The bacterium Pseudomonas viridiflava (Burkholder) Dowson is the causal agent of leaf bacterial speck (⁸8 Jones, JB; Jones, JP; McCarter, SM; Stall, RE. Pseudomonas viridiflava: Causal agent of bacterial leaf blight of tomato. Plant Disease, Saint Paul, v.68, p.341-342, 1984. Available at: <https://www.apsnet.org/publications/PlantDisease/BackIssues/Documents/1984Articles/PlantDisease68n04_341.PDF>. Accessed on: 15 jul. 2020.
https://www.apsnet.org/publications/Plan... ) and pith necrosis (²2 Alivizatos, A.S. Tomato pith necrosis caused by Pseudomonas viridiflava. Annales de l’Institut phytopathologique Benaki, Atenas, v.15, p.41-47, 1986., ¹⁰10 Lelliott, RA; Stead, DE. Methods for the diagnosis of bacterial diseases of plants. London: Blackwell Scientific Publications, 1987. v.2, 216p.) in tomato plants (Solanum lycopersicum L.). In 1973, this species was reported by Wilkie et al. (²²22 Wilkie, JP; Dye, DW; Watson, DRW. Further hosts of Pseudomonas viridiflava. New Zealand Journal of Agricultural Research, Wellington, v. 16, p. 315-323, 1973. DOI: https://doi.org/10.1080/00288233.1973.10421110.
https://doi.org/10.1080/00288233.1973.10... ) causing pith necrosis and leaf speck symptoms and was subsequently associated with symptoms in the fruits (⁶6 Goumas, D.E.; Malathrakis, N.E.; Chatzaki, A.K. Characterization of Pseudomonas viridiflava associated with a new symptom on tomato fruit. European Journal of Plant Pathology, Berlin, v.105, p.927-932, 1999. DOI: https://doi.org/10.1023/A:1008725818334.
https://doi.org/10.1023/A:1008725818334... ). Pseudomonas viridiflava was described causing leaf bacterial speck on tomato in Greece (⁵5 Goumas, D.E.; Chatzaki, A.K. Characterization and host range evaluation of Pseudomonas viridiflava from melon, blite, tomato, chrysanthemum and eggplant. European Journal of Plant Pathology, Berlin, v.104, p.181-188, 1998. DOI: https://doi.org/10.1023/A:1008675704834.
https://doi.org/10.1023/A:1008675704834... ) and the United States (⁸8 Jones, JB; Jones, JP; McCarter, SM; Stall, RE. Pseudomonas viridiflava: Causal agent of bacterial leaf blight of tomato. Plant Disease, Saint Paul, v.68, p.341-342, 1984. Available at: <https://www.apsnet.org/publications/PlantDisease/BackIssues/Documents/1984Articles/PlantDisease68n04_341.PDF>. Accessed on: 15 jul. 2020.
https://www.apsnet.org/publications/Plan... ), as well as pith necrosis in Greece (²2 Alivizatos, A.S. Tomato pith necrosis caused by Pseudomonas viridiflava. Annales de l’Institut phytopathologique Benaki, Atenas, v.15, p.41-47, 1986., ¹²12 Malathrakis, NE; Goumas, DE. Bacterial soft rot of tomato in plastic greenhouses in Crete. Annals of Applied Biology, Wellesbourne, v.111, p.115-123, 1987. DOI: https://doi.org/10.1111/j.1744-7348.1987.tb01438.x.
https://doi.org/10.1111/j.1744-7348.1987... ), Argentina (¹1 Alippi, A.M.; Dal Bo, E.; Ronco, L.B.; Lopez, M.V.; Lopez, A.C.; Aguilar, O.M. Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse. Plant Pathology, London, v.52, p.287-302, 2003. DOI: https://doi.org/10.1046/j.1365-3059.2003.00850.x.
https://doi.org/10.1046/j.1365-3059.2003... ), Portugal (¹⁸18 Passo, V; Moura, L; Duclos, J. Detection of Pseudomonas corrugata, P. mediterranea and P. viridiflava in naturally infected tomato stems by bio-PCR. Acta Horticulture, Brussels, v.789, p.315-318, 2005. DOI: https://doi.org/10.17660/ActaHortic.2008.789.43.
