Genetic Diversity of Candidatus Liberibacter asiaticus Based on Two Hypervariable Effector Genes in Thailand

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasA I and lasA II, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasA I imply extensive variation exists within the full and partial repeat sequence while the single band from lasA II indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.

Introduction genetic diversity studies on the lasA I and lasA II genes from Florida and other global isolates revealed not only the extensive variations in the intragenic repeat numbers, repeat arrangements, and the sequences flanking the repeat region, but also suggested multiple introductions of this bacterial pathogen into Florida [13]. In this study, we characterized the lasA I and lasA II genes of the Las isolates collected from infected Citrus spp. and D. citri in different geographical regions of Thailand in an effort to define the genetic relationships amongst these Las isolates.

Sample collection
Two hundred thirty-nine symptomatic citrus leaf samples and 55 psyllids were collected from 239 HLB-affected citrus plants in different orchards throughout 22 provinces in Thailand from September 2010 to December 2012 ( Figure 1). In this study, both citrus and psyllid samples were collected from 5 different citrus varieties: mandarin (Citrus reticulata), sweet orange (C. sinensis), pummelo (C. maxima), lime (C. aurantifolia), and Kaffir lime (C. hystrix). Most of these varieties are commercial citrus species in Thailand.

DNA extractions
Total DNA of each plant sample was prepared from 0.2 g of Las-infected leaf midribs. The midribs were frozen in liquid nitrogen and quickly ground to fine powder with a mortar and pestle. The leaf powder was transferred to a 1.5 mL centrifuge tube and total DNA was extracted using a DNeasy Plant Mini Kit (Qiagen,Valencia, CA, USA) according to the manufacturer's instructions. The DNA was eluted with 100 mL of distilled water and kept in -20˚C until assayed. Individual insect DNA was extracted according to the method previously reported [20]. Briefly, a single psyllid was ground in a 2 mL screw capped tube containing 5 glass beads and 500 mL extraction buffer (0.1 M NaCl, 0.2 M sucrose, 50 mM EDTA, 50 mM Tris-HCl pH 8.0, and 1.25% SDS) using FastPrep-24 Homogenizer (MP Biomedicals, LLC, OH USA). Ground samples were incubated at 65˚C for 15 minutes followed by the addition of 160 mL of solution III (5 M potassium acetate, 3 M glacial acetic acid), and 500 mL of 24:1 chloroformisoamyl alcohol mixture. After being mixed by inversion, the samples were placed on ice for 10 minutes before centrifugation at 14,000 rpm for 10 minutes. The aqueous phase was transferred to a new 1.5 mL tube and the DNA was precipitated by using an equal volume of isopropanol. The DNA pellets were washed twice with 75% ethanol and dried using a speed vacuum. The DNA pellets were then resuspended in 40 mL of distilled water and kept at 220˚C until assayed.

Detection of Ca. Liberibacter asiaticus by Real Time PCR
To evaluate the titer of Las bacteria in plant and psyllid DNA, the extracted DNA was initially analyzed by real-time PCR using primers and probes targeting the 16s rRNA gene and the intergenic tandem-repeat regions of lasA I and lasA II gene in Las genome. TaqMan real-time PCR amplification was performed in a Master Cycler Realplex Real-Time PCR System (Eppendorf Inc., USA) using specific primers HLBasf, HLBr and probe HLBp as listed in Table 1. The 15 mL TaqMan PCR reaction mixture contained 7.5 mL of TaqMan PCR master mix (Applied Biosystems), 250 nM of each primer, 150 nM of probe, and 100 ng of DNA template. The real-time PCR amplification setting included 95˚C denaturation for 5 minutes, followed by 40 cycles of 94˚C for 3 seconds and 60˚C for 30 seconds. SYBR Green1 real-time PCR was used to detect amplicons produced with primer set LJ900f/r (Table 1), which targets the repeat region of lasA I and lasA II . The 15 mL reaction mixture contained 7.5 mL of SYBR Green master mix, 600 and 900 nM of LJ900f and LJ900r respectively, and 100 ng of DNA template. The thermal cycling conditions were as follows: 95˚C denaturation for 3 minutes followed by 40 cycles at 95˚C for 3 seconds, and 62˚C for 30 seconds, with fluorescence signal capture at the end of each 62˚C step followed by a default melt (dissociation) stage.

