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ORIGINAL RESEARCH published: xx December 2020 doi: 10.3389/fpls.2020.617664 1 58 2 59 3 60 4 61 5 62 6 63 7 64 8 65 9 Resistance to ‘Candidatus 66 10 67 11 Liberibacter asiaticus,’ the 68 12 69 13 Huanglongbing Associated 70 14 71 15 Bacterium, in Sexually and/or 72 16 73 Q11 17 Graft-Compatible Relatives 74 18 75 19 Mônica N. Alves1, Silvio A. Lopes1, Laudecir L. Raiol-Junior2, Nelson A. Wulff1, 76 20 Eduardo A. Girardi2, Patrick Ollitrault1,4 and Leandro Peña3* 77 Q1 21 78 22 1 Fundo de Defesa da Citricultura, Araraquara, Brazil, 2 Empresa Brazileira de Pesquisa Agropecuária, Cruz das Almas, Brazil, 79 Q2 3 Centre de Coopération Internationale en Recherche Agronomique pour le Développement, BIOS Department, UPR 23 80 Q12 amélioration génétique des espèces à multiplication végétative, Montpellier, France, 4 Instituto de Biologia Molecular y 24 81 Celular de Plantas – Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Valencia, Spain 25 82 26 83 27 Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding 84 Edited by: 28 85 Agata Gadaleta, sources of genetic resistance to HLB-associated ‘Candidatus Liberibacter asiaticus’ 29 86 University of Bari Aldo Moro, Italy (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes 30 87 Reviewed by: exist. Some Citrus relatives of the family , subfamily , were 31 88 Ed Stover, described as full-resistant to Las, but they are phylogenetically far, thus incompatible 32 Horticultural Research Laboratory 89 33 (USDA-ARS), United States with Citrus. Partial resistance was indicated for certain cross-compatible types. 90 34 Muqing Zhang, Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft- 91 University of Florida, United States 35 compatible with Citrus, may provide new rootstocks able to restrict bacterial titer 92 Q3 36 *Correspondence: 93 37 Leandro Peña in the canopy. Use of seedlings from monoembryonic species and inconsistencies 94 38 [email protected]; in previous reports likely due to Las recalcitrance encouraged us to evaluate more 95 39 [email protected] accurately these Citrus relatives. We tested for Las resistance a diverse collection 96 40 97 Specialty section: of graft-compatible Citrinae species using an aggressive and consistent challenge- 41 98 This article was submitted to inoculation and evaluation procedure. Most Citrinae species examined were either 42 99 Breeding, susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New 43 a section of the journal 100 44 Frontiers in Plant Science Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for 101 45 Received: 16 October 2020 the first time as full-resistant, opening the way for using these underutilized genotypes 102 46 Accepted: 10 December 2020 as Las resistance sources in breeding programs or attempting using them directly as 103 47 Published: xx December 2020 104 possible new Las-resistant Citrus rootstocks or interstocks. 48 Citation: 105 49 106 Alves MN, Lopes SA, Keywords: HLB, Rutaceae, Microcitrus, Citrus breeding, Eremocitrus, Aurantioideae, greening Q13 50 Raiol-Junior LL, Wulff NA, 107 51 Girardi EA, Ollitrault P and Peña L 108 52 (2020) Resistance to ‘Candidatus INTRODUCTION 109 Liberibacter asiaticus,’ 53 110 the Huanglongbing Associated 54 111 Q14 Bacterium, in Sexually and/or Huanglongbing (HLB) is the most destructive disease of citrus worldwide. Its occurrence is 55 Graft-Compatible Citrus Relatives. associated with the infection of trees with one of the following Gram-negative intracellular 112 56 Front. Plant Sci. 11:617664. α-proteobacteria, ‘Candidatus Liberibacter asiaticus’ (Las), ‘Ca. L. americanus’ (Lam) or ‘Ca. L. 113 57 doi: 10.3389/fpls.2020.617664 africanus’ (Laf), which colonize the phloem of their host (Bové, 2006). Las and Lam are 114

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Q6 115 naturally transmitted by the Asian citrus psyllid Diaphorina environmental conditions, plant ages, number of replicates, 172 116 citri Kuwayama (Sternorrhyncha: Liviidae) (Capoor et al., 1967; field vs. greenhouse tests, seedlings vs. mature plants and 173 Q9 117 Yamamoto et al., 2006), while Laf is transmitted by the African grafted vs. rooted stocks (Hung et al., 2000; Folimonova et al., 174 118 citrus psyllid Trioza erytreae Del Guercio (Sternorrhyncha: 2009; Shokrollah et al., 2009; Feng et al., 2015; Ramadugu 175 119 Triozidae) (McClean and Oberholzer, 1965). In Brazil, Las and et al., 2016; Miles et al., 2017). For example, Poncirus trifoliata 176 120 Lam have been detected (Coletta-Filho et al., 2004; Teixeira et al., has been reported as resistant (Albrecht and Bowman, 2012), 177 121 2005), Las being the most common HLB-associated bacterium, partially resistant (Folimonova et al., 2009) or showing different 178 122 currently present in over 99.9% of all ‘Liberibacter’-positive field levels of resistance, recovery or delayed infection depending on 179 123 samples analyzed at Fundecitrus (Bassanezi et al., 2020). Las the accession (Ramadugu et al., 2016). The monoembryonic, 180 124 tolerates higher temperatures, reaches higher titers in Citrus and Australian native Eremocitrus glauca (Lindl.) Swingle as well as 181 125 is more efficiently transmitted than Lam (Lopes et al., 2009, Microcitrus australasica (F. Muell.) Swingle and other Microcitrus 182 126 2013), all this making the former associated with the most severe species have been considered partially resistant, with transient 183 127 damages caused by HLB in Asia and the Americas (Gottwald replication or variable responses among seedlings, respectively 184 128 et al., 2007). HLB-infected, severely affected trees produce small (Ramadugu et al., 2016), representing the first results opening 185 129 and irregularly shaped fruits with a thick peel that remains the way for their use in breeding programs for Las resistance, 186 130 green. Leaves shows a blotchy mottle, yellowing and may become as they are cross-compatible with Citrus (Swingle and Reece, 187 131 thicker and with enlarged veins. The canopy show a premature 1967). However, the use of seedlings for Las challenge inoculation 188 132 defoliation and dieback of twigs (Bové, 2006). In areas where no in monoembryonic species implies that segregating individuals 189 133 control of the insect vector is done, the severity of symptoms and were actually evaluated, likely explaining their variable responses 190 134 disease progression in the orchards increase rapidly (Gottwald to Las (Ramadugu et al., 2016). Moreover, Las infection through 191 135 et al., 1989, 1991). D. citri in the field, being the natural challenge inoculation 192 136 The control of HLB is based on planting of healthy trees system, does not allow distinguishing resistance to the bacterium 193 137 from insect-proof nurseries, monitoring of D. citri and tree from resistance to the psyllid vector or to both vector and 194 138 flushing, application of insecticides to reduce the population of bacterium. Furthermore, the influence of other abiotic and biotic 195 139 the insect vector when reaching an unacceptable threshold and factors present in the field may affect seedling performance even 196 140 rapid eradication of symptomatic trees. In Brazil, since the disease in absence of clear Las infection (Miles et al., 2017). 197 141 was first reported in 2004, these practices have been adopted in Resistant Citrus relatives graft-compatible with Citrus may 198 142 the main citrus producing region, comprising the São Paulo State provide interstocks or new rootstocks (other than the widely 199 143 and Southwest of Minas Gerais State (Bassanezi et al., 2020). used P. trifoliata accessions and their hybrids with sweet 200 144 However, the cost of implementing these strategies is high and and ) able to restrict bacterial titer and 201 145 in spite of them, the incidence of the disease remains around 20% distribution in the scion and thus potentially reduce disease 202 146 annually in the region (Fundecitrus, 2020). These palliative and damages. Eremocitrus glauca and several Microcitrus species as 203 147 preventive measures are employed for HLB management because M. australasica and the ‘Sydney’ hybrid have performed well 204 148 there is neither cure nor resistant cultivars within Citrus, which as interstocks. Moreover, E. glauca, Microcitrus types and their 205 149 may be used to control the disease durably. hybrids warrant consideration as citrus rootstocks (Bitters et al., 206 150 It has been reported that some Citrus relatives belonging to 1964, 1977). Additionally, the most promising Aurantioideae 207 151 the family Rutaceae, subfamily Aurantioideae may be partially, genera for use as citrus rootstocks were Atalantia (some of its 208 152 transiently or totally resistant to Las. In the case of some Poncirus species previously classified in the Severinia genus), Naringi, 209 153 trifoliata (L.) Raf. accessions the bacterium reaches inconsistent Citropsis, Limonia (previously known as Feronia) and Swinglea 210 154 infections with low titers and uneven distribution (Folimonova (Bitters et al., 1964, 1969, 1977). Responses to Las infection have 211 155 et al., 2009). For Murraya paniculata (L.) Jack or Swinglea been tested for several species from these genera (Koizumi et al., 212 156 glutinosa (Blanco) Merr. (Cifuentes-Arenas et al., 2019) infection 1996; Hung et al., 2000; Feng et al., 2015; Ramadugu et al., 2016; 213 157 is just transient and after a few months plants become Las-free. Cifuentes-Arenas et al., 2019). However, the use of seedlings 214 158 In Clausena excavata Burm. F., Glycosmis pentaphylla (Retz.) from monoembryonic species (Citropsis), inconsistencies in 215 159 Corr. and other Aurantioideae Citrus relatives the bacterium is results of different reports (Limonia), and low number of 216 160 undetectable and thus unable to replicate in the genotype under plant replications in some cases (Naringi and Atalantia), make 217 161 test (Ramadugu et al., 2016). it advisable to perform a more accurate evaluation of these 218 162 Consistent resistance to Las characterized by lack of detectable Citrus relatives for resistance to Las. In addition to the 219 163 bacterial replication in germplasm sexually compatible with identification of promising germplasm to be used as parents in 220 164 Citrus is of major interest because it may be used in sexual sexual breeding programs or as rootstocks/interstocks, a better 221 165 breeding programs aimed to generate hybrid and backcross knowledge of the distribution of the response to Las in the gene 222 166 citrus-like populations, which could provide new rootstocks pool within Citrinae considering phylogenetic relations, would 223 167 or scion varieties resistant to HLB. Moreover, segregating provide valuable orientation to decipher the determinants of 224 168 progenies could be useful to identify genetic loci involved in resistance/susceptibility. 225 169 the resistance trait. However, claims for resistance to Las in To gain more insight into Las resistance irrespective of the 226 170 close relatives to Citrus are discrepant depending on the use insect vector within close Citrus relatives, we have selected cross- 227 171 of different genetic backgrounds, challenge inoculation systems, and/or graft-compatible Aurantioideae species of the Citrinae 228

