rpoD Gene Research Articles

First Report of Robbsia andropogonis Causing Bacterial Blight of Danae racemosa (Poet’s Laurel) in a Virginia Ornamental Nursery

Danae racemosa (L.) Moench (Asparagaceae, Monocots) is a shade-tolerant, evergreen shrub native to Persia. On 02 July 2024, at an ornamental nursery in Hanover County, Virginia, 300 plants in 7.6 L containers were observed with blotchy, rust-colored, water-soaked lesions on the cladophylls and stems. Heavy bacterial streaming was observed from the lesions under the microscope, and sterilized tissue was cultured on King's B medium at 22 ± 2°C. After 72 hours, cream-colored colonies developed that were oxidase and gram negative and did not produce fluorescent pigments on King B medium. PCR amplification and sequencing of the partial 16S rRNA, gyrB, and rpoD genes from strain GS24-DSB16 were deposited into the NCBI Genbank database (PQ181184, PQ182786, PQ182787). Based on the morphological and molecular characteristics, the causal agent was identified as Robbsia andropogonis (Smith) Lopes-Santos et al. Koch’s postulates were fulfilled by wounding twenty cladophylls from each plant with a sterile needle, and 10 μl of an aqueous bacterial suspension was pipetted on each wound. R. andropogonis has a wide global distribution from a host range spanning >15 plant families, including maize, sorghum, sugarcane, citrus, coffee, and carnation. To the authors knowledge, this is the first report of the bacteria causing blight on D. racemosa, and the first host in Asparagaceae.

First Report of Xanthomonas campestris pv. raphani Causing Leaf Spot Disease on Broccoli in China.

Broccoli (Brassica oleracea L. var. italica) is one of the important cruciferous vegetables in China, known for its considerable economic and nutritional value (Li et al. 2021). In September 2023, leaf spot disease was observed on broccoli seedlings in the commercial fields in Weifang City, Shandong Province, China (119°15'E, 36°70'N). Further investigation revealed that the disease incidence was approximately 60% in 4.5 square hectometers (hm2) broccoli field, resulting in substantial economic losses. This disease primarily affects the leaves, manifesting distinct symptoms such as circular, dark necrotic spots that gradually lighten and are encircled by a chlorotic halo. Some lesions further develop a black or purplish border, exhibit concentric zonation, and eventually fall off, leaving behind holes, as shown in Figure S1B and C. In severe cases, decay originates from the central perforation and spreads outwards, as shown in Figure S1A. To identify the causal agent of this disease, infected leaf tissues were collected and surface disinfected by immersing in 75% ethanol for 30 seconds, followed by three rinses with sterile water. The samples were grinded in sterile deionized water, and the extract was plated on NA. After incubation at 28°C for 48 h, individual colonies were transferred to fresh NA plates. A total of 12 strains with the similar morphological characteristics were isolated from diseased samples collected from the three plots. After 48 hours of growth on NA medium at 28℃, each colony attained a diameter ranging from 3 to 5 mm. These colonies appeared yellow, slightly elevated, nearly circular in shape, with a smooth and moist edge. Three representative strains were selected for further investigation. All strains were Gram-negative, aerobic, and rod-shaped. The analysis of BIOLOG GENIII microplate system revealed the capability of three isolates using cellobiose, trehalose, glucose, mannose, galactose, and sucrose. Furthermore, the isolates were unable to hydrolyze arginine and utilize rhamnose and inositol. The 16S rRNA gene was amplified and sequenced to confirm that three isolated bacteria belong to the genus Xanthomonas (OR772321-OR772323), followed by PCR amplification for 4 housekeeping genes atpD, dnaK, gyrB, and rpoD (Young et al. 2008; Saux et al. 2015). The obtained sequences were submitted to the GenBank under accessions OR789628-OR789630, OR785471-OR785473, OR789631-OR789633, and OR785468-OR785470. Gene sequences were aligned, concatenated, and used to generate a phylogenetic tree using the neighbor-joining method in MEGA11 (Tamura et al. 2021). The phylogenetic analysis revealed that all the three isolates were clustered with Xanthomonas campestris pv. raphani strains 5055 and 576, respectively (Figure S2). (Dubrow et al. 2022). These results were consistent with those of the reported X. campestris pv. raphanin (Cruz et al. 2015). To verify the pathogenicity of these strains, we used a spray inoculation method. In detail, bacterial suspensions (30 mL per treatment) containing approximately 108 CFU/ml were sprayed onto healthy, four-week-old broccoli plants and incubated in a phytotron at 28°C and above 90% RH. Negative controls were performed using sterile distilled water. Each isolate underwent three trials and each treatment included 12 broccoli seedlings. Leaf spot symptoms were observed on 5 days post inoculation, as shown in Figure S1E, F and G. Negative control plants showed no symptoms (Figure S1D). We further re-isolated the bacterium from the symptomatic plants and verifying the bacteria as X. campestris pv. raphanin with the aforementioned sequence analysis, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of X. campestris pv. raphani causing leaf spot disease on broccoli in China. This study enhances our understanding of the pathogenic bacterium on broccoli and lays the groundwork for developing targeted management strategies.

A single rareσ70 variant establishes a unique gene expression pattern in the E. coli pathobiont LF82.

