Istituto Sperimentale per la Frutticoltura, Via di Fioranello, 52, I-00040 Ciampino aeroporto (Roma), Italy
Correspondence
Marco Scortichini
mscortichini{at}hotmail.com
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ABSTRACT |
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This author is a staff member of the Istituto Sperimentale per la Patologia Vegetale, Roma, Italy, temporarily assigned to ISF.
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INTRODUCTION |
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Genetic variability among this bacterium was ascertained by plasmid analysis (Janse et al., 1996) and repetitive PCR, using enterobacterial repetitive intergenic consensus (ERIC), BOX A subunit of the BOX element of Streptococcus pneumoniae (BOX) and repetitive extragenic palindromic (REP) primer sets, and P. avellanae strains from northern Greece are clearly differentiated from those isolated in central Italy by two PCR products of approximately 300 and 800 bp using ERIC primers (Scortichini et al., 1998
, 2002b
). Primers based on the 16S rRNA gene sequence (Scortichini & Marchesi, 2001
) or on the hrpW gene sequence (Loreti & Gallelli, 2002
) can be used for the rapid detection of this pathogen.
Where genetic variability within P. avellanae has been studied, nothing is known about the population structure in terms of genetic diversity and linkage disequilibrium. To study these fundamental aspects, we have used the multilocus enzyme electrophoresis (MLEE) method combined with statistical treatment of the data. MLEE analysis enables investigation of whether the alleles at different loci within a population of bacteria are randomly associated, as in the case of linkage equilibrium, or whether a significant association exists between the alleles, as for linkage disequilibrium (Maynard Smith et al., 1993). Several bacterial species, mainly of medical (Whittam et al., 1983
; Caugant et al., 1987
; Johnson et al., 1994
; Farfan et al., 2000
) or environmental (Wise et al., 1995
; Rius et al., 2001
) interest, have been investigated by means of MLEE. Such studies have shown that the population structure of the species studied can vary from clonal to panmictic, with cases of epidemic structure (Maynard Smith et al., 2000
). Concerning bacterial plant pathogens, there has only been one study performed with MLEE (Denny et al., 1988
) and information about these bacteria is lacking.
The objective of this study was to extend our knowledge on the genetic structure of P. avellanae strains isolated from hazelnut showing symptoms of decline in Greece and Italy by using MLEE. Similar to medical or environmental bacteria (Maynard-Smith et al., 2000), the assessment of population structure of phytopathogenic bacteria is important for understanding their epidemiology as well as for checking the persistence of particular clones in time and space. Such studies, by revealing the genetic composition of the pathogen, can also contribute to more appropriate detection and control of the disease. The results of our analysis would indicate a clonal structure for the entire population of P. avellanae as well as for the population subsets studied. Some clones have persisted over decades both in northern Greece and in central Italy.
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METHODS |
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Electrophoresis and specific enzyme staining.
Non-denaturing vertical PAGE (Mini Protean; Bio-Rad) was used for all enzymes. The acrylamide concentration in the gels depended on the enzyme studied (6 % for continuous polyacrylamide gels and 10 %/8 % or 8 %/5 % for discontinuous polyacrylamide gels). Tris/HCl (0·8 M, pH 8·8) buffer was used in continuous gels and 0·125 M Tris/HCl (pH 6·8) stacking buffer and 0·4 M Tris/HCl (pH 8·8) resolving buffer were used in discontinuous gels. Tris/glycine (0·19 M, pH 8·3) buffer was used for the electrode compartment. A constant voltage, depending on the acrylamide concentration in the gel, was applied until the bromophenol blue band reached the bottom of the gel. All strains were run twice. The following 10 enzymes, representing a sample of structural genes of the bacterial genome and showing polymorphism (Selander et al., 1986; Denny et al., 1988
; Rius et al., 2001
), were assayed: isocitrate dehydrogenase, shikimic dehydrogenase, superoxide dismutase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, esterase, leucine aminopeptidase, glutamate dehydrogenase-NADP, phosphoglucose isomerase and phosphoglucomutase. The staining of the gel to reveal specific enzyme activity was carried out following the method described by Selander et al. (1986)
. For each enzyme, distinct mobility variants were designated as electromorphs and numbered in order of decreasing anodal migration. Displacement of the electromorphs was expressed in terms of relative electrophoretic mobility with respect to the bromophenol blue band. Absence of enzyme activity was attributed to a null allele and designated as 0. Distinct combinations of alleles over the 10 loci assayed were named as electrophoretic types (ETs).
Data treatment.