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229 subtribe as well as intergeneric hybrids and inoculated them with also added the ‘Mountain’ (C. halimii B.C. Stone), which 286 230 Las. A challenge and evaluation system allowing unequivocal was the less Las-susceptible type within Citrus according to 287 231 demonstration of either resistance or susceptibility was used. Ramadugu et al.(2016)( Table 1 and Figure 1). 288 232 For this, we have selected mature buds from each genotype of 289 × 233 interest, and grafted them onto ‘’ (Citrus limonia Plant Material, Grafting and Las 290 234 Osbeck), a well-known, Las-susceptible rootstock. Challenge 291 235 inoculation was performed by grafting Las-infected budwood Challenge Inoculation 292 236 pieces both onto the rootstock and onto the scion, so infection In a preliminary experiment, the 25 accessions from Citrus 293 237 may come to the scion under test directly from the infected and Citrus relatives shown in Table 1, belonging to the 294 238 grafted budwood or from the bacterial flow moving up from family Rutaceae, subfamily Aurantioideae, subtribe Citrinae, 295 239 the susceptible ‘Rangpur’ lime rootstock. Evaluation for Las were selected from the virus/viroid-free Fundecitrus germplasm 296 240 multiplication was done regularly by qPCR over 24 months collection and propagated by grafting onto ‘Rangpur’ lime 297 × 241 after inoculation (MAI). By using such method, we identify (Citrus limonia Osbeck) nucellar rootstocks to study 298 242 here which Citrinae species are the most indicated to be graft-compatibility. The Las-susceptible, graft-compatible sweet 299 × 243 used as sources of full-resistance to Las either in breeding orange varieties ‘Pera’ and ‘Tobias’ [C. sinensis (L.) Osbeck] 300 244 programs or directly as potential Citrus rootstocks/interstocks. 301 245 The distribution of susceptibility/resistance responses to Las 302 246 within the Citrinae germplasm is discussed according to TABLE 1 | Citrinae genotypes/accessions tested for resistance to ‘Candidatus 303 247 chloroplast-based phylogeny. Liberibacter asiaticus.’ 304 248 305 Genotype/Accessiona Common namea 249 306 250 MATERIALS AND METHODS Citrus × sinensis (L.) Osbeck ‘Pera’ ‘Pera’ sweet orange 307 c 251 (P ) 308 252 Plant Genotypes C. × sinensis ‘Tobias’ (P) ‘Tobias’ sweet orange 309 d 253 The cross-compatible Citrus relatives selected were four Poncirus Citrus halimii B.C. Stone (M ) ‘Mountain’ citron 310 254 trifoliata (L.) Raf. accessions, ‘Pomeroy,’ ‘Rubidoux,’ ‘Barnes,’ Poncirus trifoliata (L.) Raf. ‘Pomeroy’ (P) ‘Pomeroy’ 311 255 and ‘Benecke,’ commonly used as rootstocks and in breeding P. trifoliata ‘Benecke’ (P) ‘Benecke’ trifoliate orange 312 Q16 256 programs with Citrus to generate new hybrid rootstocks P. trifoliata ‘Barnes’ (P) ‘Barnes’ trifoliate orange 313 257 (Phillips and Castle, 1977; Soares-Filho et al., 2003; Bowman P. trifoliata ‘Rubidoux’ (P) ‘Rubidoux’ trifoliate orange 314 258 et al., 2016) and belonging to two different genetic groups, Microcitrus australasica (F. Muell.) Australian finger lime 315 Swingle (M) 259 ‘Rubidoux’ and ‘Barnes’ to group 3 and ‘Pomeroy’ and 316 M. australasica ‘Sanguinea’ (M) ‘Sanguinea’ Australian finger lime 260 ‘Benecke’ to group 4, according to Fang et al.(1997); the 317 M. australasica ‘True Sanguinea’ (M) ‘True Sanguinea’ Australian finger lime 261 Australian limes, including (1) the pure species: Microcitrus 318 M. australasica × (Fortunella ‘Faustrimedin’ hybrid 262 australasica (F. Muell.) Swingle, M. australasica ‘Sanguinea,’ 319 sp. × Citrus reticulata) ‘Calamondin’; 263 M. australasica ‘True Sanguinea,’ M. inodora (F.M. Bail) Swingle, C. × oliveri (PMe) 320 264 M. warburgiana (F.M. Bailey) Tanaka, M. papuana Winters, M. inodora (F.M. Bail) Swingle (M) Australian large-leaf wild lime 321 265 M. australis (A. Cunn. ex Mudie) Swingle and Eremocitrus glauca M. warburgiana (F.M. Bailey) Tanaka (M) New Guinean wild lime 322 266 (Lindl.) Swingle, (2) three hybrids among them: M. virgata M. papuana Winters (M) Brown river finger lime 323 267 (M. australis × M. australasica), known as the ‘Sydney’ hybrid, M. australis (A. Cunn. Ex Mudie) Australian round lime 324 268 a Microcitrus sp. × E. glauca and an E. glauca × Microcitrus Swingle (M) 325 269 sp. hybrids, and (3) two Australian lime hybrids with Citrus: M. virgata (M. ‘Sydney’ hybrid 326 270 an Eremocitrus glauca × Citrus × sinensis hybrid (eremorange) australis × M. australasica) (PM) 327 b 271 and the ‘Faustrimedin’ hybrid [M. australasica × ‘Calamondin’ Microcitrus sp. × E. glauca hybrid (PM) Australian lime hybrid BGC 695 328 272 (Fortunella sp. × C. reticulata Blanco); C. × oliveri](Table 1 and E. glauca (Lindl.) Swingle (M) Australian desert lime 329 273 Figure 1). Among those Citrinae genotypes cross-incompatible E. glauca × C. × sinensis hybrid (PP) Eremorange 330 b 274 but graft-compatible with Citrus, we selected those recommended E. glauca × Microcitrus sp. hybrid (PM) Australian desert lime hybrid BGC 682 331 275 as suitable rootstocks and/or interstocks by Bitters et al.(1964, Atalantia citroides Pierre ex Guillaumin Cochin China atalantia 332 (PPf) 276 1969, 1977), including Atalantia citroides Pierre ex Guillaumin, 333 A. ceylanica (Arn.) Oliv. (PP) Ceylon atalantia 277 A. ceylanica (Arn.) Oliv., L., Citropsis 334 Limonia acidissima L. (P) Indian wood apple or elephant apple 278 gilletiana Swingle & M. Kellerm, and Naringi crenulata (Roxb.) 335 Citropsis gilletiana Swingle & M. Gillet’s cherry orange 279 Nicolson. Limonia acidissima is classified in the Balsamocitrinae 336 Kellerm. (M) 280 subtribe by Swingle and Reece(1967). However, it appears to be 337 Naringi crenulata (Roxb.) Nicolson (PM) Hesperethusa 281 closely related with Atalantia species according to chloroplastic 338 a 282 phylogenetic studies and clearly included in the Citrinae clade The nomenclature used follows Swingle and Reece(1967) and Bayer et al.(2009). 339 bOriginal accession number at the Citrus Germplasm Bank (BGC) of Embrapa 283 (Bayer et al., 2009). As Las susceptible controls, we used two Cassava & Fruits in Cruz das Almas, Bahia. 340 284 Citrus genotypes, the Brazilian sweet orange varieties ‘Pera’ and cP, polyembryonic; dM, monoembryonic; ePM, possibly monoembryonic; fPP, 341 285 ‘Tobias’ [C. × sinensis (L.) Osbeck] (Donadio et al., 1995). We possibly polyembryonic; according to Swingle and Reece(1967) and Bitters(1986) . 342 Q17

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343 400 344 401 345 402 346 403 347 404 348 405 349 406 350 407 351 408 352 409 353 410 354 411 355 412 356 413 357 414 358 415 359 416 360 417 361 418 362 419 363 420 364 421 365 422 366 423 367 424 368 425 369 426 370 427 371 428 372 429 373 430 374 431 375 432 376 433 377 434 378 435 379 436 380 437 381 438 382 439 383 440 384 441 385 442 386 443 387 444 388 445 Q4 389 FIGURE 1 | Representative photographs of each of the selected Citrinae genotypes/accessions [cross-compatible (A–T); and cross-incompatible, putative 446 graft-compatible (U–Y) with Citrus about 1 year after grafting onto ‘Rangpur’ lime rootstock]. (A) Citrus × sinensis ‘Pera’; (B) C. × sinensis ‘Tobias’; (C) C. halimii; Q5 390 447 (D) Poncirus trifoliata ‘Pomeroy’; (E) P. trifoliata ‘Benecke’; (F) P. trifoliata ‘Barnes’; (G) P. trifoliata ‘Rubidoux’; (H) Microcitrus australasica; (I) M. australasica 391 448 ‘Sanguinea’; (J) M. australasica ‘True Sanguinea’; (K) ‘Faustrimedin’ hybrid; (L) M. inodora; (M) M. warburgiana; (N) M. papuana; (O) M. australis; (P) M. virgata 392 hybrid; (Q) Microcitrus sp. × E. glauca hybrid; (R) Eremocitrus glauca; (S) E. glauca × C. × sinensis hybrid; (T) E. glauca × Microcitrus sp. hybrid; (U) Atalantia 449 393 citroides; (V) A. ceylanica; (W) Limonia acidissima; (X) Citropsis gilletiana; (Y) Naringi crenulata. 450 394 451 395 452 396 (Donadio et al., 1995) were also propagated on ‘Rangpur’ on this same rootstock using buds from a single donor 453 397 lime as controls. mother plant per genotype, and kept at a greenhouse in 454 398 Twenty to thirty plants per accession from Citrinae genotypes Araraquara, São Paulo State, Brazil (Figure 1). True-to-typeness 455 399 that were graft-compatible with ‘Rangpur’ lime were propagated was assessed by analyzing all Citrinae species morphologically 456

