Format for summarizing the current conditions of related matters and considering challenges and future directions of research collaboration (Reference material for greening/huanlongbing)

1. Basic data on the pest

Items Current conditions/Basic information Distribution of the causal bacteria 【】 ‘Candidatus asiaticus’ Las is spread in Sao Paulo, Minas Gerais, and Parana states. It was recently reported in Mato Grosso do Sul state (HLB was officially reported in (Las) that state in September 2019). These are among the main citrus-producing areas in Brazil, corresponding to ~ 80% of the in Brazil. 【】 In China mainland, Las is spread in 10 provinces covering most of the citrus planting areas. Guangdong, Guangxi, Fujian, Zhejiang, Jiangxi, Hunan, Yunnan, Guizhou, Hainan and Sichuan.

【France】 Las is spread in most parts in French overseas territories: -In the Caribbean: Guadeloupe and Marnique -In South West Indian Ocean: Reunion Unknown status for the French Guyana and Mayotte. Mainland France: absent, but under active survey since it has been suspected in Portugal in 2015 and because , one of the vectors, is spreading in Portugal and Spain from the northern African countries. 【Italy】 Las not known to occur in Italy nor in the EPPO region (Euro-Mediterranean region https://www.eppo.int/index)

Reference - EPPO A1 List of pests recommended for regulation as quarantine pests - version 2019-09 – https://www.eppo.int/ACTIVITIES/plant_quarantine/A1_list 【】 Las is spread in most parts of Okinawa prefecture, and a few outlying islands (Tokunoshima, Okinoerabu and Yoron) in Kagoshima prefecture. The quarantine areas are indicated in this file by Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries (PPS). (http://www.maff.go.jp/pps/j/guidance/pestinfo/pdf/PestInfo_82_01.pdf) 【USA】 Considering the U.S. as a whole, as of March 23rd, 2017, , , Georgia, Louisiana, Puerto Rico, South Carolina, Texas and the U.S. Virgin Islands have at least some regions under quarantine (positive detection of CLas) (https://www.aphis.usda.gov/plant _health/plant_pest_info/citrus_greening/downloads/pdf_files/national-quarantine-bound.pdf). The entire states/commonwealths of Florida, Georgia, Puerto Rico and the U.S. Virgin Islands are under quarantine while only certain regions of the other states have quarantine limitations. The disease is emerging in Texas and California and an updated map on the HLB and psyllid quarantine zones in California is pasted below (see Fig. 1 in the Appendix). ‘Ca . L. africanus’ (Laf) 【Brazil】 Laf does not occur in Brazil. 【China】 Laf does not occur in China. 【France】 French overseas territories: Laf has been formerly reported in Reunion Mainland France: absent 【Italy】 Laf not known to occur in Italy nor in the EPPO region.

Reference - EPPO A1 List of pests recommended for regulation as quarantine pests - version 2019-09 – https://www.eppo.int/ACTIVITIES/plant_quarantine/A1_list 【Japan】 Laf does not occur in Japan. 【USA】 Laf does not occur in USA. ‘Ca . L. americanus’ (Lam) 【Brazil】 The original report of Lam was in Sao Paulo state, Brazil. Initially, it corresponded to 80% of the causal agent of HLB in the country, but it has drastically reduced its incidence and now it is not found (or very rarely found) in the field. 【China】 Lam does not occur in China. 【France】 French overseas territories: absent Mainland France: absent 【Italy】 Lam not known to occur in Italy nor in the EPPO region

Reference - EPPO A1 List of pests recommended for regulation as quarantine pests - version 2019-09 – https://www.eppo.int/ACTIVITIES/plant_quarantine/A1_list 【Japan】 Lam does not occur in Japan. 【USA】 Lam does not occur in USA. Other species 【Brazil】 Brazil has at least two phytoplasmas associated with HLB symptoms; one belonging to the 16SrIII group and the other to the 16SrIX group. The incidences, however, are low. 【China】 Phytoplasma (Candidatus Phytoplasma ateri) was detected in some citrus samples showing HLB-like symptoms. However, HLB is highly associated with mottling and Zn-deficiency-like symptoms. 【France】 No other species 【Italy】 Some phytoplasmas are apparently associated with citrus greening in several countries, however, such phytoplasmas are not present in Italy. Instead, (CTV), is present in Italy.

References -Davino S., et al., (2003). The first Citrus tristeza virus outbreak found in a relevant citrus producing area of Sicily, Italy. Plant Disease, 87(3), 314- 314. -Ferre , L., et al., (2014). Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in southern Italy. Phytopathologia Mediterranea, 3-13. 【Japan】 Some phytoplasmas are apparently associated with citrus greening in several countries, however such phytoplasmas are not present in Japan.

【USA】 In the USA, no other species of bacteria are reported to be associated with HLB disease in citrus. Distribution of the vector insects 【Brazil】 Diaphorina citri D. citri is widely distributed in Brazil. 【China】 D. citri is distributed in most citrus planting areas except Chongqing. D. citri has been found in Guangdong, Guangxi, Fujian, Zhejiang, Jiangxi, Hunan, Yunnan, Guizhou, Hainan, Sichuan, Taiwan, Macao and Hongkong. 【France】 Present in French overseas territories where HLB occurs but absent in EU 【Italy】 D. citri not known to occur in Italy nor in the EPPO region

Reference - EPPO A1 List of pests recommended for regulation as quarantine pests version 2019-09 – https://www.eppo.int/ACTIVITIES/plant_quarantine/A1_list 【Japan】 D. citri is distributed in most parts of Okinawa prefecture, and a few outlying islands (Amamiooshima, Kikaijima, Tokunoshima, Okinoerabu and Yoron) in Kagoshima prefecture in Japan. The quarantine areas are shown in this file by Plant Protection Station, Ministry of Agriculture, Forestry and Fisheries (PPS). (http://www.maff.go.jp/pps/j/guidance/pestinfo/pdf/PestInfo_82_01.pdf) 【USA】 In the U.S. and its territories, Diaphorina citri is present in Alabama, Arizona, California, Florida, Georgia, Guam, Hawaii, Louisiana, Mississippi, Puerto Rico, South Carolina, Texas, and the U.S. Virgin Islands (https://www.aphis.usda.gov/plant_ health/plant_pest_info/citrus_greening/downloads/pdf_files/national-quarantine-bound.pdf). In late May 2008, D. citri was discovered in Jefferson and Orleans Parishes, Louisiana. On September 2, 2008, the psyllid was first detected in San Diego County, California. On October 27, 2009, the psyllid was discovered in Yuma County, Arizona. On April 21, 2010, surveys determined that the psyllid was present in the U.S. Virgin Islands. Trioza erytreae 【Brazil】 T. erytreae does not occur in Brazil. 【China】 T. erytreae does not occur in China. 【France】 Present in southern EU countries: Portugal and Spain. CIRAD sampled some Trioza erytreae for further analysis and should be able to bring more details about the situation in Reunion Island.

【Italy】 T. erytreae not known to occur in Italy but it is present in Europe (Portugal and Spain).

