Detection of Pantoea ananatis, causal agent of leaf spot disease of maize, in Mexico

Additional keywords: Enterobacteriaceae, Zea mays, phytopathogen.

Maize is the most important cereal for human nutrition in Mexico. Therefore, detection and control of diseases of this plant is particularly important since world prices for maize have risen rapidly.

Species of the genus Pantoea have clinical, economic and ecological significance. Several Pantoea strains display plant growth promoting activity (Sergeeva et al. 2007). Paradoxically, phytopathogenic species are also included in this genus. For example, P. ananatis is the causative agent of agave disease (Fucikovsky and Aranda 2006), and also of palea rice browning (Cortesi and Pizzatti 2007), blight and dieback of Eucalyptus (Coutinho et al. 2002), leaf blotch disease of Sudangrass (Azad et al. 2000), brown stalk rot of maize (Goszczynska et al. 2007), leaf spot disease of maize (Paccola-Meirelles et al. 2001), and also a post-harvest disease in melon (Bruton et al. 1991).

In 2007 in the Mexican states of Puebla and Tlaxcala, we detected maize plants showing a leaf spot disease. This paper reports on the isolation and identification of the causative agent of this syndrome in Mexican maize.

Plant samples of the native maize cultivar ‘Criollo’ showing signs of a leaf spotting condition (Fig. 1), including roots and soil were obtained from twelve maize fields in the States of Tlaxcala and Puebla.

Nonsterile leaf surfaces were sampled with sterile cotton plugs soaked with 1% sucrose. In addition, leafs and stem pieces were surface sterilised with 0.25% tosylchloramide sodium (Cloramine T), washed with sterile water, homogenised and serially diluted in a 1% sucrose solution. Dilutions of sterile and non-sterile parts of the plant parts were plated on modified MESMA agar (Fuentes-Ramírez et al. 1999).

Rhizospheric soil was diluted and plated as described above. The plates were incubated at 30°C for two days and any pale and slightly yellow colonies were selected.

Isolates were Gram stained and colony morphology observed on YDC plates (Lakso and Starr 1970). The isolates were tested for: urease, arginine dehydrolase, ornithine decarboxylase, lysine decarboxylase, β-galactosidase, catalase, oxidase, indole production, gelatin liquefaction, and citrate utilisation. Acid production was determined from D-mannose, sorbitol, L-rhamnose, melibiose, sucrose, arabinose, xylose, and meso-inositol in media supplemented with Bromothymol Blue.

Motility was determined by microscopic observation in tryptic soy broth.

Genomic DNA was extracted with a commercial kit (Wizard Genomic DNA Purification kit, Promega Co.) following manufactures’ procedures. Amplified rDNA restriction analysis (ARDRA) 16S rRNA genes were amplified according to Weisburg et al. (1991). PCR conditions consisted of an initial denaturing cycle (95°C, 3 min), 35 amplification cycles (95°C, 1 min; 55°C, 1 min; 72°C, 2 min) and a final extension cycle (72°C, 3 min). The PCR mixture contained 150 μM dNTPs, 3 mm MgCl₂, 3 μM of each primer fD1 and rD1 (Weisburg et al. 1991), 10× Taq buffer, and 1.2 U Taq polymerase (Altaenzymes, Co.). The amplification products were digested with the endonucleases DdeI, HhaI, and HaeIII and the DNA fragments separated by electrophoresis in a 3% agarose gels. In addition, the 16S rRNA gene was sequenced with primers fD1 and rD1 at the Institute of Biotechnology, UNAM, Mexico (Weisburg et al. 1991). The gene coding the ATPase domain of DNA gyrase was also partially sequenced, because this locus provides reliable phylogenetic relationships among Enterobacteriaceae (Dauga 2002). gyrB genes were amplified with gyrB 01-F and gyrB 02-R primers, and sequenced with gyrB 01-F and gyrB 08-R (Brady et al. 2008). The DNA fragments were sequenced. The sequences were analysed using BLAST (Altschul et al. 1997) and Clustal W (DNA Star, v. 5.08).

Maize seeds of the native cultivar ‘Criollo’ were surface sterilised with 0.25% tosylchloramide sodium (Cloramine T) for 5 min and washed with sterile distilled water. Disinfected seeds were planted in sterilised vermiculite. Plants were irrigated with water four times per week, and two times in total over the experiment with 20 mL of Hoagland’s solution. The stems of four week old plantlets were inoculated in the stem with 10⁶ colony forming units (CFU) of the bacterial strains UAPB01001, UAPB01002, and UAPS07100, using a sterile syringe. Each strain was replicated five times. Inoculated plants were grown for four more days, and the stems cut and observed. Bacterial strains were re-isolated from the inoculated plants in order to evaluate the authenticity of the inoculated strain and to ensure that cross contamination had not taken place.

A limited amount of bacterial diversity was observed from disinfected surface sterilised leaves and stems, which were plated on in MESMA agar. Isolates showing yellow pigmentation were obtained from the rhizosphere isolations and from maize plants showing the leaf spot symptom. ARDRA tests and phenotypical characterisation of four isolates from surface sterilised tissue (including leaf tissue close to spots, and one from the rhizosphere) showed phenotypes similar to those reported for P. ananatis (Table 1). The production of yellow pigmentation, citrate utilisation, indole production, and motility also matched the profile for P. ananatis. Those strains shared the same ribotype after digestion with DdeI, HaeIII and HhaI (Fig. 2). BLAST and Clustal W analysis of the 16S rRNA and gyrB gene from isolates UAPB01001, UAPB01002, and UAPS07100 showed an identity higher than 99% to the corresponding gene sequences for P. ananatis, indicating that these maize isolates belong to P. ananatis clade (Table 2). Inoculation of maize plants with isolates UAPB01001, UAPB01002, and UAPS07100 caused symptoms of stem rotting, including the vascular bundle (Fig. 3). Isolations from these inoculated plants yielded bacteria with phenotypic and ARDRA patterns identical to the inoculated bacteria. P. ananatis is capable of colonising diverse environments (Coutinho and Venter 2009) and its presence in the rhizosphere suggests that soil could provide an inoculum source. This work is the first report of P. ananatis causing disease of maize in Mexico.

Table 1.  Phenotypic tests of the bacterial isolates

Table 2.  Sequence similarity with Pantoea ananatis (% identity)^a