www.thaiagj.org Thai Journal of Agricultural Science 2012, 45(3): 161-170

Association of ‘Candidatus Liberibacter asiaticus’, the Causal Agent of Citrus Huanglongbing in Murraya paniculata and Diaphorina citri in

A. Jantasorn1, Y. Duan2, T. Puttamuk1, S. Zhang3 and N. Thaveechai1,*

1Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, 10900, Thailand 2USDA-ARS-USHRL, Fort Pierce, FL, USA 34945 3University of Florida, IFAS-TREC, Homestead, FL, USA 33031-3314

*Corresponding author. Email: [email protected]

Abstract Orange jasmine, Murraya paniculata, is a preferred alternative host for the Asian citrus psyllid, the primary vector of citrus Huanglongbing (HLB) disease caused by ‘Candidatus Liberibacter asiaticus’ (Las). M. paniculata plant samples and psyllids on the Murraya plants from ten diverse geographical were collected and extracted for DNA to evaluate the presence and titers of Las by conventional PCR and two different methods of real-time PCR. The data showed variation of Las levels both in M. paniculata and psyllids in Thailand. Different titers among individual psyllid sample were observed in each of Thailand. Samples from province displayed the average titer level of HLB pathogen with Ct value of 21.85. The highest titer level was found in sample from province with Ct values of 16.12. The titer of Las is therefore low in M. paniculata and associated psyllids. The results suggest that urban planting of M. paniculata may serve as a minor source of Las inoculum. Our work provides a first association of the infection frequency of Las on psyllid vector from and M. paniculata in Thailand which could be a potential source of inoculum for citrus HLB in Thailand.

Introduction are widely distributed in commercial nurseries and grown as amenity trees. There is a potential risk for Citrus Huanglongbing (HLB), previously known spreading of HLB disease through commercial as citrus greening is one of the most destructive Murraya spp. seedlings. disease on citrus in the world including Thailand. Both D. citri and Las utilize a number of host The causal agent of HLB, ‘Candidatus plants besides cultivated Citrus spp. The D. citri Liberibacter’, belongs to the alpha subdivision of has reported hosts from at least twenty-three genera the Proteobacteria, is restricted in phloem sieve within the family Rutaceae (Halbert and tubes of infected plants and transmitted by psyllids, Manjunath, 2004, Pena et al., 2006), although or by grafting or by dodder transmission (Zhou et germplasm varies in susceptibility to psyllid al., 2007). Currently, three species of the pathogen, infestations. Bergera koenigii, Citrus macrophylla, “Candidatus Liberibacter asiaticus” (Las), “commercial citrus” and M. paniculata have been ‘Candidatus Liberibacter africanus’ (Laf), and reported as preferred host plants of the psyllid ‘Candidatus Liberibacter americanus’ (Lam) are (Halbert and Manjunath, 2004, Westbrook et al., recognized based on 16S rRNA. ‘Candidatus 2011, Yang et al., 2006). In a study of four host Liberibacter asiaticus’, vectored by Asian citrus species, the intrinsic rate of increase of the D. citri psyllid (ACP, Diaphorina citri), is the most population was highest on young grapefruit (Citrus prevalent in the world. In Asia, M. paniculata, an paradisi) (Tsai et al., 2000). ‘Ca. Liberibacter ornamental rutaceous shrub, is a preferred host for asiaticus’ has been isolated from ten Rutaceous ACP. M. paniculata, and other species in this genus genera (Deng et al., 2007, Folimonova et al., 2009, 162 A. Jantasorn et al. Thai Journal of Agricultural Science

