scholarly journals First Report of Leaf Blight Caused by Pantoea agglomerans on Rice in Korea

Plant Disease ◽  
2010 ◽  
Vol 94 (11) ◽  
pp. 1372-1372 ◽  
Author(s):  
H. B. Lee ◽  
J. P. Hong ◽  
S. B. Kim
Keyword(s):  
16S Rrna ◽  
Natural Infection ◽  
Leaf Blight ◽  
Pantoea Agglomerans ◽  
Tween 20 ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Isolation And Identification ◽  
First Report ◽  
Stalk Rot

In September 2009, leaf blights were observed on rice (Oryza sativa L., variety Dongjin 1 and Hopyeong) in paddy fields located in Gwangyang and Naju, Jeonnam Province, Korea. Lesions appeared first as water-soaked stripes or light brown-to-slightly reddish spots on the upper blades of the leaves, ultimately causing leaf blight and stalk rot. Ten strains of bacteria were isolated from the blighted leaf samples and four isolates (EML-ORY1, -ORY2, -ORY3, and -ORY4) suspected to be Pantoea spp. were selected on the basis of colony types and sampling sites. The isolates readily grew at 27 to 32°C but growth was significantly lower at 35°C. Using the API 20E system, EML-ORY1, 2, and 3 showed the same reaction patterns and gave 15 positive reactions whereas EML-ORY4 gave 11 positive reactions, but results were negative for arginine dihydrolase, citrate utilization, sorbitol fermentation, and rhamnose fermentation. All strains were considerably different from Pantoea agglomerans ATCC27155, which produced nine positive reactions. The strains were identified based on the 16S rRNA gene sequence analyses. A neighbor-joining tree was generated for the four isolates using PHYLIP with the following known bacterial strains: P. agglomerans DSM3493; P. vagans LMG24199; P. eucalypti LMG24197; P. ananatis ATCC19321; and Kluyvera georgiana ATCC51603. The four isolates from rice formed a monophyletic cluster and were most closely related to P. agglomerans DSM3493 (GenBank AJ2334231) with an average 16S rRNA sequence similarity of 99.0%. GenBank Accession numbers for the four isolates are: EML-ORY1, HM854282; -ORY2, HM854283; -ORY3, HM854284; and -ORY4, HM854285. On the basis of molecular phylogenetic analyses and API 20E test, we determined that the causal pathogen might be a subspecies of P. agglomerans. Pathogenicity tests were performed on 2-week-old rice seedlings (variety Hopyeong) in duplicate with bacterial suspensions containing 1.5 × 109 CFU/ml with 0.001% Tween 20. Of the isolates, EML-ORY3 demonstrated the strongest pathogenicity to rice seedlings when evaluated by five scoring systems on the basis of symptom development and severity levels. Disease symptoms appeared 3 days after artificial inoculation. Symptoms on the inoculated leaves were similar to those of natural infection and included water-soaked stems with a light brown color, blighted leaves, and stalk rot, with no symptoms found on water-treated controls. P. agglomerans, formerly called Enterobacter agglomerans (or Erwinia herbicola), is a group of gram-negative bacteria that belong to the family Enterobacteriaceae (3). Pantoea spp. are known to cause different diseases on a broad range of host plants including gypsophila, cotton, pineapple, maize, barley, onion, melons, and eucalyptus and also have been implicated as opportunistic pathogens in humans (1,2). P. agglomerans has been widely found in nature on leaves, fruits, and the seeds of many crops and is a known endophyte (1,2). To our knowledge, this is the first report of rice leaf blight caused by a putative subspecies of P. agglomerans in Korea. The importance of this pathogen to rice production in Korea is unknown. References: (1) Y. Feng et al. J. Appl. Microbiol. 100:938, 2006. (2) S. Manulis and I. Barash. Mol. Plant Pathol. 4:307, 2003. (3) M. P. Starr. The genus Erwinia. Page 1260 in: The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria. Springer-Verlag, NewYork, 1981.

scholarly journals First Report of Pantoea agglomerans Causing Leaf Blight and Vascular Wilt in Maize and Sorghum in Mexico

Plant Disease ◽  
2007 ◽  
Vol 91 (10) ◽  
pp. 1365-1365 ◽  
Author(s):  
G. Morales-Valenzuela ◽  
H. V. Silva-Rojas ◽  
D. Ochoa-Martínez ◽  
E. Valadez-Moctezuma ◽  
B. Alarcón-Zúñiga ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Analysis ◽  
Leaf Blight ◽  
Pantoea Agglomerans ◽  
Rrna Gene ◽  
Vascular Wilt ◽  
First Report ◽  
Central Highland ◽  
The 16S Rrna Gene

