Fig. 1 Up-to-date greening quarantine zones in Southern California Fig. 2 The number of HLB positive trees detected monthly in California. From: https://www.datoc.us/the-hlb-epidemic

Fig. 3A Flow diagram for the detection of ‘Ca. Liberibacter’ spp. in material (EPPO PM 77/121 (1)).

Fig. 3B Flow diagram for the detection of ‘Ca. Liberibacter’ spp. in insect vectors (EPPO PM 77/121 (1)).

Table 1 Host Family Literature Atalantia (= Severinia) buxifolia CPC CABI, 2018 Balsamocitrus dawei Rutaceae Beattie et Barkley, 2009 Calodendrum capense Rutaceae CPC CABI, 2018 Catharanthus roseus* Apocynaceae Garnier and Bové, 1983 microcarpa Rutaceae EPPO, 2018 Citroncirus x () Rutaceae EPPO, 2018 Citroncirus webberi Rutaceae Beattie and Barkley, 2009 Citrus amblycarpa Rutaceae Beattie and Barkley, 2009 Citrus aurantifolia Rutaceae CPC CABI, 2018 Citrus hystrix Rutaceae Beattie and Barkley, 2009 Citrus jambhiri Rutaceae CPC CABI, 2018 Citrus junos Rutaceae Beattie and Barkley, 2009 Citrus latifolia Rutaceae CPC CABI, 2018 Citrus limettioides Rutaceae EPPO, 2018 Citrus limon Rutaceae CPC CABI, 2018 Citrus limonimedica Rutaceae EPPO, 2018 Rutaceae CPC CABI, 2018 Citrus maxima Rutaceae CPC CABI, 2018 Citrus medica Rutaceae CPC CABI, 2018 Citrus x nobilis Rutaceae CPC CABI, 2018 Citrus paradisi Rutaceae CPC CABI, 2018 Citrus reticulata Rutaceae CPC CABI, 2018 Citrus sinensis Rutaceae CPC CABI, 2018 Citrus x limonia Rutaceae EPPO, 2018 Citrus x Rutaceae EPPO, 2018 Clausena indica Rutaceae CPC CABI, 2018 Clausena lansium Rutaceae CPC CABI, 2018 Cleome rutidosperma** Capparidaceae Brown et al., 2014 Fortunella margarita Rutaceae EPPO, 2018 Limonia acidissima Rutaceae EPPO, 2018 Murraya paniculata Rutaceae CPC CABI, 2018; Zhou et al., 2007 Nicotiana glauca* Solanaceae Tolba and Soliman, 2015 Nicotiana tabacum* Solanaceae Tolba and Soliman, 2015

1 Pisonia aculeata** Nyctaginaceae Brown et al., 2014 Poncirus trifoliata Rutaceae EPPO, 2018 Pithecellobium Fabaceae Fan et al., 2011 (= Archidendron) lucidum** Solanum lycopersicum* Solanaceae Duan et al., 2008 Swinglea glutinosa Rutaceae Beattie and Barkley, 2009 Toddalia sp. Rutaceae EPPO, 2018 Toddalia lanceolata (= Vepris Rutaceae Graça and Korsten, 2004 undulata) octandrum** Brown et al., 2014 trifolia Rutaceae CPC CABI, 2018 * artificial transmission by cuscuta ** Brown et al. (2014) detected HLB in three non-Rutaceae weeds present in Jamaica-infected citrus orchards. It is the same for Pithecellobium lucidum in China. No psyllids were observed on these plants, which are not reported as host plants of HLB psyllids.

2

Table 2 Host Plants and Other Plants Affected (by C. Liberibacter asiaticus) (https://www.cabi.org/isc/datasheet/16565#tohostsOrSpeciesAffected) Plant name Family Context

Atalantia buxifolia Rutaceae Other

Catharanthus roseus (Madagascar Apocynaceae Other

periwinkle)

Citrus Rutaceae Main

Citrus aurantiifolia () Rutaceae Other

Citrus aurantium (sour ) Rutaceae Other

Citrus jambhiri (rough ) Rutaceae Other

Citrus latifolia (tahiti lime) Rutaceae Other

Citrus limon (lemon) Rutaceae Other

Citrus macroptera Rutaceae Other

Citrus maxima (pummelo) Rutaceae Other

Citrus medica () Rutaceae Other

Citrus nobilis () Rutaceae Other

Citrus reticulata (mandarin) Rutaceae Main

Citrus sinensis (navel orange) Rutaceae Main

Citrus x paradisi () Rutaceae Other

Clausena indica Rutaceae Other

Clausena lansium (wampi) Rutaceae Other

Cleome rutidosperma (fringed Capparaceae Wild host spiderflower)

Limonia acidissima (elephant apple) Rutaceae Other

Pisonia aculeata Nyctaginaceae Wild host

Trichostigma octandrum Wild host

Triphasia trifolia (limeberry) Rutaceae Other Table 3 List of plant evaluated in this study and their permissiveness to Candidatus Liberibacter asiaticus (CLas) or the Asian citrus psyllid (ACP). Take from: Sétamou, M., Alabi, O.J., Simpson, C.R. and Jifon, J.L., 2017. Contrasting amino acid profiles among permissive and non-permissive hosts of Candidatus Liberibacter asiaticus, putative causal agent of Huanglongbing. PloS one, 12(12), p.e0187921. (https://doi.org/10.1371/journal.pone.0187921.t001) Common name Botanical name CLas statusa ACP statusb Grapefruit Citrus x paradisi Rio Red Permissive Host Sweet orange Citrus x sinensis Marrs Permissive Host Lemon Citrus x limon Valley lemon Permissive Host Curry leaf Murraya koenigii Unknown Non-permissive Host Orange jasmine Murraya exotica Lakeview Non-permissive Host White sapote Casimiroa edulis Unknown Non-permissive Non-host Madagascar Catharanthus Unknown Permissive Non-host periwinkle roseus Table 4

