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Preserving plant samples from remote locations for detection of RNA and DNA viruses

Viral diseases in plants have a significant impact on agricultural productivity. Effective detection is needed to facilitate accurate diagnosis and characterization of virus infections essential for crop protection and disease management. For sensitive polymerase chain reaction (PCR)-based methods, it is important to preserve the integrity of nucleic acids in plant tissue samples. This is especially critical when samples are collected from isolated areas, regions distant from a laboratory, or in developing countries that lack appropriate facilities or equipment for diagnostic analyses. RNAlater® provides effective, reliable sample storage by stabilizing both RNA and DNA in plant tissue samples. Our work indicated that total RNA or DNA extracted from virus-infected leaf samples preserved in RNAlater® was suitable for reverse transcription polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, high-throughput sequencing (HTS), and enzyme-linked immunosorbent assay (ELISA)-based diagnostic analyses. We demonstrated the effectiveness of this technology using leaf tissue samples from plants with virus symptoms grown in farmers’ fields in Bangladesh. The results revealed that RNAlater® technology was effective for detection and characterization of viruses from samples collected from remote areas and stored for extended periods. Adoption of this technology by developing countries with limited laboratory facilities could greatly increase their capacity to detect and diagnose viral infections in crop plants using modern analytical techniques.


Introduction
Studies have shown that leaf tissue samples of virus-infected plants can degrade rapidly during transit or storage and require immediate appropriate sampling and preservation (Wang et al., 2018a). This is needed to obtain DNA and RNA of sufficient quality and quantity, the most important step for subsequent reliable molecular detection and characterization of plant viruses (Huang et al., 2017;Mäki et al., 2017;Patel et al., 2017). Sample preservation for Frontiers in Microbiology 02 frontiersin.org virus detection usually involves freezing plant tissues with dry ice or liquid nitrogen and then either storing the frozen samples in an ultracold freezer or shipping them on dry ice (Prendini et al., 2002). The cost of shipping with dry ice from remote areas is high and can make sample preservation an obstacle in developing countries. RNA and DNA can be quickly degraded by endogenous nucleases and by oxidative processes. Degraded DNA is of limited use because large DNA fragments may be required for downstream molecular analyses (Sirakov, 2016). RNA can also be degraded by cellular and environmental endogenous ribonucleases if transit or storage conditions are not ideal, or are of long duration (Fleige and Pfaffl, 2006;Relova et al., 2018).
RNAlater ® is a highly effective fixative for preserving biological materials (Bennike et al., 2016;Kruse et al., 2017). It is a commercially available reagent that acts as a nucleic-acid-preserving buffer, containing high concentrations of quaternary ammonium sulfates and cesium sulfates that inhibit degradation of RNA or DNA by RNases and DNases. RNAlater ® can preserve RNA and DNA in plant tissue at room temperature for several weeks and is recommended for PCR and genome sequencing analyses (Yockteng et al., 2013). To date, detection and characterization of plant viruses from leaf tissue stored in RNAlater ® has not been detailed.
We assessed the efficacy of collecting papaya (Carica papaya), banana (Musa paradisiaca), and taro (Colocasia esculenta) leaves with virus-like symptoms in RNAlater ® to protect their RNA and DNA. We then analyzed the tissues using enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), complementary DNA (cDNA) synthesis, reverse transcription (RT)-PCR, single-tube nested PCR (STNP), and high-throughput sequencing (HTS). We identified complete and partial genomes of viruses and subviral agents using this approach. These findings could be useful in surveillance programs during virus outbreaks in agricultural systems of developing countries.

