Introduction

Potato is the fourth largest staple crop in the world. Biotic stress caused by a pathogen such as virus (Wang et al. 2011) and abiotic stress such as salinity (Hasegawa et al. 2000; Fita et al. 2015) have been/are the two major constraints for sustainable development of the world’s agricultural production including potato.

Potato leafroll virus (PLRV), Potato virus S (PVS) and Potato virus Y (PVY) are among the most damaging viruses attacking potato (Valkonen 2007). PLRV is a single-stranded RNA and phloem-limited Polerovirus (Valkonen 2007). Symptoms induced by PLRV on potato crops include upright rolling and yellowing or reddening leaves, leathery leaves, phloem necrosis, stunting of the plant, and necrosis in tubers. PVS is a single-stranded and belongs to the genus Carlavirus (Adams et al. 2004). PVS has two strains namely ordinary strain (PVSO) and Andean strain (PVSA) (Salari et al. 2011). Most PVS isolates do not induce apparent symptoms on many of potato cultivars, but some isolates do more severe symptoms on susceptible cultivars (Rose 1983). PVY is a single-stranded virus and the type species of the genus Potyvirus. This virus causes typical symptoms on potato such as mosaic patterns on leaves, leaf distortion, necrosis on veins or shoots and tubers, and stunted plant growth, with the symptoms varying from the cultivars infected and virus strains (Singh et al. 2008). Several strains have been found in PVY, including the common strain (PVYO), the necrosis strain (PVYN) and the ringspot necrosis strain (PVYNTN), PVYN-Wi, PVYNTN-NW, PVYNE-11 and PVYZ (Glais et al. 2002; Lorenzen et al. 2008; Singh et al. 2008). According to Singh et al. (2008), PVYo and PVYN were the primary potato-infecting PVY strains. A more recent study performed by Funke et al. (2017) in the Columbia Basin of North America found that occurrence frequency of PVYO strain dropped from 63% in 2011 to less than 7% in 2015 in all PVY-positive plants, while that of recombinant PVYN-Wi strain increased from less than 27% to 53% during the same period.

Existing studies showed single virus infection caused yield losses of 40–60% by PLRV, 10–20% by PVS and 20–50% by PVY (Hamm and Hane 1999; Hane and Hamm 1999; Wang et al. 2011). Mixed infection resulted in a much greater loss of the tuber yield than single infection (Wright 1970; Manzer et al. 1978; Wang et al. 2011; Nyalugwe et al. 2012). Control of potato viruses is at present time effectively achieved by use of certified potato seed tubers (Wang et al. 2011). Results reported from China, the biggest potato-growing country of the world, showed use of virus-free potato plants increased tuber yield by 30–50%, depending on the virus types and potato cultivars infected (Wang et al. 2011). Notably, at present time, certified potato seed tubers are planted only in 10–30% of China’s total potato-growing area, much lower than 90% in developed countries (Wang et al. 2011).

Salinity is one of the major abiotic stresses causing server damage to agricultural production (Hasegawa et al. 2000; Fita et al. 2015). At present time, at least more than 20% of the world’s irrigated cropland is affected by salinity (Flowers 2004) and the situation is even worse in developing countries including China (Fita et al. 2015). Numerous studies have demonstrated salt stress resulted in considerable reductions in potato tuber yield (Nadler and Heuer 1995; Zhang et al. 2005; Shaterian et al. 2005, 2008; Elkhatib et al. 2006; Khalid and Aftab 2016).

In order to better understand the mechanisms involved in the reduced tuber yield caused by either viral diseases or salt stress, great efforts have been exerted to investigate influences of either virus diseases (Wright 1970; Manzer et al. 1978; Hamm and Hane 1999; Hane and Hamm 1999; Li et al. 2013; Nyalugwe et al. 2012) or salt stress (Nadler and Heuer 1995; Zhang et al. 2005; Shaterian et al. 2005, 2008; Elkhatib et al. 2006; Khalid and Aftab 2016) on potato plants, but those of co-stress by virus and salt are quite limited.

Crops grown in field are often exposed simultaneously to abiotic and biotic stress, and responses of plants to co-stress by two or more factors may differ from those to each of the multiple stresses (Suzuki et al. 2014). Mixed infection by viruses frequently occurs in the commercial potato field all over the world (Wang et al. 2011). Therefore, studies on effects of co-stress by virus (including single- and co-infection) and salt on plants are of significance.

We previously reported PLRV- and PVY-infection, singly and in combination, reduced root development, vegetative growth and microtuber production, and altered physiological metabolism and endogenous levels of hormones (Li et al. 2013). The present study further studied growth, microtuber production and physiological metabolism in virus-free and virus-infected potato in vitro plantlets grown under NaCl-induced salt stress. Three major potato viruses, either singly or in combination, were included in the present study. Results reported here would help us to better understand possible mechanism of reduced tuber yield caused by co-stress by virus and salt.

Materials and methods

Plant materials

Potato ‘Zihuabai’ (Solanum tuberosum), a major cultivar grown in China and susceptible to potato viruses, including PLRV, PVS and PVYo, was employed in the present study. Virus-free (hereinafter referred to as healthy) in vitro stock plantlets that had been produced by cryotherapy (Wang et al. 2006) were cultured on a shoot maintenance medium (SMM) composed of Murashige and Skoog (1962) medium (MS) supplemented with 30 g/l sucrose and 7 g/L agar (pH, 5.8), as described by Li et al. (2013). The cultures were grown at a consistent temperature of 23 ± 2 °C under a 16-h photoperiod with a light intensity of 50 μmol/s/m2 provided by cool-white fluorescent tubes. Subculturing was done every 3 weeks.

