Molecular diagnosis approaches have widescale applications in detecting pathogens alongside conventional diagnosis methods.²² They can be used for the successful detection of Xanthomonas from symptomatic as well as latent infection in host tissues. Detection of symptomless propagation material is of importance because latent infection populations can lead to disease development and severe epidemics under favourable conditions. In the present investigation, a survey was undertaken to detect the pathogen causing bacterial blight (oily spot) in pomegranates by using bacterial pathogen-specific primers.

Microbial and biochemical tests
Microbial phenotype and biochemical characterization of the collected bacterial isolates revealed that the pathogenic bacteria were Xanthomonas spp. The collected isolates when grown on NA at 28 °C had smooth, round, yellow-coloured colonies with entire margins (Figure 1a). They were Gram-negative bacteria as they gave pinkish staining with counter stain safranin; which was confirmed by string formation of the culture when using 3% KOH test. All eleven Xcp isolates were weakly positive in the oxidase test, positive in the catalase test, hydrolyzed starch as indicated by a clear zone around colonies when iodine stained media was used and showed gelatin liquefaction. Similar test results were earlier reported in Xcp¹⁶ and Xcc²⁷, they tested negative for the arginine dihydrolase test: as they turned to the orange-pink colour in the presence of the pH indicator phenol red. No growth was observed on asparagine medium; typical of the presence of Xanthomonas; which is a characteristic feature that distinguishes it from closely related pseudomonads.²⁸

Pathogenicity test
The Xcp isolates were shown to be pathogenic. After artificially inoculating the pomegranate plants, typical water-soaked angular lesions appeared after 7 days. Isolation of the pathogen from artificially infected plants proved Koch’s postulate, thereby confirming their pathogenicity. Sharma et al. evaluated various Xcp inoculation methods on pomegranates, of which the spray method yielded the most reproducible symptoms.²⁹ The host specificity of Xanthomonas species or pathovars is generally limited.³⁰

Molecular characterization
Genomic DNA isolated from thirteen isolates of Xanthomonas species [including eleven of Xcp from Maharashtra and two Xcc isolates (from Tamil Nadu and Nagpur)] as well as twenty infected leaf samples [from four locations of Nashik district, Maharashtra, India] were diluted to a uniform concentration of 10 ng/µl for use as PCR templates (Figure 1a and b).

In the present study, molecular detection of the bacterial pathogen was undertaken using different primers, specifically SCAR-based markers from the Xanthomonas repetitive intergenic consensus (XRIC) sequence as well as those reported previously in literature for Xcp and related Xanthomonas. Six of the seven primers amplified the products.

16S ribosomal RNA gene-specific PCR amplification studies
The 16S rRNA gene-specific primer pair (16F27 + 16R1525) amplified a 1500 bp fragment in all bacterial samples (except the three unpurified cultures of bacterial samples numbers 7 to 9) (Figure 2) and among 17 of 20 bacterial blight-infected plant DNA samples (except plant samples numbers 2, 4, and 19) (Figure 3) confirming the presence of bacteria.

Initially, a 1500 bp 16S rRNA gene band from single isolate Xcp Deola-Nashik was cloned and sequenced and a 1018 bp sequence was obtained (GenBank accession FJ827773.1). In a sequence similarity analysis using NCBI megablast (Highly similar sequences), 100 BLAST hits were observed with 98%-99% coverage and 98.53%-99.51% identity. These BLAST results were further subjected to Distance tree results by selecting the Maximum Fast Evolution Method. It revealed that the closest accessions were six Xcp strains; which together with 32 Xcc accessions formed a subcluster (Figure 4). Another subcluster was comprised of seven X. citri pv. malvacearum (Xcm) and seven X. citri pv. glycines (Xcg) accessions. This high sequence identity observed in the 16S rRNA gene suggests that Xcp and Xcc are closely related to Xcm and Xcg. Another close X. citri cluster comprised of accessions of X. pv. phaseoli; X. pv. vignifola, and X. pv. perforans.