Conventional PCR
All primers for conventional PCR are listed in Table 1. Las-infected samples were confirmed using the CGO3F/CGO5R primer pair targeting 16S rRNA [21]. DNA samples that tested Las positive by RT-PCR were used for amplification of lasA I and lasA II genes. The primer pair LJ730/LJ729 were used to amplify the fragment including the lasA I full gene and its flanking region; the lasA II full gene and its flanking regions were amplified by the primer pair LJ812/LJ1089. The PCR reaction was performed in a 20 mL reaction mixture containing 10 mL of 2x buffer D (Epicentre Biotechnology, Madison, WI, USA), 250 nM forward/reverse primers, 1.25 U Taq-DNA polymerase (New England BioLads Inc., Ipswich, MA, USA) and 1-2 mL of template DNA. The PCR cycles were initiated with 95˚C for 3 minutes, follow by 40 cycles of 94˚C for 20 seconds, 50-54˚C for 30 seconds, 72˚C for 2 to 3 minutes according to different primer sets, and the final extension at 72˚C for 10 minutes. The amplified PCR products were separated by electrophoresis in 1% agarose gels (1x TAE buffer) containing ethidium bromide (0.5 mL/mL) and photographed under an UV illuminator.

Cloning and sequencing
Conventional PCR products were ligated into TOPO TA vector pCR2.1 and the Escherichia coli TOP10 chemical competent cells were transformed with the

Sequence and phylogenetic analysis
BLAST program was used to compare the similarity between the newly obtained DNA sequences with the homologous sequences published in the NCBI GenBank. Phylogenetic relationships for protein sequences were inferred using parsimony and maximum likelihood approaches. For each protein, a multiple alignment was produced in Mesquite v.2.73 using ClustalW v.2.0.12 with default settings [22,23], followed by manual adjustments. This data was analyzed in ProtTest v.2.4 with Akaike Information Criterion, resulting in a best fit substitution model for amino acid replacement (JTT+G+F; [24][25][26]). Maximum likelihood inference was conducted using RaxML v7.2.6 on the CIPRES teragrid portal with default settings and JTT, followed by 1000 bootstrap replicates [27]. These 1000 trees were used to construct a majority rule consensus tree in PAUP. Using maximum parsimony, a heuristic search with random stepwise addition and tree bisection-reconnection was implemented in PAUP. Support was assessed using NJ bootstrap (1000 replicates).