All virus-infected in vitro potato plantlets were provided by Potato Genetic Resource Center, Heilongjiang Academy of Agricultural Sciences of China. Virus-infected in vitro potato plantlets included single-infected with PLRV (China isolate, Genebank accession: KR051195; Han et al. 2016), PVYo (China isolate Genebank accession: U09509; Sun and Zhang 2005) and PVSo (China isolate, Genebank accession: AY687337; Zhou et al. 2007) and co-infected with PLRV and PVYo, and with PLRV and PVS. Virus(es) was(were) transmitted from each of the virus infected plantlets to the healthy in vitro stock plantlets by micrografting, as described by Hao et al. (2017). Briefly, shoots (1.0–15 cm in length) containing two nodes were excised from each type of the virus-infected plantlets and used as scions. A ‘V’ shape (about 3–5 mm in length) was cut at the base of scions. Shoots (1.5–2.0 cm in length) containing three nodes were taken from the healthy stock plantlets of ‘Zihuabai’ and used as rootstocks. A vertical cut (about 5–8 mm in length) was made at the top of rootstocks. In vitro micrografting was conducted by inserting the ‘V’ shape of scions into the vertical cut of rootstocks, with a silicon tube to hold the micrografting union. Micrografts were grown on SMM under the light conditions and developed into plantlets after about two weeks of micrografting. Subculturing was done every 3 weeks. After three months of micrografting, rootstocks were removed from the micrografts and cut into two segments. One was used for confirmation of sanitary status by reverse transcription-polymerase chain reaction (RT-PCR), as described below. Another one was used for micropropagation to establish virus-infected stock plantlets only when its corresponding segment was confirmed to be virus-infected, and otherwise was discarded. All virus-infected in vitro stock plantlets were micropropagated under the same conditions as used for the healthy in vitro stock cultures. Thus, all stock plantlets with different sanitary status were established in the same potato ‘Zihuabai’. Subculture was performed every 3 weeks.

Virus detection by RT-PCR

Sanitary status of the rootstocks, on which specific virus-infected scions had been micrografted for three months, was analyzed by RT-PCR. RT-PCR was conducted according to Li et al. (2013). Total RNA was extracted from shoots with leaves (0.5 g fresh weight) by the Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Following RT reaction, PCR amplification was conducted in a C1000TM Thermal Cycler (BIO-RAD, Singapore). The forward primer (5’-ATGAGTACGGTCGTGGTTAA-3′) and the reverse primer (5’-CTATTTGGGGTTTTGCAAAG-3′) were used to amplify approximately 600 bp bands for PLRV (Yan et al. 2010); the forward primer (5’-GGCATACGGACATAGGAGAAACT-3′) and the reverse primer (5’-CTCTCTGTGTTCTCCTCTTGTGT-3′) to amplify 447 bp bands for PVYo (Sun and Zhang 2005); and the forward primer (5‘-ACCRGATCCGACAAGCTCAGG-3′) and the reverse primer (5‘-GCCATTTGCTCRGTGTTGG-3′), which were designed by us, to amplify a product of approximately 380 bp bands for PVSo. The PCR products were run on a 1% agarose gel electrophoresis in Tris-acetate buffer, and visualized under ultraviolet light, following staining with ethidium bromide.

Vegetative growth

Shoot segments, each being 0.5 cm in length containing a single node, were removed from each of the 3-weeks-old healthy and virus-infected stock plantlets. The segments were cultured on SMM containing four different NaCl concentrations (0, 0.05, 0.15 and 0.25 M) and placed in the same cultural conditions as used for the in vitro stock plantlets. After 3 weeks of culture, vegetative growth was recorded by measuring length and node number of shoots, and dry weight (DW) of the plantlets, while root growth by root number and the longest root length. DW was measured, according to Piwowarczy et al. (2016). The whole plantlets containing both shoots and roots from each treatment were separately oven-dried at 120 °C for 24 h. Percentage of dry weight of in vitro plantlets was calculated and presented.

Microtuber production

From the above experiment, we found NaCl at 0.25 M led to severe damage to the healthy plantlets and almost killed the virus-infected plantlets. Therefore, NaCl concentrations of 0, 0.05 and 0.15 M were used in this experiment. Shoot segments, each being 1.0 cm in length containing two nodes, were removed from each of the healthy and virus-infected in vitro stock plantlets and cultured on a microtuber production medium (MPM) contained in vessels (6.5 × 9 cm, 10 shoots per vessel), to induce microtuber formation. MPM was composed of SMM supplemented with 60 g/L sucrose and 4 mg/L kinetin (KT), according to Li et al. (2013). The cultures were placed under the same cultural conditions, as used for the in vitro stock plantlets. After 8 weeks of culture, number of microtubers and percentage of microtubers ≥5 mm in diameter were recorded.

Physiological metabolism

The same NaCl concentrations (0, 0.05 and 0.15 M) were used in this experiment. The healthy and virus-infected in vitro shoots were prepared, as described above for vegetative growth. After 3 weeks of culture, all the in vitro plantlets were harvested and used in the following analyses.

Total chlorophyll content

Fresh leaves (1 g) were used for measurements of total chlorophyll (T chl) content, according to a method described by Li et al. (2013). The samples were placed into 10 ml culture tubes filled with 5.0 ml of 80% acetone and maintained at 22 ± 2 °C in the dark for 24 h. Contents of Chl a and Chl b were measured in the extracts by reading at 663 nm and 645 nm with a spectrophotometer (Thermo Multiskan MK3,USA). T chl was calculated as described by Li et al. (2013).