Diagnostic techniques for Candidatus Liberibacter spp. causal agent of citrus greening

PCR based

Year Reference Specific for Type of test Notes 1996 Jagoueix et al., 1996 Las, Laf end point PCR EPPO, NAPPO. Distinction between Las, Laf by XbaI digestion of the amplicons. 1996 Tian et al., 1996 Las end point PCR NAPPO not tested on Laf and Lam 1999 Hocquellet et al., 1999 Las, Laf end point PCR EPPO, NAPPO

2001 Hoy et al., 2001 Las Long PCR, which long PCR better than end incorporates poin PCR a second DNA polymerase with proof-reading activity 2005 Teixeira et al., 2005 Lam end point PCR EPPO, NAPPO

2005 Okuda et al., 2005 Las*. § TAIL-PCR and § Li et al., 2007 showed Las+Laf LAMP that Laf is also detected 2006 Wang et al., 2006 Las end point & 2 real SYBR Green (best time PCR sensitivity); TaqMan (best specificity) 2006 Li et al., 2006 Las, Laf, TaqMan qPCR EPPO, NAPPO; F Lam primers species-specific (3 different F). R and probe the same for all. HLBas & HLBaf detect both. Detection & Identification 2008 Teixeira et al., 2008 Las, Lam SYBR Green Different primers pair PCR; end point PCR

2008 Manjunath et al., 2008 Las single & the new primers/probe multiplex are only for the internal TaqMan qPCR control wingless (wg) 2010, Bertolini et al., 2010, Las, Laf, TaqMan qPCR EPPO 2014 2014 Lam (tissue-print & squash) 2012, Fujikawa & Iwanami Las end point PCR, in Fujikawa et al ., 2013 2013 2012; Fujikawa et al., direct PCR from comparison of 6 sample 2013 midribs preparation methods (best.: DNA extraction and Biomasher-pellet) 2012 Morgan et al., 2012 Las TaqMan & SYBR EPPO PCR 2013 Ananthakrishnan et Las,Laf,Lam real time PCRs validated in al., 2013 both SYBR Green and TaqMan 2014 Rigano et al., 2014 Las LAMP +Lateral Target gene Flow CLIBASIA_05175 Dipstick (LFD) 2014 Kogenaru et al., 2014 Las a field detection kit for testing psyllids for Las. LAMP conducted in a Smart-DART™ detection unit 2015 Keremane et al., 2015 Las LAMP 2015 Orce et al., 2015 Las, Laf, Syber Green real can differentiate Las or Lam time PCR Laf (same curve) from singleplex Lam by their reaction characteristic melting curves 2016 Meena & Baranwal, Las multiplex PCR 2016 also for viruses ICRSV, CVVCV, CYMV, CTV 2016 Zheng et al., 2016 Las SYBR Green and more sensitive than Li et TaqMan PCR al 2006 and Morgan et al., 2012 2017 Ding et al., 2017 Las Immune Tissue Print and Immune Capture-PCR 2017 Qian et al., 2017 Las LAMP +visual detection of the results 2018 Choi et al., 2018 Las LAMP 2018 Park et al., 2018 Las real time PCR root HLB test is more sensitive than the leaf HLB test 2018 Zhong et al., 2018 Las droplet digital more sensitive than PCR qPCR 2018 Selvaraj et al 2018 Las duplex droplet duplex ddPCR and qPCR digital PCR assay are more robust, accurate and sensitive than the singleplex

2019 Hong et al., 2019 Las nested PCR Nested: attention to contaminations

Other techniques (Non-PCR based)

Year Reference Type of test notes 1983 Garnier & Bové, Electron microscopy EPPO 1983 1991 Roistacher 1991 Biological indexing EPPO, NAPPO 2017 Pagliaccia et al., Antibody-based 2017 diagnosis using a Las secreted protein as the detection marker 2019 Wang et al., 2019 Non-Destructive detection method based on microscopic confocal raman 2019 US 10, 408, 740 B2 Method for detecting trees whose leaves possess over - quantities of starch. A portable and real - time sensing system based on polarized light 2019 Tran et al., 2019 Detection of a secreted protein biomarker for citrus greening using a single-walled carbon nanotubes-based chemiresistive biosensor

Internationally agreed diagnostic protocols

2012 NAPPO DP 02: end point PCR Jagoueix et al., 1996; Tian et al., 1996; Hocquellet Citrus et al., 1999, Teixeira et al., 2005 Huanglongbing real time PCR Li et al., 2006, 2007

2014 EPPO PM 7/121 end point PCR Jagoueix et al., 1996; Hocquellet et al., 1999, (1) Laf Lam Las Teixeira et al., 2005 a,b real time PCR Li et al., 2006; Bertolini et al., 2014; Morgan et al., 2012

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