LF82, an adherent-invasive Escherichia coli (AIEC) pathobiont, is associated with Crohn's disease, an inflammatory bowel disease of unknown etiology. Although AIEC phenotypes differ from those of 'commensal' or pathogenic E. coli, work has failed to identify genetic features accounting for these differences. We have investigated a natural, but rare, single nucleotide polymorphism (SNP) in LF82 present within the highly conserved rpoD gene, encodingσ70 [primary sigma factor, RNA polymerase (RNAP)]. We demonstrate thatσ70 D445V results in transcriptomicand phenotypic changes consistent with LF82 phenotypes, including increased antibiotic resistance and biofilm formation and increased capacity for methionine biosynthesis. RNA-seq analyses comparingσ70 V445 versus σ70 D445 identified 24 genes upregulated byσ70 V445 in both LF82 and the laboratory E. coli K-12 strain MG1655. Using in vitro transcription, we demonstrate thatσ70 D445V directly increases transcription from promoters for several of the up-regulated genes and that the presence of a 16 bp spacer and -14 G:C is associated with this increase. The position of D445V within RNAP suggests that it could affect RNAP/spacer interaction. Our work represents the first identification of a distinguishing SNP for this pathobiont and suggests an underrecognized mechanism by which pathobionts and strain variants can emerge.

First Report of Pseudomonas amygdali pv. morsprunorum causing Bacterial Canker in Sweet Cherry Orchards in Washington State.

In 2023, an outbreak of bacterial canker disease (BCD) in sweet cherry orchards caused significant economic losses to growers and nurseries in the Pacific Northwest, USA (Fig. S1). The cherry industry in Washington State alone is valued at over $800 million (USDA NASS, 2022). BCD poses a recurring threat to the state's sweet cherry [Prunus avium (L.) L.] orchards, especially young and newly planted orchards. Three Pseudomonas species, including P. syringae pv. syringae (Pss), P. amygdali pv. morsprunorum (Pam) (formerly P. syringae pv. morsprunorum Race 1, Psm1), and P. avellanae pv. morsprunorum (formerly P. syringae pv. morsprunorum Race 2, Psm2), have been reported to be associated with BCD in sweet cherries (Hulin et al. 2019). While Pss is widely prevalent in the United States, Pam has only been reported in Michigan (Renick et al., 2008) as well as in Europe, Central America, South Africa and Australia (Hulin et al. 2019) . In 2023, we surveyed more than 60 cherry orchards and collected hundreds of canker samples from newly planted up to 8-year-old trees. BCD prevalence ranged from 40-100% in cherry orchards, leading to the removal of hundreds of thousands of trees. Affected cherry trees exhibited characteristic bacterial canker symptoms, including dead bud, canker, and gummosis (Fig. S1). Bacteria were isolated from canker tissues or ooze on King's B (KB) agar plates (King et al., 1954) and more than 300 fluorescent Pseudomonas isolates were obtained from 12 symptomatic sweet cherry cultivars. PCR results using Pss- and Pam- specific primers (SyrB and Psm1, Table S1) (Sorensen et al., 1998; Kałużna et al., 2016) revealed that 91.9% and 8% isolates were tested positive for SyrB and Psm1, indicating that these isolates potentially belong to Pss and Pam, respectively. Pathogenicity tests using immature cherries cv. Sentina showed that all isolates caused typical necrotic lesions and could be re-isolated and re-identified as Pss and Pam, thus completing Koch's postulates. The identity of three Pam representative isolates (S79, S158, S202) was further confirmed by comparing gyrD and rpoD housekeeping genes as well as 16S rRNA gene sequence with other Pam strains in GenBank (Parkinson et al., 2010; Gomila et al., 2017). Blast searches against GenBank using gyrB (GenBank accession numbers PP357444-PP357446), rpoD (PP357447-PP357449) and 16S rRNA (GenBank accession numbers PP421223-PP421225) gene sequences, ranging from 520 to 859bp, matched those of the Pam isolates (GenBank accession numbers CP026558 or PP218075) with 100% homology and 100% query coverage, further indicating that these isolates are indeed Pam. This represents the first documented record of Pam causing BCD in the Pacific Northwest, USA, suggesting the complexity of the disease, which underscores the need for effective management strategies for cherry growers in the region.

Biomarker Identification for Preterm Birth Susceptibility: Vaginal Microbiome Meta-Analysis Using Systems Biology and Machine Learning Approaches.

The vaginal microbiome has a substantial role in the occurrence of preterm birth (PTB), which contributes substantially to neonatal mortality worldwide. However, current bioinformatics approaches mostly concentrate on the taxonomic classification and functional profiling of the microbiome, limiting their abilities to elucidate the complex factors that contribute to PTB. A total of 3757 vaginal microbiome 16S rRNA samples were obtained from five publicly available datasets. The sampleswere divided into two categories based on pregnancy outcome: preterm birth (PTB) (N = 966) and term birth (N = 2791). Additionally, the samples were further categorized based on the participants' race and trimester. The 16S rRNA reads were subjected to taxonomic classification and functional profiling using the Parallel-META 3 software in Ubuntu environment. The obtained abundances were analyzed using an integrated systems biology and machine learning approach to determine the key microbes, pathways, and genes that contribute to PTB. The resulting features were further subjected tostatistical analysis to identify the top nine features with the greatest effect sizes. We identified nine significant features, namely Shuttleworthia, Megasphaera, Sneathia, proximal tubule bicarbonate reclamation pathway, systemic lupus erythematosus pathway, transcription machinery pathway, lepA gene, pepX gene, and rpoD gene. Their abundance variations were observed through the trimesters. Vaginal infections caused by Shuttleworthia, Megasphaera, and Sneathia and altered small metabolite biosynthesis pathways such as lipopolysaccharide folate and retinal may increase the susceptibility to PTB. The identified organisms, genes, pathways, and their networks may be specifically targeted for thetreatment of bacterial infections that increasePTB risk.

