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Grapefruit [Citrus ×aurantium (synonym C. ×paradisi)] is an important citrus commodity that originated in Barbados in the 17th century. Grapefruit is the youngest member of the genus Citrus. Most commercially important grapefruit cultivars arose through natural and induced mutations, not traditional breeding, of the white-fleshed and seedy Duncan grapefruit. Now, cultivars with a range of flesh colors exist; the pigmentation is correlated with lycopene content. A bud sport mutant of grapefruit discovered in Texas has a deep golden-colored flesh, significantly different from the typical reddish pigmentation. In this review, we discuss grapefruit’s journey from its origin in Barbados and its global establishment including production, marketing, drug interactions, cultivar development, genetic diversity, and commercially significant cultivars.

Asiatic citrus canker (ACC) foliar lesions were evaluated on progenies of 84 seed-source genotypes (“parent genotypes”) from the Citrus Variety Collection (CVC) of the University of California at Riverside (UCR) of Citrus trifoliata and hybrids between C. trifoliata and other Citrus species and hybrids. Trees were planted Aug. 2013 in a completely randomized design at the Fort Pierce U.S. Department of Agriculture (USDA) grove. Plants were assessed visually Aug. 2017, Sept. 2019, and Sept. 2020 for distinctive ACC lesion incidence and severity. Progeny were compared by parent genotypes using nonparametric analysis. Incidence of ACC [percentage of leaves displaying symptoms, verified by quantitative polymerase chain reaction (qPCR) to be associated with Xanthomonas citri pv. citri] across parent genotypes ranged from 8% to 80% (mean, 49%) of leaves affected in 2017, from 4% to 58% (mean, 29%) in 2019, and 8% to 46% (mean, 25%) in 2020. In 2017, of 49 C. trifoliata parent genotypes, only four separated from the two highest ACC-incidence statistical categories [Citrus Research Center (CRC) 3345, 3484, 3888, and 4017]. whereas 29 of the 35 C. trifoliata hybrids displayed lower ACC incidence, which separated from the two highest statistical categories. In 2019, of the C. trifoliata, only six separated from the highest ACC-incidence statistical category (CRC 3330, 3484, 3547, 3549, 3876, and 3888), whereas all 35 C. trifoliata hybrids displayed lower ACC incidence and separated from the highest statistical category, and 26 hybrids separated from 18 of the C. trifoliata. In 2020, only three C. trifoliata separated from the highest ACC-incidence statistical category (CRC 2861, 3549, and 3888) and 20 hybrids separated from 18 of the C. trifoliata. By parent genotype, ACC incidence correlated substantially between each pair of the 3 years, with r2 values of 0.39, 0.57, and 0.65. Of 34 hybrids validated, similar numbers had C. trifoliata, grapefruit (C. ×aurantium var. racemosa), and sweet orange (C. ×aurantium var. sinensis) chloroplasts. Chloroplast type affected ACC incidence and severity, but not in a consistent manner. Near-isogenic groups within C. trifoliata, as determined by DNA markers, were associated with some statistically different ACC sensitivity. Overall, hybrids of C. trifoliata with other citrus types displayed markedly reduced ACC sensitivity compared with C. trifoliata, indicating that this trait is readily overcome through breeding.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses inCitrusand wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in two subtribes, the Balsamocitrinae and Clauseninae, can recognize all tested microbial features (flg22, csp22, chitin), including one fromCandidatusLiberibacter species (csp22CLas), the bacterium associated with citrus greening disease aka Huanglongbing. We investigated differences at the receptor level for flagellin perception (FLS2 receptor) and chitin perception (LYK5 receptor) in citrus genotypes. We were able to characterize two genetically linkedFLS2homologs from ‘Frost Lisbon’ lemon (responsive) and ‘Washington navel’ orange (non-responsive). Surprisingly,FLS2homologs from responding and non-responding genotypes were expressed in citrus and functional when transferred to a heterologous system. ‘Washington navel’ orange weakly responds to chitin, but ‘Tango’ mandarin exhibits a robust response. LYK5 alleles were identical or nearly-identical between the two genotypes and able to complement theArabidopsis lyk4/lyk5-2mutant with respect to chitin perception. Collectively, our data indicates that differences in chitin and flg22 perception in these citrus genotypes are not the result of sequence polymorphisms at the receptor level. These findings shed light onto the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Citrus huanglongbing (HLB) disease associated with the ‘Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.

