A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.


Introduction
Citrus is infected by a wide range of graft-transmissible pathogens, including citrus exocortis viroid (CEVd). CEVd is classified as belonging to the genus Pospiviroid within the family Pospiviroidae and has a 371 nucleotide-long circular, single-stranded RNA genome (Di Serio et al. 2021). CEVd has a global distribution and a broad host range including woody plants such as citrus, grape and fig (Semancik et al. 1973;García-Arenal et al. 1987;Yakoubi et al. 2007), as well as herbaceous plants such as tomato, eggplant, carrot, broad bean and petunia (Mishra et al. 1991;Fagoaga et al. 1995;Fagoaga and Durán-Vila 1996;van Brunschot et al. 2014). The viroid is mechanically transmissible (Barbosa et al. 2005) and, importantly for perennial crops such as citrus, is disseminated in asymptomatically-infected, vegetative propagules such as budwood. Seed transmission is reported in impatiens, verbena and tomato but not in citrus (Semancik 1980;Singh et al. 2009).
CEVd is the causal agent of exocortis or scalybutt disease of Citrus trifoliata, trifoliate hybrids and Rangpur lime rootstocks. The symptoms of this disease include the development of gummy pitting, scaly bark and corky pustules on the rootstock but not on the scion (Fraser et al. 1976). Affected trees exhibit canopy yellowing and stunting, leading to reductions in yield, but fruit quality is not affected (Vernière et al. 2004). Scalybutt was first observed in Australia in the 1930s and was originally attributed to a viral infection, as the disease was able to be transmitted by graft inoculation (Benton et al. 1950;Fraser and Levitt 1957). The viroid-like nature of the infectious agent was determined by Sänger (1972) and Semancik and Weathers (1972), and the first genome sequences obtained a decade later, including the sequence of an Australian isolate of the viroid (Visvader et al. 1982). Subsequent studies of scalybutt in Australia showed that trees are infected by complex mixtures of sequence variants of CEVd, and these sequence variants can be divided into two classes according to their biological properties in tomato and Gynura aurantiaca. Class A sequence variants cause severe stunting and epinasty in these hosts, whereas Class B variants only induce mild symptoms (Visvader and Symons 1985). Mutations in the P L and P R regions located in the pathogenic and variable domains, respectively, differentiate the two sequence classes, but whether mutations in one or both domains are responsible for the changes in the biological properties of the viroid has not been determined. The differences in symptom expression between the sequence classes was slight in a trial of clementine mandarin grown on C. trifoliata rootstocks (Vernière et al. 2004), where the severe Class A isolate subtly reduced tree height and rootstock growth, and induced increased scaling of the rootstock. There was also a greater (although not statistically significant) growth reduction in trees of Washington navel on Carrizo citrange infected with a severe Class A isolate than was recorded in trees infected with a Class B isolate (Murcia et al. 2015).
To protect the Australian citrus industry from graft-transmissible diseases, a budwood and seed scheme is operated by the Australian Citrus Propagation Association (trading as Auscitrus). To ensure propagation materials supplied by Auscitrus are of high-health status, the budwood and rootstock seed source trees are pathogen tested by an independent laboratory (Donovan et al. 2015). Equivalent schemes are operated elsewhere in the world, such as in the United States of America, Spain and South Africa (Vidalakis et al. 2010;Pina et al. 2015;Jooste et al. 2019). Methods to detect CEVd have evolved over the years, and have included biological indexing, sequential polyacrylamide gel electrophoresis (sPAGE), imprint hybridisation, reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Roistacher et al. 1977;Rivera-Bustamante et al. 1986;Palacio-Bielsa et al. 1999;Bernad and Durán-Vila 2006;Osman et al. 2017). RT-qPCR is currently the method of choice for re-testing of Auscitrus high-health status supply trees but, for determining the initial pathogen status of a tree, it is standard practice to use a secondary method to independently confirm the results from the pathogen testing.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a well-established diagnostic technology that is based on auto-cycling strand displacement DNA synthesis performed by Bst DNA polymerase, which has high strand displacement activity. RT-LAMP is an attractive alternative to many older diagnostic technologies, as it allows rapid and highly sensitive detection of an RNA template, while avoiding the need for thermocycling. Bst DNA polymerase is also a robust enzyme that remains active in the presence of contaminants that would otherwise inhibit a PCR, thereby allowing crude extracts to be used as templates for the reaction (Francois et al. 2011). The RT-LAMP amplification products can be visualised by gel electrophoresis, by observing turbidity changes in the reaction or by using colour changing dyes such as SYBR™ Green I, PicoGreen or hydroxy naphthol blue. Real-time detection, using fluorescent dye such as SYBR™ Green I, can be combined with portable instruments such as the Genie systems (Optigene, Horsham, UK) allowing rapid confirmation of the infection status of a field sample, permitting management strategies to be implemented immediately. RT-LAMP assays have been developed for citrus tristeza virus (CTV) (Warghane et al. 2017;Selvaraj et al. 2019), citrus leaf blotch virus (Liu et al. 2019) and Indian citrus ringspot virus (Kokane et al. 2021) but not so far for any citrus-infecting viroids. A RT-LAMP assay has also been developed to allow the simultaneous detection of six pospiviroids, but CEVd is not among the target pathogens (Tseng et al. 2021).
In this study, we describe the development of a novel onestep RT-LAMP method for the sensitive, robust and rapid field-based detection of CEVd in citrus plants using six primers, including two loop primers, specifically designed to amplify multiple strains of CEVd.

