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Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats
The Plant Pathology Journal 2015;31:25-32
Published online March 31, 2015
© 2015 The Korean Society of Plant Pathology.

Ji-Hye An1, Young-Hee Noh1, Yong-Eon Kim1, Hyok-In Lee2, and Jae-Soon Cha1,*

1Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea, 2Animal and Plant Quarantine Agency, Anyang 430-016, Korea
Correspondence to: Phone) +82-43-261-2554, FAX) +82-43-271-4414, E-mail) jscha@cbnu.ac.kr
Received September 24, 2014; Revised February 7, 2015; Accepted February 7, 2015.
This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0) which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.
Abstract

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

Keywords : detection, halo blight, oat, PCR, TaqMan PCR
Materials and Methods

Bacterial strains and cultures

Strains of P. coronafaciens isolated in Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany and New Zealand were obtained from LMG (Belgian Coordinated Collection of Microorganisms, Laboratory of Microbiology, University of Gent, Belgium) and from KACC (Korean Agricultural Culture Collection, Rural Develpoment Administration, the Republic of Korea). For the non-target bacteria, strains related to P. coronafaciens in genus Pseudomonas or the representative strains of common plant pathogenic bacteria in genus Acidovorax, Pectobacterium, Ralstonia, Rhodococcus, Xanthomonas, and Clavibacter were collected from the various culture collections (Table 1). The bacteria were routinely grown in nutrient agar media containing 8 g of nutrient broth and 15 g of agar per liter.

Genome sequencing and PCR primer design

Whole genome shotgun sequencing of P. coronafaciens LMG 5060 was performed using the Roche/454 pyrosequencing method on a genome sequencer with FLX titanium (Margulies et al., 2005). ORF prediction was performed with Glimmer v. 3.02 (Delcher et al., 2007), Prodigal (Hyatt et al., 2010), and GeneMark.hmm-P (Lukashin and Borodovsky, 1998). ORFs from the draft genome sequence of P. coronafaciens LMG 5060 with more than 500 base pairs were BLASTed in the gene bank. To design the primers, candidate ORFs were selected from the ORFs with lower than 80% nucleotide homology to any known genes. The PCR primers were designed with Primer 3 (v. 0.4.0). The specificity of the primers for P. coronafaciens was analyzed by PCR. Nested primers and the nucleotide sequence for the TaqMan probe were designed from amplicon sequence of P. coronafaciens-specific primers. All the primers and the TaqMan probe used in the study were synthesized by Bioneer Co., Ltd (Daejeon, Korea) or NeoProbe Co., Ltd. (Daejeon, Korea). The TaqMan probe was labeled at the 5′ end with FAM and at the 3′ end with TAMRA.

PCR and TaqMan PCR assays

The genomic DNA was extracted from the bacterial cells using the GeneAll ExgeneTM Cell SV kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s protocol. DNA concentrations were determined with a QubitTM fluorometer (Invitrogen®, Carlsbad, CA, USA).

The PCR assays used 1 μl of template DNA in a 25 μl reaction mixture containing 2 mM of Tris-HCl (pH 8.0), 10 mM of KCl, 10 uM of EDTA, 100 uM of DTT, 0.05% Tween 20, 0.05% Nonidet P-40, 5% glycerol, 2.5 mM of dNTP, 5 units of Ex Taq DNA (TaKaRa Bio Inc., Shiga, Japan), and 10 pM of each primer. The reactions were performed in a T Gradient Thermal Cycler (Biometra, Göttingen, Germany) programmed for one cycle of 10 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, with a final extension step for 7 min at 72°C. The amplicons were analyzed by electrophoresis in a 2% agarose gel in TAE buffer.

The TaqMan PCR assay was carried out using 2 μl of DNA template, 8.5 μl of Premix Ex Taq (TaKaRa Bio Inc., Shiga, Japan), 1 μl of TaqMan probe (10 pmoles/μl), 0.5 μl (10 pmoles/μl) of each nested primer, and 12.5 μl of DEPC-DW (Bioneer, Daejeon, Korea). Real-time PCR amplifications were performed in a Smart CyclerTM System (Cepheid, Sunnyvale, CA, USA) with 40 cycles at 95°C for 30 sec and 60°C for 20 sec after initial incubation for 30 sec at 95°C.