Materials and methods
Effect of extended storage times in RNAlater ® on nucleic acid quality, quantity and virus detection and characterization by PCR and ELISA Papaya leaf samples with typical virus-like symptoms such as mosaic, ringspots, and leaf distortions as well as asymptomatic leaf samples, were collected from the island of Oahu, Hawaii. The samples were cut into small pieces and 500 mg added to 2 ml of RNAlater ® solution (Qiagen, Germany). After 7, 15, or 30 days at room temperature, immersed leaf tissues were kept at 4°C for 3 days before being transferred to 20°C for 1 week and then stored at-80°C for 1 month.
Leaf samples selected for plant virus detection were cleaned and dried with a paper towel, and then ground into powder with liquid nitrogen in a sterile mortar and pestle. Total RNAs were extracted from 100 mg of the sample with the RNeasy ® Plant Mini Kit (Qiagen Inc., Germany). The RNAs were eluted in 80 μl of RNase-free water and stored at-80°C for later use. Yield and quality of the total RNAs were assayed using 1% agarose gel electrophoresis and spectrophotometry.
Papaya total RNA samples were tested for viruses by RT-PCR assays using universal potyvirus-specific nuclear inclusion body (NIb) primers (Zheng et al., 2010), papaya ringspot virus (PRSV)specific coat protein (CP) primers (Bateson et al. 1994) and PRSVspecific STNP assay (Hamim et al., 2018), resulting in amplicons of 350 bp, 905 bp and 128 bp, respectively. The reaction mixture consisted of 2 μl RNA, 1 μl random hexamer primers (50 μg/ml) and 6.5 μl RNase-free H 2 O. The reaction was incubated for 10 min at 72°C and quickly chilled on ice. A cocktail of 5 μl dNTP mixture (2.5 mmol/l each), 4 μl 5 × MMLV reaction buffer, 1 μl MMLV reverse transcriptase (200 U/μl) and 0.5 μl RNase inhibitor (40 U/μl) was then added (Promega, United States). The RT reaction was incubated at 25°C for 10 min, and then 42°C for 50 min. Reactions were chilled on ice and stored at-20°C prior to potyvirus NIb (Zheng et al., 2010) or PRSV-CP (Bateson et al. 1994) RT-PCR assay and PRSV-specific STNP (Hamim et al., 2018). Specific primer pairs reported by Noa-Carrazana et al. (2007;  Efficacy of RNAlater ® for determining whole-genome sequences of PRSV isolates by HTS from papaya leaf samples collected in Bangladesh PRSV-specific STNP (Hamim et al., 2018), and ELISA (Agdia Inc., United States) analyses were carried out on 11 papaya leaf Frontiers in Microbiology 03 frontiersin.org Candidates included contigs that matched viral genomes and had a total E-value of 10 −4 or below. To provide the annotation finally required for viral agent identification, the narrowed list of viral hits was then evaluated against the complete nonredundant GenBank databases using BLASTx to match sequences against protein (nr) and BLASTn to compare sequences against nucleotides (nt). Full-length sequences of PRSV genomes BD-1 and BD-2 obtained by HTS from composite samples pap-1 and pap-2, respectively, were verified by direct sequencing of PCR-amplified overlapping DNA fragments using virus-specific primers designed from the HTS-derived sequences (Table 1). PCR amplifications were performed on cDNA synthesized from total RNA as described above and amplified using the following conditions: 4 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 50 to 65°C depending on the primers, and 2 min at 72°C; with a final elongation at 72°C for 7 min (Hamim, 2019;Hamim et al., 2019a). The PCR products were resolved on 1% agarose gel in 1X TAE buffer. Gels were stained with ethidium bromide and visualized in a UVP transilluminator. PCR products of the predicted size were purified, cloned into the PGEM-T Easy ® cloning vector (Promega, United States), and sequenced at the University of Hawaii's 1 https://www.ncbi.nlm.nih.gov/genome/viruses/ Advanced Genomic and Sequencing Services and at Genewiz, in California.
Efficacy of RNAlater ® for detection of isolates of PRSV and Begomoviruses from papaya leaf samples collected from different remote regions in Bangladesh The presence of specific PRSV isolates was detected by RT-PCR assays from RNAlater ® -preserved papaya leaf samples with virus-like symptoms: mosaic, ring spots, leaf curl and leaf distortion collected from eight geographical districts in Bangladesh (Table 3). Total RNAs were extracted from the samples using the RNeasy ® Plant Mini Kit, and cDNAs synthesized as reported (Hamim, 2019;Hamim et al., 2019a).
Total DNAs were extracted from 100 mg of the papaya leaf samples from Bangladesh stored in RNAlater ® with the DNeasy ® Mini extraction kit (Qiagen Inc., Germany). The DNAs were eluted in 50 μl of DNase-free water and stored at-20°C for later use. Yield and quality of the total RNAs were assayed using spectrophotometry (Thermo Fisher Scientific, Waltham, MA, United States). DNA extracted from papaya leaf samples was used to test for the presence of begomoviruses using PCR with begomovirus-specific primer pairs (Table 1), accompanied by Sanger sequencing (Hamim et al., 2020). From these analyses, full-length genome sequences of specific begomoviruses as well as the cognate betasatellites or associated components and defective betasatellites were obtained by direct sequencing of PCR-amplified overlapping DNA fragments using virus-specific primers (Table 1). In addition, RNAlater ®stored papaya leaf samples were used to examine the geographic distribution of various begomoviruses by PCR using virusspecific primers.

