Effect of Yield and Quality of Erythropalum Scandens Bl. Under Different Dosage and Proportion of Chicken Manure and Cow Manure

Erythropalum scandens Bl. is a kind of leafy vegetable which has high edible and medicinal value in Southern China. However, it has been treated as a kind of wild vegetable for a long time and there is still little researches on its yield and quality after fertilization. This study aimed to assess the effect of yield and quality of E. scandens after using two kinds of organic fertilizer: chicken manure and cow manure, which nd out the responses of the edible part in E. scandens after using different dosage and proportion of chicken manure and cow manure and nd out a best fertilization treatment. We investigated the yield and quality of the edible part in biennial E. scandens including average yield, titratable acid content, sugar acid ratio, crude protein content, ascorbic acid content, tea polyphenols content, nitrate content, general avone content and macronutrient content (including total nitrogen, phosphorus and potassium) after using different dosage and proportion of chicken manure and cow manure, which to nd out a best fertilization treatment via the yield, nutrient and taste. Compared with CK group, proper fertilization can increase the average yield of E. scandens and promote its growth and macronutrient accumulation at the same time. The content of all kinds of nutrients are higher than CK group after using organic fertilizers and they can be accumulated during the process of fertilization. The E. scandens seedlings had the highest yield and best quality under T 6 treatment (0.8kg/plant chicken manure+1.2 kg/plant cow manure). However, nitrate can be accumulated in the edible part of E. scandens during the process of fertilization. Compared with CK group, organic fertilization can increase the yield and quality of E. scandens, but excessive fertilization can lead to a signicant increase in nitrate levels in plants, even exceeding food safety standards. The nitrate level of E. scandens is a little bit high in this research and better solution is still needed to ensure the quality and food security of E. scandens in the near future.


Introduction
Proper fertilization is important during the process of plant growth and vegetable production, which can improve vegetable's yield, quality and increase soil fertility. But the soil residual of excessive fertilizers can cause lots of problem, such as greenhouse gas emission, crop yield decrease, soil texture recession (Reay et al., 2012;Wang et al., 2019a), ground water pollution, surface water eutrophication (Gu et al., 2013;Le Afolayan, 2015). People treated them as a kind of food when famine and food shortage came because it can provide vitamins, microelements, ber and more nutrient to human (Łukasz and Katija, 2015;Sajid and Joachim, 2015). Fertilization is rarely studied on these plants, which has been found in some kinds of wild leafy vegetables. For example, organic fertilizers can improve the yield of pumpkin and nightshade (Azeez et al., 2010). Some other kinds of leafy vegetables (such as Moringa oleifera (Sarwar et al., 2020), Calamus gracilis (Wang et al., 2019b) and so on) also have some researches on fertilizers. There are a number of resources survey (Łukasz and Katija., 2015), phytochemistry and pharmacological studies review (Bello et al., 2019), nutrient composition testing (Garcı ´a-Herrera, 2014) researches about these plants. Erythropalum scandens Bl. is a kind of perennial woody lianas distributed naturally in the south and southeast of China (Xi et al., 2009). Its young leaves and stems is always used as a kind of eatable wild leafy vegetable in tropical and subtropical China because of its unique avor. The content of its nutrients are higher than those common vegetables in Guangxi Zhuang Autonomous Region in China (Long et al., 2017). Besides, its leaves, roots and stems can be used as a kind of medicine, which can treat gout, relieve edema for human beings . Besides, it also has the function of being interlaminar plant, conserving soil and water and so on (Qi and Tang, 2008;Jiang et al., 2014.). Zhu et al (2018) extracted its DNA and analyzed its genetic and genomic information in order to have a further promotion of its medicinal and edible value; Besides, there are some researches about its essential oil extraction (Feng et al., 2014). But there has been no research on fertilization, growth regulation and other aspects up till now.
According to few fertilization researches in leafy vegetables nowadays, chicken manure and cow manure were selected as two kinds of organic fertilizers to increase the yield and quality of E. scandens seedlings in this experiment. A series of eld experiments were conducted to measure the average yield, titratable acid content, sugar acid ratio, crude protein content, vitamin C content, tea polyphenols content, nitrate content, general avones, amino acid content in its eatable part and total N, P, K content in its tender buds to re ect the change of E. scandens seedlings after using different dosage and ratio of these two kinds of organic fertilizers. These all indices can also be the basis of E. scandens cultivating under different fertilizers, so as to promote its salesment and economic development in the countryside. To some extent, the enhancement of its sale can even improve farmers' annual income in these areas.

