Embrapa Maize and Sorghum, Sete Lagoas, MG, Brazil; Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Detection and Molecular Diversity of Pantoea ananatis Associated with White Spot Disease in Maize, Sorghum and Crabgrass in Brazil Ubiraci biraci Gomes omes De Paula aula Lana ana 1,2 ,Eliane liane Aparecida parecida Gomes omes 1 ,Dagma agma Dionısia ionı ´sia Silva ilva 1 ,Rodrigo odrigo Veras eras Costa osta 1 , Luciano uciano Viana iana Cota ota 1 ,Douglas ouglas Ferreira erreira Parreira arreira 1 ,Isabel sabel Regina egina Prazeres razeres Souza ouza 1 and Claudia laudia Teixeira eixeira Guimaraes uimara ˜es 1,2 AuthorsÕ addresses: 1 Embrapa Maize and Sorghum, PO Box 151, 35701-970 Sete Lagoas, MG, Brazil; 2 Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, PO Box 486, 31270-901 Belo Horizonte, MG, Brazil (correspondence to C. T. Guimara˜es. E-mail: claudia@cnpms.embrapa.br) Received September 19, 2011; accepted April 18, 2012 Keywords: Pantoea ananatis, maize white spot disease, molecular detection, genetic diversity, Brazil Abstract Bacteria of the genus Pantoea have become important plant pathogens worldwide in recent years. Pantoea ananatis was reported as the cause of maize white spot, a serious maize disease in Brazil, causing significant yield losses. However, very little information is avail- able about how to detect this pathogen, its genetic variability and the putative alternative hosts in maize- growing areas. To address these issues, we implemented a rapid and efficient PCR-based method to identify P. ananatis isolated from leaves showing white spot symptoms and evaluated its genetic diversity in maize, sorghum and crabgrass. Of the 29 bacteria isolated from typical water-soaked lesions of white spot disease that produced yellow colonies, 15 isolates were identi- fied as P. ananatis by 16S rDNA sequencing and cor- rectly detected by the PCR reaction, amplifying a specific fragment of the ice nucleation gene (ina). These P. ananatis isolates included 13 from maize, one from sorghum and one from crabgrass, while the other 14 yellow colony isolates were from other bacterial species, including two Pantoea species (Pantoea dispersa and Pantoea agglomerans) that were not amplified by the ina primers. These results indicate that the optimized PCR assay can be used to detect P. ananatis isolated from white spot lesions and could be used as a large- scale and cost-effective method of detecting this patho- gen in leaf lesions on maize and other grasses. All isolates were evaluated for hypersensitive response (HR) on tobacco, revealing that some P. ananatis were able to induce HR. The high genetic variability revealed by rep-PCR did not differentiated the P. ananatis isolates based on their hosts or HR reaction. The detection, characterization and diversity of P. ananatis from maize, sorghum and crabgrass in our study can be applied in understanding epidemiology and designing control strategies for maize white spot disease in Brazil. Introduction The high incidence and severity of maize white spot disease has contributed to a significant reduction in maize yield in Brazil since the 1990s (Fernandes and OIiveira 1997). Under favourable environmental condi- tions, this disease causes leaf senescence and a reduc- tion in grain size and weight, resulting in yield losses up to 63% in susceptible genotypes (Pinto 1995; Pinto et al. 1997). Disease symptoms begin with slight chlo- rosis and the development of water-soaked spots on the leaf surface, which become necrotic and straw- coloured (Paccola-Meirelles et al. 2001). Currently, the most sustainable and effective method for white spot disease control is the development of maize resistant varieties (Schuelter et al. 2003). The cause of this disease was initially attributed to Phaeosphaeria maydis, a filamentous fungus, which was detected in late lesions of maize white spot (Fantin 1994). However, in later studies, the bacterium Pan- toea ananatis was isolated from lesions at initial stages of development, the so-called water-soaked lesions (Paccola-Meirelles et al. 2001; Bomfeti et al. 2008). Pantoea ananatis was also isolated from maize white spot lesions in Mexico (Pe´rez-y-Terro´n et al. 2009), Argentina (Alippi and Lo´pez 2010) and Poland (Kra- wczyk et al. 2010), and this bacterium is now consid- ered the causal agent of white spot disease. Pantoea ananatis infects a wide range of economi- cally important crops worldwide and is emerging as a pathogen of increasing importance due to its high viru- lence in new monocotyledonous and dicotyledonous hosts (Coutinho and Venter 2009). Pantoea ananatis was reported in asymptomatic crabgrass leaves (Gita- itis et al. 2002) and in sorghum leaf lesions showing white spot symptoms (Cota et al. 2010). This species was also isolated from maize kernels (Rijavec et al. 2007) and from the rhizosphere of P-efficient maize J Phytopathol 160:441–448 (2012) doi: 10.1111/j.1439-0434.2012.01924.x Ó 2012 Blackwell Verlag GmbH