J. Phytopathology 156, 281–286 (2008) 2008 The Authors Journal compilation 2008 Blackwell Verlag, Berlin
doi: 10.1111/j.1439-0434.2007.01355.x
Fars Research Center for Agriculture and Natural Resources, Fars, Zarghan, Iran
Characterization of Iranian Pectobacterium carotovorum Strains from Sugar Beet by Phenotypic Tests and Whole-cell Proteins Profile A. Fassihiani1 and R. Nedaeinia2 AuthorsÕ addresses: 1Fars Research Center for Agriculture and Natural Resources, PO Box 73415-111, Fars, Zarghan, Iran; 2 Microbiology Department, Undersecretary for Food and Drug, Hazar Jarib, PO Box 81686-34171, Isfahan, Iran (correspondence to A. Fassihiani. E-mail:
[email protected]) Received April 3, 2007; accepted July 26, 2007 Keywords: Pectobacterium, Erwinia, sugar beet, soft rot, Iran
Abstract Thirty isolates of Pectobacterium carotovorum from soft rot-affected sugar beet plants in the Fars province of Iran were characterized phenotypically and by analysis of whole-cell protein electrophoresis patterns. The isolates were found to be heterogeneous based on the results of physiological and biochemical tests and protein profiles. The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 27% of the collected isolates (phenon 4) could be identified as P. betavasculorum when compared with reference strains. Strains of the first, second, third and fifth phenon shared similar characters with those of P. carotovorum subsp. carotovorum, P. betavasculorum and P. carotovorum subsp. odoriferum, but were distinct from these subspecies. Inoculation of phenon 4 isolates into wounded sugar beet petioles led to black streaking, root rot and vascular necrosis. Other isolates were incapable of causing systemic symptoms in inoculated plants.
Introduction The areas under cultivation of sugar beet (Beta vulgaris L.) in Iran during the last 4 years were 174 000 ha yielding 5 397 000 tons of root and 660 000 tons of sucrose (Sheikholeslami, 2006). One of the factors contributing to low yield is root rot. It has long been known that root rot in sugar beet is caused by several soil-borne pathogens including Pythium aphenidermatum (Fatemi, 1971; Fassihiani, 1991; Banihashemi, 1998), Phytophthora drechesleri (Ershad, 1971), Ph. cryptogea (Banihashemi, 1998) and Rhizoctonia solani (Hedjaroude and Alizadeh, 1970). A Gram-negative bacterium was recently isolated from rotting sugar beet roots from the Fars province of Iran. It was associated with symptoms induced by root rot pathogens such as Pythium and Phytophthora species while there is no previous report of involvement of bacteria in sugar beet root rot in Iran. Erwinia carotovora subsp.
betavasculorum (Ecb) has been known as one of the most important causes of vascular necrosis and soft rot of sugar beet in other countries (Thomson et al., 1977 & Saleh et al., 1996). The latter causes vascular necrosis in the roots and black streaking running up the petioles, black internal rot of the tap root, foamy white exudates on the crown and petioles later in the season; the area surrounding the necrotic bundles turn pink to reddish brown after exposure to air for a few minutes. Bacterial root rot of sugar beet was first reported in 1973 in California (Thomson et al.,1973). Subsequently, it was found in Washington, Arizona and Idaho (Ruppel et al., 1975; Stanghellini et al., 1977; Thomson et al., 1977). The E. carotovora is divided into the five subspecies: atroseptica, carotovora, betavasculorum, odorifera and wasabiae (De Boer and Kelman, 2001). E. c. betavasculorum, E. c. atrosepticum (Eca) and E. c. wasabiae (Ecw) have been elevated to species status and renamed Pectobacterium betavascularum (Pb), P. atrosepticum and P. wasabiae, while the name of other two subspecies remained unchanged (Gardan et al., 2003). This study describes the use of phenotypic tests in combination with sodium dodecyle sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole-cell proteins for: (i) identification of this bacterium in southern Iran and differentiation of other isolates of P. carotovorum associated with sugar beet root rot.