Ethics statement
The Erythropalum scandens Bl. is a common kind of leafy vegetable in China. It's not concluded in the "IUCN Red List of Threatened Species". This experiment was conducted in Nanning Arboretum and has been approved. The process of plant transplanting, maintenance and leaves collecting are conducted in accordance with ethical guidelines, in order to ensure its normal growth.

The introduction of test site, plants, soil and fertilizers
The eld experiment was conducted at Liangfengjiang Forest Research Center, Nanning Arboretum (22°43'N, 108°17'E), Guangxi Zhuang Autonomous Region, China. The total area of the test site is about 4863.7 hactare. The average annual temperature is 21.6℃, the average annual temperature of the hottest and coldest months is 28.3℃ and 12.8℃ respectively, and the average annual precipitation is about 1300.6mm. The climate is the south Asian tropical monsoon climate, which is suitable for the growth of E. scandens. The soil sample which is used for this study was partial acid red soil, with a pH of 5.5. The other basic physical and chemical properties of the tested soil were: total nitrogen (N) 0.68g/kg, total phosphorus (P) 0.26 g/kg, total potassium (K) 4.09 g/kg, available nitrogen (N) 87.25mg/kg, available phosphorus (P) 10.14mg/kg, available potassium (K) 85.12mg/kg, organic matter 14.67g/kg. The biennial Erythropalum scandens Bl. seedlings (43.9±7.4cm in the length of branches and 9.43±1.35mm in diameter at the base) were provided by Liangfengjiang Forest Research Center and Nanning Arboretum. In January 2018, a at open eld was chosen to plant the annual E. scandens seedlings and they were planted as a row spacing of 60cm ( Figure 1). Before planting the seedlings, the soil in the eld should be loosen, and the planting pattern is shown in Figure 1 below. Before the o cial experiment, the tested seedlings should be carefully managed, the frequency of irrigation should be formulated properly according to the soil condition and weeding should be done once a week. Automatic spraying 2-3 times per day in drought weather to ensure the healthy growth of the tested seedlings.
The chicken manure was obtained from Nanning Liangfeng Agriculture and Animal Husbandry Co. LTD, which contained total nitrogen 2.25%, total phosphorus 2.92%, total potasium 2.13%, organic matter 29.82% and its ratio of carbon and nitrogen was 8.44; the cow manure was obtained from cattle farm in Guangxi University, which contained total nitrogen 1.41%, total phosphorus 0.97%, total potasium 1.04%, organic matter 43.69% and its ratio of carbon and nitrogen was 17.97. Both of these manures were fully rotten before the formal experiment. Besides, some of the harmful elements, such as arsenic (As), chromium (Cr), cadmium(Cd) and plumbum (Pb) are also tested before the experiment. The chicken manure contained arsenic (As) 2.17mg/kg, chromium (Cr) 6.81mg/kg, cadmium (Cd) 0.83mg/kg and plumbum (Pb) 3.84mg/kg; The cow manure contained arsenic (As) 1.04mg/kg, chromium (Cr) 5.63mg/kg, cadmium (Cd) 0.44mg/kg and plumbum (Pb) 1.22mg/kg.

