Glucose Nutrient Agar Research Articles

Elucidating the physiological and molecular characteristics of bacterial blight incitant Xanthomonas auxonopodis pv. punicae; a life threatening phytopathogen of pomegranate (Punica granatum. L) and assessment of H2O2 accumulation during host-pathogen interaction

Bacterial blight of pomegranate caused by Xanthomonas auxonopodis pv.punicae (Xap) threaten the existence of a group of farmers for the past few decades who rely on pomegranate cultivation for their livelihood since it will cause huge yield loss. The primary focus of this study was to conduct a thorough analysis of the characterization of this blight incitant Xap. Physiological, biochemical, and molecular characteristics of six phytopathogenic strains of Xap, designated as PBF1 (PBF: Pomegranate Blight Fruit), PBF2, PBF3, PBF4, PBF5, and PBF6, isolated from the infected fruits were examined. Bacterial colonies were featured as gram-negative, yellow-pigmented circular with a glistening appearance. An attempt to determine the best culture medium, favouring bacterial proliferation was successfully done with four distinct medium, Nutrient Glucose Agar (NGA), Nutrient sucrose Agar (NSA), Yeast Dextrose Calcium Carbonate Agar (YDCA) and Yeast Glucose Calcium Carbonate Agar (YGCA) and comparatively, significant growth was found in NGA (66.66%) followed by YDCA (33%). According to the antibiotic susceptibility results, both ampicillin and streptomycin were determined as potentially effective drugs in preventing the proliferation of Xap (P 0.05). The reactive oxygen species-mediated plant immune response during host-pathogen interaction was confirmed by accessing the presence of H2O2 accumulation in infected leaves via 3,3 - diaminobenzidine (DAB) -staining technique. Bacterial isolates from this study were confirmed by two universal constitutive genes such as gyrB and 16S rRNA. From the BLAST analysis, the isolates were identified as Xap with base pair lengths of 1408bp, 1180bp, and 1159bp, which correspond to PBF1, PBF2, and PBF3, respectively. A neighbor-joining phylogenetic tree study explaining a strong phylogenetic relationship between the query sequence and closely related bacterial species.

Characterization of Xanthomonas hortorum isolates causing leaf spot on Primula vulgaris

AbstractThe bacterial leaf spot of Primula vulgaris was reported from forest locations in Mazandaran province, Iran in 1995. The causative bacterium was initially identified as Xanthomonas campestris based on sequences of ITS and 16S rRNA regions. On the basis of sequences of gyrB and dnaK housekeeping genes, the isolates resembled strains of Xanthomonas hortorum. The present study was performed to clarify the taxonomic status of the incitant bacterium. Samples of P. vulgaris with leaf spot symptoms were collected from the Darabkola forest (Mazandaran province) and the bacterial strains were isolated on plates of glucose nutrient agar. Some isolates only induced hypersensitive reaction on geranium (Pelargonium × hortorum) leaves but a few isolates incited leaf spot symptoms when inoculated on P. vulgaris, P. heterochroma and P. × polyantha plants. The isolates were Gram stain, oxidase and urease negative but positive in tests for catalase and hydrolysis of gelatin, aesculin and Tween 80. Several unique nonpathogenic isolates were recovered with varying carbon source utilization profiles and different molecular features, including distinct ERIC‐PCR profiles. Representative gyrB, rpoD, dnaK and fyuA housekeeping genes were amplified by PCR and sequenced for comparison with Xanthomonas species in the GenBank database using maximum‐likelihood, maximum‐parsimony and Bayesian algorithms to construct phylogenetic trees. The pathogenic and nonpathogenic strains were closely related to X. hortorum pathovars. Therefore, it is likely that these isolates belong to a new pathovar of X. hortorum. Based on previous studies and our findings, we propose naming the strain causing primrose leaf spot as X. hortorum pv. primulicola.

Reduction in Rumen Tetracycline-Insensitive Bacteria during a Grain Challenge Using the Isoflavone Biochanin A.