Materials and Methods Isolation
Thirty isolates were obtained from diseased sugar beet during 2001–2003 growing seasons from different counties in the Fars province of Iran. Isolation was made from the margin of necrotic vascular bundles of diseased roots. Infected tissues were surface sterilized with 70% ethanol and ground in 5 ml sterilized-distilled water (SDW) and a loopful of the suspension was streaked on Eosin Methylen Blue Agar and nutriwww.blackwell-synergy.com
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ent agar. Colonies were further purified on nutrient agar. All strains were maintained on nutrient agar during this study. Hypersensitivity tests on tobacco were performed for all strains according to Kelman et al. (1964). Pathogenicity tests
All bacterial strains were grown on nutrient agar for 72 h at 28C. Two-month-old sugar beet seedlings (cv. P22) were inoculated by inserting a small amount of bacterial cells into the petioles using a sterile toothpick (Thomson et al., 1977). The plants were placed in clear polyethylene bag for 48 h at 28C in a growth room and then unbagged. Control plants were treated with SDW. Ten plants were inoculated with each isolate. Six-month-old sugar beet plants were also inoculated by the same method under greenhouse conditions. Plants were maintained in the greenhouse at 25–30C. All Plants were harvested 3–5 weeks after inoculation, sliced and examined for vascular necrosis and root rot.
had reached the separation gel. The current was then increased to 30 mA until the dye had migrated approximately 100 mm through the separation gel. The gels were removed and fixed in a mixture of 40% methanol, 10% acetic acid and 50% deionized water. The gels were stained with 0.1% Coomassie Brilliant Blue R-250. They were observed on a white light viewing illuminator and then photographed. Relative masses of protein band were calculated by interpolation from the known standards.
Results Pathogenicity tests
Tests used for identification of Pectobacterium species isolated from sugar beet are listed in Table 1. Tests were carried out according to methods described previously (Dye, 1969; Stanghellini et al., 1977; Thomson et al., 1981; Schaad, 1988). Acid production from various substrates, reducing substances from sucrose, growth in 7–9% NaCl and growth at 37C was determined according to methods of Dye (1968). Characteristics were included in a numerical taxonomy analysis for 30 strains. A distance matrix was calculated using the Jaccard coefficient and a cluster analysis was performed using the UPGMA algorithm (Sneath and Sokal, 1973).
Bacterial vascular necrosis of sugar beet caused both wilt and root rot symptoms in sugar beet. Disease symptoms were induced on all inoculated 2-month-old sugar beet plants. Black streaking on petioles was observed 3–5 days after inoculation at 28C in a growth room (Fig. 1b) and root rot and vascular necrosis were developed in 3–5 weeks in a greenhouse at 25–30C (Fig. 1c). Leaf symptoms included black streaking of the petioles and froth in the centres of the crowns, which is produced by escaping gases produced by bacterial fermentation (Fig. 1d). Root symptoms varied from soft rot to dry rot (Fig. 1a). Disease symptoms on sugar beets in the Field and on artificially inoculated plants were consistent with those described elsewhere (Thomson et al., 1977). Not all P. carotovora strains were able to cause vascular necrosis and root rot on sugar beet plants. Only 27% of the strains (eight isolates) were able to cause disease symptoms described above. Other isolates failed to cause typical vascular necrosis and root rot, although they induced restricted rot and non-systemic symptoms at the inoculation sites on the petioles. The same bacteria were re-isolated from diseased sugar beet roots in all cases.
