DIVERSITY OF Pectobacterium STRAINS BY BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR CHARACTERIZATION DIVERSIDADE DE ISOLADOS DE Pectobacterium spp. PELA CARACTERIZAÇÃO BIOQUÍMICA, FISIOLÓGICA E MOLECULAR

Pectobacterium is a complex taxon of strains with diverse characteristics. It comprises several genera, including Erwinia, Brenneria, Pectobacterium, Dickeya, and Pantoea. Pectobacterium and Dickeya cause diseases in a wide range of plants, including potatoes, where they are causative agents of soft rot in tubers and blackleg in field-grown plants. Characterizing Pectobacterium species allows for the analysis of the diversity of pectinolytic bacteria, which may support control strategies for plant bacterial diseases. The aim of this study was to perform biochemical, physiological, and molecular characterizations of Pectobacterium spp. from different sites and host plants. The isolated strains were characterized by the glucose fermentation test, Gram staining, catalase activity, oxidase activity, growth at 37 oC, reducing substances from sucrose, phosphatase activity, indole production, acid production from different sources (sorbitol, melibiose, citrate, and lactose), pathogenicity in potato, and hypersensitivity reactions. Molecular characterization was performed with species-specific primers ECA1f/ECA2r and EXPCCF/EXPCCR, which identify P. atrosepticum and P. carotovorum subsp. carotovorum (Pcc), respectively, and with primers 1491f/L1RA/L1RG and Br1f/L1RA/L1RG that differentiate Pcc from Dickeya chrysanthemi and from P. carotovorum subsp. brasiliensis. The strains were identified as belonging to the genus Pectobacterium, though they did not fit the biochemical nor the molecular classification standards for subspecies differentiation, indicating significant diversity among the strains.


INTRODUCTION
Potato propagation is potentially associated with the dissemination of pathogens, including bacteria belonging to the genus Pectobacterium. These bacteria, the causative agents of blackleg and soft rot diseases (LOPES; QUEZADO-DUVAL, 2001), are capable of producing pectinolytic enzymes that lead to plant death in the field and rot in potato tubers under either field or storage conditions. Controlling the diseases is difficult, since the bacteria present large genetic variability and can survive in a large number of host plants. This makes it difficult to select resistant cultivars (BRISOLLA et al., 2002).
In the potato crop, there is the possibility that other subspecies or even yet unidentified species of Pectobacterium are involved in blackleg and soft rot diseases (FESSEHAIE;LEVESQUE, 2002). This is a fact that may have important epidemiological implications; it may influence how specific control strategies are chosen to manage the diseases. Therefore, it is of great importance to correctly identify Pectobacterium strains.
There is a need for studies involving the identification and characterization of pectobacteria in potato tubers. Therefore, our aim was to perform biochemical, physiological, and molecular characterization of Pectobacterium strains isolated from potato plants at different sites and compare them to bacteria isolated from other hosts.
Genomic DNA from bacterial strains was extracted according to published protocol (SAMBROOK; FRITSCH; MANIATIS, 1989). The PCR reaction used primers ECA1f (5' -GAA CTT CGC ACC GCC GAC CTT CTA -3') and ECA2r (5' -GCA CAC TTC ATC CAG CGA -3') (DARRASSE et al., 1994) that amplify a 690-bp fragment particular of P. atrosepticum. PCR amplification was performed with an initial denaturation step at 94 °C for 5 min followed by 25 cycles of 94 °C for 30 s, 65 °C for 45 s, 72° C for 45 s and a final extension of 72 °C for 5 min. For all primers, the amplification reactions were performed in a final volume of 50 μL, containing 1X Taq buffer (Invitrogen), 0.2 mM of each dNTP, 2.5 mM MgCl2, 100 ng of each primer, 2.5 U of Taq DNA polymerase (Invitrogen, 5 U μL -1 ) and 100 ng of DNA template, in the GeneAmp 9700 thermocycler (Applied Biosystems). PCR products were loaded onto a 1.5% agarose gel with 0.20 μg 100 mL -1 of ethidium bromide and electrophoresed at 100 V for 40 min. Images were taken using a computerized gel analysis system (Kodak Digital Science 1D).