Halbert and Manjunath, 2004), and can also be paniculata as alternate host and insect vector transmitted to non-Rutaceaous plant species psyllid (D. citri) in Thailand and to determine the including dodder (Cuscuta campestris), tobacco importance of M. paniculata plant and D. citri as a (Nicotiana tobacum), tomato (Solanum inoculum source of citrus HLB. The infection status lycopersicum) and periwinkle (Catharanthus of Las in M. paniculata and D. citri that dwell on roseus) in the laboratory using dodder transmission M. paniculata was collected from central, eastern (Halbert and Manjunath, 2004, Zhou et al., 2007 ). and some samples from northern parts of Thailand. Host range of the HLB disease may be limited by We documented total infection and seasonal trends feeding preferences of the D. citri rather than by the using conventional PCR and two different qPCR physiological potential of the bacteria (Halbert and detection methods for Las. Manjunath, 2004). In Indonesia, M. paniculata seedling exposed to D. citri obtained from field- Materials and Methods grown citrus tree showed typical external and internal symptom 10 months post-inoculation Collection Murraya paniculata Plants (Tirtawidjaja, 1981). In Thailand, however, no Characterization on symptom expression of abnormalities were detected on orange jasmine and Huanglongbing naturally infected citrus and curry leaf (Berhgera koenigii) plants 8 months after relative plants were determined. The Murraya infectious psyllids fed on them (Koizumi et al., samples from diverse geographical regions were 1996). These studies should be interpreted with collected in Thailand. The collection sites of caution since only visible symptom was evaluated Murraya plants were selected in of the but no specific technique for HLB detection was Northern Region, the Central Plain and the Eastern used for inoculated plant evaluation. In China, Las Region. All of the Murraya plants were established was successfully detected in M. paniculata trees from cutting or macrottings. Symptomatology of with the used of nested but not standard PCR (Li Huanglongbing on Murraya appeared as the same and Ke, 2002). In USA, successful HLB symptoms reported elsewhere including vein transmission was reported from citrus to M. yellowing and the leaf symptom similar to zinc paniculata via psyllids (Damsteegt et al., 2011) and deficiencies and symptomless . from M. paniculata to citrus by the parasitic plant, dodder (Zhou et al., 2007). One alternative host Collection of Psyllids species that has the potential to be especially Adults of D. citri were collected from both problematic for Citrus production is the ornamental visually healthy and symptomatic plants belonging plant M. paniculata (Bove, 2006). M. paniculata is to M. paniculata, and related genera from diverse an excellent host for D. citri. (Halbert and geographical regions in Thailand, such as Manjunath, 2004, Tsai et al., 2000, Tsai et al., commercial groves, retail, resident sites and discount 2002). Because M. paniculata may flush frequently, garden centers in different . or in the case of regular hedging nearly Adult psyllids were collected using an aspirator. The continuously, D. citri are able to reproduce on this insects were catalogued and stored in 75% ethanol in plant at times when they could not reproduce on 2 mL tube for further analysis of Las and non-Las Citrus, which may increase the psyllid population infections. The psyllids were assigned unique in nearby Citrus plantings (Pluke et al., 2008, Yang sample number and stored at -20°C until processed. et al., 2006). Concern about the potential for spread of the vector and disease has led the state of Florida DNA Extraction Methods to restrict the conditions for propagation and sale of All DNA extractions were performed in a sterile M. paniculata in the State (Clark, 2007), but no laminar flow hood. Crude extractions of psyllid programs to manage existing plantings have been DNA were made using a modification of the implemented. There are less information of citrus method described in De Barro and Driver (1997). HLB on an alternate host especially M. paniculata Psyllids were stored in 70% ethanol at 4°C from and psyllid vector (D. citri) in Thailand. Therefore, the