Re-isolation Of Bacteria Research Articles

First Report of Enterobacter cloacae Causing Stem, Leaf and Fruit Rot on Tomato in China.

Tomato (Solanum lycopersicum L.), as one of the most economically important and highly nutritious vegetable crops across the world, is widely cultivated in China, one of the largest tomato-concuming countries in the world (Ye et al., 2020; Wang and Liu, 2021). At present, major bacterial diseases in tomato include bacterial speck disease,tomato bacterial wilt and bacterialcanker, all of which affect the tomato production around the world (Rosli et al., 2021; Peritore-Galve et al., 2021; Wang et al., 2022). In April 2022, a new bacterial disease was discovered on leaves, stems and fruits of tomato in a farmer's greenhouse located in Longfeng District in DaQing (125°07`-125°15`E, 46°28`-46°32`N), Heilongjiang Province, China. This field had tomato disease incidences approximately 50%. Apparent brown discolorations were found on fruits, leaves and stems in tomato plants. Symptoms were similar to fungal brown spots caused by Phytophthora infestans of tomato (Zhi et al.,2021; Liu et al.,2021) (Supplementary Figure S1). To isolate and identify the pathogen, the tissues of infected fruits, leaves and stems with typical symptoms were excised from diseased plants separately, and were disinfected with 75% ethanol for 10 s followed by 2% NaClO for 3 min and then washed five to eight times with sterile water (Wang et al., 2017). Afterwards, the samples were plated on nutrient agar (NA) solid medium and incubated. After incubation at 30°C for 2-3 days, bacterial colonies were isolated, then purified on nutrient agar (NA) solid medium at least twice by a streak plate method (Dou et al., 2019; Li et al, 2021; Zhao et al., 2022). White colonies grew on the NA medium after incubating for 2 days, showing round, opaque and smooth, which was similar to characteristics describedas Enterobacter cloacae (García-González et al., 2018; Li et al, 2021).To further confirm the speculation on the identity of the isolated bacterium, the fragments of 16S rRNA were amplified and sequenced. The sequence of 16S rRNA was uploaded into GeneBank with accession numbers (OP077195.1). BLAST analysis of the sequence showed 97.68% identity with one corresponding sequence of E. cloacae in GeneBank (namely MK937637.1). Furthermore, a phylogenetic tree based on the sequence of 16S rRNA gene revealed that the isolate was grouped in the same clade as E. cloacae (Supplementary Figure S2). Based on Kochpostulates to test pathogenicity of isolated bacteria, bacteria were inoculated on 30 day-old healthy tomato plants with three leaves stages, and the re-isolation of bacteria were carried out after 2 days of inoculation. To confirm pathogenicity, the isolates were cultured on LB medium at 30℃ for 2 days to prepare suspensions and adjusted to an optical density (OD) of 0.2 at A600, with a final concentration of 1ⅹ108 CFU/ml. Eight pottedtomato plants were sprayed with bacteria suspensions, and eight control potted plants were sprayed with sterile distilled water. These seedlings were incubated in a chamber at 30°C with a 12 h light/dark photoperiod, with 85% relative humidity. After 2 days, inoculated tomato seedlings showed irregular small spots in leaves and brown necrosis at blade tips, and 8 to 10 days later, the leaves of tomato plants browned and died. The symptoms were the same with those of the initial diseased leaves of tomato plants (Supplementary Figure S1). No symptoms were observed on the control leaves (Supplementary Figure S3). Pathogenicity tests were repeated three biological times with same results. Meanwhile, the bacteria strains were re-isolated from symptomatic inoculated seedlings and confirmed as E. cloacae by culture and sequence methods as above. In China, there are no detailed records about the causal agent of this disease on tomato in a published paper in Chinese and English. To our knowledge, this is the first report of Enterobacter leaf brown necrosis caused byE. cloacaeon tomato in China. Those results are of great significance for the production and management of tomato in greenhouse and control of the disease.

First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi.

On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country.

INVOLVEMENT OF PAENIBACILLUS POLYMYXA IN THE ETIOLOGY OF BACTERIAL SOFT ROT OF CALLA LILY

We studied bacterial isolates 15M and 16M from calla lily tubers showing soft rot in commercial plantations in Poland. Colony morphology differed from that of known agents of bacterial soft rot of this plant. The use of Microgen Bacillus ID system allowed to identify both isolates as Paenibacillus polymyxa. This was confirmed by sequence analysis of 16S rRNA gene fragments. The isolates showed pectolytic activity on crystal violet pectate medium and on potato tubers. They caused strong soft rot on calla tubers and leaf petioles already 48 h after inoculation. Re-isolation of bacteria from rotted tissue confirmed their identity with those used for inoculation. This is the first report that P. polymyxa can cause soft rot on calla lily.