Total soluble sugar content

Total soluble sugar content was analyzed according to Li et al. (2013). Fresh leaves (1 g) were extracted with 10 ml 80% ethanol in a water bath at 70 °C for 1 h. The ethanol extracts were transferred into glass tubes and further extracted two times with 5 ml 80% ethanol in at 70 °C for 45 min for each time. The ethanol extracts from each time were collected and evaporated at 60 °C, followed by adding 3 ml double-distilled water to each tube and then centrifugation a 10,000 rpm for 10 min. The supernatant was filtrated through a 0.45 μm high performance liquid chromatography (HPLC) certified syringe filter and measured in an HPLC 360 Autosampler (Waters, Milford, MA, USA).

Free proline content

Free proline content was determined, using the method of Bates et al. (1973). Shoots (0.5 g fresh weight, FW) were extracted with 10 ml of 3% (w/v) sulfo-salicylic acid. The extract was centrifuged at 3000 rpm for 20 min. The supernatant was collected and reacted with acid ninhydrin in a boiling water bath for 1 h, followed by transfer to room temperature for 10 min to terminate the reaction. The reaction solution was again extracted with 4 ml of toluene and then used for measurement of free proline by reading at 520 nm in a spectrophotometer (Thermo Multiskan MK3, USA) for measurement of free proline using L-proline as a standard.

Lipid peroxidation

Lipid peroxidation was determined by measuring the amount of methane dicarboxylic aldehyde (MDA) produced by the thiobarbituric acid reaction as described by Heath and Packer (1968). Leaves (1.0 g FW) were extracted with 10 ml of 10% trichloroacetic acid, and the crude extract was mixed with the same volume of a 0.5% (w/v) thiobarbituric acid solution containing 20% (w/v) tricholoroacetic acid. The mixture was heated at 95 °C for 30 min and then quickly cooled in an ice-bath. The mixture was centrifuged at 10,000 rpm for 10 min and the absorbance was recorded at 450 nm, 532 nm and 600 nm, respectively, in a spectrophotometer (Thermo, Multiskan MK3, USA). The MDA concentration was calculated using the following formula: 6.45 × (OD 532–OD 600)–0.56OD450.

Analysis of superoxide anion (O2 .-) and H2O2 localization in situ

Superoxide anion (O2.-) was analyzed, as described by Cui et al. (2016). Briefly, the whole plantlets were immersed in 0.1% solution of nitroblue tetrazolium (NBT) in 10 mM K-phosphate buffer (pH, 7.6), vacuum-infiltrated for 30 min and illuminated for 2 h, followed by bleaching in 95% boiling ethanol for 5 min. Relative quantity of O2.- signal was calculated on the base of a function of standard signal, which was obtained from Mn, according to Kim et al. (2007). H2O2 localization in situ was conducted, according to the method of Cui et al. (2016). The whole plantlets were immersed in a 0.1% filtered solution of 3, 3′-diaminobenzidine (DAB) in 10 mM MES buffer (pH, 5.0), vacuum-infiltrated for 30 min and then incubated at room temperature for 4 h in the dark, followed by bleached in 95% boiling ethanol for 5 min. Production of H2O2 in the plantlets was visualized with DAB staining to give rise to brown spots. Photographs were captured by a digital camera (PowerShot G9, Canon, Japan).

Statistical analysis

A complete randomized design was used in the present study. Ten samples were included in each of experiments. All experiments contained three replicates and were conducted 3 times. Data were subjected to analysis of variance (ANOVA) and the least significant difference (LSD) was calculated at P ≤ 0.05 by Student’s t-test. Two-way ANOVA, including virus infection and salt stress as factors, was performed to analyze effects of virus infection (single- and co-infection), salt stress and co-stress by salt and virus (single- and co-infection) on growth, microtuber production and physiological metabolism. Significant differences were analyzed at P ≤ 0.05 and P ≤ 0.01, respectively.

Results

Virus detection by RT-PCR

Virus detection by RT-PCR showed each of the positive controls produced a specific band: approximately 600, 447 and 380 bp for PLRV, PVYo and PVS, respectively, while negative control did not show such bands (Fig. 1). Such bands were never detected in the healthy stock plantlets that were produced by cryotherapy (Fig. 1). For PLRV, single- and co-infected in vitro stock plantlets obtained by micrografting showed a specific band of approximately 600 bp (Fig. 1). For PVYo, single- and co-infected in vitro stock plantlets gave a specific band of 447 bp (Fig. 1). For PVS, single- and co-infected in vitro stock plantlets produced a specific band of 380 bp (Fig. 1). Only stock cultures that were positive responses to RT-PCR were used as virus-infected samples in the following experiments.