The isoflavone biochanin A was previously shown to promote weight gain in growing steers by selectively inhibiting rumen bacteria-like growth-promoting feed antibiotics. The hypothesis that biochanin A inhibited the action of drug efflux pumps was tested by enumerating tetracycline-insensitive bacteria from steers in a subacute rumen acidosis (SARA) challenge. Steers (n = 3/group) treatment groups were forage only, SARA control, SARA with monensin (0.2 g d-1), and SARA with biochanin A (6.0 g d-1). As the steers were stepped up from the forage-only basal diet to 70% cracked corn, the number of rumen bacteria enumerated on two tetracycline-containing media types (nutrient glucose agar and tetracycline, and bile esculin azide and tetracycline) increased (p < 0.05) from as little as 1.7(105) to as great as 6.7(106) cfu mL-1 on the nutrient glucose agar in the SARA and monensin control groups. The biochanin A group maintained the same number of tetracycline-insensitive bacteria as the forage-only controls (p > 0.05). The effects were similar to the more selective media type, but the differences were smaller. These results support the hypothesis that biochanin A inhibits the activity of drug efflux pumps in vivo.

Growth Kinetic Study of Bacterial Pathogens of Pomegranate Crop

The activities of microorganisms are greatly affected by the chemical and physical conditions of their environments. Different organisms react to their environment in different ways. An environment that is harmful to one microorganism may be beneficial to another. Sometimes an organism can tolerate an adverse condition in which it is unable to grow. In the present study, optimization of growth conditions of Xanthomonas axonopodis VMB13, Xanthomonas campestris VMB15 and Xanthomonas vesicatoria VMB17 bacterial pathogens of Pomegranate crop were studied. It is observed that the maximum colony number of the pathogens was observed at 300C which was followed by 25 0C, 350C. The minimum colony number of the pathogen was observed at 400C, 200C which was followed by 150C. No growth of the pathogens was observed at 100C and 500C on sterile nutrient glucose agar. The large number of development of colonies was obtained at pH 7.0. Further increase in pH was found to decrease the number of colonies on the agar medium. The maximum number of colonies of the pathogen was observed on agar medium with 0.5% sodium chloride (NaCl) concentration followed by 0.25% and 1.0% salt concentration. Lowest colonies were observed on NG medium with 1.5%, 0.05% sodium chloride concentration respectively.

Uji Efektivitas Daya Antibakteri Ekstrak Alga Terhadap Pertumbuhan Bakteri Patogen Streptococcus Mutans

Background: Steptoccocus mutans is one of flora normal bacteria in the human body which can cause infections in the mouth cavity. Algae are plant-like organisms that may be found in a marine ecosystem. Recents studies have reported that algae contains numerous pharmacological activities compounds, such us: antibiotic, cytotoxic, antifungal, anti virus and antitumor. Purpose: this research aimed to know the antibacterial activity of algae extract against the growth of pathogen bacteria Streptococcus mutans. Methods: Algae were extracted by using aquades.The inhibition test was performed by agar diffusion method on Glucose Nutrient Agar (GNA) using three types of algae extracts: Eucheuma cottoni, Turbinaria decurrens and Ulva reticulata on the incubation period 1x24 and 2x24 hours at 37◦C. Data collection technique was based on the results of measuring the diameter of the inhibition zone (clear zone) for the growth of Streptococcus mutans bacteria using a caliper. The data obtained were analyzed using quantitative descriptive analysis methods. This data analysis was processed to see if there was a difference in antibacterial power from extract algae samples. Results: U. Reticulate algae extract formed a clear zone 11.2 mm in diameter at 1x24-hour incubation, and 10.3 mm at 2x24-hour incubation. T. deccurens formed clear zones 12.7 mm in diameter at 1x24-hour incubation, and 11.4 mm at 2x24-hour incubation. E. cottonii formed clear zones 10.1 mm in diameter at 1x24-hour incubation, and 9.3 mm at 2x24-hour incubation. Conclusion: Extracts of T. decurrens have the most potential in inhibiting the growth of pathogenic microbes of S. mutants.

