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Grafting a scion onto a rootstock results in physical and physiological changes in plant growth and development, which can affect tree vigor, productivity, and tolerance to stress and disease. Huanglongbing (HLB) is one of the most destructive citrus diseases and has become endemic in Florida since its introduction in 2005. It is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), which cause severe metabolic disruptions in affected plants. Although most scion cultivars are highly susceptible, some rootstock cultivars are tolerant and allow the grafted tree to cope better with the disease. The objectives of this study were to identify rootstock traits that can be used to assess cultivars under controlled greenhouse conditions in advance of longer-term field trials. We used 10 commercially important rootstocks with different genetic backgrounds and known field performance in graft combination with ‘Valencia’ sweet orange scion. Trees were graft-inoculated with CLas and compared against mock-inoculated trees. Tree health and CLas populations were assessed regularly, and root growth was monitored using a minirhizotron imaging system. Plants were excavated and destructively sampled 21 months after inoculation to assess biomass distributions and other CLas-induced effects. We found significant differences between healthy and infected trees for most variables measured, regardless of the rootstock. In contrast to leaf CLas titers, root titers were significantly influenced by the rootstock, and highest levels were measured for ‘Ridge’ sweet orange and sour orange. Root growth and root biomasses were reduced upon infection but differences among rootstocks did not always agree with reported field performances. Despite severe biomass reductions plants maintained their relative distribution of biomass among different components of the root system, and no dead roots were observed. Root respiration was reduced by CLas infection and was overall higher in tolerant cultivars suggesting its potential as a physiological marker. This study improves our knowledge about the strengths and weaknesses of assessing rootstock traits of grafted trees in a controlled greenhouse setting. Results from the study suggest that in addition to HLB tolerance, other rootstock traits will ultimately have major contributions to field survival and productivity of the grafted trees in an HLB endemic production environment.

Huanglongbing (HLB) is a devastating disease of citrus that is found in most citrus production areas around the world. The bacterium associated with HLB resides in and damages the phloem, restricting the movement of photosynthates throughout the plant and leading to tree decline. Considerable root loss can be observed in affected trees even when few disease symptoms are visible aboveground. Root traits can substantially influence tree performance and use of superior rootstocks is one strategy to manage tree health and reduce production losses in a disease-endemic environment. Citrus rootstocks are typically propagated by seed, but due to the increased demand for some of the best-performing cultivars, propagation by other methods is being used to overcome seed shortages. In this research, differences in root architecture and root growth of six different rootstocks propagated by seed, cuttings, and tissue culture, and their influence on the grafted ‘Valencia’ (Citrus sinensis) scion were investigated. A field trial was established in southwest Florida in 2017. Trees were evaluated for their performance during the first 2 years after planting and a subset of trees was excavated for detailed analysis of root architectures and biomass distribution. Significant differences among propagation methods were found for the rootstock trunk diameter and the lateral (structural) root length, which were largest in seed-propagated rootstocks. Most of the other horticultural and root architectural traits were not significantly influenced by the rootstock-propagation method; however, many of the measured variables were significantly influenced by the rootstock cultivar regardless of the propagation method. The results showed that rootstocks propagated by cuttings and tissue culture were similar to seed-propagated rootstocks in their influence on the grafted tree during the early years of growth in the field.

Modern citrus nursery production makes use of potted-tree propagation in greenhouses. Supplemental lighting is one method by which nursery tree growth and profitability may be significantly improved, but limited specific information is available. Five replicated experiments were conducted to determine the utility and effects of increasing daylength during the winter months by supplemental illumination from light-emitting diode (LED) or high-pressure sodium (HPS) lights in citrus nursery propagation. Studies used ‘Valencia’ sweet orange scion, the most common citrus cultivar grown in Florida, and the commercially important rootstocks sour orange, ‘Cleopatra’ mandarin, ‘US-812’, ‘US-897’, ‘US-942’, and ‘US-1516’. Comparisons used the three common types of citrus rootstock propagation: seed, stem cuttings, and micropropagation. Six responses were measured in the lighting experiments, including vegetative growth before budding, scion bud survival, and scion bud growth after budding. Supplemental HPS or LED light to extend daylength to 16 h in the citrus nursery during short-day winter months was observed to be effective in increasing unbudded rootstock liner growth and ‘Valencia’ scion growth on all rootstocks and propagation types. Generally, the positive effect on vegetative growth from an increased daylength was stronger with the HPS light than with LED light, while increasing daylength with LED light, but not HPS light, provided some increased bud growth initiation. Use of HPS or LED supplemental lighting to extend daylength offers significant growth advantage for the citrus nursery industry in winter.