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457 (Swingle and Reece, 1967) and in the case of Australian lime ‘True Sanguinea,’ M. warburgiana, M. papuana, M. australis, 514 458 hybrids also by using SSR molecular markers (Fanciullino et al., Microcitrus sp. × E. glauca hybrid, E. glauca × C. × sinensis 515 459 2005; Froelicher et al., 2008; Luro et al., 2008; Ollitrault et al., hybrid and E. glauca × Microcitrus sp. hybrid as well as 516 460 2008)(Supplementary Table 1). Plants were grown in 4 L C. × sinensis ‘Tobias’ and ‘Pera’ controls. 517 461 polyethylene bags filled with coir, irrigated and fertilized twice To be sure that Las challenge-inoculated plants were actually 518 462 a week, and sprayed monthly with insecticides and miticides. infected, the fibrous root tissue from the susceptible ‘Rangpur’ 519 463 Las challenge-inoculation experiments were conducted in a lime rootstock was evaluated at 12 MAI as well as its bark 520 464 greenhouse in which the mean daily air temperature varied at 12 and 24 MAI. Only plants with Las-positive roots at 12 521 ◦ ◦ 465 between 18.5 C to 34.4 C and illumination was natural. MAI had their leaves evaluated. Las graft-infection success was 522 466 The original source of inoculum was Las-positive sweet orange calculated as the percentage of plants with Las-positive rootstocks 523 467 budwood from a farm located in São Paulo (Brazil), propagated per the total number of Las-graft-inoculated composite plants 524 468 and kept at Fundecitrus since 2006 (Lopes et al., 2009). Las-free (Supplementary Table 2). 525 469 and Las-positive D. citri populations are continuously reared at To assess movement of Las from the rootstock to the scion 526 470 Fundecitrus, as described in Parra et al.(2016). Las-free psyllids through the vascular system, bark samples were collected at 5 and 527 471 are reared on healthy Murraya paniculata (L.) Jack seedlings 30 cm above the bud union and at the scion canopy (21–152 cm) 528 472 while Las-positive psyllids are reared on Las-positive ‘Valencia’ from each plant that remained without Las-multiplication in leaf 529 473 sweet orange plants grafted on ‘Rangpur’ lime. For this work, samples including M. warburgiana, M. papuana, M. australis, 530 474 greenhouse-grown ‘Rangpur’ lime seedlings were inoculated by Microcitrus sp. × E. glauca hybrid, Eremocitrus glauca, E. 531 475 exposing them to Las-infected D. citri insects. Budwood from glauca × C. × sinensis hybrid and E. glauca × Microcitrus 532 476 these seedlings was then used to inoculate ‘Valencia’ sweet orange sp. hybrid as well as C. × sinensis ‘Tobias’ and ‘Pera’ as Las 533 477 nursery plants grafted on ‘Rangpur’ lime, which were used as positive controls. 534 478 source of inoculum for the 25 Citrinae accessions. Las-infection Samples were all subjected to DNA extraction. Total DNA was 535 479 in ‘Rangpur’ lime seedlings and ‘Valencia’ sweet orange plants extracted from 0.5 g of leaf midribs, 0.3 g of fibrous roots or 0.3 g 536 480 was confirmed by qPCR (Li et al., 2006). Seven to sixteen plants of bark tissue and processed using a cetyltrimethylammonium 537 481 per genotype were selected based on regular and homogeneous bromide (CTAB) extraction buffer (Murray and Thompson, 538 482 growth within each accession to be graft-inoculated with Las 1980) as described by Teixeira et al.(2005). DNA quality was 539 483 using two budwood pieces ca. 3 to 5 cm long per plant. checked with the NanoDrop (Thermo Scientific) (Desjardins and 540 484 In all plants to be tested, one Las-infected (qPCR-positive) Conklin, 2010). Real-time polymerase chain reactions (qPCR) 541 485 budwood piece was grafted on the ‘Rangpur’ lime rootstock, for detection of the 16S rDNA from Las were ran using 1 µL 542

486 and another one on the scion variety, both at 5 cm below and of total DNA (100 ng/µL), TaqMan R PCR Master Mix (1x) 543 487 above the scion-rootstock junction, respectively. At the same (Invitrogen, Carlsbad, CA, United States) and HLB as Las-specific 544 488 time, 4 to 14 uniform plants of each genotype were grafted with primer/probe (0.5 µM/0.2 µM) in a StepOnePlus thermocycler 545 489 Las-negative budwood from healthy greenhouse-grown ‘Valencia’ (Applied Biosystems) as described by Li et al.(2006). A positive 546 490 sweet orange plants grafted on ‘Rangpur’ lime, which were used and a negative sample were included as quality controls during 547 491 as negative controls. In the case of ‘Pera’ sweet orange, 41 DNA extractions and qPCR assays. As an internal control, the 548 492 plants were Las graft-inoculated as described above and 33 plants mitochondrial gene cytochrome oxidase (COX) was used (Li 549 493 were grafted with healthy budwood pieces. Three months after et al., 2006). As plant housekeeping controls, primers from the 550 494 graft challenge inoculation, plants were pruned at 0.5–1.5 m Actin and Rubisco small subunit genes were designed based on 551 495 from the rootstock to promote new shoot growth and thus Las homologous sequences of six and nine different plant species, 552

496 translocation from the infected budwood pieces and rootstock respectively, that were aligned using DNA MAN R , and were also 553 497 to the scion in the Las-challenge inoculated plants (Johnson used (Supplementary Tables 3, 4). 554 498 et al., 2014; Raiol-Junior et al., 2021). Control plants were pruned Leaf, bark and root samples were considered Las-positive 555 499 likewise. Only those plants showing Las infection in the ‘Rangpur’ when their qPCR cycle threshold (Ct) was lower than 34.0. Las 556 500 lime rootstock at 12 months after graft-inoculation (MAI) were quantification was done based on the linear relation among Ct 557 501 considered as successfully Las-inoculated. The exact number of and the 16S rRNA log, according to y = −0.2998Ct + 11.042, 558 2 502 plants used per genotype for resistance evaluation as well as that R = 0.9981 (Lopes et al., 2013). Target gene concentrations below 559 503 of healthy controls are detailed in Supplementary Table 2. 0.9 16S rRNA log, which corresponded to a Ct value of 34.0, 560 504 produced variable results (Lopes et al., 2013), so this was used 561 505 as the lower detection limit for Las-positive samples. However, 562 506 Sample Collection and Evaluation by Ct values between 34.0 and 36.0 were considered as suspicious to 563 507 qPCR be positive, so only when all samples from other remaining time 564 508 A random but representative sampling of 16–20 leaf pieces from points showed Ct values over 36.0, the plant was considered as 565 509 actively growing shoots per plant scion were collected at 4, Las-negative. For root samples, a standard curve was generated 566 510 6, 8, 10, and 12 MAI with Las-infected or Las-free (control) using the amplified PCR product of the gene 16S rRNA from 567 511 budwood. Those accessions that remained with few or no Las. Eight- to ten-fold serial dilutions were prepared in triplicate 568 512 Las-positive scions at 12 MAI, and continued to grow well, and mixed in total DNA at 100 ng/µl for healthy ‘Rangpur’ lime 569 513 were re-evaluated at 24 MAI, including Microcitrus australasica roots (Supplementary Figure 1). The linear relation among Ct 570

