Comparison of root tolerance to drought and aphid (Myzus persicae Sulzer) resistance among different potato (Solanum tuberosum L.) cultivars

This study was conducted to determine the root system architecture and biochemical responses of three potato (Solanum tuberosum L.) cultivars to drought and aphid (Myzus persicae Sulzer) infestation under greenhouse conditions. A factorial experiment comprising three potato cultivars (Qingshu 9, Longshu 3, and Atlantic), two levels of water (Well watered and drought) application and aphid infestation (Aphids and no aphids) was conducted. The results show that drought stress and aphid infestation significantly increased the root-projected area, root surface area, number of root tips, and number of root forks of all cultivars, relative to their corresponding control plants. The least root projected area, root surface area, number of root tips, and number of root forks occurred on DXY under both drought and aphid infestation. Nevertheless, the greatest root projected area, root surface area, number of root tips and number of root forks occurred on QS9 plants. Moreover, increased SOD, CAT, and POD activities were observed across all cultivars, under drought and aphid stress. The highest SOD, POD, and CAT activities occurred in QS9; under drought and aphid stress, while the least SOD, POD, and CAT activities was observed in DXY. The Atlantic cultivar, which possesses a root system sensitive to water deficit, demonstrated greater resistance to aphid infestation under well-watered and drought-stressed conditions. Conversely, Qingshu 9, which possesses a root system tolerant to water deficit, was highly susceptible to aphids. This study shows that the root architectural and biochemical traits that enhance potato tolerance to drought do not necessarily correlate to a plant’s tolerance to aphids.

Plant roots are the main organs for transporting diverse resources from the soil, and thereby control plant efficiency 1 . The mechanism by which plant roots obtain water and nutrients from soil is complex and it involves several abiotic and biotic interactions 2 . Plants may develop deep or fibrous root systems in order to obtain available soil moisture for survival 3 . The increase in root length and proliferation of roots for drought tolerance has been reported in several crops, including Oryza sativa 4 , Zea mays 5 , Hordeum vulgare 6 , Triticum aestivum 7 , Brassica napus 8 , and Glycine max 9 . Root diameter and number of root forks that determines root conductivity haves also been found to improve drought tolerance in legumes 10 . Smaller root diameter efficiently increases hydraulic conductance by increasing the root surface area that can be used for water uptake 11 . Hence, a decrease in root diameter has been suggested as a trait for improving plant transmission of resources under stress conditions 7 . Root crossing (root branching) governs the bearing of vertical and horizontal distribution of roots in the soil, and is predicted as a vital trait for drought tolerance in Sorghum bicolor 12 , Triticum aestivum 13 , and Oryza sativa 4 . Plants under stress condition can exhibit signs of tissue dehydration, confirmed by a reduction in their root moisture content 14 . This was reported in Rosmarinus officinalis by Sánchez-Blanco et al. 15 and in Nerium oleander by Bañón et al. 16 .
Sap-sucking insects severely decreases potato production worldwide and are important economic pests of crops 17 . Globally, potato farmers consider aphids to be of greater economic importance than defoliators or tuber pests 18 . The green peach aphid, Myzus persicae (Sulzer) is a common aphid species that attacks close to 400 crops from diverse families as secondary hosts 19 . These aphids cause major damage to potato plants through feeding, honeydew production, and transmission of viruses 20 . It is reported that severity of drought-stress could cause outbreaks of insect pests 21 . Koricheva et al. 22 , in a review, reported insect response to induced water-deficit in woody plants, indicated that the population of sap-sucking insects increases more rapidly on drought-stressed plants than on well-watered plants. However, Huberty and Denno 23 who worked on drought-stress and its impacts on herbivorous insects reported contrasting results.
Potato (Solanum tuberosum L.) is an important global food source 24,25 . Potato plants have comparatively shallow root systems and are sensitive to stress conditions 21 . The impact of drought is a major factor affecting the sustainable production of the crop 26 Climate change has increased the incidence of irregular weather patterns, comprising low and erratic rainfall patterns that can causes drought and increases pest populations, thereby adversely affecting crop production 27,28 . Thus, the development of potato varieties with high yield, improved quality, and drought and pest resistance is imperative. The use of aphid-resistant potato cultivars has also been proposed as one of the most important strategies for aphid control in China 25,28 .