Six new hybrid rootstocks, ‘US-1279’, ‘US-1281’, ‘US-1282’, ‘US-1283’, ‘US-1284’, and ‘US-1516’, were released from the U.S. Department of Agriculture (USDA) citrus breeding program to provide improved tree tolerance to huanglongbing (HLB), the most destructive disease facing the citrus industry in the United States and many other parts of the world. Five of these new rootstocks were released based on field performance in trials with the rootstocks propagated by stem cuttings, rather than the traditional propagation using nucellar seedlings. In this study, we evaluated the fruit, seed, and seedling characteristics of these new rootstocks, along with four other USDA rootstocks of commercial importance. The study included a determination of the percentage of true-to-type and off-type seedlings by both plant morphology and simple sequence repeat (SSR) markers. All 10 rootstocks produced an acceptable number of seeds and good seedling emergence from those seeds. The rootstocks ‘Swingle’, ‘US-802’, ‘US-812’, ‘US-1283’, ‘US-1284’, and ‘US-1516’ had a high percentage of true-to-type seedlings and correspondingly good potential to be propagated by seeds. However, no true-to-type plants were observed among seedlings from the rootstocks ‘US-1279’, ‘US-1281’, and ‘US-1282’, indicating that economical seed propagation will be impossible for these cultivars. The 10 SSR marker sets used in this study were observed to easily differentiate the 10 rootstocks studied, and readily distinguished true-to-type and off-type seedlings among progeny from all 10 rootstock clones. This study presents information of significant value for commercial nurseries involved in propagation of citrus rootstocks, and those involved in citrus rootstock breeding and development around the world. We propose the use of these 10 SSR marker sets as readily applicable for accurate identification of most citrus rootstock cultivars and their true-to-type seedlings.

Citrus crops have a long history of cultivation as grafted trees on selected rootstock cultivars, but all current rootstocks have significant limitations and traditional methods of rootstock breeding take at least 2–3 decades to develop and field test new rootstocks. Citrus production in the United States, and other parts of the world, is impaired by a wide range of biotic and abiotic problems, with especially severe damage caused by the disease huanglongbing (HLB) associated withCandidatusLiberibacter asiaticus. All major commercial citrus scion cultivars are damaged by HLB, but tree tolerance is significantly improved by some rootstocks. To overcome these challenges, the USDA citrus breeding program has implemented a multi-pronged strategy for rootstock breeding that expands the diversity of germplasm utilized in rootstock breeding, significantly increases the number of new hybrids evaluated concurrently, and greatly reduces the time from cross to potential cultivar release. We describe the key components and methodologies of this new strategy, termed “SuperSour,” along with reference to the historical favorite rootstock sour orange (Citrus aurantium), and previous methods employed in citrus rootstock breeding. Rootstock propagation by cuttings and tissue culture is one key to the new strategy, and by avoiding the need for nucellar seeds, eliminates the 6- to 15-year delay in testing while waiting for new hybrids to fruit. In addition, avoiding selection of parents and progeny based on nucellar polyembryony vastly expands the potential genepool for use in rootstock improvement. Fifteen new field trials with more than 350 new hybrid rootstocks have been established under the SuperSour strategy in the last 8 years. Detailed multi-year performance data from the trials will be used to identify superior rootstocks for commercial release, and to map important traits and develop molecular markers for the next generation of rootstock development. Results from two of these multi-year replicated field trials with sweet orange scion are presented to illustrate performance of 97 new hybrid rootstocks relative to four commercial rootstocks. Through the first 7 years in the field with endemic HLB, many of the new SuperSour hybrid rootstocks exhibit greatly superior fruit yield, yield efficiency, canopy health, and fruit quality, as compared with the standard rootstocks included in the trials.