16S rRNA gene (1500 bp) sequencing has been extensively used for bacterial characterization and it can assist with bacterial taxonomic resolution.^31-33 Earlier the same primers were used to establish the phylogenetic relationships in the genus Xanthomonas which had high sequence similarity (98.2%) of the 16S ribosomal RNA gene.²³ High sequence similarity (95%-98%) was also reported in the 16S-23S rDNA intergenic region between X. oryzae pv. oryzae and X. campestris pathovars.³⁴

Extensive molecular studies have reviewed the classification of Xanthomonas based on the genomic diversity and relationships therein.^35,36 X. citri have been grouped into the subspecies Xcc and Xcm (both included pathovars from diverse hosts).³⁷ It was proposed that Xcp is very close to X. citri subsp. malvacearum.³⁸ Recently, genome sequences (4.94 Mb) of Xcp were found to have 98.78% to >99% nucleotide identity with X. citri pv. citri.^39,40

XRIC (Xanthomonas Repeat Intergenic Consensus) box amplification and sequence study
Previously, a 145 bp XRIC sequence Box was reported, and was suggested to be Xanthomonas specific, and that over a hundred copies of the box may be present in the genome in immediate upstream or downstream regions of various genes.¹⁸ The XRIC box sequence formed a hairpin-like loop structure, with multiple copies including those in the pigmentation locus. They further suggested that the 64% G+C content of these XRIC elements was characteristic of Xanthomonas. Previously repetitive extragenic palindromic sequence PCR was used for the diagnosis of X. euvesicatoria.¹⁵

During the current investigation, a uniformly monomorphic pattern: with twin fragments of sizes 159 bp and 216 bp were observed in eight Xcp pure isolates and two Xcc isolates, as well as in twenty infected field samples. The 159 bp and 216 bp product lengths were observed in all three unpurified bacterial cultures (Figure 5). Among the twenty infected plant samples, eighteen of them amplified 159 bp and 216 bp bands, but samples no. 2 and 20 did not amplify (Figure 6). Similar profile was also observed in two crude extracts of naturally infected and the artificially inoculated plant sample (Figure 6). Amplification was not observed in either of the negative controls, i.e., healthy plant DNA or without template DNA. The amplification profile of X. vesicatoria isolates varied both in the number of amplicons as well as their polymorphic profile, with their size ranging from 180 bp to 700 bp.

PCR amplification derived 216 bp XRIC band was cloned and sequenced (GenBank accession #FJ827773). In the initial sequencing study, only a single Xcc (Strain 603) accession (Hairpin loop forming Xanthomonas specific region) showed a significant match. However, during subsequent sequence identity studies using NCBI megablast (Highly similar sequences), fifty-four BLAST hits were observed with 98%-100% coverage and 94.91%-97.18% identity. These 54 similar accessions included 33 sequence accessions from Xcc, seven from Xcp strains, seven from Xcg, six from Xcm, and one unclassified Xanthomonas. This indicates that the Xcp, Xcc, and Xcm accessions are closely related. In the same analysis (highly similar BLAST), another 32 accessions showed limited sequence similarity with the 3’end region of this accession (for the last 162-216 bp region), with 12%-25% coverage and 85.7%-100% identity; with 18 of those accessions being X. oryzae strains and the other 14 accessions being X. campestris. The option of somewhat similar sequences (BlastN) yielded a total of 100 BLAST hits with a complete 100% coverage and 76.44%-96.76% identity in various Xanthomonas.

Thus, in the present study, the XRIC sequence (accession no. FJ82777) from an Xcp isolate revealed that the initial 170 bases were conserved/shared only with closely related Xcp, Xcc, Xcg and Xcm; while in contrast, the last 50 bases of the accession were shared by various other Xanthomonas. This suggests that the initial 170 bp region is a good candidate for designing Xcp specific primers. As a result, it was proposed that the XRIC based PCR diagnosis can be used in the identification or detection of Xanthomonas in field diseased plant samples. Most of the conserved sites were intergenic regions either related to genes encoding pathogenesis-related or hypothetical proteins.

