Effect of Boron Fertilizer on the Root Growth Characteristics and Nutrient Contents of the Tuberous Root of Yacon

Mengchun ZHOU Xinyan ZHOU Mao TAN Yaling DUAN Yanzi HE Maoying XIANG Hualun ZHOU

Abstract [Objectives] This study was conducted to investigate the effects of boron fertilizer on the root system and nutrient contents of yacon. [Methods] By the field test method， high- and low-dose of boron fertilizer （Na2B8O13， 9 000 and 3 000 g/hm2） and equal amount of clean water （CK） were sprayed 3 times in the soil area where plants were grown in the early， middle and late stages of yacon growth， and the effects of applying boron fertilizer on the growth and fruit quality of yacon were analyzed. [Results] The indexes of the root system of yacon treated with boron fertilizer were significantly higher than those of the CK. The yield and total sugar， vitamin C， ash， Ca， Fe， Zn and other nutrients of the boron fertilizer treatments increased significantly compared with the CK. The yields of the low- and high-dose treatments increased by 77.2% and 211.2%， respectively， compared with the CK; and the contents of total sugar， vitamin C， ash， Ca， Fe and Zn in the high-dose treatment increased by 28.4%， 163.6%， 33.2%， 73.3%， 41.2% and 56.2%， respectively， compared with the CK. The nutrients in yacon treated with the low dose of boron fertilizer were lower than those with the high dose. The application of boron fertilizer could increase the yield of yacon， improve its quality and increase the contents of nutrients such as Ca， Fe， Zn and total sugar. [Conclusions] This study provides a reference for the reasonable application of boron fertilizer in the production of yacon and the improvement of the quality of yacon.

Key words Boron fertilizer; Yacon; Root system; Quality; Nutrient composition

Yacon， also known as Smallanthus sonchifolius， is a perennial herb of Helianthus in Asteraceae. It is native to the Andean Plateau in the south of Peru and the west of Bolivia， and has been a traditional food of the local Indians for more than 500 years[1-5]. The growth period of yacon has a greater elasticity[6]. It is strongly tolerant to light， susceptible to waterlogging and drought， and grows better at a low temperature. It is especially suitable for growing in sandy soil at an altitude of about 1 000 m[6-7]. It likes light and moist soil， and places where there is cloud and mist are the most beneficial to the growth and nutrient storage of yacon. Boron （B） is one of the indispensable trace elements in the process of plant growth. It has effects on photosynthesis， sugar transport， pollen germination， fertilization and seed setting in plants. Boron can enhance the drought resistance and disease resistance of crops and promote the early maturity of crops[8]. Meanwhile， boric fertilizer can promote root growth and significantly increase crop yields[9].

Yacon is widely grown in the world， but it is mainly distributed in Sichuan， Guizhou， Shaanxi， Xinjiang， Shandong， Yunnan and other regions in China. The crop has excellent health-care and therapeutic effects， strong commerciality， good storage resistance， and cultivation techniques easy to grasp[10]. Although the economic value of yacon is great， its growth is retarded below 15 ℃， it is not cold-resistant， and the stems and leaves are easy to die in frost. In addition， the overall yield， quality and appearance of yacon are generally not ideal. Specifically， the cracking rate of fruit peel is about 30%， which affects the economic benefit of yacon production. How can we improve the yield， quality and reduce the emergence of rotten roots and peel cracking of yacon has become a key technical problem that needs to be solved urgently[3].

It is rainy all year round in some areas of China， and the moisture content in the actual production field is not easy to control， which is easy to cause rotten roots of yacon[11]. Reasonable application of boron fertilizer to change the yield of yacon from low to high is a problem worthy of attention. At present， there are few reports on the effects of boron fertilizer on the growth characteristics， nutrient content and quality of yacon. In view of this， in order to improve the yield and quality of yacon， the effects of boron fertilizer on the root system and nutrient contents of yacon were investigated by applying boron element. This study will provide a reference for the reasonable application of boron fertilizer in yacon production and the improvement of yacon quality .

