Detection of Pantoea stewartii from sweet corn leaves by loop-mediated isothermal amplification (LAMP)by Hiroshi Uematsu, Yasuhiro Inoue, Yasuo Ohto

Journal of General Plant Pathology


Agronomy and Crop Science / Plant Science


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Detection of Pantoea stewartii from sweet corn leaves by loop-mediated isothermal amplification (LAMP)

Hiroshi Uematsu • Yasuhiro Inoue •

Yasuo Ohto

Received: 17 June 2014 / Accepted: 9 November 2014  The Phytopathological Society of Japan and Springer Japan 2015

Abstract To improve the diagnosis of Stewart’s wilt, we developed a loop-mediated isothermal amplification (LAMP) assay, based on two conserved sequences of the cpsD and pstS–glmS gene regions. The detection limit of the LAMP assay was 104 colony-forming units/mL. No cross reaction was observed with other Pantoea spp., other genera associated with sweet corn diseases, or strains isolated from the surface of maize leaves in Japan. The LAMP reaction was not inhibited by leaf contents. The simplicity of sample preparation and short processing time make this

LAMP assay useful for field surveillance of Stewart’s wilt.

Keywords Pantoea stewartii  Loop-mediated isothermal amplification (LAMP)  Diagnostics


Stewart’s wilt, caused by Pantoea stewartii subsp. stewartii (syn. Erwinia stewartii [Mergaert et al. 1993], hereafter abbreviated Pnss), is one of the most important diseases of sweet corn (Zea mays L. var. rugosa) and inbred field corn.

Young seedlings infected with Pnss become severely wilted and die in most cases. The organism is transmitted by the corn flea beetle (Chaetocnema pulicaria). Seed transmission is also possible (Pataky and Ikin 2003). Pnss is endemic throughout a large portion of the maize-growing regions of the eastern and midwestern United States, and it occurs intermittently in Canada.

More than 60 countries have placed quarantine regulations on maize seed produced in Pnss-affected regions to prevent the introduction of the pest (Pataky and Ikin 2003).

Because Pnss is technically difficult to detect from seed at a port-of-entry inspection but can be easily detected in the field during the growing season, importation of maize seeds is approved under the condition that the plants concerned are inspected at the growing site in exporting countries (MAFF Plant Protection Station 2014). Therefore, also in noninfested countries and areas, including Japan, field surveillance is essential to eliminate the disease at an early stage. To implement field surveillance effectively, proper and rapid methods are needed to detect Stewart’s wilt from questionable plant materials in the fields.

Several methods to detect and identify P. stewartii have been reported (Coplin et al. 2002; Lamka et al. 1991;

Tambong et al. 2008; Wensing et al. 2010), including subspecies-specific methods (Gehring et al. 2014; Xu et al. 2010). Some conventional techniques, such as enzymelinked immunosorbent assay (Lamka et al. 1991), are timeconsuming and relatively insensitive compared with nucleic acid-based methods. Molecular techniques include conventional PCR (Coplin et al. 2002; Gehring et al. 2014;

Wensing et al. 2010), ‘miniprimer’ PCR (Xu et al. 2010) and a specific real-time PCR assay (Tambong et al. 2008;

Wensing et al. 2010). Some of these methods use the cpsD gene region and the region between the pstS and glmS genes. The cpsD region, part of the cps gene cluster, is required for the production of the exopolysaccharide stewartan, and plays an important role in pathogenicity and virulence (Coplin and Majerczak 1990). The region between pstS and glmS seems to be specific to this species (Wensing et al. 2010). However, while PCR-based methods are rapid, specific and highly sensitive, their practical use is hindered by the need for complex and expensive

H. Uematsu (&)  Y. Inoue  Y. Ohto

National Agriculture and Food Research Organization,

Agricultural Research Center, Kannondai, Tsukuba,

Ibaraki 305-8666, Japan e-mail: 123

J Gen Plant Pathol

DOI 10.1007/s10327-015-0580-4 thermal cycling equipment and time-consuming processes.

Inhibition of PCR by contaminant plant components also makes it difficult to detect Pnss in plant materials.

Loop-mediated isothermal amplification (LAMP) is a new molecular diagnostic technique that is simple, rapid and sensitive (Notomi et al. 2000). It does not require expensive thermal cycling equipment because DNA amplification uses only one enzyme, Bst DNA polymerase, under an isothermal condition of 60 to 65 C (Notomi et al. 2000). The LAMP reaction uses a set of four specially designed primers (FIP, BIP, F3, B3) that recognize six distinct sequences on the target (Notomi et al. 2000). The reaction is accelerated by the addition of loop primers (Nagamine et al. 2002). The LAMP reaction can be read by measuring turbidity caused by a white precipitate of magnesium pyrophosphate without the need for gel electrophoresis. Moreover, its sensitivity is less affected than that of PCR by leaf components (Kaneko et al. 2007). For these reasons, the LAMP assay has been used to detect a range of plant pathogens (Harper et al. 2010; Kubota et al. 2008;

Okuda et al. 2005; Oya et al. 2008).

In this study, we developed a LAMP assay for the detection and diagnosis of Stewart’s wilt from plant material in the field.

Materials and methods

Bacterial strains

We tested 26 strains of Pantoea spp., 17 strains of other genera (Table 1), and 57 unidentified strains isolated from the surface of maize leaves throughout Japan, the colonies of which all resemble Pantoea spp. on Luria–Bertani broth agar, Miller (Nakalai Tesque, Kyoto, Japan). All strains were used to test the specificity of the LAMP reaction. A pure culture of Pnss ICMP 5929 was used for optimizing the temperature for the LAMP reaction and for sensitivity and inoculation experiments. Sequence analysis of 5 strains of Pnss (ATCC 8199, ICMP 270, 722, 5929, 5930) was used to design LAMP primer sets. All strains were cultured in Luria–Bertani broth agar, Miller.

Sequencing analysis and primer design for LAMP

To obtain DNA sequences for designing LAMP primers, we performed conventional PCR. We used primer pair

CPSL1/CPSR2c (Coplin et al. 2002) to amplify the cpsD region and PST3581/PST3909c (Wensing et al. 2010) to amplify the pstS–glmS region. To prepare template solutions, we suspended the bacteria in 1 mM 2-[4-(2hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) solution (Nakalai Tesque). The bacterial cell suspensions (107 cfu/mL) were incubated at 98 C for 10 min and then chilled on ice. The PCR using 5 lL of template solution was performed in a total volume of 20 lL containing 1 9 Ex Taq buffer (20 mM Mg2?; Takara Bio, Ohtsu,