Citrus is a host to several pathogens many of which have an economic impact on the crop. Viroids are among of these induced annual worldwide losses in fruit trees (Nemeth, 1986; Roistacher, 1992). They are known as causal agents of many diseases on herbaceous and woody plants (Flores et al., 2005) causing symptoms on leaves (deformation, vein discoloration, epinasty, mottling, chlorotic and necrosis), stems (cankers, bark scaling and/or cracking), fruits malformation, and in severe cases plants death (Kovalskaya & Hammond, 2014).
Potato spindle tuber viroid was the firstly detected on potato plants in 1971 by Danier (Owens et al., 2012), before many viroids were reported infecting several plant crops worldwide, and particularly in warm climate regions (El-Dougdoug et al., 2017).
To date, eight of viroid species belonged to the Pospiviroidae family were reported in citrus: Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III), Citrus viroid-IV (CVd-IV; syn. Citrus bark cracking viroid (CBCVd)), Citrus viroid-V (CVdV), Citrus viroid-VI (CVd-VI), Citrus viroid-VII (CVd-VII), Citrus dwarfing viroid (CDVd), and Citrus bent leaf viroid (CBLVd) (Serra et al., 2008; Ito et al., 2001; Di Serio et al., 2017; Seigner et al., 2020; Duranvila et al., 1988; Chambers et al. 2018).
Viroids possess unique genome structure with few hundreds of nucleotides (246 to 433) in a single-strand circular RNAs; capable to multiply within the host cells (Flores et al., 2015; Serra et al., 2018). Although viroids do not encode any protein, they can replicate autonomously in higher plants (Duranvila, 2019). They are able to reproduce and accumulate in their host nucleus and chloroplast, respectively (Murcia et al., 2015; Flores et al., 2005). The two known families Pospiviridae and Avsunviridae employ asymmetric and symmetric rolling-circle replication mechanism, respectively (Hammann & Steger, 2012).
This study was to report the prevalence of citrus viroids in Palestinian germplasm collection.
Field inspections were carried out on citrus germplasm collection that found in National Agriculture Research Center (NARC), Jenin-Palestine. Putatively viroid like symptoms such as epinasty, stunting, vein clearing, discoloration, distortion of leaves, mottling, necrotic or chlorotic spots, scaling, cankers, and bark cracking were reported (Fig.1).
Lack of specific symptoms; and sometimes symptomless or similar to those of virus sources, it could be hard to determine viroid-infected trees (Aviña-Padilla et al., 2022). Leaves from different forty-two citrus plants (Clementine, Grapefruit, Lemon, Orange, Kumquats, Pomelo, and two rootstocks Volkameriana and Trifoliata) were collected and conserved for molecular assays.
Two-step-RT-PCR was applied to detect citrus viroids: CEVd, CVd-IV, and CVd-III, using specific sets of primers as shown in Table 1.
Table 1: Sets of primers used for one step RT-PCR detection of citrus viroids.
Primer
|
Sequence 5à-3
|
Size bp
|
Position
|
Reference
|
CEVd-R
CEVd-F
|
CCGGGGATCCCTGAAGGACTT
GGAAACCTGGAGGAAGTCGAG
|
371
|
78–98
99–119
|
Gross et al. (1982)
|
CVd-III-R
CVd-III-F
|
CGTCACCAACTTAGCTGCCTTCGT
GTCTCCGCTAGTCGGAAAGACTCCG
|
269
|
91–112
135–159
|
Sieburth et al. (2002)
|
CVd-IV-R
CVd-IV-F
|
CCGGGGATCCCTCTTCAGGT
GGTGGATACAACTCTTGGGTTGT
|
138
|
52–71
217–239
|
Puchta et al. (1991)
|
18sa-R
18sa-F
|
TTCAGCCTTGCGACCATACT
CGCATCATTCAAATTTCTGC
|
844
|
|
Gambino & Gribaudo (2006)
|
(R) antisense primer, (F) sense primer; (a) Internal control of plant rRNA.