Oat seed preparation and extraction

Prior to inoculation, the oat seeds (cultivar Samhan, provided by the National Honam Agricultural Experiment Station in Korea) were surface sterilized by soaking in 70% ethanol for 30 minute and 1.2% sodium hypochlorite solution for 1 hour. The oats seeds were washed 3 times with sterilized water and dried completely in clean bench. Twelve grams of the surface-sterilized oat seeds were artificially inoculated by shaking (120 rpm) in 45 ml of P. coronafaciens LMG 5060 suspension for 24 h. The seeds were dried by leaving them in a clean bench for 20 h. The bacterial pathogen was extracted from the artificially inoculated oat seeds as described previously (Maes et al., 1996). The seeds (12 g) were shaken in 12 ml of cold aqueous saline (0.85% NaCl) containing a drop of Tween 20 for 5 min on a rotator shaker (180 rpm) at room temperature. The extracts were treated at 95°C for 7 min and used in the PCR assay or stored at −20°C.

Results

Primer and PCR specificity

To obtain specific primers of the P. coronafaciens, the draft genome sequence of P. coronafaciens LMG 5060 was constructed by genome shotgun sequencing in this study. The draft genome sequence was deposited at NCBI GenBank (accession number: JSED00000000). The 36 primer sets were designed from the ORFs in total 2028 contigs which showed low homology to that of known genes in GenBank and the primer specificities for P. coronafaciens were checked by PCR assays with target and non-target bacterial strains. Among the primers tested, the Pc-12-F (5′-GATTGCGTCATATGCAACAT-3′) and Pc-12-R (5′-AATAGCAGATCCAGCCAAAG-3′) primer sets amplified a 498 bp in all 13 strains of P. coronafaciens (Fig. 1), whereas target-size DNA was not amplified in non-target bacteria, including 30 P. syringae pathovars, 3 P. savastanoi pathovars, and 16 other pathogenic bacteria (Fig. 2). The nested primers, Pc-12-ne-F (5′-AACGACGGGCTGCAGTTTAT-3′) and Pc-12-ne-R (5′-AACGTGATAGCAGCCCCACT-3′), were designed from the Pc-12-F/Pc-12-R amplicon. The PCR assay was conducted with the Pc-12-ne-F/Pc-12-ne-R nested primer set and genomic DNA amplified the 298 bp in all 13 strains of P. coronafaciens (supplement Fig. 1), whereas target-size DNA was not amplified in any non-target bacteria, including 30 P. syringae pathovars, 3 P. savastanoi pathovars, and 16 other pathogenic bacteria (Supplement Fig. 2).

TaqMan PCR specificity

To reduce the rate of false-positives and quantitatively detect P. coronafaciens, a TaqMan probe, Pc-taqman (5′-TGAAACCGCCGAAACGGTCT-3′), was designed from the sequence of the Pc-12-ne-F/Pc-12-ne-R amplicon. The real-time PCR with Pc-12-ne-F/Pc-12-ne-R and Pc-taqman generated Ct values in a dose-dependent for 10 ng-10 pg of P. coronafaciens LMG 5060’ genomic DNA. The r2 of the linear regression was 0.994 (Fig. 3A).

The specificity of the TaqMan PCR was checked with the target and non-target bacteria. In the TaqMan PCR assays, 10-fold diluted DNAs (10 ng to 10 pg) of P. coronafaciens LMG 5060 were used as standard, and 10 ng of DNAs of the other bacterial strains were used. The Ct values of the 13 P. coronafaciens strains were 22–28 cycles (Fig. 3B and Table 2), whereas the average Ct values of the DNA of 49 non-target bacterial strains were more than 37 cycles (Table 2). The Ct values of the target and non-target bacteria were well separated into two different groups.

