Methyl Red Reaction Research Articles

Isolation and Diagnosis of Acinetobacter baumannii Isolates from Patients with Meningitis in Child Protection Hospital in Baghdad

Environmental contamination has been estimated to account for up to 20% of all hospital-acquired illnesses. Acinetobacter baumannii has recently become the predominant pathogen, posing a significant risk to human health. In present study, two hundred fifteen CSF samples were collected from children between the ages of new born to 14 years who were visiting child protection hospital in Baghdad. For the period from first of Oct 2023 to the end of Jan 2024, and the samples were sent for diagnosis. Isolates were identified after culturing them on MacConkey agar, blood agar, and mannitol salt agar. A number of biochemical tests were performed to detect Acinetobacter baumannii bacteria. The results indicated a negative oxidase reaction, a positive catalase reaction, a negative indole reaction, a negative methyl red reaction, a negative Voges-Proskauer reaction, and a positive citrate reaction, which were performed for the samples and for final diagnosis the Vitek-2 Compact system was used. After cultivation and diagnosis, 7 isolates of Acinetobacter baumannii were obtained out of 40 samples that showed positive culture results with bacterial growth.

First Report of Pantoea agglomerans Causing Leaf Blight on Schisandra chinensis in China.

Schisandra chinensis (Turcz.) Baill is a perennial liana, which is widely cultivated and used in China. In August 2022, Schisandra chinensis leaves with small light brown spots were found on plants growing in Fusong (127°28'E, 42°33'N) of China. There was 15% disease incidence and 50% disease severity of Schisandra chinensis in 2-ha fields of S. chinensis. As the disease progressed, the spots become darker and form round or irregular concentric circles. Leaves with brown spot symptoms were collected from the field. Leaf pieces (5 mm × 5 mm) were excised from lesion margins, surface disinfected with 75% ethanol for 1 min, followed by 1.5% sodium hypochlorite for 3 min, and incubated on Luria Bertani (LB) solid medium at 28°C for 24 hours. Eight cultures were isolated, and representative single colony (XWWZH) was selected from the pure cultures according to colony characteristics for observation The purified colonies were round, yellow, and slimy, cells were straight rod-shaped (0.40 to 0.52 × 1.12 to 1.69 µm) were observed. The isolate was Gram negative. It was positive for methyl red reaction, lysine decarboxylase reaction, gelatin hydrolysis reactionand sucrose utilization. It was negative for indole reaction and produced H2S. The bacterium was preliminarily identified as Pantoea agglomerans based on morphological and biochemical tests (Baird et al. 2007). The 16S rDNA and a portion of rpoB of strain XWWZH were amplified and sequenced. The sequences were submitted to GenBank. (Accession OP763753 and OQ813505, respectively). Phylogenetic trees were constructed based on the 16S rDNA and rpoB gene sequences. The sequences of strain XWWZH clustered with strains P. agglomerans deposited in GenBank. The pathogenicity was verified with non-wounded S. chinensis seedlings by punching holes with sterile needles and injecting a solution of 1 × 108 CFU/ml solution. Sterile ddH2O was injected in the control experiment. The inoculated seedlings were incubated in a greenhouse at 25°C with a relative humidity of 65 to 70%. Five to eight days after inoculation, inoculated leaves, exhibited symptoms which were morphologically identical to those of the originally infected leaves whereas control plants remained asymptomatic. The pathogenicity assays were repeated twice with the same results. The re-isolated pathogen had the same morphology and DNA sequences as the original isolate obtained from the field samples, completing Koch's postulates. Strains of P. agglomerans have been reported to severely infect many plants (Ren et al.2008; Lee et al. 2010; Yang et al. 2011; Guo et al. 2019; Gao et al, 2022), but to the best of our knowledge, this is the first report of a strain of P. agglomerans causing leaf blight on Schisandra chinensis in China. The identification of leaf blight caused by P. agglomerans will enable farmers to prevent and manage it ahead of time to reduce losses.

Oricola nitratireducens sp. nov., a novel nitrate reducing bacterium isolated from marine sediment in East China Sea.