Fig. 1
figure 1

Detection of virus by RT-PCR in in vitro stock plantlets of potato ‘Zihuabai’. M = molecular marker; Lane 1 = negative control of PLRV, PVYo and PVS; Lane 2 = positive control of PLRV; Lane 3 = positive control of PVYo; Lane 4 = positive control of PVS; Lanes 5–7 = PLRV-infected samples in stock shoots single-infected with PLRV, co-infection with PLRV and PVYo, and PLRV and PVS, respectively; Lane 8 = PLRV-free samples; Lanes 9 and 10 = PVYo-infected samples in stock shoots singled-infected with PVYo, and co-infected with PLRV and PVYo; Lane 11 = PVYo-free samples; Lanes 12 and 13 = PVS-infected samples in stock shoots single-infected with PVS, and co-infected with PLRV and PVS; Lane 14 = PVS-free samples. All virus-free samples were taken from healthy plantlets derived from cryotherapy

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Vegetative growth

Without salt stress, the healthy cultures produced much longer shoot length than all virus-infected ones (Figs. 2 and 3a). Although shoot length was similar in the cultures single-infected with PVYo and PVS, and co-infected with PLRV and PVS, and PLRV and PVYo, it was significantly shorter in those co-infected with PLRV and PVYo than single-infected by PLRV (Figs. 2 and 3a). With salt stress induced by 0.05 M NaCl, shoot length was much longer in the healthy cultures than all virus-infected ones (Figs. 2 and 3a). The cultures single-infected with PLVR and PVYo produced similar shoot length, which was significantly longer than those found in the cultures single-infected with PVS, co-infected with PLRV and PVY, and PLRV and PVS. NaCl at 0.15 M resulted in almost no shoot elongation of the healthy cultures and completely inhibited shoot elongation of all virus-infected cultures (Figs. 2 and 3a). NaCl at 0.25 M caused serious damage to the healthy cultures and almost killed the virus-infected cultures (Fig. 2). Without salt stress, the healthy and single PLRV-infected cultures produced similar number of nodes, significantly higher than those single-infected with PVYo and PVS, and co-infected with PLRV and PVYo, and PLRV and PVS, with the least number of nodes produced in the last two types of the culture (Fig. 3b). When stressed by NaCl at 0.05 M, the healthy cultures produced the greatest number of nodes among all the treatments (Fig. 3b). Co-infection with PLRV and PVYo, and PLRV and PVS resulted in the least number of nodes. Without salt stress, the number of roots produced by the healthy cultures was similar to that by PLRV single-infected cultures, but was significantly higher than those by other types of cultures tested (Fig. 3c). When stressed by NaCl at 0.05 M, the healthy cultures and those single-infected with PLRV and PVYo produced similar number of roots, which were markedly higher than those in cultures single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS (Fig. 3c). When stressed by 0.15 M NaCl, cultures single-infected with PLRV produced much higher number of roots than the healthy cultures and those single-infected with PVYo. Cultures single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS failed to produce any roots (Figs. 2 and 3c). Without salt stress, the longest root length produced by the healthy cultures was similar to those produced by cultures single-infected by PLRV, PVYo or PVS, but was greater than those produced by cultures co-infected with PLRV and PVYo, and PLRV and PVS (Figs. 2 and 3d). When grown at 0.05 M NaCl, the significant differences in the longest root length were found only between the healthy cultures and those co-infected with PLRV and PVYo (Figs. 2 and 3d). When stressed by NaCl at 0.15 M, the longest root length was greater in the healthy cultures than in those single-infected with PLRV and PVYo (Figs. 2 and 3d).

Fig. 2
figure 2

Vegetative growth in the healthy and virus-infected in vitro plantlets of potato ‘Zihuabai’ grown under NaCl-induced salt stress. NaCl concentrations at 0.00 M (1), 0.05 M (2), 0.15 M (3) and 0.25 M (4). Healthy plantlets (I); Plantlets single-infected with PLRV (II); Plantlets single-infected with PVYo (III); Plantlets single-infected with PVS (IV); Plantlets co-infected with PLRV and PVYo (V); Plantlets co-infected with PLRV and PVS (VI). Photos were taken after 3 weeks of culture. Bars = 5 cm

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Fig. 3
figure 3

Shoot length (a), node number (b), root length (c) and the longest root length (d) of the healthy and virus-infected in vitro plantlets of potato ‘Zihuabai’ grown under NaCl-induced salt stress. Data were presented as means ± SE and with different letters in the treatments receiving the same NaCl concentration indicated significant differences at P ≤ 0.05 by Student’s t-test. Healthy plantlets (I); Plantlets single-infected with PLRV (II); Plantlets single-infected with PVYo (III); Plantlets single-infected with PVS (IV); Plantlets co-infected with PLRV and PVYo (V); Plantlets co-infected with PLRV and PVS (VI)

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Without salt stress, significant difference in dry weight existed only between healthy plantlets and PVYo single-infected ones, while all other treatments gave similar data of dry weight (Fig. 4a). Salt stress induced by 0.05 M NaCl did not cause significant differences in dry weight, regardless of their sanitary status (Fig. 4a). When stressed by NaCl at 0.15 M, healthy plantlets and those single-infected by PLRV or PVYo produced similar dry weight, which was significantly higher than those single-infected by PVS, and co-infected by PLRV and PVYo, and PLRV and PVS (Fig. 4a).

Fig. 4
figure 4

Dry weight (a), and levels of total chlorophyll (b), total soluble sugar (c), free proline (d), methane dicarboxylic aldehyde (MDA) (e) and superoxide anion (O2·-) (f) in the healthy and virus-infected in vitro plantlets of potato ‘Zihuabai’ grown under NaCl-induced salt stress. Data were presented as means ± SE and with different letters in the treatments receiving the same NaCl concentration indicated significant differences at P ≤ 0.05 by Student’s t-test. Healthy plantlets (I); Plantlets single-infected with PLRV (II); Plantlets single-infected with PVYo (III); Plantlets single-infected with PVS (IV); Plantlets co-infected with PLRV and PVYo (V); Plantlets co-infected with PLRV and PVS (VI)

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Two-way ANOVA showed that in both single- and co-infected plantlets, virus infection, salt stress, and co-stress by salt and virus all had significant influences (P ≤ 0.01) on growth of shoots and roots (Table 1).