ISOLASI MIKROBA PENGHASIL ANTIBIOTIK DARI PASIR PANTAI LEMO-LEMO KABUPATEN BULUKUMBA DALAM MENGHAMBAT BEBERAPA BAKTERI PATOGEN

Research on the isolation of antibiotic-producing microbes from the sand of Lemo-lemo beach in Bulukumba Regency has been carried out in inhibiting several pathogenic bacteria. This study aims to obtain microbes from beach sand that can produce antibiotics. The first stage of microbial isolation was carried out by diluting 10-1 to 10-7 using the pour method on Glucose Nutrient Agar (GNA) and Potato Dextrosa Agar (PDA) medium, then fermented using Maltose Yeast Broth (MYB) medium. The activity was tested using agar diffusion in Nutrient Agar (NA) medium against the test microbes. The results obtained 7 bacterial isolates and 3 fungal isolates showing the clear zone around them. The bacterial isolate that provides the best activity is AB6 isolate which can inhibit many pathogenic microbes, namely Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella thypi, Staphylococcus aureus, Sthapylococcus epidermis and Vibrio colera. Meanwhile, all fungal isolates provided good activity against the tested microbes, namely isolates AJ1, AJ2, and AJ3. The next step was macroscopic morphological observations by looking at bacterial growth on the medium NA upright, NA slanted, NB while microscopically Gram staining was carried out, where isolates AB1 to AB7 is a gram-positive bacillus. Furthermore, the biochemical activity test was carried out which included motility test, catalase test, citrate test, and temperature variation growth test. Based on the results of the study, it was found that AB1 to AB7 isolates belonged to the genus Bacillus and had Bacillus firmus species identified using the vitek2 compact tool. Meanwhile, the fungal isolates, namely AJ1 and AJ2, were included in the penicillium fungus and AJ3 included the Apergillus fungus.

Reactions of banana plantlets Musa acuminata L. to extracellular polysaccharides from Ralstonia zyzygii subsp. celebensis causal agent of blood diseases

Extracellular polysaccharides generated by bacteria play an important role in inhibiting the translocation of nutrients and water, protecting bacteria in extreme conditions and neutralizing compounds released by plants. Giving filtrate pathogens with different concentrations causes different plant reactions in each variety. This study was aimed to determine the effect of various concentrations of extracellular polysaccharides (EPS) obtained from Ralstonia zyzygii subsp celebensis (rzc) towards the growth of banana plantlets in-vivo. A randomized complete design was used with parameters of two varieties (Barangan and Ambon) and EPS in different concentration of 0; 10; 11.5; 13, and 14.5%. EPS filtrate culture was obtained from initially growing bacteria on NGA (Nutrient Glucose Agar) medium and subculture (mineral media) and centrifuged at 6000 rpm for 15 minutes then sterilized with a membrane filter. Extracellular polysaccharides were put into MS media, then planting 3-month-old banana plantlets with uniform growth. Culture were placed for inoculation that controlled in a culture room at a temperature 26 ± 20C with relative humidity of 55 ± 5% and were exposed to 16 h photo period. Observation parameters were number of shoots and leaves, time of appearance of shoots and leaves (days). The results showed that response of Barangan gave better results on number of shoots than ambon in tolerating the effect of extracellular polysaccharides filtrate. Concentration of 13% is tolerance limit for the growth number of banana plantlets. Barangan variety can produce the highest number of shoots at a concentration of 13% extracellular polysaccharides filtrate.

