experiments, each with three replicate plants per treatment. Four isolates were grown on KMB for 48 h at 27°C, suspended in 0.01M potassium phosphate buffer (pH 7.0), and adjusted to 0.6 optical density at 600 nm (approximately 1 × 108 CFU/ml). Five- to six-week old plants were spray-inoculated until run-off, incubated in a humidity chamber for 48 h, and then placed in a greenhouse at 20 to 25°C for symptom development. For negative and positive control treatments, a similar number of plants each were sprayed with sterile buffer or P. cannabina pv. alisalensis strains CFBP1637 and CFBP 6866PT, respectively. Water-soaked and brown/tan lesions similar to the original symptoms appeared on plants inoculated with the arugula isolates and P. cannabina pv. alisalensis strains 7 to 14 days postinoculation. No symptoms developed on plants treated with sterile buffer. The bacterial strains re-isolated from surface-disinfested symptomatic tissue were identical by rep-PCR to the isolates used to inoculate the plants, thus, confirming Koch's postulates. Identical replicated experiments conducted on broccoli raab indicated that the arugula isolates were also pathogens of broccoli raab (Brassica rapa subsp. rapa, the original host from which P. cannabina pv. alisalensis was isolated). To our knowledge, this is the first report of bacterial blight of crucifers caused by P. cannabina pv. alisalensis in Minnesota. Arugula germplasm is being evaluated for resistance to this pathogen as an acceptable management method for organic cropping systems.

First Report of Pseudomonas syringae pv. coriandricola Causing Bacterial Leaf Spot on Carrot, Parsley, and Parsnip in Serbia.

During the spring of 2014, a severe leaf spot disease was observed on carrot (Daucus carota), parsley (Petroselinum crispum), and parsnip (Pastinaca sativa) on a 0.5-ha vegetable farm in Vojvodina Province, Serbia. The disease appeared under wet and cool conditions with 5 to 25% of plants infected for each of the three crops. Symptoms were characterized as brown angular leaf spots, ~2 mm in diameter, often limited by veins. Collected symptomatic leaves were rinsed and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in sterile phosphate buffer and streaked onto nutrient agar with 5% (w/v) sucrose (NAS). After isolation, whitish, circular, dome-shaped, Levan-positive colonies consistently formed. Five strains from each host (carrot, parsley, and parsnip) were used for further study. Strains were gram-negative, aerobic, and positive for catalase and tobacco hypersensitive reaction but negative for oxidase, rot of potato slices, and arginine dihydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (3). Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the REP, ERIC, and BOX primers (4) were identical for all strains. Sequence typing of the housekeeping genes gyrB and rpoD (1) was performed for three representative strains (one from each host). Sequences were deposited in the NCBI GenBank database as accessions KM979434 to KM979436 (strains from carrot, parsnip, and parsley, respectively) for the gyrB gene and KM979437 to KM979439 (strains from parsnip, parsley and carrot, respectively) for the rpoD gene. Sequences were compared with pathotype strain Pseudomonas syringae pv. coriandricola ICMP12471 deposited in the Plant Associated and Environmental Microbes Database ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ). BLAST analysis revealed 100% homology for gyrB and 99% homology for rpoD. Pathogenicity was tested with five representative strains from each host on four-week-old plants of carrot (cv. Nantes), parsley (cv. NS Molski), and parsnip (cv. Dugi beli glatki) using two methods: spraying the bacterial suspension (108 CFU ml-1) on the leaves until runoff (5) and injecting the bacterial suspension into leaves with a hypodermic syringe (2). Four plants were used per strain and method. Sterile distilled water was applied as a negative control treatment for each plant species. All plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse at 25°C and 80% relative humidity and examined for symptom development over a period of three weeks. For all strains, inoculated leaves first developed water-soaked lesions on the leaves 5 to 7 days after inoculation (DAI); 14 DAI lesions became dark brown, often surrounded by haloes. No symptoms were observed on control plants inoculated with sterile distilled water. For fulfillment of Koch's postulates, re-isolations were done onto NAS. Re-isolated bacteria were obtained from each inoculated host and confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR fingerprinting profiles. Based on the pathogenicity test accompanied by completion of Koch's postulates, sequence analysis, and bacteriological tests, the strains were identified as P. s. pv. coriandricola. To our knowledge, this is the first report of bacterial leaf spot of carrot, parsley, and parsnip in Serbia. It may present a threat to production due to quality requirements for fresh market.

Draft Genome Sequence of a Hypersensitive Reaction-Inducing Pantoea agglomerans Strain Isolated from Olive Knots Caused by Pseudomonas savastanoi pv. savastanoi.

Pantoea agglomerans strains inducing a hypersensitive reaction in tobacco leaves are frequently isolated inside olive knots caused by Pseudomonas savastanoi pv. savastanoi. Here, we report the draft genome sequence of the Italian P. agglomerans strain, which is able to increase olive knot disease severity when coinoculated with P. savastanoi pv. savastanoi.

