Introduction

Pakistan is a citrus-growing country where 0.198 million hectares area is under citrus fruits cultivation that is higher than other horticultural fruit crops grown in the country [1]. Being an important export crop of Pakistan, Punjab produces more than 97% citrus as compared to other provinces. The cultivated area of citrus in Punjab is 183,210 hectares due to adequate water, and favorable environmental conditions for the citrus growth [2]. Citrus crop is threatened by many pathogens and among them Viroids are notorious. Viroids are the smallest, low molecular weight, infectious obligate endo-parasites having circular, single-stranded RNAs. Viroids have no protein coat, they multiply and replicate through RNA in the host plant and cause-specific diseases in plants, that’s why known as an important viral like phytopathogenic agent [3,4,5,6]. All reported viroids from different host plants are divided into two different families named Pospiviroidae (having specific central conserved region and replicate in the nucleus) and Avsunviroidae (having no specific central conserved region and replicate in the chloroplast) [7,8,9].

Citrus plants are infected by different seven viroid species belong to the family Pospiviroidae and divided into different distinct genera based on their biological and physiological properties, such as Citrus exocortis viroid (CEVd, Pospiviroid), Citrus hop stunt viroid (CHSVd, Hostuviroid) [10], Citrus bark cracking viroid (CBCVd, Cocadviroid), Citrus bent leaf viroid (CBLVd, Apscaviroid), Citrus viroid III (CVd-III, Apscaviroid), Citrus viroid V (CVd-V, Apscaviroid) and Citrus viroid VI (CVd-VI, Apscaviroid) [11,12,13]. Potato spindle tuber viroid (PSTVd) was the first known viroid which belongs to the family Pospiviroidae. The likely secondary structure of the member of Pospiviroidae consists of five structural domains, the right terminal, central conserved, pathogenicity, variable, and left-terminal domain [14]. CVd-V belongs to the genus Apscaviroid of family Pospiviroidae [15,16,17]. The species of Apscaviroid do not produce well-defined disease but the effect was observed on the plant in the form of stunting and yield reduction [18, 19]. Genomic study of CVd-V indicates 293–294 nucleotides bases, higher GC contents, with strong TCR (Terminal Conserved Regions) and characterized by a rod-like conformation with 68.7% paired nucleotides. CVd-V has a single unique character as compared to a member of the genus Apscaviroid with a lower Central Conserved Region (CCR) and strand of CVd-V have C197-U transition that changes the C-G base pair with G-U base pair [20].

Recently, CVd-V was reported in citrus cultivars from Pakistan by Cao et al. [20] with 294 nucleotide bases. It was also reported from Japan due to its unusual ability to replicate in Atalantia citroides (a citrus relative resistant to or unaffected by all previously known citrus viroids including CVd-I, CEVd, CVd-IV, CVd-II, and CVd-III [21]. It induces the symptoms of mild necrotic lesions and fissure (cracks), occasionally chockfull with gum in the stems of indicator plants Etrog citron (Citrus medica L.). Inoculated indicator plants were-placed in controlled conditions where the temperature was 28 °C ± 2, and showed symptoms of bark scaling, cracking of stems, leaf curling, and lesions on the mid-vein of a leaf [22]. Synergistic effect of CVd-V with other viroids in mixed infection on indicator plant (Etrog citron) pronounced the leaf symptoms and dwarfing [23, 24]. Synergistic effect of CVd-V with CBLVd and CVd-III on Etrog citron showed severe epinasty and stunting with multiple lesions in the mid vein of the plant [25]. Although CVd-V was reported in citrus cultivars from different regions of the world, however, recently it was reported from Turkey, Nepal, Pakistan [20, 26], California [27], and Australia [17].

Recent spread of CVd-V in different parts of the world raised interest in conducting a study that may cover the geographic distribution of CVd-V in Pakistan, particularly, in Punjab, the most contributing province in citrus production. It is also pertinent to mention here that now it has been proved that citrus viroids are among the major problems for the citrus industry hampering per acre yield [1, 28, 29]. To evaluate the existence and dissemination of CVd-V in core and non-core citrus cultivated areas of Punjab, a survey was conducted to identify the new citrus hosts of CVd-V using RT-PCR with reported and newly designed primers.

