Single-colony Isolates Research Articles

First Report of Wilt Disease of Ginger Caused by Achromobacter xylosoxidans in China

Ginger (Zingiber officinale) is an important commercial crop that has been widely cultivated in China for more than 2500 years. One variety, Tongling white ginger, has been grown in the Yi'an District of Tongling city, Anhui province (30°45 N, 117°43 E), China. In August 2022, symptoms of yellowing and wilting were observed on ginger plants, with a disease incidence rate exceeding 20% in the field. The stem base of the diseased plants became soft, brown and water-soaked. Additionally, the rhizomes displayed symptoms of browning and water-soaked rot, resembling those caused by Ralstonia solanacearum and Enterobacter cloacae (Yu et al. 2003; Nishijima et al. 2004; Liu et al. 2021). Consequently, ginger bacterial wilt disease may potentially emerge from a combination of infections by diverse pathogenic bacteria. To identify novel pathogens causing the wilt disease, stem tissues of the diseased plants from different locations were sterilized with 1% sodium hypochlorite (NaOCl) for 10 min, followed by at least three time rinses with sterile water. The sterilized samples were then ground with 0.9% saline solution and left at room temperature for 30 min. A 20 μL aliquot of the suspension was serially diluted and cultured on Luria-Bertani (LB) medium at 28°C. A total of 217 isolates was picked and purified for taxonomic identification by 16S rRNA gene analyses with the primer 27F/1492R (Weisburg et al. 1991). Among these isolates, 23 single colony isolates were identified as A. xylosoxidans through NCBI BLASTn analyses. Furthermore, three single isolates from different locations were randomly selected for further experiments. The growing colonies appeared opaque white and round. Microscopic evaluation revealed that cells were rod-shaped with an average length of 1.95 µm and average width of 0.46 µm. The three isolates shared identical 16S rRNA sequences, displayed 99.72% identity with the sequence from A. xylosoxidans strain SeqID2 (GenBank accession NO. MH266081.1). The glutamate synthase (gltB), GTP-binding membrane protein (lepA), NADH:ubiquinone oxidoreductase subunit L (nuoL), RNA polymerase beta-subunit (rpoB), and the enolase (eno) genes of the three isolates were amplified by PCR using primer pairs gltB-F/gltB-R, lepA-F/lepA-R, nuoL-F/nuoL-R, rpoB-F/rpoB-R and eno-F/eno-R, respectively (Spilker et al. 2012; Vandamme et al. 2016). The gene sequences of 16S rRNA (OQ711945, OQ740153 and OR230037), gltB (OR242732, OQ737692 and OR262112), lepA (OR233727, OQ737693 and OR262113), nuoL (OR233726, OQ737694 and OR262114), ropB (OR233725, OQ737695 and OR262115) and eno (OR242733, OQ737696 and OR262116) for the isolates ZOR02, ZOR05 and ZOR12 were deposited in GenBank database. The gltB, lepA, nuoL, rpoB and eno sequences of the isolates ZOR02, ZOR05 and ZOR12 showed 98.66-99.16%, 98.9-100%, 96.28%-97.34%, 98.47-99.44% and 99.27-99.82% similarity to A. xylosoxidans strain AX27, respectively. Phylogenetic trees were constructed based on the 16S rRNA and gltB-lepA-nuoL-rpoB-eno multilocus using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA11.0 software (Álvarez et al. 2018). For pathogenicity tests, bacterial suspensions were initially prepared in sterile water at a final concentration of 108 CFU mL-1. Subsequently, 10 μL of bacterial suspensions was injected into the stem base of two-month-old ginger plants, while sterile water was used as a control (Wang et al. 2022). These plants were then incubated at 28°C and 70% relative humidity. There were three replicates for each treatment, and each replicate contained five plants. After six days of inoculation, the ginger plants injected with bacterial suspensions alone exhibited severe wilting symptoms similar to those observed in the field. However, water-soaked symptoms were not observed on rhizome tissues from the pathogen-infected plants. Bacterial pathogens were re-isolated from the diseased plants and identified using the morphological and molecular methods to meet Koch's hypothetical tests. To our knowledge, this is the first report of A. xylosoxidans causing wilt disease of ginger in China. In 2022, the average yield loss due to wilt disease in the Yi'an District of Tongling exceeded 20%, posing a major threat to local ginger cultivation. Effective disease management strategies are needed to develop for the control and prevention of the disease.