In our previous research, we found that drought-tolerant potato cultivar was more susceptible to green peach aphids compared to drought-sensitive cultivar 29 . However, the root architectural and biochemical reactions of potato plants to drought-stress and green peach aphid has not been reported. The current research was therefore based on the hypothesis that the responses of root tolerance to drought stress in potato cultivars vary from the responses of root tolerance to aphid infestation. Our specific goal was to determine the root system architecture and biochemical reactions of three potato cultivars to drought stress and green peach aphid infestation under greenhouse conditions. This research will provide a scientific basis for breeding cultivars adapted to regions with frequent drought stress and aphid infestation.

Results
Aphid performance. The results showed a significant (P < 0.01) variety × drought interaction effect on aphid population abundance. Relative to the initial population, the number of green peach aphids at 28 days post-infestation associated with Qingshu 9, Longshu 3, and Atlantic was increased by 71.2, 68.7, and 43.3%, respectively, under well-watered conditions, and by 64.2, 60.2, and 35.8% under drought stress (Fig. 1). Moreover, green peach aphids reared on Atlantic plants exhibited a 53.1 and 44.4% decrease in population compared with those reared on Qingshu 9 and Longshu 3 plants, respectively, under well-watered and drought-stressed conditions, at 32 days post-infestation. There was also a significant (P < 0.01) variety × drought interaction effect on aphid fresh weight, dry weight, and mortality rate (Fig. 2). Drought stress significantly decreased the fresh weight of aphids reared on Qingshu 9, Longshu 3, and Atlantic by 32.7, 36.1, and 39.4% in comparison to the respective controls (Fig. 2a). Drought stress also decreased aphid dry weight of Qingshu 9, Longshu 3, and Atlantic by 48.8, 49.7, and 50.9% in comparison to the control plants (Fig. 2b). Generally, aphid mortality rate was higher on drought-stressed plants. Aphid mortality rate on drought-free (58.6%) and those under drought (68.7%) were highest on Atlantic and least on Qingshu 9 (12.2 and 32.7%, respectively) (Fig. 2c). This suggests that Atlantic and Qingshu 9 were considered the most resistant and susceptible cultivars to aphids, respectively.
Effect of drought stress and aphid infestation on root conductivity and distribution. There was a significant (P < 0.02) variety × drought × aphid interaction effect on root crossings. However, total root length, root volume, and average root diameter were not affected (P = 0.06). Drought stress increased total root length of Qingshu 9, Longshu 3, and Atlantic by 75.7, 58.7, and 38.5%, respectively, in respect to the control plants. Under aphid infestation, total root length of Qingshu 9, Longshu 3, and Atlantic also increased by 43.4, 31.4, and 21.7% relative to the control plants. Drought and aphid infestation increased total root length of Qingshu 9, Longshu 3, and Atlantic by 79.4, 60.3, and 43.2%, respectively, over the corresponding control plants. The least increase in total root length under drought and aphid stress occurred on Atlantic (Fig. 3a). Drought stress   (Fig. 3d). Com-  www.nature.com/scientificreports/ paratively, Atlantic had the least total root length, root volume, and root crossings, and the largest average root diameter under both drought stress and aphid infestation. The greatest total root length, root volume, and root crossings, and the least average root diameter among the cultivars occurred on Qingshu 9 (Fig. 3).