Electromorphs and ETs were equated with alleles and allelic combinations, respectively, for statistical analysis. Computer programs written and kindly provided by Professor T. S. Whittam (Department of Microbiology and Molecular Genetics, Michigan State University, USA) were used to analyse the data for ET designation, genetic diversity calculations and ET clustering. The genetic diversity (h) for a locus was calculated according to Nei (1978). The probability that two isolates differ at the j locus is hj=n(
)/(n-1), where Xi is the frequency of the i allele at the j locus and n is the number of individuals in the sample (Maynard-Smith et al., 1993
). Mean genetic diversity (H) is the arithmetic mean of h over all the loci examined. Clustering of data obtained by MLEE was performed by using the unweighted pair-group method with arithmetic averages (UPGMA). Distance is measured as the proportion of mismatched loci between pairs of ETs. The cophenetic correlation was calculated using NTSYS-PC version 1.80 (Rohlf, 1993
). Multilocus linkage disequilibrium was estimated on the basis of distribution of allelic mismatches between pairs of bacterial strains among all the loci examined. The ratio of the observed variance in mismatches (VO) to the expected variance at linkage equilibrium (VE) provides a measure of multilocus linkage disequilibrium that can be expressed as the index of association (IA)=(VO/VE)-1 (Brown et al., 1980
; Maynard Smith et al., 1993
). For populations in linkage equilibrium, VO=VE, and IA is not significantly different from zero, whereas values of IA greater than zero indicate that recombination has been rare or absent. To test if IA differed significantly from its expected value of zero, the ETLINK program version 3.0, kindly provided by Professor T. S. Whittam, was used. The maximum variance obtained was compared to that expected in a freely recombining population (linkage equilibrium) by using 1·000 randomization of the dataset (P=0·001). The null hypothesis of a random association of alleles (i.e. the population is at linkage equilibrium) was rejected if this probability was smaller than the selected significance level. In addition, to test whether the population structure of P. avellanae and subsets was epidemic, sensu Maynard Smith et al. (1993)
, the IA value was also calculated by taking into consideration only the ETs. For multilocus linkage disequilibrium analysis, the 102 P. avellanae strains were also studied as three population subsets according to their geographic origin. The three population subsets were composed as follows: 20 strains from northern Greece, 57 strains from central Italy (province of Viterbo) and 25 strains from central Italy (province of Rome). This last group of strains was isolated from one single hazelnut orchard established using propagative plant material obtained from the province of Viterbo. A GT statistic was used to compare the mean genetic diversity of the three P. avellanae population subsets (Sokal & Rohlf, 1981
).
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RESULTS |
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Genetic relationships among multilocus genotypes
The genetic relationship among the 53 ETs of P. avellanae and the ETs of P. s. pv. actinidiae and P. s. pv. theae is shown in Fig. 1. The cophenetic correlation coefficient of the total sample was R=0·79. The shortest genetic distance observed between ETs (0·10) corresponds to a single locus difference. At a genetic distance of 0·71, two main groups of P. avellanae ETs were found, namely A and B. Group A included 18 ETs and contained strains isolated in central Italy, from the provinces of Viterbo or Rome. Group B contained the remaining 35 ETs. This group, in turn, can be divided into two subgroups, B1 and B2 (Fig. 1
). Subgroup B1 contained all the strains isolated from northern Greece and some ETs from central Italy; subgroup B2 contained strains isolated from the provinces of Viterbo and Rome. P. s. pv. actinidiae and P. s. pv. theae clustered apart from the P. avellanae ETs, showing 97 % genetic distance.
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DISCUSSION |
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Previous studies on genetic diversity in P. avellanae based on the assessment of short interspersed elements of the bacterial genome were carried out by using repetitive PCR with ERIC, BOX and REP primer sets. These studies also revealed differences between the populations found in Greece and Italy. In fact, ERIC primer sets clearly indicated that all the strains isolated in Greece give two PCR products of 300 and 800 bp that have never been found in the strains isolated in Italy (Scortichini et al., 1998; 2002b
). It is interesting to note that the value of H found here is rather high for a bacterial species that appears to be strictly associated only with a single host plant, namely C. avellana. Until now, P. avellanae has never been isolated from environments (i.e. soil, water, alternative host plants) other than its host plant. Studies are under way to verify the possible presence of this phytopathogen in other ecological niches. However, it is worth noting that P. avellanae, in either Greece or Italy, is more virulent in hazelnut orchards established on very acidic soils (pH<4·5). Stress conditions occurring on plants could select different bacterial genotypes able to subsequently propagate in a clonal manner.
This study demonstrated a high frequency of occurrence of null alleles. A high frequency of occurrence of null alleles has already been reported for P. stutzeri (Rius et al., 2001) as well as for Helicobacter pylori (Go et al., 1996
), other species with a high genetic diversity. Hypotheses to explain this result, such as the occurrence of enzyme-inactivating mutations or the absence of the structural gene, have still to be verified.