Experimental design
The biennial E. scandens seedlings were recovered for about two months after transplanting and were carefully managed during the period of their recovery. The whole process of the experiment was conducted from September 2018 to September 2019. A pre-experiment was done before the formal experiment began. 5-level treatments of chicken manure and cow manure (0.4g/plant, 0.8 g/plant, 1.2 g/plant, 1.6 g/plant, 2.0 g/plant) were set respectively to test the effect of fertilization. After the preexperiment, we found that the growth of E. scandens seedlings will be inhibited under the treatment of 1.6 g/plant, 2.0 g/plant. Combined with the previous pre-experiment, some examples in some wooden plants and the calculation of the reasonable amount of fertilization applied, we set the nal 3 levels of the two kinds fertilizer (0.4kg/plant, 0.8kg/plant and 1.2kg/plant).
While doing formal experiment, treatments were arranged in a randomized block test design, being an exploratory research, three replications were used in each treatment, 20 plots were repeated for each plot, and a total of 600 plants were used in the whole experiment. Two kinds of organic fertilizers-chicken manure and cow manure-were used as fertilizers and set at 3 levels (0.4kg/plant, 0.8kg/plant and 1.2kg/plant, respectively). A total of 10 groups of treatments were set up, with a control treatment to which no fertilizers were added (CK). Fertilizer formula of each group is shown in the table below (Table  1).

Experimental procedures
Two rows of E. scandens seedlings without any treatment were set between different groups. At the same time, trenches were dug in the soil and plastic lm was embedded in order to prevent the interference and in uence among different fertilization treatments. Two fertilization treatments were conducted in March and June 2019. During fertilization, dig pits with a size of 35cm×25cm at the corresponding plants of each plot, weigh the organic fertilizer used by each group, mix it with the excavated soil, and ll the pits again. After the completion of land ll, level the corresponding positions. During the experiment, the watering frequency in summer and cloudy and rainy days was 2-3 times a day and 1 to 2 times a day in turn (the soil and seedlings in the eld of watering frequency were appropriately ne-tuned).

Indices determine
During the experiment period, the edible parts of tender stems and leaves of E. scandens after each fertilization treatment were cut over 15cm each month and placed on the electronic balance, and their fresh weight (accuracy was 0.01g) was measured and the value was recorded. After the recording was completed, the total output increment was calculated and recorded; Titratable acid was measured according to the method described by (

Data statistics and analysis
All data were statisticed by Excel 2016 and analyzed by SPSS version 17.0 (IBM Corp., Armonk, NY, USA). Analysis of variance was used to analyze the difference between different groups under different fertilizer treatments. Duncan's new complex range method was used for multiple comparisons. Generalized linear models are used to determine the main factors in uencing these same parameters and to quantify the effect of the interactions between two different kinds of organic fertilizers (chicken manure and cow manure).

Average yield
It can be seen from Figure 2 that different types of fertilization treatments can improve the average yield of E. scandens, and the average yield of E. scandens has an extremely signi cant improvement than CK group after using chicken manure and cow manure (p < 0.01). The average yield of CK group has a decrease due to the lack of fertilization. Besides, under the condition of the same application amount of chicken manure (0.4kg/plant and 0.8kg/plant), the total increase yield of E. scandens with the increase of the application amount of cow manure (T 3 >T 2 >T 1 , T 6 >T 5 >T 4 ). However, when the application amount of chicken manure was 1.2kg/plant, the average yield of E. scandens rst increased and then decreased with the increase of the application amount of cow manure. Under the condition of the same application amount of cow dung, the increased yield of E. scandens rst increased and then decreased with the increase of application amount of chicken manure (T 4 >T 7 >T 1 , T 5 >T 8 >T 2 , T 6 >T 9 >T 3 ). Among all the treatments, T 6 group (chicken manure 0.8kg/plant+ cow manure 1.2kg/plant) has the maximum average yield: 20.58g/area ( Figure 2).

Titratable acid content
It can be seen from Table 2 that the titratable acid content has no signi cant difference at rst, but the titratable acid content of E. scandens can be signi cantly improved under different types of fertilization treatments (p < 0.05) during the period of the experiment (the metaphase and the anaphase). The content of titratable acid increased in the same set of treatment, but the degree of increments varies with the amount of fertilizer applied. At the metaphase and anaphase, titratable acid content increased with the increase amount of cow manure when the amount of chicken manure is the same (T 3 >T 2 >T 1 , T 6 >T 5 >T 4 , T 9 >T 8 >T 7 ), and vice versa(T 7 >T 4 >T 1 , T 8 >T 5 >T 2 , T 9 >T 6 >T 3 ). In other words, when the amount of one fertilizer is the same, increasing the use of another fertilizer enhances the accumulation of titratable acid. Among all the treatments, at the metaphase and anaphase, T 9 group (chicken manure 1.2kg/plant+ cow manure 1.2kg/plant) has the maximum titratable acid content among all the treatments, and the results are: 0.751% and 0.995%, and these two results were 27.94% and 66.67% higher than the CK group, respectively ( Table 2). Table 2 Effects of different fertilization treatments on titratable acid content of E. scandens seedlings.