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.

An insect trap constructed using three-dimensional (3D) printing technology was tested in potato (Solanum tuberosum Linnaeus; Solanaceae) fields to determine whether it could substitute for the standard yellow sticky card used to monitor Bactericera cockerelli (Šulc) (Hemiptera: Psylloidea: Triozidae). Sticky cards have shortcomings that prompted search for a replacement: cards are messy, require weekly replacement, are expensive to purchase, and accumulate large numbers of nontarget insects. Bactericera cockerelli on sticky cards also deteriorate enough that specimens cannot be tested reliably for the presence of vectored plant pathogens. A prototype trap constructed using 3D printing technology for monitoring Diaphorina citri Kuwayama (Hemiptera: Psylloidea: Liviidae) was tested for monitoring B. cockerelli. The trap was designed to attract B. cockerelli visually to the trap and then funnel specimens into preservative-filled vials at the trap bottom. Prototype traps were paired against yellow sticky cards at multiple fields to compare the captures of B. cockerelli between cards and traps. The prototype trap was competitive with sticky cards early in the growing season when B. cockerelli numbers were low. We estimated that two or three prototype traps would collect as many B. cockerelli as one sticky card under these conditions. Efficacy of the prototype declined as B. cockerelli numbers increased seasonally. The prototype trap accumulated nontarget taxa that are common on sticky cards (especially Thysanoptera and Diptera), and was also found to capture taxa of possible interest in integrated pest management research, including predatory insects, parasitic Hymenoptera, and winged Aphididae (Hemiptera), suggesting that the traps could be useful outside of the purpose targeted here. We believe that 3D printing technology has substantial promise for developing monitoring tools that exploit behavioural traits of the targeted insect. Ongoing work includes the use of this technology to modify the prototype, with a focus on making it more effective at capturing psyllids and less susceptible to capture of nontarget species.

Maintenance of virus-free citrus in nurseries and orchards is essential to control the spread of aphid-transmitted Citrus tristeza virus (CTV). To this end, a lateral flow immunoassay (LFIA) was developed to detect CTV that was user-friendly and field-deployable. Polyclonal antisera was generated in rabbit against purified virions from Nicotiana benthamiana inoculated with an infectious recombinant cDNA clone of CTV (rCTV). Affinity purified rCTV IgG (1 mg/ml) and Mouse IgG (0.5 mg/ml) were conjugated with gold nanoparticles and coated onto a glass fiber membrane conjugate pad. Affinity purified rCTV IgG and anti-mouse IgG were used as test line and control line, respectively on the test strip. The LFIA detected CTV within 10 min and was sensitive up to a 1:80 dilution of crude plant sap extract. The LFIA was validated using CTV strains T30, T36, VT and RB from greenhouse and field sources and results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. The immunostrip readily detected CTV after six months in storage in a sealed bag with silica gel at room temperature indicating good stability. The LFIA test strips offered a cost-effective tool to detect CTV in the field or nursery by non-skilled personnel without laboratory equipment.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied, thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host&ndash, virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

In budwood certification programs, routinely used, technology-based laboratory methods for detecting known citrus pathogens need to be complemented with biological indexing to confirm the absence of known and unknown graft transmissible pathogens (GTPs). We report here a procedure for biological indexing using very young indicator plants, 75–90 days old. Assay results of traditional biological indexing and mini-plant indexing conducted for 51 quarantine accessions and 29 positives infected with known pathogens were compared. The evaluation indicated similar sensitivity of detection between the two methods. Traditional bioindexing requires premium greenhouse resources and a lengthy incubation period; the use of mini-plants for indexing can provide a viable alternative since the technique can be conducted with a fraction of the resources typically needed. In southern California, we were able to index for GTPs requiring cooler temperatures during summer by placing mini-bioindicator plants near the cooling pads in an evaporatively-cooled greenhouse. Indicator plants kept under favorable conditions expressed symptoms throughout the year. Limited greenhouse space is required due to the small size of the indicator plants, and they can be used for biological indexing within 3 months as compared to 9–12 months with traditional indicator plants. Young plants require much less space with 240 plants/m2 of bench space as compared to 30 plants/m2 using the conventional approach for biological indexing. The use of mini-plant biological indexing will enable citrus ‘clean stock’ and certification programs to function more efficiently with limited resources and facilities.