Table 1 Two-way ANOVA of effects of virus-infection and NaCl-induced salt stress, and their combing effects on vegetative growth, microtuber production and physiological metabolism of in vitro plantlets of potato ‘Zihuabai’
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Microtuber production

Without salt stress, healthy shoots and single-infected shoots with PLRV or PVYo produced similar number of microtubers per vessel, but shoots single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS gave much fewer number of microtubers (Table 2; Fig. 5). Percentage of microtubers ≥5 mm in diameter was significantly higher in the healthy shoots than in those single-infected with PLRV or PVYo. No microtubers ≥5 mm were produced in shoots single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS. When stressed by 0.05 M NaCl, the healthy shoots produced the greater number of microtubers than all virus-infected shoots, in either single- or co-infection (Table 2; Fig. 5). Number of microtubers in shoots single-infected with PLRV or PVYo was greater than those in shoots single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS. Shoots co-infected with PLRV and PVS almost failed to produce microtubers. Healthy shoots produced much higher percentage of microtubers ≥5 mm than PLRV and PVYo single-infected shoots (Table 2; Fig. 5). Shoots single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS completely failed to produce any microtubers ≥5 mm. When stressed by 0.15 M NaCl, percentage of microtubers ≥5 mm was higher in healthy shoots than PLRV single-infected shoots (Table 2, Fig. 5). None of shoots single-infected with PVYo or PVS, co-infected with PLRV and PVYo, and PLRV and PVS were able to produce microtubers.

Table 2 Effects of virus infection and NaCl stress on microtuber production of in vitro plantlets of potato ‘Zihuabai’
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Fig. 5
figure 5

Microtuber production in the healthy plantlets and virus-infected in vitro plantlets of potato ‘Zihuabai’ grown under NaCl-induced salt stress. No microtubers were produced in the healthy plantlets and virus-infected in vitro plantlets grown at 0.25 M NaCl. Healthy plantlets (I); Plantlets single-infected with PLRV (II); Plantlets single-infected with PVYo (III); Plantlets single-infected with PVS (IV); Plantlets co-infected with PLRV and PVYo (V); Plantlets co-infected with PLRV and PVS (VI). Photos were taken after 8 weeks of culture. Bars = 5 mm

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Two-way ANOVA showed that in single virus-infected shoots, virus infection, salt stress, and co-stress by salt and virus infection had significant influences (P ≤ 0.01) on microtuber production (Table 1). In virus co-infected shoots, only salt stress had, while virus-infection and co-stress by salt and virus infection did not, significant influences (P ≤ 0.01) on microtuber production (Table 1).

Physiological metabolism

Total chlorophyll (T chl) content

Without salt stress, no significant differences were detected in T chl levels among the healthy plantlets and those single-infected with PLRV, PVYo or PVS (Fig. 4b). However, co-infection with PLRV and PVYo, and PLRV and PVS caused markedly reductions in T chl levels. When salt stressed by NaCl at 0.05 M, content of T chl was not significantly different among the healthy plantlets and those single-infected with PLRV or PVYo, and co-infected with PLRV and PVYo, and PPLRV and PVS (Fig. 4b). The healthy plantlets had much higher content of T chl than PVS single-infected ones. When stressed by NaCl at 0.15 M, significantly lower levels in T chl, than the healthy plantlets, were found only in plantlets co-infected with PLRV and PVYo (Fig. 4b).

Total soluble sugar

Without salt stress, no significant differences in total soluble sugar were found among the healthy plantlets and those single-infected with PLRV, PVYo or PVS (Fig. 4c). Compared with the healthy plantlets, co-infection with PLRV and PVYo, and PLRV and PVS caused markedly increased content of total soluble sugar. When stressed by NaCl at 0.05 M, total soluble sugar was much lower in the healthy plantlets than all virus-infected ones, including single- and co-infection (Fig. 4c). Co-infection with PLRV and PVYo, and PLRV and PVS resulted in the highest levels of total soluble sugar. When stressed by NaCl at 0.15 M, the healthy plantlets gave the highest content of total soluble sugar among all treatments, followed by plantlets single-infected with PLRV, PVYo and PVS (Fig. 4c). Not enough samples were available for analysis of total soluble sugar in plantlets co-infected with PLRV and PVYo, and PLRV and PVS.

Free proline

Without salt stress, no significant differences in levels of free proline were detected in all treatments, regardless of their sanitary status (Fig. 4d). When stressed by NaCl at 0.0 5 M, levels of free proline were also similar in all treatments (Fig. 4d). When stressed by NaCl at 0.15 M, levels of free proline were similar between healthy plantlets and those single-infected with PLRV, PVYo or PVS, but were significantly higher in healthy plantlets than in those co-infected with PLRV and PVYo, and PLRV and PVCS (Fig. 4d).