In a grafted citrus tree, the leaf and root metabolic composition and the response to the phloem-limited pathogenic bacterium Candidatus Liberibacter asiaticus are influenced by the rootstock. Eleven different citrus rootstock varieties were investigated for their influence on the grafted tree response to Candidatus Liberibacter asiaticus (CLas), the pathogen associated with Huanglongbing (HLB). Rootstocks included mandarin and sweet orange cultivars, sour orange, and hybrids of citrus and trifoliate orange. Leaves and roots of healthy and CLas-infected greenhouse-grown plants were collected 6 months after graft inoculation and metabolite profiles were assessed by untargeted gas chromatography–time-of-flight-mass spectrometry (GC–TOF-MS). The genetic origin of the rootstock determined their metabolic profiles in the healthy state and influenced the root metabolic response to CLas. In the leaves, metabolic differences were determined by the tree response to CLas and to a lesser extent by the rootstock cultivar on which the plants were grafted. Among the leaf metabolites that responded most consistently to infection were citrulline, ornithine, and quinic acid, which were upregulated in infected plants. The reverse was found for cerotinic acid, cyanoalanine, hexadecane, and raffinose. In both leaves and roots, several metabolites associated with plant stress and disease tolerance such as benzoic acid and proline responded differently in plants on the mandarin × trifoliate hybrid rootstocks compared with the other rootstocks. This corresponds with the different HLB field tolerance of grafted trees on different rootstocks, which is generally higher in combination with some mandarin × trifoliate hybrids. Our results demonstrate the influence of rootstock on the grafted tree response to CLas and underscore the importance of rootstocks for disease management in an HLB-endemic environment.

The devastating citrus disease Huanglongbing (HLB), associated with bacterial pathogens of the genus Candidatus Liberibacter, has spread across many citrus production areas worldwide causing devastating economic losses. In Florida, infection rates in most commercial citrus orchards approach 100 percent by the time trees are 3–4 years old. Most scion cultivars are highly susceptible to Ca. Liberibacter asiaticus (CLas), the pathogen prevalent in Florida, but significant tolerance has been identified within the species Poncirus trifoliata (trifoliate orange) and some of its hybrids that are commonly used as rootstocks. In this study we investigated the relative influence of rootstock and scion in citrus plants composed of susceptible and tolerant cultivars in both the scion and the rootstock position on HLB disease progression and expression of stress and disease-related genes. Experiments were conducted in the greenhouse using the susceptible cultivars ‘Valencia’ orange (Citrus sinensis) and ‘Cleopatra’ mandarin (C. reticulata), and the tolerant trifoliate hybrid cultivars ‘US-802’ (C. grandis × P. trifoliata), ‘US-897’ (C. reticulata × P. trifoliata), and ‘US-942’ (C. reticulata × P. trifoliata). Plants were either mock-inoculated or graft-inoculated with CLas. The incidence of CLas infection at 6 and 12 months after inoculation and the severity of the disease was different among scion/rootstock combinations. Typically, trees with a tolerant cultivar in the scion position contained a lower number of bacteria and were less damaged by infection than trees with a susceptible cultivar in the scion position, regardless of rootstock. Previous observations of significant influence from rootstock on field tree tolerance to CLas may be partially the result of differential rootstock ability to tolerate other stresses, or respond favourably to therapeutic treatments, under the weakened condition caused by the infection. Differential expression of defense-related and other genes found largest fold-differences between non-infected and infected plants when a susceptible cultivar was in the scion position. Fewer genes responded in roots than in leaves, and genes associated with starch metabolism responded strongly and in an opposite direction when comparing roots and leaves, probably indicative of carbon depletion occurring in roots of infected plants.