Effect of drought stress and aphid infestation on root proliferation. Drought stress and aphid
infestation caused an increase in root projected area, root surface area, and number of root tips, of all cultivars relative to their corresponding control plants (    (Fig. 6d). Comparatively, Atlantic had the highest decrease of root fresh weight, root dry weight, root moisture content, and root mass fraction under drought stress. Nonetheless, the least decrease of root fresh weight, root dry weight, root moisture content, and root mass fraction of the cultivars occurred on Qingshu 9 plants. In contrast, under aphid infestation, the greatest decrease of root fresh weight, root dry weight, root moisture content, and root mass fraction under drought stress occurred on Qingshu 9, whereas the least decrease occurred on Atlantic (Fig. 6). www.nature.com/scientificreports/ Root biomass response to drought stress and aphid infestation. Drought and aphids stress significantly (P < 0.01) influenced root biomass accumulation of the potato cultivars. The highest biomass accumulation (45.9%) occurred on the Qingshu 9 cultivar under drought stress (Fig. 7a). However, under aphid infestation, the highest biomass occurred on Atlantic plants (Fig. 7b). Moreover, under the effect of both drought and aphid stress, Qingshu 9 had the highest (39.1%) biomass accumulation (Fig. 7c). Plant root biomass was greater in Atlantic (89.1%), but lower in Qingshu 9 (57.5%), under aphid stress. Accordingly, the Atlantic and Qingshu 9 cultivars were considered the most tolerant and susceptible cultivars to aphids, respectively.     www.nature.com/scientificreports/ Effect of drought stress and aphid infestation on antioxidant enzyme activities. To evaluate whether variances can be related with the cultivars' resistance to drought and aphid stress, the changes in the scavenging activity of ROS such as SOD, POD, and CAT were examined. The results indicate that SOD, POD, and CAT activities increased in all cultivars under drought stress and aphid infestation compared to their corresponding controls (Fig. 8d-f). Under drought stress, SOD activity in Qingshu 9, Longshu 3, and Atlantic increased by 75.5, 51.7, and 24.1%, respectively, in respect to control plants. Under aphid infestation, SOD activity in Qingshu 9, Longshu 3, and Atlantic also increased by 51

Discussion
The responses of aphids to drought stress reported in previous studies have indicated contrasting findings 30,31 . Koricheva et al. 22 reported that aphid population increased on drought-stressed cultivars than on well-watered cultivars, whereas Huberty and Denno 23 found decreases in their population under similar condition. Few field trials support the notion that aphid population increases on drought-stressed cultivars; experimentally imposed water deficit, nonetheless, often negatively influences aphid population abundance 23 . In this study, aphid population was higher on the drought-free plants compared with the drought-stressed plants across all cultivars. Drought stress negatively affected aphid population, which led to greater mortality rate and decreased biomass of the aphids in all the cultivars. Other studies have produced contradictory or varying results 32,33 . However, the response of insect pest population to drought stress is suggested to hinge on the variety of plant and the intensity of stress 34 . An experiment on Brassica oleracea showed that aphid population increased on drought-free plants compared with the drought-stressed plants 35 . Moreover, Floater 36 recorded greater aphid mortality and lower aphid biomass on drought-stressed plants. In the present study, the drought-sensitive cultivar showed more resistance to aphid infestation under both water treatments. This cultivar also exhibited low aphid population abundance and a high aphid mortality rate. Conversely, the drought-tolerant cultivar was susceptible to the aphids and showed greater aphid population abundance and a low aphid mortality rate. It appears that aphid performance correlates positively with high water content in the host plant, under drought condition. The loss of water of all cultivars under drought stress increased mortality rate and decreased the fresh weight of aphids. Moreover, the tolerant cultivar (Qingshu 9) exhibited wide variation in root hydraulic conductivity traits, confirmed by increasing root fresh weight, root dry weight, root moisture content, root mass fraction, and root biomass, which led to greater availability of sap for the aphids to feed on. Accordingly, Qingshu 9 was the most susceptible host because the aphids survived better on it. In contrast, the sensitive cultivar (Atlantic) that possesses poor root hydraulic conductivity, exhibited signs of tissue dehydration, confirmed by a reduction in root fresh weight, root dry weight, root moisture content, root mass fraction, and root biomass that possibly starved the aphids to death over time. Noticeably, the moderately tolerant cultivar, Longshu 3, was not extremely susceptible, neither was it extremely resistance to the green peach aphid. However, comparatively, the green peach aphid performed better on it than the Atlantic cultivar, under drought stress. This was confirmed by the high aphid population abundance and low aphid mortality rate, compared with the sensitive cultivar. This is probably due to its ability to maintain turgor pressure by improving its hydraulic conductivity, compared with the Atlantic cultivar. The Longshu cultivar can also be utilize in areas where both drought and aphids are major concern. It is reported that, variations in host plant physical and chemical composition can have important consequences on herbivore population dynamics 37 . Potato varieties differ in the volatile profiles in their headspace and these differences elicit different behavior from the green peach aphid 38 . Although the high dehydration of Atlantic plants greatly increased aphids mortality rate under drought condition, Atlantic also exhibited higher resistance to aphids under well-watered condition. This suggest that the drought-sensitive cultivar may contain secondary metabolites that act as repellants to the peach aphid. We therefore speculate that plants natural defense against aphid attack and water availability contributes significantly to the outcome of aphid population abundance. Thus, host plant response to stress condition should be assessed when considering the response of herbivore insects to drought stress. The Atlantic cultivar can be utilized to protect against losses in potato production in regions where peach aphids are a key pest of potato as proposed by Xu et al. 39 .