Enhancing the Conventional Culture: the Evaluation of Several Culture Media and Growth Conditions Improves the Isolation of Ruminal Bacteria

The rumen microbiota is critical in cattle digestion. Still, its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested comprised eight media cultures, three sample dilutions (10−2, 10−6, 10−12), and two incubation times (3 and 7 days). Bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates. The results indicate that none of the culture media recovered all rumen populations and that there was an altered relative abundance of the dominant phyla. In the rumen, Bacteroidetes and Firmicutes comprised 75% and 15% of the relative abundance, respectively, while in the culture media, these were 15% and 60%, respectively. Principal coordinate analysis (PCoA) of the bacterial community revealed significant shifts in population composition due to dilution, with 10−2 and 10−6 dilutions clustered closely while the 10−12 dilution differed markedly. In contrast, incubation duration did not influence population diversity. According to the results, two media, CAN and KNT, were selected based on their ability to recover more similar populations compared to the rumen sample. The metataxonomic study showed that CAN media had consistent reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate. From these, 64 pure cultures were obtained and 54 were identified through 16S rRNA gene sequencing. Being Streptococcus the most frequently isolated genus, this prevalence contrasts with the liquid media composition, underscoring the importance of refining single colony isolation strategies. Although no culture medium could replicate the native rumen bacterial population perfectly, our findings highlight the potential of CAN and KNT media in recovering populations that are more closely aligned to natural rumen conditions. In conclusion, our study emphasizes the importance of integrating molecular approaches in selecting suitable cultivation media and parameters to depict rumen bacteria accurately.

Core genome multilocus sequence typing of Clostridioides difficile to investigate transmission in the hospital setting

PurposeTraditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission.MethodsWe analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed.ResultsFrom January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters.ConclusionAmong clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.

In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.

Standardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and/or conditions and generate meaningful information for clinically relevant settings. IMPORTANCE Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

First report in South Korea of a bacterial leaf blight of oats caused by Pseudomonas salomonii.

In March 2020, approximately 20% of leaves in a commercial oat field growing Avena sativa (cv. Samhan) in Jeongeup, Korea (35.3859°, 126.5607°), displayed leaf blight in the seedling stage of development. The lesions observed in 2020 were of a yellow discoloration that spread from the leaf tip downward with minimal brown delineation (Fig. S1A). These symptoms differed from the haloes delineated at the edges by extensive brown necrosis caused by Pseudomonas coronofaciens (Kim 2020a; Fig. S1C). To isolate the causal agent, 3-cm-long pieces of symptomatic leaves from ten different oat plants were disinfected by submersion in 70% ethanol for 5 min followed by immersion in 1% sodium hypochlorite for 5 min and rinsing extensively with sterile distilled water. The air-dried segments were transferred intact to nutrient agar, and only one colony type (yellow-colored, wet, with a shiny convex surface) was observed. After single colony isolation, three isolates from different 2020 field-grown diseased leaves, termed 2007, 2009, and 2011, were selected at random. The isolates produced fluorescent siderophores on King's medium B and triggered a hypersensitive response (HR) when infiltrated into tobacco (cv. Xanthi) leaves (Fig. S1D). Multilocus sequence typing analysis with four housekeeping genes was used for taxonomic identification of these isolates (Maiden et al. 1998). The 16S rRNA sequences were amplified with the 27F/1492R universal primers (Weisburg et al. 1991). The primers for three housekeeping genes were designed using genome sequence of P. coronaficiens X-1 causing halo blight disease in Korea (NZ_CP050260.1, Kim, 2020b). The products obtained had sizes of 640 bp for gltA (using primers F: 5'-CCT GGT AGC CAA GAT GCC GAC-3'; R: 5'-CAA AGA TCA CGG TGA ACA TGC TGG-3'), 710 bp for gyrB (with primers F: 5'-TCG GCA GCC GAG GTC ATC ATG AC-3'; R: 5'-TTG TCT TTG GTC TGC GAG CTG AA-3'), and 870 bp for rpoD ( with primers F: 5'-GTG AAG GCG AAA TCG AAA TCG-3'; R: 5'-CCG ATG TTG CCT TCC TGG ATC AG-3'). The concatenated sequences (2,353 bp/2,376 bp) had 99% identity with the gene sequences from P. salomonii type strain (AY091528.1, NZ_FNOX01000003, LC486864.1, LC486849.1), but lesser identity (90%) with P. coronafaciens (Fig. S2). Pathogenicity of the Korean isolates was confirmed by fulfilling Koch's postulates using leaves of 2 week-old greenhouse-grown 'Samhan' seedlings. Plants (n=50) were sprayed with 108 cfu/ml bacterial suspensions in water or with sterile water as controls. The plants were incubated for a week at 23 °C in 100% relative humidity under a 10 h light/14 h dark photoperiod. Five days after bacterial inoculation, yellow discoloration appeared at the leaf tips which progressed downward with time (Fig. S1B). Three bacterial isolates extracted from yellowed, inoculated leaves had 16S rRNA gene sequences identical to that of P. salomonii Korean isolates, 2007, 2009 and 2011, and they caused the anticipated symptoms when inoculated into oat leaves. These findings indicate that P. salomonii should be added to the potential pathogens of oats grown in Korea. Understanding whether spring weather conditions (warmth and humidity) boost this oat disease will help devise disease alert systems for farmers (Anderson 2004; Chakraborty 2005).