A novel strain designated NBU1457T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Cells of strain NBU1457T was Gram-negative, facultatively anaerobic, non-motile and ovoid-shaped. Strain NBU1457T grew optimally at 37°C, NaCl concentration of 2.0-3.0% (w/v) and pH 6.5-7.5. Catalase and oxidase activities, urease, nitrate reduction and H2S production were positive. Indole production, methyl red reaction, hydrolysis of starch, gelatin, casein, Tweens 20, 40, 60 and 80 were negative. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Oricola thermophila MEBiC13590T (98.8%), Oricola cellulosilytica CC-AMH-0T (97.9%) and Oricola indica JL-62T (97.9%). Phylogenetic analyses indicated that strain NBU1457T clustered with the genus Oricola and closely related to strains Oricola thermophila MEBiC13590T, Oricola cellulosilytica CC-AMH-0T and Oricola indica JL-62T. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1457T and related species of genus Oricola were well below the threshold limit for prokaryotic species delineation. The DNA G + C content was 63.2%. The major cellular fatty acid was summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. The only respiratory quinone was ubiquinone-10 (Q-10). Combining results of our phenotypic, chemotaxonomic and genotypic data, strain NBU1457T is considered to be a representative in the genus Oricola, which the name Oricola nitratireducens sp. nov. is proposed. The type strain of the new species is NBU1457T (= KCTC 82225T = MCCC 1K04764T).

First report of bacterial wilt disease caused by Pantoea agglomerans on the ornamental perennial Oxalis articulata in China.

Oxalis articulata is now widely cultivated in China as an ornamental species, and thus found in abundance in agricultural farms, gardens, and lawns. In December 2021, some severely infected Oxalis articulata were observed at many places at Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. Yellow was first observed on the margin of the leaves, leading to light brown and wilting at a later stage. To identify the pathogen, symptomatic leaves were collected and cut into small pieces, surface disinfected in 1% sodium hypochlorite solution for 3 min, followed by 75% alcohol for 0.5 min, then rinsed in sterile distilled water thrice. Then they were transferred onto Luria-Bertani medium and incubated at 28°C for 3 days. The colonies were round, yellow, viscous and smooth, which was consistent with the characteristics of Pantoea agglomerans (Li et al. 2020; Zhang et al. 2022). The bacteria tested gram-negative, negative for indole test and Voges-Proskauer reaction, and positive for methyl red reaction, lysine decarboxylase and ornithine decarboxylase. In addition, the bacteria can utilize D-xylose, sorbitol, adonitol, and glucose, but can't utilize raffinose, urea, and Simmons. Meanwhile the bacteria can not produce H2S, and can not produce gas from D-glucose as well. These results of physiological and biochemical characteristics were consistent with those of Pantoea agglomerans (Gavini et al. 1989). To identify the strain, the 16S rDNA gene fragment was amplified by polymerase chain reaction (PCR) using universal primers 8F and 1510R, and sequenced. The BLAST results indicated that the 16S rDNA sequence of the strain OAPB-1, deposited under GenBank accession LC709256, showed 99.93%(1376/1377) and 99.49% (1370/1377) identitytothecorresponding sequence of Pantoea agglomerans FC2948 (MH532498.1) and the type strain Pantoea agglomerans DSM 3493 (AJ233423) respectively. The Neighbor-Joining phylogenetic tree generated using MEGA11 indicated that it formed a clade with the other P. agglomerans. Furthermore, the phylogenetic analysis based on sequences of housekeeping genes (atpD, rpoB and infB; GenBank accession LC722492 to LC722494) showed the same result. Based on the above results, the strain OAPB-1 from Zhejiang was identified as P.agglomerans. To test the Koch's postulates, bacterial suspensions (2×108CFU/mL) were injected into the middle of healthy leaves of mature plants with sterile water as a control. Then the plants were placed at 28°C in a light incubator with 12-h-light/12-h-dark photoperiod and approximately 60% humidity. Leaves in the inoculated group showed symptoms similar to those observed on the naturally infected leaves, while leaves in the control group showed no symptoms. The pathogen was reisolated from inoculated leaves, and its morphological characteristics and molecular identification results were consistent with those of the original isolate. P. agglomerans is a bacterium associated with plants, and also infects humans and animals (Dutkiewicz et al. 2016). In China, it has been reported to infect many kinds of plants (Fan et al. 2022; Guo et al. 2020; Han et al. 2020; Li et al. 2020; She et al. 2019; Zhang et al. 2022). As far as we know, this is the first report of P. agglomerans causing bacterial wilt on Oxalis articulata in China. These results further expand the range of plants that can be infected by P. agglomerans, and help to establish an effective control strategy against the disease.