First Report of a Leaf Blight in Rice (Oryza sativa) Caused by Pantoea ananatis and Pantoea stewartii in Thailand

In 2014 to 2017, the prevalence of leaf blight disease was surveyed in various rice fields located in northern Thailand with 40% disease incidence. Rice leaves with leaf blight symptoms (30 to 50% of the lesion area) ranging from yellow, light brown, to slightly reddish spots were collected and used for bacterial isolation, identification, and storage. Pieces of rice leaf were cut, surface sterilized, and then placed on nutrient glucose agar (NGA). After incubation at 28°C for 24 to 48 h, a single colony of each isolate was picked and restreaked on the NGA plate two additional times. Round, yellow, smooth, nonflat, mucoid colonies were observed on the NGA plate. Bacterial isolates that were gram-negative and rod-shaped were selected for molecular identification. The positive bacterial isolates were used for DNA extraction and PCR amplification of the 16S-23S rDNA spacer region of Xanthomonas oryzae pv. oryzae (∼470 bp) (Adachi and Oku 2000). An unexpected PCR product (∼700 bp) using the XOR primer pair was observed. The PCR product was gel purified for DNA sequencing analysis. The sequencing data were subjected to a BLASTn search of the NCBI database. Results showed 694/700 (99%) identity of our suspected sequences to the gyrB gene of Pantoea ananatis (GenBank no. KT203549.1). Additionally, four pairs of primers specific to housekeeping genes of P. ananatis were designed from P. ananatis strain LMG 20103 (GenBank no. CP001875.2), which are gyrB, rpoB, infB, and atpD. The result showed all four primers successfully amplified all four amplicons in 11 bacterial isolates, and all the sequencing data after BLASTn showed 99 to 100% similarity to P. ananatis (GenBank no. KT203549.1 with 666/670 [99%] identity [gyrB], KT735144.1 with 564/564 [100%] identity [rpoB], KT429228.1 with 499/499 [100%] identity [infB], and KT203523.1 with 593/594 [99%] identity [atpD]). Seven bacterial isolates were shown to be positive only for gyrB and rpoB gene amplifications, and the sequencing data indicated that they belonged to P. stewartii (GenBank no. KT203549.1 with 670/670 [100%] identity [gyrB] and KT735140.1 with 564/564 [100%] identity [rpoB]). Moreover, to validate the species identification, biochemical characterization was performed using the API 20E system, and the result showed all 18 bacterial isolates were identified as Pantoea spp. 2 (urease negative and indole positive). The partial sequencing data of gyrB gene and rpoB genes of all bacterial isolates conducted in this study were deposited in the NCBI database with accession numbers MH244161 to MH244200. Two bacterial isolates from each species were used to confirm for the infectivity of these pathogens in rice via the clipping method. The bacterial suspension (10⁸ CFU/ml) in sterile distilled water was inoculated into rice plants. Inoculation of rice plants with sterile distilled water was used as a negative control (five individual plants per group). The yellow, orange to brown stripes were observed at day 7 postinoculation as expected. To fulfill Koch’s postulates, the bacteria were reisolated from symptomatic leaves for DNA extraction and PCR amplification. The sequence alignment of rpoB gene showed 100% identity between the reisolated sample and the original one. This study is the first report of leaf blight in rice caused by P. ananatis and P. stewartii in Thailand. Toward this end, it has become increasingly evident that P. ananatis and P. stewartii are among the most important and newly emerged leaf blight disease causing pathogens in rice.

Miniaturized fermentation in eppendorf tubes for the detection of antagonistic actinomycetes

Actinomycetes are recognized as a prolific source for bioactive metabolites. Screening adequate number of strains by appropriate high quality screening protocol determines success of drug discovery programs. To accomplish screening for antagonistic actinomycetes at higher rate, the fermentation stage of the initial screening needs to be miniaturized. The present study is attempted for miniaturized production of bioactive compounds from 40 actinomycetes isolated from less explored ecosystems. Actinomycetes were cultivated in eppendorf tubes (2 ml) using 0.5 ml of yeast extract malt extract agar and modified nutrient glucose agar medium for 10 days and extracted using 1 ml of methanol. The crude extracts were tested against S. aureus, B. subtilis and E. coli by disc diffusion method. All the isolates were also tested for antagonistic activity by conventional cross streak method and disc diffusion method using extracts prepared by plate culture method. Crude bioactive compounds produced from five selective isolates by shake flask method showed good activity. Antagonistic activity, requirements, time duration and cost effect of all the three methods were compared. Miniaturized production process in eppendorf tubes was found to be more advantageous and suitable to develop as high throughput screening (HTS) method for the detection of antagonistic actinomycetes particularly in resource poor laboratories settings.