Health, growth, fruit production, and fruit quality of citrus crops are severely affected by tree infection with Candidatus Liberibacter asiaticus (CLas) and subsequent development of the disease huanglongbing (HLB). The use of HLB-tolerant rootstocks is one strategy that is used to ameliorate the effects of HLB in commercial production. Although there is a clear long-term rootstock effect to improve tree performance, such field trials take many years for establishment and evaluation, and this long time-period is very limiting to expeditious evaluation of new rootstocks. In this study, we have conducted a 50-week greenhouse experiment to evaluate rootstock influences on Valencia sweet orange tree response to CLas infection. The infection of trees with CLas reduced scion and rootstock growth, increased leaf yellowing, and reduced the number of leaves per tree and leaf area, regardless of rootstock. There were clear rootstock influences on some traits during the 50-week study. In general, infected trees on US-942 rootstock had lower CLas root titers, less reduction of the number of leaves, less reduction of leaf area, and less leaf yellowing, as compared with some of the other rootstocks. The 50-week greenhouse evaluation method provided results that corresponded well with results from long-term field testing, indicating this may be a useful tool to accelerate evaluation and selection of new rootstocks, as well as in testing other HLB management strategies.

Summary Huanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium, Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB‐tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB‐tolerant citrus hybrid (Poncirus trifoliata × Citrus reticulata) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB‐tolerance. These regulators display similar expression patterns in another HLB‐tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid (Microcitrus virgata). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus‐induced gene silencing in N. benthamiana, this innovative and cost‐effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1‐Associated Protein, and the negative regulator Vascular Associated Death Protein.

This updated 4th edition of the Florida Citrus Rootstock Selection Guide (FLCRSG) is a revision of the 2016 publication. The guide is a convenient, easy-to-use reference to 21 characteristics of 49 rootstocks. Of those, 12 are time-honored commercial rootstocks (highlighted in blue), which are the most reliably characterized. The next 13 rootstocks are minor commercial ones (highlighted in green) that are less frequently used today in Florida but may have been prominent at one time. The third group consists of the most recently released 24 rootstocks (highlighted in yellow) for which there is limited commercial experience. The new addition includes three new USDA rootstocks and updates information on a few traits. Written by William S. Castle, Kim D. Bowman, Jude W. Grosser, Rhuanito Soranz Ferrarezi, Stephen H. Futch, and Steve Rogers, and published by the UF/IFAS Horticultural Sciences Department.https://edis.ifas.ufl.edu/hs1260

Rootstock variety influences leaf metabolic profiles in a grafted citrus tree, but influence also depends on the scion. Proper selection of rootstocks in tree fruit crops such as citrus is important for successful production. Despite a large number of commercially available rootstocks, studies have mostly been limited to basic horticultural observations. We used untargeted gas chromatography-time of flight-mass spectrometry (GC-TOF MS) based metabolomics to understand the biochemical influence of rootstock on 2-year-old field-grown ‘Cara Cara’ navel orange (Citrus sinensis L. Osbeck) and ‘Hirado Buntan’ pummelo (C. grandis L. Osbeck) trees grown on four rootstock cultivars with different genetic background. Five hundred unique metabolites were quantified in all trees, of which 147 were chemically identified. In navel orange trees, 48 root metabolites differed significantly in concentrations among rootstocks, compared with 29 metabolites in pummelo trees. In navel orange trees, raffinose, conduritol beta-epoxide, allantoic acid, myo-inositol, gamma-tocopherol, and beta gentiobiose were among the compounds that contributed most to this variation. In pummelo trees, hexitol, allantoic acid, glucoheptulose, tryptophan, gamma-tocopherol, glycerol-3-galactoside, and raffinose were among the most discriminating metabolites, but only allantoic acid passed significance criteria. Rootstock was also found to influence the quantities of 226 metabolites in leaves of the navel orange scion. Conduritol-beta-epoxide and myo-inositol were among the metabolites most influenced by rootstock. In contrast, the influence of rootstock on the pummelo scion was less prominent, with only six metabolites displaying significant differences. Our findings demonstrate that rootstock variety can influence the metabolic profile of the leaves in a grafted tree, but that the extent of the effect is also influenced by the scion. The majority of root metabolites that discriminated most between rootstocks did not display the same rootstock-specific discrimination in the leaves, suggesting tissue specificity or limited movement across the graft union.