RESULTS AND DISCUSSION
All bacterial strains showed a round shape, smooth edges, 1 to 3 mm diameter convex colonies, smooth texture, brilliant appearance, opaque optical property, and yellowish cream color. They were Gram-negative, fermentative, catalase positive, and oxidase negative, meeting the phenotypic criteria to be classified as Pectobacterium KELMAN, 2001). Inoculated strains developed soft rot symptoms in potatoes and positive hypersensitivity reactions in tobacco leaves (Table  2).
Strains UFU A6 and UFU A47 (Table 2) did not produce acid from citrate, whereas strain UFU A7 was sorbitol positive. Strains UFU A7 and UFU A20 showed no reduction of substances from sucrose, but the latter showed phosphatase activity. UFU A14 was melibiose-positive for acid production. Based on phenotypic characteristics, it was not possible to differentiate the strains into species nor subspecies level, indicating wide biochemical diversity among them.
Researchers were able to detect biochemical diversity between Pectobacterium strains in Chinese cabbage (ALVARADO, 2006), potato tubers, ornamental (KANG; KWON;GO, 2003) and medicinal plants (HU et al., 2008). Alvarado (2006) reported strains of Pcc capable of growing at 37 ºC and producing acid from lactose, but some of them were ineffective at producing acid from sorbitol and had no phosphatase activity. Moreover, 10.2% of the strains evaluated by the author showed a reduction of substances from sucrose and 12.8% were unable to produce acid from melibiose. Opposite results were obtained in our study: the majority of the Pectobacterium strains did not reduce substances from sucrose but all of them showed positive reaction to acid production from melibiose.
It can be inferred that there is a great diversity among Pectobacterium species, making it difficult to identify them based only on biochemical and physiological parameters. The occurrence of strains with intermediate characteristics impairs correct classification of these bacteria PALMA, 2006 In the physiological and biochemical characterization of Pectobacterium strains isolated from a cold climate region of Brazil, the strains that grew at 37 °C did not necessarily fit the standard characteristics of P. atrosepticum . Those were later classified as a new subspecies, named P. carotovorum subsp. brasiliensis . In a study regarding the occurrence and diversity of pectinolytic bacteria in potato seed tubers, 119 strains were classified as P. carotovorum subsp. brasiliensis by biochemical tests, 96 as Pcc, and eight did not correspond to any species or subspecies previously described . Similar results were found in our study, in which strains recovered from different sites and host plants did not fit into any of the proposed subspecies, suggesting great genetic and phenotypic diversity among the strains imposed by evolutionary factors. Thus, continuous research is needed to characterize and categorize these microorganisms to provide information that can be applied in disease control strategies in agriculturally important crops. Since some strains did not fit within known subspecies characteristics, more tests that link the genetic, biochemical, and physiological heterogeneity of pectinolytic bacteria are necessary.
PCR products of 480 and 581 bp for strain UFU A9 (Figure 1) and absence of the 581 bp fragment for strain UFU A20 indicate that they belong to a new subspecies. No DNA amplification was visualized for strain UFU A6.
Primers Br1f/L1RG/L1RA are used to identify P. carotovorum subsp. brasiliensis by a 322 bp fragment. They generated amplicons only for strain UFU A33 (obtained from lettuce) of 352, 420, and 690 bp sizes (Figure 2). Those fragments cannot be associated to P. carotovorum subsp. brasiliensis.
Molecular analysis did not confirm the identification of the biochemically characterized strains, reinforcing the hypothesis that they belong to other species / subspecies or that the primers were not able to amplify the expected fragments.
Biosci. J., Uberlândia, v. 36, n. 2, p. 316-323, Mar./Apr. 2020 http://dx.doi.org/10.14393/BJ-v36n2a2020-46256    Hu et al. (2008), in the characterization of Pcc from a medicinal herb (Pinelli ternata), found two strains apparently classified as Pcc and P. carotovorum subsp. odoriferum, sharing 97-99% similarity. After conducting more precise studies, they concluded that the strains were Pcc. The authors reported that the differences between both strains and other Pcc species in Europe and North America are related to the geographic distribution and diversity of host plants. Several studies show that pectobacteria are genetically distinct, forming heterogeneous groups even within subspecies (DARRASSE et al., 1994).
Knowledge of diversity is an important prerequisite for the identification of phytobacteria, as well as for taxonomic classifications, to support epidemiological studies and the development of strategies for plant diseases control, especially regarding selection of resistant varieties in breeding programs. This is particularly important when different closely related species and subspecies cause diseases in the same host, as is the case of pectobacteria in potatoes DUARTE, 2006).

CONCLUSIONS
The strains evaluated in our study were characterized as belonging to the genus Pectobacterium. No further classification was accomplished using biochemical and molecular techniques, demonstrating their great diversity.