gyrB Gene Sequences Research Articles

Pathology and drug susceptibility study of an outbreak of bacterial pathogen infecting the Chinese soft‐shelled turtle ( Pelodiscus sinensis )

The Chinese soft-shelled turtle (Pelodiscus sinensis) is a freshwater softshell turtle native to East Asia. It is a choice of food and tonic in China, Taiwan and other Asian countries and is commonly cultured in ponds, lakes and canals. This study aimed to study the aetiology of a deadly disease outbreak among these turtles in Jiangxi Province of China in 2020 and 2021. By performing an aseptic dissection of symptomatic and moribund P. sinensis, a dominant strain (No.: ZF2) was isolated from livers, and studies were carried out to ascertain the biological status, pathogenicity and pathological characteristics of the isolate. For isolate identification, a morphological observation, physiological and biochemical analysis, 16S rRNA and gyrB gene sequencing, and phylogenetic analysis were performed. Other experiments were also carried out simultaneously, such as infection experiments, histopathological observation, virulence gene detection and drug sensitivity. Through traditional and molecular biological methods study with the pathogenic strain, ZF2 was identified as Aeromonas veronii. The results of the virulence gene test showed that ZF2 carried three virulence genes, aer, ast and act. Histopathological observation revealed that various internal organs were severely damaged after infection and manifested as liver ischemia and whitening, intestinal chorion shedding, glomerular congestion and splenic lymphocyte proliferation. After the drug sensitivity test results, the ZF2 strain was identified to be sensitive to nine drugs, including cefradine, neomycin, minocycline and enrofloxacin. It was intermediate or resistant to other drugs.

Isolation, identification, and characterization of pathogenic Aeromonas hydrophila from critically endangered Acipenser baerii

The Siberian sturgeon (Acipenser baerii) is a long-lived and late-maturing fish; its natural populations are considered endangered and listed in the first class of protected animals in Kazakhstan. To date, attempts have been made to increase the number of sturgeons through artificial reproduction in aquaculture and a release of the resultant juveniles into the Ural River, which flows into the Caspian Sea. This species faces an increased risk of Aeromonas infections. Aeromonas hydrophila is the most important sturgeon pathogen in Kazakhstan, but studies on A. hydrophila infection in Kazakhstan are still inconclusive. In the present work, our purpose was to isolate and characterize dominant bacteria in diseased A. baerii. This isolate, tentatively named AB005, was identified as A. hydrophila in an analysis of its morphological, physiological, and biochemical features and 16S ribosomal-RNA and gyrB gene sequences. A pathogenicity test was carried out for the isolate on healthy Oreochromis niloticus and Acipenser ruthenus via intraperitoneal injection along the caudal peduncle of the fish. Half-lethal doses (LD50) of isolate AB005 for O. niloticus and A. ruthenus were determined: 8.37 × 105 and 2.89 × 106 colony-forming units per milliliter, respectively. Virulence gene profiling revealed the presence of seven virulence genes related to pathogenicity (acyltransferase, phospholipase A, serine protease, heat-stable cytotonic enterotoxin, nuclease, and aerolysins A and B) in this A. hydrophila isolate. Drug sensitivity testing showed that the isolate is sensitive to quinolones, aminoglycosides, nitrofurans, amphenicols, and tetracyclines. The present findings will lay the foundation for future research on this pathogen in Siberian-sturgeon aquaculture.

Isolation and identification of an endophytic bacteria Bacillus sp. K-9 exhibiting biocontrol activity against potato common scab.