Number
Treatment experiment (the metaphase and the anaphase), but the sugar acid ratio of E. scandens seedlings showed a trend of rst increasing and then decreasing in CK group. After fertilization, the sugar acid ratio increased in the same set of treatment differently. At the metaphase and anaphase, there was no obvious relationship between the sugar acid ratio and the consumption of chicken manure and cow manure. Among all the treatments, at the metaphase and anaphase, T 5 group (chicken manure 0.8kg/plant+ cow manure 0.8kg/plant) has the maximum sugar acid ratio among all the treatments, and the results are: 6.03 and 7.29, and these two results were 53.05% and 92.35% times higher than the CK group, respectively (Table 3).

Crude protein content
It can be seen from Table 4 that the crude protein content has no signi cant difference at rst, but it can be signi cantly improved under different types of fertilization treatments (p < 0.05) during the period of the experiment (the metaphase and the anaphase), but the crude protein content of E. scandens seedlings showed a trend of rst increasing and then decreasing in CK group. At the metaphase and anaphase, the crude protein of E. scandens seedlings increased with the increasing of cow manure under the condition of the same treatment of chicken manure (T 3 >T 2 >T 1 , T 6 >T 5 >T 4 , T 9 >T 8 >T 7 ), but there was no signi cant relationship between the amount and increasing of fertilizer and the crude protein content in other treatments. At the metaphase, T 6 treatment had the maximum content of crude protein (26.34%), and at the anaphase, T 6 and T 9 treatments had the maximum value (27.79%). These two results were 8.93% and 19.12% higher than CK group, respectively (Table 4).

Ascorbic Acid (Vitamin C) content
It can be seen from Table 5 that the content of vitamin C can be signi cantly improved under different treatments of chicken manure and cow manure (p < 0.05), but in CK group the Vc content increased at rst and then decreased. With the process of fertilization, Vc in E. scandens seedlings can be accumulated in the same treatment except CK group. When using 0.4g/plant chicken manure and cow manure, the Vc content of E. scandens seedlings can be improved with the increasing of cow manure and chicken manure respectively, but with the increasing of chicken manure and cow manure, there was a trend of rst rising then falling, even continually decline at last with the increasing dosage of another manure. For example, at metaphase, when using 0.8g/plant chicken manure, the Vc content showed a trend of rst rising and then falling (T5>T6>T4), but when the dosage of chicken manure was up to 1.2g/plant, the Vc content of E. scandens decreased continuously with the increasing of cow manure (T 7 >T 8 >T 9 ). At the metaphase and anaphase, T 5 treatment (0.8g/plant chicken manure+0.8g/plant cow manure) had the maximum of Vc content (25.02mg/100g and 35.05mg/100g), and they were 134.88% (34.88%) and 189.25% (89.25%) higher than CK group (Table 5).

Tea polyphenols content
It can be seen from Table 6 that the tea polyphenols content increased continually in the same group with the process of fertilization. Although the nal content at anaphase can be signi cantly different under different fertilization combination (p < 0.05), in CK group the content of tea polyphenols showed a trend of increasing rst and then staying the same. At the metaphase and anaphase, the content of tea polyphenols increased continually with the increasing dosage of chicken manure when using the same dosage of cow manure. The trend of change in E. scandens seedlings with the increasing of cow manure is similar at metaphase when using the same dosage of chicken manure, but when using 1.2g/plant chicken manure, the tea polyphenols content would stay the same. But there was no obvious relationship between tea polyphenols content and the dosage change of fertilizers in other conditions. At the metaphase and anaphase, the tea polyphenols content had the maximum under T 5 treatment (0.8g/plant chicken manure+0.8g/plant cow manure), which the values were 0.34% and 0.45% respectively, and they were 78.95% and 136.84% higher than CK group (Table 6).