Electrophoresis of whole-cell protein
Phenotypic tests
Pectobacterium species were characterized by one dimensional SDS–PAGE of whole-cell protein according to the method of Laemmli (1970). They were grown in nutrient agar medium at 28C for 48 h. Bacterial cells were harvested and washed by centrifugation (6700 · g for 10 min) twice in sterile-deionized water. The suspension was adjusted to A600 = 1.8 to 2 with a spectrophotometer and mixed with lysis buffer (1.25 mm Tris–HCl, pH 6.8; 4% SDS; 10% 2-mercaptoethanol; 20% glycerol; and 0.01% bromophenol blue). Suspensions were heated at 95C for 10 min before loading on a discontinous gel. The discontinous gel was comprised of 40 mm of stacking gel on top of more than 100 mm of separation gel. The total acrylamide concentration in stacking and separating gel was 4% and 10%, respectively. The standard was a low-range protein marker (14 400–97 400 Da). Electrophoresis was carried out in discontinuous system of Laemmli (1970) at 10C in a vertical slab filled with electrophoresis buffer (25 mm Tris, 192 mm glycine and 0.1% SDS, pH 8.3). A constant current of 20 mA per gel was applied until the bromophenol blue
The results of biochemical and physiological properties of the bacterial isolates are presented in Table 1. The bacterial strains isolated from sugar beet were Gramnegative, oxidase-negative, catalase-positive and fermented glucose. They possessed peritrichous flagella, were rod shaped, motile, and able to rot potato slices and grew at 37C. All isolates gave positive hypersensitive reaction on tobacco leaves. Therefore, they were identified as member of the species P. carotovorum. The dendrogram of phenotypic distances among 30 strains is shown in Fig. 2. At a distance of 0.77, five phenons and one unclustered strain were observed. Phenon 1 corresponded to 6 strains and was most similar to Pcc in regard to acetoin and methyl red production, acid production from melibiose and cellobiose, H2S production, and inability to form reducing substances from sucrose but they had common characteristics with Pb (Ecb479) and Pco in regard to the ability to form acid from a-methyl glucoside, palatinose and maltose. Phenon 2 corresponded to seven strains and was similar to P. c. subsp. carotovorum (Pcc) in regard to
Identification Phenotypic tests
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Table 1 Physiological properties of Iranian Pectobacterium carotovorum strains from sugar beet compared with other reference P. carotovorum subspecies Erwinia carotovora subspeciesb
Iranian strains groups Characteristic Gram reaction Catalase production Phosphotase activity Oxidase production Growth at 37C Nitrate reduction Flourescent production on KB Fermentative metabolism Aesculin hydrolysis Arginine dihydrolae Pigment production H2S production from thiosulphate Potato soft rot Gelatin liquefaction Litmus milk Acidification Coagulation Reduction litmus Alkali form Methyl red(MR) Acetoin production (VP) Gas from glucose Reducing substance from sucrose H2S from cystein Indole production Erythromycine sensitivity Motility Growth on 5%NaCl Growth on 8%NaCl Growth on 9%NaCl Urease production Acid production from Ribose Xylose Glucose Lactose Sucrose Maltose a-methyl glucoside Melibiose Cellobiose Trehalose Sorbitol Palatinose Utilization of Citrate Malonolate
1 (6)a
2 (7)
3 (5)
4 (8)
5 (4)
Eca
Ecb
Ecc
– + – – + + – + + + – + + +
– + – – + + – + + + – + + +
– + – – + (+) – + + + – + + +
– + – – + + – + + + – + + +
– + – – + + – + 75 + – (+) + 50
– + – – – + – + + + – + + +
– + – – + + – + + + – + + +
– + – – + + – + + + – + + +
+ + – – 83 + – 17 – – R + + 67 – –
+ 43 – – 100 + – – – 14 R + + + 29 –
+ 80 – – 40 + – + 20 20 R + + + + 20
+ 75 – – + + – + – – R + + + + –
+ 75 – – 50 75 75 75 25 25 R + + + 75 25
+ + – – + + – – – – R + + + + –
+ + – – + + – – – – R + + + + –
+ + – – + + – – – – R + + + + –
+ + + + + 50 + + + + 33 +
+ + + + + 43 – + + + 43 –
+ + + + + + + + + + + +
+ + + + + 88 + 75 38 + – +
+ + + + + + 50 + + + + 50
+ + + + + + + + + + – +
+ + + + + + + – – + – +
+ + + + + + – + + + – –
17 –
43 –
80 –
25 38
25 –
+ –
– –
+ –
a
Numbers of isolates in each group shown in parentheses. Eca = E. carotovora subsp. atroseptica, Ecc = E. c. subsp. carotovora, Ecb = E. c. subsp betavasculorum. +, positive reaction; ), negative reaction; (+), delayed positive reaction. b
the ability to form acid from melibiose and cellobiose, ability to use citrate, inability to form reducing substances from sucrose, and inability to form acid from palatinose. Phenon 3 corresponded to five strains and was most similar to Pco with regard to the ability to form reducing substances from sucrose, acid formation from a-methyl glucoside, melibiose, cellobiose, sorbitol, maltose, palatinose, acetoin formation and citrate consumption.