Potato scab is an important soil-borne disease that can significantly reduce the quality and economic value of potatoes. The purpose of this study was to isolate, screen and identify endophytic bacteria that have antagonistic and control effects on potato scab disease, and to determine the control effect and yield traits of the selected strains on potato scab disease in field conditions. A bacterial strain K-9 was isolated from the junction between scab spot and healthy epidermis of potato tuber. The K-9 strain was identified as Bacillus sp. through morphological, physiological and biochemical characterization, and 16S rDNA and gyrB gene sequence analysis. The diameter of the inhibition zone of strain K-9 against Streptomyces scabies on the YME plate was 3.82cm. The K-9 strain could inhibit eight types of crop pathogens, with the highest inhibition rate (70.39%) against another soil-borne potato disease (potato black scurf). In the field test, the control effect of K-9 strain against potato scab was not significantly different from that of mixed bacteria or chemical agents, but the disease index and the scab index in the K-9 treatment were significantly lower than in the control. The potato yield in the K-9 treatment was 12.44% higher than the control. In summary, the K-9 strain can prevent not only potato scab, but also increase potato yield. Therefore, the endophytic bacterial K-9 strain may be a potential biological control agent.

Pathogenesis of Aeromonas caviae in Clariasmagur

Aeromonas spp. is a pathogenic bacteria that potentially cause infection in farmed fish, including Catfishes. In the present study, dominant bacteria were isolated from diseased Clarias magur and tentatively named BLBM-05. Based on morphological, physiological, and biochemical features as well as 16S rRNA gene sequence and gyrB gene sequences (Gen Bank accession number: MT973994.1 and MZ398017.1), the bacteria in the isolate was found to be Aeromonas caviae. Further, the isolate was screened for five known virulence genes, namely β-hemolysin, lafA, exu, ompA1 and ascV. Among them, three virulence genes related to pathogenicity, including aerolysin (aer), outer membrane protein (ompA1), lateral flagella (lafA), were identified in the A. caviae isolate. The median lethal dosage (LD50) of the BLBM-05 isolate for magur was determined as 1.53x106 CFU/mL. The histopathological analysis showed that the BLBM-05 isolate induced considerable histological lesions in the magur fish, including necrosis, hemolysis of erythrocytes, myolysis, hemorrhage, and desquamation in the intestinal tissue, tissue loosening, and infiltration of inflammatory cells. Drug sensitivity test showed that the isolate was susceptible to Gentamicin, Ceftazidine, Ceftrioxone, Amikacin, Tetracycline, Meropener and Oxytetracycline. The present results provide a scientific basis to identify A. caviae further, a line of treatment for magur infected by this pathogen.

Diversity and antimicrobial susceptibility profiles of Aeromonas spp. isolated from diseased freshwater fishes in Thailand.

Motile Aeromonas septicemia (MAS), a disease caused by Aeromonas spp., is recognized as a major disease in freshwater aquaculture. This study aimed to investigate the distribution and diversity of Aeromonas spp. and their antimicrobial susceptibility patterns. A total of 86 isolates of Aeromonas spp. were recovered from diseased freshwater fishes from 13 farms in Thailand. All isolates were identified using biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), polymerase chain reaction assays, and the gyrB gene sequence analysis. The result of MALDI-TOF MS showed 100% (86 isolates) accuracy at genus-level identification, and 88.4% (76 isolates) accuracy at species-level identification. Six species of Aeromonas were confirmed through nucleotide sequencing and phylogenetic analysis of the gyrB gene Aeromonas veronii (72.1%), Aeromonas jandaei (11.6%), Aeromonas schubertii (9.3%), Aeromonas diversa (3.5%), Aeromonas hydrophila (2.3%), and Aeromonas punctata (1.2%). Antimicrobial susceptibility tests for all isolatesrevealed resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). The multiple antibiotic resistance (MAR) index varied between 0.14 and 0.86, with MAR values more than 0.2 in 99% of isolates. Furthermore, four diverse multidrug-resistant (MDR) patterns were found among Aeromonas isolates. Our finding show that A.veronii is the most abundant species in Thai cultured freshwater fish with the highest MDR patterns.

Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8.

