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Correspondence to: J. Camargo Martinati, Laboratório de Biologia. Celular e Molecular, Centro de .... using CHEF and RAPD analysis. The other plum strain.

CURRENT MICROBIOLOGY Vol. 50 (2005), pp. 190–195 DOI: 10.1007/s00284-004-4405-5

Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2005

Phylogenetic Relationships of Xylella fastidiosa Strains Based on 16S–23S rDNA Sequences Juliana Camargo Martinati,1 Flµvia Tereza Hansen Pacheco,1 Vitor Fernandes Oliveira de Miranda,2 Siu Mui Tsai1 1 2

Cell and Molecular Biology Laboratory, Agriculture Nuclear Energy Center (CENA/USP), Piracicaba, S¼o Paulo, Brazil Herbarium Mogiense (HUMC), Mogi das Cruzes University (UMC), S¼o Paulo, Brazil

Received: 10 July 2004 / Accepted: 18 October 2004

Abstract. In spite of the lack of resolution of Xylella fastidiosa phylogenetic relationships, parsimony analysis of the 16S–23S rDNA sequence from a wide range of hosts has been evaluated in this research. In order to establish an easier method for sequencing the spacer region completely, a new primer pair was designed. The sequences obtained revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.80 to 1.00. The cladogram constructed allowed the clustering of two major clades. From these results it has been possible to recognize the monophyletic grouping of some strains belonging to the same host, possibly representing only one infection process. However, for other hosts there is paraphyletic and polyphyletic grouping. This methodology followed from promising results regarding strain–host clustering. With the parsimony approach, hypothetical genealogical relationship among Xylella strains may be inferred.

Xylella fastidiosa [35] is a xylem-limited bacterium, responsible for several diseases in plants such as grapevines, peach, plum, almond and others, as well as in ornamental plants. It is transmitted by xylem sapfeeding insects such as leafhoppers Cicadellidae [17]. Studies on differentiation among X. fastidiosa strains are being carried out with an eye to discovering methods for characterizing the main X. fastidiosa strains and possibly the establishment of pathovars. All X. fastidiosa strains are currently classified as one species but differ in important respects. Previous studies have differentiated X. fastidiosa strains on the basis of pathogenicity, nutritional requirements [18], DNA homology [20], structural protein analysis [4], RFLP [5], RAPD [1] and VNTR analysis [9]. Although X. fastidiosa is well characterized, there is uncertainty regarding the phylogenetic relationship among the strains. Studies analyzing genetic variability using the neighbor joining method have been done [23]; however, this method does not Correspondence to: J. Camargo Martinati, Laboratório de Biologia Celular e Molecular, Centro de Energia Nuclear na Agricultura (CENA/USP), 13400-970, P.O. Box 96, Piracicaba, S¼o Paulo, Brazil; email: [email protected]

necessarily reflect the evolutionary position of the organisms, being a useful tool to infer distance (for a theoretical discussion see [27]). Ribosomal DNA has been widely used to infer phylogenetic relationships in microorganisms [16, 38]. The rDNA genetic locus is a genetic unit of broad evolutionary interest since it is extremely important in all organisms and is sufficiently conserved for it to be used in a universal organization of evolutionary relationships [3, 13, 21]. The sequence analysis of the small subunit, 16S rDNA, has frequently been used for determining inter- and intraspecific relationships. However, as evolutionary distances decrease, insufficient diversity is often found in the 16S gene and thus genetic relationships of closely related species cannot be well defined [30]. It has been proposed that the spacer region 16S–23S (ITS) could overcome this problem due to its higher variation in length and sequence. Indeed the analysis of this region has successfully differentiated strains of many groups of bacteria [2, 19], and sequences of this spacer region of many species have become available for comparison. A shortage of information about phylogeny among the main X. fastidiosa strains has encouraged thorough

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Table 1. Xylella fastidiosa strains used for phylogenetic analysis based on the rDNA spacer region and their 16S–23S rDNA sequence accession numbers