In a Fort Pierce, FL, field planting, plant growth, and Huanglongbing (HLB) severity were assessed as indicators of HLB tolerance on progenies of 83 seed-source accessions of Citrus and Citrus relatives mainly from the Riverside, CA, genebank. The HLB-associated pathogen [Candidatus Liberibacter asiaticus (CLas)] and vector [asian citrus psyllid (ACP), Diaphorina citri] were abundant, and trees were naturally challenged for 6 years before metrics (leaf mottle, percent canopy mottle, overall health, canopy density, canopy width, canopy height, and trunk diameter) were collected in Oct. and Nov. 2015. The healthiest trees with low or no HLB symptoms were distant citrus relatives: Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, and one accession of Severinia buxifolia. Within Citrus, most of the healthiest trees with densest canopies, little leaf loss, and greater growth were those with pedigrees that included Citrus medica (citron). These included progenies of Citrus hybrid (‘Limon Real’), Citrus limetta, Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in this category exhibited distinct leaf-mottle characteristic of HLB and substantial pathogen titers, but maintained dense canopies and exhibited good growth. Trees from seed-source accessions in the genus Citrus without citron in their background were generally among the least healthy overall with less dense canopies. The exceptions were progenies of two Citrus aurantium accessions, which were markedly healthier than progenies of other Citrus seed-source accessions not derived from citron. Linear regression analysis, between metrics collected and pedigree of seed parent, indicated that percentage of citron in the pedigree significantly correlated with measures of tolerance. Although no commercial Citrus genotypes yielded progenies with strong HLB resistance, in this field experiment several progenies maintained dense canopies and good growth, and may be useful for breeding HLB tolerant cultivars.

Citrus huanglongbing (HLB) is a destructive disease with no known cure. To identify sources of HLB resistance in the subfamily Aurantioideae to which citrus belongs, we conducted a six-year field trial under natural disease challenge conditions in an HLB endemic region. The study included 65 Citrus accessions and 33 accessions belonging to 20 other closely related genera. For each accession, eight seedling trees were evaluated. Based on quantitative polymerase chain reaction analysis of the pathogen titers and disease symptoms, eight disease-response categories were identified. We report two immune, six resistant, and 14 tolerant accessions. Resistance and tolerance observed in different accessions may be attributed to a multitude of factors, including psyllid colonization ability, absence of pathogen multiplication, transient replication of the bacterium, lack of pathogen establishment in the plant, delayed infection, or recovery from infection. Most citrus cultivars were considered susceptible: 15 citrons, lemons, and limes retained leaves in spite of the disease status. Resistance and high levels of field tolerance were observed in many noncitrus genera. Disease resistance/tolerance was observed in Australian citrus relative genera Eremocitrus and Microcitrus, which are sexually compatible with citrus and may be useful in future breeding trials to impart HLB resistance to cultivated citrus.

International audience; In citrus, the production of anthocyanin pigments requires the activity of the transcriptional activator Ruby. Consequently, loss-of-function mutations in Ruby result in an anthocyaninless phenotype [1]. Several citrus accessions, however, have lost the ability to produce these pigments despite the presence of wild-type Ruby alleles. These specific mutants have captivated the interest of botanists and breeders for centuries because the lack of anthocyanins in young leaves and flowers is also associated with a lack of proanthocyanidins in seeds and, most notably, with an extreme reduction in fruit acidity (involving about a three-unit change in pH). These mutants have been defined collectively as "acidless" [24]. We have identified Noemi, which encodes a basic helix-loop-helix (bHLH) transcription factor and which controls these apparently unrelated processes. In accessions of Citron, limetta, sweet lime, lemon, and sweet orange, acidless phenotypes are associated with large deletions or insertions of retro- transposons in the Noemi gene. In two accessions of limetta, a change in the core promoter region of Noemi is associated with reduced expression and increased pH of juice, indicating that Noemi is a major determinant of fruit acidity. The characterization of the Noemi locus in a number of varieties of Citron indicates that one specific mutation is ancient. The presence of this allele in Chinese fingered Citrons and in those used in the Sukkot Jewish ritual [5] illuminates the path of domestication of Citron, the first citrus species to be cultivated in the Mediterranean. This allele has been inherited in Citron-derived hybrids with long histories of cultivation.

Potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a key pest of potato (Solanum tuberosum L.) and other solanaceous crops (Solanales: Solanaceae) as a vector of “Candidatus Liberibacter solanacearum” (Lso), the pathogen associated with zebra chip disease of potato. Potato psyllid populations typically are monitored using sticky card traps, and psyllids collected from sticky traps often are subjected to polymerase chain reaction (PCR) to monitor the incidence of Lso within psyllid populations. Psyllids collected from sticky traps are often mangled, desiccated, and coated with sticky residue, which may interfere with detection of Lso by PCR. A recently developed prototype 3-dimensional-printed trap that captures insects directly into a preservative (70% ethanol) was previously tested for monitoring psyllid populations. The capture of psyllids directly into a preservative may reduce degradation of DNA or protect specimens from PCR-inhibiting contaminants, thus improving the detection of Lso by PCR. Our goal was to compare the detectability of Lso in psyllids captured into preservative (prototype trap) to that in psyllids removed from sticky card traps. Overall, detection rates were higher in psyllids from the prototype trap than from sticky card traps. This improvement in Lso detection appeared to be partly due to the specimens yielding more DNA of higher quality. Results of this study demonstrate that compared with sticky card traps, a trap that captures psyllids directly into a preservative provides higher quality specimens for collection of molecular data, including pathogen diagnosis, population genetics, and molecular gut content analysis.

The phylogeny of Rutaceae subfamily Aurantioideae has previously been estimated only using plastid and repetitive nuclear sequences. We added sequences of two low copy nuclear loci to allow further diagnosis of phenomena that may mislead phylogenetic inference. After testing for patterns expected under recombination, positive selection, and hybridization, we excluded data sets or sequences accordingly and then inferred the species tree using the multispecies coalescent. We then reconstructed the ancestral area using parsimony and the dispersal-extinction-cladogenesis model to test the hypothesis that Citrus s. l. may have originated in Australasia and migrated or rafted to Eastern Asia. The ancestral area of Citrus s. l. inferred under either method and several models was west of Wallace's line. Therefore, Citrus s. l. did not appear to have rafted west on what became the Halmahera Islands (Indonesia). Our findings are also consistent with previously reported ages for the origin of this group tha...

We report the detection of the huanglongbing (HLB)-associated bacterium ‘Candidatus Liberibacter asiaticus’ from both plants and insects in Pakistan and the seasonal variability in the numbers of ‘Ca. L. asiaticus’-positive psyllid vector, Diaphorina citri. Our studies showed that ‘Ca. L. asiaticus’ was detectable from trees in areas with maximum temperatures reaching nearly 50°C (average maximum of 42°C). However, the bacterium was present at very low levels in psyllids both in summer (June to August) and autumn (September to November) in contrast to reports from Florida, where the bacterium was detectable at very high levels during October to November. We hypothesize that hot summer temperatures in Pakistan may interfere with acquisition and replication of ‘Ca. L. asiaticus’ in psyllids and may lead to dead or nontransmissible ‘Ca. L. asiaticus’ in plants. Psyllid counts were very low in both summer and winter, showed a population peak (‘Ca. L. asiaticus’-positive vectors) in spring, and showed a larger peak (‘Ca. L. asiaticus’-free psyllids) in autumn. Natural thermotherapy during hot summers and a low vector population during environmental extremes may have played a major role in long-term survival of the citrus industry in Pakistan. These results may be useful in developing management strategies for U.S. citrus industries in Texas and California.

Genetic traits in Poncirus trifoliata (Rutaceae) are known to confer resistance to colonization by the Asian citrus psyllid, but field surveys indicated these traits were not expressed in any of 24 simple hybrids between P. trifoliata and sweet orange. No resistance to colonization by the psyllid was observed in 14 other types of P. trifoliata hybrids. Three citrumelo hybrids appeared to be resistant in 1 survey, but in another survey, none of 6 citrumelos exhibited resistance. Interestingly, colonization was somewhat reduced on a hybrid with P. trifoliata in the pedigree of both parents, suggesting the possibility that traits suppressing oviposition may be recovered through similar crosses.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus. Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Ruby, a regulatory gene encoding a MYB transcription factor, is essential for anthocyanin production, and differences in its activity determine most of the natural variation in pigmentation in Citrus and related genera.