Luteolibacter marinus sp. nov., a novel bacterium in the family Verrucomicrobiaceae, isolated from marine sediment.

A Gram-negative, rod-shaped, non-motile and strictly aerobic bacterium, designated NBU1238T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1238T was able to grow optimally at 28-32 °C, at pH 7.5 and with no NaCl. Catalase and oxidase activities, H2S production and hydrolysis of Tweens 40 and 60 were positive. Methyl red reaction, Voges-Proskauer test and hydrolysis of starch, casein and Tweens 20 and 80 were negative. The major cellular fatty acids were iso-C14 : 0, C16 : 0, C16 : 1 ω9c and C14 : 0. The only respiratory quinone was menaquinone-9. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine. The 16S rRNA gene sequence of strain NBU1238T showed 95.6, 95.6, 94.8 and 93.8% sequence similarity to Luteolibacter flavescens GKXT, Luteolibacter luteus G-1-1-1T, Luteolibacter arcticus MC 3726T and Luteolibacter pohnpeiensis A4T-83T, respectively. Phylogenetic analyses indicated that strain NBU1238T clustered with the genus Luteolibacter and was closely related to strains L. flavescens GKXT, L. arcticus MC 3726T and L. luteus G-1-1-1T. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1238T and related species of genus Luteolibacter were well below the threshold limit for prokaryotic species delineation. The DNA G+C content of the genomic DNA was 65.0 mol%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1238T is considered to be a representative of a novel species in the genus Luteolibacter, for which the name Luteolibacter marinus sp. nov. is proposed. The type strain is NBU1238T (=KCTC 82227T=MCCC 1K04772T).

Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov., two novel species in the family Flavobacteriaceae isolated by a modified in situ cultivation technique from marine sediment.

A modified in situ cultivation technique was developed and applied to resource mining of uncultured microbes from marine sediments of Meishan Island in the East China Sea. Two novel strains NBU2968T and NBU2984T were isolated by this method but not standard Petri dish, which indicated the modified technique was more effective compared to conventional approaches for isolating uncultured microbes and could be popularized and applied to other aquatic environments. The two novel strains were identified by the polyphasic taxonomic approach. Cells of both strains were observed to be Gram-staining-negative, rod-shaped, nonmotile, aerobic, and yellow-pigmented. Catalase and oxidase activities and hydrolysis of Tweens 40, 60, and 80 of two novel strains were positive. Methyl red reaction, H2S production, and hydrolysis of Tween 20 were negative. According to 16S rRNA gene sequence analysis, two novel strains shared the highest similarities (96.4–97.7%) to the species with a validated name in the genus Hanstruepera, while shared lower sequence similarities (<95.6%) to all other genera. Phylogenetic analysis revealed that strains NBU2968T and NBU2984T were affiliated with the genus Hanstruepera. ANI and dDDH values between the two novel strains and Hanstruepera species were 77.4–78.3% and 20.4–20.9%, respectively, which were below the thresholds for species delineation. The 16S rRNA gene sequence similarity, ANI, and dDDH values between the two novel strains were 99.3, 88.9, and 36.3%, respectively, indicating that the two strains represent different species. The genomes of NBU2968T and NBU2984T were 3.28 Mbp with a G+C content of 34.2% and 3.09 Mbp with a G+C content of 34.4%, respectively. The only respiratory quinone was menaquinone-6 (MK-6). The major cellular fatty acids were iso-C15:0, iso-C15:1G, and iso-C17:0 3-OH. The major polar lipids of the two strains were phosphatidylethanolamine, unidentified amino lipids, and unidentified lipids. Based on the above polyphasic characteristics, strains NBU2968T and NBU2984T represent two novel species within the genus Hanstruepera, for which the names Hanstruepera marina sp. nov. and Hanstruepera flava sp. nov. are proposed. The type strains are NBU2968T (= MCCC 1K06392T= KCTC 82913T) and NBU2984T (= MCCC 1K07472T= KCTC 92511T), respectively.