Nitrate content
It can be seen from Table 7 that the nitrate content increased continually in the same group under different fertilization, but the content of nitrate decreased without any fertilizers (CK group). There was also a signi cant difference between groups in nitrate content at metaphase and anaphase (p < 0.05). At the metaphase and anaphase, the nitrate content increased continually with the increasing dosage of chicken manure and cow manure when using the same dosage of another manure. The content of nitrate had the maximum under T 9 treatment (1.2g/plant chicken manure+1.2g/plant cow manure) at both metaphase and anaphase, which the values were 1638.92mg/kg and 2087.50 mg/kg respectively, and they were 73.16% and 152.75% higher than CK group (Table 7). Therefore, nitrate content increases with the increase of fertilizer application.

General avone content
It can be seen from Table 8 that the general avone content can be continually improved with the process of using organic fertilizers, even there was a little bit increase of the general avone content with the growth of E. scandens seedlings in CK group. At the metaphase and anaphase, when using the same amount of cow manure, the content of general avone rst increasing, then decreasing and then increasing, and nally gradually decreasing with the increasing of the chicken manure (T 3 >T 2 >T 1 , T 6 >T 4 >T 5 , T 7 >T 8 ≥T 9 ). Accordingly, when using the same dosage of cow manure at metaphase, the content of general avone increased with the increasing of chicken manure (T 7 =T 4 >T 1 , T 8 >T 5 >T 2 , T 9 >T 6 >T 3 ), and when it came to the anaphase, the general avone content showed a trend of rst increasing, then decreasing with the increasing of cow manure (T 7 >T 4 >T 1 , T 8 >T 5 >T 2 , T 6 >T 9 >T 3 ). The general avone content of E. scandens had the maximum under the T 7 treatment (1.2g/plant chicken manure+ 0.4g/plant cow manure) in both metaphase and anaphase, which the values were 0.075% and 0.091%, and they were 31.58% and 54.24% higher than CK group, respectively.

Total nitrogen(N), phosphorus(P) and potassium(K) content
It can be seen in Table 9 that the total content of nitrogen(N), phosphorus(P) and potassium(K) in tender buds of E. scandens seedlings can be improved gradually with the process of fertilization and they can accumulate in the buds. There were no signi cant difference on these three macronutrient content at rst (p > 0.05), but different organic fertilization can affect the content of them signi cantly (p < 0.05). At metaphase and anaphase, the content of total phosphorus in the tender buds of E. scandens seedlings can be improved gradually with the increasing dosage of chicken manure and cow manure when using the same dosage of the other kind of fertilizer (T 3 >T 2 >T 1 , T 6 >T 5 >T 4 , T 9 >T 8 >T 7 , T 7 >T 4 >T1, T 8 >T 5 >T 2 , T 9 >T 6 >T 3 ), and the trends in total kalium was similar. But when using 1.2g/plant chicken manure or cow manure at anaphase, there was a trend of rst increasing then decreasing with the increase of the other fertilizer of the total kalium content in the tender buds of E. scandens seedlings (T 8 >T 9 >T 7 , T 6 >T 9 >T 3 ).
But as the content of total nitrogen, the situation was quite different. When using the same dosage of cow manure at metaphase and anaphase, the total nitrogen content in tender buds of E. scandens seedlings increased with the increased of chicken manure (T 9 >T 6 >T 3 at metaphase, T 9 ≥T 6 >T 3 at anaphase), but when using the same dosage of chicken manure, there was no signi cant relationship between the increment of cow manure and total nitrogen content in the tender buds of E. scandens seedlings. It can be seen in the Table 9 that the total nitrogen content in the tender buds of E. scandens seedlings had the maximum under T 6 treatment (0.8g/plant chicken manure+ 1.2g/plant cow manure) at both metaphase and anaphase, and as for total phosphorus and total kalium can be best improved under T 9 treatment (1.2g/plant chicken manure+ 1.2g/plant cow manure), and the maximum value of total N, P, K at anaphase were 44.47g/kg, 1.21g/kg and 26.29g/kg, respectively.