There are generally inadequate supplies of seed for the newest rootstocks to satisfy the growing demand for the propagation material to be used in commercial citrus nurseries. Consequently, rootstock propagation, which is traditionally by seed, now often makes use of alternative methods such as cuttings and tissue culture (TC). Propagation through cuttings and TC will generate a root system that is largely composed of adventitious or lateral roots, compared with seed propagation, which will generally promote the formation of a well-defined taproot. In this study, we compared root architecture and growth of seven different rootstock plants, generated from seed, stem cuttings, or TC, during the early weeks of growth in the greenhouse. Based on total dry biomass, root mass fraction of plants generated from cuttings ranged from 11% to 16%, and from 16% to 29% and 21% to 30% for micropropagated plants and seedlings, respectively. Plants propagated through cuttings had the most primary roots (7–10), followed by tissue culture–propagated plants which developed an average of 2–6 primary roots. As expected, plants grown from seeds mostly developed a single and well-defined taproot during the first weeks. The total number of first order lateral roots was highest in the plants propagated as cuttings (108–185) compared with 53–103 and 43–78 for tissue culture–propagated plants and seedlings, respectively. Similarly, specific root length (SRL) was highest in plants derived from cuttings (21–43 m·g−1) and lowest in plants grown from seed (7–20 m·g−1). It is suggested that the larger number and length of roots on rootstock plants propagated through vegetative methods may be better suited for resource acquisition as compared with seed propagated plants.

A simple multicomponent system is described that is effective for rapid propagation of a diversity of citrus rootstock genotypes by single node cuttings, including new hybrids and cultivars that are most commonly used as rootstocks. Efficiency of this system for rooting shoot explants of six important rootstock cultivars, Swingle, Cleopatra, US-802, US-812, US-897, and US-942 is compared in a repeated study. Many of the cuttings began to grow within 2 weeks after planting into potting mix. Growth for the resultant plants of different genotypes was compared through 20 weeks, and significant differences were observed. US-802 had the highest success in establishing growing plants at 8 weeks, with 82–91% of single node cuttings successfully rooted and growing, while Cleopatra was the least efficient with a 42–45% recovery of growing plants. Comparison of plant weight for cuttings and seedlings indicated that 12 week old nucellar and true-to-type seedlings are similar in size to 12–16 week old cuttings of the same cultivars. Plants of all rootstocks, whether cuttings or seedling-propagated, were observed to approach a dry weight ratio of 80% shoot and 20% root. Pronounced differences in the number and length of roots were identified among the rootstocks, indicating large differences in root structure that might be important in relation to eventual field plant health and growth. The commercial utility of the described methods is discussed.

The objective of this experiment was to compare fruit-quality parameters of ‘Ray Ruby’ grapefruit grown on seven rootstocks. Four recent releases from the United States Department of Agriculture (USDA) rootstock breeding program, ‘US-852’, ‘US-897’, ‘US-942’, and ‘US-812’ (all Citrus reticulata × Poncirus trifoliata hybrids), ‘x639’ (C. reticulata × P. trifoliata), along with industry-standard ‘Sour Orange’ and ‘Swingle’ citrumelo were evaluated in a commercial orchard trial in Indian River County, FL. Fruit-quality data were collected in 2011–12 (eight harvests), 2012–13 (five harvests), and 2014 (single harvest). In each season, rootstock effects on fruit size, total solids, and solids acid ratio were significant. ‘Sour orange’ and ‘Swingle’ produced the largest fruit, whereas ‘US-897’ (a semidwarfing rootstock) produced the smallest fruit. Peel thickness (measured only in the 2011–12 season) was greatest in ‘Sour Orange’ early in the season, but not toward the end of the season. Misshapen (“sheep-nosed”) fruit occurred more frequently on ‘Sour Orange’ than on other rootstocks, although the incidence of sheep-nosing was minor. Analysis of variance (ANOVA) for fruit-quality data collected in January of each of the 3 years confirmed that ‘Sour Orange’ and ‘Swingle’ produced the largest fruit and ‘US-897’ produced the smallest fruit. Total solids were the highest in ‘US-897’ and the lowest in ‘x639’ and ‘US-852’. Taken together, our data indicate that ‘US-942’ and ‘US-897’ rootstocks produced fruit with quality characteristics that equaled or exceeded ‘Sour Orange’ and ‘Swingle’, the two most common rootstocks used in the Indian River district.