time of collection until DNA extraction was this study is to document the incidence of Las in M. performed. Individual psyllids were place in a 2 Vol. 45, No. 3, 2012 Causal agent of citrus huanglongbing in Thailand 163 mL screw-cap tube (USA Scientific, Ocala, FL) hood. Samples were resuspended in 100 µL containing approximately twelve 1.6-1.8 mm nuclease-free water (Qiagen, Valencia, CA). zirconium silicate beads (Ceroglass, Columbia, TN) Prior to qPCR analysis, the nucleic acid and 150 µL of lysis buffer (5%1M KCl, 5%1M Tris concentration of all plant samples was determined at pH 8.4, 0.45% Tween20, 0.45%NP40, 89.1% by spectrophotometry at a wavelength of 260 nm autoclaved deionized water). Tubes were placed in a using a Nanodrop-1000 detector (NanoDrop FastPrep-24-system (MP Biomedicals, Solon, OH) Products, Wilmington, DE). Samples were diluted and homogenized for 30s at 6 m s-1. following in nuclease-free water so that 100 ng of DNA was homogenization, 100 µL of liquid was transferred to loaded per reaction for the rest of M. paniculata a clean 1.5 mL centrifuge tube (USA Scientific), and samples. Plant samples were stored at -20°C until placed in a water bath at 65°C for 15 min. Samples qPCR was performed. were immediately placed on ice for at least 10 min, and then centrifuged at 14,000 rpm for 5 min. the Conventional PCR Amplification supernatant was removed and stored at -20°C until The CGO3F (RGG GAA AGA TTT TAT TGG qPCR was performed. AG) and CGO5R (GAA AAT AYC ATC TCT Leaf midribs were used for DNA extraction from GAT ATC GT) primers set were used for M. paniculata because this tissue contains the conventional PCR in this study. DNA amplification highest titers of Las in Citrus trees (Li et al., 2009). was performed in a final volume of 20 µL M. paniculata samples were extracted using a buffer containing 10 µL of 2X buffer D (Epicentre based extraction method as follows: 900 µl of Biotechnologies, Madison, WI, USA), 250 nmol of extraction buffer (for 1000 mL buffer: 12.1 g Tris- each forward/reverse primer, 1.25 units of Taq Base, 18.61 g EDTA (pH of solution adjusted to 8.0 DNA polymerase (New England BioLabs Inc., using 5 M NaOH to facilitate EDTA dissolution), Ipswich, MA, USA), and 1 to 2 µL of genomic 29.22 g NaCl, 25 g PVP; β-mercaptoethanol added template DNA from Las which was extracted from to buffer just before use at 0.7 mL mL-1) was added midribs of orange jasmine, psyllid vectors and Las to the tubes containing slingshot pellets and plant cultures. Conditions of PCR preparation were sample, and the tubes were homogenized in a denaturation at 95°C for 3 min, followed by 35 FastPrep-24-system for 40 sec at 6 m s-1. following cycles of amplification with denaturation at 94°C homogenization, 40 µL of 20% SDS was added and for 45 sec, annealing at melting temperature at the samples were vortexed. Samples were placed in 52°C for 30 sec, and DNA extension at 72°C for 1 a 65°C water bath for 30 min, and stirred by min with a final extension at 72°C for 10 min using inverting the tube every ten minutes. Samples were a thermocycler (Perkin- Elmer 9600/Applied removed from the water bath, centrifuged at 14,000 Biosystem, Bedford, MA). The PCR products were rpm for 10 min, and 700 µL of supernatant for each separated by 1% agarose gel electrophoresis in sample was transferred to a new 1.5 mL centrifuge TAE buffer, the gel was stained with ethidium tube. A volume of 220 µL of 5M KoAC was added bromide and amplified DNA bands were viewed to the supernatant, and the samples were placed on under UV-transilluminator. ice for at least 20 min. After removal from the ice, the samples were centrifuged at 14,000 rpm for 10 PCR Detection of ‘Candidatus Liberibacter min, and 650 µL of the upper aqueous layer was Asiaticus’ transferred to a new 1.5 mL tube. A volume of 460 All samples were assayed by qPCR with the µL of cold 70% isopropyl alcohol was added to the LJ900f/r series primers (5’-3’ sequences: forward samples, the samples were vortexed, and placed on GCCGTTTTAACACAAAAGATGAATATC, ice for at least 5 min. After removal from the ice, reverse ATAAATCAATTTGTTCTAGTTTACGAC) samples were vortexed at 14,000 rpm for 30 min at described in Zhou et al. (2011) LJ900f/r primers

