• Notas Científicas Erwinia chrysanthemi : pectolytic bacterium ...

  • FileName: 41n10a15.pdf [read-online]
    • Abstract: Index terms: Arracacia xanthorrhiza, Pectobacterium, peruvian carrot, disease, etiology. ... Pectobacterium. betavasculorum sp. nov. and Pectobacterium wasabi sp. nov. International ...

Download the ebook

Erwinia chrysanthemi and soft rot outbreaks of arracacha 1567
Notas Científicas
Erwinia chrysanthemi: pectolytic bacterium causing
soft rot outbreaks of arracacha in Brazil
Gilmar Paulo Henz(1), Francisco José Becker Reifschneider(2) and Valmir Duarte(3)
(1)Embrapa Hortaliças, Caixa Postal 218, CEP 70359-970 Brasília, DF, Brazil. E-mail: [email protected] (2)Consultative Group on
International Agricultural Research, 1818 H Street NW, 20433 Washington, DC, EUA. E-mail: [email protected] (3)Universidade
Federal do Rio Grande do Sul, Fac. de Agronomia, Caixa Postal 776, CEP 91501-970 Porto Alegre, RS, Brazil. E-mail: [email protected]
Abstract – 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’).
Index terms: Arracacia xanthorrhiza, Pectobacterium, peruvian carrot, disease, etiology.
Erwinia chrysanthemi: bactéria pectolítica envolvida na “mela”
da mandioquinha-salsa no Brasil
Resumo – O objetivo deste trabalho foi identificar as bactérias pectolíticas envolvidas na podridão-mole de
raízes de mandioquinha-salsa no Brasil. De 1998 a 2001, 227 isolados de Erwinia spp. foram obtidos de raízes de
mandioquinha-salsa e identificados por testes bioquímicos e fisiológicos (atividade pectolítica, lecitinase,
α-methyl glucosídeo, fosfatase, sensibilidade à eritromicina, crescimento a 37oC). Destes isolados, 89,9% foram
identificados como E. chrysanthemi (Ech), 9,7% como E. carotovora subsp. carotovora (Ecc) e somente 0,5%
como E. carotovora subsp. atroseptica. A identidade de 20 isolados representativos de Ech e Ecc foi confirma-
da por PCR (primers ‘149f’, ‘L1r’, ‘ADE1’, ‘ADE2’), com exceção de dois isolados de Ech e um de Ecc.
Termos para indexação: Arracacia xanthorrhiza, Pectobacterium, batata-baroa, podridão-mole, etiologia.
In tropical regions, postharvest losses of vegetable be placed in a separate genus (Pectobacterium) on the
crops can easily reach 30% due to poor handling basis of the 16S rDNA sequences. Gardan et al. (2003)
practices, diseases and inadequacy in packing and elevated three subspecies of Pectobacterium
refrigeration. Deterioration caused by pectolyctic bacteria carotovorum to species level (P. atrosepticum,
is one of the main causes of postharvest losses of P. betavascularum and P. wasabi). Presently, the
perishable products worldwide. taxonomy of Erwinia is in a state of flux (Yap et al.,
Soft rot of fleshy plant organs of vegetables is typically 2004), and the proposed name Pectobacterium by
caused by the Erwinia carotovora group, particularly, Hauben et al. (1998) has not yet been accepted by many
E. carotovora subsp. carotovora (Ecc), E. carotovora researchers working with this group of bacteria.
subsp. atroseptica (Eca) and E. chrysanthemi (Ech). In Brazil, Ecc was considered the most important
Identification of pectolytic erwinias is traditionally based species of the pectolytic group, causing losses in more
on biochemical and phenotypic characteristics (De Boer than fifty crop plants, such as lettuce, garlic, potato, sweet-
& Kelman, 2000), and more recently molecular potato, eggplant, zucchini, onion, carrot, cauliflower,
techniques have also been applied. As a result, Hauben arracacha, melon, cucumber, sweet-pepper, okra,
et al. (1998) suggested that the pectolytic bacteria should cabbage, tomato, chicory, collard, among others