Four new citrus rootstocks developed by USDA, ARS and released between 2001 and 2010 have gained considerable commercial popularity in Florida and have been used for propagation of more than 2 million trees over the last 2 years. Results from three new field trials are presented comparing these rootstocks with other important rootstocks, and discussion is presented to summarize field performance in these trials and from numerous other sources. The rootstocks vary widely in their effect on tree vigor. When used as a rootstock for sweet orange, ‘US-802’ typically supports strong vigor and development of a large tree, ‘US-812’ and ‘US-942’ provide moderate vigor, and ‘US-897’ induces a relatively dwarf tree. Other characteristics and attributes of the four rootstocks are also discussed, including effects on cropping, fruit quality, disease and pest resistance, and tolerance of abiotic factors. Of special relevance, relative tolerance of these rootstocks to huanglongbing (HLB) disease can be surmised from a combination of data from several sources, with ‘US-942’ and ‘US-802’ typically providing the best yields per tree in trials containing many rootstocks and affected by HLB. ‘US-812’ and ‘US-897’ have appeared to have most utility in locations where HLB is not present or effectively managed, or where trees are being planted at high density to optimize production per unit area. To facilitate continuing expansion of commercial use, information on seed production, and the relative ease of nursery propagation is also presented.

Asian citrus psyllid (Diaphorina citri Kuwayama) transmits a bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) putatively responsible for a devastating citrus disease known as Asiatic huanglongbing (HLB) (citrus greening disease). The psyllid and disease have invaded many citrus-growing regions including the United States, where the disease is seriously jeopardizing the Florida citrus industry. We recently concluded research that showed CLas transmission rates are increased when citrus flush is present. Flush is any new leaf growth ranging in development from first emergence up until the leaves are fully expanded yet still tender. In an experiment with seedlings of a rootstock cultivar ‘US-942’, a 1-wk infestation of 20 Asian citrus psyllids from an infected colony resulted in 53–60% of seedlings becoming infected when flush was present compared with only 7% when no flush was present. In a second experiment with ‘US-942’, 77–97% of seedlings became infected when flush was present compared with 40% when no flush was present. A similar experiment with ‘Valencia’ sweet orange resulted in 23, 80, and 3% seedlings becoming infected when young, older, or no flush was present, respectively. Young plants are therefore more likely to contract HLB if flush is present, with older flush promoting higher infection rates under the conditions of this study. Based on this finding, healthy citrus should be protected from Asian citrus psyllid infestations throughout a flush. To evaluate germplasm for CLas resistance, inoculations using infected Asian citrus psyllid would best be achieved if flush is present.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Candidatus Liberibacter asiaticus (Las) is a phloem-limited bacterium associated with huanglongbing (HLB), one of the most destructive diseases of citrus in Florida and other citrus-producing countries. Natural transmission of Las occurs by the psyllid vector Diaphorina citri, but transmission can also occur through grafting with diseased budwood. As a result of the difficulty of maintaining Las in culture, screening of citrus germplasm for HLB resistance often relies on graft inoculation as the mode of pathogen transmission. This study evaluates transmission efficiencies and HLB progression in graft-inoculated and psyllid-inoculated citrus under greenhouse and natural conditions in the field. Frequencies of transmission in graft-inoculated greenhouse-grown plants varied between experiments and were as high as 90% in susceptible sweet orange plants 6 to 12 months after inoculation. Transmission frequency in a tolerant Citrus × Poncirus genotype (US-802) was 31% to 75%. In contrast, transmission of Las after controlled psyllid inoculation did not exceed 38% in any of four experiments in this study. Whereas the time from inoculation to detection of Las by polymerase chain reaction (PCR) was faster in psyllid-inoculated US-802 plants compared with graft-inoculated US-802 plants, it was similar in graft- and psyllid-inoculated sweet orange plants. HLB symptom expression was indistinguishable in graft- and psyllid-inoculated plants but was not always associated with the number of bacteria in affected leaves. The highest number of Las genomes per gram leaf tissue measured in sweet orange plants was one to four × 107 in graft-inoculated plants and one to two × 107 in psyllid-inoculated plants. Highest numbers measured in tolerant US-802 plants were one to three × 106 and two to six × 106, respectively. Compared with artificial inoculation in a greenhouse setting, natural inoculation of field-grown sweet orange trees occurred at a much slower pace, requiring more than 1 year for infection incidence to reach 50% and a minimum of 3 years to reach 100%.