4°C, the supernatant was discarded, and the pellet target the 1-12 tandem repeats of the hyvI and hyvII was washed by placing 700 µL cold 70% ethanol in genes of Las prophages, and were used because the tubes for 15 min the liquid phase was discarded, they provide a high degree of sensitivity in a 1- and the pellet was allowed to dry in a laminar flow strep qPCR reaction. Briefly, 2 µL of the DNA 164 A. Jantasorn et al. Thai Journal of Agricultural Science template from psyllid or 100ng of Murraya DNA then by 60°C for 30 sec. All samples were assigned were used in a qPCR reaction totaling 15 µl using at Ct value based on the cycle when fluorescence the PerfeCTa SYBR Green FastMix 2x master mix exceeded 0.2∆Rn. (Quanta Biosciences, Inc., Gaithersburge, MD), and a reaction concentration of 600 nM forward Results and 900 nM reverse primer, and nuclease-free water. Reactions were run on Mastercycler Murraya paniculata Surveys Realplex Real time PCR system (Eppendorf Inc., Total of 134 orange jasmine (Murraya Hauppaugeny, NY, USA) with amplification paniculata) trees were collected from diverse setting of: initial denaturation at 95°C for 5 min, geographical regions in Thailand. Mostly the then 40 cycles of 95°C for 3 sec, followed by 62°C samples located in Central plain and some samples for 30 sec. at the end of each run a disassociation located in Northern and Eastern plain of Thailand. cycle of 95°C for 15 sec, 62°C for 1 min, and a ‘Ca. Liberibacter asiaticus’ was detected by gradual ramp to 97°C for 15 s was performed. All conventional PCR in ten samples from Pathum run included positive and at least four no template Thani, Lop Buri, Bangkok, and controls. All samples were assigned at Ct value Chanthaburi provinces respectively. All the based on the cycle when fluorescence exceeded remaining trees were infected by only on species of 0.2∆Rn. samples that did not produce the correct liberibacter that positive with Las specific primer. melt curve were assigned a value of undetermined. ‘Ca. Liberibacter asiaticus’ was present in 7.46% Any sample that amplified (at any cycle) and had of all conventional PCR positive orange jasmine the correct melt profile was re-run in triplicate. trees (Table 1). The highest incidence of Las Samples that amplified and had the correct melt infected Murraya trees was observed from Rayong profile in at least two of the three triplicate runs province (30%) in Eastern region whereas the were considered positive detections. lowest incidence of Las was from Bangkok and As a check for our results using LJ900f/r, all provinces (6.6%) in the Central plain psyllid and plant samples that tested positive by the region (Table 1). The positive samples of Las LJ900 primer as well as 50 randomly selected infection from conventional PCR methods were negative samples of each type were run on the shown in Figure 2. Quantitative determination of HLBaspr primer 5’-3’ sequences: forward Las population for potential of infected Murraya TCGAGCGCGTATGCGAATACG, reverse trees as a reservoir of liberibacter, was done by GCGTTATCCCGTAGAAAAAGGTAG, probe qPCR to further analyse the DNA samples of all AGACGGGTGAGTAACGCG with 6- surveyed trees. No obvious differences in size and carboxyfluorescein (6-FAM) reporter dye on the 5’ symptom severity of ‘Ca. Liberibacter asiaticus’ end and Iowa Black FQ on the 3’, IDT, infected Murraya tree were observed between www.idt.com), with target the 16S rRNA of Las locations and survey period at the time of sample (Li et al., 2006). These primers are among the most collection. Also, no visible differences were common methods for diagnostic detection of Las, detected between PCR positive and PCR negative and have been used to detect Las in D. citri (Lopes trees. However, visible differences in symptom et al., 2010) and M. paniculata (Damsteegt et al., severity were found in samples infected by ‘Ca. 2011). For HLBaspr, we ran a 15 µL reaction, Liberibacter asiaticus’ from and including TaqMan Universal PCR Mastermix Chanthaburi provinces. The symptom shown (Applied Biosystems, Beverly, MA, USA), a yellow and wavy leaves which was similar to reaction concentration of 0.4 mM each primer, and nitrogen deficiency (Figure 1). Mottled leaves and a reaction concentration of 500nM probe, and fruits with aborted seeds, both characteristics of nuclease-free water. qPCR reaction were run on the HLB in citrus were not observed in the PCR Mastercycler Realplex Real time PCR system positive Murraya trees. The yellowing of Murraya (Eppendorf Inc., Hauppaugeny, NY, USA) with leaves included a variety of chlorotic patterns, most amplification setting of: initial denaturation at 95°C frequently that resembling nitrogen deficiency. for 5 min, followed by 40 cycles of 95°C for 3 sec, Vol. 45, No. 3, 2012 Causal agent of citrus huanglongbing in Thailand 165