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571 and the log 16S rRNA was y = 1.073Ct + 25.098, R2 = 0.9929. Ct after propagating them and once they had been already Las- 628 572 values higher than 34.0 were also inconsistent for roots. When Ct inoculated. For L. acidissima, progressive overgrowth of the 629 573 values were 34.0 or close to 34.0, bark pieces from the rootstock scion resulted in five of the 10 Las challenge-inoculated plants 630 574 were further analyzed at 12 and 24 MAI by qPCR to confirm dying before the twelfth month of evaluation (Figure 2B). Even 631 575 Las-infection of the ‘Rangpur’ lime. considering that two plants were positive (Ct ≤ 34.0) and another 632 576 one was considered suspicious (Ct > 34 and ≤36.0) at 10 633 577 Data Analysis MAI, the insufficient number of replications alive at 12 MAI, 634 578 Data were analyzed with the statistical software RStudio (RStudio led us to disregard this species in our analysis. At the end of 635 579 Team, 2015). The data were assessed for homoscedasticity the experiment, all grafts of these accessions were affected by 636 580 (Levene, 1960), and normality (Shapiro and Wilk, 1965). All overgrowth of the scion. For C. halimii, incompatibility reaction 637 581 multiple comparisons were first subjected to analysis of variance took almost 1 year to start appearing, but in this case, it affected 638 582 (ANOVA). Contrast analysis was performed to compare means to 100% of the grafts of ‘Valencia’ sweet orange budwood pieces 639 583 between groups (Rosenthal and Rosnow, 1985) and when on the scion but not on the ‘Rangpur’ lime rootstock, neither 640 584 significant differences were found, the means were compared by on the bud union between C. halimii and ‘Rangpur’ lime. It 641 585 the t-test (p < 0.05). Analysis was done using the Las titer average was characterized by a profuse exudation of gum at the graft 642 586 at 12 MAI in log10 of amplicon copies per gram of plant tissue union in all plants (Figure 2C). Therefore, only after being 643 587 estimated based on a standard curve described by Lopes et al. propagated, inoculated, and analyzed by qPCR for Las infection 644 588 (2013). during months, we realized that these plants had a problem which 645 589 was seriously affecting their growth and general aspect and which 646 590 Phylogenetic Tree Construction finally killed all of them between 11 and 15 MAI (Supplementary 647 591 Table 6). The ‘Rangpur’ lime rootstocks that survived resulted 648 To establish the phylogenetic tree, we used the sequences of eight 592 Las-positive at 12 MAI. Moreover, all their L. acidissima and 649 chloroplastic regions (atpB-coding region, rbcL-atpB spacer, 593 C. halimii scions were also Las-positive at 12 MAI (results not 650 rps16 spacer, trnL-F region, rps4-trnT spacer, matK-50trnK 594 shown; Supplementary Table 6). A possible role of Las in these 651 spacer, psbM-trnDGUC spacer and trnG intron) published 595 incompatibility reactions could be discarded because uninfected 652 by Bayer et al.(2009) selecting those accessions for which 596 budwood controls suffered the same incompatibility problems 653 informations on HLB resistance were available from our study 597 with identical frequencies for both genotypes (results not shown). 654 and that from Ramadugu et al.(2016). We added a few accessions 598 655 within the True Citrus group to cover more species as well as 599 656 some representatives of the Triphasiinae subtribe to maintain 600 Response of Graft-Compatible Citrinae 657 the global structure of the Aurantioideae phylogenetic tree. 601 Species to Challenge-Inoculation of Las 658 According to the Swingle and Reece(1967) classification, the 602 All graft-compatible accessions were propagated on ‘Rangpur’ 659 Clauseneae tribe was represented by three subtribes: Clauseninae 603 lime rootstock and challenge-inoculated by grafting two 660 (six accessions), Merrilliinae (one accession) and Micromelinae 604 budwood pieces, one on the rootstock and another one on 661 (one accession). The tribe was represented by three 605 the scion, from Las-infected ‘Valencia’ sweet orange trees. 662 subtribes: Triphasilinae (nine accessions), Balsamocitrinae (five 606 Uninfected, healthy ‘Valencia’ trees were used as a source of 663 accessions) and Citrinae (37 accessions). For the 59 selected 607 budwood pieces to be grafted in the corresponding controls for 664 accessions (Supplementary Table 5), the sequences were 608 each genotype. Las infection in budwood pieces was confirmed 665 obtained from the National Center of Biotechnology Information 609 by testing a small piece from each one by qPCR. Using this 666 (NCBI). For each genome fragment, the sequences were aligned 610 challenge inoculation system, Las infection in the scion may 667 to the C. × sinensis reference chloroplast genome sequence 611 come from the infected budwood pieces grafted on it and/or 668 (GenBank: NC008334) using BioEdit software (Hall, 1999) and 612 from the ‘Rangpur’ lime stock once infected. In any case, 669 curated manually in InDel areas. The resulting alignment was 613 infection of the rootstock would ensure a continuous flow of 670 used to establish a Neighbor Joining (NJ) tree according to 614 bacteria moving up to the scion. Root infection was evaluated 671 Kimura(1980) genetic distances and considering deletions as 615 just at 12 MAI, because root sampling damaged the composite 672 missing data, using DarWin 6 software (Perrier and Jacquemoud- 616 plants and at that time-point scion Las-infection outcomes were 673 Collet, 2006). One thousand bootstraps were performed to test 617 already obtained. To confirm consistent rootstock infection, 674 the robustness of each branching. 618 ‘Rangpur’ lime bark was also evaluated at 12 MAI. However, Las 675 619 infection in the rootstock was successful in 63 to 100% of the 676 620 graft-inoculated plants, irrespective of the phylogenetic relation 677 621 RESULTS of scion types with Citrus (Supplementary Table 2). As all scions 678 622 grafted on challenge inoculated but Las-negative ‘Rangpur’ lime 679 623 Graft-Compatibility Onto ‘Rangpur’ Lime rootstocks resulted to be Las-negative, for further analyses of 680 624 Rootstock Las-resistance we only considered those plants with Las-positive 681 625 Most genotypes used were graft-compatible on ‘Rangpur’ lime rootstocks. This result revealed largely inefficient Las-infection 682 626 (Figure 2A). Incompatibility reactions were observed in two of the scions through graft-inoculation of infected budwood 683 627 species, L. acidissima and C. halimii, but only about 1 to 2 years and how difficult was to get 100% infection of a well-known 684

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685 742 686 743 687 744 688 745 689 746 690 747 691 748 692 749 693 750 694 751 695 752 696 753 697 754 698 755 699 756 700 757

701 FIGURE 2 | (A) An Eremocitrus glauca × Citrus × sinensis scion propagated on the ‘Rangpur’ lime as an example of good rootstock/scion compatibility between 758 702 two genotypes. (B) Limonia acidissima scion propagated on ‘Rangpur’ lime showing overgrowth incompatibility at the graft union. (C) Profuse gum exudation in the 759 703 graft union between ‘Valencia’ sweet orange budwood and Citrus halimii scion. 760 704 761 705 762 706 Las host as ‘Rangpur’ lime even under controlled experimental Category 2. Partially Resistant 763 707 conditions and forced challenge-inoculations, and therefore Eight Citrinae accessions were considered as partially resistant, 764 708 how important is to use proper controls of infection to avoid because they showed Las infection in just part of the clonally 765 709 getting false negatives when testing genotypes for resistance propagated scions. In Las-positive propagations, infection was 766 710 to this bacterium. usually delayed and bacterial titers were generally lower than 767 711 Based on Las infection results, we classified the Citrinae those found in sweet orange controls (p < 0.0377). The three 768 712 genotypes used as scions in three categories (Table 2): accessions of Australian finger lime (Microcitrus australasica, M. 769 713 australasica ‘Sanguinea’ and M. australasica ‘True Sanguinea’), 770 714 Category 1. Susceptible ‘Faustrimedin,’ M. inodora, M. virgata, the cherry orange Citropsis 771 715 This comprised all genotypes with 100% of the clonal gilletiana and Naringi crenulata were included in this group 772 716 propagations resulting Las-positive at 12 MAI, when their (Table 2 and Supplementary Table 7). Such variable responses 773 717 evaluation finalized. It included nine genotypes: the two sweet may be considered as a genetic resistance trait, which was 774 718 orange controls ‘Pera’ and ‘Tobias,’ C. halimii, the four accessions partially overcome in some clonal propagations by aggressive 775 719 of P. trifoliata (‘Pomeroy,’ ‘Benecke,’ ‘Barnes,’ and ‘Rubidoux’), Las challenge inoculation. However, no correlation was found 776 720 and the two Atalantia species (A. citroides and A. ceylanica). between higher bacterial titer in the rootstock and resistance 777 721 Ct values in the scion canopy leaves and ‘Rangpur’ lime roots breaking in the scion (Supplementary Table 8). 778 722 and bark for each plant and evaluation date are detailed in The three M. australasica accessions showed similar response 779 723 Supplementary Table 6. HLB-like symptoms on the infected patterns, with six out of 11 scions being Las-positive at 8 MAI, 780 724 scions at 10–12 MAI were masked by nutritional deficiencies and the other five by 12 MAI. However, Las infection was 781 725 probably due to the severe root loss caused by the bacterium inconsistent because some scions that were infected at 8 MAI 782 726 (Figure 3), so sensitivity to Las infection in the scion was not resulted qPCR-negative at 12 MAI. Moreover, five scions resulted 783 727 evaluated. Because of the severe root damage, one A. citroides Las-positive, with Ct > 32.0, just at 1–2 out of five time points 784 728 composite plant died about 10 MAI. As mentioned above, evaluated (Supplementary Table 7). As Las titers were generally 785 729 C. halimii scions were seriously affected by an incompatibility low, with Ct > 30.0 at 10–12 MAI, inconsistent detection may 786 730 problem with ‘Valencia’ budwood pieces, irrespective of being be derived from uneven distribution of a few bacterium cells 787 731 infected or not with Las, which finally killed four infected infecting those scions. The ‘Sanguinea’ accession showed four 788 732 composite plants at 11–12 MAI and the remaining infected ones out of nine Las positive scions but the ‘True Sanguinea’ showed 789 733 before 15 MAI. C. halimii non-inoculated control composite just one (Ct > 30.0) out of 11 scions (Supplementary Table 7). 790 734 plants, which had been grafted with Las-free ‘Valencia’ budwood Because of its apparent higher resistance compared to the other 791 735 pieces, died between 11 and 14 months after grafting. accessions, we decided to maintain and re-evaluate the ‘True 792 736 The contrast analysis showed not significant differences in Sanguinea’ accession again at 24 MAI. At this time, three out 793 737 bacterial titers when comparing all Category 1 accessions together of 10 were Las-positive (Ct > 32.0). The remaining one died 794 738 (p < 0.0884), and Citrus with P. trifoliata accessions (p < 0.12). possibly due to the severe damages caused by the bacterium to the 795 739 However, they were significant when comparing P. trifoliata ‘Rangpur’ lime rootstock (Table 3 and Supplementary Table 10). 796 740 versus Atalantia (p < 0.000183) and also when comparing Citrus The trigeneric hybrid ‘Faustrimedin,’ with half of its genome 797 741 versus Atalantia accessions (p < 1e−04). coming from M. australasica, showed only three scions out of 10 798