TaqMan PCR detection of P. coronafaciens from the artificially inoculated oat seeds

The TaqMan PCR assay developed in this study was applied to detect P. coronafaciens from the oat seeds. Since the seeds naturally infested with the pathogen were not available, the artificially inoculated oat seeds were used. TaqMan PCR with the seed extracts generated a range of mean Ct values from 21.2 for 108 cfu/ml-inoculated seed extracts to 32.2 for 10 cfu/ml-inoculated seed extracts (Table 2). Since Ct values of all the non-target bacteria used in this study were larger than 37 cycles, the TaqMan PCR can detect P. coronafaciens from the seed extract of the artificially inoculated oat seeds above 10 cfu/ml inoculation level.

Estimation of the recovery number of P. coronafaciens from the artificial inoculated seed extract was tried with several different ways, but the accurate enumeration of P. coronafaciens from the seed extract failed because the many non-target-shape colonies were cultured on the culture plate and the selective medium for P. coronafaciens was not available. Surface sterilization of the oat seeds prior to artificial inoculation could not prevent growth of some non-target bacteria on the recovery culture plates.

Discussion

Highly specific PCR and TaqMan PCR assays have been developed in this study to detect P. coronafaciens, the halo blight pathogen of oats. Since P. coronafaciens is a plant quarantine bacterium in many countries and using of the certificated seed is important for the disease control, developments of these assays are significant for the management of halo blight of oats.

To obtain specific primers of the P. coronafaciens, a genome-wide search was conducted from the draft genome sequence of P. coronafaciens which was constructed by genome shotgun sequencing in this study. A specific primer, Pc-12-F/Pc-12-R, was designed from ORF 7 in contig 7 of the draft genome sequence of P. coronafaciens (NCBI GenBank accession number: JSED00000000). ORF 7 was 1341 nucleotides long, and no significant homologous gene was found in the NCBI GenBank database. PCRs with Pc-12-F/Pc-12-R and Pc-12-ne-F/Pc-12-ne-R generated the target size DNA from all 13 strains of P. coronafaciens isolated in seven countries. The target-size DNA was not amplified in 49 strains of non-target bacteria. The Ct values of the target and non-target bacteria were well separated in the TaqMan PCR. The homology of the ORFs in which PCR was designed and the results of the PCR and TaqMan PCR indicate that the primer sequences and PCR assays developed in this study are highly specific to P. coronafaciens.

TaqMan PCR was applied to detect the target pathogen from artificially inoculated oat seeds. TaqMan PCR generated the P. coronafaciens–positive Ct values in the seed extracts obtained from oat seeds inoculated in 10 cfu/ml and above. Although detection sensitivity of this TaqMan PCR cannot compared to the previously published results because detection of P. coronafaciens from oat seeds has not been published, positive detection of the seed extracts obtained from oat seeds inoculated in 10 cfu/ml P. coronafaciens LMG 5060 suspension and above is thought to be a quite high sensitivity and can apply to seed test and plant quarantine service.

