Citrus greening disease, or Huanglongbing (HLB), has devastated citrus crops globally in recent years. The causal bacterium, 'Candidatus Liberibacter asiaticus', presents a sampling issue for qPCR diagnostics and results in a high false negative rate. In this work, we compared six metabolomics assays to identify HLB-infected citrus trees from leaf tissue extracted from 30 control and 30 HLB-infected trees. A liquid chromatography-mass spectrometry-based assay was most accurate. A final partial least squares-discriminant analysis (PLS-DA) model was trained and validated on 690 leaf samples with corresponding qPCR measures from three citrus varieties (Rio Red grapefruit, Hamlin sweet orange, and Valencia sweet orange) from orchards in Florida and Texas. Trees were naturally infected with HLB transmitted by the insect vector Diaphorina citri. In a randomized validation set, the assay was 99.9% accurate to classify diseased from nondiseased samples. This model was applied to samples from trees receiving plant defense-inducer compounds or biological treatments to prevent or cure HLB infection. From two trials, HLB-related metabolite abundances and PLS-DA scores were tracked longitudinally and compared with those of control trees. We demonstrate how our assay can assess tree health and the efficacy of HLB treatments and conclude that no trialed treatment was efficacious.

HomePlant DiseaseVol. 106, No. 7First Report of Citrus Virus A in Texas Associated with Oak Leaf Patterns in Citrus sinensis PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Citrus Virus A in Texas Associated with Oak Leaf Patterns in Citrus sinensisJong-Won Park, John V. da Graça, Marissa Gonzalez, Eliezer S. Louzada, Olufemi J. Alabi, and Madhurababu KuntaJong-Won Parkhttps://orcid.org/0000-0002-1936-8230Texas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author, John V. da GraçaTexas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author, Marissa GonzalezTexas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author, Eliezer S. LouzadaTexas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author, Olufemi J. Alabihttps://orcid.org/0000-0002-2471-7052Texas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author, and Madhurababu Kunta†Corresponding author: M. Kunta; E-mail Address: Madhura.Kunta@tamuk.eduhttps://orcid.org/0000-0002-2545-4071Texas A&M University-Kingsville Citrus Center, Weslaco, TX 78599Search for more papers by this author AffiliationsAuthors and Affiliations Jong-Won Park John V. da Graça Marissa Gonzalez Eliezer S. Louzada Olufemi J. Alabi Madhurababu Kunta † Texas A&M University-Kingsville Citrus Center, Weslaco, TX 78599 Published Online:22 May 2022https://doi.org/10.1094/PDIS-03-21-0628-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleIn 2018, two new negative sense coguviruses in citrus were identified, citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) (Navarro et al. 2018a, b). Since then, members of the genus Coguvirus have also been detected in other plant species (Svanella-Dumas et al. 2019; Wright et al. 2018; Xin et al. 2017). In 2016, leaf flecking with oak leaf patterns was observed in five embryo-rescued navel orange (NO) (Citrus sinensis [L.] Osbeck) trees grafted on C22 (C. sunki × Poncirus trifoliata) rootstock that were maintained in a shade house. Madam Vinous (MV) sweet orange trees graft-inoculated with blind buds from the symptomatic NO plants developed the same symptoms in the new growth. These symptoms were similar to those on the citrus concave gum (CG) source tree of the California isolate CG301, one of the standard citrus disease isolates used as a positive control for biological indexing (Roistacher et al. 2000). None of the trees with oak leaf symptoms tested positive with reverse transcription (RT)-PCR for a panel of viruses and viroids commonly infecting citrus. In this study, CG301 leaf RNA-Seq data were used as a platform to identify any viral agents associated with the oak leaf symptoms observed in the symptomatic NO trees. Of ∼162.8 million paired-end CG301 RNA-Seq reads (150 bp), the de novo assembly of ∼9.6 million reads, not mapped to the C. sinensis genome (v.1.1), yielded 5,375 contigs. BLASTn using the NCBI virus database (txid 10239) identified two contigs, #49 (6,715 nt) and #20 (2,764 nt), which exhibited ∼96% sequence identity to, respectively, RNA1 and 2 of the CiVA isolate W4 (MG764565; MG764566) and 71 to 73% identity to that of the CCGaV isolate CGW2 (KX960112; KX960111). A 5′-Nuclease assay based on contig #20 detected coguvirus sequences in the five symptomatic NO and graft-inoculated MV trees as well as in CG301, but not in 44 asymptomatic