Pelagibius marinus sp. nov., a novel bacterium isolated from marine sediment, and emended description of the genus Pelagibius.

A Gram-stain-negative, rod-shaped, motile and aerobic marine bacterium, designated strain NBU2595T, was isolated from marine sediment sampled on Meishan Island, located in the East China Sea. Strain NBU2595T grew at 10-40 °C (optimum, 37 °C), at NaCl concentration of 0-10.0 % (w/v; optimum, 0.5 %) and at pH 6.0-8.0 (optimum, pH 7.0). Catalase and oxidase activities and H2S production were positive. Methyl red reaction and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were negative. The major cellular fatty acids were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C18 : 1 2-OH and C18 : 0 3-OH. The sole respiratory quinone was ubiquinone 10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. Comparative analysis of 16S rRNA gene sequences showed highest similarity to Pelagibius litoralis CL-UU02T (97.9%), and low similarities (<92.9 %) to other species. Phylogenetic analyses indicated that strain NBU2595T clustered with the genus Pelagibius and was closely related to P. litoralis CL-UU02T. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU2595T and the related species of the genus Pelagibius were well below the thresholds for prokaryotic species delineation. The DNA G+C content was 66.5 mol%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU2595T should be placed in the genus Pelagibius as representing a novel species, for which the name Pelagibius marinus sp. nov. is proposed. The type strain is NBU2595T (=MCCC 1K04773T=KCTC 82223T).

Nisaea sediminum sp. nov., a heavy metal resistant bacterium isolated from marine sediment in the East China Sea.

A Gram-negative, rod-shaped, motile and strictly aerobic bacterium, designated NBU1469T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1469T grew optimally at temperature of 40°C, NaCl concentration of 2.0% (w/v) and pH 7.5. Catalase and oxidase activities, H2S production, nitrate reduction and hydrolysis of Tween 20 were positive. Indole, methyl red reaction, urease, hydrolysis of gelatin, starch, casein, Tweens 40, 60 and 80 were negative. The major cellular fatty acids were C16:0, C19:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The only respiratory quinone was ubiquinone-10 (Q-10). The major polar lipids were phosphatidylglycerol, two unidentified amino-phospholipids and two unidentified phospholipids. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Nisaea nitritireducens DR41_18T (98.1%) and Nisaea denitrificans DR41_21T (97.6%). Phylogenetic analyses indicated that strain NBU1469T formed a distinct lineage with strains Nisaea nitritireducens DR41_18T and Nisaea denitrificans DR41_21T within the genus Nisaea. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1469T and related species of genus Nisaea were well below the threshold limit for prokaryotic species delineation. The DNA G + C content was 63.6%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1469T is considered to be a representative of a novel species in the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed. The type strain is NBU1469T (=KCTC 82224T =MCCC 1K04763T).

Shimia sediminis sp. nov., a bacterium isolated from marine sediment in the East China Sea.

A Gram-stain-negative, ovoid-shaped, motile and strictly aerobic bacterium, designated ZQ172T, was isolated from marine sediment sampled on Zhoushan Island located in the East China Sea. Strain ZQ172T grew optimally at temperatures of 25-30 °C, at NaCl concentration of 2.0 % (w/v) and at pH 7.0-8.5. Catalase and oxidase activities, urease, and nitrate reduction were positive. Indole production, methyl red reaction, H2S production and hydrolysis of gelatin, starch, casein and Tweens 20, 40, 60 and 80 were negative. The major cellular fatty acid was summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The only respiratory quinone was ubiquinone-10. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to Shimia aestuarii JC2049T (97.5 %), Shimia abyssi JAMH 043T (97.5 %) and Shimia thalassica CECT 7735T (97.3 %). Phylogenetic analyses indicated that strain ZQ172T clustered with the genus Shimia and was closely related to Shimia thalassica CECT 7735T. The average nucleotide identity and digital DNA-DNA hybridization values between strain ZQ172T and related species of the genus Shimia were well below the threshold limit for prokaryotic species delineation. The DNA G+C content was 59.6 mol%. Based on its phenotypic, chemotaxonomic and genotypic data, strain ZQ172T is considered to be a representative of a novel species in the genus Shimia, for which the name Shimia sediminis sp. nov. is proposed. The type strain is ZQ172T (=KCTC 62578T=MCCC 1K03531T).