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799 being Las-positive, but just at one time point and with Ct > 32.0 by qPCR and all were confirmed as Las-infected (Table 3 and 856 800 in the three cases. When re-evaluated at 24 MAI, four scions Supplementary Table 10). 857 801 resulted to be Las-positive, having been three of them Las- Regarding the cross-incompatible Citrus relatives within this 858 802 negative until 12 MAI (Tables 2, 3 and Supplementary Tables 7, category, Citropsis gilletiana had three plants out of 13 that 859 803 10). M. virgata, another hybrid with half of M. australasica were Las-positive at 12 MAI, and other three with Ct ranging 860 804 genome, showed three out 10 scions being Las-positive at 12 MAI. between 34.8 and 35.7 at least at one time point. Likewise, 861 805 Only five of them could be re-evaluated at 24 MAI, being Las- five out of nine Naringi crenulata scions were Las-positive but 862 806 negative, and the other five died due to Las-induced damages only at 12 MAI, and the bacterial titers were usually low, with 863 807 to the rootstock (Tables 2, 3 and Supplementary Tables 7, 10). Ct > 30.0 (Table 2 and Supplementary Table 7). As in Category 864 808 M. inodora also was partially resistant, as six out of 13 scions 1, HLB-like symptoms resembling mineral deficiencies appeared 865 809 resulted Las-positive at least at one time point, but as in the case in infected scions likely due to the severe root loss caused by 866 810 of the M. australasica accessions and hybrids mentioned above, the bacterium, which precluded evaluating sensitivity to Las 867 811 infections were inconsistent though bacterial titers were not so infection in the scion. 868 812 low as compared to those of the other genotypes. Las-induced Contrast analysis among Category 2 accessions showed that 869 813 damages in the rootstock killed two plants at 8 and 12 MAI and differences observed in bacterial titers were not significant within 870 814 other two before 24 MAI, with three out of 10 scions resulting the group (p < 0.08), but they were significant when comparing 871 815 Las-positive. All accessions that were re-evaluated at 24 MAI had Category 1 and 2 (p < 0.001). Moreover, there were significant 872 816 the bark tissue from the ‘Rangpur’ lime rootstocks also analyzed differences in bacterial titers when specifically comparing ‘Pera’ 873 817 874 818 875 819 TABLE 2 | ‘Candidatus Liberibacter asiaticus’ infection in the Citrinae genotypes evaluated as determined through detection of the 16S DNA by qPCR. 876 820 877 Category Accession Freq.a Scion Rootstock 821 878 822 Leaves Root Bark 879 823 880 824 Ct avgb ± SEMc Log avgd ± SEM Ct avg ± SEM Log avg ± SEM Ct avg ± SEM Log avg ± SEM 881 825 882 1 Citrus × sinensis ‘Pera’ 41/41 25.7 ± 0.6 4.7 ± 0.2 30 ± 0.6 3.4 ± 0.2 27.4 ± 0.4 4.2 ± 0.1 826 883 C. × sinensis ‘Tobias’ 09/09 22.3 ± 0.4 5.7 ± 0.1 29.9 ± 1.1 3.4 ± 0.3 29.6 ± 0.9 3.5 ± 0.3 827 884 Citrus × halimii 05/05 30 ± 1.1 3.4 ± 0.3 31.4 ± 0.9 3.0 ± 0.3 30.6 ± 0.3 3.2 ± 0.1 828 885 Poncirus trifoliata ‘Pomeroy’ 06/06 27.9 ± 1.4 4.0 ± 0.4 30.6 ± 1.5 3.2 ± 0.5 31 ± 0.4 3.1 ± 0.1 829 886 P. trifoliata ‘Benecke’ 06/06 31.2 ± 1.0 3.0 ± 0.3 29.7 ± 1.4 3.5 ± 0.4 29.5 ± 0.8 3.5 ± 0.2 830 887 P. trifoliata ‘Barnes’ 08/08 32.0 ± 1.1 2.8 ± 0.3 31.0 ± 0.9 3.1 ± 0.3 29.6 ± 1.0 3.5 ± 0.3 831 888 P. trifoliata ‘Rubidoux’ 06/06 26.2 ± 1.4 4.5 ± 0.4 30.6 ± 1.1 3.2 ± 0.3 29.4 ± 0.9 3.6 ± 0.3 832 Atalantia citroides 10/10 29.9 ± 0.7 3.4 ± 0.2 26.0 ± 0.9 4.6 ± 0.3 26.2 ± 0.6 4.5 ± 0.2 889 833 A. ceylanica 12/12 29.4 ± 1.2 3.6 ± 0.4 25.8 ± 0.4 4.7 ± 0.1 26 ± 0.7 4.6 ± 0.2 890 834 2 Microcitrus australasica 06/11* 31.62 ± 0.7 2.9 ± 0.2 27.4 ± 0.6 4.1 ± 0.2 30.8 ± 0.5 3.2 ± 0.2 891 835 M. australasica ‘Sanguinea’ 04/09* 26.0 ± 0.9 4.6 ± 0.3 29.0 ± 1.4 3.7 ± 0.4 28.7 ± 0.6 3.6 ± 0.1 892 836 M. australasica ‘True Sanguinea’ 01/11* 33.8 ± 0.0 2.34 ± 0.0 26.8 ± 0.0 4.4 ± 0.0 29.3 ± 0.0 3.6 ± 0.0 893 837 Faustrimedin hybrid; C. × oliveri 02/10* 33.1 ± 0.9 2.5 ± 0.3 33.5 ± 0.6 2.4 ± 0.2 30.8 ± 0.2 3.2 ± 0.1 894 838 Microcitrus inodora 04/12* 25.4 ± 2.9 4.8 ± 0.9 31.6 ± 1.4 2.9 ± 0.4 28.0 ± 1.0 4.0 ± 0.3 895 839 Microcitrus virgata hybrid 03/10* 30.0 ± 1.7 3.4 ± 0.5 30.1 ± 1.9 3.4 ± 0.6 31.9 ± 1.1 2.8 ± 0.4 896 840 Citropsis gilletiana 05/13* 32.2 ± 1.9 2.7 ± 0.6 24.8 ± 0.3 5.0 ± 0.1 27.6 ± 0.5 4.1 ± 0.4 897 841 Naringi crenulata 05/09* 32.1 ± 0.7 2.7 ± 0.2 26.8 ± 0.3 4.4 ± 0.1 28.2 ± 0.7 3.8 ± 0.2 898 842 3 Microcitrus warburgiana 00/09 nde nd 31.9 ± 0.6 2.8 ± 0.2 30.1 ± 0.6 3.4 ± 0.2 899 843 Microcitrus papuana 00/08 nd nd 29.1 ± 0.8 3.7 ± 0.2 30.0 ± 0.5 3.4 ± 0.1 900 844 Microcitrus australis 00/10 nd nd 32.5 ± 0.5 2.7 ± 0.2 30.7 ± 0.7 3.2 ± 0.2 901 845 Microcitrus × Eremocitrus hybrid 00/07 nd nd 31.8 ± 0.7 2.8 ± 0.2 29.4 ± 0.7 3.6 ± 0.2 902 846 Eremocitrus glauca 00/07 nd nd 25.3 ± 0.3 4.8 ± 0.1 29.8 ± 0.8 3.5 ± 0.2 903 847 E. glauca × C. × sinensis hybrid 00/12 nd nd 30.7 ± 0;4 3.2 ± 0.1 28.9 ± 0.5 3.7 ± 0.1 904 848 Eremocitrus × Microcitrus hybrid 00/08 nd nd 33.1 ± 0.3 2.5 ± 0.1 31.1 ± 0.3 3.1 ± 0.1 905 849 906 Average obtained from all plants of each accession at 12 MAI. 850 aFreq., number of scion Las-positive (Ct ≤ 34.0)/total of plants evaluated (with Las-positive Rootstock Ct ≤ 34.0). 907 851 bCt avg, cycle threshold average determined through the detection of the 16S DNA by qPCR. 908 c 852 SEM, standard error of the mean. 909 dLog, Las titer average in log10 of amplicon copies per gram of plant tissue estimated based on a standard curve as described by Lopes et al.(2013). 853 910 end, non-detected. 854 *Rootstock roots and bark avg ± SEM from accession in the category 2 were obtained exclusively from plants with positive (Ct ≤ 34.0) or suspicious to be positive 911 855 (34.0 < Ct ≤ 36.0) leaves. Details on values of each plant are in the Supplementary Tables 6, 7, 9. 912

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913 970 914 971 915 972 916 973 917 974 918 975 919 976 920 977 921 978 922 979 923 980 924 981 925 982 926 983 927 984 928 985 929 986 930 987 931 988 932 989 933 990 934 991 935 992 936 993 937 994 938 995 939 996 940 997 941 FIGURE 3 | HLB-like symptoms in roots from ‘Rangpur’ lime rootstocks graft-inoculated either with Las-infected or healthy control budwood in composite plants 998 with infected vs. healthy control scions from three susceptible, the partially resistant Microcitrus australasica and the full-resistant E. glauca × C. × sinensis 942 999 genotypes at 12 months after challenge-inoculation. (A) Citrus × sinensis ‘Pera’ Las-infected; (B) C. × sinensis ‘Pera’ control; (C) C. × sinensis ‘Tobias’ 943 Las-infected; (D) C. × sinensis ‘Tobias’ control; (E) Atalantia citroides Las-infected; (F) Atalantia citroides control; (G) Microcitrus australasica Las-infected; 1000 944 (H) Microcitrus australasica control; (I) E. glauca × C. × sinensis hybrid Las-inoculated; (J) E. glauca × C. × sinensis hybrid control. 1001 945 1002 946 1003 947 and ‘Tobias’ sweet orange controls versus Category 2 (p < 0.0377) hybrid evaluated in this work for the first time, showed the best 1004 948 and when comparing P. trifoliata versus Category 2 accessions graft-compatibility with ‘Rangpur’ lime as scion growth was 1005 949 (p < 0.001). vigorous. Considering their genetic backgrounds, all the seven 1006 950 accessions are probably sexually compatible with Citrus. 1007 951 Category 3. Resistant To confirm that these accessions were truly resistant, namely 1008 952 The seven accessions included into this group were Microcitrus that there was vascular connection at the grafts and there 1009 953 warburgiana, M. papuana, M. australis, a Microcitrus was bacterial movement from rootstock to scions through the 1010 954 sp. × E. glauca hybrid, Eremocitrus glauca, an eremorange hybrid vascular system, bark tissue from each scion was evaluated by 1011 955 (E. glauca × Citrus × sinensis), and an E. glauca × Microcitrus qPCR at 5 and 30 cm above the grafting propagation line and at 1012 956 sp. hybrid. A variable number of plants from these accessions the canopy (21–152 cm, depending on the accession) at 24 MAI 1013 957 showed Las-infected ‘Rangpur’ lime rootstocks, confirming the to assess the presence of the bacterium. Las was detected in the 1014 958 partial success of Las inoculation (Supplementary Table 1), but scion bark close to the bud union (5 cm) in most plants but as 1015 959 none of the leaf samples from scions grafted on Las-positive sampling was performed farther from the grafting line, Las was 1016 960 rootstocks resulted positive for Las at 12 MAI (Table 2 and found in very few plants at low concentration (30 cm) or was not 1017 961 Supplementary Table 9). Because of this, they were classified detected (canopy) (Table 4 and Supplementary Table 11). 1018 962 as resistant to Las. Although E. glauca was graft-compatible 1019 963 with ‘Rangpur’ lime, its growth and development was generally 1020 964 poor, being this an intrinsic characteristic of the accession and Distribution of the Response to Las 1021 965 not related to bud union problems (Bitters et al., 1964, 1969). Categories According to Phylogeny 1022 966 The other six accessions were further evaluated at 24 MAI and A phylogenic tree was established using available data for eight 1023 967 the full resistance was confirmed for all of them (Table 3 and genic and intergenic chloroplastic sequences (Bayer et al., 2009). 1024 968 Supplementary Table 10). The eremorange, the Microcitrus Sequence alignment for the 59 accessions was performed with 1025 969 sp. × E. glauca hybrid and the E. glauca × Microcitrus sp. BioEdit software and manually curated for InDel regions. We 1026