MDA

Without salt stress, levels of MDA in the healthy plantlets and those single-infected with PLRV or PVYo were similar to those in plantlets co-infected with PLRV and PVS, but were significantly lower than those in plantlets single-infected with PVS, and co-infected with PLRV and PVY (Fig. 4e). There were no significant differences in levels of MDA produced between plantlets co-infected with PLRV and PVYo, and those co-infected by PLRV and PVS (Fig. 4e). When stressed by NaCl at 0.05 M, the healthy plantlets, and PLRV and PVYo single-infected plantlets produced similar levels of MDA, which were significantly lower than those in plantlets single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS (Fig. 4e). When stressed by NaCl at 0.15 M, the level of MDA in the healthy plantlets was similar to those in PVYo single-infected plantlets, but was significantly lower than those in plantlets single-infected with PLRV or PVS, and co-infected with PLRV and PVYo, and PLRV and PVS (Fig. 4e). The highest levels of MDA were found in plantlets single-infected with PLRV, and co-infected with PLRV and PVYo, and PLRV and PVS (Fig. 4e).

Superoxide anion (O2 ·-) and H2O2 localization in situ

Without salt stress, similarly levels of O2·- was detected in the healthy plantlets, and PVYo single-infected and PLRV and PVYo co-infected plantlets, which were lower than in those single-infected with PLRV or PVS, and co-infected with PLRV and PVS (Fig. 4f). The highest level of O2·- was found in plantlets single-infected with PVS, followed by PLRV and PVS co-infected plantlets, and PLRV single-infected plantlets. When stressed by NaCl at 0.05 M, levels of O2·- were similar among the healthy plantlets and those single-infected with PLRV or PVYo (Fig. 4f), but were much lower than those in plantlets single-infected with PVS, and co-infected with PLRV and PVYo, and PLRV and PVS. When stressed by NaCl at 0.15 M, levels of O2·- in the healthy plants were not significantly different from those in PVY, PVYo or PVS single-infected plantlets, and in PLRV and PVYo co-infected plantlets (Fig. 4f). The highest levels of O2·- were found in plantlets co-infected by PLRV and PVS.

H2O2 accumulation was measured using DAB staining to give rise to brown spots. Without salt stress, H2O2 accumulation was hardly seen in the healthy plantlets (Fig. 6a). Intensity and areas of brown spots increased in virus infected plantlets (Fig. 6b, c, d, e and f), and were much stronger and larger in the plantlets single-infected with PVS (Fig. 6d), and co-infected with PLRV and PVYo (Fig. 6e) and PLRV and PVS (Fig. 6f). When stressed by NaCl at 0.05 M, H2O2 accumulation was easily observed in the healthy plantlets (Fig. 6a). Intensity and areas of brown spots obviously increased in all virus infected plantlets (Fig. 6b, c, d, e and f), compared with the corresponding virus-infected plantlets without salt stress.

Fig. 6
figure 6

H2O2 in situ localization in the healthy and virus-infected in vitro plantlets of potato ‘Zihuabai’ grown under NaCl-induced salt stress. Healthy plantlets without (A) and with NaCl (a); Plantlets single-infected with PLRV without (B) and with NaCl (b); Plantlets single-infected with PVYo without (C) and with NaCl (c); Plantlets single-infected with PVS without (D) and with NaCl (d); Plantlets co-infected with PLRV and PVYo without (E) and with NaCl (e); Plantlets co-infected with PLRV and PVS without (F) and with NaCl (f). Bars = 1 cm

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Two-way ANOVA showed that in both single- and co-infected plantlets, virus infection, salt stress, and co-stress by salt and virus all had significant influences (P ≤ 0.01) on physiological metabolism of in vitro plantlets (Table 1).

Discussion

Although negative effects of either salt stress (Suzuki et al. 2014; Fita et al. 2015) or virus infection (Wang et al. 2011) on plants including potato have been well-documented, studies on effects of co-stress by both salt and virus have been quite limited. Compared with field-grown plants, in vitro culture techniques have advantages that can avoid influences exerted by light, temperature and nutrients, and attacks by other pathogens, thus facilitating studies of co-stress by biotic and abiotic stress to plants (Li et al. 2013; Cui et al. 2015, 2016). In addition, most of the previous studies on effects of virus focused on single virus infection (Hamm and Hane 1999; Hane and Hamm 1999), with few on co-infection (Wright 1970; Manzer et al. 1978; Nyalugwe et al. 2012; Li et al. 2013), mainly due to difficulties in establishment of mix infection in the same plant. In vitro culture techniques established in the present study allowed us to carry out such studies. To the best of our knowledge, this is the first report addressing effects of co-stress by salt and virus, in single- and co-infection, on plants.

Results obtained in the present study clearly demonstrate that the three major potato viruses including PLRV, PVY and PVS, both in single- and co-infection, significantly inhibit vegetative growth of in vitro plantlets of potato ‘Zihuabai’. Results of inhibitory effects of single virus infection on vegetative growth found in the present study were totally consistent with those previously reported by us using the same host-virus combination (Li et al. 2013) and by other groups using different potato host-virus combinations such as ‘Igor’ infected with PVYNTN (Petrovič et al. 1997; Dolenc and Deermastia 1999) and ‘Pentland Squire’ infected with PVYNTN (Anžlovar et al. 1996). Negative effects of single virus infection on plant growth have also been reported in other host-virus combinations including Grapevine leafroll disease-infected grapevine (Cui et al. 2015, 2016), Citrus tristeza virus-infected citrus (Wang et al. 2009), Banana bunchy top virus-infected Musa (Haq et al. 2012), Tobacco streak virus-infected raspberry (Tsao et al. 2000), Raspberry bushy dwarf virus-infected raspberry (Tsao et al. 2000) and Tomato ringspot virus-infected raspberry (Tsao et al. 2000). Significantly inhibited vegetative growth by co-infection by PLRV and PVY found in the present study confirmed the results previously reported by us using the same host-virus combination (Li et al. 2013). In addition, we further demonstrated co-infection with PLRV and PVS exerted markedly negative effects on plant growth of potato.

