Effect of infection of potato plants by Potato virus Y (PVY), Potato virus S (PVS), and Potato virus M (PVM) on content and physicochemical properties of tuber starch

Abstract Potato virus Y (PVY), Potato virus S (PVS), and Potato virus M (PVM) infection of potato plants leads to decreased dry matter and starch content in tubers. Starch samples from potato tubers infected with PVY, PVS, and PVM had higher amylose content. Granules of starch isolated from potato tubers infected by PVS exhibit larger granules than starch granules isolated from tubers of healthy plants. In contrast, in the case of PVM and PVY infection, starch granules were significantly smaller in diameter. A decrease in the degree of crystallinity has been observed in all samples of starches obtained from the tubers of infected plants compared to starch isolated from tubers of healthy plants. A slight decrease in gelatinization temperature was noted for starch samples isolated from tubers infected by PVY and PVM, and a slight increase in gelatinization temperature for starch samples isolated from tubers infected by PVS compared to starch isolated from tubers of healthy plants. In all samples of starch obtained from tubers of infected plants, an increase in the value of gelatinization enthalpy was observed. Thus, it can be concluded that damage to potato plants by PVM and PVY leads to a significant decrease in the quality of starch in tubers. At the same time, infection by PVS had practically no considerable effect.


| INTRODUC TI ON
Among the plethora of diseases affecting potato plants that viral etiology is the most common and causes significant damage to seed and tuber production. According to numerous sources, there are 40-50 viruses that infect potato plants all over the world (Bashir et al., 2021;Kreuze et al., 2020;Loebenstein et al., 2001;Valkonen, 2007;Wang et al., 2011), of which the following are  (Bai et al., 2007;Rogozina et al., 2019;Valkonen, 2015;Wang et al., 2011;Zhang et al., 2010). Viral infection of potato plants affects both the yield and quality of potatoes (Kotzampigikis et al., 2008). Contemporary research suggests that the loss of yield associated with viral infection of potato plants varies from 10 to 80% (Awasthi & Verma, 2017;Bashir et al., 2021;Hao et al., 2007;Huang et al., 2009;Kumar et al., 2020;Kumar et al., 2019;Wang et al., 2011;Wang et al., 2005;Whitworth et al., 2006). Most damaging effects are observed in the plants infected by PLRV and PVY (Kotzampigikis et al., 2008;Weidemann, 1988). PLRV and PVY infection have a fast rate of spread by vectors such as aphids (Gray et al., 2010), which explains their highly damaging effect on potatoes causing severe and moderate symptoms in plants (leaf curl, wrinkle and stripe mosaics, and leaf mosaic curl) that, if plants are propagated vegetatively, will almost always infect the propagated plants through tubers. Losses in yields after a combined infection by viruses can be even more severe (Struik & Wiersema, 1999).
Viruses, being obligate parasites and affecting the metabolism of plants, cause a violation of physiological processes, resulting in a decrease not only in the productivity of plants but also affecting the biochemical composition of tubers. As a result, plants grown from previously infected seed tubers (secondary infection) usually produce tubers unsuitable for sale (Bashir et al., 2021).
Infection by plant viruses leads to biochemical and physiological changes in photosynthesis, CO 2 assimilation, and starch accumulation (Trethewey & Smith, 2000). It is noted that viruses usually suppress the accumulation of starch in the leaves of plants at an early stage of infection (Zhao et al., 2022). Infection of potato plants with PLRV, PVY, PVS, and PVM leads to a decrease in the starch content in tubers by 0.5%-9% compared to uninfected tubers (Blotskaya, 2000). It is noted that various viruses, for example, sweet potato leaf curl virus (SPLCV) and Tomato leaf curl New Delhi viruspotato (ToLCNDV-potato) lead to a significant decrease in the starch content in the sweet potato (Hou et al., 2020) and potato tubers (Solanum tuberosum L.) (Lal et al., 2021), respectively.
Potato starch possesses some unique physicochemical properties compared to starches from other sources (e.g., high phosphate content, lack of internal lipids, and proteins in the granules) (Burlingame et al., 2009;Romano et al., 2016). The properties of starch are affected by the ratio of amylose to amylopectin, granule size, phosphate content, and other parameters (Tang et al., 2002;Vasanthan et al., 1999). It is noted that among the effects of ToLCNDV-potato on the potato plant, it reduces the content of starch and amylose (Lal et al., 2021). However, there are no studies on the properties of starch obtained from potato tubers infected with PVY, PVS, and PVM. Therefore, this study will systematically reveal the structure and physicochemical properties of potato starch isolated from tubers infected by PVY, PVS, and PVM, and the obtained results of this study hope to provide a basis for potato cultivation and potato starch processing and utilization.

| Starch isolation
Starch was extracted from fresh potato tubers by repeated washing with deionized water at room temperature, according to Richter et al. (1968).

| Dry matter and starch content
Dry matter (Thermo-ventilated oven at 105°C) and starch content were determined by the Evers polarimetric method.