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1027 identified 6430 positions with single nucleotide polymorphism value = 67%) and differentiated from the other species of the 1084 1028 and used them to establish the NJ tree (Figure 4). The True Citrus Balsamocitrinae and Triphasiinae subtribes and the Clauseneae 1085 1029 clade grouping the Oceanian genera Microcitrus, Eremocitrus, tribe (Figure 4). 1086 1030 and and the Asian genera Citrus, Poncirus Resistance to Las (category 1 here and categories 6 to 1087 1031 and Fortunella was very well defined (bootstrap value = 100%). It 8 for Ramadugu et al., 2016) appeared to be concentrated 1088 1032 was linked with lower support to a cluster joining three Atalantia in the clade joining Atalantia species with the True Citrus 1089 1033 species and L. acidissima and then a third cluster including two (Figure 4). Within this clade, there was a very strong phenotypic 1090 Q18 1034 Citropsis species, N. crenulata and Pleiospermium laliatum. These differentiation between the Oceanian species tested that were 1091 1035 three clusters constitute a clade quite well defined (bootstrap all classified as full resistant or partially resistant while all 1092 1036 1093 1037 1094 1038 TABLE 3 | ‘Candidatus Liberibacter asiaticus’ infection in the Citrinae genotypes re-evaluated at 24 months after inoculation (MAI), as determined through detection of 1095 the 16S rDNA by qPCR. 1039 1096

1040 Category Accession Freq.a Scion Rootstock 1097 1041 1098 1042 Leaves Bark 1099 1043 1100 Ct avgb ± SEMc Log avgd ± SEM Ct avg ± SEM Log avg ± SEM 1044 1101 1045 1 Citrus × sinensis ‘Pera’ 15/15 25.5 ± 0.7 4.7 ± 0.2 27.9 ± 0.5 4.0 ± 0.1 1102 1046 C. × sinensis ‘Tobias’ 09/09 23.8 ± 1.4 5.2 ± 0.4 29.1 ± 0.5 3.7 ± 0.2 1103 1047 2 M. australasica‘True Sanguinea’ 03/10* 33 ± 0.1 2.5 ± 0.0 30.9 ± 0.7 3.1 ± 0.2 1104 1048 Faustrimedin hybrid; C. × oliveri 04/10* 30.3 ± 1.1 3.3 ± 0.3 31.2 ± 1.1 3.0 ± 0.3 1105 1049 Microcitrus inodora 03/07* 25.3 ± 1.7 4.8 ± 0.5 27.4 ± 1.3 4.2 ± 0.4 1106 1050 Microcitrus virgata hybrid 00/09* nde nd 29.0 ± 0.4 3.7 ± 0.1 1107 1051 3 Microcitrus warburgiana 00/06 nd nd 30.1 ± 0.0 3.4 ± 0.0 1108 1052 Microcitrus papuana 00/04 nd nd 31.2 ± 0.8 3.0 ± 0.2 1109 1053 Microcitrus australis 00/08 nd nd 30.3 ± 0.1 3.3 ± 0.0 1110 1054 Microcitrus × Eremocitrus hybrid 00/07 nd nd 31.0 ± 0.9 3.1 ± 0.3 1111 1055 E. glauca × C. × sinensis hybrid 00/11 nd nd 28.5 ± 0.4 3.8 ± 0.1 1112 1056 Eremocitrus × Microcitrus hybrid 00/08 nd nd 33.4 ± 0.1 2.4 ± 0.0 1113 1057 1114 Average obtained from all plants of each accession. 1058 aFreq., number of scion Las-positive (Ct ≤ 34.0)/total of plants evaluated (with Las-positive Rootstock Ct ≤ 34.0). 1115 1059 bCt avg, cycle threshold average determined through the detection of the 16S DNA by qPCR. 1116 c 1060 SEM, standard error of the mean. 1117 dLog, Las titer average in log10 of amplicon copies per gram of plant tissue estimated based on a standard curve as described by Lopes et al.(2013). 1061 1118 end, non-detected. 1062 *Rootstock bark avg ± SEM from accession in the category 2 were obtained exclusively from plants with positive (Ct ≤ 34.0) or suspicious to be positive (34.0 < Ct ≤ 36.0) 1119 1063 leaves. Details on values of each plant are in the Supplementary Table 10. 1120 1064 1121 1065 TABLE 4 | ‘Candidatus Liberibacter asiaticus’ presence in the Citrine genotypes evaluated in different distances from the rootstock at 24 months after the 1122 1066 inoculation (24 MAI). 1123 1067 1124 Accession Freq.a 05 cm Freq. 30 cm Freq. Apex (21–152 cm) 1068 1125 1069 Ct avgb ± SEMc Log avgd ± SEM Ct avg ± SEM Log avg ± SEM Ct avg ± SEM Log avg ± SEM 1126 1070 1127 Citrus × sinensis ‘Pera’ 41/41 25.5 ± 0.4 4.8 ± 0.1 41/41 25.4 ± 0.3 4.8 ± 0.1 41/41 25 ± 0.3 4.8 ± 0.1 1071 1128 Citrus × sinensis ‘Tobias’ 09/09 26.7 ± 1.1 4.4 ± 0.33 09/09 25.2 ± 0.9 4.8 ± 0.3 09/09 23.8 ± 0.6 5.3 ± 0.2 1072 1129 Microcitrus warbugiana 04/06 31.7 ± 0.6 2.9 ± 0.2 00/06 nde nd 00/06 nd nd 1073 1130 Microcitrus papuana 02/04 32.5 ± 0.4 2.6 ± 0.1 00/04 nd nd 00/04 nd nd 1074 1131 Microcitrus australis 07/08 32.8 ± 0.6 2.6 ± 0.2 00/08 nd nd 00/08 nd nd 1075 1132 Microcitrus × Eremocitrus hybrid 06/07 30.8 ± 0.5 3.2 ± 0.1 03/07 27.8 ± 1.0 4.1 ± 0.3 00/07 nd nd 1076 1133 Eremocitrus glauca 06/07 31.6 ± 0.8 2.9 ± 0.3 00/07 nd nd 00/07 nd nd 1077 1134 E. glauca × C. × sinensis hybrid 11/11 31.2 ± 0.4 3 ± 0.1 04/11 30.6 ± 0.4 3.2 ± 0.1 00/11 nd nd 1078 1135 Eremocitrus × Microcitrus hybrid 08/08 32.1 ± 0.3 2.8 ± 0.1 00/08 nd nd 00/08 nd nd 1079 1136 a 1080 Freq., number of bark scion samples Las-positive (Ct ≤ 34.0)/total of plants evaluated (with Las-positive Rootstock Ct ≤ 34.0). 1137 bCt avg, cycle threshold average determined through the detection of the 16S DNA by qPCR. 1081 1138 cSEM, standard error of the mean. 1082 dLog, Las titer average in log10 of amplicon copies per gram of plant tissue estimated based on a standard curve as described by Lopes et al.(2013). 1139 1083 end, non-detected. 1140

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1141 1198 1142 1199 1143 1200 1144 1201 1145 1202 1146 1203 1147 1204 1148 1205 1149 1206 1150 1207 1151 1208 1152 1209 1153 1210 1154 1211 1155 1212 1156 1213 1157 1214 1158 1215 1159 1216 1160 1217 1161 1218 1162 1219 1163 1220 FIGURE 4 | Distribution on the phylogenetic tree of the Aurantioideae sub-family (NJ tree based on eight chloroplast genome fragments) based on accession 1164 responses to Las challenge inoculation according to this study (red text: susceptible, blue text: partially resistant, green text: full resistant) and Ramadugu et al. 1221 1165 (2016) (red rectangle: susceptible –categories 6 to 8; blue rectangle: tolerant –categories 3, 4; green rectangle: resistant –categories 1, 2). 1222 1166 1223 1167 1224 1168 Asian citrus species analyzed here or previously assessed by involved in the resistance trait for direct modification of well- 1225 1169 Ramadugu et al.(2016) were found to be susceptible. In the known elite Citrus cultivars using modern biotechnology tools, 1226 1170 Oceanian clade, all resistant species were grouped in a same especially those which cannot be improved by sexual breeding 1227 1171 sub-clade, differentiated (bootstrap value = 66) from another due to their high heterozygosity, or (iii) to be used promptly 1228 1172 one grouping the partially resistant M. australasica, C. × oliveri as new rootstocks or interstocks to potentially alleviate HLB- 1229 1173 and the non-tested M. garrawayi. Intriguingly, within the induced damages. 1230 1174 Citrinae subtribe, C. gilletiana and N. crenulata were partially However, the characteristics of Las infection in Citrus 1231 1175 resistant to Las (Figure 4). During their field evaluation under genotypes, cultivars and relatives have generally rendered 1232 1176 natural Las inoculation through D. citri, Ramadugu et al.(2016) confounding results, mainly because symptoms appear many 1233 1177 found that all analyzed species of the Balsamocitrinae and months after infection which delays disease development (Hung 1234 1178 Triphasiinae subtribes as well as those of Clauseneae tribe were et al., 2001; Folimonova et al., 2009; Cifuentes-Arenas et al., 1235 1179 resistant or tolerant to HLB (categories 1–2 and 3–4 of their 2019), they can be mistaken with those derived from nutritional 1236 1180 study, respectively). deficiencies, at least at the beginning of infection (da Graça, 1237 1181 1991; Bové, 2006), the uneven distribution of the bacterium 1238 1182 within the infected trees (Tatineni et al., 2008; Li et al., 2009; 1239 1183 DISCUSSION Raiol-Junior et al., 2021), its active multiplication quite restricted 1240 1184 to new flushes and developing roots (Hilf and Luo, 2018; 1241 1185 Searching for resistance to HLB within Citrus and its relatives of Raiol-Junior et al., 2021), the environmental influences on 1242 1186 the family Rutaceae, subfamily Aurantioideae, has been an active bacterial multiplication and plant colonization (Lopes et al., 1243 1187 area of research due to the severe damages caused by the disease 2009; Gasparoto et al., 2012) and largely unknown plant host- 1244 1188 on tree performance, production and fruit quality. Citriculture pathogen molecular interactions which certainly may affect 1245 1189 costs in HLB-affected regions have been increased due to the their outcome. To further complicate this interplay, the psyllid 1246 1190 implementation of treatments to keep citrus groves economically vector D. citri shows preference for specific colors and volatile 1247 1191 productive but curative methods capable of overcoming losses are compounds emitted by the host plants (Wenninger et al., 1248 1192 lacking (Bassanezi et al., 2020). 2009; Patt and Sétamou, 2010; Hall et al., 2011), and once 1249 1193 Therefore, there is a need for confident and reliable sources of settled it prefers to feed and reproduce on young shoots 1250 1194 resistance to either the Las bacterium, the insect vector D. citri rather than mature leaves (Hall et al., 2016; Cifuentes-Arenas 1251 1195 or both, which could be used for introgression into the Citrus et al., 2018). Moreover, psyllids exhibit a clear preference for 1252 1196 germplasm (i) to generate new Citrus-like cultivars that may be some hosts within Rutaceae, subfamily Aurantioideae (Bergera, 1253 1197 useful as rootstocks or scions, (ii) to map and identify the genes Murraya) over others (Citrus), while some Aurantioideae species 1254

