Fig 1.
Annual ryegrass (Lolium rigidum) florets with a seed, a nematode gall, or a bacterial gall inside.
(A) i) healthy seed inside floret, ii) healthy seed isolated from floret; (B) i) nematode gall (pointed and black in color) inside floret in place of seed, ii) nematode gall isolated from floret; and (C) i) bacterial gall (pointed and bright yellow in color) inside floret in place of seed, ii) bacterial gall isolated from floret.
Table 1.
Isolates recently collected in 2013–14 from South Australia and old isolates received from different culture collections, universities and institutes were used to determine the population structure of Rathayibacter toxicus.
Table 2.
Primers designed and used to amplify MLST genes for Rathayibacter toxicus.
Table 3.
Primer sequences and results for ISSR analysis of 54 isolates of Rathayibacter toxicus collected from South and Western Australia.
Fig 2.
A phylogenetic tree was generated using consensus partial 16S ribosomal RNA gene sequence (about 1015 bp) of Rathayibacter toxicus, R. tritici, R. agropyri, R. rathayi, R. iranicus, R. caricis and R. festucae.
Clavibacter michiganensis subsp. nebraskensis was included as an outgroup. The tree was constructed using neighbor-joining method and the Tamura-Nei genetic distance model. Detail of isolates and accession numbers of submitted sequences are given in Table 1 and S1 Table, respectively. A consensus tree was generated through bootstrap analysis using Geneious Tree Builder program with 1000 cycles; the obtained values labeled at the forks indicate the confidence limits for the grouping. The scale bar at the bottom indicates the substitution rate.
Fig 3.
The ISSR profiles of 54 isolates of Rathayibacter toxicus.
(A) Primer P15 produced an unique locus of 1.2 kb to differentiate population RT-I (locus absent) from RT-II and RT-III populations (locus present); (B) primer P16 produced an unique locus of 1.7 kb only with population RT-II; (C) primer UBC 810 produced an unique locus of 3.5 kb only with population RT-III of R. toxicus. The numbers above the gel images correspond to the individual isolates listed in Table 1.
Fig 4.
An ISSR phylogenetic tree of 54 isolates of Rathayibacter toxicus achieved using the neighbor-joining method.
Isolates were grouped into three clusters named as population RT-I, RT-II and RT-III. The scale bar at the bottom indicates the dissimilarity among the isolates. Details for all isolates are listed in Table 1. This consensus tree was generated using bootstrap resampling method in Resample module of NTSYSpc with 1000 replicates.
Table 4.
ISSR loci data for the three Rathayibacter toxicus populations: RT-I, RT-II and RT-III.
Fig 5.
A phylogenetic tree of 54 isolates of Rathayibacter toxicus was generated using concatenated consensus partial gene sequences six genes.
A total of 5,182 nucleotides from vancomycin resistant protein vanA, CRISPR-associated protein cse4, secA ATPase, chromosome partition protein SMC, tRNA dihydrouridine synthase, and cysteine desulfurase genes, were analyzed to generate this tree. Three distinct groups RT-I, RT-II and RT-II were formed. The tree was constructed using neighbor-joining and Tamura-Nei genetic distance model. A consensus tree was generated through bootstrap analysis using Geneious Tree Builder program with 1000 cycles; the obtained values labeled at the forks indicate the confidence limits for the grouping. The scale bar at the bottom indicates the substitution rate. Detail for all isolates and gene accession numbers submitted to NCBI GenBank are given in Table 1 and S1 Table.
Table 5.
Nucleotide differences in the partial gene sequences used for multi-locus sequence typing (MLST) analyses of Rathayibacter toxicus populations RT-I, RT-II, and RT-III.
Fig 6.
NJ phylogenetic trees were generated using consensus partial sequences of the individual genes used in the MLST analysis.
(A) chromosome partition protein SMC; (B) vancomycin resistant protein vanA; (C) CRISPR-associated protein cse4; (D) tRNA dihydrouridine synthase; (E) cysteine desulfurase; and (F) secA ATPase. RT-I, RT-II and RT-III are the three populations of R. toxicus identified by MLST and ISSR. FH-100 is a single isolate that did not group consistently with all genes.