Citrus Tristeza Virus Infection Research Articles

DAS-ELISA-based detection and RT-PCR validation for precise and timely analysis of Citrus tristeza virus infection in Nepalese citrus species

Citrus viral infections pose a substantial threat to the global citrus industry, resulting in multitudinous economic losses annually. Timely and precise identification of citrus viruses offers a promising avenue for proactive insights preemptively before the instigation of extensive infections. The present study was devised to expedite the prompt identification of the presence of Citrus tristeza virus (CTV) within Nepalese citrus species utilizing a Double Antibody Sandwich-Enzyme-Linked Immunosorbent Assay (DAS-ELISA), followed by its validation using reverse transcription PCR (RT-PCR). Samplings extracted from plants harvested from 74 out of the 77 administrative districts within Nepal provide substantial evidence of extensive propagation of viral contagion among citrus cultivars across 64 districts (86.48%) of the nation. A comprehensive analysis of the samples obtained from diverse geographical localities revealed that 36 districts (48.64%) exhibit mild infestation, while 9 districts (12.16%) evince moderate infestation. Moreover, samples harvested from 19 districts (25.67%) were highly infected; intriguingly, specimens from 10 districts (13.51%) manifest an absence of viral intrusion. The results have been validated with RT-PCR assay. Given the profound significance of citrus as a pivotal repository of vitamin C and its essential role in conferring advantageous health attributes to humankind, alongside its substantial contribution to the economic paradigm, a meticulous examination is imperative to safeguard these plants against an array of viral infections. Collectively, the DAS-ELISA methodology along with RT-PCR approaches constitutes an exquisitely sensitive and meticulously specific modality, poised to accelerate the expeditious detection of citrus-associated viruses within burgeoning economies like Nepal. This advancement supports timely and precise detection and diagnosis of agriculturally virulent conditions, thereby forestalling the eruption of viral epidemics and concomitantly curbing their financial repercussions.

Stem-pitting caused by Citrus tristeza virus is associated with increased phloem occlusion

Stem-pitting (SP) disease results from disruption of normal phloem and xylem development. In citrus, a characteristic manifestation of SP caused by Citrus tristeza virus (CTV) is phloem regeneration. We hypothesized that phloem regeneration occurs due to reduced functionality of CTV infected phloem cells. To examine phloem cell occlusions in CTV-SP, we analyzed callose and phloem-protein (PP) accumulation in Citrus macrophylla trees infected with CTV mutants exhibiting different SP phenotypes from very mild (CTVΔp13) to severe (CTVΔp33), in addition to full-length CTV and healthy plants. CTV infection was accompanied by callose and PP accumulation in the phloem. With the increase in the SP symptoms from very mild to severe, there was a constant increase in the levels of callose and PP, accompanied by an increase in PHLOEM-PROTEIN 2 and a decrease in BETA-1,3-GLUCANASE gene expression levels. These results indicate that SP symptom development is associated with increased phloem occlusion.

Callose synthase and xyloglucan endotransglucosylase gene expression over time in Citrus × clementina and Citrus × sinensis infected with citrus tristeza virus

Citrus tristeza virus (CTV) is a virus that already caused great losses in citrus producing regions. The cell wall of plant cells plays an important role in the defence response to viruses. Following several studies indicating that cell wall enzyme transcripts of callose synthase 7 (calS7) and xyloglucan endotransglucosylase 9 (xth9) are modified during a viral infection, transcript expression of calS7 isoform x5 (calS7x5) and xth9 was evaluated over time in Citrus × sinensis ‘Valencia Late’ (VL) and Citrus × clementina ‘Fina’ (CL), infected with the severe CTV isolate T318A, by quantitative (q) PCR. qPCR analysis of healthy and CTV infected citrus was performed at 15 days, 10 months and at 31 months post-inoculation (dpi/mpi), respectively. The CTV titer, evaluated at the three time-points by qPCR, increased over time in bark tissues, with VL plants exhibiting a titer about 5 times higher than CL 31 mpi. CTV infection did not cause significant changes in calS7x5 gene expression over time in both citrus cultivars. However, CTV infection was associated with significant up-regulation of xth9 in VL compared to controls 31 mpi. This study highlights that CTV infection can affect the expression of specific cell wall-associated genes over time and that this influence was distinct for VL and CL. This study provides further insight into the CTV-citrus host interaction, with the long-term response of VL to a severe CTV isolate involving a high expression of the xth9 gene.

