Improving citrus bud grafting efficiency.

Commercial citrus trees are composed of a scion grafted onto a rootstock. Because grafting is one of the most expensive methods of plant propagation, grafting efficiency is of large practical importance. The purpose of this study was to improve citrus bud-grafting efficiency. The effects of six factors that included BA, Tween-20, DMSO, type of solvent (water or EtOH), cardinal orientation of grafted bud, and type of supplemental light (LED, metal halide, none) on forty-four bud-grafting measures were determined using a multifactor design of experiment approach. Four measures useful for identifying treatments of practical value included the number of rootstock axial buds that formed shoots, the percentage of grafted buds that formed shoots, the length of the longest shoot formed from the grafted buds, and the total leaf area of the grafted bud shoots. The factors that most affected these responses were no supplemental light to minimize the number of shoots from rootstock axial buds, a south orientation and 5 mM BA to maximize the percentage of grafted buds that formed shoots, a north orientation and 5 mM BA to maximize the length of the longest grafted bud shoot, and 5 mM BA to maximize the leaf area of the grafted bud shoots.

Genetic modulation of Valencia sweet orange field performance by 50 rootstocks under huanglongbing-endemic conditions.

Although the citrus scion cultivar primarily determines the characteristics of the fruit, the rootstock cultivar of the graft combination has a major role in determining the horticultural performance of the tree. The disease huanglongbing (HLB) is particularly devastating to citrus, and the rootstock has been demonstrated to modulate tree tolerance. However, no existing rootstock is entirely suitable in the HLB-endemic environment, and citrus rootstocks are particularly challenging to breed because of a long life cycle and several biological characteristics that interfere with breeding and commercial use. This study with Valencia sweet orange scion documents the multi-season performance of 50 new hybrid rootstocks and commercial standards in one trial that forms the first wave of a new breeding strategy, with the aim of identifying superior rootstocks for commercial use now, and mapping important traits to be used in selection for the next generation of outstanding rootstocks. A large assortment of traits were quantified for all trees in the study, including traits associated with tree size, health, cropping, and fruit quality. Among the quantitative traits compared between rootstock clones, all except one were observed to have significant rootstock influence. Multiple progeny from eight different parental combinations were included in the trial study, and significant differences between parental combinations of the rootstocks were observed for 27 of the 32 traits compared. Pedigree information was integrated with quantitative trait measurements to dissect the genetic components of rootstock-mediated tree performance. Results suggest there is a significant genetic component underlying rootstock-mediated tolerance to HLB and other critical traits, and that integration of pedigree-based genetic information with quantitative phenotypic data from trials should enable marker-based breeding approaches for the rapid selection of next-generation rootstocks with superior combinations of traits that are needed for commercial success. The current generation of new rootstocks included in this trial is a step toward this goal. Based on results from this trial, the new hybrids US-1649, US-1688, US-1709, and US-2338 were considered the four most promising new rootstocks. Release of these rootstocks for commercial use is being considered, pending the evaluation of continuing performance in this trial and the results from other trials.

Steam Explosion (STEX) of Citrus × Poncirus Hybrids with Exceptional Tolerance to Candidatus Liberibacter Asiaticus (CLas) as Useful Sources of Volatiles and Other Commercial Products.

Florida citrus production has declined 75% due to Huanglongbing (HLB), a disease caused by the pathogenic bacterium Candidatus Liberibacter asiaticus (CLas). Methods to combat CLas are costly and only partially effective. The cross-compatible species Poncirus trifoliata and some of its hybrids are known to be highly tolerant to CLas, and thus can potentially serve as an alternative feedstock for many citrus products. To further investigate the commercial potential of citrus hybrids, three citrus hybrids, US-802, US-897, and US-942, were studied for their potential as feedstocks for citrus co-products using steam explosion (STEX) followed by water extraction. Up to 93% of sugars were recovered. US-897 and US-942 have similar volatile profiles to that of the commercial citrus fruit types and as much as 85% of these volatiles could be recovered. Approximately 80% of the pectic hydrocolloids present in all three hybrids could be obtained in water washes of STEX material. Of the phenolics identified, the flavanone glycosides, i.e., naringin, neohesperidin, and poncirin were the most abundant quantitatively in these hybrids. The ability to extract a large percentage of these compounds, along with their inherent values, make US-802, US-897, and US-942 potentially viable feedstock sources for citrus co-products in the current HLB-blighted environment.

SuperSour: A New Strategy for Breeding Superior Citrus Rootstocks.

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 with Candidatus Liberibacter 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.

Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system.

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.

Epifluorescence and Stereomicroscopy of Trichomes Associated with Resistant and Susceptible Host Plant Genotypes of the Asian Citrus Psyllid (Hemiptera: Liviidae), Vector of Citrus Greening Disease Bacterium.