The Asian citrus psyllid, Diaphorina citri Kuwayama, was reported for the first time in Florida in June 1998, and huanglongbing (HLB, citrus greening), vectored by D. citri, was detected in Florida for the first time in Aug 2005. In Florida, the only known HLB pathogen is ‘Candidatus Liberibacter asiaticus’ (Las). After HLB was known to be established in Florida, the psyllid vectors found in regulatory samples from plants for sale were tested for the pathogen by real-time qPCR. Approximately 1,200 regulatory samples were tested between Aug 2005 and Aug 2009. Samples came from venues in 44 of Florida's 67 counties. Most of the samples came from citrus, but about 11% came from Murraya exotica, a popular ornamental plant and close relative of citrus. Approximately 9.7% of the psyllid samples tested were positive for Las. Numbers of samples and proportion of positive samples varied by year and by county.

Huanglongbing (citrus greening) is one of the most serious diseases of citrus. Movement of the disease occurs as a result of natural transmission by the insect vector and by movement of infected plant material. We demonstrate here that Diaphorina citri Kuwayama, the vector of citrus greening pathogens, can be transported in trailers of unprocessed fruit. Several samples of D. citri collected from trailers of oranges were positive for citrus greening pathogens, indicating that the disease can spread widely with the movement of the mature fruit as a result of moving the vectors. While movement of disease and the vector through plant materials into new areas has been recognized, our findings emphasize the need to evaluate the importance of long distance movement of psyllids on unprocessed fruit, even in the absence of vegetative tissue.

Author(s): Ramadugu, C.; Keremane, M.; Stover, E.; Halbert, S.; Duan, Y. P.; Lee, R. F. | Abstract: Huanglongbing (HLB), a devastating disease of citrus, has become a serious problem for the citrus industries in Brazil and Florida, and both the disease and its psyllid vector, Diaphorina citri continue to spread to other citrus growing regions. Host resistance or tolerance to the pathogen would be extremely valuable to the citrus industry. A field trial was established in Fort Pierce, Florida where HLB has become endemic to assess the HLB tolerance level of different cultivars of citrus and citrus relatives. Over 800 seedlings representing over 100 accessions (8 replications of each) belonging to 18 genera of the subfamily Aurantioideae and family Rutaceae were evaluated over a period of four years. Leaf samples were collected at 6 month intervals during the spring and fall seasons and tested for the presence of HLB associated bacterium, Candidatus Liberibacter asiaticus (LAS) by real time PCR. While most accessions were found to be susceptible to HLB, the bacterium (LAS) was not detectable in about 20 accessions for up to four years of analysis. These include many trifoliates and trifoliate hybrids, some species of Bergera, Casimiroa, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Naringi, and Zanthoxylum. Information on varietal tolerance of citrus and its relatives to HLB is very important for management of the disease.n While most accessions in Citrus were susceptible, partial resistance was observed in some clonal populations of Citrus latipes. The probable basis of resistance is being investigated.

Author(s): Wang, J.; Roose, M.; Ramadugu, C.; Lee, R.; Manjunath, K.; Lin, H.; Chen, J.; Shatters, R.; Polek, M.; LeVesque, C.; Vidalakis, G. | Abstract: Huanglongbing (HLB, citrus greening) associated with ‘Candidatus Liberibacter’ species is a widespread devastating citrus disease not previously reported in California (CA). In March 2012, ‘C. Liberibacter asiaticus’ (CLas) was detected from an Asian citrus psyllid (ACP, Diaphorina citri) sample from Los Angeles, CA at the Jerry Dimitman Laboratory of the Citrus Research Board. Subsequent citrus plant surveys within a 400m area of the CLas-positive ACP sample performed by the California Department of Food and Agriculture identified an infected multi-grafted citrus tree at a residence in Los Angeles, CA. The CLas-positive tree was removed and nucleic acids from different plant tissues (i.e. roots, trunk, stems, and leaves) were extracted and distributed to several federal, state, and university laboratories nationwide for preliminary evaluation. Labs attempted to identify the species and graft types of the infected citrus, study the genetic characteristics and genome diversity of the detected bacterium, as well as test for other graft-transmissible citrus pathogens (GTCP). Preliminary data suggested that one type of lemon was the original rootstock that received over 20 citrus grafts. CLas DNA population analysis suggested a possible single Asian origin. Preliminary tests indicated the possible presence of other GTCP. Further evaluations on the CA CLas-positive find are ongoing.