Materials and methods

Survey of citrus orchards and samples collection

A survey was conducted from April 2018 to May 2019 for the collection of viroid-infected samples from citrus cultivated areas of Punjab, Pakistan. A total of 154 samples were collected from seven Districts (Sahiwal, Sargodha, Khanewal, Rahim Yar Khan, Multan, Layyah, Toba Tek Singh) of Punjab, Pakistan. Samples were collected from 8 to 25 years old citrus cultivars, i.e., 12 from ‘Feutrell’s Early’, 53 from ‘Kinnow’ mandarin (Citrus reticulata), 32 from sweet orange (C. sinensis), 21 from sweet lime (C. limettioides), 21 from grapefruits (C. paradisi), 4 from lemon (C. jambhiri), 8 from tangerines (C. tangerina) and 3 from tangelos (C. tangelo). All collected samples were showing characteristic symptoms including bark cracking, mild leaf bending, lesions on the mid-vein of leaf, and severe stunting.

Biological indexing

Biological indexing was done by collecting CVd-V infected bud woods from three infected citrus cultivars of sweet orange, sweet lime, and Kinnow, and stored in a refrigerator. One percent solution of sodium hypochlorite was used to disinfect the buds and other grafting related helping material (scissors, forceps, and surgical blades). Each pot contained one indicator Arizona 861-S1 Etrog citron plant. The T-grafting method was performed to fix the infected CVd-V buds on an indicator plant Arizona 861-S1 Etrog citron already grafted on rough lemon rootstock [30, 31]. The indicator plants were given a T-cut 30 cm above from the soil surface; bud was adjusted into the cut and wrapped carefully for 2 weeks. Grafted plants were maintained at hot temperature in the greenhouse at 32–40 °C in day and 24–32 °C at night. The symptoms of CVd-V were observed 2–3 months after grafting.

Total RNA extraction and RT-PCR assays

100 mg of citrus plant leaf/bark sample was expunged in the centrifuge tube and grounded with plastic micropestle with the help of liquid nitrogen. 500 µl of Plant Triazol RNA Regent (Invitrogen Inc., USA) was added into crushed sample and mixed well. The sample tubes were incubated at room temperature for few minutes. Tubes were centrifuged for 2 min at 12000 rpm after incubation and supernatant was transferred carefully to the new 1.5 ml tube. 100 µl of 5 M NaCl and 300 µl of chloroform were added in the supernatant, mixed thoroughly and centrifuged at 12000 rpm. The top separated phase was transferred to new 1.5 ml tube. 2-Propanol was added in an equal volume and incubated for 10–15 min at room temperature to precipitate RNA. After incubation, the tubes were centrifuged at maximum speed for one minute and pellet of RNA was suspended and dried. Nuclease free water was added to dissolve the pellet and stored at − 20 °C. DNase treatment was given to extracted RNA samples. The RNA concentration and purity was checked by Nano-drop.

cDNA synthesis

cDNA was synthesized by RevertAid™ First-Strand cDNA kit (Applied Bio-system, USA) using the protocol of Fiore et al. [32]. A reaction mixture for each sample contained 5 µl of RNA, 1 µl 10 mM dNTPs, 1 µl random DNA hexanucleotides, 1 µl reverse transcriptase and 3 µl nuclease-free water was prepared. The reaction profile included denaturation at 65 °C for 5 min, reverse transcription with M-MLVRT at 42 °C for 60 min, and the terminal temperature of 85 °C for 10 min.

Primer designing

Specific primers of viroids were-designed using the software Mega X. The primers were designed to test CVd-V having complete sequences with Accession Numbers retrieved from NCBI. Designed primers were analyzed by BLAST search to confirm the specificity for detection. Newly designed back-to-back specific primers were CVd-V AF1 (AGGAGWAGAAAGTACTCACCTG) and CVd-V AR1 (CTWCTCCTCTGCTTTTATT) with the amplicon product size of 294 bp (Table 1).