Microbulbifer hainanensis sp. nov., a moderately halopilic bacterium isolated from mangrove sediment.

A new bacterium was successfully isolated from a mangrove sediment sample in Haikou City, Hainan Province, China. The organism is a Gram-negative, rod-shaped, non-motile and strictly aerobic bacterium, named NBU-8HK146T. Strain NBU-8HK146T was able to grow at temperatures of 10-40°C, at salinities of 0-11% (w/v) and at pH 5.5-9.5. Veoges-Proskauer, methyl red reaction and hydrolysis of Tween 20 were negative. Catalase and oxidase activities, H2S production, hydrolysis of starch, casein, Tweens 40, 60 and 80 were positive. The major cellular fatty acids were C16:0, iso-C15:0 and summed feature 9. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and two unidentified glycolipids. According to 16S rRNA gene sequence similarities, strain NBU-8HK146T shared 98.0%, 97.9%, 97.7%, 97.6% and 97.3% similarities to the species with validated name Microbulbifer taiwanensis CC-LN1-12T, Microbulbifer rhizosphaerae Cs16bT, Microbulbifer marinus Y215T, Microbulbifer donghaiensis CN85T and Microbulbifer aggregans CCB-MM1T, respectively. Phylogenetic analyses indicated that strain NBU-8HK146T formed a distinct lineage with strains Microbulbifer taiwanensis CC-LN1-12T and Microbulbifer marinus Y215T. Both digital DNA-DNA hybridization values (19.5-22.7%) and average nucleotide identity values (73.2-78.9%) between strain NBU-8HK146T and related species of genus Microbulbifer were below the species delineation cutoffs. The DNA G+C content was 58.9mol%. Many proteins involving in the adaption of osmotic stress in the salt environment of mangrove were predicted in genome of strain NBU-8HK146T. From phenotypic, genotypic, phylogenetic and chemotaxonomic characteristics, strain NBU-8HK146T can be regarded as a new Microbulbifer species for which the name Microbulbifer hainanensis. The type strain is NBU-8HK146T (= KCTC 82226T = MCCC 1K04737T).

Rheinheimera mangrovi sp. nov., a bacterium isolated from mangrove sediment.

A Gram-stain-negative, rod-shaped, motile and strictly aerobic bacterium, designated LHK132T, was isolated from a mangrove sediment sample collected in Haikou, Hainan Province, PR China. Strain LHK132T was able to grow at temperatures of 10-45 °C, at salinities of 0-7.0 % (w/v) and at pH 6.0-9.0. Catalase and oxidase activities, H2S production, urease and methyl red reaction were positive. Indole, nitrate reduction, hydrolysis of gelatin, starch, casein and Tweens 20, 40, 60 and 80 were negative. The major cellular fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The only respiratory quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. According to 16S rRNA gene sequence analysis, strain LHK132T had 98.3, 97.5, 97.4, 97.2 and 97.1% similarities to Rheinheimera soli BD-d46T, Rheinheimera sediminis YQF-1T, Rheinheimera tangshanensis JA3-B52T, Rheinheimera mesophila IITR-13T and Rheinheimera arenilitoris J-MS1T, respectively. Phylogenetic analyses indicated that strain LHK132T formed a distinct lineage with R. soli BD-d46T within the genus Rheinheimera. The average nucleotide identity and digital DNA-DNA hybridization values between strain LHK132T and related species of the genus Rheinheimera were well below the thresholds for species delineation. The DNA G+C content was 46.7 mol%. On the basis of its phenotypic, chemotaxonomic and genotypic data, strain LHK132T is considered a representative of a novel species in the genus Rheinheimera, for which the name Rheinheimera mangrovi sp. nov. is proposed. The type strain is LHK132T (=KCTC 62580T=MCCC 1K03529T).