During an in silico simulation study of the available database using available prokaryotic genomes, none of the 25-genus other than Xanthomonas yielded any amplification, even under less stringent conditions with XRIC primers (results are not presented). These twenty-five-genera included five genera of order Xanthomonadales, i.e., Stenotrophomonas; Xylella; Pseudoxanthomonas; Frateuria, and Dyella, as well as common pathogenic bacteria like Pseudomonas, Erwinia, and Ralstonia. Under stringent conditions simulation (no mismatch allowed), XRIC box primer in silico amplification was observed in seven species/pathotypes amongst the panel of 14 Xanthomas; including three Xcc strains (amplified a 144 bp amplicon); three X. oryzae pv. oryzae (128 bp and 144 bp amplicons) and a X. oryzae pv. oryzicola (125 bp and 1156 bp amplicons). However, under less stringent in silico amplification conditions (two base mismatches allowed with none at the 3’ end), all 14 species/pathotypes of Xanthomonas yield amplification, with all of them yielding multiple bands, including common amplicons in the range of 142-146 bp.

SCAR based Xcp primers for specifically detecting Xcp
Based on these findings, that this region is highly pathovar specific, two new pairs of SCAR primers, Xcp1-20 + Xcp133-152 and XcpF + XcpR, were designed using Primer3 software at the website www.ncbi.nlm.nih.gov (Figure 7). Xcp1-20 + Xcp133-152 was designed to amplify the 152 bp region by excluding the ~60 bp terminal region shared with different Xanthomonas species; while XcpF/R was designed to amplify the almost complete region. Further, in silico PCR amplification under stringent conditions using both newly designed SCAR primer sets revealed no amplification in any other member of Xanthomonas (including three Xcc strains among the panel of 14 members) or Pseudomonas. With XcpF + XcpR primers, under less stringent conditions (two mismatches allowed with one in the last 3 nucleotides at the 3’ end): an amplicon of size 129 bp was observed in only two members of Xoo (KACC10331 and MAFF 311018). The size of those two amplicons observed in this study was significantly smaller than that of 129 bp specific to Xcp.

The current investigation was undertaken to further confirm/validate the in silico amplification results by further in vitro PCR amplification with the designed primer sets. The newly designed SCAR-based primer pair Xcp1-20+Xcp133-152 amplified a 152 bp band in all Xcp isolates and bacterial blight-infected pomegranate samples (Table 4; Figure 8). In no. 10 Pune-Daund Xcp isolate, additional 371 bp and 720 bp bands were amplified. In the two Xcc isolates (Tamil Nadu and Nagpur isolates) (no. 12 and no. 13) yielded 371 bp bands. Three unpurified cultures, 7, 8 and 9, lacked amplification, likely due to the absence of Xanthomonas. All the twenty-disease infected pomegranate leaf samples used for the amplification amplified a 152 bp product (Figure 8).

Table (4):
Detailed PCR amplification pattern

Figure 8. Bacterial and plant DNA amplified using SCAR Xcp1-20 + Xcp133-152 primer pair
M: 100 bp DNA ladder (GeneRuler^TM); Lanes 1 to 6, 10-11: Xcp DNA amplified products; Lanes 7 to 9: Unpurified culture DNA; Lanes 12-13: Xcc DNA amplified products; Lanes 15 to 34: BB infected plant DNA amplified products

Another SCAR Xcp F/R primer pair amplified a 200 bp band in all blight-infected plant samples, as well as seven of eight Xcp, isolates collected from western Maharashtra. Pune-Daund Xcp isolate no. 10 amplified a 350 bp band along with both Xcc isolates nos. 12-13, while three unpurified bacterial cultures did not amplify (Table 4; Figure 9). All twenty infected pomegranate DNA samples amplified a 200 bp band with primer pair XcpF + XcpR (Figure 10). Under in silico conditions, the pathovar-specific primer set, Xcp-F/R and Xcp1-20 + Xcp133-152 didn’t amplify in any other Xanthomonas except Xcc.