Quick Decline and Stem Pitting Citrus tristeza virus Isolates Induce a Distinct Metabolomic Profile and Antioxidant Enzyme Activity in the Phloem Sap of Two Citrus Species

Susceptibility to the severe Citrus tristeza virus (CTV), T36, is higher for Citrus macrophylla (CM) than for C. aurantium (CA). How host-virus interactions are reflected in host physiology is largely unknown. In this study, the profile of metabolites and the antioxidant activity in the phloem sap of healthy and infected CA and CM plants were evaluated. The phloem sap of quick decline (T36) and stem pitting (T318A) infected citrus, and control plants was collected by centrifugation, and the enzymes and metabolites analyzed. The activity of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), in infected plants increased significantly in CM and decreased in CA, compared to the healthy controls. Using LC-HRMS2 a metabolic profile rich in secondary metabolites was assigned to healthy CA, compared to healthy CM. CTV infection of CA caused a drastic reduction in secondary metabolites, but not in CM. In conclusion, CA and CM have a different response to severe CTV isolates and we propose that the low susceptibility of CA to T36 may be related to the interaction of the virus with the host's metabolism, which reduces significantly the synthesis of flavonoids and antioxidant enzyme activity.

Simple template-based reverse transcription-recombinase polymerase amplification assay for routine diagnosis of citrus tristeza virus.

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32min), although the best conditions were 38°C for 30min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.

Citrus Tristeza Virus: From Pathogen to Panacea.

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus. During the past century, CTV induced grave epidemics in citrus-growing areas worldwide that have resulted in a loss of more than 100 million trees. At present, the virus continues to threaten citrus production in many different countries. Research on CTV is accompanied by distinctive challenges stemming from the large size of its RNA genome, the narrow host range limited to slow-growing Citrus species and relatives, and the complexity of CTV populations. Despite these hurdles, remarkable progress has been made in understanding the CTV-host interactions and in converting the virus into a tool for crop protection and improvement. This review focuses on recent advances that have shed light on the mechanisms underlying CTV infection. Understanding these mechanisms is pivotal for the development of means to control CTV diseases and, ultimately, turn this virus into an ally.

Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange.

Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No significant trends of CTV infection on summed amounts of all sugar, phenolic, or terpenoid compounds were observed. However, individual compound levels were affected by CTV infections. Subsequent canonical discriminant analysis (CDA) that utilized profiles of individual amino acids, terpenoids, or phenolics successfully distinguished leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study reveals biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance.

A Comprehensive Analysis of Citrus Tristeza Variants of Bhutan and Across the World.

Mandarin orange is economically one of the most important fruit crops in Bhutan. However, in recent years, orange productivity has dropped due to severe infection of citrus tristeza virus (CTV) associated with the gradual decline of citrus orchards. Although the disease incidence has been reported, very limited information is available on genetic variability among the Bhutanese CTV variants. This study used reverse transcription PCR (RT-PCR) to detect CTV in collected field samples and recorded disease incidence up to 71.11% in Bhutan’s prominent citrus-growing regions. To elucidate the extent of genetic variabilities among the Bhutanese CTV variants, we targeted four independent genomic regions (5′ORF1a, p25, p23, and p18) and analyzed a total of 64 collected isolates. These genomic regions were amplified and sequenced for further comparative bioinformatics analysis. Comprehensive phylogenetic reconstructions of the GenBank deposited sequences, including the corresponding genomic locations from 53 whole-genome sequences, revealed unexpected and rich diversity among Bhutanese CTV variants. A resistant-breaking (RB) variant was also identified for the first time from the Asian subcontinent. Our analyses unambiguously identified five (T36, T3, T68, VT, and HA16-5) major, well-recognized CTV strains. Bhutanese CTV variants form two additional newly identified distinct clades with higher confidence, B1 and B2, named after Bhutan. The origin of each of these nine clades can be traced back to their root in the north-eastern region of India and Bhutan. Together, our study established a definitive framework for categorizing global CTV variants into their distinctive clades and provided novel insights into multiple genomic region-based genetic diversity assessments, including their pathogenicity status.

