(Abstract) To characterize the major pathogens of onion rot in Korea, 139 bacteria were isolated from rotten onions collected from main producing districts, Chang-Nyung, Eui-Ryung, and Ham-Yang. The 18% (25 strains) of bacterial isolates have carboxymethylcellulase (CMCase) activity and the 53% (74 ...
(Abstract) To characterize the major pathogens of onion rot in Korea, 139 bacteria were isolated from rotten onions collected from main producing districts, Chang-Nyung, Eui-Ryung, and Ham-Yang. The 18% (25 strains) of bacterial isolates have carboxymethylcellulase (CMCase) activity and the 53% (74 strains) have polygalacturonase (PGase) activity. Thirty one among randomly selected 45 strains of PGase producing bacteria have pathogenicity to onions. The isolates were classified into Pseudomonas sp. (18 strains), Bacillus sp. (11 strains), Yersinia sp. (7 strains), and others (9 strains) on the basis of FAME patterns. Eighteen strains of Pseudomonas sp. were mainly divided into three cluster in the dendrogram and only the two clusters of them showed pathogenicity to onions. CMCase and PGase activities of Pseudomonas sp. appeared to be weaker than those of Bacillus sp.. However, the pathogenicity of Pseudomonas sp. to soften onions was stronger than that of Bacillus sp. Inoculation of 102 cfu of Pseudomonas sp. gave rise to softening of onions. One of these Pseudomonas sp., which shows the strongest pathogenicity, was selected (designated as CH1 strain), and was subjected to identification. Although the FAME analysis identified CH1 as Pseudomonas gladioli, the Biolog system failed to accurately recognize this strain. Since Pseudomonas gladioli was reclassified as Burkholderia gladioli by Yabuuchi et al. and since the 16S rDNA sequence of this strain showed 99% similarity to B. gladioli according to a blast search for homologous sequence, CH1 was identified as B. gladioli. Further investigation of its morphological and biochemical characters determined CH1 as B. gladioli pv. alliicola. After the successful inoculations, the pathogenicity of CH1 was established and Koch's postulates were satisfied. This is the first record of soft rot of onion caused by B. gladioli pv. alliicola in Korea. And I suggest the name 'bacterial soft rot of onion' to the symptom caused by this bacterium. B. alliicola CH1 was able to produce PGase but did not produce pectin (pectate) lyase and CMCase. The PGase produced from CH1 appeared as three activity bands such as 45 kDa, 35 kDa, and 29 kDa by the active staining on the SDS-PAGE. No enzyme activity could be produced in a culture medium containing glucose as a sole carbon source, but another carbon souces such as galactose, polygalacturonic acid, especially pectin increased PGase production of B. alliicola CH1. Since the addition of calcium ion to the medium inhibits the enzyme production, it seems reasonable to use the calcium fertilizer in the field to elevate the storage of onions. To elucidate whether the PGase is involved in pathogenicity of of B. alliicola CH1, the PGase gene was cloned and characterized, and then the relation between the PGase and pathogenicity was investigated. A genomic library of PGase-producing B. gladioli pv. alliicola CH1 was constructed in E. coli DH5α with pUC19 vector. Screening of this library with a pitting activity on a PEC-SSA medium resulted in the isolation of a clone that shows the PGase activity. From the clone, the recombinant plasmid that carries a 20 kb HindIII fragment insert was isolated. Based on the restriction map of the plasmid, the subcloning of the 2.6 kb ApaI-XhoI fragment that carries the PGase gene of B. alliicola CH1 was performed with pBSK(-) vector. The entire nucleotide sequence of the PGase gene was determined. The gene, designated as pehA, encodes a protein of 460 amino acid residues with a molecular mass of 46,405 Da. The deduced amino acid sequence of the PGase revealed a typical amino-terminal signal sequence with a potential cleavage site between Ala and Thr residues at positions 27 and 28 of the protein sequence. Since the PehA mature protein appeared to contain 433 amino acids with a calculated molecular mass of 43,653 Da and a pI of 4.87, it seems that the cloned pehA gene encodes the 45 kDa PGase among the three PGases that could be detectable from B. alliicola CH1 by active staining on the SDS-PAGE. The nucleotide sequence had some putative functional sites at appropriate positions, a potential Shine-Dalgarno sequence, a -35 and -10 E. coli-like