Pesq. agropec. bras., Brasília, v.41, n.10, p.1567-1571, out. 2006
1568 G.P. Henz et al.
(Jabuonski et al., 1986; Michereff & Mariano, 1993). Arracacha roots with typical soft-rot symptoms were
Ech affects many hosts and is considered more important collected from wholesale and retail markets in São Paulo,
in tropical and warm areas, regarded as highly aggressive Paraná, Minas Gerais and Rio de Janeiro states and also
in temperatures above 30oC. Eca causes black-leg of in Brasília, DF. Bacterial isolates were obtained both
potatoes and is less prevalent in tropical regions, being straight from the rotted host tissues or by inoculating
considered as a cool temperature variant of Ecc. Recently, sweet pepper fruits with toothpicks previously
atypical strains causing blackleg of potato in Southern Brazil impregnated into rotted tissue in order to avoid secondary
were proposed as a new subspecies (E. carotovora subsp. bacterial growth (Takatsu et al., 1980).
brasiliensis) by Duarte et al. (2004). Bacteria were isolated in nutrient agar (NA) and Kado
During summer, the erwinia soft rot of arracacha roots & Heskett ‘523’ media and then incubated at 28oC for
can cause losses of up 100% in only three days after two days (Klement et al., 1990; De Boer & Kelman,
harvest (Henz, 2001). In Brazil, the problem was first 2000). Single bacterial colonies resembling Erwinia were
described by Silva (1967) as “mela”, the Portuguese word harvested and tested for pectolytic activity on arracacha
that better describes the sticky soft rot of the roots, but roots and sweet pepper fruits kept on moist chambers.
the author was unable to properly identify the causal Isolates tested positive for pectolytic activity were
agent. During the seventies, Camino & Díaz Polanco submitted to the following physiological and biochemical
(1972) described Erwinia amylovora as the causal tests: Gram test; growth at 37oC; reducing substances
agent of soft rot in arracacha plants in Venezuela, and from sucrose; phosphatase; lecithinase; α-methyl
later on Zapata & Pardo (1974) reported the occurrence glucoside; erythromycin sensitivity; growth in NaCl 5%
of Erwinia sp. in arracacha plants in Colombia. Romeiro (Klement et al., 1990; De Boer & Kelman, 2000). Based
et al. (1988) published the first paper identifying the on these test results, the isolates were categorized into
causal agent of the disease at the subspecific level, species of Erwinia and subspecies of Erwinia
associating Erwinia carotovora subsp. carotovora with carotovora. After that, all Erwinia isolates were kept
soft rot of arracacha roots in Minas Gerais State, Brazil. in sterile distilled water for further identification.
The same group presented the report in an annual meeting, To confirm the identity, forty representative
and later on a survey on bacterial diseases of vegetable isolates were chosen by hazard and sent for additional
crops (Gomide & Romeiro, 1992). In the Distrito Federal identification to the Centre of Expertise for Potato
area, the three most important pectolytic erwinias (Ecc, Diseases, Canadian Food Inspection Agency, in
Eca and Ech) were found associated to the soft rot of Charlottetown, Canada. The IGS region of the DNA of
arracacha (Henz, 2001). Lopes & Quezado-Soares (1997) Erwinia isolates were amplified with primers IGS
considered Ecc as the most important subspecies causing ‘1491f’ and ‘L1r’ (Fessehaie et al., 2002) and by primers
soft rot in arracacha roots in Brazil. ‘ADE1’ and ‘ADE2’ (Nassar et al., 1996). After
Although diseases caused by Erwinia spp. are very 25 cycles, the results of PCR were separated by agarose
harmful and occur in a large number of agricultural gel (1%) electroforese for one hour. The PCR-IGS were
crops in Brazil, there are few papers published on performed at the following conditions: 94oC/2 min, (94oC/
the pathogen recently. In a survey on potato plants 45 sec, 62oC/45 sec, 72oC/90 min) 25X, 72oC/10 min,
showing blackleg symptoms in 22 fields of nine 4oC/5 min (Duarte et al., 2004). Two representative
counties in Rio Grande do Sul State, 55% of the strains isolates of Ech from arracacha roots were also
were identified as Eca, 44% as Ecc and only 1% as submitted to the Biolog system, based on the utilization
Ech (Oliveira et al., 2003). Arracacha soft rot of 95 sources of carbon.