field trees located near the shade house where the symptomatic NO trees had been kept. A full genomic sequence of the coguviruses present in CG301 and a symptomatic NO tree was reconstructed by RT-PCR. Both the CG301 and NO isolate have a 6689-nt long negative sense RNA1 (MT922052; MK689372) encoding RNA-dependent RNA polymerase (RdRp) and a 2739-nt long ambisense RNA2 (MT922053; MK689373) encoding movement protein (MP) and nucleocapsid protein (NP). CG301 and NO isolates share ∼96% nucleotide sequence identity. The genomes of both CG301 and NO isolates share 95.4 to 97.8% sequence identity to that of CiVA isolate W4 and 70 to 72.9% sequence identity to CCGaV isolate CGW2. BLASTp showed that RdRp of CG301 and NO isolates have 96.3 to 97.7% sequence identity to CiVA W4 RdRp and ∼77% to CCGaV CGW2 RdRp. These data indicated the presence of CiVA in the symptomatic NO trees and in the concave gum source tree CG301. Recent reports of CiVA in South Africa and Greece indicated a potentially wider distribution of CiVA in various citrus growing regions that may be associated with two graft-transmissible citrus diseases, CG and impietratura disease (Beris et al. 2021; Bester et al. 2021; Roistacher et al. 2000; Velázquez et al. 2019). Although the source of CiVA in the symptomatic NO trees and the degree of CiVA prevalence in Texas has not been determined, a possible involvement of vectors or other means of spread (e.g., seed transmission) cannot be ruled out (Timmer et al. 2017). The current study demonstrated the need for further studies to determine the level of threat of coguviruses for citrus production in Texas.The author(s) declare no conflict of interest.References:Beris, D., et al. 2021. Phytopathology 111:1782. https://doi.org/10.1094/PHYTO-01-21-0027-R Link, ISI, Google ScholarBester, R., et al. 2021. J. Citrus Pathol. 8:1. https://doi.org/10.5070/C481049000 Crossref, Google ScholarNavarro, B., et al. 2018a. Mol. Plant Pathol. 19:1075. Crossref, ISI, Google ScholarNavarro, B., et al. 2018b. Front. Microbiol. 9:2340. Crossref, ISI, Google ScholarRoistacher, C. N., et al. 2000. Int. Organ. Citrus Virol. Conf. Proc. 14:144. Google ScholarSvanella-Dumas, L., et al. 2019. Plant Dis. 103:2703. https://doi.org/10.1094/PDIS-01-19-0028-PDN Link, ISI, Google ScholarTimmer, L. W., et al. 2017. Compendium of Citrus Diseases, 2nd Ed. American Phytopathological Society, St. Paul, MN. https://doi.org/10.1094/9780890545850.002 Google ScholarVelázquez, K., et al. 2019. J. Citrus Pathol. 6:11. Google ScholarWright, A. A., et al. 2018. Arch. Virol. 163:3339. https://doi.org/10.1007/s00705-018-3999-z Crossref, ISI, Google ScholarXin, M., et al. 2017. Front. Microbiol. 8:1514. https://doi.org/10.3389/fmicb.2017.01514 Crossref, ISI, Google ScholarThe author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 7 July 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Downloaded 382 times Article History Issue Date: 8 Jul 2022Published: 22 May 2022First Look: 28 Dec 2021Accepted: 24 Dec 2021 Page: 2005 Information© 2022 The American Phytopathological SocietyKeywordscitrus concave gum-associated viruscitrus virus ARNA virussweet orangeThe author(s) declare no conflict of interest.PDF download

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as 'Candidatus Liberibacter africanus'. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five 'Ca. L. africanus' subspecies have been identified in various rutaceous species, with 'Ca. L. africanus subsp. clausenae' the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.

Citrus canker caused by Xanthomonas citri subsp. citri (syn. X. axonopodis pv. citri) was first reported in South Texas during the early 1900s, prompting quarantine and eradication programs by the federal government. Its last reported incidence in Texas was from two trees from Corpus Christi in 1943. In May 2016, USDA Animal and Plant Health Inspection Service-Plant Protection and Quarantine (PPQ) confirmed the presence of the Asiatic A strain, which is by far the most widespread and severe form infecting most citrus varieties, on two sour orange trees in a park in Houston. Subsequently, infected trees were detected in a nursery in Richmond (Fort Bend Co.) in August 2016 and in dooryard trees in Pearland (Brazoria Co.) in 2018. PPQ and Texas Department of Agriculture collected a combined 1,949 positive A strain samples from Harris, Fort Bend, and Brazoria Counties during May 2016 to January 2021. These findings resulted in seven active citrus canker quarantine zones being established in the Upper Gulf Coast area. The Texas Department of Agriculture is removing positive trees, and surveys are continuing.