First Report of Pseudomonas syringae pv. syringae Causing Bacterial Leaf Spots of Oil Pumpkin (Cucurbita pepo) in Serbia.

Oil pumpkin (Cucurbita pepo L.) is commonly used for oil production, mainly in central and eastern Europe (1). In Serbia, it grows only in the north (Vojvodina Province), up to 1,500 ha. In June 2008, typical bacterial spot symptoms (dark green, water-soaked, transparent and greasy spots with yellow margins) were observed for the first time, cultivated at the experimental fields near Backi Petrovac. Since then, bacterial spots were regularly observed on oil pumpkin in the beginning of the growing seasons and during rainy weather, with disease incidence ranging from 5 to 20%. Bacteria isolated from 40 diseased leaves formed white, round, convex, and mucoid colonies on nutrient sucrose agar (NSA). Eight representative strains were aerobic, gram-negative, non-spore-forming rods. All strains produced fluorescent pigment and catalase. In levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests (3), they induced a hypersensitive reaction in tobacco leaves, did not cause soft rot of potato tubers, and were positive for levan and negative for oxidase and arginine dihydrolase. According to the LOPAT profile, they were classified in the Ia subgroup of pseudomonads (3). Strains hydrolyzed aesculin, but were unable to hydrolyze starch or reduce nitrates to nitrites. Negative reactions were obtained with hydrogen sulfide and indole. Reactions were identical to those of reference strain Pseudomonas syringae pv. syringae CFBP 1582, which was included in all biochemical, physiological, and molecular tests for comparison. To identify the pathogen, PCR and DNA sequencing were employed. Fragments of 752 bp for the syrB gene and 1,040 bp for the syrD gene were amplified from all strains, using B1/B2 and SyD1/SyD2 primer sets, respectively (2). The pathogenicity was tested on seeds and seedlings of oil pumpkin cv. Olinka. Strains were grown for 48 h on nutrient broth (NB) at 28°C and bacterial suspensions of ~108 CFU ml-1 were used for inoculations. Sterile water was used as negative control. Seeds (at the BBCH-1-0 stage) allowed to imbibe water were wounded by needle, immersed in the bacterial suspensions, and maintained in humid petri dishes to allow symptom development. The cotyledons of seedlings at the BBCH-10 stage were inoculated by hypodermic needle and potted plants were maintained at 25 ± 1°C and 75% relative humidity. Symptoms, including dark green, water-soaked spots, appeared 5 to 7 days after inoculation of both seeds and seedlings. The bacterium was re-isolated from spots of all seeds and seedlings tested, fulfilling Koch's postulates (the identity of re-isolated strains was confirmed by pathogenicity, morphology, and biochemical features). No symptoms were observed on controls. 16S rDNA amplicons obtained from representative strain Tk21 and re-isolated strain Tk21R with fD1/rD1 primers (4) were sequenced and deposited in GenBank under accession nos. KF305578 and KF735064, respectively. The sequences showed 100% similarity to each other and P. syringae pv. syringae from pepper (KC816630.1) (China), Ficus carica (JQ071937) (Serbia), and culture-collection ICMP:3023 (HM190217). On the basis of the symptoms, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on oil pumpkin in Serbia.

First Report of Tomato Pith Necrosis Caused by Pseudomonas mediterranea in the United States and P. corrugata in Ohio