Las population dynamics in infected citrus plants and psyllids in Thailand.
A total of 239 DNA samples extracted from Las-infected citrus leaves of different geographical origins collected throughout Thailand were used in this study ( Figure 1).  (Table 1) to amplify a 534 bp amplicon of 16S rDNA (data not shown).
In this study, evaluation of the genetic diversity of the Las positive Thai isolates was based on two homologous tandem repeat genes, lasA I and lasA II, which are found within the prophage region sequences FP1 (prophage 1) and FP2 (prophage 2), respectively. RT-PCR primers LJ900f/r (Table 1), which target an identical region found in both the lasA I and lasA II genes [17], were used initially to investigate the presence/absence of the Las prophage sequence in the Thai isolates. All 239 citrus samples and 55 psyllid samples tested positive by RT-PCR using LJ900f/r primers with Ct values from 14-30, indicating the presence of lasA I and/ or lasA II genes in all Las positive Thai isolates.
Further in depth evaluation of the genetic diversity within the Thai isolates was conducted using conventional PCR to individually amplify the lasA I and lasA II genes and their flanking regions from all 239 infected citrus and 55 psyllid DNA samples using primer set LJ729/LJ730 and LJ812/LJ1089 as described previously (  Table 1). The representative PCR results from the different provinces are shown in Figure 2. The different size and/or multiple sized amplicons observed in the agarose gel reveal the genetic diversity of the prophage region sequences amongst the Thai isolates. The dynamics of the Las prophage populations in the different citrus varieties and psyllids collected from Thailand are indicated in Table 2. Of the 294 Las positive DNA samples from Thailand, 142 (48.29%) contained lasA I , 186 (63.26%) contained lasA II , and 57 (19.38%) contained both lasA I and lasA II . Interestingly, more Las isolates from HLB-affected citrus plants contained lasA II (71.12%) than lasA I (37.23%). In contrast, Las isolates from infected psyllids contained lasA I more frequently (96.36%) than lasA II (29.09%).
Of note is the fact that 23 samples (7.82%) were undetectable using both the lasA I and lasA II primers in conventional PCR despite an average Ct value of 21.65 for the 16S rDNA using RT-PCR (data not shown) and a positive result using the LJ900f/r primer pair. Among these 23 samples, 10 contained a low titer of lasA I or lasA II with a Ct value ranging from 25-30 by SYBR Green real-time PCR using primers LJ900f/r, which may explain why the 10 samples tested negative by conventional PCR using both lasA I and lasA II region primers. However, 13 contained a higher titer of lasA I or lasA II with a Ct value from 19-24 by LJ900f/r, thus eliminating low titer as a reason why the lasA I and lasA II primers could not amplify in these samples.
The association of the Las genetic diversity with regard to their geographical origins in Thailand was also investigated in this study (Table 3). Except for the samples from Lampang, Bangkok, Nakornnayok, Chai Nat, Kamphaeng Phet and Phichit province, Las isolates from other provinces contained more lasA II than lasA I . Furthermore, all samples from 4 provinces (Chiang Rai, Pathum Thani, Ubon Ratchathani and Buriram) were positive for lasA II only.