BACKGROUND AND SIGNIFICANCE: Foliar trichomes (tiny hair-like structures) are part of the plant defense mechanisms that may confer resistance to some herbivore pests. Trifoliate orange, Poncirus trifoliata, is a genotype resistant to infestations by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), vector of the economically important citrus greening (huanglongbing) disease bacterium. We discovered that dense trichomes are associated with young leaves of trifoliate orange plants and hypothesized that these might be responsible for reduced infestation by this psyllid. MATERIALS AND METHODS: Epifluorescence and stereomicroscopy were used to study the density and structure of trichomes associated with young flush leaves and stems of trifoliate orange and of five other plant genotypes that are highly susceptible to colonization by the psyllid: lemon, grapefruit, sweet orange, curry leaf, and orange jasmine. RESULTS: Simple unicellular trichomes were observed at moderate-to-large densities on young leaves and stems of each genotype except lemon and sweet orange, which had considerably fewer trichomes. Trichomes were generally abundant on young leaves of curry leaf and orange jasmine, two genotypes that are often heavily colonized by the psyllid. Although we did not quantify oviposition rates on these genotypes, we observed that psyllid females deposited eggs on young leaves, buds, and stems regardless of the density of trichomes present, sometimes directly within or close to a dense bed of trichomes. CONCLUSIONS: While trichomes were moderately abundant on young leaves of trifoliate orange, our results strongly suggest that these trichomes may play little or no role in reduced colonization by the psyllid on this genotype.

Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids.

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.

Metabolic variations in different citrus rootstock cultivars associated with different responses to Huanglongbing.

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.

Transmission Rates of 'Ca. Liberibacter asiaticus' by Asian Citrus Psyllid Are Enhanced by the Presence and Developmental Stage of Citrus Flush.

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 53­60% of seedlings becoming infected when flush was present compared with only 7% when no flush was present. In a second experiment with 'US-942', 77­97% 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.

Gene regulatory networks elucidating huanglongbing disease mechanisms.

Next-generation sequencing was exploited to gain deeper insight into the response to infection by Candidatus liberibacter asiaticus (CaLas), especially the immune disregulation and metabolic dysfunction caused by source-sink disruption. Previous fruit transcriptome data were compared with additional RNA-Seq data in three tissues: immature fruit, and young and mature leaves. Four categories of orchard trees were studied: symptomatic, asymptomatic, apparently healthy, and healthy. Principal component analysis found distinct expression patterns between immature and mature fruits and leaf samples for all four categories of trees. A predicted protein - protein interaction network identified HLB-regulated genes for sugar transporters playing key roles in the overall plant responses. Gene set and pathway enrichment analyses highlight the role of sucrose and starch metabolism in disease symptom development in all tissues. HLB-regulated genes (glucose-phosphate-transporter, invertase, starch-related genes) would likely determine the source-sink relationship disruption. In infected leaves, transcriptomic changes were observed for light reactions genes (downregulation), sucrose metabolism (upregulation), and starch biosynthesis (upregulation). In parallel, symptomatic fruits over-expressed genes involved in photosynthesis, sucrose and raffinose metabolism, and downregulated starch biosynthesis. We visualized gene networks between tissues inducing a source-sink shift. CaLas alters the hormone crosstalk, resulting in weak and ineffective tissue-specific plant immune responses necessary for bacterial clearance. Accordingly, expression of WRKYs (including WRKY70) was higher in fruits than in leaves. Systemic acquired responses were inadequately activated in young leaves, generally considered the sites where most new infections occur.

Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis.

Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented susceptibility conferred by the Arabidopsis ndr1-1 mutant to Pseudomonas syringae strains and also led to enhanced resistance to an oomycete pathogen Hyaloperonospora arabidopsidis. Such heightened resistance is associated with increased SA production and expression of the defense marker gene PATHOGENESIS RELATED 1 (PR1). In addition, we found that expression of PR1 and accumulation of SA were induced to modest levels in citrus infected with Candidatus Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that CsNDR1 is a functional ortholog of Arabidopsis NDR1. Since Ca. L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production.

Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus huanglongbing disease.

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

BACKGROUND: Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. METHODOLOGY/PRINCIPAL FINDINGS: Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. SIGNIFICANCE: The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

Transcriptome profiling of citrus fruit response to huanglongbing disease.

Huanglongbing (HLB) or "citrus greening" is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production.

Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus.

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.

Is there meaningful plant resistance to diaprepes abbreviatus (Coleoptera: Curculionidae) in citrus rootstock germplasm?

Host plant resistance to the Diaprepes root weevil, Diaprepes abbreviatus (L.) was assessed for seedlings of 54 Poncirus trifoliata (L.) Raf. selections and two families of progeny from crosses between Citrus and P. trifoliata. Weight gain was consistently lower when larvae were reared in pots containing the progeny of Citrus reticulata Blanco 'Sunki' x P. trifoliata 'Flying Dragon' compared with larvae reared on progeny of 'Pearl' (C. reticulata x C. paradisi Macf.) x 'Flying Dragon'. This is the first evidence of genetic control of resistance to the Diaprepes root weevil within sexually compatible citrus rootstock germplasm. There was a significant positive correlation between percentage root loss and larval weight gain within the resistant progeny, indicating a possible antixenotic effect. Two varieties of P. trifoliata were identified as more resistant than 'Flying Dragon' based on larval weight gain.