promoter sequence, and a KdgR repressor binding site which is usually found in pectolytic bacteria. The deduced amino acid sequence of PehA showed 81% identity to the PGase PehA of B. cepacia plasmid pPEC320 and 79% identity to the PGase Pg-A of B. glumae. However, PehA showed low identity of 12-22% to the pectolytic enzymes from other sources including bacteria Erwinia species, fungi, and plants. The fact that the sequence identity of PGase genes is relatively high between the same genus and is low in other genus may lead to a development of a similar disease symptoms in onion following infection with bacterial pathogens that belong to the same genus. However, although the similarity of PGase genes is low in each other, the putative enzyme active sites, such as GHG, NTD, DD, and RIK motifs, are very conserved in prokaryotes and eukaryotes. Therefore, the basic reaction mechanism of PGases, such as substrate binding, catalytic activity, may be very similar among the enzymes from different origins. The analysis of amino acid sequence of PehA using Pfam protein family database showed that B. alliicola PGase belongs to glycosyl hydrolase family 28 that contains most of plant cell wall degrading enzymes. B. alliicola PGase has the β-sheet structure which is a common feature of the plant cell wall degrading enzymes and is linked with α-helices. As an effective plant virulence factor, the most important advantage of the parallel β-sheet fold structure in the B. alliicola PGase is thought to confer the stability that helps maintain the enzyme activity under the hostile extracellular environment. In onion bulb inoculations of B. alliicola or its cultural filtrates, the tissues around the inoculation points became soft rot, whereas the inoculations of the E. coli transformant harboring pheA gene appeared to nibble the tissues, demonstrating that the pehA gene play an important role, at least in part, in development of soft rot in onion plants following infection of B. alliicola CH1. Since the cloned pehA gene encodes the 45 kDa PGase among the three PGases, such as 45 kDa, 35 kDa, and 29 kDa of B. alliicola CH1, it seems likely that the soft rot activity of B. alliicola may be due to the combined activity of these three PGases. However, a possiblity of involvement of other factors in the induced soft rot caused by the B. alliicola could not be excluded.
(Abstract) To characterize the major pathogens of onion rot in Korea, 139 bacteria were isolated from rotten onions collected from main producing districts, Chang-Nyung, Eui-Ryung, and Ham-Yang. The 18% (25 strains) of bacterial isolates have carboxymethylcellulase (CMCase) activity and the 53% (74 strains) have polygalacturonase (PGase) activity. Thirty one among randomly selected 45 strains of PGase producing bacteria have pathogenicity to onions. The isolates were classified into Pseudomonas sp. (18 strains), Bacillus sp. (11 strains), Yersinia sp. (7 strains), and others (9 strains) on the basis of FAME patterns. Eighteen strains of Pseudomonas sp. were mainly divided into three cluster in the dendrogram and only the two clusters of them showed pathogenicity to onions. CMCase and PGase activities of Pseudomonas sp. appeared to be weaker than those of Bacillus sp.. However, the pathogenicity of Pseudomonas sp. to soften onions was stronger than that of Bacillus sp. Inoculation of 102 cfu of Pseudomonas sp. gave rise to softening of onions. One of these Pseudomonas sp., which shows the strongest pathogenicity, was selected (designated as CH1 strain), and was subjected to identification. Although the FAME analysis identified CH1 as Pseudomonas gladioli, the Biolog system failed to accurately recognize this strain. Since Pseudomonas gladioli was reclassified as Burkholderia gladioli by Yabuuchi et al. and since the 16S rDNA sequence of this strain showed 99% similarity to B. gladioli according to a blast search for homologous sequence, CH1 was identified as B. gladioli. Further investigation of its morphological and biochemical characters determined CH1 as B. gladioli pv. alliicola. After the successful inoculations, the pathogenicity of CH1 was established and Koch's postulates were satisfied. This is the first record of soft rot of onion caused by B. gladioli pv. alliicola in Korea. And I suggest the name 'bacterial soft rot of onion' to the symptom caused by this bacterium. B. alliicola CH1 was able to produce PGase but did not produce pectin (pectate) lyase and CMCase. The PGase produced from