Materials and Methods

General situation of the experimental field

The experiment was carried out in the experimental field of Guizhou Normal University， Guiyang City， Guizhou Province in 2018. The experimental field has yellow soil， and has the characteristics of flat terrain， convenient irrigation， good and uniform soil fertility. It was not planted with a previous crop.

Experimental material

Tested varieties： The yacon used was the conventional variety common in production.

Fertilizers tested： Instant boron （Na2B8O13·4H2O， content≥99%）， Liaoning Pengda Technology Co.， Ltd.

Experimental design

According to the properties of the instant boron fertilizer and the reference instructions for using the product， the reference dose was used as the low-dose treatment level of the T1 group， and the three-fold dose of the T1 group was used as the high-dose treatment level. The randomized block design was used. The set three treatments were as follows： T1 Na2B8O13 3 000 g/hm2 （water 10 L， the same below）， T2 Na2B8O13 9 000 g/hm2， and control check （CK）， spraying the same amount of clean water. Each treatment was repeated 3 times， and the area of each plot was 30 m2.

Experimental methods

The row spacing of yacon was 130 cm and the plant spacing was 90 cm. The conventional fertilization was nitrogen-phosphorus-potassium compound fertilizer 750 kg/hm2， in which 2/3 was used as base fertilizer， and 1/3 was applied by top dressing. The boron fertilizer was applied by the soil spraying method. On the basis of conventional fertilization， the boron fertilizer was applied in the early， middle and later stages of the growth of yacon， three times in total， at an equal amount. The tubers of yacon were collected by plot during the maturation stage and dried after harvesting， and the soil was removed from the surface of the tubers. The cleaned tubers were sealed in vacuum bags and stored in a freezer at -25 ℃ for testing. Meanwhile， the whole root system of yacon was collected， sealed in vacuum bags after washing and placed in a refrigerator at 4 ℃ for temporary storage， in order to analyze the relevant indicators of the root system. Then， the root soil samples were collected corresponding to the growing area， in order to detect the background value of boron content in the soil.

Root sampling method and material treatment

Sampling was performed at the maturation stage. Five plants with uniform growth and complete roots were randomly selected in each plot. After cleaning these roots， they were scanned and analyzed with a root system analysis system （model： GXY-A） and a scanner MICROTEK （model： MRS-9600TFU2L） for relevant indicators. The rhizosphere soil was collected， added in sealed bags and stored in a refrigerator at 4 ℃， in order to determine the background value of boron content in the rhizosphere soil.

Yield determination

Sampling was repeated 3 times in each test plot， and all the tubers of three yacon plants were collected each time. The soil on the surface of the tubers was cleaned， and the tubers were accurately weighed.

Boron content in yacon

The boron content in yacon was determined by curcumin spectrophotometry.

Preparation of boron standard stock solution： Boric acid （H3BO3， spectrally pure） was dried at 40-50 ℃ for 3 h， and 0.572 0 g was weighed and dissolved in warm water. The solution was transferred to a 1 000 ml quartz volumetric flask， diluted to constant weight， and shaken well. The solution contained boron at 100.0 μg/1 ml.

Preparation of curcumin-oxalic acid solution： Curcumin （0.10 g） and oxalic acid （superior purity， 12.50 g） were weighed in 250 ml of 95% ethanol. The solution was stored in plastic bottles or quartz volumetric flasks， which were then wrapped with black paper. This solution was prepared 1 d before use， and could be stored in a refrigerator for 1 week after being sealed.

Preparation of boron standard solution： First， 10 ml of boron standard stock solution （100.0 μg/ml） was pipetted and diluted to a volume of 1 000 ml， obtaining a 1.0 μg/ml boron standard solution containing. Then， 0， 0.20， 0.40， 0.60， 0.80 and 1.00 ml of the boron standard solution were added into a 50 ml volumetric flask and diluted to constant weight with water， respectively.