First report of stem blight caused by Diaporthe eres on highbush blueberry (Vaccinium corymbosum) in Michigan.

The U.S. is the world's leading producer of highbush blueberries (Vaccinium corymbosum L.), and Michigan is ranked in the top five production states (USDA NASS, 2022). In June and July of 2021, 268 blueberry stem blight samples were collected for a pathogen survey across 22 total fields in Van Buren and Ottawa counties in Michigan. Current season stems with symptoms of necrosis and wilting were collected. Stems were cut just below the necrotic area and cross-sections (2-3 mm long) were surface disinfested in 10% bleach for 1 min, rinsed twice in sterile distilled water, and dried on sterile paper towels. Stem cross-sections were plated onto potato dextrose agar (PDA) amended with 100 µg/ml streptomycin sulfate and 50 µg/ml ampicillin. Plates were incubated at 21°C under a 12-h photoperiod for 5-6 days. Outgrowing fungi with morphology similar to Diaporthe spp. were transferred to new PDA plates 2 consecutive times after 7 days of similar incubation to ensure single colony isolation. After 7 days, colonies consisted of white and light brown mycelia that were mostly flat, with some isolates that had partially raised mycelia towards the center of the plate. After 3-4 weeks, colonies turned brown and gray and produced dark brown pycnidia. Aseptate, hyaline, fusiform to ellipsoid, biguttulate alpha conidia measuring 5.4 to 7.6 x 2.6 to 3.7 µm (n = 60) were produced. No beta conidia were observed. In total, 3 isolates, representing 3 different farms (37-95 km apart) and cultivars ('Duke', 'Jersey', and 'Bluecrop'), as well as 2 counties, were identified as Diaporthe through colony morphology (Gomes et al. 2013, Udayanga et al. 2014). Amplification and subsequent Sanger sequencing were performed for the internal transcribed spacer (ITS) region and portions of the translation elongation factor (TEF) 1-α, β-tubulin (TUB), and histone H3 (HIS) genes using primers ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), T1/Bt-2b (Glass and Donaldson 1995), and CYLH3F/H3-1b (Glass and Donaldson 1995), respectively. Representative sequences were deposited in NCBI GenBank (accession no. OQ507870-OQ507872 for ITS, and OQ550272-OQ550278 for TEF, HIS, and TUB). BLASTn results revealed 97-100% identity for all 4 genes across other established D. eres isolates reported in Gomes et al. (2013). For example, JMK047 had 99.8% (577/578 bp), 99.7% (327/328 bp), 100% (701/701 bp), and 100% (439/439 bp) homology with ITS, TEF, TUB, and HIS sequences, respectively, of D. eres CBS 439.82 (accession no. KC343090, KC343816, KC344058, KC343574). Koch's Postulates were fulfilled via pathogenicity tests on 2-year-old potted 'Blueray' plants with 2 isolates. Stems were surface sterilized with 1% bleach then 8-mm long pieces of bark were removed using a sterile razor blade to expose the cambium. Plugs of sterile PDA (negative control) or mycelia from 7-day old cultures on PDA (5-mm diameter) were placed onto the cambium layer and sealed with Parafilm. Six stems on unique plants were inoculated per treatment. Plants were grown in a 20.5°C greenhouse with a 14-hr photoperiod. After 3 weeks, the stems inoculated with D. eres isolates showed similar stem blight symptoms to those observed in the field while control stems remained healthy. Re-isolation and sequencing of the ITS region of 3 replicates per treatment with the protocol described above confirmed symptoms correlated with D. eres isolates. This is the first report of D. eres associated with stem blight of highbush blueberry in Michigan, and the second report in the U.S. (Lombard et al. 2014). Increasing prevalence of D. eres in U.S. blueberries may affect disease management programs. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. 10.1080/00275514.1999.12061051. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. 10.1128/aem.61.4.1323-1330.1995. Gomes, R. R., et al. 2013. Persoonia 31:1. 10.3767/003158513x666844. Lombard, L., et al. 2014. Phytopathol. Mediterr. 51(2):287. 10.14601/Phytopathol_Mediterr-14034. Udayanga, D., Castlebury, L. A., Rossman, A. Y., Chukeatirote, E., and Hyde, K. D. 2014. Fungal Divers. 67:203-229. 10.1007/s13225-014-0297-2. USDA NASS. 2022. Noncitrus Fruits and Nuts 2021 Summary. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, California, USA.