Table 1 Primers of CVd-V used during the study
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RT-PCR optimization with newly designed primers

After RT reaction, PCR amplification of CVd-V was done by using 1 µl of cDNA as a template with mixture containing 1 µl of each target specific reported primers (10 mM) CVd-V by Cao et al. [20], 0.5 µl of 10 mM dNTPs, 1.7 µl of 25 mM MgCl2, 0.3 µl of Taq DNA polymerase (Invitrogen), 2.5 µl of 10× buffer and make up the total volume of 25 µl with nuclease free water. Conditions for the amplification of CVd-V were; initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 45 s and a final extension at 72 °C for 10 min. The amplified product was confirmed by gel electrophoresis in 1.5% agarose with the expected 294 bp band size of CVd-V.

Purification of PCR products, sequencing, and phylogenetic analyses

Purification of PCR products was performed by FavorPrep PCR Clean-up mini-kit (Favorgen Bioteck Corp; cat. no: FAPCK001-1). Purified PCR products were sequenced using Sanger sequencing technology. The sequences were aligned with Mega X software and compared with other isolates of GenBank using online BLAST on NCBI [33]. After cleaning, the sequences were submitted to database, and accession numbers were obtained. Configuration was done using MUSCLE expertise and maximum probability phylogenetic trees of citrus viroid were constructed using Mega X software version 10.1.8 [34]. The evolutionary distances were computed using the Maximum Composite Likelihood method [35] and are in the units of the number of base substitutions per site, the percent nucleotide sequence identity was calculated. For the construction of the phylogenetic tree of CVd-V, seventeen other CVd-V sequences were obtained from GenBank. Apple scar skin viroid ASSVd; DQ362906) was used as outgroup in the analysis. All isolates of citrus viroid were reported with their accession number, country name, and citrus cultivar. Percentage identities with other viroids were also noted.

Results

Symptoms of observation

During the survey, many characteristic symptoms of viroids including bark cracking, browning of leaf tips, petioles necrosis, mild leaf bending, lesions on the mid-vein of leaves, and severe stunting were observed on all samples of citrus from several locations (Multan, Sahiwal Citrus germplasm, Sargodha, Toba Tek Singh, Layyah, Rahim Yar Khan, and Khanewal) of Punjab, Pakistan. It was observed during field surveys that Kinnow mandarin plants were showing severe bark scaling as compared to other citrus cultivars, while others were showing yellowing, severe stunting, gummosis, and leaf curling.

Molecular detection of Citrus viroid V (CVd-V) through RT-PCR

For the detection of CVD-V, reported primers were used [20]. However, those primers were unable to produce specific PCR products. Therefore, newly designed specific viroid primers by MEGA X were used for the detection. Newly designed primers detected CVd-V successfully with the amplification product of 294 bp (Fig. 1). CVd-V was detected in 102 out of 154 samples from different citrus cultivars through RT-PCR, with an average of 66.66% disease incidence. Cultivar-wise disease incidence of CVd-V was also calculated from all collected samples. Maximum disease incidence was observed on grapefruit (85.71%) as compared to other cultivars, tangerine (75%), Feutrell’s Early “mandarin” (75%), sweet lime (71.42%), tangelos (66.66%), Kinnow mandarin (62.26%), sweet orange (53.12%), and lemon (50%) (Table 2). Palestine Sweet lime (C. limettioides), Roy Ruby (Citrus paradisi), Olinda Valencia (C. sinensis), Kaghzi lime (C. aurantifolia) and Dancy (C. reticulata) were identified for the first time from Pakistan as new citrus host plants of CVd-V, using newly designed primers. Furthermore, the co-infection of CVd-V was also established with other viroids in most tested samples.