Effects of Two Citrus Tristeza Virus Isolates on Sweet Orange (Citrus sinensis) Propagated on a Citrus Tristeza Virus Tolerant Rootstock: A Metabolomics and Transcriptomics Approach

Citrus tristeza virus (CTV) was once the most economically important citrus pathogen in the world. In regions where CTV is endemic, CTV tolerant rootstocks have been widely adopted. Despite these changes, CTV can still affect plant health and fruit quality. To assess the impact of CTV infection on scions grown on CTV tolerant rootstock, Washington navel [Citrus sinensis (L.) Osbeck] scions grafted onto Carrizo rootstock (Poncirus trifoliate × Citrus sinensis) received grafts from citrus infected with a mild isolate (T30 isolate 07539) or a stem-pitting isolate (CCTEA-107) of CTV, and metabolomics and transcriptomics analyses were performed over a period of 10 months after grafting of CTV-infected material. The phenotypic response to either isolate was weak. The largest difference in the leaf metabolome was seen 10 months postgrafting for both isolates, with a stronger response observed for plants infected with the stem-pitting isolate. Analysis of differentially expressed genes revealed that plants infected with the milder isolate exhibited a difference in gene expression 4 months after infection, whereas plants infected with the stem-pitting isolate exhibited a difference in gene expression 10 months after infection. This study highlights differences and similarities in the metabolome and transcriptome response of Washington navel trees on Carrizo rootstock to two CTV isolates.

First Report of Citrus tristeza virus Infecting Citrus Trees in Georgia, USA.

Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including 'Flying Dragon') located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer's instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5'-ATGTCAGGCAGCTTGGGAAATT-3') and AR18R (5'-TTCGTGTCTAAGTCRCGCTAAACA-3') which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.

High throughput sequencing from Angolan citrus accessions discloses the presence of emerging CTV strains

BackgroundCitrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use.MethodsDouble-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3).ResultsFrom the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs.ConclusionPhylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).

Citrus miraculin-like protein hijacks a viral movement-related p33 protein and induces cellular oxidative stress in defence against Citrus tristeza virus.

SummaryTo defend against pathogens, plants have developed a complex immune system, which recognizes the pathogen effectors and mounts defence responses. In this study, the p33 protein of Citrus tristeza virus (CTV), a viral membrane‐associated effector, was used as a molecular bait to explore virus interactions with host immunity. We discovered that Citrus macrophylla miraculin‐like protein 2 (CmMLP2), a member of the soybean Kunitz‐type trypsin inhibitor family, targets the viral p33 protein. The expression of CmMLP2 was up‐regulated by p33 in the citrus phloem‐associated cells. Knock‐down of the MLP2 expression in citrus plants resulted in a higher virus accumulation, while the overexpression of CmMLP2 reduced the infectivity of CTV in the plant hosts. Further investigation revealed that, on the one hand, binding of CmMLP2 interrupts the cellular distribution of p33 whose proper function is necessary for the effective virus movement throughout the host. On the other hand, the ability of CmMLP2 to reorganize the endomembrane system, amalgamating the endoplasmic reticulum and the Golgi apparatus, induces cellular stress and accumulation of the reactive oxygen species, which inhibits the replication of CTV. Altogether, our data suggest that CmMLP2 employs a two‐way strategy in defence against CTV infection.