References - Update on the situation of Trioza erytreae in Portugal. (2017) EPPO Reporting Service no. 09 - 2017 https://gd.eppo.int/reporting/article-6135 - Siverio F, et al., (2017) Survey of huanglongbing associated with ‘Candidatus Liberibacter’ species in Spain: analyses of citrus plants and Trioza erytreae. Phytopathologia Mediterranea 56(1), 98-110

【Japan】 T. erytreae does not occur in Japan. 【USA】 T. erytreae does not occur in the USA. Other species 【Brazil】 D. citri is the only vector insect, and no other species transmits the citrus greening agent in Brazil. 【China】 D. citri is the only vector insect, and no other species transmit the citrus greening agent in China. 【France】 Other species of psyllium, almost all of which are absent in the EU area, are observed in species of the family, but knowledge on these species is variable and most often unknown. They are potentially vector but no acquisition and transmission tests have been performed to date: this is the case of Diaphorina auberti , D. murrayi , Trioza litsae , Powellia vitreoradiata (present in Europe), Leuronota fagarae , Cacopsylla heterogena , C. murrayi . Some species such as Cacopsylla citricola and C. citrisuga , Diaphorina communis , D. punctulata , D. zebrana may acquire HLB but are unable to multiply and inoculate it. There is strong doubt for the following species: Trioza citroimpura, Cacopsylla evodiae, C. fagarae, C. toddaliae, Psylla loranthi, Mesohomotoma lutheri, Agonoscena cisti . Finally, a widespread Pseudococcidae, Ferrisia virgata in tropical and subtropical regions, is capable of acquiring and transmitting HLB under experimental conditions, but the strain of the bacterium would be different from that found in psyllids because it does not cause no symptoms. 【Italy】 There is no evidence of pathogen transmission by other psyllid species. CLas has been detected in Diaphorina communis and Cacopsylla citrisuga, but the two species are absent in Europe.

References -Donovan N.J. et al., (2012) First report of ’Candidatus Liberibacter asiacus’ in Diaphorina communis. Australasian Plant Disease Notes, 7, 1–4. -Cen Y., Zhang L., Xia Y., Guo J., Deng X., Zhou W., Sequeira R., Gao J., Wang Z., Yue J., Gao Y. (2012) Detecon of ’Candidatus Liberibacter asiacus’ in Cacopsylla (Psylla) citrisuga (Hemiptera: Psyllidae). Florida Entomologist, 95, 304–311.

【Japan】 D. citri is the only vector insect, and no other species transmit the citrus greening agent in Japan. 【USA】 D. citri is the only citrus greening vector insect in the USA, and no other species transmit the citrus greening bacteria in the USA and its territories. Disease distribution in various land 【Brazil】 categories Greening occurs in citrus trees in home gardens in Brazil, but the disease is spread in several production areas of the above-mentioned states. Residential areas HLB in gardens is considered a problem only when it occurs near commercial orchards that are effective in controlling ACP and HLB, as infected trees can be a constant source of vector and inoculum for those commercial fields. 【China】 Greening occurs in citrus and its relatives trees in home gardens in China. 【France】 HLB occurs in citrus trees in home gardens and public gardens in territories where HLB can be found: Martinique, Guadeloupe, and Reunion.

【Italy】 The species of Candidatus Liberibacter causal agent of citrus greening are not known to occur in Italy nor in the EPPO region (see above)

【Japan】 Greening mainly occurs in citrus trees in home gardens in Japan. 【USA】 Citrus greening occurs in door yard citrus trees in residential areas in the USA in all states where the disease has been detected. In California, the detection of citrus greening in residential trees has skyrocketed since the summer of 2017 (see Fig. 2 in the Appendix). A website is available for the public to track the epidemic in residential areas (https://www.datoc.us/the-hlbepidemic ).

Commercial citrus field 【Brazil】 Greening occurs in both small-scale and large-scale commercial citrus fields in the above-mentioned states. However, tree removal is performed since the first report of the disease in the country, in 2004, and has been law-enforced since 2008 (https://www.defesa.agricultura.sp.gov.br/legislacoes/instrucao-normativa-mapa-53-de-16-10-2008,830.html). 【China】 HLB has infected most commercial citrus field in China mainland, except Chongqing area, and destroyed numerous citrus trees. In Ganzhou area of Jiangxi province, more than 45 million citrus trees were eliminated since 2012, causing economic loss of 9 billion RMB. of China is facing big threat of HLB. 【France】 HLB is widely distributed in commercial citrus orchards in territories where HLB can be found: Martinique, Guadeloupe, and Reunion.

【Italy】 N/A

【Japan】 Some small-scale commercial citrus fields in the vicinity of residential areas are affected with greening. Occurrence of greening in large-scale commercial citrus fields is rare. 【USA】 In the USA, the majority of citrus grown in Florida is processed for juice. HLB is extensive throughout commercial citrus production in Florida and emerging in Texas. In California, commercial production is focused on fresh market produce. To date, no trees have been confirmed positive by PCR in commercial citrus groves in California. Nurseries 【Brazil】 Nursery plants have been produced under screen in Brazil since 1990s and, therefore, there are no reports of greening in nurseries in the country. 【China】 HLB once occurred a lot in almost nurseries in China. 【France】 Nurseries are under official certification schemes guaranteeing the absence of disease such HLB.

【Italy】 N/A

【Japan】 Occurrence of greening in nurseries are not known in Japan.