Phenon 4 corresponded to eight strains exactly similar to Pcb in regard to ability to form reducing compounds from sucrose, acid formation from a-methyl glucoside, maltose, and palatinose and inability to form acid from sorbitol. Phenon 5 corresponded to five strains and was most similar to Pco. One main characteristic, seen solely in this group, was gas formation from glucose in 75% of strains. With regard to phenotype characteristics, a high variability was found.
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(a)
(b)
(c)
(d) Fig. 1 Disease symptoms of bacterial vascular necrosis and root rot of sugar beet caused by Pectobacterium betavasculorum in field grown and artificially inoculated plants: (a) Cross section of a naturally infected root showing soft rotting in the centre and vascular necrosis. (b) Artificially inoculated seedling showing blackened-wilted petioles. (c) Artificially infected root showing soft rot and vascular necrosis near the periphery. (d) Field grown sugar beet with long black streaking along the petioles and frothing in the crown area
Fig. 2 Dendrogram of phenotypic characteristics of 30 Iranian sugar beet Pectobacterium carotovorum strains based on UPGMA algorithm of utilized carbon sources. Phenon 1, Pc12-55; Phenon 2, Pc43-79; Phenon 3, Pc51-81; Phenon 4, Pc61-74; Phenon 5, Pc70-36 and Pc 71 (unclustered)
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Electrophoresis of whole-cell protein
The SDS–PAGE whole-cell protein profiles of 30 P. carotovorum strains showed some Variation in banding patterns. Major differences were observed in protein bands between 29.7- and 50-kDa molecular mass range, and these were used to differentiate the strains. The 30 strains could be subdivided into five groups according to the presence or absence or difference in intensity of protein bands (Table 2). Not all the 30 strains originally obtained from sugar beet in Iran had identical protein profiles. Only 27% of the strains isolated from sugar beet produced diseased symptoms, typical of those reported by Thomson et al. (1977). These isolates (comprising the fourth group) and the reference strain Ecb 479 had identical protein profiles with three intensively stained bands at estimated molecular weight (MW) of 29 700, 40 000 and 47 600 (Fig. 3). The fourth group contained the strains that did not possess the band at 33 700 MW. This band was unique to groups 1 and 2. The third group was closely similar to the fourth group except for having an additional band at 39 000 MW, which was absent in strains of the fourth group. The first and second groups consisted of 13 isolates of P. carotovorum subspecies, which had the same three bands as in Ecb 479 and an intense band at 33 700 MW (Table 2). Some other strains had a faint band at this MW. These isolates were similar in major protein bands to standard strain of P. carotovorum subsp. carotovorum (Pcc SCRI 1035). Groups 2 differed from group 1 by having a minor protein band at 39 000 MW. The fifth group consisted of miscellaneous strains that did not match with any other groups and the reference strains. They all had a unique protein band at 50 000 MW. P. atrosepticum could be identified from Pb and Pcc by lack of an intense protein band at 40 000 MW (Table 2). This protein band was present in other groups at 40 000 MW except in group 5 (Fig. 3).
