Effects of Flooding Stress on Growth and Root Physiology and Bioche-mistry of Grafted Red-seed Watermelon Seedlings

Ke ZHANG, Siliang LUO, Tangjing LIU, Wu QIN, Suping WU

Abstract ［Objectives］ This study was conducted to explore how to improve the waterlogging tolerance of red-seed watermelon through grafting， to provide a theoretical basis for its cultivation in rainy season.

［Methods］ The effects of flooding stress on the growth and root physiological and biochemical characteristics of grafted and own-rooted red-seed watermelon seedlings were studied using Luffa as rootstocks and "Zhongxin 1" red-seed watermelon as scions.

［Results］ After flooding stress， the biomass and root activity of grafted seedlings of red-seed watermelon were significantly higher than those of own-rooted seedlings. With the prolongation of flooding stress time， the soluble sugar and proline content showed a trend of first increasing and then decreasing， and the grafted seedlings had a larger increase and a smaller decrease， and were always significantly higher than own-rooted seedlings in the same period. The content of malondialdehyde in the root system of grafted seedlings increased first and then decreased， while it Continued to increase in own-rooted seedlings， and the increase in own-rooted seedlings was significantly greater than that in grafted seedlings during the same period.

［Conclusions］ Grafting on Luffa rootstocks could improve waterlogging tolerance of red-seed watermelon.

Key words Red-seed watermelon; Grafted seedlings; Flooding stress; Root system; Physiological metabolism

Received： December 30， 2022  Accepted： March 1， 2023

Supported by "Watermelon and Muskmelon Germplasm Innovation and Genetic Improvement" Post of Guangxi Bagui Scholars （2016A11）.

Ke ZHANG （1984-）， female， P. R. China， assistant research fellow， devoted to research about genetic breeding and cultivation physiology of cucurbit crops.

*Corresponding author. E-mail： gxaas_ltj@163.com.

Red-seed watermelon （Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao） is a seed-using watermelon［1-3］， commonly known as "Hongda Gua"， "Xizi Gua" and "Guazi Gua". It is a seed-using watermelon variety in Citrullus lanatus ssp. vulgaris of Cucurbitaceae［4-5］. It is mainly distributed in more than 10 provinces （regions）， including Gansu， Xinjiang， Inner Mongolia， Ningxia， Guangxi， Anhui， Jiangxi， Hunan， Heilongjiang， and Guangdong. Red-seed watermelon is a kind of economic crop with high edible nutrition and medical health value. The seeds of red-seed watermelon are rich in high-quality vegetable oil and protein， as well as a variety of amino acids， vitamins， mineral elements， polysaccharides， and other substances， and thus have important medical and health effects. However， the basic research work on red-seed watermelon is very weak［1］. Currently， with the development of the commodity economy， the research on red-seed watermelon is gradually being paid attention to. The Horticultural Research Institute of Guangxi Academy of Agricultural Sciences and other units have successively carried out the cultivation and breeding of southern ecotype red-seed watermelon， and have taken the lead in conducting space mutation breeding of red-seed watermelon and the selection of new functional healthcare varieties. Liu and He et al.［6-15］ conducted long-term and systematic research on major diseases such as Fusarium wilt and anthrax in the production of red-seed watermelon in southern China， and proposed new solutions to solve or alleviate the diseases of red-seed watermelon in continuous cropping. It has played a positive role in promoting the scientific research and production of red-seed watermelon. In recent years， the rapid rise and development of red-seed watermelon seed processing industry at home and abroad has led to an increasing demand for red-seed watermelon seeds in the market. Therefore， there is an urgent need to cultivate new varieties of red-seed watermelon with high yield， high quality， and disease resistance， as well as to solve the problem of unstable yield caused by excessive rainfall in the production of red-seed watermelon in the southern ecological region.