Table 1 Detection of ‘Candidatus Liberibacter asiaticus’ by conventional PCR of Murraya paniculata collected from different provinces of Thailand.

No. Infected by ‘Ca. Location (province) Number of sample Liberibacter asiaticus’ (%) Central plain Bangkok 30 2 (6.6) Chainat 3 0 Lop Buri 5 1 (20) Nakhon Pathom 20 0 Pathum Thani 30 2 (6.6) 3 0 Samut Songkhram 2 0 5 0 Eastern plain Chanthaburi 2 0 Chon Buri 15 3 (13.3) Rayong 10 4 (30) Trat 6 0 Northern plain 2 0 Kamphaeng Phet 1 0 Total 134 10 (7.46)

presence of Las and to evaluate the Las population levels. The number collected for each site and date was varied. Only 39 (16.8%) psyllids from 10 provinces were positive for Las by the LJ900 method. The Ct values for the psyllids where Las was detected using the LJ900 primers ranging from 17.57-29.87 (Table 2). The psyllids from which we isolated the highest number of copies of

hyvI/hyvII also had a detectable number of copies of the 16S rRNA gene of Las. Fourty-two (18.1%) psyllids were positive for Las by using HLBaspr primers. The Ct values were highly variable with Ct values ranged from 16.12 to 36.74 using primers and probe targeting on 16S rRNA gene (Table 2).

Figure 1 Murraya paniculata leaves with various degree of yellowing symptom of huanglongbing that tested positive for Prevalence of Las in Murraya paniculata ‘Candidatus Liberibacter asiaticus’ by Conventional PCR. A and B the samples collected from Rayong province and C and Samples D the samples collected from . The Murraya samples covering all fourteen provinces in Thailand collected from the month of January, March, July, and August 2010 were Prevalence of Las in Psyllid Samples retained in the analysis. Of these 44 samples, six

Over the course of the experiment, 232 psyllids tested positive for the hyvI/hyvII target DNA samples collected from Murraya plants at different sequence. The positive samples represented in locations in Thailand were tested to determine the Pathum Thani, Nakhon Pathom, Chanthaburi,

166 A. Jantasorn et al. Thai Journal of Agricultural Science

(A) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

(B) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 2 PCR detection of ‘Candidatus Liberibacter asiaticus’ isolated from Murraya paniculata leaves using Las-specific primers targeting the 16S rDNA. Lane 1 water control; lane 2 healthy plant control; lane 3 and 4 the samples collected from ; lane 5 and 6 the samples collected from province; lane 7 and 8 the samples collected from ; lane 9 the samples collected from Lop Buri province; lane 10 and 11 the samples collected from Chanthaburi province; lane 12 and 13 the samples collected from province; lane 14 and 15 ; lane 16 ; lane 17 positive control; lane M 1kb DNA Ladder (Promega) (A). Lane M 1kb DNA Ladder (Promega); lane 1 and 2 the samples collected from Bangkok province; lane 3 and 4 the samples collected from ; lane 5, 6 and 7 the samples collected from Rayong province; lane 8 and 9 the samples collected from Chon Buri province; lane 10 and 11 the samples collected from ; lane 12, 13 and 14 the samples collected from Bangkok (B).

Table 2 Detection of ‘Candidatus Liberibacter asiaticus’ from psyllids (Diaphorina citri) collected from different provinces of Thailand by two primer set of real-time PCR. LJ900f/r1/ HLBaspr2/ Location No. of positive Range of Ct values No. of positive Range of Ct values Totals (Province) sample (Ct ≤30) sample (Ct ≤36.9)