Fim3-dependent autoagglutination of Bordetella pertussis

Autoagglutination (Agg) of Bordetella pertussis is often observed in clinical laboratory. However, its causal factors and frequency in circulating strains are unknown. Repeated single colony isolation enabled us to detect an Agg- mutant in the supernatant of an Agg+ strain of B. pertussis. Whole-genome sequencing and immunoblot analysis disclosed that the Agg- mutant had a single C-deletion in its fim3 promoter region (Pfim3) which abolished Fim3 fimbriae production. A B. pertussis fim3-knock out mutant also lacked the Agg+ phenotype. Agg+ clinical isolates were detected a higher production of Fim3 than Fim3-producing Agg- isolates. B. pertussis is known to harbor multiple Pfim3 poly(C) lengths within a single strain culture and our newly developed PCR/LDR assay revealed that Agg+ isolates harbor the highest Pfim3 poly-14C abundance. We evaluated the frequency of autoagglutination in clinical B. pertussis isolates collected in Japan between 1994 and 2018 (n = 203). Fim3 production was confirmed for 190 isolates and 74.7% of them displayed the Agg+ phenotype. The Agg+ phenotype was strongly associated with Pfim3 poly-14C abundance. Taken together, our findings demonstrated that B. pertussis autoagglutination occurs in response to high Fim3 levels and the Agg+ strain has predominated in Japan over the past two decades.

Multigene phylogeny reveals a cryptic diversity in the genus Dinobryon (Chrysophyceae) with integrative description of five new species.

The genus Dinobryon is one of the most recognizable chrysophyte genera, characterized by dendroid colonies with a biflagellate inside each cellulosic lorica. The representative forms of lorica are cylindrical, conical, vase, or funnel shaped, with undulation on the lorica wall. Traditionally, the morphological characteristics of the lorica and the colony organization have been used for the delimitation of Dinobryon species. To understand the taxonomy and phylogeny of colonial Dinobryon species, we performed molecular and morphological studies using 39 unialgal cultures and 46 single colony isolations from environmental specimens collected in Korea. We used a nuclear internal transcript spacer (ITS1-5.8S-ITS2) to find the genetic diversity of Dinobryon from environmental samples and a combined dataset from six gene sequences (nuclear SSU and LSU rRNA, plastid LSU rRNA, rbcL and psaA, and mitochondrial CO1 genes) for phylogenetic analysis. We found 15 different lineages based on the genetic diversity of the nuclear ITS sequences. The phylogenetic tree of the colonial species based on the combined multigene dataset were divided into 18 subclades, including five new species, each with unique molecular signatures for the E23-5 helix of the V4 region in the nuclear SSU rRNA and the E11-1 helix of D7b, and the E20-1 helix of D8 regions in the nuclear LSU rRNA. Morphological studies were focused on lorica dimension and shape, and stomatocyst morphology. The Dinobryon species showed similarities or differences in lorica morphologies between and within species, and also differences in lorica size between culture and environmental samples. Five Dinobryon species formed distinctive stomatocysts, their stomatocyst morphologies, including collar structure, surface ornamentation, and cyst shape, showed unique characteristics in each species and were useful for identification. Here, we propose five new species based on morphological and molecular evidences: D. cylindricollarium, D. exstoundulatum, D. inclinatum, D. similis, and D. spinum.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

First report of rice bacterial leaf blight and grain discolouration caused by Pantoea agglomerans in India

