Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

Weller, Simon A.; Stead, D. E.

doi:10.1046/j.1365-2672.2002.01506.x

Cited by 41 publications

(26 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Real-time quantitative PCR (RTQ-PCR) has been shown to be a powerful tool for quantifying bacterial abundance in many different kinds of complex environmental samples down to the genus and species levels (3,17,44,48). In recent studies, the abundance of a genus or species in an environmental sample has also been related to the overall eubacterial abundance in the analyzed system (1,5,32,42).…”

mentioning

confidence: 99%

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Skovhus

Ramsing

Holmström

et al. 2004

Appl Environ Microbiol

View full text Add to dashboard Cite

A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from doublestranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

show abstract

mentioning

confidence: 99%

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Skovhus

Ramsing

Holmström

et al. 2004

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…These strains appeared to be related to the genus Agrobacterium, and thus it was unclear whether these strains (35). belonged to a previously undescribed Agrobacterium species or were in fact genuinely not Agrobacterium strains.…”

Section: Discussionmentioning

confidence: 99%

“…Two PCR assays were employed to demonstrate the presence of pRi in these strains, one targeted to the conserved virD2 gene (11) found in all pTi and pRi and one targeted to the T-DNA of pRi commonly associated with RMA crops (35). We have assumed that positive results in both of these assays and the subsequent results in the in vitro pathogenicity test indicate the presence of pRi in the nine ␣-Proteobacteria strains, although the presence of pRi was not demonstrated by other techniques, such as electrophoretic isolation of plasmids (14).…”

Section: Discussionmentioning

confidence: 99%

“…DNA was extracted from resulting cultures by heating a 100-l suspension of each culture (6 min, Ͼ95°C). A supernatant was obtained from this suspension by centrifugation (14,500 rpm, 3 min) (Eppendorf MiniSpin plus; Eppendorf AG, Hamburg, Germany) and used as template in two PCR assays, the conventional PCR of Haas et al (11), testing for the virD2 region found in all pTi and pRi, and the rol TaqMan PCR (35), testing for a gene commonly found in the T-DNA of RMA pRi.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other α- Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

Weller¹,

Stead²,

Young

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring ␣-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring ␣-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%

show abstract

“…2). Suzaki et al, 2004）；2） Ti プラスミド特異的プライマー（Cubero et al, 2006Haas et al, 1995;Picard et al, 1992;Pionnat et al, 1995;Pulawska and Sobiczewski, 2005;Puopolo et al, 2007;Sobiczewski et al, 2005）；3）Ri プラスミド特異的プライマー（伊予住・市川，1999）；4）特定の系統（オパイン型など）の病原性プラスミドのみを対象としたプライマー（Bini et al, 2008Dong et al, 1992;Eastwell et al, 1995;Kaufmann et al, 1996;Szegedi and Bottka, 2002;Tan et al, 2003;Weller and Stead, 2002;Yakabe et al, (Table 2) to determine which pathogenic plasmid respective strains carry and which pathogenic state they belong to. Amplification of the DNA fragments (780-784 bp) from the 16S rRNA gene (16S rDNA) (internal control) of the respective strains indicated that the respective PCR reactions were completed properly.…”

Section: Te Buffer 340unclassified

Simultaneous plasmid profiling of phytopathogenic <i>Rhizobium</i> species (former <i>Agrobacterium </i>species) using multiplex colony-direct PCR.

宏之

浩一

章

2016

Jpn. J. Phytopathol.

View full text Add to dashboard Cite

(2016). Simultaneous plasmid profiling of phytopathogenic Rhizobium species (former Agrobacterium species) using multiplex colony-direct PCR. Jpn. J. Among Rhizobium species, six species include plant pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Based on their pathogenic states, the members belonging to the six species can be divided into three types, namely, crown gall bacteria carrying Ti plasmid, hairy root bacteria carrying Ri plasmid, and nonpathogenic bacteria carrying neither pathogenic plasmids. In this study, we developed a plasmid-profiling method using a multiplex colony-direct PCR to identify any pathogenic plasmid present in the respective strains and thus determine their pathogenic state. For that purpose, a universal primer set for both pathogenic plasmids and a specific primer set for the Ri plasmid were designed based on the sequences of virC1 and rolC, respectively. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as a plasmid-profiling tool was then validated using the Rhizobium species containing plant pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from virC1-specific amplification (278 bp) and the other (780-784 bp) from 16S rDNA as the internal control, were stably obtained from all crown gall bacteria used. For hairy root bacteria, three fragments derived from virC1 (278 bp), rolC (438 bp) and 16S rDNA were always observed. On the other hand, only a fragment derived from 16S rDNA was amplified from the other nonpathogenic bacteria. Therefore, this method is useful for rapid plasmid profiling of the Rhizobium species containing plant pathogens, which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies of the pathogens.

show abstract

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

Cited by 41 publications

References 19 publications

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other α- Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

Simultaneous plasmid profiling of phytopathogenic <i>Rhizobium</i> species (former <i>Agrobacterium </i>species) using multiplex colony-direct PCR.

Contact Info

Product

Resources

About