Detection of Citrus Tristeza Virus in Mandarin Orange Using a Custom-Developed Electronic Nose System

Olfaction is one of the primary senses of a living organism and has been used as first aid for inspecting freshness, edibility, and the quality of food. This article addresses a technique where the biological olfaction process has been mimicked by electronic nose (E-Nose) to detect a pathogen named Citrus Tristeza Virus (CTV) in Khasi Mandarin Orange plants. The proposed technique may be used as an alternative to currently used traditional serological or molecular tests, which requires costly and elaborate laboratory infrastructures. In this article, leaves from 62 plants were collected and examined for viral infection using the gold-standard polymerase chain reaction test. A commercial E-Nose system, Alpha MOS Fox 3000, was used for hypothesis testing, resulting in an accuracy of 95.30% using random forest classifier. These results were used to identify the most prominent sensors and their target gases responding to the CTV infection and were considered as a base for sensor selection for custom developing a prototype. The prototype was designed based on simulations for optimum airflow in the sensor chamber. Bootstrap ensemble of $k$ -nearest neighbors and adaptive boost ensemble of decision tree classifier distinguished the data generated from the prototype with an accuracy of 99.36% and 97.58%, respectively. The performance of the classifiers was visualized graphically using dimensionality reduction techniques. These results indicated that the E-Nose technique employed could differentiate the healthy and infected samples and the prototype could serve as an effective first-hand diagnosing tool for large-scale orchards.

A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners.

The RNA genome of citrus tristeza virus (CTV), one of the most damaging viral pathogens of citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the virus infection cycle and is needed for virus ability to infect the extended host range, with other CTV proteins shown to mediate virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in virus ability to infect the extended host range is discussed.

Molecular characterization of the gibberellin-stimulated transcript of GASA4 in Citrus

A Citrus clementina gene, CcGASA4, which is involved in the responses of citrus to stress, was characterized. The gene was induced by Citrus tristeza virus infection, wounding and gibberellic, salicylic and abscisic acid treatments. A qRT-PCR analysis showed that CcGASA4 had a very high basal expression in flowers yet was still able to be further induced independently in giberellic, salicylic and abscisic acid-treated flowers. Subcellular localization analysis revealed that the CcGASA4 protein localized to the cell membrane and nucleus. A yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays showed that CcGASA4 interacted with two proteins, the large proline-rich protein bag6-A (PRPBAG6-A) and the general negative regulator of transcription subunit 3 (CNOT3). PRPBAG6 has been reported to be involved in disease resistance. Replacing some of CcGASA4's conserved cysteines with alanines (Cys → Ala) abolished the protein’s interaction with CNOT3 but did not show any effect on the protein’s interaction with PRPBAG6-A. Thus, CcGASA4 appears to play multiple roles in Citrus, probably by interacting with different proteins and/or by localizing to different subcellular compartments.

Exploring the genetic diversity and recombination analysis of Citrus tristeza virus isolates prevalent in Northeast India

Citrus tristeza virus (CTV) is associated with three manifestations: citrus decline, stem pitting and seedling yellows of almost all citrus species and hybrids. Infected citrus plants harbour a large number of distinct CTV strains due to multiple and frequent inoculations by aphids and the fallible nature of its RNA polymerase. As such, it is important and necessary to identify different strains of CTV for proper management and disease control. In this study, we report the identification of 15 CTV isolates prevalent in three states of Northeast (NE) India: Assam, Meghalaya and Nagaland. A total of 50 samples were collected, out of which 15 showed CTV infection based on enzyme linked immuno-sorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. RT-PCR products of 674 bp and 407 bp corresponding to the coat protein (CP) gene and 5′ORF1a region respectively were sequenced and their phylogenetic analysis was carried out. Analysis of putative recombination events detected recombination in four isolates in the 5′ORF1a fragment and in one isolate in the CTV-CP gene. Furthermore, CP monomer structure of four CTV isolates was predicted by Phyre2 online structure prediction tool. Phylogenetic studies involving more number of CTV isolates reveal better understanding of the recombination or evolutionary pattern of the virus which would aid in developing advanced CTV diagnostic methods.

The Detection and Surveillance of Asian Citrus Psyllid (Diaphorina citri)-Associated Viruses in Florida Citrus Groves.