【USA】 Nurseries within, and within a 5-mile radius of, a quarantine zone are prohibited from selling all host nursery stock and the movement of all host plants and fruits. This restriction applies to residents and commercial operations. Host range 【Brazil】 Citrus The main citrus species produced in Brazil (more than 80% of the production) is sweet (Citrus sinensis), which is severely affected by HLB. Other cultivated citrus are limes and mandarins, and hybrids like . All of them are affected by HLB. 【China】 In China, Las can infect species in the genus Citrus, Fortunella and Poncitrus, including C. sinensis, C. reticulata, C. unshiu, C. paradisi, C. limonum, etc (Miyakawa and Zhao, 1990). Miyakawa, T., Zhao, X. Y. Citrus host range of Greening disease. Proceedings of the 4th International Asia Pacific Conference on Citrus Rehabilitation. FAO-UNDP, 1990: 118-121. 【France】 See Table 1 in the Appendix. 【Italy】(potential hosts) In Italy, the main cultivated Citrus species are: •Sweet orange- Citrus sinensis •Clemen ne- Citrus clemenna • - Citrus limon L. •Mandarin - Citrus deliciosa Ten. •Bergamot - Citrus bergamia Risso • - Citrus paradisi Macf. •Cedar - Citrus Medica •Chino o - Citrus myrfolia the following species are also cultivated: •Bi er orange - Citrus auranum (rootstock, now banned where CTV is present) • oFortunella margarita (Lour) swingle (ornamental and for fruit) oFortunella crassifolia Swingle (ornamental) oFortunella obovata Tan. (ornamental) oFortunella hindsii (Champ.) swingle (ornamental) • Wester (rootstock) • - Poncirus trifoliata (L.) Raf. (CTV resistant rootstock) •Poncirus trifoliata varietà monstruosa (T. Ito) swingle (ornamental) • - Citrus sinensis x Poncirus trifoliata (CTV resistant rootstock) •Citrus Wolkameriana Pasq. (rootstock) •Calamondino - Citrus mis (o madurensis) Lour (ornamental) • - Citrus limonia (Osbeck) (ornamental) •Finger - Microcitrus papuana winter (ornamental) •Citrus paradisi x Poncirus trifoliata ( Swingle) (CTV resistant rootstock) •Sastuma - Marc. •Cleopatra mandarin - Citrus reshnii Ort. ex Tan. (ornamental) •Citrus medica cv sarcodactylis (ornamental) 【Japan】 In Japan, species in the genus Citrus, Poncirus, and Fortunella are the only known host plants of Las (Miyakawa, T. 1980, Ann. Phytopath. Soc. Japan 46: 224-230). Main natural citrus hosts of Las are flat lemon ( ), tankan (Citrus tankan ), (Citrus reticulata ), and satsuma (Citrus unshiu ), all of which are common in the sub-tropical islands in Okinawa and Kagoshima, Japan （https://www.jstage.jst.go.jp/article/kyubyochu1955/52/0/52_0_66/_pdf）. 【USA】 In the CABI Compendium, plants that could serve as hosts include citrus species and a number of other genera (https://www.cabi.org/isc/datasheet /16565#tohostsOrSpeciesAffected), Table 1. However, how well some hosts serve as sources of inoculum for the psyllid to acquire the bacterium and subsequently transmit to new hosts is still unclear (Cifuentes-Arenas, J.C., Beattie, G.A.C., Peña, L. and Lopes, S.A., 2019. Murraya paniculata and Swinglea glutinosa as short-term transient hosts of ‘Candidatus Liberibacter asiaticus’ and implications for spread of huanglongbing. Phytopathology, (ja) and 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. In the US and in 1980 specifically, Citrus and citrus related species including Citrus, Poncirus, and Fortunella were thought to be the only known citrus/close citrus relatives that are host plants of CLas (Miyakawa, T. 1980, Ann. Phytopath. Soc. Japan 46: 224-230). However, it is likely that, given the array of Rutaceae plants that can function as hosts (see Table 1), other near citrus relatives could also be present in the U.S. and function as hosts. There are clearly other plant hosts including Murraya species that may at least transiently harbor the bacterium (see Tables 2 Citrus relatives 【Brazil】 In Brazil, although Murraya paniculata is not a good host for Las, it is an excellent host for the Asian citrus psyllid (ACP). Because of that, some municipalities have specific laws reinforcing the elimination of such plants near citrus production areas. 【China】 In China, CLas was also detected on Murraya paniculate, Atalantia buxifolia, Clausena and Citrus medica var. sarcodactylis (Deng et al., 2008; Zhou et al., 2007). Deng, X., Lou, Z., Feng, Z., Li, H., Chen, J., Civerolo, E. L. First report of 'Candidatus Liberibacter asiaticus' from Atalantia buxifolia in Guangdong, China. Plant Disease, 2008, 92 (2): 314-314. Zhou, L. J., Gabriel, D. W., Duan, Y. P., Halbert, S. E., Dixon, W. N. First report of dodder transmission of Huanglongbing from naturally infected Murraya paniculata to citrus. Plant Disease, 2007, 91 (2): 227-227. 【France】 In French overseas territories, Murraya paniculata is known to be a transient HLB host where insect vector populations are present. Murraya koenigii has been proved not to be a host for the HLB. 【Italy】 Murraya paniculata

【Japan】 In Japan, Murraya paniculata is apparently a transient host. Natural occurrences of greening disease on other citrus relatives (Toddalia asiatica , Feronia limonia , etc. ) are not reported. 【USA】 In the USA, Murraya paniculata and M. koenigii are transient and somewhat resistant hosts. M. koenigii is reported to be more resistant. The breadth of natural occurrence of greening disease in other citrus relatives remains unclear, but infections can occur in other plant genera and even in periwinkle using dodder infection methods (a parasitic plant) (see Tables 2 and 3 in the Appendix).

Table2 : https://www.cabi.org/isc/datasheet/16565#tohostsOrSpeciesAffected Table3 : taken 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. Other species 【Brazil】 N/A

【China】 N/A 【France】 Unknown. 【Italy】 N/A

【Japan】 N/A 【USA】 N/A

Virulence 【Brazil】 There are no reports in variation of virulence among Brazilian isolates of Las. A recent work revealed population homogeneity and new genomic organization in prophages type 1 and 2 within Las isolates in Brazil (https://doi.org/10.1094/PHYTO-08-18-0295-R).

【China】 There is no difference of virulence found among the population of Las in China.

【France】 French overseas territories: different aggressiveness in Guadeloupe and Martinique, but not in Reunion.

【Italy】 N/A

【Japan】 Difference in virulence (mild, moderate, severe, etc.) is not found among Japanese isolates of Las. A Japanese isolate Ishi-1 lacking the DNA polymerase region (Kato, H. https://doi.org/10.1371/journal.pone.0106109) shows severe virulence as the isolate Kin-1 retaining the DNA polymerase region. 【USA】 There have been no peer reviewed published papers demonstrating different CLas strains with respect to symptom severity (Virulence) in identical plant genotype backgrounds. However, there are clearly different symptoms that develop in different trees of the same genotype of citrus (i.e. blotchy mottled , corky veins, vein yellowing; branch die-back etc.). Preliminary results are suggesting that there are strain variants of CLas that cause these varying symptoms; however, this has not been verified. It is also clear that symptoms developing in citrus in Texas are less severe than those observed in Florida. In this case, also, the reason for this difference has not been elucidated, but it is presumed to be the result of environmental difference. For example, the Texas region where they grow citrus has hotter summers and CLas is known to be sensitive to heat. So, reduced symptoms in Texas may be linked to the exposure to increased temperatures. 2. Pest management technologies

Items Diagnostic techniques 【Brazil】 Under field conditions, very well-trained teams identify the disease visually. That is normally enough for tree removal. Under laboratory conditions, the most used detection tool is qPCR (normally based on Li et al., 2006 - Journal of Microbiological Methods, v. 66, n. 1, p. 104-115).