Huanglongbing (HLB), or Citrus Greening, is one of the most devastating diseases affecting agriculture today. Widespread throughout Citrus growing regions of the world, it has had severe economic consequences in all areas it has invaded. With no treatment available, management strategies focus on suppression and containment. Effective use of these costly control strategies relies on rapid and accurate identification of infected plants. Unfortunately, symptoms of the disease are slow to develop and indistinct from symptoms of other biotic/abiotic stressors. As a result, diagnosticians have focused on detecting the pathogen, Candidatus Liberibacter asiaticus, by DNA-based detection strategies utilizing leaf midribs for sampling. Recent work has shown that fibrous root decline occurs in HLB-affected trees before symptom development among leaves. Moreover, the pathogen, Ca. Liberibacter asiaticus, has been shown to be more evenly distributed within roots than within the canopy. Motivated by these observations, a longitudinal study of young asymptomatic trees was established to observe the spread of disease through time and test the relative effectiveness of leaf- and root-based detection strategies. Detection of the pathogen occurred earlier, more consistently, and more often in root samples than in leaf samples. Moreover, little influence of geography or host variety was found on the probability of detection.

Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5' and 3' region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

This chapter, updating a review written for the 50th anniversary of the International Organization of Citrus Virologists (Bové et al., 2010), covers citrus diseases caused by ca. 30 viruses and viroids while briefly describes graft-transmissible diseases of unknown etiology. Prokaryote-associated graft-transmissible diseases such as Huanglongbing, stubborn, witches’ broom of lime, and citrus variegated chlorosis are not included here. The description of the diseases and viral pathogens includes history, symptomology, epidemiology, and latest research developments. The chapter also includes general sections on diagnostics, control, and concluding remarks on graft-transmissible viral diseases of citrus.

Huanglongbing (HLB, citrus greening disease) in the major citrus-producing states of the United States is associated with Candidatus Liberibacter asiaticus (CLas), which is vectored by the Asian citrus psyllid (ACP). Surveys were conducted in Texas from 2007 to 2017 to assess the prevalence and titer of CLas in ACPs and citrus trees. ACP and citrus leaf tissue samples were collected from suspect trees in residential areas and commercial groves (orchards) and assayed for CLas by quantitative PCR. CLas detection in ACPs (2011) preceded that of citrus trees (2012) by several months. Annual incidences of CLas-positive ACPs and leaf tissue followed an exponential growth pattern over the survey period, varying from 0.03 to 28.7% in ACPs and 0.6 to 36.5% in citrus trees. There was a significant and positive relationship between the monthly incidences of CLas-positive ACP and leaf tissue samples. The proportion of HLB detection sites also increased with time, reaching 26 and 40% of commercial groves and residential sites, respectively, by 2017. Seasonal variations were observed in the incidences of CLas-positive ACPs and citrus trees such that significantly more CLas-positive ACPs and trees were recorded during the fall and winter of a given year relative to the hot summer. A temporal analysis of the class distribution of cycle threshold values revealed a trend of increased bacterial accumulation in ACPs and trees over time, with the trend more pronounced for the former than the latter host type. These findings provide a comprehensive insight into the ongoing CLas/HLB epidemic in Texas, with potential lessons for California and other citrus-producing areas where the disease is not yet established.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylumNegarnaviricotawas amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy ofNegarnaviricotaas now accepted by the ICTV.