Bangkok 27 2 28.69-28.98 6 33.47-36.66 Lop Buri 23 1 29.85 3 33.95-35.06 Nakhon Pathom 22 2 19.39-27.93 1 16.12 Pathum Thani 24 6 27.16-29.65 3 35.90-36.74 Samut Sakhon 20 3 25.45-29.31 1 24.79 Saraburi 24 2 28.61-29.40 4 29.33-36.63 Chanthaburi 27 4 17.57-29.87 6 21.85-36.44 Chon Buri 20 4 24.36-29.93 2 33.02-33.43 Nakhon Nayok 22 4 25.65-29.44 2 33.93-34.83 Kamphaeng Phet 23 11 24.40-29.76 14 29.83-36.65 Total 232 39 (16.8%) 42 (18.1%) 1/ Primers LJ900f/r target the hyvI/hyvII gene of Las prophages. 2/ Primer HLBaspr target 16S rRNA of Las. Vol. 45, No. 3, 2012 Causal agent of citrus huanglongbing in Thailand 167

Rayong and Trat provinces. We were able to positive with high Ct value by 16S rRNA primer. amplify 16S rRNA of Las from 20 of the 44 This suggests that ‘Ca. Liberibacter asiaticus’ either samples that some samples tested positive for was decreased multiplying in plant or that M. hyvI/hyvII (Table 3). paniculata is not a good alternate host for Las which may contain some inhibitors. Discussion The rate of Las infection of D. citri that developed on M. paniculata is low relative to that The presence of ‘Ca. Liberibacter asiaticus in of D. citri that develop on citrus, and also lower Murraya in urban area was demonstrated. DNA than infection rates published for the potato-tomato sequence comparison revealed no variation in the psyllid, Bactericera cockerelli and another 16S rRNA gene within each species, suggesting Liberibacter species, ‘Candidatus Liberibacter that the Murraya and citrus associated with solanacearum’ (Liefting et al., 2008). Using liberibacters are probably the same. Although both conventional and quantitative PCR, the infection citrus and Murraya was found to be host of ‘Ca. level of D. citri collected from infected citrus in the Liberibacter asiaticus’ in the cities, their responses field is 45-82% (Ammar et al., 2011; Hung et al., to infection seemed to differ considerably. A few 2004; Subandiya et al., 2000). The infection rate of Murraya trees found infected in 2010 were Bactericera cockerelli with ‘Ca. Liberibacter observed for subsequent symptom development in solanacearum’ was 19-20% when the psyllids were the Rayong and Chanthaburi provinces. The collected directly from their host plants (Wen et al., damage to Murraya was much less severe than that 2010). The low rate of infection and level of on citrus. The presence of Las infection in M. bacterial titer in both plant and a psyllid sample paniculata samples using a detection method with raises the possibility that M. paniculata may have a the LJ900fr primers targeting on hyvI/hyvII unique resistance trait for Las. Some citrus prophage region was extremely low. Twenty of M. cultivars do not display severe symptoms when paniculata plant samples that amplified using the they contact Las but they have a bacterial load 16S rRNA primers had a high Ct value, similar to similar to diseased citrus (Folimonova et al., 2009) what we have observed in running these primers and several orders of magnitude higher than we against other species of bacteria (unpublished). The found in M. paniculata. The fact that M. paniculata only some sample in this experiment that would be has both a low level of infection and a low titer of considered positive with low Ct value by 16S bacteria in infected plants despite psyllid movement rRNA detection method was psyllid where to and from citrus and high levels of HLB incidence amplification was detected using the HLBaspr in the local citrus population indicates that a trait of primers. There are conflicting reports about the plant may be restricting reproduction or whether transovarial transmission of Las occurs in movement of the bacteria. The bacterium appear to D. citri (Pelz-Stelinski et al., 2010 and Xu et al., remain at a low titer in the psyllids originated on M. 1988). If transovarial transmission occurs, it is paniculata despite the fact that Las has been possible that this infection originated from an reported to replicate in D. citri (Hung et al., 2004, infected female that migrated to M. paniculata from Inoue et al., 2009). citrus. M. paniculata infections were detected when In addition to citrus, M. paniculata (orange using primers targeting the bacterial 16S rRNA jasmine) is a preferred host of D. citri, and retail gene than with using primers targeting the hyvI/hyvII trade in this ornamental shrub is strongly implicated tandem repeat sequence. The discrepancies between in the distribution of D. citri. M. paniculata is the two primer sets could occur for several reasons. reported to be a cryptic or largely asymptomatic The most likely is that more infections were host of “Ca. Liberibacter” (Clark, 2007), but detected using the 16S rRNA primers because of another report concludes that the bacteria cannot higher sensitivity of that method and 16S rRNA replicate in M. paniculata (Anonymous, 2011). The regions are the best characterized regions, highly epidemiological significance of Murraya as a host conserved among the species of ‘Candidatus for the HLB pathogen is therefore unclear. The Liberibacter spp. The M. paniculata plants were taxonomy of Murraya paniculata is uncertain wild 168 A. Jantasorn et al. Thai Journal of Agricultural Science