Fusarium pseudograminearum causes crown rot in wheat. This study aimed to assess the effects of the bacterial strain QTH8 isolated from Cotinus coggygria rhizosphere soil against F. pseudograminearum. Bacterial strain QTH8 was identified as Bacillus halotolerans in accordance with the phenotypic traits and the phylogenetic analysis of 16S rDNA and gyrB gene sequence. Culture filtrates of bacterial strain QTH8 inhibited the mycelial growth of F. pseudograminearum and resulted in mycelial malformation such as tumor formation, protoplast condensation, and mycelial fracture. In addition, bacterial strain QTH8 also inhibited the mycelial growth of Hainesia lythri, Pestalotiopsis sp., Botrytis cinerea, Curvularia lunata, Phyllosticta theaefolia, Fusarium graminearum, Phytophthora nicotianae, and Sclerotinia sclerotiorum. The active compounds produced by bacterial strain QTH8 were resistant to pH, ultraviolet irradiation, and low temperature, and were relatively sensitive to high temperature. After 4 h exposure, culture filtrates of bacterial strain QTH8—when applied at 5%, 10%, 15%, 20%, 25%, and 30%—significantly reduced conidial germination of F. pseudograminearum. The coleoptile infection assay proved that bacterial strain QTH8 reduced the disease index of wheat crown rot. In vivo application of QTH8 to wheat seedlings decreased the disease index of wheat crown rot and increased root length, plant height, and fresh weight. Iturin, surfactin, and fengycin were detected in the culture extract of bacterial strain QTH8 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Bacterial strain QTH8 was identified for the presence of the ituC, bacA, bmyB, spaS, srfAB, fend, and srfAA genes using the specific polymerase chain reaction primers. B. halotolerans QTH8 has a vital potential for the sustainable biocontrol of wheat crown rot.

Pathogenicity of Aeromonas veronii causing mass mortalities of Odontobutis potamophila and its induced host immune response

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. However, few cases of A. veronii infection were reported in Odontobutis potamophila, which has been becoming a promising fish species in China in recent years. In this study, the dominant bacteria were isolated from diseased O. potamophila showing signs of hemorrhage on fins, ulceration on the dorsal and abdomen. The representative isolate Stl3-1was identified as A. veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. The median lethal dosage (LD50) of the isolate Stl3-1 for O. potamophila was determined as 4.5 × 105 CFU/mL. Histopathological analysis revealed that the isolate Stl3-1caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Detection of virulence-related genes showed that A. veronii Stl3-1 was positive for exu, ompA, lip, flaH, hlyA, aer, flgM, tapA, act, flgA, gcaT and flgN. Additionally, quantitive real-time PCR (qRT-PCR) was also undertaken to analyses the host defensive response in O. potamophila infected by A. veronii. The immune-related gene expressions in O. potamophila during experimental infection were monitored at different point of time, and the results showed that the expression levels of MHC II, Myd88, TLR, and SOD were significantly up-regulated in liver, gill, spleen, and head kidney. The results revealed that A. veronii was a pathogen causing mass mortalities of O. potamophila and will contribute to better understanding the host defensive response against A. veronii infection.

Reclassification of Enterobacter sp. FY-07 as Kosakonia oryzendophytica FY-07 and Its Potential to Promote Plant Growth

Precise classification of bacteria facilitates prediction of their ecological niche. The genus Enterobacter includes pathogens of plants and animals but also beneficial bacteria that may require reclassification. Here, we propose reclassification of Enterobacter FY-07 (FY-07), a strain that has many plant-growth-promoting traits and produces bacterial cellulose (BC), to the Kosakonia genera. To re-examine the taxonomic position of FY-07, a polyphasic approach including 16S rRNA gene sequence analysis, ATP synthase β subunit (atpD) gene sequence analysis, DNA gyrase (gyrB) gene sequence analysis, initiation translation factor 2 (infB) gene sequence analysis, RNA polymerase β subunit (rpoB) gene sequence analysis, determination of DNA G + C content, average nucleotide identity based on BLAST, in silico DNA–DNA hybridization and analysis of phenotypic features was applied. This polyphasic analysis suggested that Enterobacter sp. FY-07 should be reclassified as Kosakonia oryzendophytica FY-07. In addition, the potential of FY-07 to promote plant growth was also investigated by detecting related traits and the colonization of FY-07 in rice roots.