A certain amount of each of the above-mentioned series of boron standard solutions （1.00 ml） was pipetted into a 50 ml evaporating dish， respectively. Then， 1 ml of distilled water and 4 ml of curcumin-oxalic acid solution were added into each evaporating dish， followed by mixing well. Each solution was heated in a 55 ℃ water bath to evaporate to dryness until brown crystals occur. After continuing to steam for 20 min and cooling to room temperature， the solid was dissolved with 95% ethanol and transferred to a 10 ml colorimetric tube. The solution was mixed well and stood for 10 min. With the reagent blank as the original reference solution， the absorbance was measured sequentially at a wavelength of 540 nm. The regression equation was obtained as following：

Y=0.440 1X+0.139 4， （R2=0.994 2）.

Preparation and determination of test sample： A certain amount of yacon （5.00 g） was weighed and added with 5.00 ml of 1% Na2CO3 solution， followed by measuring the pH value. If the sample is acidic， 1% Na2CO3 solution should be added continuously until it is alkaline. The solution was heated in a water bath and evaporated to dryness and then carbonized to smokeless. The residue was put a muffle furnace to 550 ℃ and ashed for 8 h until it was completed ashed. Next， 5 ml of 10% hydrochloric acid was added to dissolve the sample in the porcelain crucible， and the mixture was diluted to 25 ml with distilled water. Then， 1.00 ml of the filtrate was transferred into a 50 ml evaporating dish， and color development and absorbance detection were performed according to the steps the same as those of the standard solutions.

Determination of nutrients in yacon

Water： The moisture content was determined by vacuum drying method. A certain amount of yacon sample （10 g） was weighed from each group in a constant-weight weighing bottle， and then put into a vacuum drying box at 70 ℃. The pressure in the box was reduced to 3 KPa， and dry air was then introduced into the box， so as to maintain a certain temperature and pressure in the box. After 4 h of drying， the sample was taken out and put in a dryer for 0.5 h. After weighing， the sample was put back in the vacuum drying box and dried for 1 h. It was also cooled and dried for 0.5 h， and then weighed. Drying and weighing was repeated until the difference between the two weights obtained before and after did not exceed 0.001 g， and the weight at this time was the constant weight.

Wherein X is the moisture content （%） in the sample; m1 is the weight （g） of the weighing bottle and sample; m2 is the weight （g） of the weighing bottle and sample after drying; and m3 is the weight （g） of the weighing bottle.

Ash： The dried sample was carbonized in a crucible until smokeless， then transferred to a muffle furnace， and ashed at 525 ℃ for 4 h. It was cooled and weighed， until the difference between the two weights obtained before and after was less than 0.5 mg.

Wherein Y is the ash content in the sample （g/100g）; a1 is the weight of the crucible and ash （g）; a2 is the weight of the crucible （g）; and a3 is the weight of the crucible and sample （g）.

Ca， Fe and Zn： The determination of Ca content referred to GB 5009.92-2016 National Food Safety Standard： The Determination of Calcium in Foods. Fe content was determined according to GB 5009.14-2017 National Food Safety Standard： The Determination of Iron in Foods. The determination of Zn content referred to GB 5009.90-2016 National Food Safety Standard： The Determination of Zinc in Foods. Flame atomic absorption spectrometry was applied to determine the absorbance values of Ca， Fe and Zn. Standard curves were prepared with the mass dose in Ca， Fe and Zn standard solutions as the abscissa and the corresponding absorbance as the ordinate. The regression equations of the three elements were YFe=0.069 3X-0.006 2 （R2=0.998 1）， YCa=0.056 2X+0.044 6 （R2=0.999 7 ） and YZn=0.340 6X-0.002 6 （R2=0.999 4）.

Total sugar： The determination of total sugar content referred to Food Analysis and Inspection Techniques[12].