Reduced growth has been reported in in vitro potato plantlets grown under NaCl stress (Alhagdow et al. 1999; Khalid and Aftab 2016) and other in vitro cultures such as Ipomoea batatas (Ekanayake and Dodds 1993), and Rosa chinensis (Wahome et al. 2001) and Lathyrus sativus (Piwowarczy et al. 2016). Similar results were also observed in field-grown potato (Ghosh et al. 2001; Shaterian et al. 2005, 2008), and other crops (Ayodele and Ojoghoro 2007; Bharti et al. 2016; Liu et al. 2016). Reduced plant growth by salt stress was attributed to inhibition of cell elongation due to decreases in cell turgor (Piwowarczy et al. 2016). Notably, we further found co-stress by salt and virus (including single- and co-infection) had significantly negative effects on vegetative growth of in vitro-grown potato plantlets.

Effects of virus infection on tuber yield of field-grown potato plants have been well-documented (Wright 1970; Manzer et al. 1978; Hamm and Hane 1999; Hane and Hamm 1999; Wang et al. 2011; Nyalugwe et al. 2012). Using in vitro plantlets in the present study, we clearly demonstrated virus infection, both in single- and co-infection, considerably reduced tuber yield. Consistent results were also obtained in our previously report using in vitro cultures of the same host-virus combination (Li et al. 2013). Significantly reduced tuber yield by co-infection found in the present study confirmed the results reported in field-grown potato (Wright 1970; Manzer et al. 1978; Nyalugwe et al. 2012).

Information on effects of salt stress on tuber yield of in vitro potato plantlets has been quite limited (Zhang et al. 2005). Most of the previous studies on effects of salt stress on yield used field-grown plants and found salt stress markedly reduced tuber yield of potato (Nadler and Heuer 1995; Ghosh et al. 2001; Elkhatib et al. 2006; Shaterian et al. 2005, 2008; Khalid and Aftab 2016), Manihot esculenta (Nair et al. 1980) and Ipomoea batatas (Ekanayake and Dodds 1993) as well as other field crops such as Oryza sativa (Zeng et al. 2001), Triticum aestivum (Azadi et al. 2015) and Hordeum vulgare (Bagheri and Sadeghipour 2009). In the present study, we not only confirmed negative effects of salt stress on tuber yield, but also revealed co-stress by salt and virus infection, both in single- and co-infection, considerably reduced tuber yield in in vitro potato plantlets.

Most of the previous studies on the effect of plant virus or salt stress on physiological metabolism used field- or greenhouse-grown plants, while the in vitro cultures have been used much less. A high accordance has been found between the in vitro and in vivo chlorophyll fluorescence measurements in monitoring changes in photosynthetic activity in response to drought stress (Faraloni et al. 2011). Anžlovar et al. (1996) found that PVYNTN infection caused marked reduction in contents of total chlorophyll in in vitro cultured ‘Desiré’ and ‘Pentland Squire’. Virus infection caused significant reductions in chlorophyll contents in the present study and also in our previous study using the same host-virus combination (Li et al. 2013), as well as in other host-virus combinations in both in vitro (Haq et al. 2012) and in vivo cultures (Gonçalves et al. 2013; Mederos et al. 2009; Sinha and Srivastava 2010). Decreased chlorophyll contents by salt stress have been well-documented in a number of field-grown plants (Radi et al. 2013; Abbas and Mobin 2016; Ma et al. 2016), but with a few using in vitro cultures including potato (Demir and Kocacaliskan 2002;Zhang et al. 2005; Piwowarczy et al. 2016). The typical symptoms such as yellowing, necrosis and mosaic induced by PLRV, PVY or PVS, indicated ruptures of chloroplasts (Shalitin and Wolf 2000; Mederos et al. 2009; Nyalugwe et al. 2012). Photosynthetic apparatus is sensitive and susceptible to abiotic stresses including salt (Hasegawa et al. 2000; Chattopadhyay et al. 2011; Singh et al. 2013), which may result in chlorophyll degradation and/or damage to the photosynthetic apparatus (Radi et al. 2013). A great reduction in chlorophyll contents represented noticeable decreases in photosynthetic capability in the infected plants or salt-stressed plants, which we believe causes losses of tuber yield of potato found in the present study.

Although results split in influences of virus infection on contents of total soluble sugar (Sinha and Srivastava 2010), marked high concentrations of total soluble sugars have been observed in Solanum tuberosum infected with PLRV and PVY, in single- and co-infection (Li et al. 2013), and other host-virus combinations such as Cucumber mosaic virus-infected Cucumismelo (Shalitin and Wolf 2000) and Sugarcane yellow leaf virus-infected Saccharum (Gonçalves et al. 2013). These results were consistent with ours. Sugar allocation from source organs to sink organs is through sieve elements. Virus infection and movement through phloem tissues result in modification of sieve elements (Hull 2002; Lalonde et al. 2003), which in turn inhibits sugar transport from the source leaves to other organs, consequently leading to accumulation of high sugar concentrations in the infected leaves. Increased accumulation of total soluble sugar has been widely reported in plants including potato grown under salt stress (Zhang et al. 2005; Zheng et al. 2008; Radi et al. 2013; Cheng et al. 2016; Khalid and Aftab 2016). Accumulation of total soluble sugar was considered as one of the most notable consequences for osmotic adjustment under salt stress, thus protecting the cell from damage by stabilizing cellular membranes and maintaining cell turgor (Cheeseman 1988; Jouve et al. 2004). Increased accumulations of protein in plants under salt stress were also suggested to overcome an enhanced production of reactive oxygen species (ROS) by such up-regulated proteins (Khalid and Aftab 2016). Over-expression of stress-responsive proteins (StDREB1 gene) has been found to be associated with stress tolerance in potato, thus demonstrating protection against ROS in plants under stress was through regulation of the stress-responsive signaling pathway (Bouaziz et al. 2013).