Transgenic solutions are being widely explored to develop huanglongbing (HLB) resistance in citrus. A critical component of a transgenic construct is the promoter, which determines tissue specificity and level of target gene expression. This study compares the characteristics of five promoters regulating the beta-glucuronidase (GUS) reporter gene in the trifoliate hybrid rootstock US-802. Two of the selected promoters direct high levels of constitutive transgene expression in other dicotyledonous plants: 2X35S, the tandem-repeat promoter of the cauliflower mosaic virus 35S gene and bul409S, a truncation of the potato polyubiquitin promoter. Because Candidatus Liberibacter, the Gram-negative bacterium associated with HLB, infects only the phloem tissue, it may be advantageous to limit transgene expression to the vascular tissue and reduce expression in the fruit. Thus, we also tested three promoters that demonstrate phloem specificity when transformed and expressed in other plants: WDV, from wheat dwarf geminivirus; AtSUC2, the sucrose-H+ symporter gene promoter from Arabidopsis; and CsSUS, the sucrose synthase promoter from citrus. Histochemical staining for GUS activity was observed throughout leaf and stem tissues for the constitutive promoters, while the three phloem-specific promoters largely showed the expected tissue-specific staining. Expression of GUS in some individual transformants with promoters CsSUS and WDV appeared leaky, with some laminar tissue staining. Relative quantification of qRT-PCR data revealed a wide range of mRNA abundance from transgenics with each of the five promoters. Fluorometry also revealed that GUS activity differed depending on the promoter used, but mRNA levels and enzyme activity were not highly correlated.

Huanglongbing (HLB) is one of the most destructive bacterial diseases of citrus. No resistant cultivars have been identified, although tolerance has been observed in the genus Poncirus and some of its hybrids with Citrus that are commonly used as rootstocks. In this study we exploited this tolerance by comparing five different tolerant hybrids with a cultivar that shows pronounced HLB sensitivity to discern potential contributing metabolic factors. Whole leaves of infected and non-infected greenhouse-grown seedlings were extracted and subjected to untargeted GC-TOF MS based metabolomics. After BinBase data filtering, 342 (experiment 1) and 650 (experiment 2) unique metabolites were quantified, of which 122 and 195, respectively, were assigned by chemical structures. The number of metabolites found to be differently regulated in the infected state compared with the non-infected state varied between the cultivars and was largest (166) in the susceptible cultivar Cleopatra mandarin (Citrus reticulata) and lowest (3) in the tolerant cultivars US-897 (C. reticulata ‘Cleopatra’ × Poncirus trifoliata) and US-942 (C. reticulata ‘Sunki’ × P. trifoliata) from experiment 2. Tolerance to HLB did not appear to be associated with accumulation of higher amounts of protective metabolites in response to infection. Many metabolites were found in higher concentrations in the tolerant cultivars compared with susceptible Cleopatra mandarin and may play important roles in conferring tolerance to HLB. Lower availability of specific sugars necessary for survival of the pathogen may also be a contributing factor in the decreased disease severity observed for these cultivars.