CEVd isolates
The isolates used in this study, representative of the two classes of CEVd, were CL156 (Class A) and CL189 (Class B). Both isolates were maintained in 'Etrog' citron (Citrus medica) as part of the pathogen collection held by the New South Wales Department of Primary Industries (NSW DPI) at the Elizabeth Macarthur Agricultural Institute (EMAI).

RNA extraction methods
Total nucleic acids were extracted from midrib tissue of fully expanded leaves from the most recent flush using a MagMAX Plant RNA extraction kit (Applied Biosystems; Thermo Fisher Scientific, MA, USA), according to the manufacturer's instructions except that the DNase I digestion step was omitted. Samples extracted using this method include the CL189 CEVd isolate and 69 Australian field samples used to compare RT-qPCR and RT-LAMP assays for CEVd detection (Supplementary Table 1). This same extraction method, including DNase I digestion, was used to extract the CL156 isolate used for the assay validation (different extraction methods were used due to a change in preferred laboratory procedure). The RNA concentration of the purified CL156 extract was determined using a Nano-Drop 2000 (Thermo Fisher Scientific). A ten-fold (1:10) serial dilution of CL156 RNA extract in nuclease free water (Applied Biosystems; Thermo Fisher Scientific) from undiluted to 1:10,000, was prepared to evaluate assay sensitivity. RNA extracts of other citrus graft-transmissible pathogens (Supplementary Table 2), used to verify the assay, were accessed from the pathogen collection held at EMAI. These extracts were obtained using various commercial kits with phenol-based, column or magnetic bead extraction methods.
A crude nucleic acid extraction method suitable for use in the field was also done using the Plant Material Lysis Kit (Optigene), where leaf material was homogenised and diluted as per the manufacturer's instructions, for use as a template in the CEVd RT-LAMP assay. The crude extract and 1:10, 1:100 dilutions of the extract in dilution buffer were tested in triplicate using the Genie ® III (Optigene) over three runs.

Cloning and sequencing
Full genomes of CEVd isolates, CL156 and CL189, were amplified from RNA extracts using the outward-facing adjacent primers CEVd-F1 and CEVd-R1 (Bernad and Durán-Vila 2006), and the PCR products were then cloned using the pGEM-T easy vector system (Promega, WI, USA), as per the manufacturer's instructions. Crude DNA extracts from five clones of each isolate were prepared by resuspending the bacteria in water and heating to 95 °C for 10 min followed by a 1 min centrifugation at 15,000 × g to clarify the extract. CEVd cDNA was confirmed in the clones by RT-PCR using the CEVd-F1 and CEVd-R1 primers and Sanger sequencing of the amplified products.