Efficacy of RNAlater ® for detection of viruses in taro and banana leaf samples collected from Bangladesh
Total RNA was extracted from taro leaf samples in RNAlater ® using the RNeasy Plant Mini Kit and synthesis of cDNA was performed using M-MLV reverse transcription (Promega, United States; Wang et al., 2017). A universal potyvirus-specific ELISA (Agdia Inc., USA) and a universal potyvirus-specific RT-PCR (Zheng et al., 2010) were used to test two samples with symptoms kept in RNAlater ® using CTAB method and were tested by PCR using BBTV-Rep gene-specific primers and CP-specific primers BBTV-HAF1 and BBTV-HAR1 . Six samples were also tested for BBTV using a BBTVspecific antibody in a triple-antibody sandwich-ELISA (Agdia, Elkhart, Inc.).

RNAlater ® on detection and characterization of RNA viruses
Average RNA yields obtained from virus-infected papaya leaves collected on Oahu, Hawaii, and stored for 7, 15, and 30 days in RNAlater ® were 315.17 ± 62.23 ng/μl, 355.1 ± 86.5 ng/ μl and 211.03 ± 61.03 ng/μl, respectively, compared to 670.17 ± 216.18 ng/μl total RNA from fresh samples. A 260/280 ratio greater than 2.0 was obtained for RNAs isolated from both RNAlater ® -stored and fresh papaya leaf samples. Gel electrophoresis analysis of total RNA in agarose gels confirmed the presence of bands corresponding to the 28S and 18S ribosomal RNA (rRNA) for all storage times and for fresh samples, providing a qualitative assessment of the rRNA integrity in RNAlater ® -stored samples (Figure 1). The absence of smearing in the agarose gel indicated the total RNAs were intact and not degraded. Leaves stored in RNAlater ® tested positive in potyvirus-specific RT-PCR with potyvirus NIb primers NIb2F and NIb3R (Figure 2A). Samples with virus-like symptoms stored in RNAlater ® solution for different lengths of time, and fresh samples, were also positive in STNP reactions specific for PRSV, and RT-PCR reactions using primers specific for PRSV CP (Figures 2B,C, respectively) as were the ELISA assays. In addition, we successfully amplified eight overlapping DNA fragments (F1 to F8) correspond to 3′ to 5′ of the fulllength PRSV genome of a Hawaiian PRSV isolate (HA) sequentially from a virus-infected papaya leaf sample that had been collected from Oahu, Hawaii and stored for 1 month in RNAlater ® . These DNA fragments were amplified using previously reported primers (Figure 3; Table 1; Noa-Carrazana et al., 2007). Sizes of these amplified products varied from 1,071 to 2,026 bp.
Papaya leaf samples with virus-like symptoms collected from Bangladesh were used to study the efficacy of RNAlater ® in combination with HTS technology for detecting viruses. The 11 samples stored in RNAlater ® , and tested with PRSV-specific STNP and ELISA were positive for PRSV. These samples were divided into two separate composite samples and full-length genomes of the two PRSV isolates, BD-1 and BD-2 identified by HTS ( Table 2). The complete genome sequences of BD-1(MH444652) and BD-2 (MH397222) were verified by Sanger sequencing of Gel electrophoresis of total RNA extracted from fresh leaves or leaves harvested and stored in RNAlater ® for 7, 15 or 30 days.
Bands corresponding to intact ribosomal RNA (rRNA) species are shown. overlapping DNA fragments obtained by RT-PCR using virusspecific primers and total RNA extracts from papaya leaf samples GAZ-52 and CD-129, respectively, which were stored in RNAlater ® (Figure 3). The effectiveness of RNAlater ® technology in routine RT-PCR assays for RNA viruses was also tested. Of the 118 papaya samples tested from eight different districts of Bangladesh, 22 were positive for PRSV BD-1, 81 were positive for PRSV BD-2, and both BD-1 and BD-2 were found in 13 of the samples (Table 3).
We investigated the efficacy of RNAlater ® technology for detecting dasheen mosaic virus (DsMV) in RNAlater ® -stored, symptomatic taro leaf samples. The two symptomatic samples tested positive for potyvirus with RT-PCR and ELISA, while an asymptomatic taro leaf sample tested negative (Figure 4). Sanger sequencing of the resulting PCR amplicons confirmed the presence of DsMV (accession MH036416; Wang et al., 2018b). DsMV CI-specific primers were used with RT-PCR to check for the presence of virus in total RNA extracts from 13 RNAlater ®stored taro leaf samples ( Figure 5; Wang et al., 2017). Eight of the samples produced target virus-specific bands. DsMV-specific triple-antibody sandwich-ELISA also confirmed that these eight samples were positive for DsMV.