Soil flooding or excessive humidity is a very common phenomenon. Flooding stress often affects the growth and development of crops. Excessive water impedes gas exchange between crops and the atmospheric environment， causing hypoxia in the flooded tissues of crops， making it difficult to maintain normal physiological metabolism and growth and development. Therefore， flooding stress often leads to serious crop yield reduction. Watermelon， originating in Africa， is a drought-tolerant plant that is very sensitive to flooding and lacks an effective defense mechanism against flooding. In southern China， continuous overcast and rainy spring and high-temperature and rainy summer have become one of the constraints on the production of watermelon and red-seed watermelon in southern China. Liu et al.［16-17］ studied the changes in physiological and biochemical indexes such as leaf morphology， cell protective enzymes， soluble protein and membrane lipid peroxidation product malondialdehyde of watermelon seedlings under flooding stress， providing a theoretical basis for watermelon breeding and watermelon cultivation in rainy areas in the south. Zhang et al.［18］ compared the waterlogging tolerance of own-rooted cucumber seedlings， pumpkin rootstock-grafted seedlings， and Luffa rootstock-grafted seedlings under waterlogging stress. The results showed that using Luffa as rootstocks could better improve the waterlogging tolerance of cucumber. Liu et al.［19］ conducted a preliminary study on the morphology and waterlogging resistance of watermelon grafted on Luffa as rootstocks. However， the effect of flooding on the growth of red-seed watermelon， especially on grafted plants， has not been reported yet. In this study， the effects of flooding stress on the growth and root physiological and biochemical characteristics of grafted and own-rooted red-seed watermelon seedlings were studied with Luffa as rootstocks and "Zhongxin 1" red-seed watermelon as scions， aiming to provide a theoretical basis for the grafting cultivation of red-seed watermelon.

Materials and Methods

Experimental materials

The tested rootstock variety was "Luffa aegyptica Miller"， a local variety of Luffa in Hezhou City， Guangxi， and the red-seed watermelon scion variety was "Zhongxin 1"， both provided by Research and Development Center of Xindu Red-seed Watermelon in Guangxi.

Experimental design

The experiment was conducted in the spring of 2022 in greenhouses of Guangxi Academy of Agricultural Science. Seedlings were raised in a plug tray substrate without soil， and were grafted by the top grafting method. The seedlings were divided into grafted seedlings and own-rooted seedlings （control） without grafting treatment. After the grafted seedlings survived， grafted seedlings and own-rooted seedlings with consistent growth were selected and transplanted into plastic boxes （10 cm×10 cm×10 cm）， according to 2 plants per pot. The transplanted seedlings were pre-incubated for 7 d， and then placed in a water culture tank to carry out flooding treatment. A total of 4 experimental treatments were set up： ① own-rooted seedlings （OS1）， ② own-rooted seedlings+flooding stress （OS2）， ③ grafted seedlings （GS1），  and ④ grafted seedlings+flooding stress （GS2）. Each treatment had 30 plants and was repeated three times， and random arrangement was adopted.

Experimental methods

Biomass measurement： On day 10 after the flooding treatment， the plants were taken out of the cultivation boxes， washed with tap water and dried by sucking. They were divided into the aboveground and underground parts， and their fresh weights were measured. After that， they were placed in an oven at 105 ℃ for 15 min， and then dried to constant weight at 75 ℃， and their dry weights were measured.

After 0， 2， 4， 6， 8， and 10 d of flooding treatment， 5 seedlings of red-seed watermelon were taken from each treatment， and a 2 cm segement was cut from the peripheral roots of the plants to determine their physiological indexes. Each treatment was repeated three times.

Root activity was measured by the TTC method［20］. Soluble sugar content was measured by the anthrone colorimetry［20］. Proline and malondialdehyde （MDA） contents were measured according to the method of Wang［20］.

Data processing

The data were analyzed using SAS software for variance analysis， and Microsoft Excel 2021 software was used for data statistics and plotting.

Results and Analysis

Effects of flooding stress on biomass of grafted and own-rooted seedlings of red-seed watermelon

Flooding stress significantly inhibited the growth of both grafted and own-rooted seedlings of red-seed watermelon， resulting in a significant decrease in the biomass accumulation of GS2 and OS2 （Table 1）.  However， the fresh and dry weights of the aboveground part of GS2 was 1.70 and 2.16 times higher than those of OS2， respectively; and the fresh weight and dry weights of the underground part were 2.11 and 2.07 times higher than OS2， respectively， indicating that the growth of grafted seedlings was less inhibited under flooding stress. Moreover， after flooding stress， the root-to-shoot ratios of the grafted and own-rooted seedlings of red-seed watermelon and dry matter ratios of their aboveground and underground parts were significantly increased， with GS2 being 1.05， 1.27， and 1.05 times higher than OS2， respectively， indicating that the adaptability of grafted seedlings to flooding stress was stronger than that of own-rooted seedlings.

