Erwinia chrysanthemi : bactéria pectolítica envolvida na “ mela ” da mandioquinha-salsa no Brasil

The objetive of this work was to identify the pectolytic bacteria associated with soft rot of arracacha roots in Brazil. From 1998 to 2001, 227 isolates of Erwinia spp. were obtained from arracacha roots and identified by biochemical and physiological tests (pectolytic activity, lecithinase, α-methyl glucoside, phosphatase, erythromycin sensivity, growth at 37oC). Of these isolates, 89.9% were identified as E. chrysanthemi (Ech), 9.7% as E. carotovora subsp. carotovora (Ecc) and 0.5% as E. carotovora subsp. atroseptica. The identity of seventeen out of twenty representative isolates of Ech and Ecc was confirmed by PCR (primers ‘149f’, ‘L1r’, ‘ADE1’, ‘ADE2’).

In tropical regions, postharvest losses of vegetable crops can easily reach 30% due to poor handling practices, diseases and inadequacy in packing and refrigeration. Deterioration caused by pectolyctic bacteria is one of the main causes of postharvest losses of perishable products worldwide.
Soft rot of fleshy plant organs of vegetables is typically caused by the Erwinia carotovora group, particularly, E. carotovora subsp. carotovora (Ecc), E. carotovora subsp. atroseptica (Eca) and E. chrysanthemi (Ech). Identification of pectolytic erwinias is traditionally based on biochemical and phenotypic characteristics (De Boer & Kelman, 2000), and more recently molecular techniques have also been applied. As a result, Hauben et al. (1998) suggested that the pectolytic bacteria should be placed in a separate genus (Pectobacterium) on the basis of the 16S rDNA sequences. Gardan et al. (2003) elevated three subspecies of Pectobacterium carotovorum to species level (P. atrosepticum, P. betavascularum and P. wasabi). Presently, the taxonomy of Erwinia is in a state of flux (Yap et al., 2004), and the proposed name Pectobacterium by Hauben et al. (1998) has not yet been accepted by many researchers working with this group of bacteria.
In Brazil, Ecc was considered the most important species of the pectolytic group, causing losses in more than fifty crop plants, such as lettuce, garlic, potato, sweetpotato, eggplant, zucchini, onion, carrot, cauliflower, arracacha, melon, cucumber, sweet-pepper, okra, cabbage, tomato, chicory, collard, among others (Jabuonski et al., 1986;Michereff & Mariano, 1993). Ech affects many hosts and is considered more important in tropical and warm areas, regarded as highly aggressive in temperatures above 30 o C. Eca causes black-leg of potatoes and is less prevalent in tropical regions, being considered as a cool temperature variant of Ecc. Recently, atypical strains causing blackleg of potato in Southern Brazil were proposed as a new subspecies (E. carotovora subsp. brasiliensis) by Duarte et al. (2004).
During summer, the erwinia soft rot of arracacha roots can cause losses of up 100% in only three days after harvest (Henz, 2001). In Brazil, the problem was first described by Silva (1967) as "mela", the Portuguese word that better describes the sticky soft rot of the roots, but the author was unable to properly identify the causal agent. During the seventies, Camino & Díaz Polanco (1972) described Erwinia amylovora as the causal agent of soft rot in arracacha plants in Venezuela, and later on Zapata & Pardo (1974) reported the occurrence of Erwinia sp. in arracacha plants in Colombia. Romeiro et al. (1988) published the first paper identifying the causal agent of the disease at the subspecific level, associating Erwinia carotovora subsp. carotovora with soft rot of arracacha roots in Minas Gerais State, Brazil. The same group presented the report in an annual meeting, and later on a survey on bacterial diseases of vegetable crops (Gomide & Romeiro, 1992). In the Distrito Federal area, the three most important pectolytic erwinias (Ecc, Eca and Ech) were found associated to the soft rot of arracacha (Henz, 2001). Lopes & Quezado-Soares (1997) considered Ecc as the most important subspecies causing soft rot in arracacha roots in Brazil.
Although diseases caused by Erwinia spp. are very harmful and occur in a large number of agricultural crops in Brazil, there are few papers published on the pathogen recently. In a survey on potato plants showing blackleg symptoms in 22 fields of nine counties in Rio Grande do Sul State, 55% of the strains were identified as Eca, 44% as Ecc and only 1% as Ech (Oliveira et al., 2003). Arracacha soft rot continues to be a very important postharvest disease during summertime in Brazil, causing severe losses. Since some new methods to control the soft rot of arracacha roots are being tested, it is important to determine the prevailing bacterial species or subspecies.
The objective of this research was to identify the pectolytic erwinias associated to the outbreaks of the soft rot on arracacha in Brazil.