The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC)

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the pathogenicity of avian pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host–pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

Pathogenicity of Bordetella bronchiseptica local isolate in piggeries

Bordetella bronchiseptica causes respiratory tract infections in many animals species. The bacteria were isolated and identified from piggeries in Indonesia. The aim of this study was to define the pathogenicity of B. bronchiseptica local isolate. Thirty piglets less than a week old were divided into 6 treatment groups. Each group of five was infected with Bordetella bronchiseptica BS9 (BCC B2455) local isolate with dose of 4.2 x 10 5 , 4.2 x 10 6 , 4.2 x 10 7 , 4.2 x 10 8 , 4.2 x 10 9 dan 4.2 x 10 10 CFU/ml respectively by intra nasal inoculation, and three piglets were used as the control. The clinical signs, reisolation of bacteria, and histopathological changes were observed. The result showed that all treatment groups showed clinical sign of serous nasal discharge, and sneezing only found in two groups. Reisolation of those bacteria from concha were found in all groups, from trachea in one group, from bronchus in all groups, and they were not found in the control group. Histopathological change of epithelial cells desquamation and lost of cillias on concha were found in all treatment groups. On the trachea, epithelial cells desquamation were found in 3 treatment groups and lost of cillias in 4 treatment groups. On the bronchus, epithelial cells desquamation were found in 2 treatment groups and lost of cillias were found in 3 treatment groups. Total piglet that showed clinical phenomenon, positive bacteria reisolation and histopathological change from each group were not proportion to the dose given. This study showed that the local isolate of B. bronchiseptica was pathogenic because it can cause the damage of ciliated cylindrical epithelial cells of the upper respiratory tract. Key Words: Bordetella bronchiseptica , Local Isolate, Pathogenicity, Piglet

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.

Research Article: Re-isolation of bacteria from intentionally contaminated surfaces

Abstract The natural habitat of the bacterium Staphylococcus is human skin and it can therefore be passed indirectly from person to person through fomites. An initial study investigating the metal push plates of 46 public restrooms throughout Angelo State University's campus for the presence of the bacterial species Staphylococcus aureus yielded evidence that the metal composing the push plates might be exerting a fairly rapid level of auto-disinfection. To test this hypothesis, studies were done to compare the re-isolation of S. aureus, S. capitis, and Salmonella typhimurium from metal and plastic surfaces intentionally contaminated with these organisms. All three organisms were more difficult to re-isolate from the metal surfaces as compared to the plastic surfaces.

L-form bacteria of Pseudomonas syringae pv. phaseolicola induce chitinases and enhance resistance to Botrytis cinerea infection in Chinese cabbage

Induction of chitinases in Chinese cabbage ( Brassica campestris L. ssp. pekinensis var. Granaat) plants treated with L-form bacteria of Pseudomonas syringae pv. phaseolicola during seed germination, was investigated using a fluorometric assay with 4-methylumbelliferyl substrates. The constitutive chitinase activities of seeds and seedlings were low and contrasted with much higher chitinase activities in seedlings treated with L-form bacteria. Maximum chitinase activities of seedlings treated with L-form bacteria was found 31 days after treatment. Extracts of the seedlings treated with L-form bacteria showed 4-fold and 1.5-fold endochitinase activities with, 4-methylumbelliferyl β- d- N-triacetylchitotrioside and 4-methylumbelliferyl β- d- N-tetraacetylchitotetraoside, respectively. Plant/L-form association was detected using a slide agglutination assay, enzyme linked immuno-sorbent assay and by re-isolation of bacteria from seedlings. L-form bacteria were also observed in root hairs using light microscopy. Re-isolation showed that L-forms were systemically distributed throughout the plant, with the highest population in roots. This was supported by ELISA results. Chinese cabbage seedlings treated with L-form bacteria had enhanced resistance to infection by the grey mould pathogen Botrytis cinerea.

Experimental bacterial endophthalmitis.

Experimental bacterial endophthalmitis in the rabbit was easily produced. Reisolation of bacteria by anterior chamber paracentesis correlated with clinical infection in the rabbit. Therefore, we recommend diagnostic anterior chamber paracentesis in human cases of suspect bacterial endophthalmitis.