RT-LAMP primer design
Two hundred and forty-six CEVd complete genome sequences were downloaded from the NCBI Non-Redundant Nucleotide Database and aligned using the ClustalW alignment algorithm within Geneious Prime (Version 220.1.2, Biomatters, New Zealand), operated using default settings. Representatives of different CEVd variants were selected and aligned with CEVd sequences obtained from the severe and mild Australian isolate variants from this work. Primers for use in the CEVd RT-LAMP were designed using the Premier Biosoft LAMP Designer (Version 1.16, PRE-MIER Biosoft, CA, USA) with default parameters; these sequences were manually assessed, and degenerate bases introduced into the primers (Table 1) to detect a wider range of CEVd variants.
Reaction mixes were incubated at 65 °C for 30 min using a Rotor-Gene Q real time PCR cycler (QIAGEN, Hilden, Germany) or 40 min using a Genie® III instrument. Incubation was followed by a melt step for confirmation of amplification specificity, from 98 °C to 75 °C at 0.5 °C/sec. Positive results were defined when using the Genie® III by the time taken (T amp ) by the reaction for fluorescence to exceed a 10,000-threshold value for amplification. assay. The RT-qPCRs were carried out in 10 µL reaction volumes, containing 5 µL qScript XLT 1-Step RT-qPCR ToughMix (Quantabio, MA, USA), 4.5pmol of each primer, 1.5 pmol of probe and 1 uL of template, with thermocycling conditions of 50 °C for 15 min, 95 °C for 2 min, then 40 cycles of 95 °C for 10 s and 62 °C for 20 s. The RT-qPCR assay was run in a QuantStudio 5 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific); the Cq values were determined using the QuantStudio Design and Analysis Desktop Software v1.5.1 (Applied Biosystems; Thermo Fisher Scientific).

Sensitivity and specificity of the CEVd RT-LAMP assay
The RNA concentration of the extract from the CEVd isolate, CL156, was determined to be 236 ng/µL. The RNA concentration of the linearized CL156-H1 trRNA was also determined using this instrument and found to be 15,280 ng/ µL. Using this concentration and the length of the trRNA, the estimated copy number was calculated to be 6.1 × 10 13 copies/µL. Two ten-fold dilution series were used to validate the RT-LAMP. The first contained CL156-H1 trRNA diluted with plant RNA extract (dilutions from 6.1 × 10 12 to 6.1 × 10 3 copies/µL), and the second, CL156 nucleic acid extract from undiluted (236 ng) to a 1:10,000 (24 pg) dilution. No amplification was observed from healthy plant and water controls. The assay detected 6.1 × 10 6 copies in triplicate (Fig. 2) and one of three dilutions of 6.1 × 10 5 copies of CEVd trRNA, or 236pg CEVd RNA extract (Table 2). All plasmid clones from both isolates (CL156 and CL189) were amplified with the CEVd RT-LAMP assay (data not shown).
The RT-LAMP assay was shown to be specific for the target organism given no amplification was observed in sample extracts for any citrus pathogens other than CEVd.

Sensitivity of CEVd RT-LAMP
A transcribed RNA (trRNA) control was produced from a clone to assess the sensitivity of the CEVd LAMP assay. Briefly, clone H1, derived from the Australian CEVd isolate CL156, was extracted using the Isolate II Plasmid Mini Kit (Meridian Biosciences, OH, USA). The plasmid was linearized with the restriction enzyme NdeI, and the DNA concentration of the linearized product determined using a NanoDrop 2000 following purification with an Isolate II PCR and Gel Kit (Meridian Biosciences). The linearized product was transcribed into RNA using the MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific) and remaining DNA was removed with DNase I, as per the manufacturer's instructions. The viroid trRNA copy number present was calculated from the RNA concentration and the length of the trRNA, and a ten-fold dilution series in known CEVdnegative plant RNA extract was prepared.