Figures
Fig. 1. Gel electrophoresis of the polymerase chain reaction products formed with primer Pc-12-F/Pc-12-R and bacterial DNA of Pseudomonas coronafaciens strains. Lanes 1~13, P. coronafaciens LMG 5060, KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, LMG 2330; lane 14, water as a negative control.
Fig. 2. Gel electrophoresis of the polymerase cain reaction products formed with primer Pc-12-F/Pc-12-R and total DNA of lane 1, Pseudomonas coronafaciens LMG 5060, lanes 2?31, P. syringae pvs, actinidiae KACC 10582, antirrhini ICMP 4303, aptata DSM 50252, atrofaciens ICMP 4394, berberidis NCPPB 2724, ciccaronei NCPPB 2355, delphhinii ICMP 529, dysoxyli ICMP 545, eriobotyae NCPPB 2331, helianthi NCPPB 1229, japonica ICMP 6305, lachrymans ATCC 11965, lapsa ATCC 10859, maculicola ICMP 3935, mellea ICMP 5711, mori ICMP 4331, morsprunorum ICMP 5795, myricae ICMP 7118, panici NCPPB 1498, papulans ICMP 4040, passiflorae NCPPB 1386, persicae NCPPB 2761, pisi ICMP 4433, ribicola NCPPB 963, sesami NCPPB 1016, syringae NCPPB 388, tabaci ICMP 2835, tagetis ICMP 4091, tomato NCPPB 2683, ulmi NCPPB 632; lanes 32~34, P. savastanoi pvs. glycinea NCPPB 1134, pahseolicola KACC 10575, and savastanoi NCPPB 639; lane 35, Acidovorax avenae subsp. avenae NCPPB 1011; lanes 36?38, Clavibacter michiganensis subsp. insidiosus NCPPB 1020, michiganensis NCPPB 1064, sepedonicus NCPPB 2137; lane 39, Pectobacterium carotovorum subsp. carotovorum, NCPPB 312; lanes 40?43, Rhizobium radiobacter DSM 30205, R. rhizogenes ATCC 11325, R. rubi NCPPB 1854, R. vitis NCPPB 3554; lane 44, Rhodococcus fascians LMG 3601; lane 45, Ralstonia solanacearum NCPPB 339; lanes 46?47, Xanthomonas campestris pvs. campestris KACC 10377, vesicatoria KACC 11157; lane 48, X. oryzae pv. oryzae KACC 10331; lane 1, P. coronafaciens LMG 5060, as a positive control; lane 49, water as a negative control.
Fig. 3. Sensitivity and specificity of the TaqMan real-time PCR with primer, Pc-12-ne-F/Pc-12-ne-R and TaqMan probe, Pc-taqman. (A) The linear regression generated by ten-fold dilution of DNA of Pseudomonas coronafaciens LMG 5060 and (B) TaqMan PCR with DNAs (circle dot) of P. coronafaciens LMG 5060, and P. coronafaciens strains (square dot): P. coronafaciens KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, and LMG 2330.
Tables

List of Pseudomonas coronafaciens strains, the non-target bacterial strains of Pseudomonas spp. and other plant-pathogenic bacterial strains used in this study