Effect of extended storage times in
RNAlater ® on detection and characterization of DNA viruses DNA extracted from RNAlater ® -stored papaya leaf samples was used to test for begomoviruses. A PCR test using a begomovirus-specific primer pair produced the predicted PCR product corresponding to the begomovirus DNA-A component in 43 of 51 samples examined. Of the 43 PCR products, Sanger sequencing identified 29 sequences corresponding to tomato leaf curl Bangladesh virus (ToLCBV; Table 4). The remainder were identified as being deriving from tomato leaf curl Joydebpur virus (ToLCJoV) and tomato leaf curl New Delhi virus (ToLCNDV ;  Table 4). We amplified and sequenced the full-length 2,760-bp genome of ToLCBV from RNAlater ® -stored Gaz17-Pap samples obtained from Gazipur, Bangladesh. A ToLCBV cognate betasatellite of 1,367 bp was also amplified from the DNA of the same Gaz17-Pap leaf sample (Table 5). Small defective betasatellites DefS39, DefS40, and Defs43 were also amplified from RNAlater ® -stored, ToLCBV-positive samples Gaz19-Pap, Gaz-20-Pap, and Gaz21-Pap, respectively (Table 5). The complete 2,762-nucleotide sequence of ToLCJoV (MT127782) was amplified from RNAlater ® -stored papaya leaf sample J −1 (Table 5).
Additionally, the betasatellite ToLCJoB (MT161673), 1,365 bp in length and associated with ToLCJoV, was obtained from the J1-Pap isolate ( Table 5). The full-length DNA-A and DNA-B Analysis of RT-PCR products from reactions using (A) potyvirusspecific nuclear inclusion body (NIb) primers, (B) PRSV coat protein (CP) primers or (C) PRSV-specific single-tube nested PCR (STNP) and total RNA from symptomatic (+) or asymptomatic (−) fresh papaya leaves or leaves stored in RNAlater ® for various periods.  Frontiers in Microbiology 08 frontiersin.org components of ToLCNDV were amplified and sequenced from the RNAlater ® -stored sample ND-71 (Table 5). These results indicated that DNA isolated from papaya tissues stored in RNAlater ® solutions was able to support amplification of full-length plant DNA virus genomes.
Having demonstrated the efficacy of RNAlater ® as a robust tool for sampling and follow-up partial and full-length genome amplifications of begomoviruses, we performed specific PCR assays of 51 papaya leaf samples stored in RNAlater ® for the begomoviruses ToLCBV, ToLCJoV, and ToLCNDV (Table 4). Three asymptomatic samples and three samples with PRSV-like symptoms tested negative for begomoviruses. PRSV-specific STNP and ELISA tests were also PRSV-negative in the asymptomatic samples, but positive for samples with PRSV-like symptoms. When the 45 samples with leaf curl symptoms typical of begomovirus infection were tested, however, ToLCJoV, ToLCBV, and ToLCNDV were detected, respectively, in 10, 29, and 4 of the samples (Table 4). DNA from six banana samples with symptoms of BBTV tested positive by PCR using BBTV-Rep-gene-specific primers, and CP-specific primers BBTV-HAF1 and BBTV-HAR1  Figure 6). Six PCR-positive samples also tested positive for BBTV by triple-antibody sandwich-ELISA using BBTVspecific antibodies.

