Xanthomonas citri pv. viticola Affecting Grapevine in Brazil: Emergence of a Successful Monomorphic Pathogen

Abstract

The pathovar viticola of Xanthomonas citri causes bacterial canker of grapevine. This disease was first recorded in India in 1972, and later in Brazil in 1998, where its distribution is currently restricted to the northeastern region. A multilocus sequence analysis (MLSA) based on seven housekeeping genes and a multilocus variable number of tandem repeat analysis (MLVA) with eight loci were performed in order to assess the genetic relatedness among strains from India and Brazil. Strains isolated in India from three related pathovars affecting Vitaceae species and pathogenic strains isolated from Amaranthus sp. found in bacterial canker-infected vineyards in Brazil were also included. MLSA revealed lack of diversity in all seven genes and grouped grapevine and Amaranthus strains in a monophyletic group in X. citri. The VNTR (variable number of tandem repeat) typing scheme conducted on 107 strains detected 101 haplotypes. The total number of alleles per locus ranged from 5 to 12. A minimum spanning tree (MST) showed that Brazilian strains were clearly separated from Indian strains, which showed unique alleles at three loci. The two strains isolated from symptomatic Amaranthus sp. presented unique alleles at two loci. STRUCTURE analyses revealed three groups congruent with MST and a fourth group with strains from India and Brazil. Admixture among populations were observed in all groups. MST, STRUCTURE and e-BURST analyses showed that the strains collected in 1998 belong to two distinct groups, with predicted founder genotypes from two different vineyards in the same region. This suggest that one introduction of grape planting materials contaminated with genetically distinct strains took place, which was followed by pathogen adaptation. Genome sequencing of one Brazilian strain confirmed typical attributes of pathogenic xanthomonads and allowed the design of a complementary VNTR typing scheme dedicated to X. citri pv. viticola that will allow further epidemiological survey of this genetically monomorphic pathovar.

Keywords: MLSA; MLVA; Vitis vinifera; Xanthomonas campestris pv. viticola; grapevine bacterial canker.

FIGURE 1

Neighbor-joining tree based on concatenated partial sequences of gyrB and rpoD of 28 strains of pathovar viticola and 15 type strains of most Xanthomonas species. Bootstrap values (1,000 replicates) are shown at each node. The 28 strains include 24 strains isolated in Brazil from bacterial canker-infected grapevines, two (Am-1 and Am-3) from Amaranthus plants grown in the vicinity of grapevines, and two strains from India (CFBP 7660 and CFPB 7691). CFBP 7660 is the pathotype strain of X. citri pv. viticola.

FIGURE 2

Maximum likelihood tree of 131 Xanthomonas strains based on the 4,759 bp concatenated sequences of atpD, dnaK, efp, fyuA, glnA, gyrB, and rpoD. Tree was constructed with PhyML and the bootstrap values higher than 50 (1,000 replicates) are shown at each node. Sequences of the pathotype strain (CFBP 7660) of X. citri pv. viticola were compared to sequences of 131 strains formerly assigned to X. axonopodis representing 21 pathovars (Mhedbi-Hajri et al., 2013) and all six Rademaker’s genetic groups 9.1–9.6 (Rademaker et al., 2005). Correspondence between Rademaker’s groups and the four Xanthomonas species (according to Constantin et al., 2016) are indicated. Xanthomonas campestris pv. campestris strain CFBP 5241 (ATCC 33913) was included as outgroup.

FIGURE 3

Pathogenicity test on Vitis vinifera cultivar Sauvignon carried out by leaf and stem inoculations with Xanthomonas strains. (A) Symptoms 35 days after inoculation of: CFBP 7764 on leaf and stem (a,b); CFBP 7657 (c), CFBP 7658 (d), CFBP 7659 (e) and negative control at 21 days after infiltration (f). (B) Symptom development was recorded as (+) necrosis at the point of infiltration; (++) necrosis at the point of infiltration followed by multiple necrotic spots on the leaves and leaf veins, or development of canker-like lesions on the stems; strain CFBP 7694 was received as X. campestris pv. viticola, but is related to X. hortorum according to gyrB and rpoD sequencing (as shown in Figure 1). Scale bar = 1.0 cm.

FIGURE 4

Minimum spanning trees of 107 Xanthomonas citri pv. viticola strains, comprising 105 Brazilian strains and two strains from India (CFBP 7660 and 7691), based on MLVA with 8 VNTR markers. The circles represent a MLVA type. The types that are connected by a thick solid line differed by 1 VNTR locus; MLVA types connected by thin solid lines differed by 2–3 VNTR loci, and the types that differed by 4 or more loci are connected by dashed and dotted lines. (A) The gray zone represents clonal complexes comprising MLVA types that differ from one another by one locus, (B) the gray zone groups types that differ by one or two loci.

FIGURE 5

STRUCTURE outputs for a test panel of 107 strains of Xanthomonas citri pv. viticola. Best K, the true value for number of clusters, was selected using the Evanno method (Evanno et al., 2005). Colors represent groups identifiable by Bayesian clustering.

FIGURE 6

E-BURST network based on eight VNTR markers showing single locus variants (thick dark lines) and double locus variants (light blue lines) in a collection of 107 Xanthomonas citri pv. viticola strains, comprising 105 Brazilian strains and two strains from India (CFBP 7660 and 7691). The predicted founders of each clonal complex (strains 1193, 1194 and 54) are indicated with colored solid circles. The colors correspond to their groups in the Structure analysis (Figure 5).