Our study found that either salt stress or virus infection (single- and co-infection), and co-stress by salt and virus infection (single- and co-infection) significantly increased proline contents. Increased proline contents by salt stress were found in Solanum tuberosum grown in vitro (Martinez et al. 1996) and in vivo (Shaterian et al. 2005; Bai et al. 2016), and in other plant species grown in the field (Chattopadhyay et al. 2011; Abbas and Mobin 2016; Piwowarczy et al. 2016). Increased proline contents were also found in Cucurbita pepo infected with Zucchini yellow mosaic virus (Radwan et al. 2007) and Vitis vinifera infected with GLRaV-3 (Cui et al. 2016). Proline accumulation involves in salt tolerance mechanism in plants (Hasegawa et al. 2000). Increased accumulation of proline helped to maintain cell membrane integrity and protect subcellular structures from damage in plants under salt stress (Hasegawa et al. 2000), and activate a hypersensitive response to virus infection (Radwan et al. 2007).

MDA, a product of membrane lipid peroxidation, has been considered an excellent marker of oxidative stress and is frequently used to detect cellular membrane damage by salinity stress (Radi et al. 2013). There have been many studies on drought-induced MDA, but data on virus-induced MDA have been quite limited. Bor et al. (2003) found the MDA level markedly increased in Beta plants under NaCl-induced salt stress. Similar results were also found in Oryza sativa (Demiral and Türkan 2005), Zea mays (Neto et al. 2006) and Triticum (Chen et al. 2013). Using in vitro-grown grapevine shoots, Cui et al. (2016) found GLRD infection did not significantly increase MDA, while PEG-induce stress caused significant accumulation of MDA, and effects of co-stress by GLRD and salt on the increased MDA were much greater than single salt stress, indicating extra injury to cell membranes by the interaction of drought stress and virus-infection. All of these data supported the results obtained in the present study using in vitro-cultured potato. Recently, transgenic potato lines over-expressing StnsLTP1 gene, a potential thermo-tolerance gene from potato, was found to enhance cell membrane integrity of transgenic plants under salt stress conditions by reducing membrane lipid per-oxidation and hydrogen peroxide content, and increasing antioxidant enzyme activity (Gangadhar et al. 2016).

Reactive oxygen species (ROS), particularly O2·- and H2O2, are sub-products responding to abiotic and biotic stress, and has been considered an indicator of oxidative damage (Bolwell et al. 1999; Neto et al. 2006). Abiotic stresses such as salt or biotic stress such as virus induce oxidative damages by accelerating production of ROS such as O2·- and H2O2 (Wu et al. 2015; Gill and Tuteja 2010). A great number of studies have shown that accumulations of O2·- and H2O2 considerably increased in plants stressed by salt (Bor et al. 2003; Demiral and Türkan 2005; Neto et al. 2006; Chattopadhyay et al. 2011; Chen et al. 2013; Radi et al. 2013; Khalid and Aftab 2016; Ma et al. 2016), with a few in virus-infected plants (Evans et al. 2006; Zhang et al. 2014; Cui et al. 2016). In the present study, we found that single stress by virus or salt, and co-stress by virus and salt increased the accumulation of O2·- and H2O2. ROS has been shown to influence the expression of a number of genes and therefore manipulate various processes of plant responses to abiotic and biotic stress (Gill and Tuteja 2010). The production of ROS is the first response of plants to biotic and abiotic stress (Gara et al. 2003; Evans et al. 2006). With continuation of the stress, an oxidative burst, that is massive and prolonged ROS production, occurs in the stressed cells (Gara et al. 2003). Such an oxidative burst functions in activating defense system, which can protect plants from damage caused by biotic and abiotic stress (Gara et al. 2003; Gangadhar et al. 2016). It is noteworthy that virus infection may improve tolerance of plants to abiotic stress. For example, markedly increased osmoprotectant and antioxidant levels were found in Brome mosaic virus-infected rice (Oryza sativa) and Cucumber mosaic virus-infected beet plant (Beta vulgaris), resulting in improvement of tolerance of the infected plants to drought (Xu et al. 2008).

In conclusion, results obtained in the present study indicate virus infection (including single- and co-infection) or salt stress, and co-stress by virus (including single- and co-infection) and salt cause severely oxidative cell damage and marked reduce photosynthetic ability, These negative effects may result in reductions in plant growth and eventually decrease tuber yield. In addition, virus-free in vitro potato plantlets used in the present study were produced by cryotherapy, a novel biotechnology for plant pathogen eradication (Wang and Valkonen 2009; Wang et al. 2014a). Using molecular markers including simple sequence repeat and amplified fragment length polymorphism, we previously demonstrated the genetic stability in the cryo-derived plantlets of potato ‘Zihuabai’ (Wang et al. 2014b). The present study further found increased vegetative growth and microtuber yield in the virus-free in vitro plantlets derived from cryotherapy. All of these results indicate the virus-free potato plants derived from cryotherapy have potential applications to field potato production, as those currently used by the traditional methods such as meristem culture.