continues to be a very important postharvest disease More than 400 bacterial isolates were obtained from
during summertime in Brazil, causing severe losses. arracacha roots with typical soft-rot symptoms during a
Since some new methods to control the soft rot of four-year period (1998–2001). According to the
arracacha roots are being tested, it is important to identification with the traditional biochemical tests
determine the prevailing bacterial species or (Klement et al., 1990; De Boer & Kelman, 2000),
subspecies. 204 isolates were characterized as Ech, 22 as Ecc and
The objective of this research was to identify the one as Eca. Twenty isolates of Erwinia from arracacha
pectolytic erwinias associated to the outbreaks of the roots were also submitted to PCR. Identification of six
soft rot on arracacha in Brazil. isolates of Ech was confirmed by specific primers
Pesq. agropec. bras., Brasília, v.41, n.10, p.1567-1571, out. 2006
Erwinia chrysanthemi and soft rot outbreaks of arracacha 1569
‘ADE1’ and ‘ADE2’ (Table 1). For primers IGS ‘1491f’ confirmed by the primers used (Table 1). Additional
and ‘L1r’, 13 isolates showed two close bands (600 pb), biochemical tests were performed for these isolates
typical of E. carotovora isolates, and seven showed two (phosphatase, α-methyl glucoside, lecithinase), and the
more separated bands, typical of E. chrysanthemi identity of isolate ‘Q1’ was confirmed as Ech and isolate
(Figure 1). Primers IGS ‘1491f’ and ‘L1r’ developed ‘46’ as Ecc. Isolate ‘P14’ was weakly positive for
by Fessehaie et al. (2002) are universal for phosphatase, negative for lecitinase and α-methyl
enterobacteria, including the pectolytic erwinias. Primers glucoside, and apparently belonged to E. carotovora,
‘ADE1’ and ‘ADE2’ recognize pel genes, which code as confirmed by the primers. The identity of a second
for the yield of pectolytic enzymes specific for Ech group of 22 Ech isolates from arracacha was confirmed
(Nassar et al., 1996), an important trait for separating by primers ADE1’ and ‘ADE2’.
this species from E. carotovora. Finally, the two isolates of Ech, ‘B2’ and ‘154’,
Only six isolates (‘46’, ‘53’, ‘P5’, ‘P6’, ‘Q5’, ‘Q20’) submitted to the Biolog system showed, respectively, 96.7
showed the same band as the check strain (Ech 571), and 89.1% of similarity, higher than the two check
while other isolates did not show this RNA sequence isolates, confirming their identity. The combination of
(Figure 2). Isolate ‘Q2”, identified as Ech by biochemical traditional biochemical and physiological tests with
tests and primers IGS, did not have the characteristic molecular tools seems to be the best way to confirm the
band for the species with primers ‘ADE1’ and ‘ADE2’, identity of the pectolytic erwinias, since the taxonomy
probably due to a deficiency in this specific sequence. of this group is in a flux (Yap et al., 2004). Traditional
Isolates ‘P14’ and ‘Q1’ were identified as Ech and identification of pectolytic erwinias is still useful, when
isolate ‘46’ as Ecc by biochemical tests, which was not molecular tools are not available.