【China】 In China, PCR-based diagnostic techniques were most applied, among which the quantitative PCR (qPCR) is widely used in laboratory diagnostic of HLB, while the conventional PCR and NEST PCR were used as basic tools in some local administrations. The LAMP (Loop-mediated isothermal amplification) and RPA (Recombinase Polymerase Amplification) techniques were also developed for detection of Las for both laboratory and field conditions. In addition to the tissue-extracted methods, e.g. PCR or LAMP, many detection techniques that are based on spectroscopy technique were developed for rapid, inexpensive diagnosis of HLB. 【France】 In France, official analyses are done by agreed laboratories through the official method MA 033 published by the ministry of Agriculture and based on a PCR. Conventional PCR: •Hocquellet, A., P. Toorawa, J. M. Bove, and M. Garnier. 1999. "Detecon and idenficaon of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon." Molecular and Cellular Probes 13 (5):373-9. doi: 10.1006/mcpr.1999.0263. •Teixeira, D. C., J. Luc Danet, S. Eveillard, E. Crisna Marns, W. C. de Jesus Junior, P. Takao Yamamoto, S. Aparecido Lopes, R. Beozzo Bassanezi, A. Juliano Ayres, C. Saillard, and J. M. Bove. 2005. "Citrus huanglongbing in Sao Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease." Molecular and Cellular Probes 19 (3):173-9. doi: 10.1016/j.mcp.2004.11.002. A recent EUPHRESCO Project (2016-A-232) underlined the need for a real time PCR official method in France for the early detection of the HLB disease in the case of the asymptomatic samples. Real Time PCR: •Li, W., J. S. Hartung, and L. Levy. 2006. "Quantave real-me PCR for detecon and idenficaon of Candidatus Liberibacter species associated 【withItaly citrus】 huanglongbing." Journal of Microbiological Methods 66 (1):104-15. doi: 10.1016/j.mimet.2005.10.018. In Italy, the official analyses are performed by the NPPO authorized laboratories, following the EPPO standard PM 7/121 (1), 2014. ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’. EPPO Bull, 44(3), 376-389. (See the Fig.3A and 3B in the Appendix). DNA extraction is performed from midribs or psyllids using the CTAB method or DNeasy Plant Mini Kit (Qiagen). Direct tissue-print and/or squash methods can be used for a rapid screening test of plants and psyllids. Reference for rapid screening -Bertolini E, et al. (2014) Tissue-print and squash real-me Polymerase Chain Reacon for direct detecon of ‘Candidatus Liberibacter’ spp. in citrus plants and insect vectors. , in press. 65, 1142–1158. Molecular test based on PCR End-point PCR: -Hocquellet A, et al., (1999) Detecon and idenficaon of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the b operon. Molecular and Cellular Probes 13, 373–379. -Jagoueix S, et al., (1996) PCR detecon of the two Candidatus Liberobacter species associated with greening disease of citrus. Molecular and Cellular Probes 10, 43–50 -Teixeira DC, et al. (2005) Citrus huanglongbing in S~ao Paulo, Brazil: PCR detecon of the Candidatus Liberibacter species associated with the disease. Molecular and Cellular Probes 19, 173–179. Real-time PCR: -Bertolini E, et al. (2014) Tissue-print and squash real-me Polymerase Chain Reacon for direct detecon of ‘Candidatus Liberibacter’ spp. in citrus plants and insect vectors. Plant Pathology, in press. 65, 1142–1158. -Li W, Hartung JS & Levy L (2006) Quantave real-me PCR for detecon and idenficaon of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of Microbiological Methods 66, 104–115. -Morgan JK, et al., (2012) Improved real-me PCR detecon of ‘Candidatus Liberibacter asiacus’ from citrus and psyllid hosts by targeng the intragenic tandem-repeats of its prophage genes. Molecular and Cellular Probes 26, 90–98. 【Japan】The diagnosis of the disease is mainly conducted by PCR, and the following methods have been reported by the recent papers. Fujikawa, T., Miyata, S., and Iwanami, T. (2013). Convenient detection of the citrus greening (huanglongbing) bacterium ‘Candidatus Liberibacter asiaticus’ by direct PCR from the midrib extract. PLoS ONE 8(2),e57011 (https://doi.org/10.1371/journal.pone.0057011).

Fujikawa, T., Iwanami, T. (2012). Sensitive and robust detection of citrus greening (huanglongbing) bacterium “Candidatus Liberibacter asiaticus” by DNA amplification with new 16S rDNA-specific primers. Molecular and Cellular Probe 26:194-197.

In some under-equipped extension centers, simpler techniques such as LAMP (Okuda, M. et al., 2005, Plant Dis. 89: 705-711) is used for detection using a commercially available kit (https://www.nippongene.com/kensa/products/lamp-kit/citrus/citrus.html). 【USA】 Diagnostic techniques are used in California for disease management. These methods will also be useful as the state of Florida monitors efficacy of treatments and replanted groves. Two USDA-validated multiplex TaqMan Real-time PCR assays for the detection of the HLBassociated bacteria are routinely used in plants. For screening plant samples, a CLas/CLamspecific 16S rDNA-based real-time PCR test combined with the plant cytochrome oxidase (COX) positive internal control is used (Li et., 2006). For retesting plant samples that produce a signal with the initial screening test, real-time PCR amplification of a second bacterial gene duplexed with the plant COX positive internal control is used. In addition, plant samples are retested using the singleplex CLas and singleplex CLam real-time PCR. For further confirmation of suspect positive plant samples, a conventional PCR test is used to amplify a fragment of the β-operon gene specific to CLas for sequence analysis (Hocquellet et al. 1999). For screening ACP samples, a CLas-specific 16S rDNA-based real-time PCR test combined with a psyllid glycoprotein (WG) gene-based, probe/primer set as a positive internal control is used (Li et., 2008). For retesting ACP samples that produce a signal with the initial screening test, the CLas 16S based real-time assay is repeated. In addition, the RNR gene based real-time PCR duplexed with a psyllid WG positive internal control is performed (from: https://www.cdfa.ca.gov/citruscommittee/docs/ActionPlan.pdf). For research in the US, different combinations of these approaches are used. LiFor W, example, Hartung increased JS, Levy L. sensitivity Quantitative in CLas real-time detection PCR hasfor detectionbeen reported and identification using intragenic of Candidatus tandem repeats Liberibacter from CLas species phage associated genes (Morgan with citrus et al. huanglongbing. Journal of microbiological methods. 2006 Jul 1;66(1):104-15.

Hocquellet A, Toorawa P, Bove JM, Garnier M. Detection and identification of the two Candidatus liberobacter species associated with citrus huanglongbing by pcr amplification of ribosomal protein genes of theβ operon. Molecular and Cellular Probes. 1999 Oct 1;13(5):3739.

Morgan JK, Zhou L, Li W, Shatters RG, Keremane M, Duan YP. Improved real-time PCR detection of ‘Candidatus Liberibacter asiaticus’ from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes. Molecular and Cellular Probes. 2012 Apr 1;26(2):90-8. Monitoring techniques 【Brazil】 Disease Very well-trained teams monitor HLB symptoms in citrus orchards up to six times a year, particularly from February to August, when symptoms are more evident. For older plants, the visual assessment is performed with the aid of platforms, so the top of the trees can be observed. Visually inspection of trained teams is normally enough for tree removal in endemic areas. 【China】 Visual inspection, PCR and LAMP are the most used methods in China. In some monitoring works, the leaves of citrus trees were collected and the qPCR was employed for quantitively detecting Las in laboratory. 【France】 Surveys are managed by National Plant Protection Organization (NPPO): the sampling is visually assessed and suspicious trees are subjected for analyses. 【Italy】 Regulation (EU) N. 652/2014 sets out priorities as regards the orientation of national programs for pest surveys for the protection of the Union territory. The priorities for 2019-2020 include Candidatus Liberibacter spp. the causal agent of Huanglongbing disease and its vectors (Diaphorina citri and Trioza erytreae ), that are Union quarantine pests, listed in the Directive 2000/29/EC and subsequently listed pursuant to the Regulation (EU) 2016/2031 as not known to occur in the Union territory (Annex II part A). Surveys are managed by the National Plant Protection Organization (NPPO) and performed by the Regional Plant Protection Service.

(Disease monitoring) In monitoring areas, Citrus trees are visually assessed, and suspicious trees samples are collected and tested by real-time PCR and/or end-point PCR. 【Japan】 Citrus trees in monitoring areas are visually assessed and suspicious trees are subjected for PCR or LAMP detection.