Discussion
Our research showed that RNAlater ® technology can be used to transport and store leaf samples with virus-like symptoms from remote locations for extended periods before analysis for viral pathogens. Virus-infected papaya leaf tissue from the island of Oahu, Hawaii was preserved in RNAlater ® solutions for 7 to 30 days at room temperature until RNA extraction, followed by a three-day storage at 4°C, one-week storage at-20°C, and one-month storage at-80°C. Kamitani et al. (2016) used RNAlater ® to prevent RNA degradation of Arabidopsis halleri leaf samples. Their samples were maintained in RNAlater at 4°C for 1 day and then moved to-20°C for 1.5 months before RNA extraction (Kamitani et al., 2016;Honjo et al., 2020).
In our study, RNA yields obtained from RNAlater ® -stored virus-infected papaya leaves were reduced in quantity and quality after different storage times compared to fresh samples. From zero to 30 days, total RNA recovered from samples preserved in RNAlater ® decreased gradually. Although the quantity of total RNA from RNAlater ® -stored samples was reduced compared to fresh samples, the quality was sufficient for detection of plant viruses and amplification of their genomic sequences. Storage in RNAlater ® is considered one of the best methods for preserving RNA for subsequent virus diagnosis (Rawiwan et al., 2021). According to Kohl et al. (2017), PCR and sequencing results from RNAlater ® -stored samples were equivalent to those from snapfrozen samples. Because of the additional equipment and costly transportation required, snap-freezing is often challenging in field situations. We have demonstrated that storage in RNAlater ® solutions can overcome some of these limitations. Analysis of overlapping RT-PCR DNA fragments (F1 to F8) covering full-length PRSV genomes using published PRSV primer sequences for Hawaii isolate (HA) or using primers designed based on HTS analysis for PRSV Bangladesh isolates BD1 and BD2 and total RNA from symptomatic papaya leaf samples stored in RNAlater ® for one-month. Size ranges of PCR fragments are shown at right.

FIGURE 4
Analysis of taro leaf samples stored in RNAlater ® for presence of potyvirus: (A) RT-PCR of total RNA from symptomatic (+) and asymptomatic (−) taro leaf samples, respectively using universal potyvirus primers; (B) potyvirus-specific ELISA of plant extracts from the same sample sources. RT-PCR analysis of total RNA for detection of DsMV from symptomatic (+) taro leaf samples stored in RNAlater ® or asymptomatic controls (−) using DsMV CI gene-specific primers.
Frontiers in Microbiology 09 frontiersin.org The full-length genomes of two PRSV isolates, BD-1 and BD-2, were identified by HTS in our study from composite RNAs pap-1 and pap-2, respectively. This indicates the effectiveness of storage in RNAlater ® prior to whole-genome characterizations of plant RNA viruses. In Kenya, the presence of Moroccan watermelon mosaic virus (MWMV) and three different species of Carlaviruses in papaya tissues stored in RNAlater ® was also determined using Illumina MiSeq ® nextgeneration sequencing (NGS) and validated using RT-PCR and Sanger sequencing (Mumo et al., 2020). Kamitani et al. (2016)   Results obtained from studies of leaf samples from three different plant species stabilized in RNAlater ® , whether collected in Oahu, Hawaii, or brought to Hawaii from farmer's fields in Bangladesh, were acceptable and reproducible among plants. In all cases, these samples yielded total RNA or DNA of a quality and quantity sufficient for plant virus detection using several methods. RNAlater ® was used to preserve the papaya leaf tissues collected for this investigation from several districts of Bangladesh. Then, using specific RT-PCR or PCR tests, we were able to effectively identify, examine the distribution and variability of certain PRSV isolates and begomoviruses in the Bangladeshi papaya samples. Therefore, managing viral diseases in papaya orchards in Bangladesh can benefit from the identification and study of the distribution of viruses and specific virus isolates in RNAlater ® -preserved samples collected from Bangladesh. This technology provides a simple, sensitive, and specific tool for the diagnosis and molecular characterization of plant viral pathogens isolated from plant tissues. We conclude that this technology supports the advanced diagnostic methods used to identify viral pathogens infecting crop plants by allowing samples to be collected from remote areas and stored for extended periods.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

Author contributions
IH and JH: conceptualization. IH, JH, and WB: methodology. IH: formal analysis, writing-original draft preparation, visualization, and investigation. IH, JH, WB, and MM: data curation. IH, JS, MM, MQ, and JH: writing-review and editing. JH and WB: supervision. All authors contributed to the article and approved the submitted version.

Funding
This research was funded by the United States Agency for International Development as part of the Feed the Future initiative under the CGIAR Fund (award number BFS-G-1100002) and the predecessor fund of the Food Security and Crisis Mitigation II grant (award number EEM-G-0004-00013). The research was also funded by the USDA National Institute of Food and Agriculture,