Plants with better root conductivity and distribution are able to thrive well under drought stress due to their ability to source water 40 . The roots of stressed plants tend to increase and spread into deeper soil layers to obtain resources 41 . The present results show that drought and aphids stress increased total root length, root volume, and root crossings of all cultivars relative to their corresponding control plants. However, the average root diameter decreased under drought and aphid stress across all cultivars. A similar result was reported on Oryza sativa 42 , Cicer arientinum 43 , and Sorghum bicolor 44 . The Atlantic cultivar had the least total root length, root volume, and root crossings, and the greatest average root diameter under both drought and aphis stress. However, the greatest total root length, root volume, and root crossings, and the least average root diameter Scientific Reports | (2021) 11:628 | https://doi.org/10.1038/s41598-020-79766-1 www.nature.com/scientificreports/ among cultivars occurred on Qingshu 9. Drought-tolerant varieties are known to be capable of increasing their root depth, root volume, and root crossings significantly more than sensitive varieties under stress conditions in legumes 45 . Previous studies reported that plants with thicker roots tend to penetrate deeper under drought stress 46 . These results are in contrast with previous reports that increase in root diameter is a significant trait in tolerant cultivars under water stress conditions. However, it agrees with others who reported that small root diameter support plants to significantly improve hydraulic conductance by increasing the amount of surface area in contact with soil water 47 . These results suggest that total root length, root volume, root crossings, and average root diameter contributed significantly in the Qingshu 9 plants tolerant to drought, but this did not improve the plants resistance to aphids attack. In order to access available soil moisture under drought stress, plants adapt by greater root proliferation 3 . Root proliferation is generally governed by the initiation and elongation of lateral roots, which usually refers to lateral root number, root projected area, root surface area, number of root tips, and number of root forks 42 . Plants with greater root proliferation have comparatively great water uptake efficiency under stress condition 48 . Cicer arietinum lines with greater proliferation have been reported to perform better in yield and drought tolerance related traits under water deficit environments 49 . In the present study, drought stress and aphid infestation caused significant increase in the root projected area, root surface area, number of root tips, and number of root forks of all cultivars, relative to their corresponding control plants. Branching of roots of Silene vulgaris was increased under drought stress 47 . Likewise, root surface area of Silene vulgaris was increased under drought stress 47 . The least root projected area, root surface area, number of root tips, and number of root forks occurred on Atlantic under both drought and aphid infestation. Nevertheless, the greatest root projected area, root surface area, number of root tips, and number of root forks occurred on Qingshu 9 plants. The benefit of a deep and proliferative root system for tolerant cultivars under stress conditions has been reported in various crops, including Oryza sativa (Uga et al. 4 ), Zea mays 5 , Hordeum vulgare 6 , Triticum aestivum 7 , Brassica napus 8 , and Glycine max 9 . The Atlantic cultivar, which exhibited poor root system architecture under both drought and aphid infestation, showed greater resistance to aphids with or without drought stress.
In plant cells, H 2 O 2 is produced through aerobic metabolism, and as a detrimental oxygen derivative, it can cause cellular damage 50  Antioxidant enzyme protections comprising SOD, POD, and CAT directly scavenge superoxide radicals and H 2 O 2 55 . Other studies have confirmed that SOD, POD, and CAT activities increased in response to stress in Glycine max 56 and Panicum virgatum 57 . Consistent with our results, increased SOD, CAT, and POD activities were observed across all cultivars, under drought and aphid stress. However, the amount of accumulation differed between cultivars. The highest SOD, POD, and CAT activities occurred in Qingshu 9; both under drought and aphid stress, while the least SOD, POD, and CAT activities was observed in Atlantic. The accumulation of antioxidant enzymes in plants is reported as a signal of its level of tolerance to stress 55 . The greater antioxidant enzyme activities observed in Qingshu 9 did not enhance their resistance to the aphid. The least antioxidant enzyme activities occurred in Atlantic, under aphids stress. However, it inhibited the population abundance of the aphid. This was demonstrated by the higher biomass accumulated in Atlantic under aphid stress. This could be due to the inhibitory compounds in Atlantic cultivar. Thus, the results of our study propose that the root mechanism of tolerance to drought among potato plants differ from the mechanism that may enhance aphid resistance.