【USA】 Disease Intensive efforts exist for disease monitoring in California because the state is in a disease eradication mode. Using risk modeling provided by Dr. Tim Gottwald, USDA, Agricultural Research Service, the following factors are considered when determining risk associated with HLB: ● Census Travel ● ACP Density ● CLAS+ ACP ● Potential ACP Source ● Citrus Road ● Packinghouses ● Farmer’s Markets ● Military and IR ● Organic Citrus ● Weather Suitability Using these risk factors, total risk is determined for each square mile grid, resulting in a recommended sampling density for a particular location in California. Approximately 20 leaves are sampled per tree for direct detection by qPCR. The California Citrus Research Board (CRB) has funded a variety of research projects on indirect detection of HLB in the pre-symptomatic phase, including metabolomics, proteomics, phytobiome and canine detection approaches in the past several years. These methods reliably show that HLB can be detected earlier than qPCR but there is debate on whether state agencies should accept indirect detection to compel mandatory tree removal. Private companies may use whatever method of detection they see fit to inform their management practices (from: Vector insect 【https://www.cdfa.ca.gov/citruscommittee/docs/ActionPlan.pdf).Brazil】 Normally, the number of psyllids is counted manually in yellow sticky insect traps spread throughout the orchard, placed mainly close to the edges. Inspections should be performed weekly. Visual assessment of the young shoots is recommended to evaluate the presence of nymphs after insecticide sprays to confirm its efficiency. 【China】 Visual inspection and manually counting is the most common method in investigation of vector insect of Las in China. 【France】 CIRAD is currently working on this aspect and should be able to bring more details about the situation in Reunion Island (additional informations will be provided by CIRAD). 【Italy】 Visual inspection of the canopy. Vector insects are collected by chromotropic traps (yellow sticky traps) and identified by morphology. Further molecular identification can be performed by PCR amplification and sequencing of the Cytochrome c oxidase I (COX1) gene, and by sequence comparison with references in GenBank (https://www.ncbi.nlm.nih.gov) or BOLDSYSTEMS (http://www.boldsystems.org/). 【Japan】 Mostly, the number of vector insects are counted manually after they are collected by beating or sweeping citrus trees and orange jasmine trees. It is also possible to detect DNA of the vector insects recovered from the surface of sticky trap plates (Fujiwara, K. et al., 2016, Journal of Applied Entomology 141:61-66). 【USA】 Vector Insect In California, an intensive effort is being made to monitor for the insect vector. These efforts include urban and residential trapping and visual inspection surveys and commercial grove trapping surveys. Traps are removed and inspected once a month, on average. If sites test positive for the Asian citrus psyllid, 5-20 mile transect surveys may be conducted to determine the extent of the infestation (from: https://www.cdfa.ca.gov/citruscommittee /docs/ActionPlan.pdf). Research conducted by the USDA ARS has shown that not all D. citri populations are competent to transmit CLas. Diversity in CLas acquisition and transmission by D. citri in insect populations may impact local or long distance spread of the pathogen (Ammar et al. 2017).

Ammar ED, Hall DG, Hosseinzadeh S, Heck M. The quest for a non-vector psyllid: natural variation in acquisition and transmission of the Border control for preventing 【huanglongbingBrazil】 pathogen ‘Candidatus Liberibacter asiaticus’ by Asian citrus psyllid isofemale lines. PloS one. 2018 Apr 13;13(4):e0195804. introduction HLB is considered an A2 quarantine disease. Therefore, movement of citrus trees from HLB-affected areas (i.e. Sao Paulo, Minas Gerais, Parana, and Mato Grosso do Sul states) to the other areas in Brazil is controlled by the plant quarantine laws. 【China】 Citrus import and movement should be under the permission of the local quarantine administration according to the plant quarantine law established by the MOA. http://www.moj.gov.cn/government_public/content/2019-10/22/657_3234232.html The Chonqing city also established its own quarantine law for the citrus HLB free area. http://www.moj.gov.cn/Department/content/2008-05/30/595_215355.html 【France】 In France and the French overseas territories, the importation of citrus plant for planting is forbidden by phytosanitary legislation. The importation of basic citrus plant material is allowed in EU under quarantine structures Imported citrus fruits from outside the EU are under phytosanitary controls; imported plants and then citrus fruits or citrus plants by passengers coming from the outside of EU or French overseas territories are forbidden, but appears difficult to control. 【Italy】 There is no Italian legislation on the matter. Italy carries out import checks (pursuant to Directive 2000/29 / EC of the plant material referred to in Annex IV, Part A, Section I, in points 18.2 and 18.3; Starting from December 14th 2019 the EU regulation 2016/2031 and its annexes will come into force) with the official finding that the plant material is originating in a country recognized as free from Candidatus Liberibacter spp., causal agent of HLB. However, since there is still no written declaration to the European Commission of third countries declared free from HLB, no HLB specific sensitive plant materials are imported in Italy from outside EU. 【Japan】 Living plants and plant parts (excluding seeds and fruits) of Citrus sp., Fortunella sp. and Poncirus sp. imported from the greening-affected countries to Japan are subject to post entry quarantine (approximately one year) for greening and other plant diseases inspections under Plant Protection Act. The living plants and plant parts (excluding seeds and fruits) of the other hosts (Toddalia asiatica, Feronia limonia, etc.) imported from the greening-affected countries to Japan are prohibited. Domestic movements of citrus trees from greening-affected prefectures (i.e. Okinawa and Kagoshima) are slightly complicated. Special inspection or prohibition is required for the movements from the greening-affected prefecture (Okinawa and Kagoshima) to greening free prefectures and another greening-affected prefecture (Okinawa or Kagoshima) by the Plant Protection Act. 【USA】 Border Control for Preventing Introduction The Department of Homeland Security, Customs and Border Protection (CBP) employs agriculture specialists at U.S. ports of entry and international mail facilities to target, detect, intercept, and thereby prevent the entry of invasive pest and disease threats. The US border with Mexico is particularly vulnerable to importation of infested citrus materials. The CBP agriculture specialists work with specialized x-ray machines that detect organic materials. They utilize agricultural canines specifically trained to sniff out meat and plant material, including citrus, at international ports of entry. Measures for prevention and control 【Brazil】 Eradication HLB inspection and symptomatic tree elimination are reinforced by law since 2008 (https://www.defesa.agricultura.sp.gov.br/legislacoes/instrucao-normativa-mapa-53-de-16-10-2008,830.html) and should occur at least every three months. Twice a year, a report on HLB incidence in every orchard should be submitted to the Secretary of Agriculture according to a law from 2011 (https://www.defesa.agricultura.sp.gov.br/legislacoes/portaria-cda-21-de-15-12-2011,940.html) 【China】 In the citrus planting areas of China, strict eradication policy has been executed for years, and especially in the Guangxi province where the first local law for control HLB was established. This law allows mandatory eradication of HLB diseased trees and encourages planters to eliminate the infected trees by governmental subsidy. https://www.gxrd.gov.cn/html/art166098.html 【France】 In French overseas territories where HLB occurs, growers are in the obligation to destruct HLB infected trees, but this measure in not well accepted and the impact is limited. 【Italy】 There is no Italian legislation on the matter. However, if HLB-affected plants were found they would be eradicated.

【Japan】 Eradication was tried in some islands in Kagoshima prefecture. So far, greening was successfully eradicated from Kikai Island (www.jppa.or.jp/shiryokan/pdf/66_06_52.pdf). Another eradication program is now in progress in Tokunoshima Island (http://amamishimbun.co.jp/2018/09/02/12952/ ). 【USA】 Eradication efforts are still ongoing in California. Due to the endemic nature of the infection in Florida, growers there are no longer in a disease eradication mode. In California, any tree that tests positive for CLas is required by State law to be removed and destroyed. Recently, townships near the Los Angeles area have announced a program to give away fruit trees to residents in low-income neighborhoods. The initiative will put additional citrus trees in the critical zone between residential areas and the iconic commercial citrus groves in Ventura County, which is already under quarantine for the Asian citrus psyllid. This initiative raises concern that there is a lack of communication between local government officials and state ag agencies and highlights a need for additional extension education to better inform local public officials about citrus greening disease. Control on contaminated budwoods 【Brazil】 and trees Movement of budwoods and trees from HLB-affected areas is banned by the plant quarantine laws.