The dendrogram obtained in the present study shows no clear relationship between the geographic origin of the strains and their grouping. The value of the cophenetic correlation obtained (R=0·79) falls into the range (0·740·90) of most frequently occurring cophenetic correlation (Sneath & Sokal, 1973). The strains from central Italy are distributed in both groups A and B. However, all 20 strains from Greece clustered into subgroup B1, thus indicating again the diversity of this population from the others. It is also interesting to note that no single ET includes strains obtained from both Greece and Italy. The dendrogram based on repetitive PCR and UPGMA analysis indicated two distinct groups of strains, related at 90 % similarity, that could be separated according to their geographic origin, namely central Italy and northern Greece (Scortichini et al., 2002b
). P. s. pv. actinidiae and P. s. pv. theae clustered apart from P. avellanae, thus indicating again that differences exist among these taxa.
The linkage disequilibrium analyses clearly indicated that P. avellanae is a clonal species when analysed at population subsets level also. The IA value for all P. avellanae strains was 0·90±0·15 (P<0·001). The IA values were always significantly different from zero for all the population subsets studied. In addition, the stepwise elimination of the most closely related ET pairs revealed that the remaining ETs were also in linkage disequilibrium (Table 3). In addition, when only the ETs were analysed, the corresponding IA value again indicated a clonal structure, sensu Maynard-Smith et al. (1993)
. Similar results were obtained by Denny et al. (1988)
with other phytopathogens, such as P. s. pv. syringae and P. s. pv. tomato. Other examples of clonal species are found in environmental bacteria such as P. stutzeri (Rius et al., 2001
) as well as in bacteria of medical and veterinary interest such as Salmonella sp. (Selander et al., 1990
), Haemophilus influenzae (Maynard Smith et al., 1993
) and Mycobacterium intracellulare (Feizabadi et al., 1997
).
The clonal structure showed by P. avellanae and its genetic diversity found here and in previous studies can be explained by hypothesizing the possible occurrence of adaptive mutations conferring selective advantages to the clones during the colonization and adaptation of ecological niches (Cohan, 1994). Since P. avellanae did not show gene recombination, its variability might be explained, alternatively, by means of sequential evolution (Levin & Bergstrom, 2000
). The clonal structure also indicates that horizontal gene transfer and recombination processes, possibly occurring also in P. avellanae, are not currently sufficient to disrupt the allelic association of the ten loci studied here. Alternatively, selection for an epistatic combination of alleles might maintain linkage disequilibrium in the presence of frequent recombination (Souza et al., 1992
). Like in other pseudomonads (i.e. P. stutzeri) (Rius et al., 2001
), the P. avellanae clones can be genetically different, but the phenotypic resemblance is sufficient to identify the species (Scortichini et al., 2002a
). However, P. stutzeri, a species adapted to soil and water, shows considerable genetic diversity and consists of at least eight genomovars as inferred by DNADNA hybridization studies (Rossellò et al., 1991
), whereas P. avellanae, a species colonizing a single host plant, shows very slight variability when assessed by DNADNA hybridization (Gardan et al., 1999
). The clonality of P. avellanae can also be explained by remembering the recent outbreaks of hazelnut bacterial decline in Greece or in Italy. In fact, the disease was first observed in Greece in 1976 on young hazelnut plantations in sites where this crop had never been cultivated before (Psallidas & Panagopoulos, 1979
). Similarly, in central Italy, the first foci of decline were observed in new areas of hazelnut cultivation in 1977 (Scortichini, 2002
). Afterwards, the disease spread quite rapidly in the two areas, destroying thousands of trees. It is interesting to note that the exchange of hazelnut propagative material between Italy and Greece never occurred. Pathogenicity tests performed with strains of the three population subsets studied revealed their high level of virulence (Scortichini et al., 2002b
). Probably, these highly virulent clonal complexes persist over decades.
Finally, we stress the divergence of the P. avellanae population subsets in the context of ecotypic adaptation. In fact, the strains isolated in central Italy either from Viterbo or Rome, are very similar to each other and many ETs contain strains from the two provinces. In fact, the hazelnut orchard in the province of Rome was established with propagative material taken from Viterbo. The population subsets of northern Greece appear to be different from these. As in the case of another plant-associated bacterium, Rhizobium leguminosarum bv. phaseoli (Souza et al., 1992), geographic separation might contribute to the allele diversity and would appear as an important factor for differential adaptation of bacterial phytopathogens to the same host plant cultivated in different areas.
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ACKNOWLEDGEMENTS |
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Received 31 March 2003;
revised 26 June 2003;
accepted 30 June 2003.
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