Strain 9a5c* P3* MUL-1* ALS-BC* ELM-1* RGW-R* PWT-22*

Host Citrus Coffee Mulberry Almond Elm Ragweed Periwinkle Grapevine*

Geographical origin Macaubal, SP Pindorama, SP Massachusetts, USA California, USA Washington, USA Florida, USA Florida, USA USA

Source IAC CCT CCT CCT CCT CCT CCT CCT

6740 6744 6746 6748 6749 6751 6068

ATCC

ATCC ATCC ATCC ATCC ATCC

35868 35870 35873 35876 35878

GenBank accession numbers AF 237651 AY 388464 AY 388467 AY 388465 AY 388468 AY 388469 AY 388470 AY 388466

ATCC, American Type Culture Collection (Manassas, VA, USA); L.W. Moore, Oregon State University (Corvallis, OR, USA). CCT, Coleżo de Culturas Tropicais do Acervo da Fundażo AndrØ Tosello, Campinas/SP. *Strains sequenced in this study.

investigation. As no comparison of the main X. fastidiosa strains in the same phylogeny study exists, we decided to carry out a phylogenetic analysis with a large number of ITS sequences of several hosts. These results should allow the phylogenetic relationships of X. fastidiosa strains to be determined and may lead to improved similar management methods for related strains. The knowledge of the origin of X. fastidiosa could help to clarify how this bacterium selects its hosts, why the same strain behaves in a pathogenic manner in one host but not another, and its role in crop yields and in the ecosystem. Materials and Methods Bacterial strains, culture conditions, and DNA extraction. Strains of X. fastidiosa from different hosts that were sequenced in this study are listed in Table 1. Each strain was donated by Fundażo AndrØ Tosello and was grown in solid PW agar medium and incubated at 28°C for 7–10 days. Bacterial DNA was extracted using CTAB buffer according to Doyle and Doyle’s [11] protocol. PCR primers and conditions. The 16S–23S spacer region was amplified by polymerase reaction (PCR) using primers designed in our laboratory (16S–23SF [5¢-GAT GAC TGG GGT GAA GTC GT-3¢]; 16S–23SR [5¢-GAC ACT TTT CGC AGG CTA CC-3¢]). Amplifications were performed in a 25 lL reaction mixture containing 100 ng of DNA template, 0.5 lM of each primer, 100 lM of dNTPs, 2.5 mM of MgCl2, 0.5 U of Taq DNA polymerase, 1 · buffer. A DNA Thermal Cycler (GeneAmp PCR System 9700 Applied Biosystems) was used with 1 cycle of 4 min at 94°C, 40 cycles at 94°C for 40 s, 52°C for 40 s and 72°C for 1 min, and a final extension of 2 min at 72°C. Sequencing of the 16S–23S rDNA spacer region. PCR-amplified ITS regions were purified using GFX PCR DNA and Gel Purification Kit (Pharmacia Biotech). Both strands of the spacer region were sequenced 10 times each (to avoid sequencing mistakes) by the dideoxy chain terminator method using a thermocycler (Perkin-Elmer Applied Biosystems) The PCRs for sequencing were performed in a total volume of 10 lL containing 30–50 ng DNA, 5 lM of each primer, 2 lL of the ABI PRISM Big Dye terminator cycle sequencing ready