Author(s): Keremane, Manjunath L.; Ramadugu, Chandrika; Kubota, Ryo; Duan, Yongping; Hall, David; Jenkins, Daniel; Lee, Richard F. | Abstract: Management of citrus huanglongbing (HLB) requires rapid detection of infected psyllids and trees in an orchard. Detection of HLB associated bacteria (LAS) can be done using psyllids since detection in infected trees is usually delayed (Manjunath et al., 2008). We have developed an easy to use, rapid and affordable detection kit for grower use for testing psyllids for LAS at a reasonable price for initial investment and an operating cost of about $2 per sample.. Eight psyllid samples can be simultaneously tested within 45 minutes. The psyllid DNA extraction and detection of LAS are conducted using a SmartDART™ unit which is operated by software installed on any android device for visualizing real time results. The test results can be e-mailed for both storage and analysis. The DNA prepared can be stored refrigerated and sent to a laboratory for validation. No other equipment (even pipets) is required for the test. The detection system was validated using a large number LAS isolates from many citrus varieties, from different countries; the results were comparable to that of traditional real time PCR data. Development of methods for multiplex detection of the pathogen and the host DNA from both psyllids and plant host are in progress. We believe the detection system will be useful for growers in intra-orchard management, for extension workers, nurserymen, and in areas where the disease has become endemic as well as in those areas where the disease has been recently introduced.

The Florida psyllid testing project (Manjunath et al. 2008, Halbert et al. 2012) showed that about 10% of regulatory samples of Diaphorina citri Kuwayama collected by Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS/DPI) inspectors from plants for sale in Florida were positive for Candidatus Liberibacter asiaticus (Las). Most of the commercial nurseries that produce the plants do not have psyllids or Las, so the most likely source of contamination is the retail venues themselves. If this is the case, great benefit could be achieved by preventing psyllid infestation in retail stores. Florida has a requirement that citrus plants for sale be treated with an imidacloprid-based soil drench (ISD). Producers are required to tag the plant with the date of treatment. The treatment expires in six months, but our data indicate that three months probably is more realistic. In 2009, there was an increase in plants infested with psyllids 30 days post-ISD treatment. In later years, this increase was not so pronounced or did not exist, suggesting that growers are getting better control.

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented.

Manjunath, K. L., Halbert, S. E., Ramadugu, C., Webb, S., and Lee, R. F. 2008. Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology 98:387-396. Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of ‘Ca. L. asiaticus’ in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of ‘Ca. L. asiaticus’ in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