A disease survey was carried out in Tamil Nadu, India during 2021 to assess the prevalence of diseases in major rice-growing areas and characterise the pathogens associated with infected plants. During the survey, rice plants at the seedling to grain-filling stage showing water-soaked, elongated, light brown lesions in the leaf margins and tips (Figure 1) were collected along with grains in a polythene bag, labelled and sealed. Leaves showing yellowing and drying from tips and margins were cut into small pieces, surface-sterilised in 1% sodium hypochlorite for one minute, rinsed three times with sterile distilled water and then allowed to air dry. After drying, the tissues were soaked for 30 minutes at room temperature in 0.5 ml of sterile distilled water and then a loop full of suspension was streaked onto nutrient agar (NA) medium. For the isolation of pathogen from infected seed, discoloured grain samples were surface sterilised in 3% sodium hypochlorite for one minute, rinsed thrice with sterile distilled water and allowed to air dry. Surface-sterilised grains were placed in a Petri plate containing NA medium. These plates were incubated at 28°C for one to two days. Upon incubation, straw to yellow-coloured colonies emerged. The colonies were further purified through single colony isolation on NA. The purified colonies were yellow in colour, translucent and undulate. (Figure 2.) The colonies exhibited umbonate surfaces with slightly concave centres (Al-Kidsawey et al., 2020; Delétoile et al., 2009). The pathogenicity of the isolates was demonstrated by inoculation of isolates on rice seedlings (cv. Co 39). For this, bacterial isolates were incubated in nutrient broth for 48 hours at 27°C. The bacterial suspension was adjusted to a concentration of 109 cfu/ml by adding sterile water. The bacterium was inoculated by cutting the tip (about 3 to 4 cm) of the fully expanded uppermost leaf with scissors and dipping in the bacterial suspension. Controls used sterile distilled water instead of the bacterial suspension. The inoculated plants were covered with polythene sheet overnight and incubated under laboratory conditions. The inoculated leaves developed progressive necrosis with colour shifts from pale green to brown within 3 to 14 days post inoculation, whereas the control plants exhibited no symptoms. Koch's postulates were fulfilled by isolating the bacterium on NA which produced yellow, flat to convex translucent colonies after 48 hours incubation. The bacterium was further subjected to biochemical tests using the KB002 HiAssorted Biochemical Test Kit (Himedia, Germany). The bacterium showed a positive reaction for citrate utilisation, phenylalanine deamination, nitrate reduction, glucose and arabinose utilization. It showed a negative reaction to lysine utilisation, ornithine utilization, urease, H2S production, adonitol, lactose and sorbitol utilization. Based on the morphological characters and biochemical tests, the bacterium was identified as a Pantoea sp. To further characterise the pathogen, DNA extraction was done, and a PCR assay was performed using universal primers 27F and 1492R targeting the 16S rRNA gene. Sequencing of the amplicon revealed that the bacterial isolates had 93.7% identity with Pantoea agglomerans type strain SGAir0210 (GenBank Accession No. CP028033.1). The partial 16S rRNA gene sequences of two strains of P. agglomerans were deposited in GenBank (OP784578 and OP784743). Pantoea agglomerans causing bacterial leaf blight and glume discolouration has been reported from Brazil, South Korea, Turkey and Venezuela (Aksoy & Boluk, 2019; Carrer Filho et al., 2018; González et al., 2015; Lee et al., 2010). Pantoea agglomerans also causes infection in humans (Al-Kidsawey et al., 2020; Delétoile et al., 2009)). Bacterial leaf blight in rice caused by P. stewartii subsp. indologenes has been reported from Tamil Nadu, India (Vinodhini et al., 2017). However, to our knowledge, this is the first report of P. agglomerans causing rice leaf blight and grain discolouration in India. Further studies are needed to ascertain the prevalence of P. agglomerans in commercial rice cultivars in other parts of India. We gratefully acknowledge the facilities provided by DST-FIST Lab, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.

Genomic diversity of Helicobacter pylori populations from different regions of the human stomach

ABSTRACT Individuals infected with Helicobacter pylori harbor unique and diverse populations of quasispecies, but diversity between and within different regions of the human stomach and the process of bacterial adaptation to each location are not yet well understood. We applied whole-genome deep sequencing to characterize the within- and between-stomach region genetic diversity of H. pylori populations from paired antrum and corpus biopsies of 15 patients, along with single biopsies from one region of an additional 3 patients, by scanning allelic diversity. We combined population deep sequencing with more conventional sequencing of multiple H. pylori single colony isolates from individual biopsies to generate a unique dataset. Single colony isolates were used to validate the scanning allelic diversity pipelines. We detected extensive population allelic diversity within the different regions of each patient’s stomach. Diversity was most commonly found within non-coding, hypothetical, outer membrane, restriction modification system, virulence, lipopolysaccharide biosynthesis, efflux systems, and chemotaxis-associated genes. Antrum and corpus populations from the same patient grouped together phylogenetically, indicating that most patients were initially infected with a single strain, which then diversified. Single colonies from the antrum and corpus of the same patients grouped into distinct clades, suggesting mechanisms for within-location adaptation across multiple H. pylori isolates from different patients. The comparisons made available by combined sequencing and analysis of isolates and populations enabled comprehensive analysis of the genetic changes associated with H. pylori diversification and stomach region adaptation.

First Report of Achromobacter Xylosoxidans Causing Stem Soft Rot on Amorphophallus Konjac in China.