Factors affecting biofilm formation as seed transmission mechanism of cowpea bacteria blight induced by Xanthomonas axonopodis pv vignicola (Burkholder) Dye

Bacteria are carried in/on seeds by biofilm formation. However relatively few studies have been focused on the factors affecting biofilm formation ability of Xanthomonas axonopodis pv. vignicola (Xav) as a mechanism of transmission. Knowing the factors that enabling plant-pathogenic bacteria to form biofilm as a means to move and establish on/in the hosts provides the necessary basis to set up appropriate management approach. The study was conducted to determine factors affecting biofilm formation as mechanisms of seed transmission of Xanthomonas axonopodis pv. vignicola by growing bacterial cells in maize, millet, sorghum, Ife brown, Sampea7 extracts and extracts of Ife brown + 0.5 g nutrient glucose agar (NGA) in a 96 microlitre wells. The seeds extract were prepared by soaking one hundred seeds each of Ife brown, Sampea7, millet, sorghum and maize in a 250 ml flask containing 100 ml SDW and for 20 h. Bacterial suspension adjusted to ca. 4.5 x 107 cfu/ml was suspended in each of the extracts and the media and filled twelve wells each of the treatment. These were incubated for 72 h, 96 h and 120 h. afterwards, the wells were emptied and the wells were stained with 1 % crystal violet (CV) solution in 33 % (V/V) acetic acid for approximately 20 minutes. Excess CV was washed with SDW. The bound CV to the wells were solubilized with 200 µl of 33 % acetic acid or acetone – ethanol and quantified spectrophotometrically using Well Reader (GF 3000 microplate Reader –Bran scientific and Instrument Company England). Specific Biofilm formations (SBF) was calculated. The experiment was replicated two times and repeated 3 times. There was statistical difference between the biofilm formation induced by the different extracts and NGA. All the seeds extract induces biofilm formation and the level of biofilm formation varies with time and the nutrient status of the media or medium.

English

Thirty isolates of Xanthomonas campestris pv. vesicatoria (Xcv) from Tanzania were evaluated for sensitivity to copper sulphate (CuSO4:5H20) using dilution plate counts. Ninety-three percent (28/30) of the isolates produced countable colonies on nutrient glucose agar amended with 0.8 mM CuSO4. All the Xcv isolates (7/7) from Arusha (northern Tanzania) grew on the copper medium. Isolates of the pathogen from Iringa (southern Tanzania) produced variable results (15/21). However, neither of the two Xcv isolates from Morogoro (Eastern Tanzania) grew on the copper medium. These results indicate long-term exposure of Xcv isolates from Tanzania to selection pressure for copper tolerance. Key words: Xanthomonas campestris pv. vesicatoria, copper resistance, Tanzania.

Isolation, screening and partial purification of antimicrobial antibiotics from soil Streptomyces sp. SCA 7

Thirty-seven actinomycetes strains were isolated from soil samples collected from an agriculture field in Vengodu, Thiruvannamalai District, Tamil Nadu, India (latitude: 12° 54′ 0033″, North; longitude: 79° 78′ 5216″, East; elevation: 228.6/70.0 ft/m). The isolates were assessed for antagonistic activity against five Gram-positive bacteria, seven Gram-negative bacteria, and two pathogenic fungi. During the initial screening, 43% of the strains showed weak activity, 16% showed moderate activity, 5% showed good activity, and 35% showed no antagonistic activity. Among the strains tested, SCA 7 showed strong antimicrobial activity. Maximum biological activity was obtained on modified nutrient glucose agar (MNGA) medium. The mycelia of SCA 7 were extracted with methanol and tested against microbial pathogens using the disc diffusion method. The crude extract was purified partially using column chromatography and assessed for antimicrobial activity. Fraction 10 showed good activity against Staphylococcus epidermidis (31.25 μg/mL) and Malassezia pachydermatis (500 μg/mL) and the active principle (fraction 10) was identified as 2,4-bis (1,1-dimethylethyl) phenol. Based on morphological, physiological, biochemical, cultural, and molecular characteristics (16S rDNA sequencing), this strain was identified as Streptomyces sp. SCA 7. It could be used in the development of new substances for pharmaceutical or agricultural purposes.