Phytophthora-induced foot rot, also known as gummosis, is an important disease affecting citrus production worldwide. In Texas, the third-largest citrus-producing state in the United States, limited information is available on the etiology and epidemiology of foot rot in commercial orchards. This study comprises a survey of foot rot incidence and severity in Texas and the characterization of Phytophthora isolates associated with the disease. Surveys in 2015 and 2017 of 30 orchards in the Lower Rio Grande Valley (LRGV) region where commercial citrus production is concentrated in the state revealed that foot rot occurred in 97% of the orchards assessed. Overall, foot rot symptoms were observed on 33.7% of the trees evaluated and the disease severity index in the region was rated at 14.2 and 16.5% in 2015 and 2017, respectively. Lesions were mostly present on the scion, while the rootstock (sour orange) was not affected. Phytophthora nicotianae was the only Phytophthora sp. isolated from the surveyed orchards and from five additional residential sites on the Texas Coastal Bend (TCB). Sporangia and chlamydospores from 34 representative LRGV isolates of P. nicotianae were larger than those of TCB isolates. In both LRGV and TCB, A1 and A2 mating types were present in the same location, albeit the A2 mating type was more prevalent. All isolates were sensitive to mefenoxam (50% inhibition in the presence of mefenoxam [EC50] < 0.5 µg/ml), except for one TCB isolate (EC50 = 143.6 µg/ml). Our research indicates that treatment for Phytophthora foot rot in the region is necessary and, although mefenoxam is still useful, alternating chemistries for resistance management are required.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Citrus tatter leaf virus (CTLV) is a graft-transmissible capillovirus that causes the tatter leaf disease of citrus and bud union incompatibility between scion and trifoliate hybrid rootstocks which can result in serious economic losses. Although several CTLV detection protocols have been described, there is a need for a highly sensitive protocol that can be used in routine diagnostic tests for virus-free budwood certification programs. To address this need, a TaqMan® chemistry based real time reverse transcription-polymerase chain reaction (RT-qPCR) assay was developed to detect CTLV from citrus plants. The nucleotide sequence analysis based on full genomic sequences of eight CTLV isolates available from GenBank showed that 3′ region of the viral genome is more conserved among different CTLV isolates than the rest of the viral genome. A new set of primers and a TaqMan® probe were designed from a conserved 500 nt-long fragment at the 3′ end of the CTLV genome for the detection of CTLV by RT-qPCR. The newly designed primers for CTLV RT-qPCR assay had ca. 98.6% amplification efficiency and showed 100% CTLV detection rate when they were tested for previously known CTLV-positive trees infected with different CTLV isolates. The sequencing of 132 bp-long RT-qPCR amplicon followed by BLASTn search showed that it had 94% to 98% sequence identity to those of various CTLV isolates available in GenBank, confirming the specificity of the assay to reliably detect CTLV.

Citrus fibrous root tissue was evaluated as an alternative source material for huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “ Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples prepared from leaf and fibrous root tissue collected from mature (> 10 years old) and young (4–5 years old) citrus trees growing in Texas and Florida confirmed that root HLB test is more sensitive than the leaf HLB test in terms of the detection rate of HLB positive trees. In addition, the current study confirmed that HLB can be diagnosed at the pre-symptomatic stage using the root HLB test to facilitate efficient removal of HLB-positive, asymptomatic trees that could serve as a source for HLB. The new HLB diagnostic method using root tissue described in the current study can assist in deploying a more efficient disease management strategy to deter HLB spread, especially at the pre-symptomatic stage.

The International Committee on Taxonomy of Viruses has ratified a taxonomic proposal to change the name of the family Ophioviridae to Aspiviridae, and to change the names of species in the family to Blueberry mosaic associated ophiovirus, Citrus psorosis ophiovirus, Freesia sneak ophiovirus, Lettuce ring necrosis ophiovirus, Mirafiori lettuce big-vein ophiovirus, Ranunculus white mottle ophiovirus and Tulip mild mottle mosaic ophiovirus. These species are all members of the genus Ophiovirus, the genus name being unchanged.

In October 2015, a Mexican lime exhibiting citrus canker symptoms was found by the USDA APHIS PPQ in a residential property in Rancho Viejo, Cameron County, Texas. Real-time PCR analysis detected the presence of Xanthomonas citri subsp. citri; USDA APHIS PPQ in Beltsville, MD confirmed this diagnosis. A delimiting survey was initiated and suspect leaf samples were collected and sent to the PPQ Beltsville lab for analysis. By October 2017, leaf samples from 197 trees were confirmed positive for citrus canker, all within a 5-mile radius; in addition, a further 59 symptomatic trees were found and all 256 infected trees (254 Mexican lime, one makrut lime, and one Ponderosa lemon) were removed. Survey data collected on stem lesions suggested the oldest lesions to be between 4 to 6 years old. A host-range study using 12 citrus varieties, including the major commercial varieties grown in Texas, were inoculated with crude leaf extracts from symptomatic leaves by leaf infiltration method. Mexican limes and alemow were the only citrus plants that developed definitive canker lesions. A combination of host range, serological, and molecular tests suggested that this isolate was different from the typical Asiatic strain and more similar to X. citri subsp. citri A(W) which was only previously reported from Florida on Mexican lime and alemow.

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

ABSTRACT We report here the draft genome sequence of “Candidatus Liberibacter asiaticus” strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.