reaction Kit (Perkin Elmer), and 2 lL of 2.5 · buffer (Tris-HCl 200 mM, pH 9.0; MgCl2 5 mM). The reactions were performed in the same conditions as the amplified PCR. Sequence reaction mixtures were recorded using the ABI Prism 3100 Genetic Analyzer (PerkinElmer Applied Biosystems). Phylogenetic analysis. Phylogenetic analysis based on maximum parsimony of the 16S–23S rDNA sequence was resolved using PAUP* (Phylogenetic Analysis Using Parsimony) 4b10 [32]. PAUP* infers phylogenies by selecting the trees that minimize tree length (number of steps) and minimize homoplasy. The sequences obtained were aligned using the Easy Align [24] program that employs the MALIGN algorithm [36, 37] with 52 other X. fastidiosa sequences available in GenBank (Table 2). Gaps were treated as fifth base [14] and transitions and transversions had the same weight. The 16S–23S rDNA nucleotide sequences obtained for the seven X. fastidiosa strains were submitted to EMBL GenBank and have been assigned the accession numbers reported in Table 1. The trees were obtained by heuristic search [34] through aleatory addition with 5000 replications. The branch swapping was made by the tbr algorithm. The strict consensus trees [31] were obtained from the most parsimonious trees. The robustness of the inferred tree was evaluated by applying bootstrap resampling [12, 34], through heuristic search with 1000 replications and aleatory addition with 100 replications. In order to root the tree we used the outgroup method [25], and the pear (PE.PLS) strain was used as the outgroup. The cladogram was constructed with the TreeView [27] program.

Results and Discussion Analysis of the 16S–23S spacer region. All seven strains tested gave the same-size PCR product (650 bp) using the primers designed in our laboratory (data not shown). The complete ITS sequence was determined by directly sequencing PCR-amplified ITS products. The ends corresponding to part of the 16S and 23S genes were eliminated according to the X0 16S–23S sequence from GenBank. A fragment of approximately 510 bases containing the ITS region of all strains was obtained. A BLAST search identified all the available 16S–23S X. fastidiosa spacer sequences in the current GenBank

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Table 2. Xylella fastidiosa strains available in GenBank included for comparison in the phylogenetic analysis based on rDNA spacer region and their 16S–23S rDNA accession numbers

Strain Maple PL.788 Plum2#4 ALS1 ALS2 ALS3 ALS4 ALS5 ALS6 ALS7 ALS9 Manteca ContraCosta Tulare CO.01 Oak 88-9 Oak 92-3 Oak 92-10 OLS#2 Stucky CI.52 CI.11067 Ann1 PF1 T1c TR1 5S2 5R1 4S3 PE.PLS GR8935 ConnCreek Stags Leap Fetzer STL Santa Cruz Meyley UCLA Preston Ranch VinoF Medeiros Traver Moore Park Douglas Oxford Hopland PD95-2 PD95-4 PD95-9 R116V3 Dixon R118V3-4

Host Acer macrophyllum Plum Plum Almond Almond Almond Almond Almond Almond Almond Almond Almond Almond Almond Coffee Oak Oak Oak Oak Oak Citrus Citrus Oleander Oleander Oleander Oleander Peach Peach Peach Pear Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine Grapevine

GenBank 16S–23S accession numbers

Geographical origin

AF073219 AF203395 AF073209 AF073240 AF073243 AF073244 AF073245 AF073246 AF073247 AF073248 AF073249 AF073241 AF073250 AF073242 AF203394 AF 073210 AF073211 AF073212 AF073213 AF073214 AF203393 AF237650 AF 073215 AF073216 AF073217 AF073218 AF 073206 AF073207 AF073208 AF203396 AF203397 AF073225 AF073226 AF073227 AF073228 AF073229 AF073230 AF073231 AF073232 AF073233 AF073234 AF073235 AF073236 AF073237 AF073238 AF073239 AF073220 AF073221 AF073222 AF073223 AF073251 AF073224

California Georgia Georgia California California California California California California California California California California California S¼o Paulo Florida Florida Florida Georgia Georgia S¼o Paulo, BR Paranµ, BR California California California California Georgia Georgia Georgia Taiwan Florida California California California California California California California California California California California California California California California California California California California California California

J. Camargo Martinati et al.: Phylogeny of Xylella Strains

Fig. 1. Cladogram (phylogenetic tree) constructed by the maximum parsimony method, based on the 16S–23S spacer sequence data of Xylella fastidiosa strains with the pear strain (PE.PLS) as the outgroup. The cladogram was generated with a heuristic search through the PAUP* program of Xylella fastidiosa strains from different hosts. Consistency index (CI) = 0.9237; Retention index (RI) = 0.9550; Homoplasy index (HI) = 0.0763. Rescaled consistency index (RC) = 0.8821.