Effects of flooding stress on root activity of grafted and own-rooted seedlings of red-seed watermelon

It can be seen from Fig. 1 that the root activity of grafted seedlings was lower than that of own-rooted seedlings before flooding treatment， but the difference was not significant. The root activity of GS2 and OS2 increased in the first 4 d after flooding stress， and began to decrease after the 4th day， with GS2 significantly higher than OS2. GS2 was significantly higher than GS1， OS1， and OS2 from the 2nd day of stress. After the 8th day of stress， OS2 was significantly lower than GS1 and OS1. For the non-flooding treatment， OS1 was higher than GS1 in the first 4 d， and GS1 was higher than OS1 after the 4th day. The root activity of grafted and own-rooted seedlings increased first and then decreased significantly under flooding stress， which might be a physiological adaptation response caused by flooding stress.

Effects of flooding stress on soluble sugar content in roots of grafted and own-rooted seedlings of red-seed watermelon

As shown in Fig. 2， there was no significant difference in the soluble sugar content in the roots of own-rooted seedlings and grafted seedlings under non-flooding treatment. Under flooding stress， the soluble sugar content in grafted seedlings was significantly higher than that in own-rooted seedlings， and the soluble sugar contents of GS2 and OS2 increased in the first 2 d of stress， and began to decrease after the 2nd day， with a greater decrease in OS2. On the 8th day， OS2 was significantly lower than GS2， GS1， and OS1.

Ke ZHANG et al. Effects of Flooding Stress on Growth and Root Physiology and Biochemistry of Grafted Red-seed Watermelon Seedlings

Effects of flooding stress on proline content in roots of grafted and own-rooted seedlings of red-seed watermelon

From Fig. 3， it can be seen that the proline content of grafted seedlings was significantly higher than that of own-rooted seedlings， and OS2 and GS2 were relatively consistent， both showing a trend of first increasing and then decreasing， and little changes were observed in GS1. OS2 was lower than GS2， GS1， and OS1 from the 8th day.

Effects of flooding stress on malondialdehyde contents in roots of grafted and own-rooted seedlings of red-seed watermelon

As shown in Fig. 4， the content of MDA in own-rooted seedlings under both flooding and non-flooding treatments was higher than that in grafted seedlings， and with the extension of stress time， except for the decrease in GS2， both treatments showed a continuous upward trend. Among the four treatments， OS2 showed the highest MDA content， and GS1 showed the lowest value.

Conclusions and Discussion

Under flooding stress， the seedlings of red-seed watermelon were damaged， which was reflected in their true leaves turning yellow and green and their roots and stems beginning to decay. In this study， the biomass accumulation of both grafted and own-rooted seedlings of red-seed watermelon under the flooding treatment decreased significantly， but the decrease of grafted seedlings was smaller than that of own-rooted seedlings， indicating that the degree of inhibition in grafted seedlings was relatively smaller and their adaptability to flooding stress was stronger than that of own-rooted seedlings.

Liu et al.［16-17］ pointed out that watermelon underwent some adaptive mechanisms under flooding stress， and produced adventitious roots， and its antioxidant system also produced a series of responses. The SOD activity of watermelon seedling leaves decreased at the early stage of flooding， and as watermelon seedlings adapted to flooding stress， SOD begun to rise， and exceeded the control after 8 d. After flooding stress， the POD activity of watermelon seedling leaves increased all the time and was higher than the control; and the soluble protein content was lower than the control. With the increase of the degree of flooding stress， the decrease of PRO increased. After flooding stress， the content of MDA first decreased and then increased， decreased again， and then significantly increased， and it was higher than the control.