First Report of Bacterial Leaf Spot Disease on Sesame (Sesamum indicum L.) Caused by Pseudomonas syringae pv. sesami in North Carolina.

In July 2022, dark brown to black, angular, water-soaked lesions were observed on sesame leaves (Sesamum indicum L.) in a research plot established to assess yield potential for eight varieties at the North Carolina (NC) Sandhills Research Station (Chavez 2023). Symptoms were indicative of a bacterial leaf spot (BLS). At early flowering stage, leaf spots were present on scattered plants; varieties ES108, SS3301, and ES201 exhibited up to 75% disease prevalence, with lower frequency in ES103, S39, S4302, S3251, and S3276. Symptomatic leaves from 3-4 plants were collected on four different dates from July through September. A section of symptomatic tissue was excised and macerated in sterile deionized water (SDW). A 10 µL aliquot was streaked onto SPA medium (15 g sucrose, 5.0 g proteose peptone, 0.50 g MgSO4 7H2O, 0.25 g K2HPO4, 15 g agar per liter of SDW) and incubated at 28ºC. After 72 h, numerous, smooth, white-cream colored, convex-shaped, colonies were individually isolated. Five randomly selected isolates from the different collection dates, designated as AHP108-AHP111 and AHP116, were genotyped. The 16S rRNA, gyrB, rpoD, and gapA genes were sequenced (Heuer et al. 1997; Hwang et al. 2005) and deposited to NCBI (GenBank Accessions: P213467- PP213470; OQ628040-OQ628042; PP214983-PP214994; and PP255798). These five isolates shared 100% sequence identity for gyrB and rpoD. AHP108-AHP111 shared 100% sequence identity for 16S rRNA and gapA, with 99.7% and 90.8% identity, respectively, for AHP116. A phylogenetic tree was inferred from a maximum-likelihood analysis of concatenated gyrB, rpoD, and gapA sequences of the five isolates and the top 11 hts from a blastn search of the NCBI nucleotide database. Those hits included closely related sequences from Pseudomonas syringae pv. sesami type strains ICMP 763T and ICMP 7459T. Based on this phylogenetic analysis AHP108-AHP111 and AHP116 are P. syringae pv. sesami. Recent genomic analysis suggests this pathovar is part of P. amygdali (Gomila et al. 2017), but an official name change has not been proposed. Each of the five isolates were infiltrated into leaves of sesame varieties ES108, ES103, and S327, consistently resulting in similar symptoms. Thus, strain AHP116, as a representative, was used to fulfill Koch's postulates using five, 30-day-old potted sesame plants (var. S3301). Plants were spray-inoculated with a bacterial suspension of ~108 CFU/ml until runoff; plants were incubated in moist chambers 24 h pre and post inoculation at 28ºC with 80% relative humidity and a 12 h photoperiod. At 13 days post inoculation, symptoms resembling those on plants at the Sandhills Research Stations in 2022 were evident. Reisolated bacteria were confirmed to be AHP116 through 16S rRNA and gyrB amplification and sequencing. No symptoms were observed on the five water-inoculated plants. BLS of sesame has been reported in Asia and is thought to be seedborne (Firdous et al. 2009; Prathuangwong and Yowabutra 1997). To our knowledge, this is the first report of P. syringae pv. sesami causing BLS on sesame in North Carolina. Sesame cultivation in the state increased from approximately 2,000 acres in 2022 to 13,000 acres in 2023 and there is interest in cultivating sesame as a rotational and alternative crop because it requires minimal input costs. Potential outbreaks of BLS in this warm, humid region could negatively affect sesame production, where little is known about the economic impact of the disease.

First report of Cassava Bacterial Blight caused by Xanthomonas phaseoli pv. manihotis in the Amazonian forest of Ecuador.

Xanthomonas phaseoli pv. manihotis (Xpm) is a plant pathogenic bacterium known as the causal agent of cassava bacterial blight (CBB). CBB is the most limiting bacterial disease affecting cassava (Manihot esculenta Crantz), characterized by diverse symptoms including angular water-soaked leaf lesions, blight, wilting, stem exudates, stem cankers and dieback. CBB has been reported in most cassava-growing regions around the world, and, under conducive conditions, crop yield losses can reach up to 100% (Zárate-Chaves et al. 2021). While Xpm genetic diversity is remarkably high in South America (Bart et al. 2012) and cassava originates and was domesticated in the Amazon basin (Allem 2002), reports of CBB in the Amazonian region are missing. To fill this gap, in October 2018 we surveyed for CBB symptoms in cassava fields of the Orellana Province, located in the Amazon forest of the Republic of Ecuador. Adult cassava plants exhibiting typical angular, water-soaked leaf lesions were found in polyculture plots, i.e. intercrops of cassava with other species such as plantains and fruit trees (a.k.a. chakras). After surface disinfection with 5% sodium hypochlorite followed by 70% ethanol, white Xpm-like colonies were isolated from diseased leaf tissues of four plants on YPGA medium (yeast extract, 5 g/l; peptone, 5 g/l; glucose, 5 g/l; agar-agar, 15 g/l) supplemented with cephalexin (40 mg/l) and cycloheximide (50 mg/l). Pathogenicity tests were performed on peat-potted, 2-month-old cassava plants of the cultivar 60444. Bacterial suspensions were adjusted to an OD600 of 0.2 (2 × 108 CFU/ml) in sterile 10-mM MgCl2 and syringe infiltrated in fully-expanded leaves. In parallel, 20 µl of each bacterial suspension adjusted to an OD600 of 0.02 (2 × 107 CFU/ml) were inoculated on stems inside a hole previously punched with a sterile needle in the junction of the third-top petiole. Sterile 10-mM MgCl2 was used for mock inoculations in both leaves and stems, and experiments were replicated in three plants. Plants were incubated in a greenhouse at 28 ± 1°C with a 12-h photoperiod. Infiltrated leaves developed watersoaking 3 days post inoculation, while wilted leaves, stem exudates, and dieback were observed 21 days after stem inoculation. Control plants remained symptomless. White Xpm-like colonies were re-isolated from symptomatic leaves (Fig S1). One colony of each of the four Xpm isolates (before and after re-isolation) was assessed using diagnostic PCRs (Bernal-Galeano et al. 2018; Flores et al. 2019), using strain Xam668 as positive control. All four candidates were positive for both diagnostic tools. The sequences of the housekeeping genes atpD, dnaK, efp, glnA, gyrB and rpoD of our isolates were extracted from full genome sequences obtained through Oxford Nanopore Technologies (ONT) (GenBank OR288194 to OR288217) and compared to their homologs in four close Xanthomonas species and a reference Xpm strain (Table S1). The sequences of the tested strains aligned with that of Xpm CIO151 (GCA_004025275.1) (Arrieta-Ortiz et al. 2013) with nucleotide identity above 99.92% (Fig S2). The four strains were named CIX4169, CIX4170, CIX4171 and CIX4172, stored in the IRD Collection of Xanthomonas, where they are available upon request. To our knowledge, this is the first report of CBB in the Amazonian region and in Ecuador, where cassava is a central element for local culture and economy. Further surveys will be necessary to evaluate the distribution and prevalence of CBB in other ecozones of Ecuador where cassava is cultivated.