The total nucleic acid (TNA) was extracted from 100 mg of citrus tissues and according to Foissac et al. (2005) procedure. Briefly, leaf tissues were homogenized in grinding buffer (pH 5.6-5.8) [4M Guanidine thiosianate, 0.2 M sodium acetate, pH 5.2, 25 mM EDTA, 1.0 M potassium acetate, 2.5% PVP-40 and 2% Sodium bisulphate] with addition of 0.5% of sodium metabisulfite and 150 µl of 10% Sodium lauroyl sarcosinate (Sarkosyl) before incubated at 70°C for10 min. On ice, 500 µl of extraction were mixed with binding solution (250 μl ethanol absolute, 500 μl NaI 6M and 35μl Re-suspended silica) and gently agitated for 10 min at room temperature. Pellet was collected and subjected to two times washing with [10.0 mM Tris-HCl, pH7.5; 0.5 mM EDTA, 50.0 mM NaCl, 50% Ethanol]. 150 μl TNA were liberated by distilled water and NanoDrop 2000/2000c UV-Vis spectrophotometers (JENWAY, Genova Nano, Fisher Scientific UK company, England.) was used to measure their quantity and quality.
Reverse transcription reaction was carried out from 500 ng TNA and 200 units of SuperScript™ III RT (Life Technologies Corporation) in a final volume of 20 µl. Firstly, TNA were mixed with 0.5µl of random hexamers primer (50ng/µl), 0.5 µl oligodT (50 µM), and 1 µL dNTPs (10 μM) in a final volume of 10 µl. The mixture was incubated at 65°C for 5 min. On ice, 2 µl of 10x RT buffer, 2 µl of 0.1M DTT, 4µl of 25mM MgCl2, 1 µL RNaseOUT(40U/µL), and 1µL SuperScriptIII RT(200U/µL) were added to the mixture, before incubation at 25°C/10 min followed by 50°C/50 min. The reaction was terminated at 85º C for 5 min and cooled at 4ºC for next use in PCR reaction.
Single PCR was performed for each viroid with its pair of primers separately (Table1). 2 μl cDNA were added to a final volume of 25 μl PCR mix composed of 2.5μl of 10x Taq polymerase buffer; 2 μl of 50 μM MgCl2; 0.5 μl of 10 μM dNTPs and 0.25 μl of Taq polymerase (5 unit/μl) (Promega Corporation, USA), with primer pairs final concentration of 0.2 μM for CVd-III and CVd-IV, and 0.5 μM for CEVd, meanwhile 18s primers was reached up to 0.14 μM. After a 5-minute denaturation stage at 94°C, 45 cycles of cDNA amplification were performed, each cycle consisted of a 50-second denaturation stage at 94°C followed by a 55°C annealing step for 50 sec, and 2 min extension at 72°C. The elongation stage was at 72°C for10 min. PCR products were visualized under UV light detector in 1.2 % agarose gel stained with GelRed (Biotium) according the manufacture recommended protocol.
For quick detection of several viroids with one reaction with high sensitivity, specificity and cost effective, multiplex PCR was performed successfully as described by Wang et al., (2009). The mix of the all viroid primer pairs at a final concentration of 0.2 μM except of CEVd (0.5 μM), and 18s (0.14 μM). Briefly, 2 μl cDNA mixture were used in PCR amplification with 2.5μl of 10x Taq polymerase buffer (Promega Corporation, USA), 2 μl of 50 μM MgCl2, 0.5 μl of 10 μM dNTPs, and 0.25 μl of Taq polymerase (5 unit/μl) in 25 μl final volume. The cDNA amplification was done with 45 cycles, after initial denaturation at 94°C for 5 min. Each cycle consisted of: denaturation at 94°C for 50 sec, annealing at 58°C for 50 sec and extension at 72°C for 2 min. The final extension was done at 72°C for 10 min
All viroids were molecularly detected in a single RT-PCRs as well as multiplex PCR which was successfully detected all tested viroids, and thus it could be recommended as quick and efficient tool for simultaneous detection of viroids at one reaction (Fig. 2).
Unexpectedly, high prevalence of citrus viroid infection was reported (52.4%), of which CVd-IV was the highest prevalence among the three detected viroids (Fig3).
CVd-IV (syn. CBCVd) was considered as minor pathogen in the genus Citrus (Duran-Vila et al. 1988). At the same time, it was associated with severe bark cracking on trifoliate orange rootstock (Vernière et al., 2011, Ito et al., 2002; Vernière.et al., 2006). Interestingly, samples from citrus rootstocks (Volkameriana and Trifoliata) were found infected with CVd-IV. This might explain high prevalence of the viroid among citrus in Palestine, beside it has wide host range (Semancik and, Vidalakis 2005). CVd-IV was reported to be one of the main viroids circulating in all citrus-growing areas worldwide (Aviña-Padilla et al., 2022; Belabess et al., 2021).