Discussions
Organic fertilization is a common way to improve the yield and quality of vegetables during the whole process of plant growing (Gai et al., 2016), but some research showed that using organic fertilizer can be bene cial to the environment but it can cause the decrease of vegetable yield . In this research, the yield of E. scandens are improved greatly after using chicken manure and cow manure, which is consistent with most studies. Reasonable using of fertilizer can promote organic growing in many kinds of plants.
Titrate acid is an important index to re ect the taste of vegetables, and it can be in uence by many aspects, such as soil conditions, planting seasons and so on . Sometimes using fertilizer (or increasing the supplement of N, P and K) can decrease the content of acids, which can improve the taste of fruit and juice (Zhang et al., 2012;Parvizi and Sepaskhah, 2015). The content of titrate acid can be improved after using organic fertilizer, which is consistent with the ndings of Cen et al., 2020 and many other researches. Organic fertilization can increase the content of titrate acid compared with common management. Some components of manure can transfer into titrate acid during the process of vegetable growing, which can in uence the taste of E. scandens.
The result indicate that the content of crude protein is higher than CK after using chicken manure and cow manure. Using pig manure can improve the crude protein content of peanut kernels . Organic fertilizer can improve the content of crude protein in seeds, vegetables and many other foods.
Ascorbic acid, also known as Vitamin C, is a main vitamin in food, fruits and many kinds of vegetables. It is a strong antioxidant and can promote the absorption of Fe in human, which is good for human's health Nitrate is very common in many kinds of vegetables and it be can transferred into nitrite by microorganisms in the saliva, which is a harmful substance for human bodies (Cassens, 1995 Vegetables can be generally classi ed into four classes according to the nitrate content according to Shen et al., 1982 andTang et al., 2007. When the content is below 432mg/kg, the vegetables can be eaten raw; Between 432mg/kg and 785mg/kg, it cannot be eaten raw but can be salted or cooked food; Between 786mg/kg and 1440mg/kg, not raw food or salt, but cooked food; Between 1441mg/kg and 3100mg/kg, or more than 3100mg/kg, the vegetables should not be eaten. Although an accurate evaluation standard has not been obtained, only T1, T2, T3 and T4 fertilization treatments can meet the edible requirements in this experiment, and all of them are suitable for cooked food. The results indicate that the content of N, P and K are higher after using the organic fertilizer. Manure application increased the content of orthophosphate and myo-inositol hexaphosphate (myoIHP), especially the orthophosphate content exceeded 95% (Qin et al., 2020.). The increment of orthophosphate can provide more phosphorus to the plant. Single and combining application of N, P, and K fertilizers could enhance fruit yield and juice content in aril and increase TSS, total sugar and vitamin C content but decrease total acid content in aril juice of pomegranate (Zhang et al., 2012). In short, although using organic fertilizer can improve the yield and quality of E. scandens, long-term organic fertilizer using can signi cantly improve the content of heavy metals, trace elements (TEs) and antibiotics (ABs) and many other kinds of harmful substances in soils, which can in uence the vegetable safety and do harm to human health (Ning et al., 2017; Margenat et al., 2020).

Conclusion
According to the result of a series of experiments, we found that in contrast to CK group, using chicken manure and cow manure appropriately can promote the growth of E. scandens and improve its yield, and the T 6 treatment (0.8g/plant chicken manure +1.2 g/plant cow manure) has the best yield and quality among all the treatments. But it was found that using too much fertilizer does not necessarily promote the growth of E. scandens. Besides, it was concluded that the content of some indices (such as nitrate) can be signi cantly increased after using too much fertilizers. In summary, how to ensure the quality and food security of E. scandens as a kind of leafy vegetable still need further research. Figure 1 Plants and soil materials and planting pattern in this experiment (A, B: the biennial E. scandens seedlings and soil material, C: planting pattern)