Variations of lasA I and lasA II in Las-infected citrus plants and psyllids in Thailand
A total of 271 of the 294 Las positive citrus and psyllid DNA samples amplified lasA I and/or lasA II genes using primer pairs LJ730/LJ729 and LJ812/LJ1089, respectively, in conventional PCR. Twenty lasA I and six lasA II amplicons from different varieties of citrus or psyllids were selected for cloning and sequence analysis. The sequencing result confirmed that the presence of the different sized amplicons using the two set of primers were indeed lasA I and lasA II and their flanking region sequences (Table 4). Sequence alignments revealed that the samples containing multiple sized amplicons and the size variants seen amongst the different samples were due to changes in the number of repeats (full or partials) and/or the arrangement of the individual repeat units within lasA I and lasA II . Overall, the lasA I gene products showed more sequence variation with regards to the partial repeat than the lasA II gene in both the citrus and psyllids samples ( Figure 2 and Figure 3). The number of full repeats varied from 1 to 9 in lasA I , with a majority having 1, 2 or 7 full repeats (Table 4), while the arrangement of the full and partial repeats in lasA I gene was highly variable as well ( Figure 3A). Each full length repeat in lasA I was 132 bp whereas the size of the partial repeats in all of the amplicons varied from 24, 33, 48, 78 and 108 bp. Commonly, a 78 bp partial repeat at the 59 end and a 33 bp partial repeat at 39 end of the entire intragenic repeat region were observed in each lasA I gene, while a majority of the other sizes (24, 48 and 108 bp) of partial repeats were observed between fulllength repeats inside the repeat region ( Figure 3). The predicted open reading frames (ORFs) of lasA I from Las Thai isolates separated into the following 12 distinct patterns when the ratio of full versus partial repeats was considered: 9/5, 8/5, 7/3, 7/2, 5/3, 5/2, 3/5, 2/5, 2/3, 2/2, 1/3, 1/2. However, the pattern of 7 full and 2 partial repeats was the dominant group from the 20 sequenced lasA I amplicons ( Table 4). The division of the ORFs of lasA I into the twelve subclasses is illustrated ( Figure 3A). Based on the sequence homology of the lasA I gene, the level of identity among the Thai isolates was 87%-99%. These isolates were more closely related to China (CHA-Cit18_pLJ316.1) and the Philippines (PHA-Psy5_pLJ313.4) isolates, sharing approximately 89%-99% identity. In contrast, Florida (FL-Psy62_pLJ108.1) and India (IND-Psy2_pLJ314.1) isolates only share 86%-97% identity with Thai isolates. The 59 end of the DNA sequences outside of the repeat region share 98-100% similarity among Thai isolates and 92-94% similarity when compared to the same sequenced region from isolates from Florida, China, the Philippines, and India. The 39 ends of the DNA sequences outside of the repeat region were more variable than 59 ends. The similarity between Thai isolates varied from 81-100%, and the 39 end of the DNA sequences outside of repeat region were more closely related to the China and Philippine isolates (92-100% similarity) when compare with those from India and Florida (88-93% similarity).
Compared to lasA I , the lasA II gene was less diversified and the sequence variations among lasA II gene amplicons were at the single nucleotide polymorphism level. All PCR products showed a single band with the expected size of the amplicon being approximately 1600 bp using the LJ812/LJ1089 primers. Most lasA II amplicons were of the expected size ( Figure 2B) despite being amplified from different samples collected from different geographic regions. The ORF of the lasA II gene from the Thai isolates contained only 2 patterns with a ratio of full to partial repeats of 4/2 or 0/1 ( Figure 3B, B1-B2).The dominant pattern present was 0 full and 1 partial repeat and was observed in 6 lasA II amplicons (Table 4). These lasA II sequences, isolated from both citrus and psyllids samples, shared 99% identity with the repeat sequence from China (CHA-Cit5_pLJ393.1) suggesting that the lasA II gene found in Las strains from Thailand possess little variation and are closely related to isolates from China. In contrast, only one clone from the THA-RE-LM3 isolate collected from Roi Et province Table 3. Evaluation of Las citrus isolates from different geographical locations in Thailand using primers targeting the lasA I and lasA II gene regions within the prophage.  (Citrus aurantifolia) contained 4 full repeat sequences and one partial repeat and was 95% identical to the Florida isolate (FL-Psy62_pLJ201.1) ( Figure 3B, B1). ORF prediction for the lasA I and lasA II gene sequences from the 26 amplicons indicated that most repeat regions were found to be in frame when the genes were translated using SEQtools 8.4 software. Therefore, deletion or insertion of the full or partial repeat unit did not disrupt the open reading frame of lasA I or lasA II genes but merely changed the length of the expected protein product.

Diversity and Phylogenetic analysis of Ca. Liberibacter asiaticus in Thailand
The lasA I and lasA II gene sequences from the Thai Las isolates were deposited to GenBank. Sequence alignments with Ca. L. asiaticus str. psy62 (Accession # NC_012985) and China (Accession # NC_020549) were subsequently performed. Based on the variable nucleotide sequences of Las isolates from citrus and psyllid samples collected from different locations in Thailand, DNA and amino acid sequences deduced from the lasA I gene and the lasA II genes were aligned and integrated into phylogenetic analyses (Figure 4 and 5). Previously identified LasA I or LasA II protein sequences from Las isolates in Florida (YP_0033084345.1, HQ263703), China (HQ263691, HQ263715), the Philippines (HQ263695), India (HQ263699) and Thailand (HQ263693) were included in these phylogenetic analyses. The leucine-rich repeat protein homolog from Colwellia psychrerythraea (Accession # AAZ26055) was used as the outgroup for both LasA proteins. Phylogenetic analyses of the lasA I gene using the maximum parsimony yielded 680 best trees with tree lengths of 1095 steps. Bootstrap support >60% was observed for the 12 nodes. The lasA I gene yielded only 6 trees with tree lengths of 581 steps. Bootstrap support >60% was observed for the 2 nodes. A comparison of the tandem repeat region of the lasA I gene from Thai isolates with other worldwide samples revealed that most of the Thai Las samples formed a well-supported sister-clade with China (CHA-Cit18_pLJ316.1) and Philippine isolates (PHA-Psy5_pLJ313.4). The Florida (FL-Psy62_pLJ108.1) and India (IND-Psy2_pLJ314.1) isolates formed a well-supported sister-clade (100% bootstrap) to this group. The lasA I gene from 4 isolates, collected from pummelo and psyllid in the Phichit province, grouped together and demonstrated a branch support of 100% bootstrap. LasA II phylogenetic inferences, containing 13 taxa, resulted in a tree with a well-supported sister-clade with 98% bootstrap to the sample from China (CHA-Cit5-pLJ393.1). Two lasA I genes from Roi Et province isolates formed a well-supported sister-clade to Florida psyllid (FL-Psy-pLJ201.10) although without bootstrap support.