Wherein W is the total sugar content; F is the amount of glucose equivalent to 10 ml of alkaline copper tartrate solution （mg）; V1 is the total volume of the total sugar sample solution （ml）; m is the sample weight （g）; V2 is the average total volume （ml） of the total sugar sample solution consumed during calibration.

Vitamin C： The vitamin C content was determined by 2，4-dinitrophenylhydrazine colorimetric method referring to GB 5009.86-2016 National Food Safety Standard： Determination of Ascorbic Acid，. The standard curve was drawn with the ascorbic acid dose （μg/ml） as the abscissa and the absorbance value （A） as the ordinate， and the regression equation was obtained： Y=0.048X-0.000 5 （R2=0.999 8）.

Determination of boron content in soil

The boron content in the soil was determined by boiling water leaching-methylimine colorimetry referring to GB7877-87 Determination of Available Boron in Forest Soil. Before and after fertilization， soil was taken from the root area of plants in treatments of difference doses with three repetitions， and was air-dried， sieved and determined for boron element as the base to distinguish the effect of boron element in the soil on yacon.

Results and Analysis

Effects of different boron fertilizer dosages on the root morphology of yacon

The content of boron fertilizer has an obvious regulation effect on the growth process of plant roots. The root hair area is the first acting part of boron fertilizer， and excessive or insufficient boron fertilizer will affect the growth and development of plant roots[13]. It could be seen from Table 1 that the low concentration foliar boron fertilizer increased the root length of yacon， which continued to grow with the further increase of boron fertilizer concentration. The root length was the largest in treatment HB and the smallest in the CK， and the difference between the treatments reached a significant level. The low-concentration boron fertilizer increased the root surface area of yacon， which increased with the further increase of the concentration of boron fertilizer. It was the largest in treatment HB， and showed a minimum in the CK. The root volumes of treatments HB and LB were significantly larger than that of the CK. However， boron fertilizer had no significant effect on the average root diameter， and there were no significant differences between the treatments.

Effects of boron fertilizer on boron content in yacon and its root soil

Soil

It could be seen from Table 2 that the average boron contents in the root soil of the CK， T1 and T2 were 0.032， 0.085 and 0.189 mg/kg， respectively. Compared with the CK， the boron content in the root soil of low-dose treatment （T1） increased 165.6%， and the boron content in the root soil of the high-dose treatment （T2） increased by 490.6%; and the boron content in the soil with boron fertilizer application was significantly greater than that in the untreated group， and the boron content in the soil increased significantly with the increase of boron fertilizer dose. It showed that the application of boron fertilizer had a positive effect on the increase of the background boron content value in the root soil， and could provide boron nutrition for the growth of yacon.

Yacon

The boron content in the soil of the test field had a certain background value. It could be seen from Table 2 that the average boron contents in the yacon of the CK， T1 and T2 were 0.49， 0.66 and 2.67 mg/kg， respectively. Compared with the CK， the boron content in yacon increased by 34.7% in low-dose treatment （T1） and 444.9% in high-dose treatment （T2）. The boron content in yacon increased significantly with the increase of boron fertilizer dose， and the differences between the treatments were significant. It showed that the application of boron fertilizer had a certain effect on the accumulation of boron content in yacon.

Effect of boron fertilizer on the yield of yacon

The yield of yacon increased with the increase of the dose of boron fertilizer. The high-dose treatment （T2） had the highest yield， with an average of 996.31 g/plant， an increase of 211.2% compared with the CK; the low-dose （T1） yield was second， with an average of 567.48 g/plant， an increase of 77.2% compared with the CK; and the yield of T2 increased by 75.6% compared with T1. Taking into account the impact of boron in food on organisms， it is ideal to spray boron fertilizer at low and medium doses for the yield of yacon. Therefore， the application amount of boron fertilizer in yacon could be controlled at 3 000-9 000 g/hm2.

