First Report of Pectobacterium aroidearum Causing Soft Rot in Lettuce and Chinese Cabbage in Brazil

Abstract

Lettuce (Lactuca sativa L.) and Chinese cabbage (Brassica pekinensis Rupr.) are leafy vegetables of great socioeconomic value in Brazil. Bacterial diseases are among the factors that might limit their yield, particularly soft rot caused by bacteria of the genus Pectobacterium. In July 2014, vegetable gardens of approximately 1.0 and 0.18 ha in the cities of Juazeiro (09° 24′ 42″ S, 40° 29′ 55″ W), state of Bahia, and Petrolina (09° 23′ 55″ S, 40° 30′ 03″ W), state of Pernambuco, Brazil, showed 5% of lettuce plants variety Elba and 44.4% of Chinese cabbage cultivar Michihilli plants with wilt of leaves, foul-smelling soft rot, and inner medulla disintegration. Bacterial isolates were selectively isolated from stem and midribs of lettuce and Chinese cabbage by transferring rot tissue to a healthy pepper fruit using a sterilized toothpick (Takatsu et al. 1981). Pepper fruits were incubated at 26°C for 36 h in a moist chamber, and after incubation bacterial isolates were isolated in Petri dishes containing casamino acid–peptone–glucose (CPG) medium. Small white colonies with “broken glass” aspect (Kelman and Dickey 1995) were observed under a stereoscope, and after that, they were preserved in sterile distilled water (SDW). Twelve isolates were obtained from lettuce and Chinese cabbage plants with soft rot symptoms, which showed the same cultural characteristics in CPG medium. Pathogenicity testing was performed using seedlings of lettuce variety Elba (30 ± 2°C; 62 ± 2% relative humidity [RH]) and detached leaves of Chinese cabbage cultivar Michihilli (28 ± 2°C; 65 ± 2% RH), which are susceptible to bacterial soft rot. Ten microliters of bacterial suspension (10⁶ CFU/ml) were injected into the intermediary portion of the leaf midrib, followed by 48 h of incubation in a moist chamber. For the negative control, seedlings and detached leaves were treated similarly with SDW. The isolate CRMP1 of Pectobacterium aroidearum (Moraes et al. 2017) was used as a reference. Typical symptoms of soft rot were observed 12 to 24 h after inoculation, followed by pathogen reisolation onto CPG medium, fulfilling Koch’s postulates. Seedlings and detached leaves used as negative control remained symptomless, whereas seedlings and detached leaves inoculated with CRMP1 showed soft rot symptoms. Both isolates (UNEB 3 and CCRMPA35) presented gram-negative cells, grew at 37°C, were erythromycin resistant, and were positive for maceration in potato tubers. The Biolog Gen III System identified the isolates as Pectobacterium sp. with a level of 70% similarity. Two isolates were selected for molecular identification, one from lettuce (UNEB3) and one from Chinese cabbage (CCRMPA35). Fragments of the 16S rDNA region (Hauben et al. 1998) and icdA, proA, and mdH genes (Ma et al. 2007) of these isolates were sequenced, and BLASTn analysis of the 16S rRNA region (GenBank MH507334 and MH507330), icdA (GenBank MH500095 and MH500096), proA (GenBank MH507331 and MH507332), and mdH (GenBank MH507329 and MH507330) genes of the isolates UNEB 3 and CCRM35 showed similarity levels of 99, 99, 98, and 99% with sequences of the 16S rRNA region and icdA, proA, and mdH genes of the type isolate of P. aroidearum (isolate SCRI109ᵀ; GenBank JN600323, HM156915, HM157162, and HM156976). Phylogenetic analysis of concatenated sequences of the icdA, proA, and mdH genes using Bayesian inference between isolates UNEB 3 and CCRM35 and species and subspecies of Pectobacterium from GenBank grouped these two isolates with SCRI109ᵀ, with a posterior probability of 100%. To our knowledge, this is the first report of P. aroidearum causing soft rot in lettuce and Chinese cabbage in Brazilian territory. In addition, the present research helps to better know the host range of P. aroidearum in Brazil, which until now was only reported in zucchini (Moraes et al. 2017).