First Report of Clavibacter michiganensis subsp. michiganensis Infecting Potato in Russia

Yellow-pigmented clavibacteria were isolated from diseased potato plants and tubers from five regions of Russia between 2011 and 2017. Severe outbreaks of the disease were recorded in the central and northwest parts of the Russian Federation. Plants exhibited yellowing and necrosis of the leaf and wilting of whole leaves and plants, and brown veins around potato tuber eyes were visible on cross sections. Vascular tissue was removed from discolored nodes, crushed in sterile 10 mM phosphate buffer, and dilution plated on yeast extract dextrose calcium carbonate agar. Creamy yellow colonies developed from the extracts after 7 days incubation at 26°C. Isolates from each diseased potato plant (35 isolates deposited to the State Collection of Phytopathogenic Microorganisms at Russian Research Institute of Phytopathology, Bolshie Vyazemy) were identified as Clavibacter michiganensis subsp. michiganensis (Cmm) using taxon-specific polymerase chain reaction (PCR) with primers CMM5/CMM6 that gave expected product of 614 bp (Dreier et al. 1995). Additionally, isolates were positive for Cmm and negative for C. michiganensis subsp. sepedonicus using a commercial ELISA test (Agdia Biofords, France). Bacteria were gram positive aerobic rods with biochemical traits uniform and similar to the type strain Cmm NCPPB2979T (Schaad et al. 2001). All isolates were positive for esculin hydrolysis, gelatin liquefaction, H₂S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose, and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)melezitose and d(–)sorbitol, and utilization of acetate, formate, lactate, propionate, and ribose. Sequencing of the 16S rRNA (Weisburg et al. 1991) and gyrB gene (Zaluga et al. 2011) was performed for all 35 isolates. 16S rRNA sequencing (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. gyrB gene analysis grouped the new potato-isolated strains (GenBank accession nos. MH042254 to MH042288) with Cmm strains from tomato obtained from the same regions. Pathogenicity of four typical isolates was tested on tomato (cv. Dubok) and potato (cv. Lion Heart) plants using suspensions of about 10⁸ cells/ml in sterile tap water. Four to six plantlets with two to four fully developed leaves were inoculated by stem infiltration (Zaluga et al. 2013) with 10 µl of bacterial suspension and were placed in a growth chamber at 20 to 25°C. The type strain of Cmm from tomato was used as a pathogenic control. The first symptoms appeared 14 days after inoculation and progressed slowly. In both tomato and potato plantlets, the four Cmm isolates displayed chlorosis of leaf margins, mottling and yellowing between veins, wilting or necrosis of individual leaf parts, and finally wilting of whole leaves. Each isolate was recovered from infected test plants and reidentified as Cmm by PCR with primers CMM5/CMM6. No symptoms were observed on negative control plants inoculated with sterile water and on potato inoculated with the Cmm type strain. 16S rRNA gene (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. This is the first report of Cmm affecting potato in Russia.

Genetic Diversity of Brenneria Nigrifluens Strains in North of Iran (Margin of Caspian Sea)