Effects of boron fertilizer on the components of yacon

It could be seen from Table 3 that the increased application of boron fertilizer could promote the increase of the content of various ingredients such as moisture， ash， total sugar， vitamin C and Ca， Fe， Zn and trace elements in yacon.

Moisture

The moisture content of yacon in each treatment was sufficient. The moisture in yacon tubers increased with the increase of boron application， but the increase in water content was not significant. The yacon tubers of the high-dose treatment （T2） had the highest moisture content of 886.01 g/kg， with an increase of 8.0% compared with the CK; and the low-dose treatment （T2） had a water content of 834.50 g/kg in yacon tubers， which increased by only 1.7% compared with the CK. The water content of yacon showed an increasing trend with the increase of boron fertilizer dosage. It showed that increasing the application of boron fertilizer was beneficial to increasing the moisture content of yacon tubers.

Ash

The ash content of yacon was higher in the treatments with the application of boron fertilizer than in the treatment without boron application， which meant that boron was beneficial to the formation of inorganic substances in yacon. The ash content of yacon showed an increasing trend with the increase of the dose of boron fertilizer， but the overall difference was not large. The best effect was achieved at the boron fertilizer dosage of 9 000 g/hm2， and the ash content was 49.58 g/kg， which increased by 33.2% compared with the CK. The ash content of the treatment with low boron fertilizer dose increased by 16.6% compared with the CK. Of the increasing rates compared with the CK， the high boron fertilizer dose was lower than the low dose treatment by nearly 1 time. It indicated that proper application of boron fertilizer had a positive effect on the ash content of yacon.

Total sugar

The total sugar content showed an increasing trend with the increase of boron fertilizer dose. Yacon is rich in sugar. Overall， the total sugar content of yacon with boron fertilizer application was higher than that without the application. The dose of 9 000 g/hm2 was the most effective， and showed a sugar content of 162.15 g/kg， which increased by 28.4% compared with the CK， and the taste and flavor of the yacon tuber were also better. The increase of total sugar in the high-dose boron fertilizer treatment was more than double that of the low-dose treatment. It showed that proper application of boron fertilizer had a positive effect on the total sugar content of yacon.

Vitamin C

The content of vitamin C in yacon was low. The content of the CK was 12.07 mg/kg， and the content of vitamin C was higher in the treatments with boron fertilizer application than in the treatment without application. The effect of boron application was optimum at 9 000 g/hm2， with which the content of vitamin C was 31.82 mg/kg， which showed an increase of 163.6% compared with the CK. The vitamin C content of the low-dose treatment increased by 137.2% compared with the CK. It showed that the application of boron fertilizer was beneficial to the increase of vitamin C content in yacon.

Trace elements Ca， Fe and Zn

The contents of Ca， Fe and Zn in the high-dose treatment of yacon were the highest， respectively， at 1 623.94， 3.53， and 2.89 mg/kg， which were 73.3%， 41.2%， and 56.2% higher than the CK， respectively. The contents of Ca， Fe and Zn in yacon of T1 were 1 073.49， 3.22 and 2.17 mg/kg， respectively， which were 14.6%， 28.8% and 17.3% higher than the CK， respectively. The contents of trace elements in yacon in each treatment ranked as Ca>Fe>Zn; and the contents of the treatments with boron fertilizer application were higher than that without the application， and the high dose treatment was greater than the low dose treatment. The effect of applying boron fertilizer on Ca content in yacon was greater than that on Zn content， and the effect on Fe content was slightly weaker. It showed that the application of boron fertilizer was beneficial to increasing the contents of trace elements in yacon， and the effects on the trace element contents were most significant in the high-dose boron fertilizer treatment.