Specificity of CEVd RT-LAMP
The specificity of the CEVd RT-LAMP assay was evaluated by testing undiluted nucleic acid extracts from plants that previously tested positive using molecular assays (data not shown) for various citrus graft-transmissible viruses and viroids (Vaira et al. 2003 Table 2) including CTV and hop stunt viroid. The RT-LAMP assay was run on a RotorGene Q thermocycler using the conditions described above.

Validation of CEVd RT-LAMP in a second laboratory
Three purified RNA extracts representing both Class A and Class B of CEVd were sent to the diagnostic laboratory at the Western Australian Department of Primary Industries and Regional Development in Perth. These samples had RNA concentrations of between 30 and 62.3 ng/µL determined using a NanoDrop 2000 and were tested on a Genie® II (Optigene) instrument using the RT-LAMP assay as described above.

Detection comparison between RT-LAMP with RT-qPCR assays
Leaf midrib or green bark samples from sixty-nine field trees, selected to represent a range of different citrus varieties and types, were tested for CEVd using RT-qPCR (Osman et al. 2017) and the newly developed RT-LAMP assay to further validate the performance of the RT-LAMP

CEVd RT-LAMP for field-based application
A ten-fold serial dilution of CL156 RNA extract (from 23.6 ng to 2.36 pg of RNA) was prepared to compare the sensitivity of the RT-LAMP assay in a RotorGene Q thermocycler and a Genie® III instrument. The results obtained show that using a Genie® III, a sample of a 1:1,000 dilution of nucleic acid extract or 236 pg RNA could be consistently detected by the RT-LAMP assay (Fig. 3a). The annealing temperature for each of the dilutions tested in the Genie® III instrument was 87.6-87.8 °C (Fig. 3b). The assay was

Detection comparison between RT-LAMP and RT-qPCR assays
A strong correlation was obtained between the diagnostic results obtained for 69 field samples from a range of citrus species and hybrids using both RT-qPCR and RT-LAMP (    The copy numbers of the trRNA dilution series amplification curves are coloured as follows; 6.1 × 10 12 -red, 6.1 × 10 11 -green, 6.1 × 10 10 yellow, 6.1 × 10 9 -blue, 6.1 × 10 8 -pink, 6.1 × 10 7 -black, 6.1 × 10 6orange, 6.1 × 10 5 -purple . The healthy plant control (purple) did not amplify in this assay in plants (Tangkanchanapas et al. 2018;Verma et al. 2020;Tseng et al. 2021). The LAMP diagnostic assay developed in this study enables the rapid detection of CEVd; testing can be completed in less than 60 min including sample preparation. This efficiency constitutes a considerable time saving when compared with RT-PCR and RT-qPCR assays that include laborious RNA extractions. Visualisation of the amplification product is based on the real-time detection of fluorescence generated from positive samples. Detection of the RT-LAMP products using this visualisation method without opening the reaction tube, reduces the risk of cross-contamination by aerosols (Panno et al. 2020) and the time taken to gain a result.
The CEVd RT-LAMP assay was able to discern CEVd from other citrus graft-transmissible pathogens. Robust specificity is essential as Australian citrus trees regularly harbour endemic pathogens; CTV is prevalent throughout Australia and other viroids, such as HSVd and CDVd, are not uncommon. The assay also successfully detected CEVd in different citrus cultivars.
The sensitivity of the CEVd LAMP assay was high and not to the same level of RT-qPCR, that was approximately ten-fold more sensitive. This result is consistent with other RT-LAMP assays, including an assay developed for cucurbit leaf crumple virus (Waliullah et al. 2020), where the sensitivity of the RT-LAMP was higher than RT-PCR but lower than RT-qPCR. In the current study, field samples in which CEVd was detected using RT-qPCR also amplified a product using the RT-LAMP assay, displaying a strong correlation of results between the two detection methods. RT-qPCR remains the gold standard for the detection of CEVd but the new RT-LAMP assay provides an additional diagnostic tool for near-field based diagnosis of CEVd to rapidly inform disease management decisions. For example, trees identified as infected with CEVd could be removed from nurseries or orchards given there is no cure for exocortis disease. Alternatively, RT-LAMP could be used to identify trees that are potentially free of CEVd to facilitate the import or commercialisation process for new varieties. However, CEVd is not the only pathogen of concern and the greater sensitivity of RT-qPCR means that RT-LAMP would not entirely replace laboratory testing of budwood supply trees but rather complement the existing citrus viroid detection tools.
In conclusion, we have successfully developed an RT-LAMP assay for the rapid detection of CEVd that is highly specific, sensitive, robust and with near-field capability. The assay, when used with the Genie® III field-based detection system, allows a rapid turnaround time from sampling to diagnostic result and will be a valuable tool to limit the propagation or dissemination of budwood from CEVdinfected plants. To the best of our knowledge, this is the repeated using the same dilution series in a RotorGene Q thermocycler with similar results obtained (Table 2; data not  shown).
To further examine the applicability and robustness of the RT-LAMP assay under simulated field conditions, a dilution series of crude nucleic acid extraction of citrus leaf material containing the CL156 isolate of CEVd and, a CL156-H1 plasmid trRNA dilution series containing from 6.1 × 10 11 to 6.1 × 10 8 copy numbers/µL were tested in triplicate using the Genie® III, an example of which is seen in Fig. 4a. The annealing temperature of the plasmid trRNA (88.6 °C) was found to be marginally higher than that of plant extracts (87.7 °C) (Fig. 4b). The results show that in all dilutions tested, RT-LAMP products were consistently amplified in all sample preparations and dilutions. A comparison of the multiple assays using purified nucleic acid extracts, crude extract or plasmid trRNA run on the Genie® III instrument is shown in Table 4.
The RT-LAMP assay produced comparable results in a second laboratory with all samples containing CEVd amplified successfully and no amplification of healthy controls.

