Effects of Magnesium Fertilizer on the Forage Crude Protein Content

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Effects of magnesium fertilizer on forage crude protein content depend upon available soil nitrogen Xiao Sun, Jihui Chen, #isheng #iu, Andrea Rosanoff, Xue Xiong, Yingjun Zhang, and Tongtong Pei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04028 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Journal of Agricultural and Food Chemistry

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Effects of magnesium fertilizer on forage crude protein content depend upon

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available soil nitrogen

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Xiao Sun1, Jihui Chen1, Lisheng Liu2, Andrea Rosanoff3, Xue Xiong1, Yingjun zhang4,

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Tongtong Pei1

6 7

8

1

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210095, China

College of Agro-grassland Science, Nanjing Agricultural University, Nanjing,

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2

11

USA

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3

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Sciences, Hengyang 421001, PR China

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4

15

China

Center for Magnesium Education & Research, 13-1255 Malama St., Pahoa, HI 96778

Hengyang Red Soil Experimental Station, Chinese Academy of Agricultural

Department of Grassland Science, China Agricultural University, Beijing 100193

16 17 18 19

20

*

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Tel & Fax:86-25-84399620.

To whom correspondence should be addressed. E-mail: [email protected],

ACS Paragon Plus Environment


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Abstract

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Magnesium (Mg) is important for both plant photosynthesis and protein-synthesis.

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Nevertheless, latent Mg deficiencies are common, and Mg addition has shown

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improved yield. Might such increasing yield cause “hidden” hunger for

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microelements and protein, and if so, what is the mechanism? We conducted two

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greenhouse experiments using low-Mg soil to investigate: (i) effects of 5 levels of Mg

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fertilizer (20 to 400 mg kg-1) on 8 elements and crude protein concentrations in annual

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ryegrass and white clover; (ii) if any protein effects of Mg fertilizer depend on soil

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nitrogen (N). Mg addition significantly increased yield in both species,

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simultaneously decreasing concentrations of crude protein, calcium (Ca), sodium,

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manganese, and potassium: Mg and Ca:Mg ratios caused by increased biomass

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dilution effects and increased [Mg]. Other mineral dilution effects of Mg fertilizer

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depended on species: the concentration of phosphorous decreased only in ryegrass

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and that of zinc only in white clover. Mg addition in soil rich with available N (from

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N fertilizer in ryegrass or biological fixation in white clover) showed increased crude

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protein content as well as increased yield in both species’ forage. These results

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suggest that Mg fertilizer can affect protein content positively or negatively

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depending on available N in soil, and that sufficient available N must be ensured

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along with Mg addition in low Mg soils.

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Key words: magnesium, acid red soil, mineral elements, protein, ryegrass, white

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clover

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Introduction

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Magnesium (Mg) is the central element of chlorophyll and a cofactor for a series of

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enzymes involved in carbon fixation. Mg also is an essential component of ribosome

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subunits and thus essential in protein-synthesis 1. Mg therefore plays important roles

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in both photosynthesis and protein-synthesis in leaves. In addition, plant Mg

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nutritional status is critical for photosynthetic products’ transportation to growing

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roots 2, an aspect of plant growth which can affect nutrient acquisition directly via

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influencing root uptake capacity, and indirectly via influencing symbiotic

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relationships between plant and microbe 3, 4. Latent and acute Mg deficiencies in

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plants are common because of Mg ions’ unique chemical property–a highly hydrated

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radius that sorbs less to colloids than other cations in soil 5. Much attention was given

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to Mg in the 1970s because of the relationship between the incidence of grass tetany

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in livestock with low Mg feeds or high potassium (K) levels in herbage 6, 7, which can

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decrease the efficiency of Mg and calcium (Ca) absorption by animals. Recent studies

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also show that low Mg diets can cause osteoporosis in humans and animals, and along

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with high Ca:Mg, can cause cardiovascular disease in humans, which has received

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considerable attention in recent years 8-10. However, few older studies have considered

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the effects of Mg fertilizer on yield and other characteristics of quality in forage 11.