The plant pathogenic bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri), have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACP-associated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reo-like virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able to help guide the direction of biotechnological pest control efforts involving a number of viruses that were detected for the first time in Florida ACP populations, including two newly identified ACP-associated viruses.

Biochemical changes in cultivars of sweet oranges infected with citrus tristeza virus.

Citrus fruit production occupies a place of considerable importance in the economy of the world including Pakistan. Tristeza disease caused by Citrus Tristeza Virus (CTV) exists in various forms that may or may not cause symptoms in the plants. The bioactive compounds and antioxidants are naturally present in plants and provide a defense mechanism that is generally accelerated in response to a stress. The objective of the present study was to target and analyze the citrus plants that were CTV positive to observe the changes in the enzymatic and non-enzymatic antioxidants of citrus (Sweet Oranges only). It was observed that in response to CTV infection, both the non-enzymatic antioxidants (total flavonoid, ascorbic acid, phenolic acid) and enzymatic antioxidants (catalase, superoxide dismutase and peroxidase) activities showed an increasing trend overall. The profiling of antioxidants in response to a viral infection may help in the discovery of new biomarkers that can be used as a monitoring tool in disease management.

CTV Vectors and Interactions with the Virus and Host Plants.

Citrus is a graft-propagated perennial crop, and Citrus tristeza virus (CTV) is readily graft-transmissible. CTV is comprised of a complex of strains and isolates and, in nature, is spread semi-persistently by aphid vectors. Therefore, citrus trees become infected with multiple CTV strains over time. An important step in characterizing a CTV field isolate is to use aphid vectors to "clean" up the CTV population of a source tree to separate strains and eliminate other graft-transmissible agents. Use of Toxoptera citricida or Aphis gossypii will expedite efficient CTV transmission. CTV vector studies require critical coordination of abundant robust and virus-free vector-competent aphid colonies and an insect-proof, climate-controlled greenhouse or growth chamber. CTV donor and healthy receptor plants with young flush growth must be available for virus acquisition and inoculation. Vector optimums for virus acquisition and inoculation are 24h for each. CTV infection is readily determined by serology using a polyclonal antiserum or a monoclonal antiserum cocktail; whereas, molecular genotyping is conducted with reverse transcription polymerase chain (RT-PCR) or real time quantitavtive RT-PCR (RT-qPCR) with strain-specific primers and probes. However, the phenotype of the aphid-transmitted isolate still requires virus indexing by graft inoculation to a citrus host range and evaluating symptoms such as stem pitting, vein clearing, stunting, and chlorosis.

A Short Note on Reflections and Publications on Citrus tristeza virus (CTV) Methodologies.

My PhD thesis work of Citrus tristeza virus (CTV) purification was aimed to develop a rapid serological assay to replace biological indexing. The task turned difficult and was achieved after a lengthy struggle, rewarded by allowing (1) the rapid diagnosis of the first incidences of natural spread of a severe CTV-VT strain in our region and (2) finding that the CTV particle isolation protocol, with some modifications, was also useful for Beet yellows virus (BYV) particles, leading to their assignment in the Closterovirus group, the first group of elongated plant viruses with different modal lengths. Later, following the introduction of ELISA for large-scale diagnosis of tristeza-infected citrus trees, the CTV infection rates through the coastal citrus production areas were continually increasing, with many ELISA-positive samples appearing symptomless, prompting the need to develop strain-specific assays. Using CTV-VT cDNA fragments, as hybridization probes, the genetic diversity among local CTV isolates was demonstrated. With the emergence of the PCR technology, we developed a CTV-dsRNA cloning method based on the ligation of known oligonucleotide molecules to dsRNA ends and the use of complementary oligonucleotides for cDNA synthesis and PCR amplification. The method allowed the cloning of a cDNA molecule complementary to a defective dsRNA of 2.4kb with intact 5 and 3 ends of the CTV-VT genome. A list of publications, resulting from continuous collaborative work with local and foreign associates and students on the development and adaptation of novel CTV methodologies, is present.