Genetic Diversity ofBrenneria NigrifluensStrains in North of Iran (Margin of Caspian Sea)Brenneria nigrifluens, the cause of shallow bark canker of Persian walnut trees (Juglans regiaL.), has become fairly widespread in Iran in recent years. It is regarded as a great threat to walnut production. To determine the diversity ofB. nigrifluensstrains, sixty strains of the causal bacterium were isolated from bark samples of infected walnut trees collected from Mazandaran, Guilan and Golestan provinces and were studied. The physiological and biochemical characteristics, electrophoretic patterns of total cell proteins and rep-PCR generated DNA fingerprints ofB. nigrifluensstrains were compared. Strains appeared to be more or less similar in phenotypic characteristics. Less than 15% of the strains differed in a few phenotypic features such as the ability in production of H2S from peptone, hydrolysis of esculin, levan production, arginine dehydrolase, nitrate reduction, indol production and methyl red reaction. These differences did not show any special distribution and therefore was not suitable for classifying the strains into distinct groups. The electrophoretic patterns of cell proteins of the strains were different from each other and were only useful for preliminary grouping of the isolates. The groups of strains were differentiated by their rep-PCR fingerprints and on which basis they were placed in six groups in similarity level 95%. Cluster analysis was performed using NTSYSpd software. The results of these studies demonstrated that the populations ofB. nigrifluensin North of Iran are genetically heterogeneous. The results can be used in selection of disease management strategies.

First Report of Bacterial Canker of Tomato Caused by Clavibacter michiganensis subsp. michiganensis in Korea.

In 2007, a new bacterial disease was observed in greenhouse-cultivated cherry tomatoes in Cheorwon and Iksan provinces, Korea. The disease caused severe wilt of tomatoes (Solanum lycopersicum cv. Koko). Infected young petioles were curled downward. Margins of the leaves rolled upward and whole leaves were distorted. Stem cankers had reddish or dark brown cavities. Vascular tissues in stems cut longitudinally were brown to deep brown, but no bird's eye lesions were observed. Eight bacterial strains recovered from the stems of wilted tomatoes produced yellow colonies on nutrient broth-yeast extract agar and pink colonies on triphenyl tetrazolium chloride. Pathogenicity of the strains (three plants per strain) on 18-day-old tomatoes (cv. Koko) was confirmed by clip inoculation of petioles of second leaves and spray inoculation with bacterial suspensions (1 × 108 CFU/ml) in sterile distilled water. Wilt and canker symptoms were observed 2 weeks after inoculation. Symptoms produced by both inoculation methods were systemic and localized. Clip inoculation of tomatoes resulted in wilt, defoliation, and open stem cankers, whereas small, white spots (2 to 3 mm in diameter) and sometimes water-soaked, dark brown-to-black lesions on the leaf margins were observed with spray inoculation. Bacteria were reisolated from stems and leaves of the inoculated plants and their identities confirmed by direct PCR using specific primer set CMM5/CMM6 (1). No symptoms were observed on negative control plants inoculated with sterile water. All strains were gram-positive aerobic rods with no polar flagella. Strains were positive for esculin hydrolysis, gelatin liquefaction, H2S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)-melezitose and d(-)sobitol, and utilization of acetate, formate, lactate, propionate, and ribose. Identification as C. michiganensis subsp. michiganensis was confirmed using 16S rDNA universal primers fD1 and rP2 (4) and internal primers (3). The 1,439-bp PCR fragment of strain BC2643 was sequenced (GenBank Accession No. EU685335) and compared with reference C. michiganensis subspecies strains in GenBank: AM410696 (C. michiganensis subsp. michiganensis), AM410693 (C. michiganensis subsp. tessellarius), AM410697 (C. michiganensis subsp. nebraskensis), AM410694 (C. michiganensis subsp. sepedonicus), and AM410695 (C. michiganensis subsp. insidiosus). The sequence had a similarity index of 0.999 calculated by Juke-Cantor model (2) with the 16S rRNA sequence of C. michiganensis subsp. michiganensis (AM410696). The fragment size of eight strains amplified by PCR using CMM5/CMM6 (1) was identical to that of the C. michiganensis subsp. michiganensis reference strain KACC20122. On the basis of the physiological, genetic, and pathological characteristics, all strains were identified as C. michiganensis subsp. michiganenesis. To our knowledge, this is the first report of C. michiganensis subsp. michiganenesis causing bacterial canker on tomato in Korea.