Pseudomonas hefeiensis sp. nov., isolated from the rhizosphere of multiple cash crops in China.

Three bacterial strains, FP250T, FP821, and FP53, were isolated from the rhizosphere soil of oilseed rape, licorice, and habanero pepper in Anhui Province, Xinjiang Uygur Autonomous Region, and Jiangsu Province, PR China, respectively. All strains were shown to grow at 4-37 °C and pH 6.0-9.0, and in the presence of 0-4.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences or housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) and phylogenomic analysis showed that strains FP250T, FP821, and FP53 belong to the genus Pseudomonas, and are closely related to Pseudomonas kilonensis DSM 13647T, Pseudomonas brassicacearum JCM 11938T, Pseudomonas viciae 11K1T, and Pseudomonas thivervalensis DSM 13194T. The DNA G+C content of strain FP205T was 59.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values of strain FP205T with the most closely related strain were 93.2 % and 51.4 %, respectively, which is well below the threshold for species differentiation. Strain FP205T contained summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as major fatty acids, and diphosphatidylglycerol along with phosphatidylethanolamine and aminophospholipid as major polar lipids. The predominant isoprenoid quinone was ubiquinone-9. Based on these phenotypic, phylogenetic, and chemotaxonomic results, strain FP205T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas hefeiensis sp. nov. is proposed. The type strain is FP205T (=ACCC 62447T=JCM 35687T).

Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative bacterium considered an emerging multi-drug-resistant pathogen. Furthermore, this bacterium can survive in extreme environmental conditions, which makes it a frequent cause of nosocomial infection outbreaks. Gene expression analyses by Reverse Transcription Quantitative real-time PCR (RT-qPCR) depend on a reference gene, also called an endogenous gene, which is used to normalize the generated data and thus ensure an accurate analysis with minimal errors. Currently, gene expression analyses in A. baumannii are compromised, as there are no reports in the literature describing the identification of validated reference genes for use in RT-qPCR analyses. For this reason, we selected twelve candidate reference genes of A. baumannii and assessed their expression profile under different experimental and culture conditions. The expression stability of the candidate genes was evaluated by using statistical algorithms such as BestKeeper, geNorm, NormFinder, Delta CT, and RefFinder, in order to identify the most suitable candidate reference genes for RT-qPCR analyses. The statistical analyses indicated rpoB, rpoD, and fabD genes as the most adequate to ensure accurate normalization of RT-qPCR data in A. baumannii. The accuracy of the proposed reference genes was validated by using them to normalize the expression of the ompA gene, encoding the outer membrane protein A, in A. baumannii sensible and resistant to the antibiotic polymyxin. The present work provides suitable reference genes for precise RT-qPCR data normalization on future gene expression studies with A. baumannii.

Occurrence of Pseudomonas cichorii Causing Midrib Rot of Lettuce Under Hydroponic Cultivation in Chile

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

First Report of Pseudomonas oryzihabitans Causing Walnut Leaf Spot Disease in China.