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According to the structure-function chain of Ågren and Weih (2012) 12, the

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concentrations of N, phosphorus (P) and manganese (Mn) need to increase to support

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continued plant growth caused by fertilizer, and the concentrations of structural

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elements (e.g. K, and Ca ) should remain relatively constant with increasing plant size

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or biomass; but a fixed amount of some microelements needed by plants cause their

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concentrations to decrease as biomass increases via a dilution effect. If Mg fertilizer

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increases biomass 11, does it also create, simultaneously, “hidden” hunger of



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microelements and protein, -- and, if so, what are the underlying mechanisms? Some

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studies and meta-analyses showed that “green revolution” or elevated CO2-induced

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increases of biomass may result in a “hidden” hunger or lower concentrations for

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some microelements and protein, which can threaten human health 9, 13-16. Also, in

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contrast to Agren and Weih’ guidelines, recent studies showed that Mg fertilizer

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decreases K and Ca concentrations because reciprocal interactions regarding uptake

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exist between Mg and K or Ca 17, 18. Also, Mg fertilizer decreases Mn concentration in

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plant not only because of the dilution effect of biomass, but also due to reduced

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uptake by roots 17. Klein et al (1982) found no significant influences of Mg addition

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on P, K, and zinc (Zn) 19, but variable influences on other elements over three crop

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years. Therefore, the concentrations of elements in plants may not only be dictated by

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yield but also can be affected by availability of nutrients in soil and competition

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between ions during uptake by plant roots.

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Recently, considerable progress has been made in studying the effect of Mg

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on yield and quality of cash crops 20, 21. A recent review reported that Mg addition

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increases crop yield, but such yields appear to depend on available nitrogen (N) in

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soils 22. In our recent study, we found that the biomass of soybean forage, but not that

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of maize, increased with Mg fertilizer 4. We attributed this difference between legume

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and grass to different functions of their symbiotic microorganisms: While both

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symbiotic microorganisms increase with Mg addition, rhizobium (in legume) can fix

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N from air, but mycorrhiza fungi (in grass) cannot. Mg may also have an important

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role in N acquisition by roots aside from microorganism enhancement, simultaneously

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controlling the processes responsible for carbohydrate and protein production. In

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several studies, N (crude protein) concentration increased in leaves of soybean, onion

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and tuber of potato with Mg fertilizer even though biomass also increased 4, 18, 19, but


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this did not occur in maize or soybean leaves where Mg fertilizer even decreased N

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concentration 4, 23. These inconsistent results among individual studies may be due to

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variable experimental environments, such as the level of available soil N, as well as

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differences in plant physiobiochemistry and symbiotic microorganisms 4, 22. Therefore,

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we speculate that the degree of protein production (an important index of food quality)

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in plants’ responses to Mg fertilizer may be affected by the available N in

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Mg-supplemented soil. Here, two greenhouse experiments were conducted to investigate (i) the

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effects of Mg fertilizer on yield and quality of forage grown in low Mg soil for a grass

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-- annual ryegrass (Lolium multiflorum L.) and a legume -- white clover (Trifolium

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repens L.), and (ii) whether the effects of Mg fertilizer in these two forage crops’

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quality depend on soil N supply provided by N fertilizer (in ryegrass) or via symbiotic

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microorganism (in white clover). Specifically, we examined mineral elements: P, K,

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Ca, Mg, iron, Mn, Zn, and sodium concentrations (hereafter [P], [K], [Ca], [Mg], [Fe],

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[Mn], [Zn], and [Na]) and crude protein concentration [crude protein] in both species.

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We hypothesized that: (i) Mg addition would increase forage yields of both grass and

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legume, (ii) [crude protein] in grass forage would show positive or negative responses

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depending on available soil N, whereas [crude protein] in legume forage would

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increase when symbiotic microorganisms exist, and (iii) if biomass increased with Mg

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addition, [K] and [Ca] would remain relatively constant, while microelement

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concentrations in both species would decrease.