Bahasa Indonesia

Pantoea stewartii subsp. stewartii is the pathogen causing stewart’s wilt disease in maize. The occurrence of the disease was recently reported in Indonesia with limited information. Research was conducted to characterize several isolates of P. stewartii subsp. stewartii isolated from maize plants collected from 6 districts in West Sumatra. Leaves showing yellow stripes symptoms along the vein was ground and suspeneded then the bacteria was isolated and incubated for 3 × 24 h at room temperature in nutrient glucose agar (NGA). Pure bacterial colonies gained from this medium was used for characterization. Assessment of bacterial characters was conducted based on morphological assay, physiological assay, biological assay (hypersensitivity, and pathogenicity) followed by polymerase chain reaction (PCR)-based identification. Unique characters of the family Enterobacteriaceae was observed on 7 isolates based on morphological and physiological characters, and only 4 isolates (PR1, PP, ST1, LA) gave positive reaction on hypersensitivity and pathogenicity test. Further molecular identification confirmed 3 out of 7 isolates had high homology to Pantoea stewartii, i.e. Pantoea stewartii strain R1-104, R1-132, and ATCC 29923 for isolates LA, PR1, and PR2, respectively.

OPTIMASI WAKTU PRODUKSI ANTIBIOTIKA ISOLAT MIKROORGANISME DARI AIR LAUT BAJOE KABUPATEN BONE DENGAN VARIASI SUMBER NITROGEN

Optimization research has been done time on the production of antibiotics from bacterial isolate from Bajoe Distric Bone waters with nitrogen sources of variation, the first stage of the selection of isolates that have the greatest obstacle zone and which inhibits microbial isolates most of the isolates tested ERB 3 which inhibited Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, Vibrio sp, Candida albicans and Aspergillus niger . Determination of the growth curve of the isolates with inoculate into a nutrient broth medium and incubated for 2 times 24 hours. Each interval of 2 hour was measured transmittance on spectrophotometer to determine the stationary phase. Results from growth curve obtained from the stationary phase at the 20 th until the 48 th hour. Determination of optimum fermentation conditions by varying the nitrogen source is peptone and tryptone. ERB 3 isolate was inoculated into the production medium and fermented using the medium of production in which every interval of 4 hours is making fermented products ranging from hour to hour-20 th to48 th . Activity was tested using agar diffusion method in the medium Glucose Nutrient Agar (GNA) against Staphylococcus aureus, Streptococcus mutans and Salmonella typhi . The results showed that the optimum time to produce antibiotics is the 24, 40 and 48hour and nitrogen source tryptone better than peptone. Keywords : Optimasi, Isolat, Antibiotika

Isolation and Identification of Bacteria Causing Tomato Leaf Spot in Yemen with Susceptibility of Certain Cultivars