【China】 The planting and movement of the budwoods that were not breed in the HLB free nursery was prohibited. All the planting and movement of the budwoods should be under the certification of local quarantine administrations. 【France】 The production of citrus plant material free of diseases such as HLB is based on certification schemes under the management of NPPO services and plant quarantine laws. 【Italy】 There is no Italian legislation on the matter. However, no HLB specific sensitive plant materials are imported in Italy from outside EU.

【Japan】 Special inspection to confirm the pathogen free of plants is required for the movements of living plants and plant parts (excluding seeds and fruits) of Citrus sp., Fortunella sp. and Poncirus sp. from the greening-affected areas (i.e. Okinawa pref. and outlying islands of Kagoshima pref.) are required by Plant Protection Act. Movements of the living plants and plant parts (excluding seeds and fruits) of the other hosts (Toddalia asiatica, Feronia limonia, etc.) from the above affected areas are prohibited by Plant Protection Act. In addition, the above plant protection measures are conducted between Okinawa and Kagoshima prefectures. 【USA】 Movement of contaminated budwood and trees is prohibited.

Chemical control 【Brazil】 HLB control in Brazil is heavily dependent on the chemical control of the ACP. There are several insecticides registered for citrus and accepted by the Brazilian and the international community and consumers.

【China】 No chemical control of HLB is available in China. Some plant immunity inducers were allegedly effective to HLB and able to prolong the production of diseased trees. However non of the reagents were proved effective under nigher laboratory nor field conditions. For controlling psyllids, 19 pesiticides have been registered in China, including some neonicotinoids and its mixtures. http://www.chinapesticide.org.cn/hysj/index.jhtml 【France】 Chemicals registered and commonly used are cypermethrine and abamectine. 【Italy】 No chemical control of citrus greening is available in Italy. No insecticides against psyllids are authorized in Italy for citrus plants.

【Japan】 No chemical control of greening is available in Japan. Some neonicotinoids (clothianidin, thiamethoxam) are registered for control of the vector insect. 【USA】 Chemical Control In the U.S., the use of ( and oxyetracycline) containing bactericides as foliar sprays has been allowed in Florida since 2016. Data supporting this use indicated limited but significant improvement in citrus yields and tree health; however the results displayed very high grove to grove variability with some groves showing no improvement. The source of this variability remains unknown, but studies are demonstrating the importance of adjuvant types used to aid penetration of bactericides across plant tissues that ultimately affects concentration that reaches the phloem system. Direct injection of formulations containing the same bactericides is being explored and has been demonstrated to be more efficacious than foliar sprays (Hu et al., 2017); however, this method is not yet approved for use. Cost of applications may be prohibitive and fruit residue concerns need to be evaluated.

Hu, J., Jiang, J. and Wang, N., 2017. Control of citrus huanglongbing via trunk injection of plant defense activators and . Phytopathology, 108(2), pp.186-195. Cultural practice 【Brazil】 HLB control in based on the production of healthy nursery trees, elimination of sources of inocula (tree removal) and reduction of the vector population density through area-wide strategies. Although insecticide sprays are commonly used, large-scale production and release is encouraged and has increased significantly in the last years. 【China】 Good trimming, fertilization and cultural management could be helpful to the pesticide application and to promote the health of the trees. 【France】 French overseas territories: citrus trees under insect proof nets (under trials)

【Italy】 As neither Candidatus Liberibacter spp. (HLB) nor their vectors are known to occur in Italy, no specific cultural practices are in place. However, in Italy, the pre-basic and basic citrus plant materials are grown in insect-proof screen-houses.

【Japan】 In some flat lemon producing areas in Okinawa, semi-disease-free-zone that is formed in the heights where few citrus psyllids occur because cliffs work as natural barrier, is maintained by restriction on the movement of budwoods/trees and chemical sprays on psyllids in case of sporadic occurrence (https://www.pref.okinawa.jp/churahome/pdf/1003/16-17.pdf), (https://sksanti.ti-da.net/c150336.html). 【USA】 There are a variety of cultural practices that can be used to control citrus greening although none are extraordinary in preventing infection. Predaceous arthropods such as lady beetles are known to consume Diaphorina citri and are found extensively where citrus is grown in the US. A strategy known as Citrus Under Protective Screens, abbreviated CUPS, provides a barrier between the tree and incoming infective psyllids. However, CUPS has some drawbacks because other pests such as thrips may enter the screen and the screen is efficient at keeping out their natural predators. Other technologies, such as reflective mulch and photonic fences are being evaluated as a means to protect new plantings. Horticultural oils are effective at limited psyllid populations but are only effective for short durations (2-3 weeks) so frequent applications are necessary (and costly). Strategies to improve tree health, such as regulating the soil and root microbiome and providing trees with enhanced formulations of micronutrients may help to minimize the impact of the disease but will not stop the spread. Use of tolerant 【Brazil】 Although there are at least two Citrus Breeding Programs with cultivars that seem to exhibit tolerance to HLB (from Embrapa and from the Citrus Research Center of the Agronomic Institute), they are not used by growers in large scale yet. 【China】 Practically tolerant or resistant cultivars are not found in China. 【France】 In the state of current knowledge, there is no total resistance, in the sense of gene-for-gene resistance, in any species of the genus Citrus, probably because the disease is historically recent (about 1 century). Breeders are therefore looking for partial resistance that decreases bacterial titer and reduces symptoms, or towards tolerances (infected trees but whose production remains compatible with industrial exploitation). The search for tolerant plants should include the possibility of finding out of orchards grown for the industry. Citrus grown from seedlings by individuals may prove to be tolerant. The existence of partially resistant or tolerant natural variants is not impossible, but would need to be verified before their introduction into breeding programs. Since bacteria associated with the HLB disease establish their infectious niche in the root system, the use of tolerant polyploid rootstocks could be an important element in the fight against HLB. The work identified for HLB relates to rootstocks and new polyploid varieties (Grosser et al., 2016, Morillon, pers.). Rootstocks are major components of orchards because they provide resistance to soil pathogens, such as spp. and nematodes, resistance to the tristeza virus, and allow adaptation to particular environmental conditions (salinity, water deficit, calcareous soils, cold, etc.). Genetic control also involves the evaluation of rootstocks not belonging to the genus Citrus. The most recent analysis (Miles et al., 2017), conducted in Florida, indicates that the least affected varieties are those that contain a part of the genome of Citrus medica (lemon). This includes hybrid varieties ('Limon Real'), , Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in these categories exhibit leaf-mottle symptoms, characteristics of HLB and important pathogen levels, but maintain dense canopy and good growth. Several varieties of Citrus reticulata rootstock show good performance with orange scions (Citrus sinensis) (Bowman and McCollum 2015, Bowman et al., 2016a and Bowman et al. These studies show that levels of resistance / tolerance exist among cultivated species and sexually compatible hybrids (Stover et al., 2015). However, the work is still far from leading to the production of graft varieties or Forimmune the moment, rootstock it todoes the not HLB. appear that tolerant varieties or varieties with a high level of resistance have been produced and used in orchards in the USA. Nothing is described in the literature concerning the situation in the Far East. Thus, to date, there is hardly any graft or combination graft / rootstock resistant, partially or totally, to CL spp. Only Tahitian lime or (Citrus latifolia), which is a natural triploid with fairly good tolerance despite very clear leaf symptoms, is widely grown in the tropics (Evans et al., 2014). Limes in general, , and are more tolerant of HLB (Lee 1996 (Halbert and Manjunath 2004), Shokrollah et al., 2009b, Ramadugu et al. 2016). 【Italy】 N/A