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Materials and methods

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Soil conditions

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Acid red-yellow soil in south China is usually poor in Mg because of soil acidification

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and leaching due to high precipitation. Therefore, we collected the acid red-yellow



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soil from Hengyang Red Soil Experimental Station in Hunan province. The soil was

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sifted with 5 cm mesh, and then spade-mixed three times for homogeneity. The five

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mixed soil samples were air-dried in a shaded and ventilated environment for

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approximately one month to achieve a constant weight for determination of soil pH

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and total elements (the method was the same as that described below to measure

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elements in leaves). The available Mg, extracted with 0.1 M BaCl2, was measured

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with ICP-OES. The soil total N, P, available Mg, and organic carbon concentrations

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and pH (with ultrapure water) were 0.082%, 0.036%, 0.0015%, 1.33% and 3.4,

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respectively.

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Experimental design

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Experiment 1

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The first experiment was designed to investigate the effects of Mg addition on yield

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and quality measured as both crude protein and mineral element concentrations. In the

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middle of October 2015, a total of 108 plastic pots (24 cm in diameter, 20 cm in depth)

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were filled with the well-mixed soil, each pot containing 4 kg soil. As in previous

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studies conducted at this site, we set 6 treatments of Mg addition (0, 20, 50, 100, 200

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and 400 mg Mg per kg soil in the form of MgO) with 9 replications (54 pots) for each

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species (ryegrass or white clover). The total MgO fertilizer (powder) for each pot was

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calculated according to each treatment concentration, molecular weight of MgO and

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soil weight. Nitrogen and P fertilizer, as base fertilizer, was applied following usual

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farmland procedures for all treatments including control soil (zero Mg addition) for

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both species (0.35 g kg-1 N and 0.25 g kg-1 P for ryegrass, corresponding to

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approximately 77 kg ha-1 N, and 55 kg ha-1 P, respectively, and 0.10 g kg-1 N and 0.15

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g kg-1 P for white clover, corresponding to approximately 22 kg ha-1 N, and 33 kg ha-1

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P).


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Experiment 2

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In the first experiment, the concentration of crude protein in both species of plants

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significantly decreased with Mg addition. Therefore, in the second experiment, we

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wanted to explore whether ample N in soil, either from fertilizer or biological fixation,

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might dampen this effect.

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Ryegrass, ample Mg and added N:

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In the middle of October 2016, ryegrass was grown in soil similar to that used in

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Experiment 1 with same base fertilizer plus 200 mg kg-1 Mg in form of MgO

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(considered “ample Mg” by Experiment 1) plus three levels of added N fertilizer as

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urea (0, 0.1, 0.7 and 1.0 N g kg-1 soil), each with 7 replications. The urea was divided

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equally and applied at tillering and at each cut. For this total of 28 plastic pots as

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above, each pot was filled with the 4 kg well-mixed soil. Yield and shoot [crude

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protein] were measured to determine if added urea in ample Mg further affected yield

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or altered [crude protein].

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White clover and ample Mg with active rhizobium:

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The white clover did not show the expected increased yield with Mg addition in

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Experiment 1. We speculated that the acidic soil (pH=3.4) might be responsible for

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less than adequate rhizobium growth resulting in inadequate N-fixation 24. We

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therefore conducted this second experiment of white clover in red soil with the pH

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improved with NaOH addition according to preliminary experimentation (pH=3.8,

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with ultrapure water) to investigate the influences of Mg addition in form of MgO (0,

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100 mg kg-1 and 200 mg kg-1) on yield and [crude protein]. Fifteen plastic pots (20 cm

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in diameter, 16 cm in depth) were each filled with 2.5 kg well-mixed soil, and each

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treatment and control had 5 replications. The base fertilizer (N and P) applied was as

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described above in Experiment 1.



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Plant materials

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Ryegrass and white clover are important potential winter forage crops in acid soil in

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south China. At the middle of October, ~10 ryegrass or white clover seeds were sown

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in each pot in the greenhouse (See above). We watered the seedlings every three days,

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and the water level was adjusted using an electronic weigher once every three days to

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maintain similar soil moisture content. After four weeks, the seedlings (~10 cm) were

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thinned in each pot to allow sufficient space for seedling growth with 5 similar

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seedlings kept in each pot. We gently removed the upper soil (~2 cm) from open

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spaces in each pot, then evenly sprinkled corresponding MgO and recovered using

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“the removed upper soil”. After fertilization, we evenly sprinkled water to help plant

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uptake. The base fertilizer (N and P) was dissolved in water, and evenly sprinkled in

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each pot. The pot position was also changed randomly once every three days to avoid

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light heterogeneity.