Colour of heterorhabditis zealandica-infected-Galleria mellonella dependent on the Photorhabdus symbiont, with two new nematode-symbiotic associations reported

Bacterial symbionts associated with entomopathogenic nematodes (EPNs) play an important role in terms of the insecticidal properties of nematodes in pest control. Galleria mellonella larvae, shortly after being infected with three different strains of Heterorhabditis zealandica, which were isolated from South African soil, changed from pale white to steel grey-blue (blue), bright red, and yellow with a green tint (green), respectively. The genetic relatedness of the bacterial symbionts that were isolated from the three strains of H. zealandica was determined by means of comparing the 16S rRNA, recA, gyrB, dnaN, gltX and infB gene sequences. Subsequently, comparing the concatenated sequences revealed the presence of three distinct Photorhabdus species. The H. zealandica strain SF41, associated with Photorhabdus heterorhabditis, produced ‘blue’ G. mellonella larvae. The H. zealandica strain MJ2C, associated with Photorhabdus thracensis, yielded ‘green’ G. mellonella larvae, while the H. zealandica strain LLM associated with Photorhabdus laumondii subsp. laumondii yielded red larvae. The colour changes in G. mellonella larvae were found to have been instigated by a particular Photorhabdus species associated with H. zealandica. The red and ‘green’ phenotypes of G. mellonella larvae were found to represent new combinations of Heterorhabditis and Photorhabdus. In future studies, the colour of infected G. mellonella larvae needs to be reported as a phenotypic character, as it indicates the different bacterial species associated with the same nematode host, as shown in the case of H. zealandica.

First Report of Bacterial Canker on Blueberry (Vaccinium corymbosum) Caused by Pseudomonas syringae pv. syringae in Serbia.

During May 2021, necrosis of young twigs and flower buds were observed on two-year-old highbush blueberry plants (Vaccinium corymbosum) cv. Draper, in a 1 hectare orchard in the municipality of Šabac, Serbia. Disease symptoms included reddish-brown to black irregularly shaped cankers developing on the shoot tips that extended downwards along the branches. In some plants, cankers surrounded the stem, causing shoot-tip dieback and necrosis of the buds. Beneath the bark, a distinct margin between diseased and healthy tissue was visible. A few weeks before symptoms development, seven freezing events with temperature from -3°C to -1°C, and five near-freezing temperatures were recorded in this area, leading to the hypothesis that symptoms were associated to the presence of ice nucleating bacteria belonging to Pseudomonas syringae. The observed disease incidence was 80%, while 10% of the plants died. Bacteria were isolated from symptomatic tissue on King's medium B (KB). After 2 to 3 days of incubation at 27°C, predominantly grey-whitish, shiny, round, convex bacterial colonies were observed on agar plates. Ten isolates producing a fluorescent pigment on KB were selected for further characterization by biochemical and molecular tests. The isolates were Gram, oxidase and arginine-dihydrolase negative, levan positive, induced hypersensitive response on tobacco leaves and showed no pectinolytic activity on potato slices. Based on the results of API 20E and API 20NE tests (BioMerieux, France), and the fact that isolates did not utilize tartrate nor had tyrosinase activity, they were preliminarly identified as Pseudomonas syrinage pv. syringae (Braun-Kiewnick and Sands 2001). Additionally, all tested isolates had ice-nucleation activity at -5°C. The syrB gene responsible for syringomycin synthesis, was amplified in all isoaltes with the specific primer pair B1/B2 (Sorensen et al. 1998). The 16S rRNA gene sequences of five selected isolates (GenBank MZ410287 to 91) showed 100% identity to P. s. pv. syringae isolated from Prunus avium in United Kingdom (GenBank CP026568) and France (GenBank LT962480). Sequences of gyrB gene (Sarkar and Guttman 2004) of two selected isolates (GenBank MZ420633 and MZ420634) showed 98,44% identity to the P. s. pv. syrinage strain isolated in France (GenBank LT962480). Pathogenicity of the isolates was confirmed on 2-year-old blueberry plants cv. Draper, by inoculating two plants per isolate. One-cm long wounds were made on branches using a scalpel and 20 µl of bacterial suspension (106 CFU/ml) was infiltrated into the tissue. The cuts were then covered with moist sterile cotton pads and wraped in parafilm for 3 days. Inoculation was also performed on two leaves per plant by needleless syringe infiltration (106 CFU/ml). Sterile distilled water was used as a negative control. Plants were maintained in greenhouse at 27°C day and 15°C night temperature. Three weeks after inoculation, the inoculated branches and leaves developed necrosis, leaves spots and cankers respectively, resembling the natural infection. Symptoms were not observed on the control plants. Bacteria were reisolated from symptomatic tissue and their identity was confirmed by amplifying the syrB gene sequence and additional biochemical tests. This is the first report of bacterial canker of highbush blueberry caused by P. s. pv. syringae in Serbia. In Europe, there was only one report on Pseudomonas spp. causing disease on blueberry leaves in Poland (Kaluzna et al. 2013). Due to market demands and export potential, blueberry production in Serbia has been rapidly increasing. In 2015, highbush blueberry was cultivated on 220 ha, while in 2020 the area increased to 1899 ha. However, under favourable environmental conditions, blueberry production might be severely affected by bacterial canker.