Tomato (Solanum lycopersicum, cvs. Mountain Fresh, Big Dena, and Trust) plants with symptoms of pith necrosis were received from six commercial high tunnels in Ohio during May and July 2012. Disease incidence ranged from 1 to 5%. Symptoms included wilting of shoots, dry, dark brown coalescent lesions on stems, brown discolored pith with a ladder-like appearance, and in some cases, adventitious root formation. Bacterial streaming was observed microscopically from necrotic stem tissue. Bacteria were isolated from surface-sterilized diseased stem tissue by plating 10-fold serial dilutions onto yeast dextrose carbonate (YDC) and Pseudomonas F (PF) agar media. The majority of the colonies recovered were similar in morphology on YDC: round and mucoid, with a greenish center that later became dry and winkled with a curly margin, and producing a yellow-green diffusible pigment. Colonies were creamy, yellow-brown in color and non-florescent on PF medium. Nine isolates from six plant samples were purified. All isolates were gram-negative, levan negative, oxidase positive, and potato rot negative. Three isolates were positive and six were negative for arginine dihydrolase activity. None induced a hypersensitive reaction in tobacco. All isolates grew at 37°C. The isolates were further identified by PCR assays using species-specific primers PC5/1-PC5/2 for Pseudomonas corrugata and PC1/1-PC1/2 for P. mediterranea (1,2). DNA of a reference P. mediterranea strain from Turkey was used as a positive control. A 600-bp band was amplified using P. mediterranea primers from the six arginine dihydrolase negative isolates recovered from four of six samples. An 1,100-bp band was amplified from the three arginine dihydrolase positive isolates from two other samples using P. corrugata primers. The 600-bp PCR products amplified from the P. mediterranea reference strain and isolate SM664-12 were purified and sequenced. The DNA sequence of SM664-12 was 99% aligned with that of the reference strain from Turkey and a BLAST search in NCBI indicated only one match with P. mediterranea strain G-229-21 (Accession No. EU117098.1), with an E-value 1e-145 and 84% identity. P. mediterranea (SM664-12) and P. corrugata (SM658-12) were each inoculated onto four 4-week-old tomato plants (cv. Mountain Fresh) by injecting a 50 μl bacterial suspension (108 CFU/ml) into the stem at the axil of the first true leaf (2). Negative control plants were injected with sterile water. Plants were kept in a mist chamber for 72 h at 25°C, then moved into a growth chamber maintained at 25/20°C day/night, 12-h light/dark, and 80% relative humidity. Plants exhibited dark brown lesions at the inoculation site after 4 weeks and brown discoloration of the pith developed, whereas no lesions were observed in control plants. The reisolated bacteria were tested by PCR and identified as P. corrugata and P. mediterranea. Therefore, we have confirmed that tomato pith necrosis in Ohio involves at least two bacteria, P. corrugata and P. mediterranea. Although tomato pith necrosis has been observed in Ohio since the 1990s, to our knowledge, this is the first confirmation of a causal agent as P. corrugata in Ohio and the first report of P. mediterranea causing tomato pith necrosis in the United States.

First Report of Bacterial Leaf Spot of Parsley Caused by Pseudomonas syringae pv. coriandricola in Ohio.

A severe leaf spot of parsley (Petroselinum crispum L. cvs. Dark Green Italian and Gigante) was observed on ∼1.5 ha in 2007 and 8 ha in 2012 on three vegetable farms in northern Ohio. Tiny, water-soaked spots that enlarged to necrotic lesions (∼5 mm wide) were first observed in June of each year. Lesions often coalesced and leaf marginal necrosis was common. Disease incidence initially ranged from 20 to 50%, and a 1.5-ha field was completely lost in 2012 as a result of the disease. Bacterial streaming was observed microscopically from leaf lesions. Diseased leaf tissue was dipped briefly in 70% ethanol, rinsed in sterile water, and blotted dry. Bacteria were isolated by plating 10-fold serial dilutions of diseased tissue extracts onto yeast dextrose carbonate and Pseudomonas F (PF) agar media. Whitish, opaque, circular colonies were isolated consistently from all samples. One isolate was purified from each of four fields. They were all gram-negative, non-fluorescent on PF medium, levan positive, oxidase negative, arginine dihydrolase negative, potato rot negative, and tobacco hypersensitive reaction positive. Repetitive extragenic palindromic sequence (Rep)-PCR fingerprint profiles using the BOXA1R primer (4) were identical for the four isolates. A pathogenicity test was conducted with strain SM69-07 isolated in 2007. A bacterial culture was suspended in sterile potassium phosphate buffer (0.01M, pH 7.4) and adjusted to 108 CFU/ml. Four 4-week-old plants each of parsley and cilantro (Ferry-Morse Seed Co.) were inoculated by spraying the bacterial suspension on the leaves until runoff. Potassium phosphate buffer was applied as a negative control treatment for each plant species. Plants were kept in a mist room with 100% humidity for 4 h, then transferred to a greenhouse with average maximum and minimum temperatures of 30 and 25°C. Leaf symptoms similar to those on the original plants were observed on the inoculated parsley and cilantro plants within 14 days of inoculation, whereas no symptoms developed on the negative control plants. One bacterial isolate obtained from each inoculated host using the isolation method described above was confirmed to be identical to the original isolates using the LOPAT tests and Rep-PCR DNA fingerprint profiles; no target bacteria were isolated from the negative control plants. Multilocus sequence typing (MLST) of the housekeeping genes gap1, gltA, gyrB, and rpoD was conducted for strain Sm69-07 (2,3). Sequence data were subjected to BLASTn searches in the Plant-Associated Microbes Database (PAMDB, http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ) (1). The sequences aligned with those of Pseudomonas syringae pv. coriandricola with 100% identity to alleles 101 (gyrB), 123 (rpoD), 7 (gap1), and 64 (gltA). Strain information and sequence alignment results for SM69-07 were submitted to PAMDB and assigned as isolate ID 1138. Based on bacterial culture morphology, LOPAT profile, pathogenicity test results, and MLST, the pathogen was confirmed as P. syringae pv. coriandricola. To our knowledge, this is the first report of bacterial spot of parsley caused by P. syringae pv. coriandricola in Ohio. Due to stringent quality requirements for fresh market parsley, this disease may pose a threat to the economic sustainability of parsley production in Ohio.