Amorphophallus konjac is one of the important commercial crops cultivated in south China and has long been used as a food source and a traditional medicine, because it is the only species with glucomannan and other trace elements (Ban et al. 2009; Melinda et al. 2010). In June of 2021, an outbreak of stem soft rot disease was observed on A. konjac plants in more than 2,000 square meters of agricultural planting fields in the Fuyuan country (25°34'50″N, 104°04'21″E), Qujing City, Yunnan Province, China. The disease incidence ranged from 30 to 35% in severely infested fields. The diseased plants displayed the first symptoms were damp brown spots. As the brown spots expanded, dark brown water stains appeared at the basal part of the stem and the bulbs were rotting with a foul smell, gradually extending to the underground parts. Progressively, the whole plants wilted and collapsed, and even the plants ultimately died. To identify the pathogen, symptomatic stems were cut into pieces, surface sterilized with 75% (v/v) ethanol, and placed on LB (tryptone/yeast extract/NaCl) medium for 24 to 48 hours at 28 ± 2°C. Six single-colony isolates were obtained from the diseased stems. The colonies on LB present a raised milky white opaque colonies moisture on the surface, round and convex in shape, with neat edges. Scanning electron microscopy showed that the cells were short rods (0.3∼0.5) × (1.9∼2.1)μm in size without any flagellum and were often arranged in pairs or clusters at certain angles. The 16S rDNA sequence of the randomly selected strain MY-G1 with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes nusA, eno, lepA and nuoL (Spilker et al. 2012) were amplified and sequenced. The 16S rDNA sequence of the 1326 bp product was deposited in GenBank (accession no. ON786717) and showed a 99.77% similarity to A. xylosoxidans strain E2 (accession no. MK849863.1). The nusA (OP680477), eno (OP680479), lepA (OP680481) and nuoL (OP680482) sequences showed 94.71%, 97.24%, 94.64% and 95.95% similarity to A. xylosoxidans strain DN002 (accession no. CP045222.1), respectively. The phylogenetic trees built based on 16S rRNA and the nusA-eno-lepA-nuoL multilocus analysis showed the isolate MY-G1 to cluster with A. xylosoxidans. Based on morphological and molecular analysis, the isolated MY-G1 was identified as A. xylosoxidans, which indicates that MY-G1 is a new strain of A. xylosoxidans. Pathogenicity tests were confirmed on the stem and petiole of one-year-old A. konjac. The wounds were made by puncturing with a MY-G1 bacteria suspension containing 108 CFU/ml (15ul/inoculation site). As a negative control, control seedlings were injected with the same amount of sterilized distilled water. Control and inoculated seedlings (each six) were kept in greenhouses and watered as needed in controlled conditions: 28°C, 75% relative humidity. Inoculated seedlings presented similar symptoms of stem soft rot, inner medulla disintegration, and wilt of leaves on developed within 3 to 9 days. The bacterial pathogen was re-isolated from inoculated seedlings and identified by morphological and molecular methods to fulfill Koch's postulates test. According to previous research, A. xylosoxidans can cross-kingdom infect animals and plants (Aisenberg et al.,2004; Ye et al.,2018). To the best of our knowledge, this is the first report of A. xylosoxidans causing stem soft rot of A. konjac in China, expanding the known pathogen for the soft rot of A. konjac, and also the host range of A. xylosoxidans.

Screening of antagonistic bacteria against the blue mold of citrus fruit from soil by a new parallel screening method without prior isolation of single strains

Antagonistic bacteria are effective in controlling fruit and vegetable diseases. However, screening for antagonistic bacteria is time-consuming. To simplify the procedure for screening antagonistic bacteria, we developed an ameliorated method for screening antagonistic bacteria against Penicillium italicum from the soil that requires no prior isolation of single colonies. The soil supernatant was directly spread on the P. italicum plate, then the antagonistic bacterial strains were isolated from the inhibition zones on the plate and further validated by dual culture assay. By using the novel method, three antagonistic bacterial strains were isolated from soil, which showed significant antagonistic effects on P. italicum. The results of 16S rDNA sequence analysis showed that the three antagonistic strains were classified as Paenibacillus sp., Bacillus sp., and Pseudomonas sp., respectively. Compared to the control, treatments with the three antagonistic trains decreased the disease incidence of blue mold by 32.5 %, 68.7 %, and 42.2 %, respectively. These results indicate that the ameliorated method is time-saving and effective for the screening of antagonistic bacteria.

Molecular Characterization and Phylogenetic Analysis of Pseudomonas aeruginosa Obtained from Wound Infection

Purpose: Wounds infection is frequent hospital acquired infections which is caused by a wide variety of microorganism. Inappropriate treatment could be more hazardous to suffering patients and could lead to death. This manuscript is aimed to identify the existence of Pseudomonas aeruginosa from the clinical samples and determine the molecular evolution and population structure of Pseudomonas species using bioinformatics tools Method: The specimens were swiped from the bandage of infected patients, who were admitted for the therapeutic intervention in the hospitals, localized in Delhi-NCR region. Primarly, specimens were cultured in cetrimide broth for specific growth of Pseudomonas sp. Single colony isolation on cetrimide agar plate and characterization of bacterial colony by producing fluorescence under UV light was done to confirm Pseudomonas aeruginosa (P. aeruginosa). Furthermore, phylogenetic analysis was performed on the basis of PCR and sequencing of 16s rRNA sequence of P. aeruginosa. Results: Assay based on bacterial culture, biochemical and 16S rRNA gene analysis methods confirm the presence of P. aeruginosa in collected wound specimen. Basic local alignment search tool analysis indicates that newly sequenced 16S rRNA gene sequence was shown 99% similarity to P. aeruginosa species, analyzed using NCBI-BLAST tool. The phylogenetic analysis and nucleotide base composition studies performed using 45 sequences of 16S rRNA gene from 18 different species of Pseudomonas, including P. aeruginosa. The phylogenetic analysis was performed using Maximum Likelihood method for evolutionary relationships. Conclusion: The biochemical and molecular characterization revealed that the clinical samples were infected with P. aeruginosa. Further, phylogenetic analysis of 16S RNA sequences revealed evolutionary conservation among different Pseudomonas species.