| Scanning electron microscopy
Scanning electron microscopy (SEM) micrographs were taken using a scanning electron microscope (JSM-6360LV, JEOL, Japan). A starch sample adhered to an SEM stub using double-sided adhesive conductive tape and coated with a thin layer of gold to make the sample conductive. The mounted sample was then placed on the SEM stage, and images were digitally captured at the accelerating voltage of 10 kV.

| Polarized light microscopy
The starch sample was suspended in a 1:1 glycerol solution (glycerol/ H2O, V/V) and was observed using light microscopy (DMBA400, Motic China Group Co., Ltd, Guangzhou, China) with the polarized light filter at 40-time magnification.

| Particle size distribution
The particle size distribution was measured using a laser lightscattering particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., England). The starch was placed in water and analyzed automatically by laser diffraction.

| Chemical characterization of the starches
Amylose levels were determined using the Juliano (1971) method.
The glucose formed is then estimated calorimetrically. From the total starch content, the amylopectin fraction is determined by difference. Replicate samples (n = 4-6) were analyzed.
Starch moisture contents were determined by drying weighed amounts of starch in predried aluminum dishes in an air oven at 103°C to reach constant weight (Roder et al., 2009). Protein contents of duplicate samples were estimated from total nitrogen measurements (N 6.25) determined by a micro-Kjeldahl method.

| Thermal properties
The thermodynamic properties of starches were determined using differential scanning calorimetry DSC 1/200 W (Mettler Toledo, USA). Samples of starch (approximately 10.0 mg dry weight) were weighed directly into an aluminum crucible (Mettler, ME-51119872), and distilled water was added in a 1:3 ratio. The crucible was sealed and equilibrated for 1 h before analysis. An empty hermetically sealed crucible was used as a reference. Then, the samples were heated from 30°C to 170°C at a rate of 10°C/min. As a result, the initial temperature (Tn), peak temperature (Tn), final temperature (Tk), and enthalpy (ΔH) were determined. The range was calculated by subtracting the initial temperature from the final temperature.

| X-ray diffraction (XRD)
Samples were analyzed using an X-ray diffractometer (D8, Bruker, Germany), equipped with a copper tube at 40 kV. The scanning range of the diffraction angle (2θ) was from 4° to 60° at a speed of 6°/min, with a step size of 0.02°. The degree of crystallinity is calculated by Jade software (Jade 5.0).

| Statistical analysis
All the experiments were the average of at least two replicates. The data were statistically analyzed using the SPSS 16.0 statistical package and expressed as mean value ± standard deviation. Statistical significance was established at p < .05.

| Diagnosis of potato viruses
In the first research stage, samples were taken from the potato tuber sprouts. To obtain healthy and monoinfected clones, a DAS-ELISA test was conducted to identify the PVY, PVX, PVS, PVM, and PLRV.

| Chemical properties and morphological characteristics of starch
As noted before, researchers found a decrease in the starch content in the tubers of potatoes infected with the virus (Blotskaya, 2000;Hou et al., 2020;Lal et al., 2021;Zhao et al., 2022;Amelyushkina, 2018;Rusetsky, 2006). The dry matter of tubers and starch content decrease in tubers of potato plants infected by PVM, PVS, and PVY. Morphological studies of potato starch samples are presented in Table 1 and Figure 2, and there is a difference in the size of granules with a spherical-oval shape. Table 1  Even though the variety of sizes and shapes of starch grains is of various origins, the internal organization of these grains in starch is practically the same. Starch macromolecules are ordered in amorphous and semicrystalline parts (Jenkins & Donald, 1995).
Double helixes are perpendicular to the growth rings and closer to the surface of the starch grains than the center. The circular orientation is due to the birefringence structure, which can be fixed using crossed polarizers. All granules exhibit a polarizing cross.

| Differential scanning calorimetry
The thermal characteristics of native potato starches determined by the differential scanning calorimetry (DSC) are shown in  (Zhou et al., 2020).
It is possible that the influence of PVM and PVY on the metabolic process of potato plants affected the crystal structure of starch granules, partially destroying them, and thereby enhancing granules' ability to swell. In addition, an increase in gelatinization enthalpy in starches isolated from virally infected tubers is observed (ΔH). ΔH increased from 5,44 J/g (virus-free) to 8,17 J/g (PVY). This points to the conclusion that more energy is needed to gelatinize them.

ACK N OWLED G M ENTS
The research team offers their deepest and most sincere gratitude to the College of Food Science of the Northwestern University of Agriculture and Forestry, Yangling, the Peoples Republic of China, for providing its laboratories and expertise during the actual research work and preparation of this article.

FU N D I N G I N FO R M ATI O N
This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan, grant number AP08857439.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting this study are available on request from the corresponding author.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M ED CO N S ENT
Written informed consent was obtained from all study participants. TA B L E . 2 XRD properties and thermal properties of «Artemis» variety potato tubers infected viruses PVM, PVY, and PVS and its starch granules.