Conclusions
The results of this study demonstrate that Atlantic, which possesses a root system sensitive to drought, showed greater resistant to the peach aphid under both water treatments. This cultivar also exhibited poor aphid population abundance, high mortality rate, and higher biomass accumulation under aphid stress. Moreover, the moderately drought tolerant cultivar, Longshu 3, was not extremely susceptible, neither was it extremely resistant to the green peach aphid. However, this cultivar showed high aphid population abundance and low aphid mortality rate, compared with the sensitive cultivar. The Qingshu 9, which possesses root system tolerant to drought, was extremely susceptible to the aphid and demonstrated high aphid population abundance, low aphid mortality rate, and low biomass accumulation under aphid stress. Moreover, dehydration of the Atlantic cultivar decreased the population abundance of the aphid under drought stress. The Qingshu 9 cultivar, which had enhanced root system, probably improved water uptake and led to greater availability of sap on which the aphid survived and increased its population. The resistance of Atlantic to the peach aphid under both water treatments may also be due to the presence of secondary metabolites in the cultivar. Thus, the Atlantic cultivar can be utilized to protect against losses in potato yield in areas where peach aphids are a major pest of potato. The Longshu 3 cultivar can also be utilized in areas where both drought and aphids are major concern. This study indicates that the root architecture and biochemical trait that enhances potato tolerance to drought do not necessarily correlate to a plant's tolerance to aphids. Experimental design and treatments. A 3 × 2 × 2 factorial experiment in a split-split plot design with three replications was conducted in a greenhouse. The treatments were: three potato cultivars, Qingshu 9 (QS 9), Longshu 3 (L 3), and Atlantic (DXY), two levels of water availability to plants (well-watered and droughtstressed), and two levels of aphid infestation (aphid infestation and no aphid infestation). The three potato cultivars were allocated to the main plots. The main plots were split into sub plots for the drought-stress and wellwatered treatments. The split-plots were further split for the allocation of the aphid and no aphid treatments. Six pots per experimental unit were assigned to a water treatment. A total of 216 pots were used for the experiment.
In each experimental unit, six plants were sampled for data collection, giving a total of six subsamples in each of the three replications for each treatment. The well-watered plants were defined as plants growing in soil with a water content of 100% of soil capacity, whereas the drought-stressed plants were defined as plants growing in a soil with a water content of 30% of soil capacity 58 . Soil capacity was determined by applying known volume of water per pot and allowing the excess water to drain through the perforated holes at the base of the plastic pot. The excess water was collected until no more water drained out. The amount of water collected was subtracted from the amount of water applied and the difference was considered the amount of water for field capacity. Prior to exposing the various cultivars to drought and aphid treatments, all the pots were maintained at field capacity by regular watering. The well-watered treatments were watered regularly to maintain the field capacity (100% soil moisture) throughout the experiment. For the 'Drought plants' , watering was withheld and monitored with soil moisture meter (Delta-T Devices, Cambridge, UK) until the water level dropped to 30%. To maintain the levels of water through time in each treatment, Delta-T Theta Probe ML2 (Delta-T Devices, Cambridge, UK) was used to measure the soil moisture content and a little amounts of water was applied to the 'Drought plants' to maintain the 30% soil capacity in each of the pots containing the drought treated plants. This procedure was repeated until the end of the experiment.
Determination of aphid population abundance, biomass, mortality rate, and tolerance index. Sixty day old potato plants were infested with aphid nymphs and monitored for 32 days. Six shoots of each cultivar in each three replications were infested with 20 nymph aphids for both levels of water availability to treatments. The aphids were introduced to the designated plants at 20 days after drought treatment. Each of the shoots was completely enclosed separately with a nylon mesh cage to prevent aphid escape. Aphid nymphs were allowed to develop into adults and reproduce on each plant for 32 days. Aphid numbers on each plant were determined on day 16, 20, 24, 28, and 32. Adults from each plant were pooled and weighed immediately after they were collected, dried at 60 °C for 24 h, and then weighed again. Aphid mortality rate was measured by counting the number of dead aphids, together with counts of their nymphs (including live and dead nymphs) on each plant. Aphid mortality rate (%) was then calculated as: