Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. ocret hs eut rvd h rteiec ntelkl as fahdeaki Saskatchewan. in dieback plant. ash host new was of a cause Kingdom adapting likely United Liberibacter the a the of on from example Keywords evidence rare sample first relatively reference a the suggest a provide they with Further, results comparison These sequence concurrent. Nearctic Confirmatory the BOLD. were from and they a isolated that to confirming KX197200) similarity information sequence sequence number: 99-100% were genes showed accession that homology ( the (GenBank for MK937570) psyllid search and number sequence of BLAST accession Liberibacter, solanacearum A combination (GenBank of Liberibacter long. a 6 bp detection used NTHA 1085 We for and and 1058 rDNA MK942379) Liberibacter. number 16S accession psyllid-transmitted the of (GenBank a of death to sequencing The due ( Sanger CO1 be and psyllid nature. could PCR ash unculturable death conventional cottony largely and the plants and symptoms with in plants the infestation Liberibacter host with of in associated Detection distribution uneven psyllids. ( its black by by specifically complicated insect-transmitted, is putatively are a pathogens of example rare relatively Candidatus a suggest they Further, Abstract Saskatchewan. provide in results dieback These ash plant. concurrent. of host was cause new Kingdom likely a psyllids United adapting the our the BOLD. Liberibacter on of from and GenBank sequencing evidence sample in gene reference first sequences Cyt-b a with the and comparisons with from CO1 comparison discrepans P. provenance. sequence were US Confirmatory they of showed that iso- homology confirming maculipennis) KX197200) for information (Bactericera search number: sequence to BLAST psyllid accession yielded Cyt-b A (GenBank Nearctic sequence and MK942379) long. the solanacearum number CO1 bp from Liberibacter 1085 accession genes lated Candidatus combination and (GenBank the a 1058 a 5 to and were NTHA similarity used that Liberibacter, sequence sequences, MK937570) We of 99-100% number two accession detection generated Liberibacter. (GenBank sequencing for 6 psyllid-transmitted discrepans). 16S rDNA NTHA a The (Psyllopsis and 16S mandshurica) to the psyllid psyllids. (Fraxinus due of of ash mancana species be sequencing cottony and determine Sanger the could nigra) and with death (Fraxinus PCR infestation and black conventional with symptoms of of associated the distribution death been that uneven The has hypothesized its Canada by We nature. Saskatchewan, complicated in unculturable is puta- plants trees are largely in pathogens ash and these Liberibacter plants of of species host Detection known in psyllids. All by diseases. specifically important insect-transmitted, economically tively severe, cause species Liberibacter Candidatus Abstract 2020 28, August 2 1 Wamonje Francis psyllids infesting species tree Liberibacter ash Candidatus from a of identification and Detection akto eerhadDvlpetCentre Development and Research Saskatoon Saskatchewan of University and rxnsnigra Fraxinus atrcr maculipennis Bactericera Cyt-b sli,Ahdisease, Ash Psyllid, : ieiatrseiscuesvr,eooial motn iess l nw pce fthese of species known All diseases. important economically severe, cause species Liberibacter odtrieseiso slis h 6 euniggnrtdtosqecs TA5 NTHA sequences, two generated sequencing 16S The psyllids. of species determine to 1 igigZhou Ningxing , n acn ( mancana and ) fU provenance. US of ) Candidatus rxnsmandshurica Fraxinus 1 aade Bamrah Ramandeep , ieiatrseis N acdn,eoytmjumping ecosystem Barcoding, DNA species, Liberibacter .discrepans P. 1 CO1 and slossdiscrepans Psyllopsis s re nSsacea,Cnd a been has Canada Saskatchewan, in trees ash ) rmcmaioswt eune nGenBank in sequences with comparisons from Cyt-b 1 ye Wist Tyler , eesqecn forpyld yielded psyllids our of sequencing gene .W yohszdthat hypothesized We ). 2 n enPrager Sean and , Candidatus 1 Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. netvcrdptoe hr h ahgnrnei ieydtrie yterneo h vector. the of range the by determined likely has is it range other unknown, pathogen a to the currently to applied where linked is been pathogen partially previously death insect-vecored least has and at ( explanation dieback al. been This canopy saliva. plant since et leaf the have this in decreasing which of toxin” syndromes a ”unknown cause psyllid-tree exhibit an precise to also the infesting attributed but found While been pseudogalls were die. and insects demonstrate eventually Edmonton the often and instances, of species all cities these later In and the Montana. trees, Saskatchewan attributed Saskatoon, was and in in that Dakota found detected 2005 South next in nigra was were and trees They ash North insect damage. black in psyllid city-owned this and found the drought 2000, of of many combination In of a of death to death. the 1900s species by tree to followed back by not Alberta dating Calgary, Feeding but States USA). US 1974) multiple MD, and (Hodkinson, Beltsville, Scotia, Nova SEL including 1988), (USDA (Hodkinson, America North into of pathogens species psyllid, plant vector ash bacterial psyllid mycoplasma-like cottony one of The than group another more of phytoplasmas, have vectors of exclusive can a vectors usually (Ethmayer species between and common primary some relationships also the but the are and being lids are, Sternorrhyncha specific to addition vector how suborder In psyllid unclear 2014). the currently and (Haapalainen, in is species insects It Liberibacter feeding 1988). given phloem (Hodkinson, Garczynski, hemipteran, Psylloidea & phytophagous superfamily (Grimm small A-F are vegetables designated Psyllids of CLso 2010). diseases Munyaneza, of other 2014; haplotypes various (Haapalainen, seven and psyllid least ZC at with Mauck are associated 2019; is There pyllaurous 2012). ’ AKA (Munyaneza, plants, (CLso): asymptomatic solanacearum with ter associated are species (Raddadi Two 2010). (Munyaneza, HLB ’ (CLas), asiaticus’ bacter of species (Garnier known techniques continent five African least at the are on There and Greece, Spain, in family Umbelliferae Tahzima the in (Alfaro-Fern´andez United crops Morocco western in in found the been of have diseases all Munyaneza almost 2012; Munyaneza, in 2018; found been (Alfaro-Fern´andez (Crosslin now Zealand Disease has New disease and chip Canada, al. of Zebra parts States, 2014). (Spreen (Munyaneza (Greenway, industry 1994 losses citrus in the of Mexico to (Batool in dollars species detected citrus of first various millions in of greening’ hundreds ’citrus worth a.k.a. (Bertolini (HLB) al. carrots Huanglongbing potato et in importantly in disease most infections (ZC) and 2018), Chip 2014) Trumble, Zebra & including Prager diseases crop 2010; devastating of series a (Crosslin with associated are species The Introduction 09.I saladprso Europe, of parts and Israel In 2009). , Candidatus BakAsh), (Black 09 rgr&Tube 08.Hr erpr e soito ewe ln otadan and host plant a between association new a report we Here 2018). Trumble, & Prager 2019; , 07 aGaa 91.Snei a rtdtce nFoiai 05 L a eutdi losses in resulted has HLB 2005, in Florida in detected first was it Since 1991). Graca, da 2007; , Fraxinus tal. et tal. et tal. et tal. et tal. et 04 Teresani 2014; , 01 n ’ and 2011) , 01 uynz,21) enGenn ies nslncoscos(Crosslin crops solanaceous in Disease Greening Vein 2012), Munyaneza, 2011; , ( 01 Jarausch 2011; , Ca rxnsmanshurica Fraxinus n sedmct uoe t ag,hwvr a nlddmlil eotdintroductions reported multiple included has however, range, Its Europe. to endemic is and 09.Alknown All 2019). , tal. et )Lbrbce pce r ru fmsl atdosapapoe-atra These proteo-bacteria. alpha fastidious mostly of group a are species Liberibacter .) slossdiscrepans Psyllopsis Ca 00 Jagoueix 2000; , tal. et Ca. .aeiau’(Lm,ad’ and (CLam), americanus’ L. . tal. et tal. et .cecn’(c)(Leonard (Lcr) crescens’ L. 02 Haapalainen 2012; , tal. et tal. et 02 07 Holeva 2017; 2012, , 2014). , Ca. Ca. MnaaAh n hi yrd.A ihother with As hybrids. their and Ash) (Mancana 09 Kaya 2019; , 05 Tahzima 2015; , Ca. ieiatr l fwihhv enietfidtruhmolecular through identified been have which of all Liberibacter, ieiatrseisaetasitdb n rmr pce of species more or one by transmitted are species Liberibacter tal. et tal. et ieiatrslncau sascae ihCro Yellows Carrot with associated is solanacearum Liberibacter Fo 81 Hmpea slia) saseils nseveral on specialist a is Psyllidae), (Hemiptera: 1861) (Flor 96 Planet 1996; , 07,weei a lorsle nmlin fdollars of millions in resulted also has it where 2007), , 2 tal. et tal. et tal. et tal. et tal. et 2016). , Psyllopsis 00 aicu ono,21;Teulon 2019; Johnson, & Kalischuk 2010; , 07 Holeva 2017; , tal. et Ca 07 uynz,21;Nelson 2010; Munyaneza, 2017; , 04.Finally, 2014). , tal. et tal. et .arcns(Lf’aeascae with associated are (CLaf)’ africanus L. . 02.Finally, 2012). , tal. et sotnascae ihpseudogalls with associated often is 05 n eey(Teresani celery and 2015) , 95.Treseis ‘ species, Three 1995). , Ca. 04.ZbaCi ies was disease Chip Zebra 2014). , tal. et ieiatrseis(Mauck species Liberibacter Ca. Ca. 07 Mawassi 2017; , Liberibacter-associated Candidatus Ca. .erpes (Leu) europaeus’ L. Psyllopsis- ieiatr psyl- Liberibacter, tal. et iue1 Figure Ca. Liberibac- Fraxinus infested tal. et tal. et tal. et Liberi- 2013; , et ) , , , Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. etn ieetLbrbce useisfo ieetgorpia oain n h TA5adNTHA and 5 NTHA the and locations geographical repre- different GenBank from from sequences subspecies using Liberibacter conducted different was senting differences evolutionary of reconstruction to Phylogenetic similarity number sequence accession 99-100% showed GenBank sequences 6; ( two psyllid NTHA these and for homology MK942379 for a number search accession BLAST showed GenBank respectively 11 A long, these, MK937570). 5; bp of (NTHA 1085 and and GenBank sequencing, 1058 gene were in that 16S were to which subjected sequences were quality trees the of from presence samples the psyllid 20 from DNA to similarity closest showed quences sequences gene 16s in homology for checked Results were (BOLD). sequences databases The Life of primers. Code appropriate Bar using and sequencing GenBank Sanger a automated to and samples H7005P CytB insect or targeting and TC-3’ our Those ACT TG-3’. from TAA TCA ATA DNA GTR AAA TAT using ATT AAA Percy GGT Diane 5’-CCC Dr. of targeting laboratory used the in and conducted Germany) reference was Mainz, PCR GmbH, Red by Biomix (Qiagen 1 2X subunit Kit template, oxidase Tissue a chrome and as DNA Blood extracted Nanodrop DNeasy the a a using quality using for done checked was extraction markers DNA MUSCLE molecular the by with identification alignment between (Muhire species sequence 1.2 differences Psyllid multiple version percentage a software Tool The by Demarcation obtained 1987). Sequence was Nei, the isolates & (Kumar in the (Saitou algorithm 7) of method (MEGA sequences MUSCLE Neighbor-Joining nucleotide 7.0 using the the done version were using Analysis alignments inferred sequence Genetics ny Multiple 5’- Evolutionary BLAST. (Jagoueix via (OA2 Molecular described GenBank previously in the primers as in gene homology Liberibacter ribosome for checked 16s ’general’ the of using region al. bp accomplished am- 1160 a that was plified GCCTCGCGACTTCGCAACCCA-3’) Liberibacter 5’- sequences OI2c of Liberibacter GCGCTTATTTTTAATAGGAGCGGCA-3’ and the detection of phylogeny laboratory. Molecular and the in comparisons ethanol sequence 70% detection, containing Molecular vials Campus glass Saskatchewan ml 4 of 5 at University to the stored transferred collected were Saskatoon, and and Rancho Traps traps of Centre. weeks products, Development City two and (Bioquip Research the every aspirators Saskatoon by approximately Canada’s hand Agri-Food managed and via and Agriculture ( trees owned and Black from trees on Scents deployed collection ash (Alpha were cards direct Hybrid traps sticky by colour Psyllid yellow and CA). specific Dominguez, OR) Psyllid Citrus Linn, Asian West or Inc., standard using trapped were Insects Psyllids of Collection Methods and Materials Ca. 09.Apiosgnrtdwr ujce oatmtdSne eunig eune eeae were generated Sequences sequencing. Sanger automated to subjected were generated Amplicons 2009). , ieiatrslncau eune(eBn ceso ubr X920 sltdfo Nearctic a from isolated KX197200) number: accession (GenBank sequence solanacearum Liberibacter atrcr maculipennis Bactericera .discrepans P. ’GAATARARTTCTTD-3.Alapiosgnrtdwr subjected were generated amplicons All 3’. TCDG- CAT TAT AAR ARR AAT GCA 5’ – R CO1 Ca. ieiatr( Liberibacter eemtd6 were apefo e a olcin( oce pcmncletdi se,U) Primers UK). Essex, in collected specimen voucher (a collection lab her from sample ( CO1 ’GAGATTGAATTATAGTC-’admtd9 and CC-3’ GTT TTA TGA AAT GGA TTT GGA 5’-GGA – F al 1 Table C19/HO18(Folmer HCO2198 LCO1490/ ) rmteUS. the from ) ® pcrpooee Tem cetfi,UA.PRwsdn using done was PCR USA). Scientific, (Thermo spectrophotometer Cyt-b .Tosmls ae TA5adNH ,gnrtdtohigh two generated 6, NTHA and 5 NTHA named samples, Two ). eeCytB were º .A ona osbe h sliswr eoee from recovered were psyllids the possible, as soon As C. Candidatus ® 3 Boie K rmxaduieslpiesfor primers universal and premix UK) (Bioline, rxnsnigra Fraxinus ’TAGNCAAATHTYTGA-3’ TTY TCH ATA CAA GGN 5’-TGA – F ieiatrslncau pyluos se- (psyllaurous) solanacearum Liberibacter tal. et rmr5-G C C C TAR ACR ACT GCT 5’-TGA primer R 94.Cnraoyidentification Confirmatory 1994). , ,Mnaa( Mancana ), Tbe1). (Table tal. et tal. et 2014). , tal. et .mandshurica F. 06 n phyloge- and 2016) , hs eedeposited were These 96 Liefting 1996; , Cyto- and ) – R et Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. fpyldasmdt etectoyahpyld olciey hswudidct httoepyld infesting psyllids species those a that with indicate infested would were the which this are UK; Collectively, trees Canada psyllid. Kent ash Western ash of in in cottony loss trees collected the the ash be sample suffer to known to assumed began a psyllid samples States to on of the United based compared the by match and and supported Canada genetic sequenced further western exact were are an that results were Percy genetic CLso. specimens Diana for These host psyllids. Dr. infested new from ash to collected a cottony sent specimens and be psyllid Saskatchewan between may homology in this genetic trees strong that indicate suggestive here is presented results it The trees, ash from collected other were multiple insects contrast, In the plants. in solanaceous trees (Leonard wild perennial CLso other with had species, of or associated and one crops sequence are vegetable CLso Liberibacter a either of with matched from species phylogenetically closely been most have grouped CLso sequences sequences of our best psyllid Further, the out BLAST. from study, and extracted in this HLB similarity better with in sequence to phenomenon generated 99-100% use known data in a now also the Communication). is is From Personal This traps Prager: extractions. 3D-printed Sean during of (Dr recovery samples design DNA psyllid new low A to provinces other identified. leading and (Horton degrade been Saskatchewan insects in has for trapped psyllids death psyllids collect the of to preserve ash deployed survey where been reaching now Canada broader have a in traps results, and these initiated, Following been trees. has ash from of collected report ) first the is here Presented Discussion discrepans P. flaxini P. The our ly). between similarity sequence 100% discrepans revealed for reference comparison the pairwise positive and The tested sample species. reference had a which sent against the identity 5, was of ranging template, NTHA be similarities DNA to samples, sequence sufficient samples these with all of Liviidae, found database One family nucleotide and GenBank Hemiptera the found order in sequences searches the similar species BLAST for of Also, search be database 98%-100%. BOLD to from A (n=10) sequenced. samples and amplified ten successfully were samples 11 by identification Species COI The similarity. sequence nucleotide 99.9% 2 of with shared those 94.4%), NTHA6) from (approximately dissimilar and similarity most (NTHA5 closest represented. study had ( subspecies sequences our distinct the CLSo two from four among with variation revealed samples clade limited sequence two same analyses revealed to The the Phylogenetic sequences Due in nucleotide sequencing. analyses. of clustered or phylogenetic comparisons samples PCR Pairwise in our the and use repeat subtypes for on to quality based able sufficient clades not of were not we were template, generated sequences the ter, ( sequences 6 ). eunigo slissget htpyld nAhtesaetectoyahpsyllid ash cottony the are trees Ash on psyllids that suggests psyllids of sequencing .discrepans P. Cyt-b tal. et Gnaki:M993.) hsdsrpnymyb u oteasneof absence the to due be may discrepancy This MG989139.1). id: (GenBank sltso wds n aainoiis(eBn d T2871adM989. respective- MF958492.1 and MT021807.1 id: (GenBank origins Canadian and Swedish of isolates .crescens L. upeetr al 1 Table Supplementary nGnak h ecnaedffrne r niaieo la pce demarcation. species clear of indicative are differences percentage The GenBank. in eune hwd8.1 iiaiyto similarity 87.21% showed sequences 02.Tesqecspeetdhr eeetatdfo slis o lns u sthe as but plants, not psyllids, from extracted were here presented sequences The 2012). , ihhmlg agso 98%10 ( 99.83%-100% of ranges homology with , COI .discrepans P. sascae ihBbc onanppy ( papaya mountain Babaco with associated is , atrcr maculipennis Bactericera Ca. eunigo sli ape olce rmtesm re a oe fthese, Of done. was trees same the from collected samples psyllid of sequencing .discrepans. P. i.aeiau apoiaey5% ( 54%) (approximately americanus Lib. COI Ca. tal., et ieiatretatdfo otn s sli ( psyllid ash cottony from extracted Liberibacter ape LS erhsof searches BLAST sample. and .Tog h te ape hwdtepeec of presence the showed samples other the Though ). 09.I a bevdta h slistapdo tcycards sticky on trapped psyllids the that observed was It 2019). Cyt-b hsseisi o nei oNrhAeiadsiehaving despite America North to endemic not is species This COI eesqecn o needn ofimto fspecies of confirmation independent for sequencing gene olce nWsigo tt S.Peiu detections Previous USA. State Washington in collected 4 .repens P. and Cyt-b al 2 Table GnakI:M994.)ad8.2 to 86.42% and MG989141.1) ID: (GenBank Rutaceae Ca. eune.Fo h al 00,cte in cities 2000s, early the From sequences. Ca. CO1 ieiatradfrwihteewas there which for and Liberibacter i oaaermsqecsadwere and sequences solanacearum Lib iue3 upeetr Table Supplementary 3, Figure ) hwdoe 95 iiaiyto similarity 99.5% over showed aiastipulata Carica Hlet&Krmn,20)and 2004) Keremane, & (Halbert iue2 Figure slossdiscrepans Psyllopsis Cyt-b × Ca. .pubescens C. Ca. ). eune for sequences Liberibacter Liberibac- P. ) Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. laoFr´ne,A,Vreur . Rodr´ıguez-le´on, M., Ib´a˜nez, Verdeguer, Alfaro-Fern´andez, Teresani, F., Hern´andez, A., D., I., Ferrandiz, spain. A., mainland Mendoza, I.(2012). in de M. Font, Hermoso & Sanju´an, J., S. C., F. J. Villaescusa, C., M. Cebrian, Alfaro-Fern´andez, A., supported was References work This identity. psyllid SMP. of to confirmation Grant for Discovery Horton NSERC David an and by Percy Diana Drs. thank We in examples multiple by evidenced Acknowledgements as trees pehenomenon required in (Merfa be jumping” process pathosystem will “ecosystem difficult HLB plants is the infected and range form this citrus to sequencing Unfortunately, host the simiar and this. their extraction is to support although, this restriction further (2016); Collectivey, al. that actual to vectors. and et suggests the Graca vector also psyllid that da it new their and by Furthermore, descrived a specific of shift. adopting host host biology pathogen dramatic particularly the mycoplasma-like rather be is a a not is of may what example species initially in rare Liberibacter plant both somewhat host were new a disease a be ash consequently the will and this (Horton psyllids correct, Montana these is and where Oregon, Alberta, infected Idaho, border while Washington, both detected, of Idaho including states and US species Montana the 2017). psyllid includes (Borges multiple that previously by range extracted geographical co-inhabited been has be CLSo to which establish from known to is bacteria which the Convolvulaceae) of ability and the ecology and maculipennis. de on disease (Chaves psyllid plant for discrepans the host western implications P. though of new possible, direct efficiency in the is have transmission in trees species the here psyllid infection ash single on presented a dependent infested by findings is species psyllid the this plant across in loss, Liberibacters observed ash of Transmission of of epidemiology. symptoms cause extreme result potential the a a that also grafting likely are of highly combination Canada a however through is overcome localizes It be bacteria known possibly the can all PCR. this where asymptomatic of and plants; remain about experiments nature in can known detection fastidious plant is complicates the the much crescens) defoliation, While psyllids, not in by 2015). and results post-inoculation infected , often days and while trees three 10-15 years in to HLB for disease two with a over also infectious progressing is defoliation This become which (Hu progressive data). death disease, (unpublished exhibit eventual HLB years and observed also to dieback, four compared are trees within be symptoms these defoliation can similar complete Canada, defoliation CLso while in western with Notably, resulting in infection eventually 1994). trees seasons, of (Burckhardt, ash result trees infested the infested swelling. in on be leaf that pseudogalls may and known borders, and in trees is leaf Canada of results of It western reddening alone. loss leaflets, in feeding rolled the loss psyllid including ash ash, of that of effect indicate context the findings than the rather These in and States. pathosystems United psyllid-Liberibacter the of population biology established the of an both either here from presented infestation introduction results the of that The Europe. result likely or therefore the is America is It States North 1988). United eastern (Hodkinson, the in years and 100 Canada over western for in America North in known been a aeaqie Lowiesaigacmo otpatwt ouainof population a with plant host common a sharing while CLSo acquired have may hssae otpatlkl ol aebe edbnwe ( bindweed field been have would likely plant host shared This i Notes Dis .maculipennis B. Ca. 96 .discrepans P. tal. et ieiatrifcintasitdb slis nadto odescribing to addition In psyllids. by transmitted infection Liberibacter 11–12. , 01.Ntby huhi a endtrie htctu lnscan plants citrus that determined been has it though Notably, 2011). , a olce nWsigo (Borges Washington in collected was tal. et tal. et is eoto Cniau ieiatrslncau’i carrot in solanacearum’ Liberibacter ‘Candidatus of report First olce rmdigahteshv motn mlctosfor implications important have trees ash dying from collected 09 Vald´es 2019; , 00.W yohsz hta nrdcdpplto of population introduced an that hypothesize We 2020). , Ca. 5 tal. et ieiatrseis(te than (other species Liberibacter tal. et 2017). , .discrepans P. 2016). , atrcr maculipennis Bactericera slossdiscrepans Psyllopsis ovluu arvensis Convolvulus edn assla yposin symptoms leaf causes feeding tal. et 07.I hshypothesis this If 2017). , nplanta in Ca. .maculipennis B. netto also infestation a known a has Liberibacter (Ann Bactericera Solanales: , tal. et tal. et Ca. , Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. rm,K .S acysi .F (2019). F. tuberosum. S. Solanum Garczynski, in solanacearum’ & bacter S. D. K. Grimm, Res Potato J Am (2014). G. Greenway, ln-ahgncbacteria. plant-pathogenic (2014). M. Haapalainen, (2000). M. V Bov´e, J. J. Graca, & capensis’. capense, da subsp. F. africanus Microbiol Calodendrum liberibacter ‘Candidatus Evol tree, H. of rutaceous Syst Proposal Roux, ornamental Africa. J South an Int Le of in province R., Cape present Western the P. liberibacter in a Cronje, of invertebrates. S., characterization metazoan Genomic Jagoueix-Eveillard, diverse M., from I Garnier, R.(1994). Vrijenhoek, subunit oxidase & c R. Austria. cytochrome Lutz, Biotechnol in mitochondrial W., trees of Hoeh, fruit (2011). tion M., stone R. Black, and H. O., pome Eisenzein, Folmer, in & phytoplasmas U. of (2010). S. vectors W. 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US seven in returns grower on costs control chip Zebra of impact Economic 50 n plBiol Appl Ann slodpsso eprt n utoia rpadonmna lns(Hemip- plants ornamental and crop subtropical and temperate of pests Psylloid irsgenn disease. greening Citrus rnsArcSci Agric Trends ilg n pdmc fCniau ieiatrseis psyllid-transmitted species, Liberibacter Candidatus of epidemics and Biology 2119–2125. , rnmsino Cniau ieiatrslncau’i artseeds. carrot in solanacearum’ Liberibacter ‘Candidatus of Transmission ln elProg Heal Plant erhfrrsror f‘Cniau ieiatrslncau and ’ solanacearum Liberibacter Candidatus ‘ of reservoirs for Search 165 2 ln Dis Plant 172–198. , 173–186. , . dnicto fanwhpoyeo Cniau Liberi- ‘Candidatus of haplotype new a of Identification nuRvPhytopathol Rev Annu 6 103 468–474. , u ln Pathol Plant J Eur eeto f‘addtsLbrbce So- Liberibacter ‘Candidatus of Detection Insects netsmin neatos“Candidatus interactions Insect-symbiont 11 29 514. , otcSci Hortic 109–136. , N rmr o amplifica- for primers DNA 147 rcNt cdSiU Sci Acad Natl Proc 34 52.European 15–20. , oaozbachip zebra Potato 159–166. , h importance The o a Biol Mar Mol Citrus Plant Bull Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. ihcro elw yposi saladtasitdb h rvln sli etrBceieatrigonica. Bactericera vector psyllid prevalent the by transmitted and Israel Phytopathology in symptoms yellows carrot with aas,M,Do,O,Paek,A,S,J . eizy . hsaa . elnn . Haviv, L., authors Meslenin, N., other Shoshana, psyllid N., & Levitzky, the M., C. of J. Porat, Sj, host A., S., native Piasezky, O., a Dror, M., umbelliferum: K.(2019). Mawassi, Solanum A. of Hansen, specimen & 49-year-old (2009). R. G. a vector. V. R. from Meduri, G. P., resurrected Clover, haplotype Sun, & crops. E., S. Solanaceous K. B. of Weir, Mauck, diseases L., with K. associated Paice, Species W., (2012). Liberibacter’ P. J. ‘Candidatus Sutherland, M. W., Davis, L. & Liefting, G. BT-1. A. crescens Davis-richardson, Liberibacter R., of J. sequence Fagen, communication T., Brief M. 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Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. apeI ODPoaiiyBATProbability BLAST Probability Ca. BOLD Lib. 100% Ca. liviidae Hemiptera Ca. 618 Ca. 373 Ca. ash 854 Mancana AO2 1-1 DowRd 450 ID AO2 Sample for searches 1096 BLAST Mancana and BOLD AO2 2: Table Mancana AO2 Mancana AO2 Y10 Mancana Y6 Mancana Y4 Y2 Northern Y1 Northern 6 NTHA Northern 5 NTHA Northern 4 NTHA Northern 2 NTHA Mancana Plant 1 Host NTHA 3 LB Sample submitted final of the Detection of approved 1: authors Table All paper. data. the and wrote reagents SP