Discussion One prerequisite for control of a plant disease is correct identification of the causal agents involved. The
1
2
3
4
5
6
7
8
M
9 10
11 97.4 kDa 66.2 kDa
47.6 kDa 40.6 kDa
39.2 kDa
29.7 kDa
36.28 kDa
26.6 kDa
26.6 kDa
21.5 kDa
Fig. 3 Sodium dodecyle sulphate–polyacrylamide gel electrophoresis whole-cell protein profiles of Iranian Erwinia carotovora strains from sugar beet . Lane 1, Ec 73; Lane 2, Ec 74; Lane 3, Ec 51; Lane 4, Ec 68; Lane 5, Ec 61; Lane 6, Ec 69; Lane 7, Ec 66; Lane 8, Ec 81; Lane M, molecular weight marker; Lane 9, reference strains Ecb SCRI 479; Lane 10, Ecc SCRI 244; Lane 11, Eca SCRI 1035
aim of this study was to identify the bacterial strains associated with sugar beet root rot in the Fars province of Iran. A collection of 30 strains of P. carotovorum isolated from sugar beet were examined for phenotypic features and whole-cell protein profiles by SDS–PAGE gel electrophoresis. Results of this investigation showed that among a heterogeneous population of P. carotovorum associated with sugar beet root rot in the Fars province of Iran, only 27% could be identified as P. betavasculorum. Comparative phenotypic properties of Iranian isolates of sugar beet root rot bacteria (Phenon 4) were similar to major characteristics with those reported for P. betavasculorum type strain SCRI 479 (NCPPB 2795) (Stanghellini et al., 1977; Thomson et al., 1981). These isolates were able to utilize a-methyl glucoside, palatinose, sorbitol and incapable of using melobiose, cellobiose, citrate and malonolate. They grew at 37–39 C and tolerated 7–9% NaCl in their culture medium. They caused soft rot and vascular necrosis as reported by Thomson et al.(1977). However, phenotypic variability has been reported among strains of Pb isolated from sugarbeet, sunflower and artichoke (Gardan et al., 2003). Other Iranian strains (Phenon 1, 2, 3, 5 and one unclustered
Table 2 Sodium dodecyl sulphate–polyacrylamide gel electrophoresis whole-cell protein profiles used to differentiate representative Pectobacterium carotovorum strains isolated from sugar beet Protein band mass (kDa)
Group 1
Group 2
Group 3
Group 4
Group 5
E. c. subsp. carotovora (SCRI 244)
E. c. subsp. betavasculorum (SCRI 479)
E. c. atrosepticum (SCRI 1035)
29.7 32.0 32.8 33.7 36.0 37.0 38.0 39.0 40.0 47.6 50.0
+ – – + – – – + + + –
+ – – + – – – – + + –
+ – – – – – – + + + –
+ – – – – – – – + + –
+ – – + – + + – – + +
+ – – + – – – – + + –
+ – – – – – – – + + –
+ – – + – – – – – + –
+, presence of band in that group; ), absence of band in that group.
286
strain) isolated from sugar beet did not exactly fit with the characteristics of subspecies of P. carotovorum in physiological and biochemical properties. For example, strains of phenon 1 differed from Pcc especially in production of acid from a-methyl glucoside, palatinose and maltose. Reports of atypical strains among subspecies of P. carotovorum are not uncommon (Seo et al., 2003; Duarte et al., 2004). Analysis of whole-cell protein by SDS–PAGE was a useful method for differentiating among some of P. carotovorum strains. Consistent banding patterns were observed with the strains evaluated especially when the reference was used on the gel next to unknown strains. There was a complete uniformity of banding profiles in the 26.6–47.6 kDa molecular mass ranges within phenon 4 isolates and this group was similar to the type strain of Ecb SCRI 479. Other sugar beet isolates did not appear to consist a single subspecies of P. carotovorum or a single pathogenic type. The eight Iranian Pb isolates that caused typical vascular necrosis and sugar beet root rot had uniform banding pattern within phenon 4. The disease was observed in