【Japan】 Practically tolerant or resistant cultivars are not found in Japan. 【USA】 Use of Tolerant Cultivars Breeding efforts in the US are intense and ongoing. Varying degrees of tolerance to HLB are well documented in certain citrus and close citrus relatives (Miles et al., 2017 and Stover et al., 2016; Stover et al., 2015). However, there are a huge number of variables that may impact the effective use of tolerant cultivars, with the biggest concern being genetic diversity of the CLas pathogen. Furthermore, tolerant varieties will continue to serve as a source of CLas inoculum for psyllids to spread the pathogen and so are not the ideal solution for citrus greening disease. Varietal pedigrees that include (Citrus medica) may be more tolerant as compared to other varieties. Poncirus trifoliata is a relatively resistant rootstock but the resistance is not transferred into the scion (Stover, E., et al., Breeding citrus for HLB resistance at the USDA/ARS U.S. Horticultural Research Laboratory, Ft. Pierce, Florida. Proceedings of the 2nd International North American Plant Protection Organization (NAPPO) Conference on HLB, 2010).

Miles, G.P., Stover, E., Ramadugu, C., Keremane, M.L. and Lee, R.F., 2017. Apparent tolerance to Huanglongbing in Citrus and Citrus-related germplasm. HortScience, 52(1), pp.31-39.

Stover, E., Inch, S., Richardson, M.L. and Hall, D.G., 2016. Conventional citrus of some scion/rootstock combinations show field tolerance under high Huanglongbing disease pressure. HortScience, 51(2), pp.127-132.

Stover, E., McCollum, G.T., Driggers, R., Lee, R., Shatters Jr, R., Duan, Y., Ritenour, M., Chaparro, J.X. and Hall, D.G., 2015. Resistance and Other control measures 【Brazil】 A lot of effot has been put on obtatining more sustainable approaches to control ACP and HLB. The first commercial biological agent to control ACP is now available for growers and other bioproducts are being tested. Tamarixia radiata release is performed in marginal areas and backyards to reduce ACP population density.

【China】 N/A 【France】 Main lead is to rely on the biological control. The biological control with natural enemies of psyllids (parasitoids, predators, and entomopathogens) is a serious method in the management of populations of these vectors. It has many advantages over chemical control. As a result, a large number of publications devoted to natural enemies and to means of biological control have been identified in bibliographic research, particularly with regard to D. citri. The review by Kondo et al. (2015) cites for example 63 species of natural enemies of this psyllid. Parasitoids have the advantage of being more or less specific and have often shown their long-term efficacy in Hemiptera. Predators are more generalist and complement parasitism in this group of insects. With respect to D. citri, its supposed origin is the Indian peninsula ( and ) with host species of the genus Murraya. It is in this subcontinent that its two primary and specific parasitoids, Tamarixia radiata (Eulophidae) and Diaphorencyrtus aligharensis (Encyrtidae) were first observed.

Information is more succinct on parasitoids of T. erytreae. Mc Daniel and Moran (1972) in Zimbabwe cite two primary species (Tetrastichus radiatus, probably an error of determination, and Psyllaephagus pulvinatus). Tamessee et al. (2002) identified seven species in Cameroon - the most effective being Tamarixia dryi (Eulophidae) and especially Psyllaephagus pulvinatus (Encyrtidae) - plus seven species of hyperparasites. In 2009, Tamassee reported seventeen species of parasitoids in this country. In southern and eastern Africa, these two species, considered to be primary parasitoids, have been known for a long time, with T. dryi showing the best efficacy (Van den Berg, 1990). Because of the limited distribution of T. erytreae outside Africa, its parasitoids have been little imported elsewhere. The well known case is the introduction in 1974 of the two primary species in Reunion (Aubert et al., 1980). P. pulvinatus did not settle on the island but T. dryi strongly parasitized the African psyllid and eradicated it from the island in the late 1970s (Aubert & Quilici, 1986). However, two factors have led to this success: the absence of T. dryi hyperparasites on this island, and especially the presence of another native psyllid host, Trioza bedsae (= aestopi) serving as alternative prey. The same success has been achieved in neighboring (Bové, 2014). In , the total rate of parasitism is 36% on average (ranges from 0 to 51%) and hyperparasitism from 1 to 46% (Catling, 1972). CIRAD has led biological control through Tamarixia radiata in Reunion Island and should be able to bring more details about the situation. (additional informations will be provided by CIRAD).

Treatment by stimulating the natural defenses of plants: results show that the injection at the trunk level of several plant defense stimulators such as salicylic acid, oxalic acid, acibenzolar-S-methyl and potassium phosphate, allows a significant control of the plant. HLB by suppressing the presence of 'Candidatus Liberibacter asiaticus' and stopping the development of disease in young plants and adult trees of sweet orange (Hu et al., 2018). Plant defense stimulator treatments induce the expression of certain PR genes, suggesting that the immune system of citrus is activatable to fight against HLB. These treatments allow an increase of the yield and a better quality of the fruits. The injection of both salicylic acid and acibenzolar-S-methyl leads to a significant induction of PR genes related to the pathogenesis PR-1 and PR-2. In addition, the injection of potassium phosphate or oxalic acid significantly induces the expression of the genes, PR-2 and PR-15, respectively. These results suggest that trees with HLB disease remain inducible to acquire systemic resistance under field conditions and are therefore able to defend themselves. Current information on HLB control via trunk injection of plant defense stimulators and antibiotics can help growers in their decision-making to develop an effective management plan. However, antibiotic or defense activator treatments in the trunk do not seem to be operational in the orchard. The efficacy of foliar spray treatments is not evaluated at this time. 【Italy】 N/A 【Japan】 Citrus psyllids are periodically monitored in the coast areas in the main Island of Kyushu, where invasion and establishment of the insect is predicted due to warm climate conditions in winter. 【USA】 Others Liberibacter crescens, a relative of CLas, can be evaluated for biological control of CLas, as it can be transformed and cultured. The genomes of some CLas strains contain varying integrated prophages, and several groups are examining the possibility of employing phage therapy for disease control. The most well studied strain of CLas, psy62, has two prophages within its genome. In addition, the psyllid endosymbiont Wolbachia encodes a repressor of a lytic phage gene promoter, and this protein is a candidate target for control of CLas in the psyllid vector. Induction of lytic prophages is a promising area for control of CLas. Bioprospecting for novel sources of antimicrobial and pesticidal compounds from nature is also a promising area of research to discover new therapeutic molecules that may be compatible with organic farming practices. Biological control of the vector is another approach to disease management. Examples include the use of predators and parasitoids of the insect vector and insect infecting fungi and viruses, which reduce vector populations. Parasitic wasps including Tamarixia radiata and Diaphorencyrtus aligarhensis are being deployed to manage D. citri. Psyllid infecting viruses have been described and may be leveraged as biocontrol tools with additional research. In particular, a novel Diaphorina citri-associated C virus and a virus in the insect-infecting densovirus family have been associated with D. citri. Entomopathogenic fungi infecting psyllids have been discovered and may be used together with novel lures and traps that may aid in fungal spore dissemination in a grove. Isaria fumosorosea can be readily cultivated in laboratory conditions and causes D. citri mortality between 4.9 and 6.1 days after exposure. Hirsutella citriformis has also been observed infecting adult D. citri in Florida citrus groves, where it causes mortality in an average of 23% of D. citri individuals. However, care must be taken in a holistic biocontrol approach, as antagonistic interactions between entomopathogens and parasitoids used to control D. citri have been documented. Research on augmentative strategies, in particular molecular regulation of entomopathogen virulence in the psyllid, will be invaluable for such efforts to reduce vector and pathogen populations to levels that are Transgenic strategies are also being developed for the control of CLas. Citrus expressing the Arabidopsis defense gene NPR1 is more tolerant to CLas. Transgenic citrus expressing defensin genes from spinach has shown increased tolerance to CLas. Evaluation of citrus varieties for CLas tolerance and QTL mapping for quantitative tolerance provides background information for development and deployment of transgenics. In addition, an asymptomatic Citrus tristeza virus strain can be used to deliver therapeutics directly into citrus phloem, including RNA interference signals that are ingested by D. citri and interfere with psyllid development. Although transgenic citrus has not been widely deployed for a myriad of reasons, transgenic tools may ultimately be an important component of a long-term and durable HLB control strategy (reviewed in Kruse et al. 2019, https://www.mdpi.com/2075-4450/10/9/300) Abandoned groves are problematic as they serve as a rich source of plant material for D. citri reproduction and HLB inoculum. States in the US vary in their approach to abandoned grove abatement.