China ranks first in the production and harvest area of walnut (Juglans regia L.) worldwide. Currently, the poor health and low yield of walnut caused by pathogen infection is of concern. In 2022, severe walnut leaf spot disease was observed on the seedlings of four walnut nurseries (0.08 to 0.23 ha) in Liaocheng, Shandong, China, with an average incidence of 48.6% (from 34.6% to 65.3% on the cultivar Xiangling). From August to October, leaf spots mainly appeared on the edges of the leaflets, and occasionally between veins. The lesions were initially soft and rotten, and then light brown, round to semi-circular. Subsequently, the adjacent lesions fused, and the edges of the leaflets and entire leaflets showed symptoms of browning and wilting. For pathogen isolation, five leaflets with representative symptoms from one of the nurseries were collected and wiped three times with sterile absorbent cotton dipped in 75% alcohol and washed with distilled water. Leaflet pieces at the junction of the lesion and healthy tissues were removed, crushed in a sterile mortar, and soaked in a small amount of distilled water for 10 min. The diseased tissue suspension was streaked on a nutrient agar medium (NA) with a sterile inoculation ring and incubated at 28°C for 24 to 72 h. The bacterial colonies obtained were further cultured on NA. The purified colonies were uniform in shape, round, and yellow, with a raised, shiny surface and smooth margin. The isolates were Gram-negative, and the electron microscope analysis showed that the pathogens were short rods (0.35 to 0.52 × 0.90 to 1.24 μm, average = 0.44 ± 0.05 × 1.08 ± 0.11 μm, n = 25). For bacterial species identification, a single-colony culture was subjected to genomic DNA extraction and gene amplification and sequencing of 16S rRNA, rpoD, and gyrB. The universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene and the specific primers 70F/70R and UP-1E/APrU (Yamamoto et al. 2000) were used to amplify the rpoD and gyrB genes, respectively. In the BLAST analysis, the 16S rRNA sequence (GenBank OR195734) of the isolate shared 99% similarity (1409/1410 bp) with Pseudomonas oryzihabitans strain IAM 1568T (AM262973.1), and the rpoD (OR709708) and gyrB (OR709707) sequences showed >98% identity to rpoD (707/717 bp; FN554494.1) and gyrB (787/801 bp; FN554210.1) of P. oryzihabitans strain LMG 7040T. Based on the above results, the isolated bacterium was identified as P. oryzihabitans. For the pathogenicity test, healthy leaflets from 10 two-year-old potted walnut seedlings (cv. Xiangling) were used as inoculation materials. The leaflets were punctured with a sterile inoculation needle of 0.4 mm, and three small holes on each leaflet at an interval of about 5 mm were covered with a piece of sterile cotton. A bacterial suspension (1 ml) at 107 CFU/ml was spread onto the cotton, and wrapped with plastic film for 24 h. Water was used as a negative control. The inoculations were performed five times. Plants were grown outdoors at a daily average temperature of 22°C with relative humidity over 45%. Two days after inoculation, the disease began to develop in the leaflets with similar symptoms to those observed in the field. In contrast, control plants remained healthy and symptomless. Bacteria were reisolated from the inoculated walnut plants, and the morphology and 16S rRNA gene sequences of the isolates were the same as those of the original strains. Since it was discovered as an opportunistic human pathogenic bacterium in the 1970s (Keikha et al. 2019), P. oryzihabitans has also been shown to cause certain plant diseases, such as panicle blight and grain discoloration on rice (Hou et al. 2020), fruit black rot on prickly ash (Liu et al. 2021), and stem and leaf rot on muskmelon (Li et al. 2021). As far as we know, this is the first report of P. oryzihabitans causing walnut leaf spot disease in China. Leaf spot caused by P. oryzihabitans may be a threat to walnut cultivation, and this report of its occurrence is the first step in determining potential spread and effective control measures.

First Report of Bacterial Leaf Spot on Muskmelon Caused by Pseudomonas syringae pv. syringae in China.

Muskmelon (Cucumis melo L.) is one of the most widely cultivated and economically important fruit crops in the world. In January 2023, muskmelon leaves of cultivar 'Sheng Gu' were observed with irregularly shaped spots in four nurseries in Wanxiang Village, Pudong District of Shanghai, China. Initial symptoms were irregular soaking on the leaves, which progressed to rotting and necrotic spots. The disease incidence of melon seedlings in different nurseries ranged from 10 to 25%. To isolate and identify the causal agent, the small pieces of lesion tissues (5×5 mm) from symptomatic leaves were sterilized in 75% ethanol for 30 s and rinsed three times with sterile water. Following that, tissues were crushed with sterile glass rod in a sterile 2.0 mL centrifuge tube containing 100 μl of sterile water. The suspension was serially diluted before being spread on Luria-Bertani (LB) medium. After 48 h of incubation at 28°C, the cream-colored bacterial colonies from the 10-4 dilution were tiny and purified by streaking on new LB plates. To confirm the species identity of the bacterial isolates, genomic DNA was extracted from four independent representative colonies from different diseased plants, and several conserved genes were amplified and sequenced, including the 16S rRNA gene with primers 27F/1492R, gyrB gene with primers gyrBFor2/gyrBRev2, and rpoD gene with primers rpoDFor2/rpoDRev2 (Lelliot et al. 1966; Murillo et al. 2011). The results showed that the four colonies were identical. Using BLAST analysis in GenBank, the 16S rDNA (accession no. OQ659765, 1,402 bp), the gyrB (accession no. OQ708618, 911 bp), and rpoD sequences (accession no. OQ708619, 798 bp) showed 99.86-100% homology with 99-100% coveage as the corresponding gene sequences in the P. syringae pv. syringae strain HS191 (accession no. CP006256.1). The bacterial isolate was designated as P. syringae pv. syringae strain PDTG. Phylogenetic tree analysis of 16S rDNA, gyrB and rpoD genes further verified that the bacteria isolate was in close proximity to P. syringae pv. syringae. Additionally, all four isolates were detected in PCR with P. syringae pv. syringae specific primers, PsyF/ PsyR (Borschinger et al. 2016; Guilbaud et al. 2016). Ten two weeks old healthy 'Sheng Gu' muskmelon seedlings were inoculated by spraying with a bacterial suspension of 108 CFU/ml, and ten additional healthy plants treated with sterilized water served as the control. The inoculated plants were maintained at 25°C and 75% relative humidity for 7 days in artificial climate room. Water-soaked rot, similar as those seen in the nurseries, appeared on leaves 7 days after inoculation (dai), while the leaves of control plants remained healthy. The bacteria were re-isolated from rot of inoculated leaves and confirmed as the original pathogen by PCR with the PsyF/ PsyR primers and the 16S rRNA gene sequences. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on muskmelon in China, and this report expands the host range of P. syringae pv. syringae.