Discussion
Ca. Liberibacter asiaticus encodes two intriguing autotransporters, LasA I and LasA II [18], which were used to evaluate the Las population diversity among Florida and international isolates [13]. In this study, the same primers were utilized to investigate the Las populations in infected citrus and psyllids from different geographical locations in Thailand. The PCR and sequencing results of these two hyper variable regions (lasA I /lasA II ) confirmed the presence of the homologous FP1 and/or FP2 prophages in Las Thai isolates (Table 2 and 3), and validated that the protocol is sufficient for the characterization and differentiation of Las populations among isolates in Thailand. Based on the previous study, most if not all Florida Las isolates contained both lasA I and lasA II , which correspond to the FP1 and FP2 prophages [6]. Although the number of Las positive samples were limited in that study, all DNA extracts from samples outside of America detected either lasA I or lasA II as describe by Zhou [6]. With an increased sample number from Thailand, most Las-infected citrus and psyllids were found to contain either FP1 or FP2, while 17.99% of the Las-infected citrus and 25.45% of the psyllids samples contained both prophages in the Thai isolates. Interestingly, 13 citrus samples containing a high titer of Las bacteria (tested by 16s rDNA based TagMan RT-PCR) and a high copy number of lasA I repeats (tested by lasA I and lasA II gene based SYBR green RT-PCR) still tested negative by conventional PCR using both lasA I and lasA II gene region primers. Since bacterial titers at this threshold are normally detectable by conventional PCR, we speculate the lack of a PCR product may be the result of a dramatic sequence variation in lasA I and lasA II and its flanking region, especially in the primer sequence's target region, which suggested the possibility of producing new prophage recombinants within the Las genome. This parallels what was seen in the Las genome from Florida [13].
Many lasA I amplicons showed multiple bands but only a single band was ever detected for the lasA II amplicon. This observation was consistent with that found in Florida isolates [13]. Although gene duplication within a single strain cannot be ruled out, the high variation of the lasA I gene from the same plant sample is thought to be indicative of a co-infection by multiple populations of Ca. L. asiaticus in a single source plant. A majority of the amplicons of the lasA I gene found in Thailand isolates (20 in total) contained 7 full and 2 partial repeats (  Table 4), a pattern consistent with both the China isolate (CHA-Cit18, HQ263691) and the Philippines isolate (PHA-Psy5, HQ263695). However, a majority of the lasA II genes found in Thailand isolates contained 0 full and 1 partial repeat (Table 4), a pattern opposite to the China isolate (CHA-Cit5, HQ263715), which contains 1 full and 0 partial repeats. Interestingly, only one isolate, THA-RE-LM3, contained 4 full and 2 partial repeats ( Figure 3B, entry B1). This isolate shared 95% identity with the Florida isolate (HQ263703). Our LasA II phylogenetic tree results agreed nicely with those previously reported by Zhou in 2011, which indicated that another major route of HLB pathogen introduction may have occurred from Thailand to Florida. Our results imply that the Las bacteria in Thailand may have been introduced from both China and the Philippines.
Historically, HLB disease, also known as yellow shoot, was first reported in southern China in 1919 [28] and the disease occurred in 1921 in the Philippines presenting as zinc deficiency symptoms [29]. HLB did not appear in Thailand until the 1960s [2,3]. Since budwood of several mandarins was introduced into Thailand from China at the same time, it was speculated that this might be the source of Las introduction [1,30]. Alternatively, transmissions of HLB bacteria might have resulted from the insect vector (Diaphorina citri) migrating from China since the psyllids found in Thailand are of the same haplotype as a majority of the psyllids in China based on the analysis of the mitochondrial cytochrome oxidase I [31].