database with similarity percentages ranging from 80% to 100%. The seven X. fastidiosa 16S–23S sequences revealed in this research were also aligned with the Easy Align program [24] with 52 other X. fastidiosa sequences available in GenBank (Table 2). The complete alignments of the sequences sequenced in this study with the other 52 sequences used for comparison are available in Martinati [22]. The similarity levels found in the 16S–23S rDNA spacer regions for different X. fastidiosa strains are not shown (for the complete alignment and all the similarity values see Martinati [22]). The results revealed that the highest level of variation was found in pear strain (PE.PLS), with similarity values ranging from 0.79 to 0.81. The closest strain to pear was the citrus (9a5c), with similarity values of 0.812, and the lowest value (0.793) was found between the pear and grapevine strains (6068).

193 Phylogenetic analysis. Unlike phenetics (e.g., neighbor joining), which uses overall similarity, cladistics (e.g., maximum parsimony) uses nested sets of share-derived characters to derive relationships among the taxa. The phylogenetic analysis by maximum parsimony using the 16S–23S rDNA sequences of the X. fastidiosa strains sequenced in this study and those available in GenBank revealed two cladograms using 367 steps (Consistency Index = 0.92; Retention Index = 0.96). From the 546 resulting aligned characters, 406 were constant (25.7% of variable characters). From the 140 variable characters, only 25 were parsimoniously informative. Two large groups were identified by our results, with the clades being shown by cladogram topology: group 1 (bootstrap 74%) includes strains GR8935 and 6068 (grapevine), 6748 (elm), 6744 (mulberry), PL.788 (plum), CI X0, CI.52, and CI.11067 (citrus), CO.01 and 6740 (coffee); and group 2 (bootstrap 69%) includes the remaining strains, except 6749 (ragweed) (bootstrap 60%) and 6751 (periwinkle) (bootstrap 74%). It was possible to subdivide group 2 into five subgroups: almond (6746); almond–plum–peach (bootstrap 70%); grapevine–maple–some almonds (bootstrap 69%); oak (bootstrap 63%); and oleander (bootstrap 89%) (Fig. 1). The cladogram analysis revealed some unexpected results. The plum strain PL.788 (a basal group of the clade formed by the coffee and citrus strains) was in a distant phylogenetic position when compared with another strain (plum 2#4) which has the same host from the same geographic region, and they differed considerably from each other when the genetic distances were considered. Others studies with the plum strains revealed similar results to those obtained here. Strain 2#4 clustered together with the peach and almond strains [15] using CHEF and RAPD analysis. The other plum strain (PL.788) grouped together with the citrus group [23] when 16S and 16S–23S sequencing were used. Although only two plum strains were used, our results showed that the both are clearly removed from the grapevine group, as found in other studies [15] (Chen et al., 1995). These two plum strains were grouped in different clades represented by a polytomy as a result of lack of cladistic information. These data demonstrate that plum strains do not belong to a monophyletic group, suggesting that infection in plum hosts occurred at least in two historical events. There are no data about these two strains in a single study. Within group 1, cladogenesis can be seen where the strains of the grapevine (6068 and GR 8935) group together with the strains of elm and mulberry (bootstrap 74%). By analyzing the data of the Chen et al.’s [7] research based on 16S and RAPD analysis, it can be seen that the divergence between grapevine and