Table 1. Identification of isolates of pectolytic Erwinia by
PCR with primers IGS ‘1491f’ and ‘L1r’ and “ADE1” and
Figure 1. Result of PCR amplification of IGS rDNA 16S-23S
regions of Erwinia with primers “149LF” and “L1ra”. Lines:
1: strain ‘571’ (Ech); 2: strain ‘31’ (Eca); 3: strain ‘71’ (Ecc);
4: strain ‘a’; 5: strain ‘B2’; 6: strain ‘B8’; 7: strain ‘B11’;
8: strain ‘B12’; 9: strain ‘C1’; 10: strain ‘C3’; 11: strain ‘C6’;
12: strain ‘C7’; 13: strain ‘C9’; 14: strain ‘C16’; 15: strain ‘D5’;
16: strain ‘D7’; 17: strain ‘D9’; 18: strain ‘I’; 19: strain ‘154’;
(1) Ecc: Erwinia carotovora subsp. carotovora; Eca: E. carotovora subsp. 20: strain ‘155’; 21: strain ‘161’; 22: strain ‘163’; 23: strain
atroseptica; Ech: E. chrysanthemi. (2)+: positive of 380 bp and 480 bp. ‘164’; 24: strain ‘166’; 25: strain ‘171’; 26: strain ‘F5’ (Eca);
(3) Ec: E. carotovora; Ech: E. chrysanthemi. (4)Isolates with discordant
27: strain ‘P1’ (Ecc); and 28: ‘tomato’ strain (Ec). IGS 16S-23S;
results: strain ‘46’: confirmed as Ech by PCR and biochemical tests;
strain ‘Q2’: confirmed as Ech but negative with primers “ADE1” and E. chrysanthemi (Ech): 354–356 (smaller) and 480 pb (larger);
“ADE2”. E. carotovora (Ec): 440–453 (smaller) and 475–490 pb (larger).
Pesq. agropec. bras., Brasília, v.41, n.10, p.1567-1571, out. 2006
1570 G.P. Henz et al.
Figure 2. Result of PCR amplification of genes pel fragments of Erwinia chrysanthemi with
primers “ADE1” e “ADE2”. (Ech isolates had one band: ‘46’, ‘53’, ‘P5’, ‘P6’, ‘Q5’, ‘Q20’; Eca, Ecc
and check (‘agua’) show no band).
The first papers on soft rot of arracacha in Venezuela is usually considered more aggressive than Ecc and Eca
(Camino & Díaz Polanco, 1972) and Colombia (Zapata (Pérombelon et al., 1995), corrobating the results of these
& Pardo, 1974) only identified correctly the genus papers.
(Erwinia). The predominance of Ech (89.8%) causing
soft rot in arracacha roots in Brazil is a novel finding, References
since previous papers reporting the disease considered
Ecc as the predominant one (Romeiro et al., 1988; Lopes CAMINO, J.M.; DÍAZ POLANCO, C. Identificación de una
& Quezado-Soares, 1997), although based on the bacteriosis en apio (Arracacia xanthorrhiza). Agronomia Tropical,
v.22, p.563-567, 1972.
identification of few isolates. Lately, Oliveira et al. (2003)
DE BOER, S.H.; KELMAN, A. Gram-negative bacteria: Erwinia
described Eca (55%) and Ecc (44%) as the predominant
soft rot group. In: SCHAAD, N.W.; JONES, J.B.; CHUN, W. (Ed.).
subspecies in potato fields in Southern Brazil, which was Laboratory guide for identification of plant pathogenic bacteria.
unexpected, and more recently, Duarte et al. (2004) St. Paul: APS, 2000. p.56-72.
reported a new, atypical potato blackleg strain called DUARTE, V.; DE BOER, S.H.; WARD, L.J.; OLIVEIRA, A.M.R.
E. carotovora subsp. brasiliensis. These recent Characterization of atypical Erwinia carotovora strains causing
findings justify the survey and identification of a large blackleg of potato in Brazil. Journal of Applied Microbiology,
v.96, p.535-545, 2004.
amount of pectolytic erwinias isolates to set control
measures and better understand disease epidemiology. FESSEHAIE, A.; DE BOER, S.H.; LÉVESQUE, C.A. Molecular
characterization of DNA encoding 16S-23S rRNA intergenic spacer
Soft rot in arracacha roots usually occurs during regions and 16S rRNA of pectolytic Erwinia species. Canadian
summertime (December to March), which coincides Journal of Microbiology, v.48, p.387-398, 2002.
with warmer temperatures in Southern Brazil (Henz, GARDAN, L.; GOUY, C.; CHRISTEN, R.; SAMSON, R. Elevation
2001). In environmental conditions such as these, Ech of three subspecies of Pectobacterium carotovorum to species level:
Pesq. agropec. bras., Brasília, v.41, n.10, p.1567-1571, out. 2006
Erwinia chrysanthemi and soft rot outbreaks of arracacha 1571
Pectobacterium atrosepticum sp. nov., Pectobacterium Characterization of Erwinia chrysanthemi by pectolytic isozyme
betavasculorum sp. nov. and Pectobacterium wasabi sp. nov. polymorphism and restriction fragment lenght polymorphism
International Journal of Systematic and Evolutionary analysis of PCR-amplified fragments of pel genes. Applied and
Microbiology, v.53, p.381-391, 2003. Environmental Microbiology, v.62, p.2228-2235, 1996.
