RESUMO
The aim of this study was to establish a link between genetic diversity and the geographic origin of Pectobacterium strains belonging to three species-P. carotovorum, P. versatile, and P. odoriferum-isolated from cabbage in Serbia by comparing their sequences with those of strains sourced from different hosts and countries in Europe, Asia, and North America. Phylogeographic relatedness was reconstructed using the Templeton, Crandall, and Sing's (TCS) haplotype network based on concatenated sequences of the housekeeping genes dnaX, icdA, mdh, and proA, while pairwise genetic distances were computed by applying the p-distance model. The obtained TCS haplotype networks indicated the existence of high intra-species genetic diversity among strains of all three species, as reflected in the 0.2-2.3%, 0.2-2.5%, and 0.1-1.7% genetic distance ranges obtained for P. carotovorum, P. versatile, and P. odoriferum, respectively. Five new haplotypes (denoted as HPc1-HPc5) were detected among cabbage strains of P. carotovorum, while one new haplotype was identified for both P. versatile (HPv1) and P. odoriferum (HPo1). None of the TCS haplotype networks provided evidence of significant correlation between geographic origin and the determined haplotypes, i.e., the infection origin. However, as haplotype network results are affected by the availability of sequencing data in public databases for the used genes and the number of analyzed strains, these findings may also be influenced by small sample size.
RESUMO
In this paper, a comprehensive overview of the 'Candidatus Liberibacter solanacearum' presence in Europe was provided. The analyzed findings revealed that, since the first appearance of this pathogen in Finland and Spain in 2008, it has spread to 13 new European countries. Therefore, 'Ca. L. solanacearum' has spread very quickly across the European continent, as evident from the emergence of new host plants within the Apiaceae, Urticaceae, and Polygonaceae families, as well as new haplotypes of this pathogen. Thus far, 5 of the 15 'Ca. L. solanacearum' haplotypes determined across the globe have been confirmed in Europe (haplotypes C, D, E, U, and H). Fully competent 'Ca. L. solanacearum' vectors include Bactericera cockerelli, Trioza apicalis, and B. trigonica; however, only T. apicalis and B. trigonica are presently established in Europe and are very important for plants from the Apiaceae family in particular. Moreover, psyllid species such as B. tremblayi, T. urticae, and T. anthrisci have also been confirmed positive for 'Ca. L. solanacearum'. Constant monitoring of its spread in the field (in both symptomatic and asymptomatic plants), use of sensitive molecular diagnostic techniques, and application of timely management strategies are, therefore, of utmost importance for the control of this destructive pathogen.
RESUMO
The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality (Backa, Vojvodina), known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented (contributing by 19.40% and 17.91%, respectively). Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes (proA, dnaX, icdA, and mdh) and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia.
RESUMO
In May 2021, a tomato producer reported an occurrence of a disease unknown so far in a greenhouse near Split, Croatia. About 30% of plants (cultivar Signora) have been affected. Symptoms resembled tomato pith necrosis, bacterial disease caused by Pseudomonas corrugata, known to occur sporadically in tomato greenhouse production in Croatia. Leaves on plants developed interveinal chlorosis, followed by necrosis and leaf collapse. When main stems were longitudinally cut, brown, disintegrated and water-soaked partly hollow pith was evident. Severely affected plants wilted. With suspicion on presence of P. corrugata, bacteria were isolated from surface-sterilized pith tissue of two tomato plants by plating onto sucrose peptone agar (SPA) and King's B medium (KB). Colonies recovered were cream-colored on SPA and non-florescent on KB. Two isolates, assigned as 1-KB and 3A, were first identified by amplification of internal transcribed spacer (ITS1) between16S rRNA and 23S rRNA using primers D21 and D22 (Manceau and Horvais 1997). The 550-bp PCR products obtained were purified and sequenced. Subsequent BLAST search showed the sequences to have 100% identity with the strain DSM 16733 isolated from tomato in Italy (Accession No. LT629790.1) and 99.77% identity with the strain SM664-12 isolated from tomato in USA (Acc. No. KC405207.1) of Pseudomonas mediterranea from NCBI. ITS sequence for one isolate 3A was deposited in GenBank under the Accession No. OP765279.1. Further identification was performed by using species-specific primers PC1/1-PC1/2 for P. mediterranea (Catara et al. 2000, 2002). Amplification of 600 bp DNA fragment confirmed the identity of isolates 1-KB and 3A as P. mediterranea. For this region sequence of isolate 3A was deposited in GenBank under the Acc. No. OP068273.1. Pathogenicity was assessed on tomato plants (cultivar Moneymaker) grown in pots in bio-chamber. Plants were grown at 25/20 °C 12h/12h dark/light regime until 8-leaves stage (BBCH 18). P. mediterranea isolate 3A was used for the inoculation. Inoculum was prepared from the isolate grown on KB medium for 48 h and suspended in sterile distilled water (concentration of 109 CFU mL-1) by dilution plate counts. Ten plants were inoculated with 10 µl of bacterial suspension injected into the stem with a syringe. Five control plants were inoculated with sterile distilled water. After 40 days of plant growth, symptoms were visible on all plants inoculated with P. mediterranea isolate 3A. Although no wilting was observed and all plants were alive, chlorosis was observed on upper leaves, chlorosis and necrosis on middle leaves, while basal leaves wilted. Longitudinal cross-sections of stems revealed brownish pith tissue with longitudinal watery pits spreading from inoculation points (Fig.S1). Symptoms were not observed on control plants. Bacterium was reisolated from three plants showing the most severe symptoms and proved to be identical to the original using species-specific primer pair PC1/1-PC1/2. To our knowledge, this is the first confirmation of P. mediterranea causing tomato pith necrosis in Croatia. Tomato pith necrosis caused by P. mediterranea may become significant bacterial disease of greenhouse tomato in Croatia.