Pseudomonas aphyarum sp. nov., Pseudomonas fontis sp. nov., Pseudomonas idahonensis sp. nov. and Pseudomonas rubra sp. nov., isolated from in, and around, a rainbow trout farm.

During a large-scale bacterial culturing effort of biofilms in the vicinity of a rainbow trout aquaculture facility in Idaho, USA, 10 isolates were identified as having pathogen-inhibiting activity and were characterized further. These isolates were shown to be Gram-negative, rod-shaped bacteria belonging to the genus Pseudomonas. Whole-genome comparisons and multi-locus sequence analysis using four housekeeping genes (16S rRNA, gyrA, rpoB and rpoD) showed that these 10 isolates clustered into four distinct species groups. These comparisons also indicated that these isolates were below the established species cutoffs for the genus Pseudomonas. Further phenotypic characterization using API 20NE, API ZYM and Biolog GENIII assays and chemotaxonomic analysis of cellular fatty acids were carried out. Based on the genomic, physiological and chemotaxonomic properties of these isolates, we concluded that these strains composed four novel species of the genus Pseudomonas. The proposed names are as follows: Pseudomonas aphyarum sp. nov. consisting of strains ID233, ID386T and ID387 with ID386T (=DSM 114641T=ATCC TSD-305T) as the type strain; Pseudomonas rubra sp. nov. consisting of strains ID291T, ID609 and ID1025 with ID291T (=DSM 114640T=ATCC TSD-303T) as the type strain; Pseudomonas idahonensis sp. nov. consisting of strains ID357T and ID1048 with ID357T (=DSM 114609T=ATCC TSD-304T) as the type strain; and Pseudomonas fontis sp. nov. consisting of strains ID656T and ID681 with ID656T (=DSM 114610T=ATCC TSD-306T) as the type strain.

Xanthomonas species causing leaf blight on eucalypt plants in Brazil and transfer of Xanthomonas axonopodis pv. eucalyptorum to Xanthomonas citri pv. eucalyptorum comb. nov.

AbstractOutbreaks of bacterial leaf blight (BLB) frequently affect eucalypt plants under nursery and field conditions in several countries. Although research has been conducted to unveil the causal agent, different bacterial species have been associated with similar disease symptoms in different countries. In order to determine the causal agent of BLB in Brazil, a survey was conducted in nine states to recover bacterial isolates from eucalypt plants exhibiting typical BLB symptoms. A total of 41 yellow‐colony isolates with varying aggressiveness towards a susceptible eucalypt clone were obtained, with 16S rDNA sequences indicating that they belong to the Xanthomonas genus. Rep‐PCR analysis separated the Xanthomonas population affecting eucalypt into six distinct groups revealing its high genetic diversity. The same population formed three clusters together with reference strains of X. citri, X. euvesicatoria and X. phaseoli in a phylogenetic tree constructed with partial dnaK, fyuA, gyrB and rpoD gene sequences. Clustering in the phylogenetic tree was clearly related to grouping based on rep‐PCR. Genome sequence comparisons of representative eucalypt isolates with type strains of validly published Xanthomonas species confirmed that the population consisted of X. citri, X. euvesicatoria and X. phaseoli. Inoculation of tomato, common bean, castor bean and eucalypt plants showed that the representative eucalypt isolates can cause disease in these plant species. Based on the results, the transfer of Xanthomonas axonopodis pv. eucalyptorum to Xanthomonas citri is proposed. These results are relevant for eucalypt BLB management under nursery and field conditions, including selection and deployment of effective plant resistance.

Identification and characterization of motile Aeromonas spp. isolated from farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

Motile Aeromonas septicaemia (MAS) caused by motile Aeromonas species is an important disease in farmed freshwater fish due to intensification of culture and improper farm practices. This study characterized and profiled motile Aeromonas species recovered from clinically sick tilapia farmed in the Philippines, with a view to identifying targeted disease prevention and control measures against MAS in farmed tilapia species. Sixteen isolates from diseased farmed Nile tilapia were identified as Aeromonas veronii (n=14), Aeromonas caviae (n=1), and Aeromonas dhakensis (n=1). Five biochemical profiles using API 20E were exhibited by the A. veronii strains giving an unreliable identification. A high level of agreement was observed in identifying the Aeromonas strains using 16S rRNA and rpoD gene sequencing, although the latter has a higher discriminatory value. Three or more virulence genes dominated by cytotoxic enterotoxin act and aerolysin aer were detected. Different genotypes based on virulence gene clustering suggested varied mechanisms used by Aeromonas to colonize and infect or to mutualistically co-exist with the fish. Acquired multiple antibiotic resistance was found in a single A. veronii isolate. All were susceptible to enrofloxacin, oxolinic acid, florfenicol, and chloramphenicol. Tetracycline and sulfonamide resistances and class 1 integron were detected in three A. veronii isolates. Several strains of motile aeromonads, especially A. veronii, which have varied genotypes based on virulence, biochemical profile, and antibiotic resistance, are involved in MAS in natural disease outbreaks in farmed Nile tilapia in the Philippines.

First Report of Xanthomonas campestris Causing Leaf Blight on Buttercup (Ranunculus asiaticus) in South Carolina, USA.