Identification of Bacillus species from ‘iru’, a fermented African locust bean product

AbstractThirty‐seven Bacillus isolates were obtained from fermenting locust bean (‘iru’) from three different locations in Nigeria. The isolates were characterized on the basis of biochemical and physiological tests. Thirty‐two of the isolates were identified as B. subtilis, two as B. pumilus and three as B. licheniformis.The Bacillus species were sub‐divided into strains. Five different strains of B. subtilis and two of B. pumilus were identified. Biochemical tests showed that the strains differ in starch hydrolysis nitrate reduction and methyl red reaction and in their ability to produce acetylmethylcarbinol. They also vary in their patterns of sugar fermentation. This study shows that various strains or dossibly subspecies of B. subtilis are the major Bacillus species involved in the fermentation of the African locust bean (Parkia biglobosa).

Development of a Multiple Inoculator System for the Identification of Aerobic Heterotrophic Bacteria

Abstract A semi-automated multiple inoculator system was modified and evaluated for use with 25-compartment petri dishes. Twenty-one species of environmental and culture collection bacteria were tested for the following characteristics: nitrate reduction, indole production, methyl red reaction, acid from glucose, gelatin hydrolysis, phenylalanine deaminase, citrate utilization, aesculin hydrolysis, reactions on skim milk agar, arginine dihydrolase, and urea hydrolysis. The agreement between the inoculator system and conventional methods was 90 to 100% for all tests except arginine dihydrolase and urea hydrolysis. The multiple inoculator described was judged a simple and versatile device for the rapid identification of large numbers of bacteria.

Nitrate reduction in Aerobacter aerogenes

Mutants of A. aerogenes blocked in aerobic and anaerobic nitrate assimilation and deficient in the reduction of nitrate and chlorate were found to give a positive methylred reaction and no gas formation from glucose. Resting cells, grown anaerobically in minimal medium with glucose, did not show gas production from formate. These results show that these mutants are also deficient in formate hydrogenylase. Revertants could readily be obtained by plating on minimal medium with nitrate as sole nitrogen source, indicating that in these mutants a pleiotropic point mutation is present, which affects both nitrate reductase and formate hydrogeny lase. It is suggested, that these mutants are deficient in the formation of an enzyme complex or particle on which these enzymes are present.

Biochemical Reactions of Leishmania Tropica 1

Summary and Conclusions Seven locally isolated strains of Leishmania tropica were uniform in their biochemical reactions, i.e. 1. No production of acid or gas from carbohydrates, 2. No liquifaction of gelatin, 3. Methyl red reaction was negative, 4. Voges Prauskauer reaction was negative, 5. Peptonization of milk, 6. Bile soluble, 7. No production of indol.

The Voges‐Proskauer and Methyl Red Reactions in the Coli‐Aerogenes Group

The Voges‐Proskauer and Methyl Red Reactions in the Coli‐Aerogenes Group

The Differentiation of the Coli and Aerogenes Groups of Bacteria

In recent years far greater importance has been attached to the separation of the coli and aerogenes divisions of the colon groups than heretofore. Rogers (1) (2) (3) and his associates showed many years ago, by a study of the gas ratios and final hydrogen ion concentration of many colon cultures, that the natural habitat of B. coli is the intestinal tract of man and animals. On the other hand, B. aerogenes is rarely found in human and animal excreta, but is predominantly a soil or grain type of organism. Therefore, these two groups of bacteria are no longer believed to have the same sanitary significance in passing on the potability of a water supply. Unfortunately under many conditions a clear-cut differentiation of these two divisions with the identification of the organisms is a difficult and long procedure. In order that this may be more easily achieved, certain disturbing factors must be eliminated or removed as far as possible, such as (a) aerobic and anaerobic spore formers capable of producing acid and gas in lactose broth and (b) bacterial symbiosis or synergism. Michaelis and Marcora (4) and later, Clark (5) showed that the final pH for cultures of B. coli are a physiological constant. This work was, also, extended to include B. aerogenes. By means of a simple medium conditions were established whereby the metabolism of one group was made to diverge very far from that of the other, resulting in a distinct difference in the final hydrogen ion concentration produced by these two main divisions of the colon group. This test is known as the methyl red reaction (6) (7). Other well known methods which have been used for the separation of the two divisions of the colon group are the Voges-Proskauer (V.-P.) (8), uric acid (9) and citrate tests (10) (11).