Molecular, Phenotypical, and Host-Range Characterization of Robbsia andropogonis Strains Isolated from Bougainvillea spp. in Mexico.

Characteristic leaf spot and blight symptoms caused by Robbsia andropogonis on bougainvillea plants were found in three locations in different provinces of Mexico from 2019 to 2020. Eleven bacterial isolates with morphology similar to R. andropogonis were obtained from the diseased bougainvillea leaves. The isolates were confirmed as R. andropogonis by phenotypic tests and 16S rRNA, rpoD, and gyrB gene sequencing. In addition to bougainvillea, the strains were pathogenic to 10 agriculturally significant crops, including maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), coffee (Coffea arabiga), carnation (Dianthus caryophilus), Mexican lime (Citrus × aurantifolia), common bean (Phaseolus vulgaris), broadbeans (Vicia faba), and pea (Pisum sativum), but not runner bean (Phaseolus coccineus). The haplotypes network reveals the genetic variability among Mexican strains and its phylogeographic relationship with Japan, the U.S.A., and China. The presence of this pathogen represents a challenge for plant protection strategies in Mexico.

Potential of an endophytic bacteria Bacillus amyloliquefaciens 3–5 as biocontrol agent against potato scab

To obtain a potential biocontrol agent for potato scab, 75 endophytic bacteria were isolated from the healthy potato tubers and strain 3-5 was selected as an optimal antagonistic bacterium against Streptomyces griseoplanus (Streptacidiphilus griseoplanus) causing potato scab. Strain 3-5 was identified as Bacillus amyloliquefaciens based on its morphological characteristics, 16S rDNA and gyrB gene sequence analysis. B. amyloliquefaciens 3–5 has biological functions of indole-3-acetic acid (IAA) production and nitrogen fixation. Polymerase chain reaction (PCR) detection revealed that B. amyloliquefaciens 3–5 had 6 diverse antibacterial substance synthesis genes, named bacD, bacAB, ituD, ituC, sfP and albF, which resulted in the production of bacilysin, iturin, surfactin and subtilosin. Field efficacy evaluation revealed that B. amyloliquefaciens 3–5 (solid fermentation) was successful in controlling potato scab with a 38.90 ± 3.2140% efficiency which is higher than other chemical bactericides except zhongshengmycin·oligosaccharins and kasugamycin·zhongshengmycin. The endophytic bacterium B. amyloliquefaciens 3–5 could be used as a biocontrol agent against potato scab due its control efficacy and environmental safety.