https://doi.org/10.17660/ActaHortic.2008... ), Turkey (³3 Aysan, Y.; Uygur, S. Epiphytic survival of Pseudomonas viridiflava, causal agent of pith necrosis of tomato, on weeds in Turkey. Journal of Plant Pathology, Pisa, v.87, p.135-139, 2005. Available at: <https://www.jstor.org/stable/41998225>. Accessed on: 15 jul 2020.
https://www.jstor.org/stable/41998225... , ²¹21 Yildiz, HN; Aysan, Y; Sahin, F; Cinar, O. Potential inoculum sources of tomato stem and pith necrosis caused by Pseudomonas viridiflava in the Eastern Mediterranean Region of Turkey. Journal of Plant Diseases and Protection, Berlin, v.111, p.380-387, 2004. Available at: https://www.jstor.org/stable/43215590. Accessed on: 15 jul. 2020.
https://www.jstor.org/stable/43215590... ), Macedonia (¹⁵15 Mitrev, S; Karov, I; Kovacevik, B; Kostadinovska, E. Pseudomonas population causing tomato pith necrosis in the Republic of Macedonia. Journal of Plant Pathology, Pisa, v.96, p.589-592, 2014. DOI: http://dx.doi.org/10.4454/JPP.V96I3.002.
https://doi.org/10.4454/JPP.V96I3.002... ) and Serbia (¹⁷17 Popović, T; Ivanović, Ž; Ignjatov, M. First report of Pseudomonas viridiflava causing pith necrosis of tomato (Solanum lycopersicum) in Serbia. Plant Disease, Saint Paul, v.99, p.1033, 2015. DOI: https://doi.org/10.1094/PDIS-01-15-0052-PDN.
https://doi.org/10.1094/PDIS-01-15-0052-... ). In Brazil, symptoms of bacterial speck were reported in Bahia and São Paulo States (¹³13 Maringoni, AC; Gioria, R; Kobori, RF; Azevedo, SM; De Pieri, C; Silva Junior, TAF. Occurrence of Pseudomonas viridiflava on tomato in Brazil. Journal of Plant Pathology, Pisa, v.91, p.504, 2009. Available at: <http://www.jstor.org/stable/41998661>. Accessed on: 15 jul. 2020.
http://www.jstor.org/stable/41998661... ), while pith necrosis was found in Santa Catarina State (¹⁶16 Monteiro, FP; Ogoshi, C; Becker, WF; Wamser, AF; Valmorbida, J. Pith necrosis associated with Pseudomonas viridiflava in tomato plants in Brazil. Plant Pathology & Quarantine, Guizhou, v.9, p.1-5, 2019. DOI: https://doi.org/10.5943/ppq/9/1/1.
https://doi.org/10.5943/ppq/9/1/1... ). In 2013, in a commercial staked tomato field in Uberlândia, Minas Gerais State, plants were observed showing leaf bacterial speck symptoms (Figure 1A), lower leaf wilt, leaf chlorosis, and severe pith necrosis (Figure 1B) including dark brown lesions and premature death. The pathogen was isolated by indirect method (¹⁹19 Romeiro, R.S. Métodos em bacteriologia de plantas. Viçosa, Editora UFV, 2001. v.1, 279p.) from infected leaves and from pith necrosis on 523 culture medium (⁹9 Kado, EI; Heskett, MG. Selective media for isolation of Agrobcterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology, Saint Paul, v.60, p.969-976, 1970. DOI: https://doi.org/10.1094/Phyto-60-969.