contributed and TW FOW research. Data. performed manuscript. the TW analysed and SP RB and NZ, FOW FOW, research; the designed SMP accession contributions (GenBank Author 6 NTHA GenBank. and in MK942379) available number publicly accession are (GenBank MK937570) 5 number NTHA samples for Sequences Accessibility Data Hybrid Treasure Hybrid Treasure Hybrid Treasure Hybrid Treasure Hybrid Treasure ash Ca. ieiatrlk eune npsyllids in sequences Liberibacter-like Primer Sequencing O 05C.Lib. Ca. Lib. Ca. 1085 Lib. Ca. 1058 Lib. Ca. AO2 478 Lib. Ca. AO2 Lib. Ca. 290 AO2 245 AO2 420 AO2 AO2 COI eune rmpsyllids from sequences 9 length Sequence sequence primer FWD using BLAST Closest Liberibacter solanacearum Liberibacter Liberibacter Liberibacter solanacearum solanacearum solanacearum solanacearum solanacearum solanacearum slossdiscrepans Psyllopsis dniy(%) identity BLAST 91%FJ914619.1 MN396643.1 99.19% FJ914619.1 FJ395219.1 99.72% 99.76% FJ914619.1 100% 98.18% 99 KX197200 KX197200 99.91 KX185608.1 100 KX185608.1 97.03 KX185608.1 99.19 MG701017.1 99.11 99.5 100% identity closest of Number GenBank Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. ue ecnae r hw nteadtoa nomto SplmnayTbe2) Table (Supplementary information additional the in of shown of are those those percentages to subspecies. puted to dissimilar similarity Liberibacter most closest were of Kimura showed They comparison box) the (red sequence Neighbor-Joining using study pairwise our computed the Percentage were using 3: distances inferred Figure Evolutionary was history MEGA7. replicates). in evolutionary (A-E) conducted (1000 clades with The 2-parameter clustered bootstrapping five ‘D’. box) in with red clade clustered the method in they in (highlighted Phylogenetically, study sequences our gene. from solanacearum ribosomal Sequences subspecies. 16s subtype. Probability Liberibacter to the according different from generated of ‘C’: were relationships Panel analysis Evolutionary arrow). 99.78 leaves (black infestation. the leaves 2: on psyllid the present Figure on with were liviidae depositions associated Psyllids Hemiptera like ‘B’: tree ‘cotton’ Panel ash characteristic trees. defoliated the ash Completely was on there disease and tree. the arrow), by ash (red caused on deformation psyllids Leaf BLAST ‘A’: and Panel 98.77% symptoms disease Probability Typical 98.77% liviidae 1: Hemiptera Figure 100% liviidae Hemiptera CAPTIONS 99.78 FIGURE 99.83% liviidae Hemiptera liviidae Hemiptera liviidae 100% unknown Hemiptera Hybrid 98.77% liviidae Treasure Hemiptera Northern BOLD 98.55% liviidae Hemiptera Ref. UK 98% liviidae ash Hemiptera Mancana 5 NTHA liviidae ash Hemiptera Mancana 3-3 DowRd ash Mancana 3-2 DowRd ash Mancana 3-1 DowRd ash Mancana 2-3 DowRd ash Mancana 2-2 DowRd ash Mancana 2-1 DowRd ash Mancana 1-3 DowRd 1-2 DowRd ID Sample A Candidatus B ieiatraeiau lgtgenclu) h com- The colour). green (light americanus Liberibacter 10 Candidatus C ieiatrslncau ge box). (grey solanacearum Liberibacter slossdiscrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis discrepans Psyllopsis eune sdi this in used Sequences Ca. eune from Sequences Liberibacter 99.5% 99.7% 100% 100% 100% 99.85% 100% 99.85% 99.83% 100% Posted on Authorea 28 Aug 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159863274.41095429 — This a preprint and has not been peer reviewed. Data may be preliminary. 11 D C B E A USA USA USA USA USA USA USA USA Canada Canada Zealand New Mexico Mexico Africa South Africa South Africa South Africa South Africa South China China China China China USA USA Country of origin Citrus sp Citrus sp Citrus sp Citrus sp. Citrus sp Citrus psyllid Nearctic Tomato Tomato Ash tree Ash tree Potato pepper Bell pepper Bell capense Calodendrum capense Calodendrum capense Calodendrum psyllid Potato psyllid Potato sp Citrus sp Citrus sp Citrus sp Citrus grandis Citrus Curry Babaco Source species