Crop roots are an important absorption and synthesis organ， and their growth and vitality directly affect the growth， nutritional status， and yield levels of the aboveground part. The root system is an important organ in crop life activities， which mainly supplies water and mineral substances required for the growth of the aboveground part， and is closely related to crop growth and yield formation. Root activity is an important physiological index used to measure root growth. The value of root activity reflects the metabolic strength of the root system. Higher root activity means more vigorous metabolism of root tissue and a stronger root system， and is more beneficial to the growth of the entire plant. In this study， before flooding stress， the root activity of grafted seedlings was lower than that of own-rooted seedlings， but the difference was not significant. The root activity of grafted and own-rooted seedlings increased in the first 4 d of the flooding treatment， and began to decrease after the 4th day， but the root activity of grafted seedlings was significantly higher than that of own-rooted seedlings. GS2 was significantly higher than GS1， OS1， and OS2 from the 2nd day of stress; and OS2 was significantly lower than GS1 and OS1 after the 8th day of stress. The change trends of grafted seedlings and own-rooted seedlings under the non-flooding treatment were consistent. Own-rooted seedlings were higher than grafted seedlings in the first 4 d of stress， and GS1 was higher than OS1 from the 4th day. During the process of waterlogging stress， the seedlings grafted on Luffa rootstocks maintained a higher root activity， which could allow the root system to maintain certain water and fertilizer absorption capacities and the leaves to maintain a high photosynthetic rate， thereby providing a material basis for the waterlogging resistance of red-seed watermelon.

In order to adapt to adverse conditions， such as drought and low temperature， crops actively accumulate some soluble sugar and reduce osmotic potential and freezing point to adapt to changes in external environmental conditions. In this study， the soluble sugar content of the grafted seedlings under flooding treatment was higher to adapt to the damage caused by flooding.

Proline is widely present in plants in a free state， and is the amino acid with the highest water solubility. It has a strong hydratability and does not carry electric charges when the pH is near neutral. During physiological drought in crop cells， its increase helps to maintain the water holding capacity of cells or tissues. Improving the adaptability of crops under drought， waterlogging and salt stress is an important way to achieve high yield and make full use of natural resources. In addition， some scholars also believe that proline is also a reactive oxygen species scavenger. In this study， the proline content of grafted seedlings was significantly higher than that of own-rooted seedlings， and GS2 and OS2 increased first and then decreased， while GS1 had no significant change. OS2 was lower than GS2， GS1， and OS1 from the 8th day.

MDA is a product of membrane lipid peroxidation and decomposition. Within a certain stress intensity， various protective mechanisms of cells maintain MDA content at a certain level， but when the stress intensity exceeds a specific threshold， intracellular metabolism is disrupted， free radicals accumulate， membrane lipid peroxidation increases， and MDA content increases. Therefore， to a certain extent， the level of MDA content can indicate the degree of cell membrane lipid peroxidation and the strength of crop response to adverse conditions. The MDA content of watermelon seedling leaves was higher than that of the control after flooding stress， indicating that after the plant was flooded， cell membrane lipid peroxidation occurred in watermelon leaves. After flooding stress， the MDA content first decreased， then increased， then decreased， and then significantly increased［16-17］. In this study， the contents of MDA in own-rooted seedlings in flooding and non-flooding treatments were both higher than those in grafted seedlings. With the extension of stress time， except for the decrease of GS2， all showed a continuous upward trend. The MDA content was the highest in OS2 under flooding stress， and the lowest value was observed in GS1， and OS1 and GS2 were between the two.

In summary， the biomass of grafted seedlings under flooding treatment was significantly higher than that of own-rooted seedlings. Grafted seedlings could significantly improve the root activity of red-seed watermelon under flooding stress. With the prolongation of flooding stress time， the soluble sugar and proline contents of both grafted and own-rooted seedlings showed a trend of first increasing and then decreasing， but the grafted seedlings exhibited a larger rising range and a smaller decreasing range， and were always significantly higher than own-rooted seedlings in the same period. The content of MDA increased first and then decreased in the root system of grafted seedlings， while it Continued to increase in own-rooted seedlings， and the increase in own-rooted seedlings was significantly greater than that in grafted seedlings during the same period. It indicated that grafting could improve the waterlogging tolerance of red-seed watermelon.

References

［1］ Zhengzhou Fruit Research Institute， Chinese Academy of Agricultural Sciences， Watermelon and Muskmelon Professional Committee of China Horticultural Society， Watermelon and Muskmelon Association of China Horticultural Society. Chinese watermelon and muskmelon［M］. Beijing： China Agriculture Press， 2000. （in Chinese）.

［2］ LIU TJ， WANG LP. Scientific research progress of seed-using watermelon［J］. Journal of Huazhong Agricultural University： Natural Science Edition， 2006， 25（S1）： 22-33. （in Chinese）.