Ecological adaptation and phylogenetic analysis of microsymbionts nodulating Polhillia, Wiborgia and Wiborgiella species in the Cape fynbos, South Africa

Polhillia, Wiborgia and Wiborgiella species are shrub legumes endemic to the Cape fynbos of South Africa. They have the ability to fix atmospheric N2 when in symbiosis with soil bacteria called ‘rhizobia’. The aim of this study was to assess the morpho-physiological and phylogenetic characteristics of rhizobia associated with the nodulation of Polhillia, Wiborgia and Wiborgiella species growing in the Cape fynbos. The bacterial isolates from root nodules consisted of a mixture of fast and intermediate growers that differed in colony shape and size. The isolates exhibited tolerance to salinity (0.5–3% NaCl) and pH (pH 5–10) and different antibiotic concentrations, and could produce 0.51 to 51.23 µg mL−1 of indole-3-acetic acid (IAA), as well as solubilize tri-calcium phosphate. The ERIC-PCR results showed high genomic diversity in the rhizobial population and grouped them into two major clusters. Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, nifH and nodC gene sequences revealed distinct and novel evolutionary lineages related to the genus Rhizobium and Mesorhizobium, with some of them being very close to Mesorhizobium australicum. However, the phylogenetic analysis of glnII and nifH genes of some isolates showed incongruency.

Pseudomonas tohonis sp. nov., isolated from the skin of a patient with burn wounds in Japan.

Strain TUM18999T was isolated from the skin of a patient with burn wounds in Japan. The strain was successfully cultured at 20-42 °C (optimum, 30-35 °C) in 1.0-4.0% NaCl (w/v) and at pH 5.5-9.5, optimum pH 5.5-8.5. The phylogenetic tree reconstructed using 16S rRNA, gyrB, rpoB and rpoD gene sequences indicated that strain TUM18999T is closely related to Pseudomonas otitidis MCC10330T. Although the partial 16S rRNA gene sequence (1412 bp) of TUM18999T exhibits high similarity to those of Pseudomonas alcaligenes NBRC 14159T (99.08 %) and Pseudomonas otitidis MCC10330T (98.51 %), multi-locus sequence analysis using 16S rRNA, gyrB, rpoB and rpoD genes reveals a clear distinction between TUM18999T and other Pseudomonas species. In addition, an average nucleotide identity >90 % was not observed in the P. aeruginosa group. Moreover, TUM18999T and P. otitidis can be distinguished based on the minimum inhibitory concentration for carbapenem. Meanwhile, the cellular fatty acids are enriched with C18 : 1 ω7c/C18 : 1 ω6c (34.35 %), C16 : 1 ω7c/C16 : 1 ω6c (24.22 %), C16 : 0 (19.79 %) and C12 : 0 (8.25 %). Based on this evidence, strain TUM18999T can be defined as representing a novel Pseudomonas species, with the proposed name Pseudomonas tohonis sp. nov. The type strain is TUM18999T (GTC 22698T=NCTC 14580T).

Phylogenomic Analysis Substantiates the gyrB Gene as a Powerful Molecular Marker to Efficiently Differentiate the Most Closely Related Genera Myxococcus, Corallococcus, and Pyxidicoccus.

Rapid and accurate strain identification of the most closely related genera Myxococcus, Corallococcus, and Pyxidicoccus can enhance the efficiency of the mining of novel secondary metabolites through dereplication. However, the commonly used 16S rRNA gene sequencing cannot accurately differentiate members of the three genera above, and the whole-genome sequencing is unable to rapidly and inexpensively provide species assignation toward a large number of isolates. To overcome the limitations, the gyrB gene was investigated as a candidate genetic marker for exploring the phylogenetic relationships of bacteria within the three genera and for developing the gyrB-based typing method. Here, the bacterial phylogeny and species affiliations of the three genera were determined based on the phylogenomic reconstruction and the analysis of digital DNA–DNA hybridization values among 90 genomes, further confirming nine novel taxa and assigning over one-third of genomes to defined species. The phylogenetic relationships of these strains based on the gyrB gene sequences were congruent with those based on their genome sequences, allowing the use of the gyrB gene as a molecular marker. The gyrB gene-specific primers for the PCR-amplification and sequencing of bacteria within the three genera were designed and validated for 31 isolates from our group collection. The gyrB-based taxonomic tool proved to be able to differentiate closely related isolates at the species level. Based on the newly proposed 98.6% identity threshold for the 966-bp gyrB gene and the phylogenetic inference, these isolates were assigned into two known species and eight additional putative new species. In summary, this report demonstrated that the gyrB gene is a powerful phylogenetic marker for taxonomy and phylogeny of bacteria within the closely related genera Myxococcus, Corallococcus, and Pyxidicoccus, particularly in the case of hundreds or thousands of isolates in environmental studies.