CH1 appeared as three activity bands such as 45 kDa, 35 kDa, and 29 kDa by the active staining on the SDS-PAGE. No enzyme activity could be produced in a culture medium containing glucose as a sole carbon source, but another carbon souces such as galactose, polygalacturonic acid, especially pectin increased PGase production of B. alliicola CH1. Since the addition of calcium ion to the medium inhibits the enzyme production, it seems reasonable to use the calcium fertilizer in the field to elevate the storage of onions. To elucidate whether the PGase is involved in pathogenicity of of B. alliicola CH1, the PGase gene was cloned and characterized, and then the relation between the PGase and pathogenicity was investigated. A genomic library of PGase-producing B. gladioli pv. alliicola CH1 was constructed in E. coli DH5α with pUC19 vector. Screening of this library with a pitting activity on a PEC-SSA medium resulted in the isolation of a clone that shows the PGase activity. From the clone, the recombinant plasmid that carries a 20 kb HindIII fragment insert was isolated. Based on the restriction map of the plasmid, the subcloning of the 2.6 kb ApaI-XhoI fragment that carries the PGase gene of B. alliicola CH1 was performed with pBSK(-) vector. The entire nucleotide sequence of the PGase gene was determined. The gene, designated as pehA, encodes a protein of 460 amino acid residues with a molecular mass of 46,405 Da. The deduced amino acid sequence of the PGase revealed a typical amino-terminal signal sequence with a potential cleavage site between Ala and Thr residues at positions 27 and 28 of the protein sequence. Since the PehA mature protein appeared to contain 433 amino acids with a calculated molecular mass of 43,653 Da and a pI of 4.87, it seems that the cloned pehA gene encodes the 45 kDa PGase among the three PGases that could be detectable from B. alliicola CH1 by active staining on the SDS-PAGE. The nucleotide sequence had some putative functional sites at appropriate positions, a potential Shine-Dalgarno sequence, a -35 and -10 E. coli-like promoter sequence, and a KdgR repressor binding site which is usually found in pectolytic bacteria. The deduced amino acid sequence of PehA showed 81% identity to the PGase PehA of B. cepacia plasmid pPEC320 and 79% identity to the PGase Pg-A of B. glumae. However, PehA showed low identity of 12-22% to the pectolytic enzymes from other sources including bacteria Erwinia species, fungi, and plants. The fact that the sequence identity of PGase genes is relatively high between the same genus and is low in other genus may lead to a development of a similar disease symptoms in onion following infection with bacterial pathogens that belong to the same genus. However, although the similarity of PGase genes is low in each other, the putative enzyme active sites, such as GHG, NTD, DD, and RIK motifs, are very conserved in prokaryotes and eukaryotes. Therefore, the basic reaction mechanism of PGases, such as substrate binding, catalytic activity, may be very similar among the enzymes from different origins. The analysis of amino acid sequence of PehA using Pfam protein family database showed that B. alliicola PGase belongs to glycosyl hydrolase family 28 that contains most of plant cell wall degrading enzymes. B. alliicola PGase has the β-sheet structure which is a common feature of the plant cell wall degrading enzymes and is linked with α-helices. As an effective plant virulence factor, the most important advantage of the parallel β-sheet fold structure in the B. alliicola PGase is thought to confer the stability that helps maintain the enzyme activity under the hostile extracellular environment. In onion bulb inoculations of B. alliicola or its cultural filtrates, the tissues around the inoculation points became soft rot, whereas the inoculations of the E. coli transformant harboring pheA gene appeared to nibble the tissues, demonstrating that the pehA gene play an important role, at least in part, in development of soft rot in onion plants following infection of B. alliicola CH1. Since the cloned pehA gene encodes the 45 kDa PGase among the three PGases, such as 45 kDa, 35 kDa, and 29 kDa of B. alliicola CH1, it seems likely that the soft rot activity of B. alliicola may be due to the combined activity of these three PGases. However, a possiblity of involvement of other factors in the induced soft rot caused by the B. alliicola could not be excluded.
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