GOMIDE, A.F.; ROMEIRO, R.S. Levantamento de doenças OLIVEIRA, A.M.R.; DUARTE, V.; SILVEIRA, J.R.P.; MORAES,
bacterianas em hortaliças na região do cinturão verde de Belo M.G. Incidence of pectolytic erwinias associated with blackleg of
Horizonte. Fitopatologia Brasileira, v.17, p.47-52, 1992. potato in Rio Grande do Sul. Fitopatologia Brasileira, v.28, p.49-
M.; MERGAERT, J.; VERDONCK, L.; SWINGS, J. Phylogenetic PÉROMBELON, M.C.M.; SALMOND, G.P.C. Bacterial soft rots.
position of phytopathogens within the Enterobacteriaceae. In: SINGH, U.S.; SINGH, R.P.; KOSHMOTO, K. Pathogenesis
Systematic Applied Microbiology, v.21, p.384-397, 1998. and host specificity in plant diseases: histopathological,
HENZ, G.P. Perdas pós-colheita e métodos de manejo da biochemichal, genetic and molecular bases. Oxford: Pergamon/Elsevier
podridão-mole causada por Erwinia chrysanthemi e Erwinia Science, 1995. p.1-20.
carotovora subspp. em mandioquinha-salsa (Arracacia ROMEIRO, R.S.; SOUSA, R.M.; MUCHOVEJ, J.J.; KIMURA,
xanthorrhiza Bancroft). 2001. 256p. Tese (Doutorado) - O. Soft rot of Peruvian carrot due to Erwinia carotovora in Brazil.
Universidade de Brasília, Brasília. Plant Pathology, v.37, p.300-302, 1988.
JABUONSKI, R.E.; TAKATSU, A.; REIFSCHNEIDER, F.J.B. SILVA, J.R. Principais aspectos da cultura da mandioquinha-salsa
Levantamento e identificação de espécies de Erwinia de diferentes ou batata-baroa. FIR, v.10, p.32-37, 1967.
plantas hospedeiras e regiões do Brasil. Fitopatologia Brasileira,
v.11, p.185-195, 1986. TAKATSU, A.; MELO, S.; GARCIA, E.J. Fruto do pimentão como
meio parcialmente seletivo para o isolamento de Erwinia carotovora.
KLEMENT, Z.; RUDOLPH, K.; SANDS, D.C. (Ed.). Methods in Fitopatologia Brasileira, v.6, p.550-551, 1980.
phytobacteriology. Budapest: Akademiai Kiado, 1990. 657p.
LOPES, C.A.; QUEZADO-SOARES, A.M. Doenças bacterianas diversity of Erwinia carotovora subsp. carotovora and its correlation
das hortaliças: diagnose e controle. Brasília: Embrapa-CNPH/ with virulence. Applied and Environmental Microbiology, v.70,
Embrapa-SPI, 1997. 70p. p.3013-3023, 2004.
MICHEREFF, S.J.; MARIANO, R.L.R. Gênero Erwinia no Brasil. ZAPATA, M.A.; PARDO, V.M. Estudios sobre el
Summa Phytopathologica, v.19, p.137-144, 1993. marchitamiento de la arracacha (Arracacia xanthorrhiza) causado
NASSAR, A.; DARRASSE, A.; LEMATTRE, M.; por Erwinia sp. Revista Facultad Nacional de Agronomia,
KOTOUJANSKY, A.; DERVIN, C.; VEDEL, R.; BERTHEAU, Y. v.29, p.39-42, 1974.
Received on November 28, 2005 and accepted on September 28, 2006
Pesq. agropec. bras., Brasília, v.41, n.10, p.1567-1571, out. 2006

Use: 0.0272