This study was conducted to isolate and identify the causal agent of bacterial leaf spot disease on tomato in district fields in Kla’abah, Taiz, Yemen. Twenty isolates were obtained from tometoes leaves and fruit of infected plants. Yellow, raised and shiny colonies were developed on four media ((1% Glucose nutrient agar (GNA), Nutrient dextrose agar (NDA), Sucrose peptone agar (SPA) and Yeast peptone glucose agar (YPGA)). Pathogenicity test results showed three isolates were highly virulent while three other isolates were moderately virulent. The pathovar Xanthomonas campestris pv. vesicatoria was identified according to visible characteristics of the colonies by (GNA, NDA, SPA and YPGA) media, cell shape, and physiological and biochemical characteristics (ability to grow at 35o C, production of H2S and ability to assimilate certain carbohydrates). Results revealed the ability of Xanthomonas campestris pv. vesicatoria to infect nine tomato cultivars (Roma V.f., Riogrand Gala, Tomato Maryam, Gala, Riogrand, Local-G 1oo3, on gy.A Seed, Ti 2 ar and RioGrand-Tezier) causing leaf spot at 26 – 29o C. Results also showed on Ti 2 ar, Local-G 1oo3 and Roma V.f. cultivars a decreased level of bacteria multiplication while Riogrand Gala, on gy. A Seed and Riogrand cultivars showed increased bacteria population.

First Description of Crown Gall Disease on Ginseng

In March of 2003, tumors (galls) were observed on ginseng seedling roots in ginseng seedbeds at Yeoju, Gyeonggi province, Korea. Symptoms were spherical or galls with about 0.5-1.0 cm in diameter formed on the upper through middle parts of the primary roots. Bacterial isolates obtained from the root galls were Gram-negative, rod-shaped with peritrichous flagella, aerobic, not forming yellow or orange colonies on nutrient glucose agar, yeast extract-dextrose CaCO3 agar and nutrient-broth yeast extract agar, nonfluorescent on King`s B agar, and non-spore forming, which were identical to characteristics of the genus Agrobacterium. They were identified as Agrobacterium tumefaciens with 0.732-0.993 similarities in 100% probability by the Biolog analyses. The 16S rRNA gene partial sequences of the six isolates tested (Genbank Accession EF486308~EF486313) were 100% homologous to those of other A. tumefaciens strains (GenBank accession AF501343, AY701900, AY701898, AY701899). The above results confirmed that this bacterium is A. tumefaciens. Pathogenicity of the bacteria was proved by the inoculation test on carrot root discs and tomato seedlings. This is the first description of A. tumefaciens causing root gall in ginseng seedling. The disease occurred locally and sparsely, but considering its appearances in seedbeds suggests that the ginseng root gall may become a threat to ginseng in Korea.

First Record of Clavibacter michiganensis subsp. michiganensis Causing Canker of Tomato Plants in Syria.

Between March and mid April of 2007, several extensive surveys for Clavibacter michiganensis subsp. michiganensis were carried out among greenhouses in the coastal strip provinces of the Mediterranean Sea in north-west Syria (Latakia and Tartous), where a large proportion of Syrian fresh-market tomatoes are produced. This bacterium causes bacterial canker of tomato and is considered an A2 quarantine pathogen by the European Plant Protection Organization (EPPO). It is currently present in all major tomato-production areas in the EPPO region (4), but has not been previously reported in Syria. The survey revealed typical canker symptoms in 7% of 150 inspected greenhouses that contained cvs. Dima, Huda, and Astona. These symptoms included stunting, dark brown-to-black lesions on the leaf margins, wilting and defoliation of whole plants, and vascular discoloration. The disease incidence in such greenhouses was estimated at 15% at the time of the survey. Diseased plants were surface sterilized and homogenized in sterile water. Serial dilutions were plated on nutrient glucose agar. Suspected colonies were further purified by repeated restreaking on new agar plates. All 10 of the suspected strains obtained from different locations were identified as C. michiganensis subsp. michiganensis on the basis of the following observations: bacterial cells of all strains had a coryneform shape, were nonmotile, gram positive according to Gram's reaction test with 3% KOH (2), oxidase-negative, and caused hypersensitive reactions on leaves of Mirabilis jalaba (1) within 24 h. PCR assays were conducted with the C. michiganensis subsp. michiganensis-specific primer set PSA-4/R (3) and template DNA prepared from in-vitro-grown bacteria with the MasterPure Gram Positive DNA Purification Kit (Epicentre Biotechnologies, Madison, WI). The expected 270-bp amplicon was observed for both reference strains as well as the Syrian strains. Pathogenicity of the strains was confirmed by artificial inoculation of 6-week-old tomato plants (Lycopersicon esculentum Mill. cv. Lyconorma). Inoculation was performed by stabbing the stem with a sterile needle through a drop (~35 μl) of bacterial suspension (~108 CFU/ml in 0.01 M MgSO4) placed in the axil of the second or third true leaf. Three tomato seedlings were inoculated with each strain. Control plants were inoculated with sterile 0.01 M MgSO4. Symptoms including lateral wilt of leaflets, stem lesions, and wilting of whole plants were observed within 10 to 15 days after inoculation, except for the negative control. To fulfill Koch's postulates, reisolation and reidentification of the pathogen was conducted as previously described. To our knowledge, this is the first record of the occurrence of bacterial canker of tomato in Syria.