Discussion
LAMP and RT-LAMP-based assays have proven to be simple, efficient, highly specific and sensitive techniques for the detection of animal and plant pathogens (James et al. 2010;Bekele et al. 2011;Rani et al. 2019;Sarkes et al. 2020). However RT-LAMP has not gained great traction for use with viroids which have small and variable genomes, providing restricted opportunities to design a generic set of primers to detect all variants of a specific viroid species (Tangkanchanapas et al. 2018). There are many sequence variants of CEVd that are known to exist (Visvader and Symons 1985;Gandía et al. 2005), and the assay developed in this study was designed to detect multiple variants of the viroid with a particular focus on the CEVd variants found in Australia. However, the assay was successful in amplifying an isolate from the United States of America (CE262, Supplementary Table 2), reflecting that sequence analysis in the development of the assay contributes to the likely detection of the majority of variants found throughout the world.
RT-LAMP assays have been designed previously to detect sequence variants using degenerate bases and mismatches to particular variants in primers with success (James et al. 2010;Park et al. 2013;Komatsu et al. 2015;Tangkanchanapas et al. 2018). A similar approach was employed in this study to cover as many CEVd variants as possible, and the results support other studies where RT-LAMP was shown to be a successful diagnostic tool for viroid detection Fig. 4 Detection of citrus exocortis viroid (CEVd) by reverse transcription loop-mediated isothermal amplification (RT-LAMP) performed using a Genie® III showing (a) amplification and (b) annealing temperature curves. Results were obtained by testing a ten-fold dilution series of the CEVd CL156-H1 plasmid trRNA from 6.1.5 × 10 11 to 6.1 × 10 8 copies and a crude extract (Optigene) of a plant containing CEVd isolate CL156 and, 1:10 and 1:100 dilutions of this extract (reactions colour coded as shown in the figure key). The healthy control (grey) shows no amplification Funding Open Access funding enabled and organized by CAUL and its Member Institutions.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest
The authors declare that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Table 4 Results from using the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for citrus exocortis viroid (CEVd) on the Genie® III, detection time (min) (based on time taken for amplification fluorescence to reach 10,000) for positive reactions of CEVd isolate, CL156, in multiple assays using either purified nucleic acid extracts, crude extract or trRNA of CL156-H1 plasmid, are shown. NA = not applicable Sample Dilution CEVd copies/ nucleic acid concentration