First Report of Postharvest Blue Mold decay caused by Penicillium expansum on Lemon (Citrus limon) fruit in China.

Lemon (Citrus limon) is one of the most important commercial (both dried and fresh) citrus fruits in China. In the spring of 2019, postharvest blue mold decay was observed at an incidence of 3-5% on lemon fruit at the local markets in Beijing, China. Fruit lesions were circular, brown, soft, and watery, and rapidly expanded at 25°C. To isolate the causal organism, small pieces (2 mm3) were cut from the lesions, surface-sterilized for 1 min in 1.5% NaOCl, rinsed three times with sterilized water, dried with sterile filter paper, placed onto potato dextrose agar (PDA) medium, and incubated at 25°C for 6 days. Eight morphologically similar single-colony fungal isolates were recovered from six lemon fruit. Colony surfaces were bluish-green on the upper surface and cream to yellow-brown one the reverse. Hyphae on colony margins were entirely subsurface and cream in color. Mycelium was highly branched, septate, and colorless, and conidiophores were 250 to 450 × 3.0 to 4.0 µm in size. Stipe of conidiophores were smooth-walled, bearing terminal penicilli, typically terverticillate or less commonly birverticillate, rami occurring singly, 16 to 23 × 3.0 to 4.0 µm, metulae in 3 to 6, measuring 12 to 15 × 3.0 to 4.0 µm. Phialides were ampulliform to almost cylindrical, in verticils of 5 to 8, measuring 8 to 11 × 2.5 to 3.2 µm with collula. Conidia were smooth-walled, ellipsoidal, measuring 3.0 to 3.5 × 2.5 to 3.0 µm. According to morphological characteristics, the fungus was identified as Penicillium expansum (Visagie et al. 2014). For molecular identification, genomic DNA of eight fungal isolates was extracted, regions of the beta-tubulin (TUB), and calmodulin (CAL) genes and ITS region, were amplified using Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 primers respectively. Obtained sequences of all isolates were identical to sequences of the representative isolate YC-IK12, which was submitted in the GenBank. BLAST results of YC-IK12 sequences (ITS; MT856700: TUB; MT856958: CAL; MT856959) showed 98 to 100% similarity with P. expansum accessions (NR-077154, LN896428, JX141581). For pathogenicity tests, 10 μl of conidial suspension (10 × 105 conidia/ml) from seven-day-old YC-IK12 culture was inoculated using a sterilized needle into the surface of each five asymptomatic disinfected lemons. As a control, three lemons were inoculated using sterile distilled water. All inoculated lemons were placed in plastic containers and incubated at 25°C for 7 days. Decay lesions, identical to the original observations, developed on all inoculated lemons, while control lemons remained asymptomatic. Fungus re-isolated from the inoculated lemon was identified as P. expansum on the basis morphology and Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 sequences. Previously, Penicillium spp. including P. expansum have been reported as post-harvest pathogens on various Citrus spp. (Louw & Korsten 2015). However, P. digitatum has been reported on lemons and P. expansum has been reported on stored Kiwifruit (Actinidia arguta), Malus, and Pyrus species in China (Tai, 1979; Wang et al. 2015). To our knowledge, this is the first report of blue mold caused by P. expansum on lemons in China. References Louw, J. P., Korsten, L. 2015. Plant Dis. 99:21-30. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pages. 8097 Visagie, C.M. et al. 2014. Studies. Mycol.78: 343. Wang, C. W. et al. 2015. Plant Dis. 99:1037.

Single-colony sequencing reveals microbe-by-microbiome phylosymbiosis between the cyanobacterium Microcystis and its associated bacteria

BackgroundCyanobacteria from the genus Microcystis can form large mucilaginous colonies with attached heterotrophic bacteria—their microbiome. However, the nature of the relationship between Microcystis and its microbiome remains unclear. Is it a long-term, evolutionarily stable association? Which partners benefit? Here we report the genomic diversity of 109 individual Microcystis colonies—including cyanobacteria and associated bacterial genomes—isolated in situ and without culture from Lake Champlain, Canada and Pampulha Reservoir, Brazil.ResultsWe identified 14 distinct Microcystis genotypes from Canada, of which only two have been previously reported, and four genotypes specific to Brazil. Microcystis genetic diversity was much greater between than within colonies, consistent with colony growth by clonal expansion rather than aggregation of Microcystis cells. We also identified 72 bacterial species in the microbiome. Each Microcystis genotype had a distinct microbiome composition, and more closely related genotypes had more similar microbiomes. This pattern of phylosymbiosis could be explained by co-phylogeny in only two out of the nine most prevalent associated bacterial genera, Roseomonas and Rhodobacter. These phylogenetically associated genera could enrich the metabolic repertoire of Microcystis, for example by encoding the biosynthesis of complementary carotenoid molecules. In contrast, other colony-associated bacteria showed weaker signals of co-phylogeny, but stronger evidence of horizontal gene transfer with Microcystis. These observations suggest that acquired genes are more likely to be retained in both partners (Microcystis and members of its microbiome) when they are loosely associated, whereas one gene copy is sufficient when the association is physically tight and evolutionarily long-lasting.ConclusionsWe have introduced a method for culture-free isolation of single colonies from nature followed by metagenomic sequencing, which could be applied to other types of microbes. Together, our results expand the known genetic diversity of both Microcystis and its microbiome in natural settings, and support their long-term, specific, and potentially beneficial associations.A9SU3YME_Rf3MWzGs5jm1TVideo

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction.