Buttercup (Ranunculus asiaticus L.) is a popular and high value ornamental species grown in landscapes and gardens and as cut flowers. It is mostly cultivated in Europe, the Mediterranean, and the Americas (Beruto and Debergh, 2004). In January 2022, leaf blight was observed on approximately 24 of forty 4-month-old R. asiaticus plants grown in a high tunnel at a cut flower farm located in Anderson County, South Carolina, USA. Symptoms included irregular, vein-limited, and necrotic leaf lesions and yellowing. Some lesions had a chlorotic halo. Two diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Symptomatic leaves were surface sterilized with 10% bleach for 1 min and rinsed in sterile water. Small leaf portions (1 × 1 cm2) were excised from the margin of lesions. They were macerated in 500 µl of sterile water and incubated at room temperature for 10 min. A loopful of suspension was streaked on nutrient agar (NA). Slightly convex, yellowish-mucoid colonies appeared after incubation at 28°C for 48 h. Two isolates, 23A and 23B, from two plants were obtained by transferring single colonies to new NA plates. Both isolates were identified as X. campestris (probability values > 0.8) using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of these two isolates were performed for housekeeping genes gyrB, rpoD, and dnaK (Young et al. 2008). The amplicon sequences (GenBank accession nos.: OR101193 and OR101194 [dnaK]; OR101195 and OR101196 [gyrB]; OR101197 and OR101198 [rpoD]) were identical between the two isolates based on sequence alignment in MEGA11 (Tamura et al. 2021). Nucleotide BLAST of these three genes showed 94.6 to 98.9% identity (dnaK: 912 of 922 bp; gyrB: 827 of 839 bp; rpoD: 803 of 849 bp) with 100% coverage with the Xanthomonas campestris pv. campestris type strain (AE008922). A neighbor joining phylogenetic tree with the concatenated sequences of these three genes showed that 23A and 23B had the closest match with X. campestris pv. campestris. However, these two isolates tested negative in the probe-based qPCR assay specific for X. campestris pv. campestris with only the positive control amplified (Köhl et al. 2011), suggesting that they may belong to a new pathovar of X. campestris. To confirm the pathogenicity of these isolates, three healthy R. asiaticus plants each were spray inoculated with suspensions of 23A and 23B in sterile tap water until runoff (OD600 = 0.1, approx. 108 CFU/ml). The non-inoculated control plants received a sterile tap water spray. The experiment was conducted twice. All plants were maintained in a growth chamber at 24°C with 10-h photoperiod. Seven to 15 days after inoculation, necrotic lesions with chlorotic halo and leaf yellowing, similar to those observed in the field, were observed on inoculated plants, while the non-inoculated control plants remained symptomless. Koch's postulates were fulfilled by reisolating the bacteria from the symptomatic plants and confirming the bacterial identity with the sequence analysis described above. The disease was first reported in California in 1996 (Azad et al. 1996) but to the best of our knowledge has not been reported again in the United States. This is the first report of X. campestris causing bacterial leaf blight in R. asiaticus in South Carolina. Since more than 50% of the flower producers/farmers grow Ranunculus in South Carolina, further work is necessary to determine how widespread the disease is and its economic impact.

Bacillus dicomae sp. nov., a new member of the Bacillus cereus group isolated from medicinal plant Dicoma anomala.

A Gram-stain-positive, endospore-forming endophytic bacterial strain designated MHSD28T was isolated from surface-sterilized leaves of Dicoma anomala collected from Eisleben, Botlokwa, Limpopo Province, South Africa. The phenotypic and phylogenetic characteristics of strain MHSD28T were consistent with those of members within the Bacillus cereus group. Comparative analysis between this strain and its relatives confirmed that it belongs to this group and forms a monophyletic branch. The digital DNA-DNA hybridization values between strain MHSD28T and its relatives were lower than the 70 % threshold for species delineation. To further determine its phylogenetic position, multi-locus sequence analysis (MLSA) based on five concatenated housekeeping gene (gyrB, atpD, DnaK, rpoB and rpoD) sequences, phenotypic analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) biotyper identification, fatty acid and polar lipid profile analyses were carried out. Phenotypic characterization, MLSA, whole genome sequence based analyses and MALDI-TOF results placed strain MHSD28T within the B. cereus group. The major fatty acids were iso-C15 : 0 and summed feature 3 and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The respiratory quinone was menaquinone-7. The cell-wall peptidoglycan structure included meso-diaminopimelic acid. Considering the above results, strain MHSD28T represents a novel species of the B. cereus group, for which the name Bacillus dicomae sp. nov. is proposed. The type strain is MHSD28T (=BD 2262T=LMG 32287T=CECT 30671T).

A Class 4-like Chromosomal Integron Found in Aeromonas sp. Genomospecies paramedia Isolated from Human Feces.

