Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months, with transformation efficiency of 30% and 22% for the respective species.

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.

Huanglongbing Pandemic: Current Challenges and Emerging Management Strategies.

Huanglongbing (HLB, aka citrus greening), one of the most devastating diseases of citrus, has wreaked havoc on the global citrus industry in recent decades. The culprit behind such a gloomy scenario is the phloem-limited bacteria "Candidatus Liberibacter asiaticus" (CLas), which are transmitted via psyllid. To date, there are no effective long-termcommercialized control measures for HLB, making it increasingly difficult to prevent the disease spread. To combat HLB effectively, introduction of multipronged management strategies towards controlling CLas population within the phloem system is deemed necessary. This article presents a comprehensive review of up-to-date scientific information about HLB, including currently available management practices and unprecedented challenges associated with the disease control. Additionally, a triangular disease management approach has been introduced targeting pathogen, host, and vector. Pathogen-targeting approaches include (i) inhibition of important proteins of CLas, (ii) use of the most efficient antimicrobial or immunity-inducing compounds to suppress the growth of CLas, and (iii) use of tools to suppress or kill the CLas. Approaches for targeting the host include (i) improvement of the host immune system, (ii) effective use of transgenic variety to build the host's resistance against CLas, and (iii) induction of systemic acquired resistance. Strategies for targeting the vector include (i) chemical and biological control and (ii) eradication of HLB-affected trees. Finally, a hypothetical model for integrated disease management has been discussed to mitigate the HLB pandemic.

Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta.

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus (CTV) of genus Closterovirus and family Closteroviridae. The disease has caused tremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595 fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 × 107-1.4 × 108/100 mg) and the lowest in partial dead twigs (1 × 103-1.7 × 104/100 mg), and shoot tip (2.3 × 103-4.5 × 103/100 mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 × 107-1.1 × 108/100 mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.

Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus.

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to 'Kinnow mandarin' hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 109) to 0.000948 amol (5.709 × 102), with detection limit of 5.709 × 102 copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

Detection and Molecular Characterization of 'Candidatus Liberibacter asiaticus' and Citrus Tristeza Virus Associated with Citrus Decline in Bhutan.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of 'Candidatus Liberibacter asiaticus' (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of 'Ca. L. asiaticus' was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The 'Ca. L. asiaticus' isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of 'Ca. L. asiaticus' and CTV isolates and their association with citrus decline in Bhutan.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus.

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the 'Kinnow mandarin', a commercial citrus crop cultivated in the northwest of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcription polymerase chain reaction (RT-PCR) that is time consuming. Here, we describe a novel, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. To standardize the RT-LAMP assay, four different primers were designed and tested to target the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. The ICRSV RT-LAMP assay developed in the present study is a simple, rapid, sensitive, specific technique. Moreover, the assay consists of only a single step and is more cost effective than existing methods. This is the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large-scale indexing of field samples in diagnostic laboratories, in nurseries, and for quarantine applications.

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus.

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15-20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Molecular detection, identification, and sequence analysis of 'Candidatus Liberibacter asiaticus' associated with Huanglongbing disease of citrus in North India.

Huanglongbing (HLB, Citrus greening), caused by a phloem-limited fastidious gram-negative bacterium, "Candidatus Liberibacter spp.", is one of the devastating diseases of citrus worldwide. The pathogen belongs to the alpha-proteobacteria group and is classified on the basis of its geographical origin and 16S rRNA sequence diversity. Although the disease has been reported from all citrus growing states of India, the status and the molecular variability among the isolates from the Northern part of the country is unknown. A total of five different HLB isolates originating from Northern India showing variable symptoms were studied. The genomic regions of four different genes, i.e., 16S rRNA, intergenic 16S/23S rRNA spacer region, rplA-rplJ, and CLIBASIA_01645 were amplified by PCR, sequenced, and variations in these sequences were assessed. Analysis of 16S rRNA clearly indicated that all five isolates fit in to 'Candidatus Liberibacter asiaticus' (CLas) group. However, 16S/23S rRNA intergenic spacer region-based analysis failed to segregate these isolates beyond species level. Sequence analysis of rplA-rplJ gene and CLIBASIA_01645 loci also confirmed the existence of diversity among the 'CLas' in the surveyed areas. Further, 16S rRNA and rplA-rplJ-based SNP analysis revealed that some isolates segregated into three new lineages, two on the basis of 16Sr (16Sr-XV and 16Sr-XVI), and one based on ß-rp (rp-IV), respectively. A tandem repeat number (TRN) at CLIBASIA_01645 region were TRN = 5, 6 and 13; with TRN = 6 being common in three 'CLas' isolates. Overall, the study demonstrated that all examined five HLB isolates belonged to 'CLas' group. However, these isolates showed distinct sequence variability in three out of four genomic regions. The results provide a robust framework for understanding differences in pathogenicity among different HLB isolates as it is plausibly related to their genomic variation, and evolutionary history.

Characterization of the heavy metal binding properties of periplasmic metal uptake protein CLas-ZnuA2.

Candidatus Liberibacter asiaticus (CLas), a phloem-limited unculturable Gram-negative bacterium, causes citrus greening disease. The proteome analysis of CLas showed the presence of a heavy metal permease and Co/Zn/Cd cation exporter system. However, there is no designated metal uptake protein specific for the heavy metal permease in CLas. One of the metal uptake proteins, designated as CLas-ZnuA2, in our previous studies, showed a lower metal-binding affinity for Mn2+ and Zn2+ and was postulated to bind and transport metals rather non-specifically. The present study focused on the characterization of the heavy metal binding properties of CLas-ZnuA2 using SPR, CD, DSC and crystallographic studies. The crystal structure analysis of Cd2+ bound CLas-ZnuA2 showed octahedral geometry for Cd2+ binding as compared to a non-preferred square-pyramidal geometry for Mn2+ and Zn2+ binding in earlier reported crystal structures. In SPR analysis, the binding affinities of 4.7 × 10-6 M, 7.2 × 10-6 M, 5.3 × 10-5 M and 4.3 × 10-5 M for Hg2+, Cd2+, Ba2+ and Co2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. Likewise, CD and DSC analysis showed relatively higher thermal stability for CLas-ZnuA2 on heavy metal binding. Taken together with the expression of the permease and exporter system for heavy metals, our results indicate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals in environmentally stressed conditions.

Crystal structures of a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus: insights into an adapted mechanism of ligand binding.

The amino acid-binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine-binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine-binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand-bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C-terminal extended loop of one chain very close to the substrate-binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C-terminal extended loop in position. Analysis of the substrate-binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg-bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix-α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand-free open state. The dissociation constant of 1.26 µm by SPR and 3.7 µm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l-cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of "Candidatus Liberibacter asiaticus" in planta.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.

Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics.

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.