Fig. 1
figure 1

Gel electrophoresis analysis shows amplified products of 293 bp from symptomatic leaves (Lane 1–9) collected from Khanewal, Sargodha, Lay Layyah, and Sahiwal. Lane M represents 100 bp Ladder (Invitrogen) and lane N: negative control

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Table 2 Percent incidence of CVd-V in citrus cultivars tested by RT-PCR
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All grafted citron plants produced moderate to severe symptoms of viroids infection (Fig. 2). Moderate symptoms related to CVd-V (leaf curling, bark scaling, necrosis of mid-veins) initiated in 6–8 weeks. Severe bark scaling symptoms by viroids infection on citrons plants were produced with an infected scion of Dancy ‘sweet orange’. Additionally, inoculated with viroid-infected scion, Etrog citron plants were tested by RT-PCR (Table 3).

Fig. 2
figure 2

Symptoms shown by grafted Etrog citron plants infected with CVd-V control plant (A) severe epinasty 3–5 months after grafting (B) severe bark cracking 3–8 weeks after grafting (C) severe stunting as compared to healthy control (D)

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Table 3 Results of biological indexing and RT-PCR analyses for detection of CVd-V
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Disease incidence of district-wise collected samples was also calculated after testing through RT-PCR with an expected amplification of 294 bp. Maximum disease incidence was found to be in core citrus-growing areas (Sargodha, Sahiwal, and Toba Tek Singh) of Punjab, Pakistan (Table 4).

Table 4 District-wise disease incidence (%) of CVd-V
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Fig. 3
figure 3

Phylogenetic analysis of Citrus viroid V (CVd-V) sequences. Shown is a Neighbor-Joining phylogenetic dendrogram based upon alignment of nucleotide sequences of the CVd-V isolates produced as a part of the study presented here with selected CVd-V

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The obtained sequences of CVd-V with their accession numbers are given in Table 5. All five isolates have shared a high nucleotide sequence identity ranging from 98 to 100%.

Table 5 List of isolates with geographic location characterized during this study
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The sequence of isolates of CVd-V characterized here were analyzed along with sequences of other isolates available in database. The phylogenetic tree was constructed keeping 1000 bootstrap value. The phylogenetic tree divided into two main groups. Group one has all sequences obtained during this study and sequences from Japan, Spain, South Africa, USA, Iran, Pakistan, China, Tunisia and North Africa. Group two has only one sequence (JQ348931) which was previously reported from Pakistan. The Neighbor-Joining tree depicted that the four out of five isolates of CVd-V characterized here are grouped with the isolate from Iran (GQ466068), while the isolate R-214 (MN885656) segregates with Spain isolate EF617306 (Fig. 3). The sequence identity of these seven isolates including five of the current study ranges from 98 to 100% (Supplementary Table). Other closely related isolates include Pakistan (JQ348930), USA (MF477859, MF477876), and Iran (KY654684). The isolates from China, Japan, Tunisia, and Africa are distantly related in the tree showing comparatively less identity with our isolates. Roy Ruby isolate (R-214; MN885656) showed 100% homology with the isolate of Spain (EF617306). The isolate from Olenda Valencia (O-250; MN885657) was also 100% identical to the isolate from Iran (GQ466068). Our isolate were assembled and analysed using a CLUSTALV algorithm of Lasergene sequence analysis software (DNA Star Inc., Madison, WI, USA), showed 85.2% sequence homology among each other (Fig. 4). Although our isolates showing close homology among each other, however comparing with the isolates from the world these isolates grouped with Iraninan isolates (GQ466068) and Spanish isolate (EF617306).