Kruse A, Fleites LA, Heck M. Lessons from One Fastidious Bacterium to Another: What Can We Learn about Liberibacter Species from Xylella fastidiosa. Insects. 2019 Sep;10(9):300 3. Others

Items Suggestions Proposed actions for international 【Brazil】 research collaboration Over the years, Brazilian research institutes as Embrapa and the Citrus Research Center of Agronomic Institute, the Fund for Citrus Protection (Fundecitrus), and growers have been very open for visits and all sorts of training. They have also sent Las and Lam positive controls to a variety of labs in different countries. With this G20 consortium we can certainly increase such activities and help/ participate in other ones proposed by the colleagues representing other countries. 【China】Our collaboration could include development and cross-checking of the detection methods, data sharing, and training growers and technicians. We could also discuss the standard and policy of eradication of HLB citrus trees. 【France】 •Detection methods and organization of proficiency tests •Epidemiology - VNTR •International newsletter on HLB 【Italy】 o Internaonal newsleer on Ca. L. spp. and their vectors

o Idenficaon, opmizaon, standardizaon, validaon and harmonizaon of diagnosc (detecon and idenficaon) methods to be used worldwide. For Italy and EU Ca. L. species associated to HLB are indicated as a priority for which to harmonize diagnostic protocols. In fact, they are among the priority of: •The EU Reference Laboratories (EURL) Plant Health consora in Bacteriology (CREA-DC is one out four-member of the EURL -Bacteriology) (CREA-DC is the Italian Reference Laboratory for plant pests) https://ec.europa.eu/food/ref-labs_en •The EU project VALITES hps://www.valitest.eu/aims_and_objecves/ (CREA-DC is a member of the VALITEST project)

Our collaboration could include data sharing on methods and the organization of wider inter-laboratory comparison. A lot of scientific papers have been published for the detection of Las, Laf, and Lam. In this regard, I collected these data reporting them in the following tables and references section. To the best of my knowledge, they should be quite all. I apologize for any errors or omissions. I hope it can be a starting point 【Japan】 Information acquired in this study group is compiled and made open to members of G20 on a website, that is linked to others useful sources provided by USDA, EPPO and others.

In order to optimize and standardize the detection methods, several reference laboratories are designated to cross-check the efficacy of the latest detection methods (Fujikawa 2012, https://www.sciencedirect.com/science/article/abs/pii/S0890850812000710) and culture methods(Fujiwara2018,https://www.frontiersin.org/articles/10.3389/fmicb.2018.03089/full), and the results are shared by networks established in the members of G20. 【USA】 i. International Grand Challenge on Citrus Greening. The USDA ARS has established a framework referred to as Grand Challenge Synergies to facilitate research coordination and problem solving for major agricultural challenges. In 2017, a team of ARS scientists led by Dr. Shatters successfully designated citrus greening disease as a USDA Grand Challenge. A team of government and university scientists established that multidisciplinary advances were needed to realize a solution to citrus greening. Research areas included tools for manipulating the genetics of the citrus greening organisms (citrus, psyllids, and Liberibacter), big data and remote environmental sensing systems and development of integrative pest management strategies that minimize impact on the environment. The Citrus Greening Grand Challenge members divided into teams based on their research areas and expertise, ranging from diagnostics to citrus horticulture. The Citrus Greening Grand Challenge changed the administrative framework allowing ARS scientists across the U.S. and with expertise on other commodities to work on citrus greening to develop technologies that could be brought to bear on a solution. In response, the agency helped to make tech transfer and funding resources available to facilitate progress. We propose that such a model may be useful in a transnational format, where the Grand Challenge teams can be expanded to include participants from other nations for information sharing and global monitoring. ii. Field trials to compare citrus greening management strategies. Another area of potential interest to international data sharing and promotion of technical exchanges may be realized through the implementation of an experimental field trial whereby participating growers select from a list of possible different management strategies. This experiment is currently ongoing in Florida to determine whether certain combinations of strategies may be more effective than others. A program funded jointly by the growers, federal government and the state of Florida called CRAFT (Citrus Research and Field Trials) is providing funds to support up to 5,000 acres of new citrus plantings through offsetting of planting and maintenance costs for 5 years. The requirement is that the replants be part of a coordinated program where the plantings represent experimental plots using combinations of current practices that have been reported to improve crop productivity. The current iteration of this experiment has only given growers options for strategies being developed and tested by US scientists but in an international conception of the experiment, growers may also be given options for iii. Productivity in the face of HLB. In Florida, there has been a continual and consistent decline in total citrus production since the appearance of HLB in that state. Only during the last growing season (and including predictions for this season) has there been a halt in this reduction and even a modest increase in total production. More intensive management practices focusing on elevated nutritional treatments and pest/pathogen control are seen as the major reasons for the halt in the production decline. International efforts to bring together research on beneficial management practices would add to the growing knowledge showing how to keep CLas infected trees productive. iv. Information sharing via web conferences. The USDA ARS has, in the past, had a monthly lab-meeting style web conference, where all researchers working on citrus greening, psyllids, or other Liberibacter diseases participated. Every month a different speaker presented their work and data. These called resulted in an enhanced cohesiveness among greening researchers, which ultimately led to the Grand Challenge initiative. Such an initiative may be expanded to include scientists from the G20 consortium to promote awareness of research on transboundary pests and pathogens. Other issues 【Brazil】 N/A 【China】 N/A 【France】 N/A 【Italy】 N/A 【Japan】 N/A 【USA】 N/A