CEVd as one of the most well-known viroid in citrus was detected in (23.8%) of tested samples (Fig3). This result was expected since it was found in many countries in Africa (South Africa, Ghana, Sudan, Morocco, Algeria, Tunisia, Libya and Egypt) and in Middle east (Turkey, Cyprus, Israel, Saudi Arabia and Oman) (da Graca and van Vuuren, 2003; Opoku, 1972; Hadidi et al., 2003; Al-Harthi et al., 2013; Abou Kubaa et al., 2016; Najar et al.,2017). CEVd was reported to have wide host range (Duran-Vila and Semancik, 2003; Duran-Vila, 2017; El-Dougdoug 2017). It was known to have symptoms on different species of citrus such as stunting, bark sloughing and cracking, leaf epinasty and cracks in the petiole (Bernard et al., 2009; Lin et al., 2015; Škorić et al., 2001; Singh et al., 2009; Palukaitis, 2014; Van Brunschot et al., 2014).
Interestingly, mixed infection was less frequent tested of samples (Table 2), where the CVd-IV was the most frequent one. Nevertheless, mixed infection could be the most likely cause which magnify the symptoms severity and productivity of citrus trees (Belabess et al., 2021).
Table 2. Number of detected samples with CVd-IV, CEVd and CVd-III in germplasm collection plot.
Citrus cultivar
|
Number of tested samples
|
Number of infected samples
|
CVd-IV
|
CEVd
|
CVd-III
|
Single
|
Double
|
Treble
|
Clementine
|
16
|
6
|
2
|
2
|
7
|
0
|
1
|
Orange
|
11
|
1
|
2
|
1
|
2
|
1
|
0
|
Pomelo
|
4
|
2
|
1
|
0
|
1
|
1
|
0
|
Grapefruit
|
3
|
2
|
1
|
1
|
2
|
1
|
0
|
Lemon
|
3
|
1
|
2
|
2
|
0
|
1
|
1
|
Kumquats
|
3
|
1
|
2
|
1
|
0
|
2
|
0
|
Volkameriana
|
1
|
1
|
0
|
0
|
1
|
0
|
0
|
Trifoliata
|
1
|
1
|
0
|
0
|
1
|
0
|
0
|
Total
|
42
|
15
|
10
|
7
|
14
|
6
|
2
|
For molecular analysis; selected isolates from the viroids (CVd-IV, CEVd, and CVd-III) were sequenced using the sequencing facilities at An-Najah National Hospital-Nablus. The obtained sequences were deposited at GenBank under the accession numbers: OP902247 (CVd-IV), OP925746 (CEVd), OP902248 (CVd-III-PS-isolate-1) and OP902249 (CVd-III-PS-isolate-2). BLASTn searching of the CVd-IV revealed high similarity to those from Turkey (MZ995261) and Oman (KC121568), in accordance of what was proposed of their middle east origin (Al-Harthi et al., 2013). Phylogenetic tree was constructed using molecular evolutionary genetics analysis across computing platforms (MEGA-11) (Tamura et al., 2021).) revealed that CVd-IV clustering with other similar viroids available in GenBank records (Fig. 4a).
Nucleotide sequences obtained from CVd-III infected samples, two isolates were revealed with (91.3%) similarity by Pairwise Sequence Alignment-EMBOSS Water provided by European Molecular Biology Laboratory (EMBL); suggesting being putatively locally mutated. Thus, these two isolates considered varied within the viroid genetic variation in the country and designated as CVd-III-PS-isolate-1 and CVd-III-PS-isolate-2. They were clustered together in a phylogenetic tree with other GenBank deposited isolates from the Middle east region as well as others isolated worldwide (Fig.4b). The same can be noticed for the CEVd as it aligned with those isolates accessions retrieved from the GenBank (Fig.4cb).
Although, this study was the first to report of citrus viroid in Palestine; their existence in relatively high prevalence is alarming, especially once rootstocks were detected infected. Knowing their main route of dissemination throughout propagating materials (Lin et al., 2015; Alkowni, 2017); phytosanitary actions are highly recommended which is the only way to prevent further spread of viroids (Papayiannis, 2014; Gergerich et al., 2015). Certification program and sanitary actions to exclude these viroids side by side with viruses are highly appealing. National germplasm collections must be subjected to strictly measurement to ensure distributing propagating plant materials free of viruses as well as virus like pathogens.