Integrons are genetic elements that store, express and exchange gene cassettes. These elements are characterized by containing a gene that codes for an integrase (intI), a cassette integration site (attI) and a variable region holding the cassettes. Using bioinformatics and molecular biology methods, a functional integron found in Aeromonas sp. 3925, a strain isolated from diarrheal stools, is described. To confirm the integron class, a phylogenetic analysis with amino acid sequences was conducted. The integrase was associated to class 4 integrases; however, it is clearly different from them. Thus, we classified the associated element as a class 4-like integron. We found that the integrase activity is not under the control of the SOS or catabolic repression, since the expression was not increased in the presence of mitomycin or arabinose. The class-4-like integron is located on the chromosome and contains two well-defined gene cassettes: aadA1 that confers resistance to streptomycin and lpt coding for a lipoprotein. It also includes eight Open Reading frames (ORFs) with unknown functions. The strain was characterized through a Multilocus Phylogenetic Analyses (MLPA) of the gyrB, gyrA, rpoD, recA, dnaJ and dnaX genes. The phylogenetic results grouped it into a different clade from the species already reported, making it impossible to assign a species. We resorted to undertaking complete genome sequencing and a phylogenomic analysis. Aeromonas sp. 3925 is related to A. media and A. rivipollensis clusters, but it is clearly different from these species. In silico DNA-DNA hybridization (isDDH) and Average Nucleotide Identity (ANI) analyses suggested that this isolate belongs to the genomospecies paramedia. This paper describes the first class 4-like integron in Aeromonas and contributes to the establishment of genomospecies paramedia.

The effect of fucoidan and inulin on the expression of Lactobacillus plantarum metabolism genes

The most stable reference gene in Lactobacillus plantarum was identified. It was found that the expression of the rpoD gene was most stable with the addition of fucoidan and inulin. The level of expression of the usp gene in L. plantarum bacteria increased by 4 times when fucoidan was added compared to the control sample, but when inulin was added to the culture medium, the expression of this gene decreased by 12 times compared to the control sample. The expression of the opuA gene with the addition of fucoidan increased by 6 times compared to the control sample, and with the addition of inulin decreased by 3 times compared to the control sample. The level of expression of the ATP gene in L. plantarum bacteria with the addition of fucoidan increased by 9 times in comparison with control sample. The expression of the lactate dehydrogenase (ldh) gene with the addition of fucoidan increased by 2 times compared with the control sample, whereas with the addition of inulin it decreased by 18 times compared to control sample. The expression level of the gacT gene with the addition of fucoidan decreased by more than 50 times compared to the control sample, and with the addition of inulin decreased by 12 times compared to the control sample. There were no significant changes in the expression of the GroEL and hsp1 genes. Thus, based on gene expression data, it can be assumed that when fucoidan is added to the culture medium, catabolic processes in L. plantarum bacteria are activated. It was found that the addition of inulin to the culture medium does not lead to an increase in the expression of catabolism genes in L. plantarum.

First Report of Xanthomonas arboricola pv. corylina Causing Bacterial Blight on Hazelnut Tree (Corylus avellana) in Montenegro.

Hazelnut is a minor but rapidly increasing commercially grown species in Montenegro. In June 2021, severe infection, affecting more than 80% of the trees, was observed on 6-year-old hazelnut plants (Corylus avellana) cultivar Hall's Giant, in a 0.3ha plantation near Cetinje, central Montenegro. Numerous, small, 2-3mm in diameter, irregular, brown, necrotic spots, sometimes surrounded by a weak chlorotic halo, were observed on leaves. As the disease progressed, the lesions coalesced and formed large necrotic areas. Necrotic leaves remained attached to the twigs. Longitudinal brown lesions developed on twigs and branches, causing their dieback. Necrotic, unopened buds were noticed as well. No fruits were observed in the orchard. From the diseased leaf, bud and twig bark tissue, yellow, convex, and mucoid bacterial colonies were isolated on yeast extract dextrose CaCO3 medium and 14 isolates were subcultured. The isolates induced hypersensitive reaction in pelargonium leaves (Pelargonium zonale), were Gram-negative, catalase positive, oxidase negative, obligate aerobic, hydrolyzed starch, gelatin and esculin, did not reduce nitrate and did not grow at 37°C and in the presence of 5% NaCl, showing so the same biochemical profile of the reference strain Xanthomonas arboricola pv. corylina (Xac) NCPPB 3037. Using primer pair XarbQ-F/XarbQ-R (Pothier et al., 2011), a 402 bp product was amplified in all 14 isolates and the reference strain, confirming their affiliation to X. arboricola species. Additionally, the isolates 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 Xac. The amplification and sequencing of the partial rpoD gene sequence of two selected isolates RKFB 1375 and RKFB 1370, were performed using a set of primers described by Hajri et al., 2012. The obtained DNA sequences showed that the isolates (GenBank Nos. OQ271224 and OQ271225) share 99.47% to 99.92% rpoD sequence identity with Xac strains CP076619.1 and HG992342.1 isolated from hazelnut in France and HG992341.1 in USA. Pathogenicity of all isolates was confirmed by spraying young shoots (20 to 30 cm long, with 5 to 7 leaves) on 2-year-old potted hazelnut plants (cv. Hall's Giant) using a handheld sprayer with the bacterial suspension (108 CFU/mL of sterile tap water), in three replicates. Sterile distilled water (SDW) and NCPPB 3037 Xac strain were used as negative and positive control, respectively. The inoculated shoots were incubated under plastic bags, providing high humidity conditions, in an acclimatized greenhouse at 22-26°C, for 72 h. Lesions surrounded by a halo appeared on leaves of all inoculated shoots within 5 to 6 weeks after inoculation, while leaves sprayed with SDW remained symptomless. Koch's postulates were confirmed by the re-isolation of the pathogen from the necrotic test plant tissue and identity checked by PCR using the primer set of Pothier et al., 2011. Based on pathogenic, biochemical, and molecular characteristics, the isolates from hazelnut plants in Montenegro were identified as X. arboricola pv. corylina. This is the first report of Xac affecting hazelnut in this country. Considering favorable environmental conditions, the pathogen can cause significant economic losses in hazelnut production in Montenegro. Therefore, phytosanitary measures have to be implemented to prevent introduction and spread of the pathogen in other areas.