https://doi.org/10.1094/Phyto-60-969... ). The isolates (Figure 1C) were biochemically characterized as Gram-negative, aerobic growth, fluorescence in King B medium, levan production-negative, oxidase-negative, pectolytic activity-positive, arginine dihydrolase-negative, tobacco hypersensitivity reaction-positive (LOPAT/- - + - +), and identified as P. viridiflava (¹⁰10 Lelliott, RA; Stead, DE. Methods for the diagnosis of bacterial diseases of plants. London: Blackwell Scientific Publications, 1987. v.2, 216p., ²⁰20 Schaad, NM; Jones, J; Chun, W. Laboratory guide for identification of plant pathogenic bacteria. Saint Paul: APS Press, 2001. 373p.). The pathogenicity of isolates was confirmed by spraying of tomato leaves until runoff and stem injection with the bacterial suspension at 1 x 10⁸ CFU.mL^-1. The plants were kept in a moist chamber 24 h before and after inoculation. Twenty days after inoculation, symptoms of leaf bacterial speck were observed on the leaf adaxial surface and petioles (Figure 1D), including small circular and water-soaked lesions, while pith necrosis symptoms were found in the stem (Figure 1E). The bacteria were then reisolated to complete Koch’s postulates. No symptoms were observed for control plants inoculated with sterile distilled water. The disease was less severe for inoculated leaves than for tomato plants in the field. According Monteiro et al. (¹⁶16 Monteiro, FP; Ogoshi, C; Becker, WF; Wamser, AF; Valmorbida, J. Pith necrosis associated with Pseudomonas viridiflava in tomato plants in Brazil. Plant Pathology & Quarantine, Guizhou, v.9, p.1-5, 2019. DOI: https://doi.org/10.5943/ppq/9/1/1.
https://doi.org/10.5943/ppq/9/1/1... ), leaf symptoms were not observed for plants inoculated with the strain obtained from the stem which, however, was reported to be capable of infecting various plant parts. Thus, P. viridiflava is less aggressive on leaves, while tomato production losses are more related to pith necrosis. The genomic DNA of bacterial strains was not amplified with the primer pair Primer 1/Primer 2, specific for P. syringae pv. tomato (Okabe) Young, Dye & Wilkie (⁴4 Bereswill, S.; Bugert, P.; Volksch, B.; Ullrich, M.; Bender, C.L.; Geider, K. Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products. Applied and Environmental Microbiology, Washington, v.60, n.8, p.2924-2930, 1994. DOI: https://doi.org/10.1128/AEM.60.8.2924-2930.1994.
https://doi.org/10.1128/AEM.60.8.2924-29... ), or with the primer pair PF/PR, specific for P. syringae pv. syringae van Hall (⁷7 Hamedan, E; Harighi, B. Genetic diversity of Pseudomonas syringae pv. syringae strains, causing bacterial stem blight disease of alfalfa in the kurdistan province of Iran. Journal of Plant Pathology, Pisa, v.96, n.2, p.303-307, 2014. DOI: http://dx.doi.org/10.4454/JPP.V96I2.030.
https://doi.org/10.4454/JPP.V96I2.030... ). Considering BOX-PCR (¹¹11 Louws, FJ; Fulbright, DW; Stephens, CT; Bruijn, FJ. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, Washington, v.60, p.2286-2295, 1994. DOI: https://doi.org/10.1128/AEM.60.7.2286-2295.1994.
https://doi.org/10.1128/AEM.60.7.2286-22... ), the isolates had the same pattern and number of bands. Sequencing of the 16S rRNA gene region using the universal pair of primers 27F/1492R (¹⁴14 Martin-Laurent, F; Philippot, L; Hallet, S; Chaussod, R; Germon, JC; Soulas, G; Catroux, G. DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and Environmental Microbiology, Washington, v.67, p.2354-2359, 2001. DOI: https://doi.org/10.1128/AEM.67.5.2354-2359.2001.
https://doi.org/10.1128/AEM.67.5.2354-23... ) was compared with sequences deposited in the GenBank and aligned closely (99.02% similarity) with P. viridiflava (GenBank No. NR_114482.1), query cover of 100% sequence. Correct characterization and identification of this bacterium can improve the disease management in the field, especially because pith necrosis symptoms can be confused with other fluorescent Pseudomonas species in tomato plants. This is the first occurrence of P. viridiflava causing tomato pith necrosis and leaf bacterial speck in Uberlândia, Minas Gerais, Brazil. The strains are maintained in the phytopathogenic bacteria collection of the Institute of Agrarian Sciences at Federal University of Uberlândia and were codified as UFU E36 and UFU E37.

Figure 1
Symptoms of leaf bacterial speck (A) and tomato pith necrosis (B), caused by Pseudomonas viridiflava. Bacterial growth on culture medium (C). Symptoms of leaf bacterial speck (D) and tomato pith necrosis (E), 20 days after inoculation.

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