Comparison of two culture media for determination of the copper resistance of Xanthomonas strains and their usefulness for prediction of control with copper bactericides

Two bacteriological culture media were compared for determination of copper resistance among strains of Xanthomonas euvesicatoria and Xanthomonas perforans from pepper and tomato and Xanthomonas campestris pv. vitians from lettuce. Of 94 strains tested, 73 grew on glucose-nutrient agar (GNA) amended with 200 μg ml −1 of copper sulfate. None of the 94 strains grew on modified casitone-yeast extract (CYE) agar amended with copper. In vitro population studies supported the preliminary classification of strains as copper resistant. Reduction in populations of strains found to be resistant on GNA+Cu were less than one log unit after 2-h exposure to copper hydroxide suspensions in vitro. Addition of mancozeb to the copper hydroxide resulted in population reductions of 4–8 log units. Therefore, GNA+Cu is more appropriate for screening of Florida Xanthomonas strains for resistance to copper. Copper hydroxide alone, mancozeb alone, or a combination of these two compounds reduced bacterial spot of tomato and pepper significantly under low disease pressure. Reductions in disease ratings were as much as ten-fold in some experiments. Only the combination of copper and mancozeb was effective at higher disease levels.

Aktivitas antibakteri flavonoid propolis Trigona sp terhadap bakteri Streptococcus mutans (in vitro) (In vitro antibacterial activity of flavonoids Trigona sp propolis against Streptococcus mutans)

A number of investigations have shown a positive correlation between the number of Streptococcus mutans (S. mutans) in dental plaque and the prevalence of dental caries. Consequently, this microorganism has been the prime target for the prevention of dental caries. Propolis being a substance made by the honeybee, is a potent antibacterial agent. The main chemical class present in propolis is flavonoids. Flavonoids are well-known plant compounds that have antibacterial property. Because S. mutans is accepted to be one of the microorganisms responsible for dental caries and flavonoids in propolis are antibacterial, the purpose of this study was to evaluate in vitro the antibacterial activity of flavonoids Trigona sp propolis against S. mutans as a first step in its possible use as an alternative anticaries agent. Extract flavonoids was purified from ethanol extract of propolis which was obtained from Bulukumba Regency South Sulawesi using thin layer chromatography. The purification of flavonoids was carried-out by UVradiation at λ max 254 nm and λ max 366 nm and treatment with ammonia. Extract flavonoids was diluted in aquadest to 0.05%; 0.075%; 0.1%; 0.25%; 0.5%; 0.75% concentrations. Aquadest and 10% Povidone iodine were also used as control solution. S mutans were grown in medium glucose nutrient agar and incubated with flavonoids for 24 and 48 hours, at 37° C. Antibacterial activity was reflected by the diameter of the inhibition zones around the stainless steel cylinder. The data were analyzed by using ANOVA followed by LSD test with significance level of 5%. The results of this study showed that after being incubated for 24 and 48 hours, all flavonoid concentrations significantly (p < 0.05) inhibited the growth of S mutans. 0.1% flavonoid was the most effective concentration to inhibit the growth of S mutans after 24 hours of incubation and 0.5% flavonoid after 48 hours of incubator.

Clavibacter michiganensis subsp. michiganensis

<i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i>