Previously seven Xanthomonas effector xop genes were evaluated for diagnosis, of which xopQ primers yielded a 190 bp amplicon in plants infected with Xcp and Xcp.^41,42 A loop-mediated isothermal amplification (LAMP) technique of Xcp was developed and validated in pomegranates using various sets of 16S rRNA primers.⁴³

Verification of previously reported gyrase (gyrB) gene-based primer for detecting Xcp
Previously, the gyrB gene was used for identifying Xanthomonas species.²⁴ They reported that Xcp isolates amplified a 491 bp band. Similarly, a specific 491 bp gyrB amplicon was also reported in bacterial blighted (Xcp) infected field samples of pomegranate.⁶ The identity of the Xap isolates was confirmed by specific gyrB and 16S rRNA gene sequences, with all isolates resulting in amplicon sizes of the PCR products were 491 bp and 1450 bp respectively.³³ Previously phylogenetic analysis of 203 isolates of Xanthomonas was done using their gyrB gene sequences.³⁶

In the present study, these gyrB gene-specific primers KM gyr-F/R amplified a 491 bp band size in all Xcp isolates; however, in both Xcc isolates (no. 11 and 12), a 375 bp band was observed (Table 4; Figure 11). In the unpurified cultures no. 7-9, there was no amplification. The 491 bp was also observed in 12 out of 20 field samples. However, in two samples from the Satana, different bands were observed, i.e., sample #8 (320 bp) and #18 (491 bp and 1088 bp bands) (Figure 12).

Verification of previously reported Xac.rpf primers
Previously, primers were developed for the detection of Xcc derived from the rpf gene region, in citrus plants.²⁵ They reported that a 581 bp band was amplified in all Xcc infected samples. From the previous sequence similarity studies conducted in our laboratory it was observed that X. citri pv. citri was the closest pathovar, specifically in terms of sequence identity to X. citri pv. punicae. The primer set for the ITS of ribosomal RNA gene sequences was highly specific for X. citri pv. citri, whereas the pthA gene primer set was common to all strains of citrus blight canker Xanthomonas.¹³ On the contrary, leucine-responsive regulatory protein (lrp) gene was informative in distinguishing groups of Xcc.⁴⁴ It was proposed that citrus canker causing Xanthomonas has self-mobilizing plasmids with pathogenicity factors, capable of transferring pathogenicity into other xanthomonad residents on the same citrus plant.⁴⁵ A highly reproducible ligation-mediated PCR technique was developed based on three insertion sequences (IS-LM-PCR) in Xcc.⁴⁶

This Xcc-specific Xac.rpf primer pair amplified a 581 bp product in almost all the Xcp/ Xcc bacterial isolates across various geographical locations, matching the previously reported size (except for three unpurified culture nos. 10, 11, and 12 from the Ahilyanagar region) (Table 4; Figure 13). It also amplified in 14 of 20 bacterial blight-infected pomegranate plant samples, yielding a 581 bp product; while six samples were from Deola and Satana tehsils (no. 6, 10, 11, 12, 17 and 19) did not amplify (Figure 14). These results in the current investigation matched expected sequence similarity studies. However, the Xac.rpf primers are not specific to Xcc only but they are also amplified in Xcp and could not differentiate them from Xcc either.²⁵

Verification using primer for specifically detecting X. oryzae pv. oryzae
Xanthomonas oryzae pv. oryzae (Xoo) based primers developed by earlier workers²⁶ were included in the current studies as the in silico PCR simulation studies with Xcp primers had shown amplicons under less stringent conditions (2 mismatches) though of varying sizes in Xoo. Insertion sequence IS1112 based PJEL1/2 primers were also used to generate specific and reproducible PCR fingerprint patterns for the Xoo isolates.⁴⁷ Similarly 16S-23S rDNA spacer-derived primers were designed by researchers that specifically amplified a 470 bp band in all strains of Xoo; as well as in other three Xanthomonas, including Xcc.³⁴

In the present study, no amplification was observed with the Xoo-specific primer pair Xoo.txt+Xoo.txt4R in any samples, either from Xcp or Xcc (Table 4). Similarly, the infected pomegranate DNA samples did not yield any amplification.