a b s t r a c t Candidatus Liberibacter asiaticus (Las) is the suspected causal agent of Huanglongbing (HLB) in Florida and other citrus producing countries around the world. Although little resistance to HLB is found within commercial citrus varieties, tolerance has been reported for some varieties that are commonly used as rootstocks. This study assesses the response of eight different rootstock varieties, which include the Citrus × Poncirus trifoliata hybrids Carrizo citrange, US-802, US-812, US-897, and US-942, Benecke trifoliate orange, Volkamer lemon, and Cleopatra mandarin, to Las under controlled conditions in the greenhouse. The percentage of plants detected positive for Las did not exceed 7% in the genotypes Benecke, Carrizo, US-802, US-942, and Volkamer during the first 6 months after graft-inoculation (mai). Pruning resulted in much increased numbers of plants with detectable Las levels and induced disease symptoms in previously asymptomatic plants. At 12 mai, percentages of Las-positive plants were 91–96% for US-802, US-812, Cleopatra, and Volkamer, and 70–78% for US-897 and US-942. Lowest percentages (44–52%) were observed for Benecke and Carrizo. The number of Las genomes per g of leaf tissue in Las-positive plants was considerably lower in Benecke and US-897 seedlings with 4.5 × 105 and 1.6 × 106 compared with

Huanglongbing (HLB), a bacterial disease of citrus, is causing substantial economic losses to the citrus industry worldwide. Sweet oranges are highly susceptible to the disease, and account for nearly 90% of all varieties grown in Florida. Rootstock is an important component of commercial citrus production, and tolerance to HLB has been reported for some rootstock varieties. Our objective was to investigate if rootstock selection has an effect on HLB disease development under natural conditions in the field. Four field trials with sweet orange scion on 15 different rootstocks were evaluated for incidence of Candidatus Liberibacter asiaticus (Las), the suspected causal agent of HLB, as well as incidence of foliar disease symptoms, canopy damage, and stem growth during the first years after Las began to spread into the trials. Trials ranged from two to nine years in age and included hybrids of trifoliate orange along with other rootstocks standard for citrus production in Florida. Fruit yield and soluble solids content were analyzed for the oldest trial. Our study showed that rootstock did not affect disease incidence and that trees on all rootstocks were considerably damaged by HLB. However, tolerance to HLB was higher in trees grafted on some rootstock selections. In the youngest trial, stem diameters on Volkamer lemon increased 53% while trees on US-852, Benton citrange and Swingle citrumelo grew the least at 21–26% from 2008 to 2010. Depending on trial and time of observation, foliar HLB symptoms on US-897 were less than on many other rootstocks, with ranks of 3.3–4.0 compared with 4.5–4.9 observed for US-812, US-852, Carrizo citrange and Kinkoji. Canopy damage ranks were 1.4–2.6 for trees on US-802 compared with 3.9–4.1 for trees on US-812, U-852, Sour orange, and Swingle in the oldest trial. In the youngest trials, trees on Volkamer showed least canopy damage while trees on US-852 and Benton were more affected. Highest fruit yields of 30–64 kg/tree were obtained from trees on US-802 and Carrizo. In 2010, highest juice soluble solids content of 2.8–2.9 kg was observed for trees on US-897 and US-812. Published by Elsevier B.V.

Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been observed in the genus Poncirus and some of its hybrids. This study compares transcriptional changes in tolerant US-897 (Citrus reticulata Blanco×Poncirus trifoliata L. Raf.) and susceptible 'Cleopatra' mandarin (C. reticulata) seedlings in response to infection with Las using the Affymetrix GeneChip citrus array, with the main objective of identifying genes associated with tolerance to HLB. Microarray analysis identified 326 genes which were significantly upregulated by at least 4-fold in the susceptible genotype, compared with only 17 genes in US-897. Exclusively upregulated in US-897 was a gene for a 2-oxoglutarate (2OG) and Fe(II)-dependant oxygenase, an important enzyme involved in the biosynthesis of plant secondary metabolites. More than eight hundred genes were expressed at much higher levels in US-897 independent of infection with Las. Among these, genes for a constitutive disease resistance protein (CDR1) were notable. The possible involvement of these and other detected genes in tolerance to HLB and their possible use for biotechnology are discussed.