StrainabHostcOrigincYearc
Acidovorax
avenae subsp. avenaeNCPPB 1011Zea maysUSA1958
Clavibacter
michiganensis subsp. incidiosusNCPPB 1020Medicago sativaCanadank
michiganensis subsp. michiganensisNCPPB 1064Lycopersicon esculentumItaly1961
michiganensis subsp. sepedonicusNCPPB 2137Solanum tuberosumCanadank
Pectobacterium
carotovorum subsp. carotovorumNCPPB 312Solanum tuberosumDenmarknk
Pseudomonas
coronafacinesKACC 12133nknknk
KACC 13262nknknk
LMG 2170Bromus sp.Canada1962
LMG 2330nknk1966
LMG 5030Lolium multiflorumJapan1967
LMG 5060bAvena sativaUnited Kingdom1958
LMG 5061Secale cerealeCanada1962
LMG 5081Avena sativaZimbabwe1971
LMG 5380Avena sativaKenya1970
LMG 5449Avena sativaGermany1959
LMG 5452Avena sativaNew Zealand1969
LMG 5536Avena sativaUnited Kingdom1965
LMG 13190Avena sativanknk
savastanoi pv. glycineaNCPPB 1134Glycine javanicaZimbabwe1961
savastanoi pv. phaseolicolaKACC 10575Phaseolus vulgarisPolandnk
savastanoi pv. savastanoiNCPPB 639Olea europaeaYugoslaviank
syringae pv. actinidiaeKACC 10582Actinidia chinensisRep. of Korea1999
syringae pv. antirrhiniICMP 4303Antirrhinum majusUnited Kingdom1965
syringae pv. aptataDSM 50252Beta vulgarisnknk
syringae pv. atrofaciensLMG 5095Triticum aestivumNew Zealand1968
syringae pv. berberidisNCPPB 2724Berberis sp.New Zealand1972
syringae pv. ciccaroneiNCPPB 2355Ceratonia siliquank1969
syringae pv. delphiniiICMP 529Delphinium sp.New Zealand1957
syringae pv. dysoxyliICMP 545Dysoxylum spectabileNew Zealand1949
syringae pv. eriobotryaeNCPPB 2331Eriobotrya japonicaUnited Kingdom1970
syringae pv. helianthiNCPPB 1229Helianthus annuusZambia1962
syringae pv. japonicaICMP 6305Hordeum vulgareJapan1951
syringae pv. lachrymansATCC 11965Cucumis sativusnknk
syringae pv. lapsaATCC 10859Triticum aestivumnk1978
syringae pv. maculicolaICMP 3935Brassica oleraceaNew Zealand1965
syringae pv. melleaICMP 5711Nicotiana tabacumJapan1968
syringae pv. moriICMP 4331Morus albaHungary1958
syringae pv. morsprunorumICMP 5795Prunus domesticanknk
syringae pv. myricaeICMP 7118Myrica rubraJapan1978
syringae pv. paniciNCPPB 1498Panicum sp.nknk
syringae pv. papulansICMP 4040Malus domesticaUSAnk
syringae pv. passifloraeNCPPB 1386Passiflora edulisNew Zealand1962
syringae pv. persicaeNCPPB 2761Prunus persicaFrance1974
syringae pv. pisiICMP 4433Pisum sativumCanada1946
syringae pv. ribicolaNCPPB 963Ribes aureumnknk
syringae pv. sesameNCPPB 1016Sesamum indicumYugoslaviank
syringae pv. syringaeNCPPB 388Oryza sativaHungarynk
syringae pv. tabaciICMP 2835Nicotiana tabacumHungary1959
syringae pv. tagetisICMP 4091Tagetes erectaZimbabwe1972
syringae pv. tomatoNCPPB 2683Lycopersicon esculentumNew Zealand1972
syringae pv. ulmiNCPPB 632Ulmus sp.Yugoslavia1958
Ralstonia
SolanacearumNCPPB 339Solanum tuberosumIsraelnk
Rhizobium
radiobacterDSM 30205Malus sp.nk1972
rhizogenesATCC 11325Malus domesticanknk
rubiNCPPB 1854Rubus ursinus var. loganobaccusUSA1942
vitisNCPPB 3554Vitis viniferaAustralia1977
Rhodococcus
fasciansLMG 3601Lillium speciosum cv. RubrumBelgiumnk
Xanthomonas
campestris pv. campestrisKACC 10377Brassica oleracea var. capitataRep. of Koreank
campestris pv. vesicatoriaKACC 11157Capsicum annuumRep. of Korea1999
oryzae pv. oryzaeKACC 10331Oryza sativaRep. of Koreank

aATCC, American Type Culture Collection, USA; DSM, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany; ICMP, International Collecti on of Micro-organisms from Plants, Landcare Research, New Zealand; KACC, Korean Agricultural Culture Collection, Rural Development Administration, Rep. of Korea; LMG, Collection of the Laboratorium voor Microbiologie en Microbiele Genetica, Ghent University, Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, United Kingdom.

bThis strains was used for genome sequencing by NGS.

cnk, not known.


The Ct values of Pseudomonas coronafaciens strains, the non-target bacterial strains and the seed extracts from artificially inoculated oat seeds with Pseudomonas coronafaciens LMG 5060 which were obtained by the TaqMan PCR assays

SamplesDNA conc.Mean Cta
Pseudomonas coronafaciensLMG 5060 (STD)10 ng22.8±0.1
1 ng28.7±2.5
100 pg31.3±1.5
10 pg36.5±3.3
KACC 1213310 ng22.5±0.1
KACC 1326210 ng22.7±0.2
LMG 217010 ng23.3±0.1
LMG 233010 ng27.1±0.7
LMG 503010 ng22.8±0.3
LMG 506110 ng28.0±1.0
LMG 508110 ng25.8±2.1
LMG 538010 ng24.7±0.4
LMG 544910 ng25.1±0.4
LMG 545210 ng25.5±2.0
LMG 553610 ng23.5±0.6
LMG 1319010 ng27.9±1.5
Non-target bacterial strains
Acidovorax avenae subsp. avenaeNCPPB 101110 ng39.1±1.7
Clavibacter michiganensis subsp. incidiosusNCPPB 102010 ng> 40
Clavibacter michiganensis subsp. michiganensisNCPPB 106410 ng> 40
Clavibacter michiganensis subsp. sepedonicusNCPPB 213710 ng> 40
Pectobacterium carotovorum subsp. carotovorumNCPPB 31210 ng> 40
Pseudomonas savastanoi pv. glycineaNCPPB 113410 ng> 40
Pseudomonas savastanoi pv. phaseolicolaKACC 1057510 ng39.5±0.9
Pseudomonas savastanoi pv. savastanoiNCPPB 63910 ng> 40
Pseudomonas syringae pv. actinidiaeKACC 1058210 ng> 40
Pseudomonas syringae pv. antirrhiniICMP 430310 ng> 40
Pseudomonas syringae pv. aptataDSM 5025210 ng> 40
Pseudomonas syringae pv. atrofaciensLMG 509510 ng> 40
Pseudomonas syringae pv. berberidisNCPPB 272410 ng37.8±1.9
Pseudomonas syringae pv. ciccaroneiNCPPB 235510 ng39.6±0.8
Pseudomonas syringae pv. delphiniiICMP 52910 ng39.5±0.9
Pseudomonas syringae pv. dysoxyliICMP 54510 ng39.3±1.3
Pseudomonas syringae pv. eriobotryaeNCPPB 233110 ng> 40
Pseudomonas syringae pv. helianthiNCPPB 122910 ng39.9±0.2
Pseudomonas syringae pv. japonicaICMP 630510 ng> 40
Pseudomonas syringae pv. lachrymansATCC 1196510 ng> 40
Pseudomonas syringae pv. lapsaATCC 1085910 ng> 40
Pseudomonas syringae pv. maculicolaICMP 393510 ng> 40
Pseudomonas syringae pv. melleaICMP 571110 ng38.7±1.3
Pseudomonas syringae pv. moriICMP 433110 ng> 40
Pseudomonas syringae pv. morsprunorumICMP 579510 ng> 40
Pseudomonas syringae pv. myricaeICMP 711810 ng> 40
Pseudomonas syringae pv. paniciNCPPB 149810 ng> 40
Pseudomonas syringae pv. papulansICMP 404010 ng> 40
Pseudomonas syringae pv. passifloraeNCPPB 138610 ng39.7±0.6
Pseudomonas syringae pv. persicaeNCPPB 276110 ng> 40
Pseudomonas syringae pv. pisiICMP 443310 ng> 40
Pseudomonas syringae pv. ribicolaNCPPB 96310 ng> 40
Pseudomonas syringae pv. sesamiNCPPB 101610 ng> 40
Pseudomonas syringae pv. syringaeNCPPB 38810 ng> 40
Pseudomonas syringae pv. tabaciICMP 283510 ng> 40
Pseudomonas syringae pv. tagetisICMP 409110 ng> 40
Pseudomonas syringae pv. tomatoNCPPB 268310 ng> 40
Pseudomonas syringae pv. ulmiNCPPB 63210 ng39.8±0.3
Ralstonia solanacearumNCPPB 33910 ng> 40
Rhizobium radiobacterDSM 3020510 ng> 40
Rhizobium rhizogenesATCC 1132510 ng> 40
Rhizobium rubiNCPPB 185410 ng> 40
Rhizobium vitisNCPPB 355410 ng> 40
Rhodococcus fasciansLMG 360110 ng> 40
Xanthomonas campestris pv. campestrisKACC 1037710 ng> 40
Xanthomonas campestris pv. vesicatoriaKACC 1115710 ng> 40
Xanthomonas oryzae pv. oryzaeKACC 1033110 ng> 40
Seed extracts from oat seeds inoculated in
 108 cfu/ml cell suspension21.2±0.5
 107 cfu/ml cell suspension21.4±0.2
 106 cfu/ml cell suspension21.3±0.2
 105 cfu/ml cell suspension22.5±0.5
 104 cfu/ml cell suspension24.2±0.7
 103 cfu/ml cell suspension25.1±1.1
 102 cfu/ml cell suspension29.2±0.3
 10 cfu/ml cell suspension32.2±0.7
 control (water treatment)> 40
References
  1. Bella, P, Licciardello, G, Tessitori, M, and Catara, V (2008). A real-time PCR quantitative detection assay for Pseudomonas savastanoi pv. nerii in Nerium oleander. Phytopathol Mediterr. 47, 204-213.
  2. Cho, JH, Jeong, MJ, Song, MJ, Yim, KO, Lee, HI, Kim, JH, Baeg, JH, and Cha, JS (2010). Development of PCR Primers to Detect Pseudomonas savastanoi pv. phaseolicola from the Bean Seeds. Res Plant Dis. 16, 129-135.
    CrossRef
  3. Collins, DJ (2010). Oat Diseases in Alabama: Circular ANR-631. Alabama Cooperative Extension Service. , .
  4. Delcher, AL, Bratke, KA, Powers, EC, and Salzberg, SL (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 23, 673-679.
    Pubmed KoreaMed CrossRef
  5. Elliott, C (1920). Halo-blight of oats. J Agric Res. 19, 139-172.
  6. Finetti-Sialer, MM, and Ciancio, A (2005). Isolate-specific detection of Grapevine fan-leaf virus from Xiphinema index through DNA-based molecular probes. Phytopathology. 95, 262-268.
    CrossRef
  7. Harder, DE, and Haber, S (1992). Oat diseases and pathologic techniques. Oat Science and Technology. 33, 307-402.
  8. Hyatt, D, Chen, GL, Locascio, PF, Land, ML, Larimer, FW, and Hauser, LJ (2010). Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 11, 119-129.
    CrossRef
  9. Lukashin, A, and Borodovsky, M (1998). GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 26, 1107-1115.
    Pubmed KoreaMed CrossRef
  10. Maes, M, Garbeva, P, and Kamoen, O (1996). Recognition and detection in seed of the Xanthomonas pathogens that cause cereal leaf streak using rDNA spacer sequences and polymerase chain reaction. Phytopathology. 86, 63-69.
    CrossRef
  11. Margulies, M, Egholm, M, Altman, WE, Attiya, S, Bader, JS, Bemben, LA, Berka, J, Braverman, MS, Chen, YJ, Chen, Z, Dewell, SB, Du, L, Fierro, JM, Gomes, XV, Godwin, BC, He, W, Helgesen, S, Ho, CH, Ho, CH, Irzyk, GP, Jando, SC, Alenquer, ML, Jarvie, TP, Jirage, KB, Kim, JB, Knight, JR, Lanza, JR, Leamon, JH, Lefkowitz, SM, Lei, M, Li, J, Lohman, KL, Lu, H, Makhijani, VB, McDade, KE, McKenna, MP, Myers, EW, Nickerson, E, Nobile, JR, Plant, R, Puc, BP, Ronan, MT, Roth, GT, Sarkis, GJ, Simons, JF, Simpson, JW, Srinivasan, M, Tartaro, KR, Tomasz, A, Vogt, KA, Volkmer, GA, Wang, SH, Wang, Y, Weiner, MP, Yu, P, Begley, RF, and Rothberg, JM (2005). Genome sequencing in microfabricated highdensity picolitre reactors. Nature. 437, 376-380.
    Pubmed KoreaMed
  12. Martens, JW, Seaman, WL, and Atkinson, TG (1984). Diseases of field crops in Canada. Canadian Phytopathological Society. , .
  13. Schena, L, Nigro, F, Ippolito, A, and Gallitelli, D (2004). Real-time quantitative PCR: A new technology to detect and study phytopathogenic and antagonistic fungi. Eur J Plant Pathol. 110, 893-908.
    CrossRef
  14. Walcott, RR (2003). Detection of seedborne pathogens. HortTechnology. 13, 40-47.
  15. Wallwork, H (1992). Cereal leaf and stem diseases. Grains Research and Development Corporation. , .
  16. Young, JM, Dye, DW, and Wilkie, JP (1978). Genus VII. Pseudomonas Migula 1894. New Zealand J Agric Res. 21, 153-177.
    CrossRef


June 2017, 33 (3)
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