Zea mays and Sorghum bicolor are important crops for animal and human nutrition worldwide. In the Central Highland Valley of Mexico, both crops are extremely important, and research is aimed toward increasing yield, disease resistance, and crop adaptation from 1,900- to 2,700-m elevation. In a 3-year field breeding experiment (2004 to 2006), leaf blight and vascular wilt symptoms were frequently observed in contiguous plots of maize and sorghum crops in Montecillo, Mexico and maize plots in Tecamac, Mexico. To identify and characterize the causal agent of these symptoms, isolations were conducted on leaves from areas where healthy and diseased tissues converged. Leaf sections of 1 cm2 from both crops were disinfested, placed on casamino acid-peptone-glucose (CPG) medium, and incubated at 28°C. After 48 h, only yellow colonies were observed and 12 isolates were selected for further characterization. Physiological and biochemical tests indicated that the isolates were nonfluorescent on King's B medium, and API 50 CHE (bioMérieux, Marcy l'Etoile, France) revealed that they were negative for gelatin hydrolysis, indole production, acid production from raffinose and positive for utilization of glycerol, D-glucose, mannitol, arbutine, esculine, salicine, cellobiose, maltose, melibiose, D-fucose, and D-arabitol; all characteristics of Pantoea agglomerans. Further identification of these isolates was accomplished by DNA analysis. For DNA analysis, 1.4-kbp fragments of the 16S rRNA gene were amplified with primer set 8F/1492R (3) and sequenced with U514F/800R universal primers (2). Five sequences were obtained and deposited in GenBank (Accession Nos. EF050806 to EF050810). A phylogenetic tree was constructed using the UPGMA method (mega version 3.1). Results of the phylogenetic analysis grouped the species P. ananatis, P. stewartti, and P. agglomerans into three clusters. The five unknown sequences were grouped into the P. agglomerans cluster. There was a 98 to 99% similarity of the five 16S rRNA gene sequences with P. agglomerans strain type ATCC 27155. Pathogenicity of the 12 isolates was confirmed by injecting 108 CFU mL–1 of inoculum into stems of 3-week-old maize cv. Triunfo and sorghum cold tolerant hybrid (A1×B5)×R1 seedlings in the greenhouse at 28°C and 80% relative humidity. Also, seedlings were inoculated with water, nonpathogenic isolates of P. agglomerans from maize (GM13, and HLA1), and not inoculated as negative controls. Three replications were included for each isolate and control. All test strains developed water-soaked lesions on juvenile leaves at 8 days postinoculation and were followed by chlorotic to straw-colored leaf streaks and then leaf blight symptoms at 3 weeks postinoculation. All negative control seedlings did not develop symptoms. In addition, the 12 isolates were infiltrated at 107 CFU mL–1 into tobacco leaves that displayed a hypersensitive response at 4 days, indicating the presence of the type III secretion system (1). Isolates were reisolated, and the 16S rRNA gene fragments were 100% similar to their original isolate sequences. P. agglomerans has been reported to affect other crops, including chinese taro in Brazil (2007), onion in the United States (2006) and South Africa (1981), and pearl millet in Zimbabwe (1997); however, to our knowledge, this is the first report of P. agglomerans associated with leaf blight and vascular wilt symptoms in maize and sorghum in the Central Highland Valley of Mexico. References: (1) J. Alfano and A. Collmer. Annu. Rev. Phytopathol 42:385, 2004. (2) Y. Anzai et al. Int. J. Syst. Evol. Microbiol. 50:1563, 2000. (3) M. Sasoh et al. Appl. Environ. Microbiol. 72:1825, 2006.

scholarly journals The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia

Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1152-1152 ◽  
Author(s):  
S. Pavlovic ◽  
M. Starovic ◽  
S. Stojanovic ◽  
G. Aleksic ◽  
S. Kojic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Natural Infection ◽  
Cytotoxic Activities ◽  
Rrna Gene ◽  
Calendula Officinalis ◽  
Aster Yellows ◽  
First Report ◽  
Pot Marigold ◽  
Stolbur Phytoplasma