Conclusions and Discussion

The root system is the vegetative organ of plants underground， which has the functions of absorption， assimilation and transportation， transmission of environmental signals and response. It is the first part of higher plants to perceive boron， as well as an important place for plants to absorb water and mineral nutrients and synthesize certain enzymes and organic substances[13]. Boron fertilizer has a significant effect on the physiological activities of the plant root system. Excessive or insufficient boron fertilizer in the root system will affect the physiological activity and development process of the plant root system[13]. The results of this study showed that a certain concentration of boron fertilizer treatment promoted the increase of root length， root surface area and root volume of yacon to a certain extent. When the concentration of boron fertilizer treatment reached a certain level （9 000 g/hm2）， various indicators increased significantly， and were all greater than those of the treatment without the application of boron， indicating that the application of boron fertilizer promoted the growth and development of the root system of yacon. However， the determination of the maximum concentration of boron fertilizer on the root system of yacon still needs further analysis and confirmation.

Total sugar is one of the main components of yacon and has a strong health care effect. The polysaccharide components of yacon are mainly inulin and fructooligosaccharides， and are a kind of excellent dietary fiber with special biological activity and chemical structure. Polysaccharides are often researched and used as a clinical application and food additive， to help bowel movements， prevention of constipation， intestinal peristalsis and reduction of the contents of heavy metal carcinogens such as cadmium and lead in the body. Polysaccharides can also be used as a multiplication factor for probiotics （mainly bifidobacteria） in the human body， which inhibits the growth of harmful microorganisms， increases the number of probiotics， and maintains the intestinal microecological balance[14-17]. The chemical name of vitamin C is ascorbic acid， which is one of the essential nutrients for the human body， but the human body cannot synthesize it by itself， and it can only be ingested from foods such as yacon， grapes， tomatoes， wheat， etc.[18]. The tuberous root of yacon contains a large amount of water-soluble plant fiber， rich fructooligosaccharides， and a variety of essential mineral trace elements such as calcium， iron and zinc and amino acids[19]. However， the supply of trace elements in the soil is often inadequate， resulting in a low content of trace elements such as Ca， Fe and Zn in yacon.

The experimental results showed that the background value of boron in the root soil of the CK was 0.032 mg/kg， and the boron contents in the yacon tubers of high-dose and low-dose treatments increased by 444.9% and 34.7%， respectively， compared with the CK. Compared with the CK， the boron contents in the high-dose and low-dose treatments increased by 490.6% and 165.6%， respectively. The length， surface area and volume of plant roots increased significantly in the treatment groups. It shows that the application of boron fertilizer could produce a positive effect on the increase of the background value of boron content in the soil and significantly promote the growth of the root system of yacon plants， thereby promoting the overall growth and development of plants and providing boron nutrition for the growth of yacon continuously， and further affect the plant's ability to resist disease and drought， as well as the yield， quality and other growth traits of yacon. Compared with the CK and the low-dose boron fertilizer treatment， the yield was the highest when the dose of boron fertilizer was 9 000 g/hm2， which significantly increased the contents of moisture， ash， total sugar， vitamin C， Ca， Fe and Zn in yacon， by 8.0%， 33.2%， 28.4%， 163.6%， 73.3%， 41.2% and 56.2%， respectively. The yield of yacon will directly affect the economic benefits of farmers， and appropriate application of boron fertilizer could make yacon produce a certain physiological effect， promote the growth of the root system of the plant， and ultimately promote the significant growth of the tuber production of yacon and improve the quality of tubers to a a certain degree.

During the planting process of yacon， the phenomenon of insufficient intake of boron and other trace elements is prone to occur， which affects the yield and quality of yacon. However， a proper dose of boron fertilizer could control the growth trend of yacon and enhance the drought resistance and disease resistance of yacon， and has a certain improvement effect on tuber yield and quality of yacon. In the production of yacon， instant boron fertilizer can be tested in a large area to provide a theoretical basis for the subsequent popularization and application of boron fertilizer in the cultivation of yacon. In addition， the physiological effects of increased boron content in yacon on humans and animals need further research.

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