［3］ Office of Agricultural Zoning Committee of Guangxi Zhuang Autonomous Region. Resources of famous and excellent agricultural varieties in Guangxi［M］. Nanning： Guangxi Peoples Publishing House， 1998. （in Chinese）.

［4］ LIU TJ， ZHANG K， LU YM， et al. In vitro screening of Fusarium wilt-resistant germplasm resources of red edible seed watermelon （Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao）［J］. Agricultural Biotechnology， 2014， 3（1）： 50-53.

［5］ Cucumber Professional Committee of Gansu Horticultural Society. Opinions on the current standard definition of some terms in the production and research of black-seed watermelon［J］. China Watermelon and muskmelon， 1999（2）： 31-33. （in Chinese）.

［6］ LIU TJ， ZHENG XG， WANG LP， et al. Heritability and correlation of yield related traits in red-seed edible seed watermelon［J］. China Cucurbits and Vegetables， 2008， 21（2）： 6-10. （in Chinese）.

［7］ LIU TJ， ZHENG XG， WANG LP. Fusarium wilt resistance of germplasm resources of red-seed watermelon （Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao） by using in vitro selection technique［J］. China Vegetables， 2007（10）： 16-19. （in Chinese）.

［8］ LIU TJ， HE SY. Chemical control experiment on anthracnose of Xindu red-seed watermelon［J］. Plant Doctor， 2007， 20（1）： 30-31. （in Chinese）.

［9］ LIU TJ. Experiment on the control of watermelon Fusarium wilt with Lvheng No. 1 （hymexazol）［J］. Guangxi Plant Protection， 2001（1）： 32-33. （in Chinese）.

［10］ LIU TJ. Experimental report on the control of watermelon Fusarium wilt with Lvheng No. 1 （hymexazol）［J］. Sichuan Agricultural Science and Technology， 2001（3）： 27. （in Chinese）.

［11］ LIU TJ， HE SY. Chemical control experiment on anthracnose of Xindu red-seed watermelon［J］. China Watermelon and muskmelon， 2004（5）： 11-12. （in Chinese）.

［12］ HE SY， LIU TJ. Experiment in the control of red-seed watermelon anthracnose［J］. Journal of Anhui Agricultural Sciences， 2004， 32（5）： 977. （in Chinese）.

［13］ LIU TJ， WANG LP， WU SP. Techniques of the stable yield and disease control of Xindu red-seeded using watermelon under continuous cropping condition［J］. China Cucurbits and Vegetables， 2006（3）： 7-11. （in Chinese）.

［14］ LIU TJ， WANG LP， WU SP. Measures for high quality and yield increase of Xindu red-seed watermelon continuous cropping［J］. Journal of Changjiang Vegetables， 2006（6）： 16-17. （in Chinese）.

［15］ LIU TJ， WANG LP. Integrated increasing production technique of high quality continuous cropping for Xindu red-seed watermelon［J］. Guangxi Horticulture， 2006（3）： 25-28. （in Chinese）.

［16］ LIU WG， YAN ZH， WANG C， et al. Response of antioxidant defense system in watermelon seedling subjected to waterlogged stress［J］. Journal of Fruit Science， 2006， 23（6）： 860-864. （in Chinese）.

［17］ LIU WG， YAN ZH， WANG C， et al. Effects of flooding stress on physiological and biochemical characteristics of watermelon seedlings［C］∥Chinese Horticultural Society. Proceedings of the 10th Member Congress and Symposium of the Chinese Horticultural Society. Changsha： Chinese Horticultural Society， 2005. （in Chinese）.

［18］ ZHANG J， LIU MY， XIAO W， et al. Study on enhancement of waterlogging tolerance of cucumber seedlings with loofah as stock［J］. Chinese Bulletin Botany， 2003， 20（1）： 85-89. （in Chinese）.

［19］ LIU XJ， ZHENG SF. The morphologic observation of watermelon grafted with loofah and evaluation of waterlogging tolerance in grafted watermelon［J］. Journal of Huazhong Agricultural University， 1995， 14（3）： 267-271. （in Chinese）.

［20］ WANG XK. Experimental principles and techniques of plant physiology and biochemistry （second edition）［M］. Beijing： Higher Education Press， 2006. （in Chinese）.