Characterization and Antimicrobial Resistance of Environmental and Clinical Aeromonas Species Isolated from Fresh Water Ornamental Fish and Associated Farming Environment in Sri Lanka.

The aims of this study were to characterize and investigate antimicrobial susceptibility and presence of integrons in 161 Aeromonas spp. isolated from ornamental freshwater fish farming environment, apparently healthy and diseased fish. Phylogenetic analyses of the gyrB gene sequences identified Aeromonas veronii as the most abundant species (75.8%) followed by Aeromonas hydrophila (9.3%), Aeromonas caviae (5%), Aeromonas jandaei (4.3%), Aeromonas dhakensis (3.7%), Aeromonas sobria (0.6%), Aeromonas media (0.6%), and Aeromonas popoffii (0.6%). Susceptibility to thirteen antimicrobials was determined and antimicrobial resistance frequencies were: amoxicillin (92.5%), enrofloxacin (67.1%), nalidixic acid (63.4%), erythromycin (26.1%), tetracycline (23.6%), imipenem (18%), trimethoprim-sulfamethoxazole (16.8%), and gentamicin (16.8%). Multi-drug resistance (MDR) was widespread among the isolates (51.6%, 83/161) with 51.6% (63/122) A. veronii isolates being MDR. In addition, 68.3% of isolates had multiple antibiotic resistance (MAR) indexes higher than 0.2, suggesting that they originated from a high-risk source of contamination where antimicrobials are often used. In all, 21.7% isolates carried class 1 integrons, with 97.1% having gene cassettes, while there were 12 isolates carrying class 2 integron gene cassettes. Our findings highlight that the aquatic environment and ornamental fish act as reservoirs of multidrug resistant Aeromonas spp. and underline the need for a judicious use of antimicrobials and timely surveillance of antimicrobial resistance (AMR) in aquaculture.

Genetic and physiological diversity of white Spanish broom (Chamaecytisus albus) endophytes.

Chamaecytisus albus (Spanish broom) is a legume shrub that can be found in only one natural locality in Poland. This specimen is critically endangered; therefore, different actions focusing on protection of this plant in the natural habitat are undertaken, and one of them involves studies of the population of Chamaecytisus albus bacterial endophytes, which in the future could be used as bioprotectants and/or biofertilizers. A collection of 94 isolates was obtained from Spanish broom nodules, and the physiological and genetic diversity of these strains was studied. A few potentially beneficial traits were detected, i.e. secretion of cellulases (66 isolates), production of siderophores (60 isolates), phosphate solubilization (25 isolates), and production of IAA (58 isolates), indole (16 isolates), or HCN (3 isolates). Twenty-nine of the 94 tested isolates were able to induce the development of root nodules in plants grown in vitro and can therefore be assumed as Chamaecytisus albus symbionts. Genome fingerprinting by BOX-PCR, as well as gyrB and nodZ gene sequencing revealed a great genetic diversity of specimens in the collection. The symbiotic isolates were classified in different clades, suggesting they could belong to different species, however, most of them revealed sequence similarity to Bradyrhizobium genus.

Enhanced Biocontrol of Cucumber Fusarium Wilt by Combined Application of New Antagonistic Bacteria Bacillus amyloliquefaciens B2 and Phenolic Acid-Degrading Fungus Pleurotus ostreatus P5.

Continuous monoculture of cucumber (Cucumis sativus L.) typically leads to the frequent incidence of Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC). As potent allelochemicals, phenolic acids are believed to be associated with soilborne diseases. This study aimed to investigate the effect of single or co-inoculation of antagonistic bacteria Bacillus amyloliquefaciens B2 and phenolic acid-degrading fungus Pleurotus ostreatus P5 on the suppression of cucumber Fusarium wilt. The strain B2 was identified as B. amyloliquefaciens based on biochemical, physiological, and 16S rDNA and gyrB gene sequence analyses. Strain B2 showed indole-3-acetic acid (IAA) and siderophore production and phosphate solubilization in in vitro assays. Scanning electron microscope (SEM) imaging showed the ability of strain B2 to adhere to the root surface of cucumber. P. ostreatus P5 could effectively degrade mixed phenolic acids as its sole source of carbon and energy for growth in liquid medium. In a pot experiment, four treatments were established as follows: (1) CK, uninoculated control; (2) B2, inoculation of strain B2; (3) P5, inoculation of strain P5; and (4) B2 + P5, co-inoculation of strain B2 and strain P5. At the end of the 60-day pot experiment, the B2, P5, and B2 + P5 treatments significantly reduced disease incidence by 48.1, 22.2, and 63.0%, respectively, compared to the CK treatment (p < 0.05). All three inoculation treatments significantly increased the growth of cucumber seedlings and suppressed the FOC population compared to the control (p < 0.05). High-performance liquid chromatography (HPLC) analysis showed that total phenolic acids were decreased by 18.9, 35.9, and 63.2% in the B2, P5, and B2 + P5 treatments, respectively. The results from this study suggest that combined application of B. amyloliquefaciens B2 and P. ostreatus P5 could be a promising strategy for suppressing Fusarium wilt and improving plant growth of cucumber seedlings under continuous cropping conditions.

Bacterial blight on Sansevieria cylindrica caused by Pseudomonas sp.

The African spear or cylindrical snake plant (Sansevieria cylindrica) is commonly used as an ornamental indoor succulent plant. In May 2019, we observed bacterial blight in S. cylindrica grown for interior decoration and air purification in Jinju, South Korea. These symptoms eventually led to leaf desiccation. Similar leaf blight symptoms have been frequently documented on social media; however, as an imported species, the pathogens of S. cylindrica have not been reported in South Korea. Based on pathogenicity tests; levan production, oxidase production, pectinolytic activity, arginine dihydrolase production, and tobacco hypersensibility tests; analytical profile index API tests; and phylogenetic analysis based on multi-locus sequence analysis using the 16S rRNA, gyrB, rpoD, and rpoB gene sequences, we identified the causative pathogen as Pseudomonas sp. This study is the first to report leaf blight caused by Pseudomonas sp. on S. cylindrica.

Identification and Genomic Characterization of Pathogenic Bacillus altitudinis from Common Pear Trees in Morocco

Bacterial burn is one of the major diseases affecting pear trees worldwide, with serious impacts on producers and economy. In Morocco, several pear trees (Pyrus communis) have shown leaf burns since 2015. To characterize the causal agent of this disease, we isolated fourteen bacterial strains from different parts of symptomatic pear trees (leaves, shoots, fruits and flowers) that were tested in planta for their pathogenicity on Louise bonne and Williams cultivars. The results showed necrotic lesions with a significant severity range from 47.63 to 57.77% on leaves of the Louise bonne cultivar inoculated with isolate B10, while the other bacterial isolates did not induce any disease symptom. 16S rRNA gene sequencing did not allow robust taxonomic discrimination of the incriminated isolate. Thus, we conducted whole-genome sequencing (WGS) and phylogenetic analyzes based on gyrA, gyrB and cdaA gene sequences, indicating that this isolate belongs to the Bacillus altitudinis species. This taxonomic classification was further confirmed by the Average Nucleotide Identity (ANI) and the in silico DNA-DNA hybridization (isDDH) analyzes compared to sixty-five Bacillus spp. type strains. The genome was mined for genes encoding carbohydrate-active enzymes (CAZymes) known to play a role in the vegetal tissue degradation. 177 candidates with functions that may support the in planta phytopathogenicity results were identified. To the best of our knowledge, this is the first data reporting B. altitudinis as agent of leaf burn in P. communis in Morocco. Our dataset will improve our knowledge on spread and pathogenicity of B. altitudinis genotypes that appears as emergent phytopathogenic agent, unveiling virulence factors and their genomic location (i.e., within genomic islands or the accessory genome) to induce trees disease.