Table 3 Detection of ‘Candidatus Liberibacter asiaticus’ from Murraya paniculata collected from different provinces of Thailand by real time PCR. LJ900f/r HLBaspr Location (Province) Total No. of positive Range of Ct No. of positive Range of Ct sample values (Ct ≤30) sample values (Ct ≤36.9) Bangkok 5 0 - 0 - Lop Buri 1 0 - 1 27.89 Nakhon Pathom 5 1 29.11 4 33.27-34.11 Pathum Thani 6 1 26.88 4 32.44-34.60 Samut Sakhon 1 0 - 0 - Samut Songkhram 1 0 - 0 - Saraburi 2 0 - 1 28.20 Chanthaburi 9 2 25.69-29.45 3 32.09-35.33 Chon Buri 2 0 - 0 - Nakhon Nayok 2 0 - 1 30.91 Rayong 4 1 29.17 3 27.36-36.45 Trat 4 1 29.25 3 32.23-36.43 Kamphaeng Phet 2 0 - 0 - Total 44 6 (13.6%) 20 (45.5%)

M. paniculata is distributed across Southeast Asia Acknowledgments including India, Ceylon, Burma, China, Taiwan, the Malay Peninsula, Philippines, and the Melanesian This work was supported financially by the Island (Swingle et al., 1967). The wild plant has Royal Golden Jubilee Ph.D. program through been brought into cultivation at least two times Thailand Research Fund (TRF). We thank USDA- (Mabberley, 1998). Although, the authors of the ARS-USHRL, Fort Pierce, FL, USA for use of study stated that further analysis was necessary research facilities, Christina Latza for technical before dividing the group into two species. support and especially Dr. Lijuan Zhou for Regardless of origin, the domesticated plant often vulnerable advice. referred to as species or cultivar exotica (Mabberley, 1998). Samuel et al., (2001) suggested References that M. paniculata and M. exotica should be separated based on differences in non-coding Ammar, E-D., R.G.J. Shatters, C. Lynch and D.G. Hall. 2011. Detection and relative titer of Candidatus plastid DNA sequence, but bootstrap support for Liberibacter asiaticus in the salivary glands and this division was low. We describe the plants that alimentary canal of Diaphorina citri (Hemiptera: we collected in this study as M. paniculata. Psyllidae) vector of citrus huanglongbing disease. This survey documents demonstrated very low Ann. Entomol. Soc. Ann. 104: 526-533. rate of Las infection in urban planting of M. Anonymous. 2011 Citrus Health Response Program, Known Distribution of Citrus Canker/Citrus paniculata and D. citri fed on these plants, much Greening (HLB) in Florida. Florida Division of Plant lower than occur in citrus and psyllids from citrus. Industry.http://www.freshfromflorida.com/pi/chrp/Ar In the few case where plant and psyllid samples cReader/ArcReader.html Accessed on 11 July 2011. tested positive, only low titers of the pathogen were Bove, J.M. 2006. Huanglongbing: a destructive, newly- emerging, century-old disease of citrus. J. Plant present. These results suggest that urban planting of Pathol. 88 (1):7-37. M. paniculata may play only a minor role as a Clark, R.A. 2007 Notice of intent to add orange jasmine direct reservoir of Las for HLB infection in citrus. to the citrus greening host list. Florida Department of The failure to find any symptoms could be Agriculture and Consumer Services Division of Plant specifically associated with low titer of liberibacter Industry.http://www.doacs.state.fl.us/pi/chrp/greenin g/Orange_jasmine_Notice_2.pdf accessed 30 August in the natural infected trees or M. paniculata 2010. contains a resistant trait to HLB.

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