The citrus industries of North and South America are endangered by Huanglongbing (HLB), also known as citrus greening, a devastating disease associated with ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two species of fastidious phloem-limited bacteria spread by the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama. The first reports of HLB from the Americas were from Brazil in 2004 followed by Florida in 2005 (3). The ACP was found in Belize in 2005 (S. Williams, personal communication) and is now present throughout Central America. On the basis of the report that the HLB-associated bacteria can be easily detected in the ACP vector (4), an initial sampling of ACP from 67 locations was collected in February 2009 from trees in the Belize, Corozal, Orange Walk, Stann Creek, and Toledo Districts of Belize, and shipped in 95% ethanol to Riverside, CA for analysis. DNA was extracted from lots containing three to five psyllids from each of the 67 samples with Fast DNA kits (MP Biomedicals, Solon, OH) and analyzed by multiplex qPCR for ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ with a Stratagene MX3005P thermocycler with primers and Taqman probes to detect the 16sRNA gene of ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ and a psyllid gene, wingless, as an internal control target (4). Nine of the sixty-seven psyllid extractions were clearly positive for ‘Ca. L. asiaticus’ with cycle threshold values of 24 to 29. ‘Ca. L. americanus’ was not detected in any of the samples. From the districts previously sampled for ACP, leaves and fruit peduncles were collected from Citrus sinensis and C. aurantifolia plants showing HLB symptoms of asymmetrical leaf mottle and lopsided fruit with aborted seeds. DNA extracted from 10 of the 12 plant samples with a Qiagen Plant DNeasy kit (Qiagen Inc., Valencia, CA) was positive for ‘Ca. L. asiaticus’ with the qPCR procedure of Li et al (3). The presence of ‘Ca. L. asiaticus’ in the positive plant and ACP samples was corroborated by amplification, cloning, and sequencing of a 1,168-bp region of the 16S rRNA gene (2) with SpeedSTAR HS DNA polymerase (TaKaRa Bio Inc., Shiga, Japan). Consensus sequences obtained from three clones each from psyllids (Accession No. GQ502291) and plants (Accession No. GU061003) showed >99% identity to corresponding regions of ‘Ca. L. asiaticus’ in GenBank. The presence of ‘Ca. L. asiaticus’ was further indicated by amplification of a 227-bp fragment from the same 10 positive plant samples using primers for the ‘Ca. L. asiaticus’ preprotein translocase subunit SecE gene (nucleotides 31418 to 31644 of the genomic DNA) (1). Presence of trees with HLB symptoms and the detection of the associated ‘Ca. L. asiaticus’ confirm the disease in the Cayo, Corozal, Stann Creek, and Toledo districts in Belize. Analyses of psyllids from limited surveys conducted from 2006 to 2008 had not detected ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’. Confirmation of HLB in Belize has significant implications to the citrus industries in Central America. References: (1) T. H. Hung et al. J. Phytopathol. 147:599, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) K. L. Manjunath et al. Phytopathology 98:387, 2008.

The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3′ poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5′ and 541 nt at 3′ termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98–99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate. Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0758-1) contains supplementary material, which is available to authorized users.

Citrus exocortis viroid (CEVd) exists as populations of heterogeneous variants in infected hosts. In vivo generated CEVd progeny variants (CEVd-PVs) populations from citrus protoplasts, seedlings and mature plants, following inoculation with transcripts of a single CEVd cDNA-clone (wild-type, WT), were studied. The CEVd-PVs population in protoplasts was heterogeneous and became progressively more homogeneous in seedlings and mature plants. The infectivity and pathogenicity of selected CEVd-PVs was evaluated in citrus and herbaceous experimental hosts. The CEVd-PVs U30C, G128A and U182C were not infectious; G50A and 108U+ were infectious but reverted back to WT and 62A+, U129A and U278A were infectious, genetically stable and more severe than WT. The 62A+ and U278A and U129A accumulated at higher levels than WT in protoplasts and seedlings respectively. The effect of specific mutations on the predicted secondary structure of the CEVd-PVs' RNA coupled with the infectivity and replication studies suggested complex structure-to-function relationships for CEVd.

Citron (Citrus medica L.) is one of the three basic species of the genus Citrus L. that have contributed to the development of cultivated citrus. We analyzed the genetic diversity of 47 citrons (32 from Yunnan Province, China; and 15 of Mediterranean origin) to understand their diversity and relationships within the species. Genetic analysis was conducted using data from microsatellite markers, single nucleotide polymorphisms generated from sequences of a nuclear malate dehydrogenase gene and a chloroplast gene, rps16. Neighbor joining and maximum parsimony analyses were conducted. All three approaches found citron to be monophyletic. Population structure analysis clustered the 47 citrons into three distinct groups. The first group consisted of wild, non-fingered citrons generally having locules, juice sacs and seeds within the fruit. The second cluster consisted mostly of fingered citrons that lacked locules, juice sacs or seeds, and some non-fingered types with smaller locules and vestigial juice sacs, but with seeds. All accessions that clustered in groups I and II originated in China. The third cluster consisted of citrons cultivated in the Mediterranean region. Genetic distance between the clusters from population structure analysis indicated considerable diversity within the species. A citron-specific microsatellite marker was identified and characterized. We observed considerable heterozygosity in certain citrons, contrary to previous reports.