Fig. 4
figure 4

Phylogenetic tree of Citrus viroid V (CVd-V) shows the interaction

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Discussion

Citrus viroids are becoming a threat to the citrus production of Pakistan. Viroid species such as CEVd, CVd-II, CVD-I, CVd-III, CVd-IV, and CVd-V have been identified from citrus cultivars of Pakistan [20]. In this study, we identified some new citrus hosts of CVd-V from Pakistan. Previously reported primers were unable for specific PCR amplification of viroids [20]. This is because one of the primer pair (reverse primer) was unable to anneal with CVd-V and did not show sequence homology with other viroids upon BLAST analysis. This was further confirmed by sequence alignment of the CVd-V available in database, in which reverse primer did not match with CVd-V sequences, attachment of forward and reverse primers are given in figure (Supplementary Fig.) Thus, new back-to-back primers (CVd-V AF1/CVd-V AR1) were designed and used for the detection which obtained an expected amplified product of CVd-V with 294 bp. Palestinia Sweet lime, Roy Ruby, Olinda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan with these newly designed primers. Whereas, Cao et al. [20] reported CVd-V from Kinnow and Feutrell’s Early, Mosambi, Saccari, sweet oranges, grapefruits, sweet limes, acid limes, sour orange, lemon, and Jatti Khatti for the first time from Pakistan. However, earlier Cao et al. [36] described most citrus cultivars in Pakistan as the host of two or three viroid species. CVd-V produces mixed infection with other viroids and indicator plants showed leaf curling on co-infection with CBLVd [12, 37]. New host Atalantia citroides a citrus host of CVd-V was also identified from Japan by Ito et al. [38] and, only this viroid can replicate in A. citroides [12]. Disease incidence of CVd-V was about 66.66%, whereas Cao et al. [20] reported very high incidence because different citrus cultivars were tested in this survey rather than testing only samples of Kinnow Mandarins, which are heavily infested. The reason for this high viroid disease incidence in Punjab is that the most of the commercial mandarin and sweet orange cultivars are grafted on rough lemon rootstock, which is highly susceptible to viroid diseases. So, it is hereby recommended to use some new disease-resistant rootstocks.

The Neighbor-Joining tree depicted that the CVd-V isolates detected from Pakistan were closely related to the isolates reported from Iran (GQ466068, KY654684), Spain (EF617306), Pakistan (JQ348930), and the USA (MF477859, MF477876). The central clade with a small branch length consisting of 11 sequences shows these closely related isolates. While, the isolates reported from China, Japan, Tunisia, and North and South Africa are comparatively farther. The closeness of the sequences is also reflected from their sequence distances and percent identity values.

Biological indexing on Etrog citron showed the characteristic symptoms of moderate to severe form and confirmed by RT-PCR results with amplification of 294 bp. Synergistic effect of CVd-V with other viroids for mixed infection on indicator plant (Etrog citron) improved leaf symptoms and dwarfing [23]. Synergistic effect of CVd-V with CBLVd and CVd-III on Etrog citron showed severe epinasty and stunting with multiple lesions in the mid vein of a plant. Because of mixed infection of viroids in the main citrus cultivars of Pakistan, the impact of CVd-V could not be assessed under normal conditions. Although numbers of the genus Apscaviroid do not prompt definite infections, rather they affect gradually in the form of stunting growth and fruit yield decline. Predictable indexing will provide more evidences of the spreading of CVd-V in the world. CVd-V has a wide host range in citrus cultivars as studied by Serra et al. [12]. During this study, it was identified from new citrus cultivars which were not previously investigated from Pakistan. Now, this viroid is distributed worldwide in citrus cultivars. Recently it was reported from Turkey, Nepal, and Pakistan [20, 26] and California [27]. The movement of CVd-V in various geographic regions is presumably the result of the proliferation of contaminated bud wood and the universal trade of plant materials. The natural effect of these movements on CVd-V needs further infectivity assay. In Pakistan, unfortunately most of the citrus nurseries were not registered by the Government and were being handled by non-technical persons. This results in the production of infected plants. Infected bud wood and infected tools are major sources of the spread of viroids. Serious quarantine measures and rules should be imposed to prevent the spread of CVd-V and all other viroids. The nursery should be propagated by technical labor. Hygienic tools should be used in the field during cultural practices. The nursery should be propagated by technical labor.

Conclusion

The infection of CVd-V is increasing in Pakistan and infecting new citrus hosts with small variation in the genome which led to the designing of new primers for its detection. Nurseries and contaminated tools are the main cause of spread of CVd-V. It is imperative to exploring sources of resistance against CVd-V and serious quarantine measures and rules should be imposed to prevent the spread of CVd-V and all other viroids.