194 mulberry strains is more recent than between grapevine and citrus strains. However, our data do not permit a chronological explanation regarding those strains diverged in different clades. The second clade groups the strains of citrus, coffee, and plum (PL 788). The strains responsible for CVC (citrus variegated chlorosis) and CLS (coffee leaf scorch) are in the same clade (bootstrap 77%), in agreement with several other studies [15, 23] which have shown close relationship between these two strains. The relatedness of citrus and coffee strains suggests a common ancestral lineage for CVC and CLS. The polytomy represented by the citrus and coffee strains does not refute the hypothesis from Quin et al.’s studies [29] that suggest that the citrus strain was originated as a clonal variant of the coffee strain because the coffee yield is older than the citrus. While these two strains of coffee (CO.01 and 6740) were a monophyletic group (bootstrap 61%), the citrus strains (CI.52, CI.11067, and CI.X0) are not in the same clade. Except for the 6068 and GR8935 strains, which group themselves with the mulberry and elm strains, all the other grapevine strains form a clade with the maple and three almond strains (Tulare, Manteca, ALS1), which have as the basal group the clade formed by all other almond strains. Some studies have reported, based on RAPD and RFPL data, that Pierce’s disease and almond leaf scorch are caused by the same X. fastidiosa strain [5, 10]. The lack of cladistic information is notable in the polytomies represented by the almond strains. The phylogenetic hypothesis shows almond strains in two distinct areas of cladogenesis, the 6746 strain representing the most basal. Our data show most almond trees in a basal position compared with grapevine strains, suggesting a more recent origin than the grapevine infection. It is possible that once most grapevine, almond, and maple strains from the same geographical region (California) have been analyzed their phylogenetic relationship will be understood (the strains grouped in the same clade). The strains isolated from oak and oleander formed monophyletic clades, that is, these strains formed separated and genetically uniform clusters as well as the oak strain. The oleander strains also showed the same grouping in other studies. Hendson et al. [15] revealed that the clustering of the oleander strains was identical even when different techniques were used, such as ERIC, RAPD, REP-PCR analysis, and 16S–23S sequencing. It would be expected that we would find the same result as Hendson et al. as the strains analyzed here are the same those that they analyzed. The fact that the oleander strains are closer to the most grapevines and

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some almond (ALS1, Manteca, Tulare) strains, could be explained by these strains having their origin in the California region as well as the strains cited above. However, only a small fraction of the genome of the oleander strain has been sequenced, not including the ITS region, and we could not compare our results. Similar results were obtained with the oak strains when the results of Chen et al.’s [39] and Hendson et al.’s [15] researches were compared. They also obtained a separate grouping by different DNA analysis techniques. The phylogenetic variability found among strains from the same host may represent interstrain/interoperon variation [6, 8]. Sequencing mistakes are improbable, given that each fragment of both strands was sequenced 10 times each to avoid this type of analysis error. Although high levels of sequence similarity were reached among the X. fastidiosa strains, these results allow a distinct grouping according to the host. Several groups could be clearly identified (the oak, peach, citrus, coffee, oleander group), perhaps representing common historical events of infection (suggested by common ancestral lineages) to the same hosts. The grouping of those strains was the same when using the cladistic method as when they were analyzed by the phenetic method, although the evolutionary relationships were not inferred when using the neighbor joining method and the topology of the tree was different and revealed distinct evolutionary relationships. A phenogram based on similarity data (distance-based data) is useful for observing relationships based on similar characters such genetic diversity, presenting the differences in the characters analyzed. However, it does not necessarily represent the evolutionary hypothesis for a certain taxonomic group. The maximum parsimony method, on the other hand, allows the hypothetical phylogenetic relationships to be evaluated so that one or more evolutionary hypotheses for the group being studied can be proposed [33]. A study on the genetic diversity of X. fastidiosa by Pooler and Hartung [28], which analyzed RAPD fragments, revealed the existence of five groups: the CVC, plum–elm, grapevine–ragweed, almond, and mulberry groups [28]. However, these data cannot be compared with ours, because RAPD data cannot be used for phylogenetic analysis when there is no evidence of homology (RAPD data are better for distance analysis). The methodology used to carry out the analysis followed from promising results regarding strain–host clustering. Despite the low nucleotide variability found in the sequenced region, the clusters were well defined when maximum parsimony was used to align and construct the phylogenetic tree.

J. Camargo Martinati et al.: Phylogeny of Xylella Strains

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