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1255 were reported as intermediate [such as Glycosmis pentaphylla field conditions, and classified ‘Simmons’ as resistant and ‘Little- 1312 1256 (Retz.) DC., Clausena harmandiana (Pierre) Guillaumin and leaf’ as showing delayed infection. Notably, in other studies these 1313 1257 Zanthoxylum ailanthoides (L.)] and other Citrus relatives as two accessions were among the most resistant Poncirus ones to 1314 1258 highly resistant to the psyllid, such as certain Poncirus trifoliata oviposition by D. citri (Westbrook et al., 2011; Richardson and 1315 1259 accessions (Westbrook et al., 2011; Richardson and Hall, 2013; Hall, 2013; Hall et al., 2015), so the field-resistance attributed to 1316 1260 Hall et al., 2015; Felisberto et al., 2019). Furthermore, the use these two accessions may be explained by a lack of preference 1317 1261 of seedlings, especially in the case of Aurantioideae relatives, by the vector, especially when exposed mixed with other more 1318 1262 introduces another factor of variation, not only due to the preferred hosts, and perhaps not to bacterial resistance. In other 1319 1263 different morphological, development and physiological features works on D. citri biology, the four Poncirus accessions used here 1320 1264 of juvenile vs. mature plants, but more importantly because many were considered partial resistant to D. citri as the female insects 1321 1265 Citrus relatives are monoembryonic, so propagation through laid significantly fewer eggs than in sweet orange controls (Hall 1322 1266 seeds does not provide clonal plants but segregating progenies et al., 2015, 2017, 2019). Therefore, P. trifoliata has interest as 1323 1267 genetically different to the mother genotype. Taking all this into a possible source of genetic resistance to D. citri rather than to 1324 1268 consideration and aiming to evaluate resistance to Las within Las. Nevertheless, other P. trifoliata accessions should be tested 1325 1269 Citrus relatives undoubtably, we decided to center our study on confidently for Las-resistance, especially those being widely used 1326 1270 the bacterium-host interaction in a way avoiding any interference in rootstock breeding programs. 1327 1271 of the above mentioned factors. Ramadugu et al.(2016) showed in their field experiment that 1328 1272 The Citrinae genotypes of interest were first propagated Microcitrus and Eremocitrus genera may be useful sources of 1329 1273 clonally onto the ‘Rangpur’ lime rootstock and then inoculated resistance to HLB, though they could not clarify whether they 1330 1274 by grafting well-controlled Las-infected wood onto the selected were resistant to D. citri or to Las. Moreover, they observed 1331 1275 plants. However, this was not trivial. In addition to difficulties variation in seedlings disease response within Microcitrus and 1332 1276 on grafting success for clonal propagation due to the genetic transient infection in E. glauca, likely due to segregation as they 1333 1277 distances among some of the Citrinae species used and Citrus, used zygotic seedlings. We confirmed here using clonal plants 1334 1278 problems in transmitting Las to them also occurred. Because that Las-resistance is widely spread within the germplasm of both 1335 1279 of the irregular distribution of Las in plant tissues, use of genera. Remarkably, our results on response to Las challenge- 1336 1280 symptomatic and qPCR-positive segments from donor plants inoculation separated the species from New Guinea, E. glauca and 1337 1281 are recommended for challenge inoculation. However, even with M. australis in the group of resistant genotypes while M. inodora 1338 1282 this, variation in Las transmission efficiencies averaging 70 to and M. australasica were included in the category of partially 1339 1283 90% within Citrus, Fortunella and Poncirus germplasm, have resistant types. Response to Las of M. australasica accessions 1340 1284 been found (Folimonova et al., 2009). In this work, we decided and hybrids reinforced generally its categorization as partially 1341 1285 to use the Las-susceptible ‘Rangpur’ lime as rootstock because resistant, with the only exception of their hybrids with E. glauca, 1342 1286 this allowed ascertaining which composite plants were actually which were full-resistant. Interestingly, all E. glauca hybrids 1343 1287 infected by Las. Although graft-inoculation was performed both used in this study were fully resistant to Las, suggesting that 1344 1288 in the scion and in the rootstock, only those plants with infected the determinants of resistance in E. glauca may have dominant 1345 1289 root system were considered for further resistance evaluation, inheritance, which is particularly engaging for introgression 1346 1290 as in no case we detected a Las-positive scion on a ‘Rangpur’ breeding schemes. Therefore, E. glauca and its hybrids as well as 1347 1291 lime rootstock free from Las. Moreover, this procedure precluded M. australis may be the most indicated ones among the Australian 1348 1292 erroneous categorization of false-negative plants as resistant. limes as parents in breeding efforts for generating Citrus-like 1349 1293 Furthermore, the upward movement of Las from the rootstock cultivars resistant to Las. Conversely, those Microcitrus species 1350 1294 to the scion 5 cm above the graft unions was confirmed in those and hybrids presenting partial resistance to Las would be less 1351 1295 accessions showing full-resistance to Las. indicated as they would probably confer incomplete resistance to 1352 1296 In more than 30 Citrus, Poncirus and Fortunella hosts their progenies. 1353 1297 tested, Folimonova et al.(2009) determined that Las was Although sexual-compatibility with Citrus is restricted to 1354 1298 unevenly distributed, with higher titers of the bacterium found some True Citrus fruit trees (Swingle and Reece, 1967; Bayer 1355 1299 in symptomatic tissue. This erratic spread within a plant led et al., 2009), graft-compatibility is widely reported, at least to most 1356 1300 to classification of P. trifoliata initially as resistant, but after a Citrinae genera (sensu Bayer et al., 2009, after Swingle and Reece, 1357 1301 subsequent test it resulted to be susceptible, thus being results 1967). There are also reports of graft-compatibility of Citrus 1358 1302 on infection of this genotype inconsistent. In our experiments, on Clausena (Bitters et al., 1964) and on Murraya paniculata 1359 1303 the four P. trifoliata accessions evaluated were categorized as (Swingle and Reece, 1967), which are farther relatives to Citrus 1360 1304 susceptible as the bacterium readily multiplied in all propagations and have been reported as resistant to Las (Ramadugu et al., 2016; 1361 1305 tested. The delay of about 2–4 months to reach 100% infection Cifuentes-Arenas et al., 2019), but being suitable hosts for D. citri 1362 1306 in P. trifoliata genotypes as compared to the two sweet orange (Felisberto et al., 2019). However, Citrus grafts on Clausena and 1363 1307 cultivars used as controls, may be attributed to the deciduous Murraya could be kept alive under greenhouse conditions but 1364 1308 nature of the former, which made them to flush much less none of them seemed recommendable in a field situation due 1365 1309 frequently than most citrus types. However, Ramadugu et al. to poor bud unions and progressive incompatibility problems 1366 1310 (2016) also tested two P. trifoliata accessions for resistance to (Bitters et al., 1969). Other promising, HLB-resistant “Remote 1367 1311 HLB, in this case by psyllid-mediated natural infection under Citroid fruit trees” (Swingle and Reece, 1967), such as Glycosmis, 1368