Arracacha roots with typical soft-rot symptoms were collected from wholesale and retail markets in São Paulo, Paraná, Minas Gerais and Rio de Janeiro states and also in Brasília, DF. Bacterial isolates were obtained both straight from the rotted host tissues or by inoculating sweet pepper fruits with toothpicks previously impregnated into rotted tissue in order to avoid secondary bacterial growth (Takatsu et al., 1980). Bacteria were isolated in nutrient agar (NA) and Kado & Heskett '523' media and then incubated at 28 o C for two days (Klement et al., 1990;De Boer & Kelman, 2000). Single bacterial colonies resembling Erwinia were harvested and tested for pectolytic activity on arracacha roots and sweet pepper fruits kept on moist chambers. Isolates tested positive for pectolytic activity were submitted to the following physiological and biochemical tests: Gram test; growth at 37 o C; reducing substances from sucrose; phosphatase; lecithinase; α-methyl glucoside; erythromycin sensitivity; growth in NaCl 5% (Klement et al., 1990;De Boer & Kelman, 2000). Based on these test results, the isolates were categorized into species of Erwinia and subspecies of Erwinia carotovora. After that, all Erwinia isolates were kept in sterile distilled water for further identification.
To confirm the identity, forty representative isolates were chosen by hazard and sent for additional identification to the Centre of Expertise for Potato Diseases, Canadian Food Inspection Agency, in Charlottetown, Canada. The IGS region of the DNA of Erwinia isolates were amplified with primers IGS '1491f' and 'L1r' (Fessehaie et al., 2002) and by primers 'ADE1' and 'ADE2' (Nassar et al., 1996). After 25 cycles, the results of PCR were separated by agarose gel (1%) electroforese for one hour. The PCR-IGS were performed at the following conditions: 94 o C/2 min, (94 o C/ 45 sec, 62 o C/45 sec, 72 o C/90 min) 25X, 72 o C/10 min, 4 o C/5 min (Duarte et al., 2004). Two representative isolates of Ech from arracacha roots were also submitted to the Biolog system, based on the utilization of 95 sources of carbon.
More than 400 bacterial isolates were obtained from arracacha roots with typical soft-rot symptoms during a four-year period (1998)(1999)(2000)(2001). According to the identification with the traditional biochemical tests (Klement et al., 1990;De Boer & Kelman, 2000), 204 isolates were characterized as Ech, 22 as Ecc and one as Eca. Twenty isolates of Erwinia from arracacha roots were also submitted to PCR. Identification of six isolates of Ech was confirmed by specific primers 'ADE1' and 'ADE2' (Table 1). For primers IGS '1491f' and 'L1r', 13 isolates showed two close bands (600 pb), typical of E. carotovora isolates, and seven showed two more separated bands, typical of E. chrysanthemi (Figure 1). Primers IGS '1491f' and 'L1r' developed by Fessehaie et al. (2002) are universal for enterobacteria, including the pectolytic erwinias. Primers 'ADE1' and 'ADE2' recognize pel genes, which code for the yield of pectolytic enzymes specific for Ech (Nassar et al., 1996), an important trait for separating this species from E. carotovora.
Isolates 'P14' and 'Q1' were identified as Ech and isolate '46' as Ecc by biochemical tests, which was not confirmed by the primers used (Table 1). Additional biochemical tests were performed for these isolates (phosphatase, α-methyl glucoside, lecithinase), and the identity of isolate 'Q1' was confirmed as Ech and isolate '46' as Ecc. Isolate 'P14' was weakly positive for phosphatase, negative for lecitinase and α-methyl glucoside, and apparently belonged to E. carotovora, as confirmed by the primers. The identity of a second group of 22 Ech isolates from arracacha was confirmed by primers ADE1' and 'ADE2'.
Finally, the two isolates of Ech, 'B2' and '154', submitted to the Biolog system showed, respectively, 96.7 and 89.1% of similarity, higher than the two check isolates, confirming their identity. The combination of traditional biochemical and physiological tests with molecular tools seems to be the best way to confirm the identity of the pectolytic erwinias, since the taxonomy of this group is in a flux (Yap et al., 2004). Traditional identification of pectolytic erwinias is still useful, when molecular tools are not available.  The first papers on soft rot of arracacha in Venezuela (Camino & Díaz Polanco, 1972) and Colombia (Zapata & Pardo, 1974) only identified correctly the genus (Erwinia). The predominance of Ech (89.8%) causing soft rot in arracacha roots in Brazil is a novel finding, since previous papers reporting the disease considered Ecc as the predominant one (Romeiro et al., 1988;Lopes & Quezado-Soares, 1997), although based on the identification of few isolates. Lately, Oliveira et al. (2003) described Eca (55%) and Ecc (44%) as the predominant subspecies in potato fields in Southern Brazil, which was unexpected, and more recently, Duarte et al. (2004) reported a new, atypical potato blackleg strain called E. carotovora subsp. brasiliensis. These recent findings justify the survey and identification of a large amount of pectolytic erwinias isolates to set control measures and better understand disease epidemiology.
Soft rot in arracacha roots usually occurs during summertime (December to March), which coincides with warmer temperatures in Southern Brazil (Henz, 2001). In environmental conditions such as these, Ech is usually considered more aggressive than Ecc and Eca (Pérombelon et al., 1995), corrobating the results of these papers.