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51′49″ N, 20°39′33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII ‘Candidatus Phytoplasma solani’ group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia. References: (1) S. A. Esmailzadeh-Hosseini et al. Bull. Insectol. 64:S109, 2011. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Prince. Phytopathology 83:1130, 1993. (4) M. Ukiya et al. J. Nat. Prod. 69:1692, 2006.

scholarly journals First Report of Bacterial Stalk Rot of Maize Caused by Dickeya zeae in Mexico

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1267-1267 ◽  
Author(s):  
B. A. Martinez-Cisneros ◽  
G. Juarez-Lopez ◽  
N. Valencia-Torres ◽  
E. Duran-Peralta ◽  
M. Mezzalama
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pathogenic Bacteria ◽  
The State ◽  
Rrna Gene ◽  
Bacterial Disease ◽  
Sterile Water ◽  
First Report ◽  
Stalk Rot ◽  
Positive Controls

A bacterial disease of maize, bacterial stalk and top rot, was found in the state of Morelos in February 2011, and in the state of Puebla in July 2013, Mexico. In both cases, the incidence of diseased plants was lower than 0.5%. The typical symptoms were a soft rot and darkening of the tissues affecting the stalk and the top of the plant, causing breaking of the stalk. The lesions progressed from the top to below nodes, leaf sheaths and blades, and rotten tissues emitted an unpleasant odor. Eleven diseased plants were collected, and bacterial colonies were isolated from fragments detached from the edges of symptomatic tissues after sterilization with a 0.5% solution of NaClO for 30 s, rinsing three times in sterile water. The sterilized fragments were macerated in drops of distilled sterile water for 10 min and the extract was streaked on King's medium B (agar 15 g, distilled water 1,000 ml, proteose peptone 20 g, K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, glycerol 10 ml). Eight representative strains from Morelos and five from Puebla were selected for identification. All strains were gram-negative, grew at 37°C, showed pectynolitic activity on potato tubers, were positive for indole production, utilized arabinose, galactose, glucose, glycerol, lactose, mannose, melibiose, rafinose, ribose, and sucrose but did not produce acid from arabitol, adonitol, and keto-methyl-glucoside (3,4). Pathogenicity tests were conducted with each strain by inoculating with a syringe four 25-day-old maize seedlings with 107 CFU ml–1 bacterial cells in the leaf collar. Plants were incubated in the greenhouse at 30°C during the day and 24°C during the night with a 12-h photoperiod, and relative humidity of 93%. The reference strains Erwinia chrysanthemi pv zeae ATTC29942 and Dickeya zeae CFBP 2052 were used as positive controls in laboratory and greenhouses tests. Sterile water was used as negative control. Two days after inoculation, soft stalk rot symptoms developed that were identical to those observed in the field. No symptoms were observed on the negative controls. Diagnostic amplification of DNA by conventional PCR was carried out and yielded the expected amplicon size of 420 bp of the Dickeya-specific pel gene with the ADE primers set (2). PCR was used to amplify the 16S rRNA gene with the universal primers 27f and 1495r (5) for molecular identification of the 13 strains (GenBank Accession Nos. KJ438941, KJ438942, KJ438943, KJ438944, KJ438945, KJ438946, KJ438947, KJ438948, KJ438949, KJ438950, KJ438951, KJ438952, and KJ438953). The strains D. zeae CFBP 2052 and E. chrysanthemi pv. zeae ATCC 29942 were sequenced as positive controls. A BLAST search with the 13 16S rRNA gene sequences of 1.4 kb were 99% identical to the sequence of D. zeae CFBP 2052 (NR_041923). D. zeae can be a major disease of maize in tropical and subtropical countries. It is particularly severe under conditions of high temperature and high humidity, but it occurs sporadically. Control of the vector, Chilo partellus, can aid disease management (1). To our knowledge, this is the first report of D. zeae causing maize stalk rot in Mexico. References: (1) CABI. Crop Prot. Compend. CAB International, Wallingford, UK, 2014. (2) A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996. (3) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. APS Press, St. Paul, MN, 2001. (5) W. G. Weisburg. J. Bacteriol. 173:697, 1991.

scholarly journals First Report of Pectobacterium carotovorum subsp. carotovorum on Spathiphyllum wallisii in Argentina

Plant Disease ◽  
2009 ◽  
Vol 93 (8) ◽  
pp. 842-842 ◽  
Author(s):  
A. M. Alippi ◽  
A. C. López
Keyword(s):  
16S Rrna ◽  
Natural Infection ◽  
Soft Rot ◽  
Unknown Etiology ◽  
Rrna Gene ◽  
Pectobacterium Carotovorum ◽  
Distilled Water ◽  
Strain Atcc ◽  
First Report ◽  
Carotovorum Subsp

Peace lily (Spathiphyllum wallisii Regel) is a popular ornamental potted plant in Argentina. During May of 2008 (austral autumn), necrotic lesions of unknown etiology were observed on S. wallisii in a nursery in Pontevedra (34°45′6″S, 58°42′42″W). Plants first showed water-soaked areas starting from the leaf tips. Infected tissue became irregular, brown, dark-to-black lesions on leaves ~12 to 14 mm in diameter surrounded by yellowish haloes. Disease incidence approached 30%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria isolated from lesions formed white-to-cream, glistening, convex colonies on yeast dextrose calcium carbonate agar. Three bacterial strains isolated from different symptomatic plants were selected for comparative analysis with Pectobacterium carotovorum subsp. carotovorum type strain ATCC 15713. All were facultatively, anaerobic, gram-negative rods, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All strains were oxidase and arginine dihydrolase negative, fermented glucose, did not hydrolyze starch, did not produce lecithinase, indole or the blue pigment indigoidine, reduced nitrates, hydrolyzed gelatin and esculin, able to rot onion slices, caused soft rot of potato tubers, resistant to erythromycin, and grew at 37°C. Acid was produced from cellobiose, d-glucose, d-melibiose, d-mannitol, d-mannose, l-rhamnose, d-sucrose, and l-arabinose but not from inositol and d-sorbitol. Bacteria utilized N-acetyl-glucosamine and citrate but not tartrate, benzoate, or propionate. Their identity was confirmed by 16S rRNA gene sequencing of strain F402Pcc (GenBank Accession No. FJ717337) showing a 99% homology with that of strain ATCC 3326 (FJ 5958691). Pathogenicity was verified on S. wallisii, Dieffenbachia picta, Aglaonema commutatum, and Anthurium andraeanum within the Araceae family by spraying two plants per strain tested with bacterial suspensions (108 CFU/ml) in sterile distilled water with and without wounding the leaves with sterile needles. Controls were sprayed with sterile distilled water. After 48 h in a humidity chamber, inoculated plants and controls were maintained at 25 ± 3°C in a greenhouse. Water-soaked areas developed from 24 to 48 h after inoculation and became necrotic within 4 to 5 days. Lesions expanded to resemble natural infection in S. wallisii within 20 days, while in the rest of the hosts tested, lesions were smaller and remained brown surrounded by yellowish haloes. All strains were reisolated from each host tested. The original and all reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (4) confirming that DNA fingerprints of the reisolated strains were identical to those of the original strains. No lesions were observed on controls. The pathogen was identified as P. carotovorum subsp. carotovorum based on biochemical, physiological, pathogenicity tests, and 16S rRNA sequencing (1–3).To our knowledge, this is the first report of this pathogen on S. wallisii in Argentina although it has been reported as causing tomato pith necrosis (1) and soft rot of vegetables after harvest (3). References: (1) A. M. Alippi et al. Plant Dis. 81:230, 1997. (2) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:381, 2003. (3) L. Halperin and L. S. Spaini. Rev. Argent. Agron. 6:261, 1939. (4) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994.

scholarly journals First Report of a Natural Infection of Stevia rebaudiana by a Group 16SrXXIV Phytoplasma in India

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1582-1582 ◽  
Author(s):  
A. Samad ◽  
S. Dharni ◽  
M. Singh ◽  
S. Yadav ◽  
A. Khan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Natural Infection ◽  
Stevia Rebaudiana ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
First Report ◽  
Wide Range

Stevia rebaudiana Bertoni (Asteraceae) is one of the most important commercial crops in the world (4). It is known to produce glycosides that are as much as 300 times sweeter than sucrose and do not affect blood sugar levels. Unlike artificial sweeteners like saccharin, they are noncarcinogenic and safe for diabetics. An unknown disease emerged during the summers of 2007 to 2009 in a field of S. rebaudiana at CIMAP Lucknow, India, where more than 20% of the plants exhibited symptoms typical of phytoplasma infection including leaf yellowing, reduced size of leaves, shoot proliferation, flower bud deficiency, as well as bushy and stunted growth. Some of these plants were potted and kept in a glasshouse for investigation. Affected plants in the field expressed a quick decline consisting of growth cessation, bronzing of mature leaves, wilting, and death, resulting in a significant reduction in biomass and quality. Typical phytoplasma-like (pleomorphic) bodies ranging from 450 to 900 nm were observed in the phloem cells of infected plants by transmission electron microscopy (1). These bodies were always found in diseased plants, but not in asymptomatic ones. No other microorganisms were noted. Total DNA was extracted from symptomatic as well as asymptomatic plants by a CTAB method. PCR was carried out with the universal phytoplasma primers P1/P6 (P1, 5′-AAGAGTTTGATCCTGGCTCAGGATT-3′; P6, 5′-CGGTAGGGATACCTTGTTACGACTTA-3′) (2) followed by nested primers R16F2n/R16R2 (R16F2n, 5′-GAAACGACTGCTAAGACTGG-3′; R16R2, 5′-TGACGGGCGGTGTGTACAAACCCCG-3′) targeting the 16S rRNA gene sequence (3). The P1/P6 and R16F2n/R16R2 primers produced the expected 1.5- and 1.2-kb amplicons, respectively, from the symptomatic plants and not from the asymptomatic ones. Seventeen symptomatic and eight asymptomatic samples were analyzed through PCR. Nested PCR products were ligated into the plasmid vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Transformation and selection of recombinant clones was carried out according to the manufacturer's recommended protocol. The sequence obtained from the final PCR product was deposited in the GenBank database (No. JF970603). It was analyzed through the iPhyClassifier ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) online tool and found to share 98.2% similarity with that of the ‘Sorghum bunchy shoot phytoplasma’ reference strain (GenBank No. AF509322) that belongs to 16SrXXIV-A subgroup. The virtual restriction fragment length polymorphism pattern of the S. rebaudiana phytoplasma 16S rRNA gene sequence showed maximum similarity to the reference pattern of AF509322 (similarity coefficient of 0.85). Although a number of phytoplasmas have been detected on a wide range of plants in India, little is known about the leafhopper that presumably transmits them to S. rebaudiana and other medicinal crops. Infections by diverse phytoplasma strains/species underscore the need for phytoplasma-free planting stock and intensification of research efforts to reduce ecological and economic impacts of these phytoplasmas. To our knowledge, this is the first report of a natural infection of S. rebaudiana by a group of 16SrXXIV-A phytoplasma. References: (1) P. V. Ajayakumar et al. Aust. Plant Dis. Notes 2:67, 2007. (2) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (3) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (4) S. M. Savita et al. J. Hum. Ecol. 15:261, 2004.

scholarly journals First Report of Pantoea agglomerans Causing Brown Apical Necrosis of Walnut in China

Plant Disease ◽  
2011 ◽  
Vol 95 (6) ◽  
pp. 773-773 ◽  
Author(s):  
K. Q. Yang ◽  
W. W. Qu ◽  
X. Liu ◽  
H. X. Liu ◽  
L. Q. Hou
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Juglans Regia ◽  
Shandong Province ◽  
Pantoea Agglomerans ◽  
The Body ◽  
Rrna Gene ◽  
First Report ◽  
Apical Necrosis ◽  
The 16S Rrna Gene

Brown apical necrosis (BAN) of walnut (Juglans regia L.) causes premature fruit drop and yield losses and has been reported to be an important walnut production problem in Spain, Italy, France, and Turkey (1,2). A number of organisms have been associated with BAN on walnut: Xanthomonas arboricola pv. juglandis, Fusarium spp., and Alternaria spp. (3). Since the spring of 2007, BAN was observed in 50 to 60% of the trees in walnut orchards in Taian City and Laiwu City, Shandong Province, China. Surface-disinfested tissue from premature walnut fruits was placed onto potato dextrose agar. Alternaria spp., X. arboricola pv. juglandis, and Pantoea agglomerans (formerly Enterobacter agglomerans) were isolated 76, 35, and 45% of the time, respectively. The P. agglomerans cultures formed a yellow lawn and were rod shaped with the body length of 1.5 to 3.0 μm, width of 0.5 to 1.0 μm, and four to six flagella. In biochemical tests, these bacteria were gram negative, lactose positive, and indole negative. Genomic DNA was extracted from one HXJ isolate and the 16S rRNA gene sequence (GenBank Accession No. HM016799) was obtained using universal primers 27F and 1492R. HM016799 had 99% sequence identity with P. agglomerans accessions in GenBank (GU477762, GQ494018, FJ756355, and AB004757). To confirm pathogenicity, HXJ isolate (108 CFU·ml–1) was inoculated at the bottom of the stigma within 5 days after florescence (DAF) and in premature fruit wounded with a needle within 30 DAF in 2008 to 2010. Stigmas injected with only sterile water served as controls. The bacteria were inoculated into three replicate 9-year-old plants of the walnut cv. Xiangling. Forty nuts on each plant were inoculated. The plants were grown in Shandong Province, China (36°09′59″N, 117°13′30″E). Ten days after inoculation, typical internal BAN symptoms were observed on all treated nuts and the controls were still healthy. In the inoculated stigmas, necrosis of stigma and style spread to internal tissues and reached the kernel. In treated premature fruit, internal tissues became necrotic and blackish and eventually led to nut drop. The same bacterium was reisolated from the inoculated tissue. On the basis of morphological, physiological, and biochemical characteristics and sequencing of the 16S rRNA gene, the bacterium was identified as P. agglomerans. To our knowledge, this is the first report of P. agglomerans causing internal type BAN of walnut in China or worldwide. References: (1) A. Belisario et al. Plant Dis. 6:599, 2002. (2) G. Bouvet. Acta Hortic. 705:447, 2005. (3) C. Moragrega and H. Özaktan. J. Plant Pathol. 92:S1.67, 2010.

scholarly journals The growth response of rice (Oryza sativa L. var. FARO 44) in vitro after inoculation with bacterial isolates from a typical ferruginous ultisol

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Musa Saheed Ibrahim ◽  
Beckley Ikhajiagbe
Keyword(s):  
16S Rrna ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Growth Response ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Rrna Gene Sequencing

Abstract Background Rice forms a significant portion of food consumed in most household worldwide. Rice production has been hampered by soil factors such as ferruginousity which has limited phosphorus availability; an important mineral component for the growth and yield of rice. The presence of phosphate-solubilizing bacteria (PSB) in soils has been reported to enhance phosphate availability. In view of this, the present study employed three bacteria species (BCAC2, EMBF2 and BCAF1) that were previously isolated and proved P solubilization capacities as inocula to investigate the growth response of rice germinants in an in vitro setup. The bacteria isolates were first identified using 16S rRNA gene sequencing and then applied as inoculum. The inolula were prepared in three concentrations (10, 7.5 and 5.0 ml) following McFarland standard. Viable rice (var. FARO 44) seeds were sown in petri dishes and then inoculated with the three inocula at the different concentrations. The setup was studied for 28 days. Results 16S rRNA gene sequencing identified the isolates as: isolate BCAC2= Bacillus cereus strain GGBSU-1, isolate BCAF1= Proteus mirabilis strain TL14-1 and isolate EMBF2= Klebsiella variicola strain AUH-KAM-9. Significant improvement in rice germination, morphology, physiology and biomass parameters in the bacteria-inoculated setups was observed compared to the control. Germination percentage after 4 days was 100 % in the inoculated rice germinants compared to 65% in the control (NiS). Similarly, inoculation with the test isolates enhanced water-use efficiency by over 40%. The rice seedlings inoculated with Bacillus cereus strain GGBSU-1 (BiS) showed no signs of chlorosis and necrosis throughout the study period as against those inoculated with Proteus mirabilis strain TL14-1 (PiS) and Klebsiella variicola strain AUH-KAM-9 (KiS). Significant increase in chlorophyll-a, chlorophyll-b and alpha amylase was observed in the rice seedlings inoculated with BiS as against the NiS. Conclusion Inoculating rice seeds with Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 in an in vitro media significantly improved growth parameters of the test plant. Bacillus cereus strain GGBSU-1 showed higher efficiency due to a more improved growth properties observed.

scholarly journals Isolation and Identification of Resistant Bacteria from Petroleum Producing Vicinity by Gene Amplification and Sequencing

2021 ◽  
pp. 1-8
Author(s):  
O. Aleruchi ◽  
O. Obire
Keyword(s):  
16S Rrna ◽  
Phylogenetic Study ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Antibiotic Resistant Bacteria ◽  
Isolation And Identification ◽  
Antibiotic Resistant ◽  
Disinfection Process ◽  
Adaptive Mechanisms

This investigation focuses on molecular identification of antibiotic resistant bacteria isolated from petroleum producing vicinity using 16S rRNA sequencing based technique. The bacterial 16s rRNA gene sequences were amplified using polymerase chain reaction, sequenced,  characterized and compared by using primers which has been compared to national center for biotechnology information (NCBI) sequence database. The presence of the plasmid mediated antibiotic resistance determinants CTX-M and QNRB genes in the bacterial isolates were analyzed. A total of four bacterial isolates that were resistant to all the antibiotic agents used were identified molecularly. The BLAST results showed 100 % similarity and phylogenetic study indicated that the genes were evolutionarily related to Morganella morganii, Pseudomonas xiamenensis, Chryseobacterium cucumeris and Staphylococcus sp., respectively. The genes obtained were submitted to the NCBI gene bank and were assigned accession number; MN094330, MN094331, MN094332 and MN094333, respectively. CTX-M and QNRB genes were however absent in the bacterial isolates. The result identified some peculiar abilities of the bacterial isolates to be resistant to antibiotics and suggests a correlation with resistance and hydrocarbon utilizing bacteria. The level of resistance could be as a result of the disinfection process during wastewater treatment procedure or the same adaptive mechanisms possessed by the isolates to control the hydrocarbon concentration in their cell. The study also clearly indicates that these wastewaters, when discharged into the environment directly may pose a risk for the spread of antibiotic resistant bacteria.

scholarly journals First Detection of Pathogenic Escherichia coli Isolates Associated With Donkey Foals’ Diarrhea in Northern China

2020 ◽  
Author(s):  
Liu Wen-qiang ◽  
Xia Nan ◽  
Zhang Jing-wen ◽  
Wang Ren-hu ◽  
Jiang Gui-miao
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Virulence Factors ◽  
Influence Factor ◽  
Genetic Relationships ◽  
Northern China ◽  
Multi Drug Resistance ◽  
Rrna Gene ◽  
Isolation And Identification ◽  
Antibiotics Sensitivity

ABSTRACTObjectiveThe aim of this study was to identify the biological features, influence factor and Genome-wide properties of pathogenic donkey Escherichia coli (DEC) isolates associated with severe diarrhea in Northern China.MethodsThe isolation and identification of DEC isolates were carried out by the conventional isolation、automatic biochemical analysis system、serotype identification、16S rRNA test、animal challenge and antibiotics sensitivity examination. The main virulence factors were identified by PCR. The complete genomic re-sequence and frame-sequence were analyzed.Results216 strains of DEC were isolated from diarrhea samples, conforming to the bacterial morphology and biochemical characteristics of E.coli. The average size of the pure culture was 329.4 nm×223.5 nm. Agglutination test showed that O78 (117/179, 65.4%) was the dominant serotype and ETEC(130/216, 60.1%) was the dominant pathogenic type. Noticeable pathogenic were observed in 9 of 10 (90%) randomly selected DEC isolates caused the death of test mice (100%, 5/5) within 6h∼48h, 1 of 10 (10%) isolates caused the death of test mice (40%, 2/5) within 72h. Our data confirmed that DEC plays an etiology role in dirarrea/death case of donkey foal. Antibiotics sensitivity test showed significant susceptibility to DEC isolates were concentrated in Nor、EFT、ENR、CIP and AMK,while the isolates with severe antibiotic resistance was AM、TE、APR、FFC、RL and CN. Multi-drug resistance was also observed. A total of 15 virulence gene fragments were determined from DEC(n=30) including OMPA (73%), safD (77%), traTa (73%), STa(67%), EAST1 (67%), astA (63%), kspII (60%), irp2 (73%), iucD (57%), eaeA (57%), VAT (47%), iss (33%), cva (27%), ETT2 (73%) and K88 (60%) respectively. More than 10 virulence genes from 9 of 30(30%) DEC strains were detected, while 6 of 30(20%) DEC strains detected 6 virulence factors. phylogenetic evolutionary tree of 16S rRNA gene from different isolates shows some variability. The original data volume obtained from the genome re-sequencing of DEC La18 was 2.55G and Genome framework sequencing was carried out to demonstrate the predicted functions and evolutionary direction and genetic relationships with other animal E.coli.ConclusionsThese findings provide firstly fundamental data that might be useful in further study of the role of DEC and provide a new understanding of the hazards of traditional colibacillosis due to the appear of new production models.

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