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1369 are graft-uncongenial with Citrus (Bitters et al., 1964). Based on determinants of susceptibility to Las, understood to mean 1426 1370 Bitters et al.(1964, 1969, 1977), our studies of graft-compatibility efficient bacterial multiplication and colonization of the whole 1427 1371 with Citrus and response to Las challenge inoculation were tree, appeared in an ancestor of the clade joining Atalantia 1428 1372 centered on Citrinae. Most species used were graft-compatible and True Citrus species. All Atalantia species have Indian 1429 1373 on ‘Rangpur’ lime, with the exception of Limonia acidissima. L. and south East Asia origin while Citropsis species are from 1430 1374 acidissima has been described as compatible with Citrus both as a Africa. It may be hypothesized that the Las susceptibility 1431 1375 scion and as a rootstock (Bitters et al., 1964; Swingle and Reece, determinants arose in a common ancestor of Atalantia and True 1432 1376 1967; Yoshida, 1996; Siebert et al., 2015), but its compatibility Citrus species, in India/South East Asia after the separation 1433 1377 with ‘Rangpur’ lime was not previously tested. Our experience of Citropsis from Asian genera of the Citrinae subtribe. Pfeil 1434 1378 with L. acidissima exemplifies that close phylogenetic relations and Crisp(2008) estimated that this separation occurred 1435 1379 could be used as an approach to foresee graft-compatibility, 12.9 My ago (with a quite large probability interval from 1436 1380 but it does not always predict successful bud unions (Bitters 7.0 to 20.7 My). 1437 1381 et al., 1977). C. halimii showed another type of unexpected The distribution of the categories of Las response to challenge 1438 1382 incompatibility, not derived from the scion-rootstock union inoculation in the True Citrus clade was also highly contrasting, 1439 1383 with ‘Rangpur’ lime, but from the use of ‘Valencia’ sweet with all Oceanian species considered as resistant or partially 1440 1384 orange budwood to challenge-inoculate Las onto C. halimii resistant while all Asian Citrus species were susceptible to Las 1441 1385 scions. At the beginning, we associated the weak growth of (Folimonova et al., 2009; Ramadugu et al., 2016; this work), 1442 1386 propagations to the high sensitivity of C. halimii to Las infection, including C. medica, which is sister species of Oceanian species 1443 1387 as indicated by Folimonova et al.(2009), but gum exudation according to chloroplast phylogeny (Pfeil and Crisp, 2008; Bayer 1444 1388 started to appear around 11 MAI at sweet orange-C. halimii et al., 2009; Carbonell-Caballero et al., 2015; this work), but allied 1445 1389 graft unions, irrespective of whether the grafted budwood was in a robust clade with C. maxima and C. for nuclear 1446 1390 Las-infected or not. We included this Citrus type in our studies phylogeny, as concluded from nuclear gene sequencing (Garcia- 1447 1391 because it had displayed some resistance to HLB in the field Lor et al., 2013), restriction site-associated DNA sequencing 1448 1392 experiments performed by Ramadugu et al.(2016), but it showed (Nagano et al., 2018) and whole-genome sequencing (Wu et al., 1449 1393 to be clearly susceptible in our challenges, as also indicated 2018). Based on chloroplastic phylogeny, Bayer et al.(2009) 1450 1394 by Folimonova et al.(2009). Regarding graft-union problems, included Microcitrus and Eremocitrus within the group of True 1451 1395 further experiments should be performed to attempt to reveal Citrus fruit trees (sensu Swingle and Reece, 1967), and proposed 1452 1396 the causes of the incompatibilities. From the cross-incompatible, to include them in the genus Citrus following Mabberley(2004). 1453 1397 graft-compatible Citrus relatives tested for resistance to Las, The monophylly of the True Citrus species and the cross- 1454 1398 the two Atalantia species used were susceptible, as already compatibility between Oceanian and Asian citrus species are 1455 1399 suggested for Atalantia citroides by Feng et al.(2015) using strong arguments sustaining this suggestion (Ollitrault et al., 1456 1400 zygotic seedlings, while Citropsis gilletiana and Naringi crenulata 2020). However, nuclear phylogeny clearly identified two sister 1457 1401 were considered as partially resistant. According to Bitters et al. clades within the True Citrus, one for the Asian species and 1458 1402 (1964), both genotypes may be excellent rootstocks for Citrus, the other for the Oceanian ones, with either full or partial 1459 1403 but clearly, those cross-compatible Citrus relatives included in resistance only present in the Oceanian clade (and observed 1460 1404 the full-resistant category offered the best alternatives to be for all Oceanian species tested). Under the hypothesis that Las 1461 1405 tested as Citrus rootstocks and interstocks, particularly those susceptibility determinants were present but not fixed in the 1462 1406 showing excellent rootstock-scion compatibility and vigor in ancestral population of the True Citrus plus Atalantia clade, 1463 1407 our studies, which were the E. glauca hybrids with Microcitrus the differentiation between Australian and Asian sub-clades may 1464 1408 and sweet orange. result from a founder effect in the two geographic regions 1465 1409 Looking at the distribution of the different types of responses or/and genetic drift. 1466 1410 to Las challenge inoculation with a phylogenetic view, we found According to Swingle and Reece(1967), Microcitrus and 1467 1411 that susceptible accessions (category 1 here and categories 6 Eremocitrus evolved from a common ancestor probably 1468 1412 to 8 for Ramadugu et al., 2016) were concentrated in a clade resembling M. warburgiana, which together with M. papuana 1469 1413 joining True Citrus and Atalantia species plus L. acidissima. are native to New Guinea. From such an ancestral form, one line 1470 1414 The sister position of L. acidissima and Atalantia species as well of evolution produced the Australian round lime (M. australis), 1471 1415 as the monophyly of True Citrus plus Atalantia species was another line culminated in M. inodora, and a third line of 1472 1416 previously described from chloroplast phylogeny by Pfeil and evolution led to the Australian finger limes, M. australasica. On 1473 1417 Crisp(2008) as well as Bayer et al.(2009), which conduced the other hand, E. glauca rapidly evolved from the common 1474 1418 them to include L. acidissima in the Citrinae subtribe. The ancestor with marked xerophytic adaptations to the Australian 1475 1419 sister position of Atalantia clade with the True Citrus clade was deserts. The chloroplast phylogeny, with the monophylly of all 1476 1420 also validated at nuclear level (Nagano et al., 2018). For the the previously mentioned species, confirm their common origin 1477 1421 subsequent node of the phylogenetic tree, with a clade including but, as previously described by Bayer et al.(2009), we observed 1478 1422 Citropsis species, N. crenulata and Pleiospermium laliatum, two main clades for the Australian and New Guinean species. 1479 1423 the two species evaluated in our study were found partially The first one included all full resistant species (M. warbugiana, 1480 1424 resistant. Even lacking data of the response of Triphasiinae M. australis, M. papuana and E. glauca) plus the partially resistant 1481 1425 subtribe to Las challenge inoculation, it seemed that the M. inodora while the species and hybrids tested for the second 1482

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1483 clade were all partially resistant (M. australasica, its hybrids with AUTHOR CONTRIBUTIONS 1540 1484 M. australis, M. virgata and with ‘Calamondin,’ C. × oliveri). 1541 1485 Considering the historical lack of interactions between HLB- LP and MA conceptualized and designed the work. MA and 1542 Q8 1486 associated pathogens/vectors and the citrus germplasm native to LR-J collected the data. MA, SL, LR-J, NW, EG, PO, and LP 1543 1487 /New Guinea, as they are still lacking in Australia and contributed to data analysis and interpretation, drafting the 1544 1488 were detected for the first time with limited spread in Papua/New article and critical revision of the article. All authors contributed 1545 1489 Guinea only in 2003 (EPPO Global Database, 2020), it may be to the article and approved the submitted version. 1546 1490 speculated that resistance to Las in Australian limes is actually 1547 1491 due to lack of functional susceptibility genes, likely derived from 1548 1492 commonly inactive genetic loci among them. 1549 FUNDING 1493 In conclusion, these results demonstrate that there is 1550 1494 consistent, complete and unequivocal resistance to Las, that 1551 This work was funded by Fundecitrus, grant No 817526 from the Q19 1495 is absence of bacterial multiplication, in a small group of 1552 European Union H2020 Innovation Action Program and project 1496 Citrus relatives including E. glauca as well as its hybrids with 1553 PID2019-104569RB-I00 from the AEI-Spain. 1497 Citrus and Microcitrus tested here, and also in some Microcitrus 1554 1498 species, which may be used directly to be assessed as possible 1555 1499 Citrus rootstocks/interstocks, to breed them with Citrus types 1556 1500 to generate new Citrus-like cultivars and to map specific ACKNOWLEDGMENTS 1557 1501 loci involved in the Las resistance (or lack of susceptibility) 1558 1502 phenotype/s. Further studies on the interaction of the Las- We thank André Luís Sanches and Miguel Antonio Mendes for 1559 1503 resistant vs. susceptible genotypes with D. citri and with Las at their excellent help with grafting for propagation, Las challenge 1560 1504 molecular level would also help in understanding host-pathogen- inoculations and plant maintenance, Lourdes Carmona and Elsa 1561 1505 vector interactions, identify effectors and metabolites prone Pons for helping with SSR marker analysis of True Citrus hybrids, 1562 1506 to genetic modulation in Citrus and therefore get full profit and Fernanda Benedito and Priscila Alves for assistance with 1563 1507 of Citrinae genetic resources to produce new citrus cultivars DNA extraction and qPCR analysis. We also thank to Embrapa 1564 1508 resistant to this ravaging bacterium. Cassava & Fruits for providing some genetic resources. 1565 1509 1566 1510 DATA AVAILABILITY STATEMENT 1567 1511 SUPPLEMENTARY MATERIAL 1568 1512 1569 Q7 The datasets presented in this study can be found in online 1513 1570 repositories. The names of the repository/repositories and The Supplementary Material for this article can be found Q10 1514 accession number(s) can be found in the article/Supplementary online at: https://www.frontiersin.org/articles/10.3389/fpls.2020. 1571 1515 Material. 617664/full#supplementary-material 1572 1516 1573 1517 1574 1518 1575 REFERENCES Bowman, K. D., Faulkner, L., and Kesinger, M. (2016). New citrus 1519 rootstocks released by USDA 2001-2010: field performance and nursery 1576 1520 Albrecht, U., and Bowman, K. D. (2012). Tolerance of trifoliate characteristics. HortScience 51, 1208–1214. doi: 10.21273/HORTSCI109 1577 1521 hybrids to Candidatus Liberibacter asiaticus. Sci. 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Batchelor, This is an open-access article distributed under the terms of the Creative Commons 1796 1740 (Berkeley and Los Angeles: Univ. Calif. Press), 191–430. Attribution License (CC BY). The use, distribution or reproduction in other forums 1797 Q21 1741 Swingle, W. (1967). The botany of Citrus and its wild relatives as a guide for their is permitted, provided the original author(s) and the copyright owner(s) are credited 1798 use in breeding. Florida State Hort. Soc. 156–164. and that the original publication in this journal is cited, in accordance with accepted Q22 1742 1799 Tatineni, S., Sagaram, U. S., Gowda, S., Robertson, C. J., Dawson, W. O., academic practice. No use, distribution or reproduction is permitted which does not 1743 Iwanami, T., et al. (2008). In planta distribution of ‘Candidatus Liberibacter comply with these terms. 1800 1744 1801 1745 1802 1746 1803 1747 1804 1748 1805 1749 1806 1750 1807 1751 1808 1752 1809 1753 1810 1754 1811 1755 1812 1756 1813 1757 1814 1758 1815 1759 1816 1760 1817 1761 1818 1762 1819 1763 1820 1764 1821 1765 1822 1766 1823 1767 1824

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