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.

Species delimitation within the colonial flagellates Uroglena, Uroglenopsis and Urostipulosphaera (Chrysophyceae)

ABSTRACT Until recently, there was no agreement on species delimitation within the morphologically similar chrysophycean genera Uroglena, Uroglenopsis and Urostipulosphaera. In this study, we aimed at a modern taxonomic revision based on the combination of morphological characters (ultrastructure of cysts, cell and colony features) and a multigene phylogeny (SSU, ITS rDNA and rbcL sequences), with ecology taken into account. Of more than 650 explored localities, only approximately one in 10 hosted a viable and detectable population of these colonial chrysophytes at the time of sampling. We established and examined 189 short-term cultures along with single colony isolates, derived mostly from blooming or encysting populations. We obtained the cyst morphology for four species and two lineages of Uroglena, two species of Uroglenopsis, and four species of Urostipulosphaera. A total of 12 resolved lineages could be attributed to previously described species or new species (Uroglena imitata sp. nov., Urostipulosphaera granulata sp. nov.). Based on our molecular analyses and morphological observations, we assign all the previously described Uroglena-like taxa to newly recognized genera and propose a key to identification. Consequently, Uroglena now includes 16 species and two varieties, Uroglenopsis contains four species and Urostipulosphaera encompasses nine species. Within Uroglena and Urostipulosphaera, species are defined by the ultrastructure of their cysts. On the contrary, as Uroglenopsis has simple cysts, species are defined by cell and colony characteristics.

Virulence structure of Blumeria graminis f. sp. tritici, the causal agent of wheat powdery mildew, in Ontario, Canada, in 2018 and 2019

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a major disease of wheat (Triticum aestivum) in Ontario, which can cause 20% yield loss. The development of resistant commercial wheat cultivars is the most economical means of controlling this disease, but only if the resistance genes used are incompatible with the virulence phenotypes present in the pathogen population. The virulence structure of Bgt in Ontario was examined in 2018 and 2019. Of the 42 single colony isolates collected in Ontario greenhouses and commercial fields, 40 virulence phenotypes, assigned VP1 to VP40, were identified on a set of 24 single-gene differential genotypes. Of the 24 resistance genes possessed by the differential genotypes, eight genes including Pm1a, Pm1b, Pm1c, Pm12, Pm16, Pm21, Pm37, and MlAG12 were effective against all of the Bgt isolates. Four genes including Pm3d, Pm29, Pm34, and NCAG13 were mostly effective with resistance reactions to more than 80% of the isolates. There were no significant differences in numbers of virulence genes per isolate between the two years, or between the greenhouse and field origins. The virulence frequencies of Bgt isolates for these effective and mostly effective genes were also not significantly affected by the year of collection and their origins, suggesting that the Bgt population is relatively stable. The effective genes identified in this study may be deployed singly or used for gene pyramiding in wheat breeding programs for developing powdery mildew-resistant cultivars in Ontario.

Where to Biopsy to Detect Helicobacter pylori and How Many Biopsies Are Needed to Detect Antibiotic Resistance in a Human Stomach.

This study aims to determine the gastric distribution, density, and diversity of Helicobacter pylori infection. Subtotal resection of the stomachs of three H. pylori-infected and asymptomatic obese patients were collected after a sleeve gastrectomy. Distribution and density of H. pylori were determined using culture and RT-PCR on multiple gastric sites (88, 176, and 101 biopsies per patient). Diversity of H. pylori strains was studied using antibiotic susceptibility testing, random amplified polymorphism DNA (RAPD) typing and cagA gene detection on single-colony isolates (44, 96, and 49 isolates per patient). H. pylori was detected in nearly all analyzed sites (354/365 biopsies, 97%). Antral density was higher in one patient only. The three stomachs were almost exclusively infected by an antibiotic-susceptible strain. One clarithromycin-resistant isolate in one biopsy was detected in two stomachs (1/44 and 1/49 isolates), while in the third one, eight (8/96 isolates) metronidazole-resistant isolates were detected. DNA typing showed infection with cagA-negative strains for one patient, cagA-positive strains for a second patient and the third patient was infected with two different strains of distinct cagA genotypes. Infection with H. pylori is shown to spread to the whole surface of the stomach, but a possibility of minor sub-population of antibiotic-resistant clones, undetectable in routine practice.