In 1998, Ohtsu used the 16S rDNA and 16S/23S intergenic regions to evaluate genetic diversity in Thailand. The sequencing data of these regions indicated that the Nepalese and Thai isolates were closely related and were slightly different from the India and Chinese isolate [32]. Based on the prophage region, Tomimura performed a phylogenetic analysis using bacteriophage-type DNA polymerase sequences. His results revealed three clusters in Southeast Asia where Thai isolates were grouped with Vietnam isolates [12]. However, multilocus microsatellite analysis of Ca. L. asiaticus indicated that Thai isolates grouped with samples from Brazil and East to Southeast Asia [33]. Results from the phylogenetic analysis of lasA I and lasA II sequences in this study also grouped the Las Thai isolates with the Chinese isolate, providing additional evidence for a possible introduction of Ca. L. asiaticus into Thailand from China. The phylogenetic analysis here indicated differences in the tandem repeat numbers and protein sequences among isolates of different geographical origins although the differences in citrus cultivars and organisms (plant versus psyllid) did not appear to influence phylogenetic placement.
Although the HLB bacteria are vectored by the psyllid, the prophage populations between psyllids and citrus appear different since 96.36% of Lasinfected psyllid samples contained FP1 compared to 37.23% of Las-infected citrus samples while 29.09% of Las-infected psyllid samples contained FP2 compared to 71.12% of Las-infected citrus samples. These results are consistent with previous studies when the two hyper-variable regions were used by Zhuo et al. [6] to investigate the prophage population dynamics. In that study, new prophage variants/types were identified from the FP1 and FP2 prophages. Type A was most abundant in Las-infected psyllids and is located in FP1, indicating that FP1 was the dominant population in Las-infected psyllids, which may be important for insect transmission.
Although Zhang et al. [15] described two largely homologous DNA sequences as the prophage region SC1/SC2 of Las-infected UF506 isolates, no discussion regarding the function of either prophage, their role in the virulence of the HLB disease, or disease transmission was provided. Many bacteriophage or prophage from bacterial pathogens encode virulence factors such as bacterial toxins, effectors, attachment proteins, and extracellular polysaccharides [34]. We hypothesize that the prophage infecting Las can change avirulent strains into virulent strains, similar to what is seen with other bacteria. For example, various temperate phages of the Myovirus and Inovirus families were able to infect Ralstonia solanacearum. Infection of Ralstonia by phage wRSS1 alters its pathogenicity by increasing the virulence of Ralstonia in host plants. Prophage genes have also been predicted to encode for pathogenicity or virulence factors such as with Xylella fastidiosa, a pathogen of grapevines and citrus. This organism contains the XfP1 prophage, which carries a gene similar to vapD, a putative virulence-associated protein from the sheep pathogen Dichelobacter [35]. Although the function of the lasA I and lasA II genes has not been completely elucidated, their similarity with other known autotransporters and translocation to the mitochondria of the host cell [18] makes them likely candidates as proteins encoded by the phage that are involved in the virulence of the Las pathogen.
Finally, this is the first study to elucidate the genetic variation of the Ca. L. asiaticus populations in Thailand using the two hypervariable effector genes, lasA I and lasA II , as molecular markers. Hypervariations in the lasA I and lasA II genes among Ca. L. asiaticus isolates implies a potential important mechanism about the adaptation ability of Ca. L. asiaticus on insect vs. plant hosts. Elucidation of the population structure, ecology, and epidemiology of the pathogen in relation to its genetic diversity and geographical location is an important step towards further effective disease management.