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Plant sampling

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Experiment 1

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At 15 weeks, leaf chlorophyll contents were measured non-destructively using a

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portable chlorophyll meter (SPAD-502, Minolta, Japan), and then forage was

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harvested near the ground to determine above-ground biomass as a measure of yield

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for each plant. Leaves and stems were then separated from each shoot (a subsample of

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ryegrass); only dried leaves were used for elemental analysis and stoichiometry

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calculations. All plant samples were oven-dried at 65℃for 72 h, and ground in

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20-inch mesh in Wiley Mill, and then kept cool and dry for chemical analysis of

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microelements.

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Ryegrass: Since shoot is important for forage, a subsample of ryegrass shoot also

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underwent elemental analysis (See Supplemental Information, Figure S1).


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White clover: White clover is generally used for pasture animals which mainly graze

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their leaves; therefore, only white clover leaves were used for elemental analysis.

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Experiment 2

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Ryegrass: After 10 weeks, the ryegrass leaves were measured for chlorophyll content,

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and shoots were cut to a ~5 cm stubble. After an additional 6 weeks, we again

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measured leaf chlorophyll contents and made a second shoot cut. All these forage cuts

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were oven-dried at 65 ℃ for 72 h, and were ground in 20-inch mesh in Wiley Mill.

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Because yield from only the second cut was significantly increased by urea treatments,

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we used only second cut shoots to analyze for elements (See Supplemental

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Information, Figure S2) as described above.

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White Clover: The white clover was harvested after 14 weeks. After cleaning, root

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nodules were removed, counted, and weighed using ten-thousandth analytical

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electronic balance after being oven-dried at 65℃for 72 h. These weights and numbers

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of nodules were used to evaluate the degree of nodule growth and N-fixation. The

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above-ground biomass was measured, and then we separated leaves from stems in a

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subsample of the white clover shoots from each pot. Leaves and the surplus shoots of

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each pot were dried and milled as described above, and measured for crude protein.

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Chemical analysis

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Subsamples of 0.5 g were digested in trace-metal-grade nitric acid, and N

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concentration (mg g-1) was determined by modified Kjeldahl wet digestion using a

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2300 Kjektec Analyzer Unit (FOSS, Hōganās, Sweden). Crude protein was calculated

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using the formula [N] × 6.25 25. For the mineral elements, the 0.1 g subsamples

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were digested using trace-metal-grade nitric and perchloric acid, and diluted in 100 ml

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of ultrapure water. We measured mineral elements using an inductively coupled

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plasma emission spectrometer (Iris Advantage 1000, USA).



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Statistical analysis

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The data were log-transformed when necessary to meet the parametric test

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assumptions of normality (Bartlett test) and homogenous variances, and then one-way

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ANOVA (Tukey HSD) was used to determine the effects of Mg treatment on forage

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yield, shoot and leaf concentrations of crude protein and mineral elements, and the

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nodule number and weight of white clover. We also used this method to determine the

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effects of urea additions under ample Mg conditions on ryegrass yield and shoot

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concentrations of crude protein and mineral elements. All statistical analyses were

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performed in R statistical software, version 3.3.2 (R Development Core Team, 2013).

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Results

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Effects of magnesium fertilizer on leaf chlorophyll and forage yield

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Chlorophyll concentration in mature leaves was significantly increased by Mg

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addition in both species (Fig.1), and further significantly increased with rate of Mg

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addition for ryegrass, but not for white clover (Fig.1). Ryegrass yields significantly

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increased by similar amounts with Mg additions (Fig.1) of 20-200 mg kg-1 Mg and

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significantly increased further with 400 mg kg-1 Mg addition. But for white clover,

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yield measured as above-ground biomass only significantly increased with 400

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mg kg-1 Mg addition (Fig.1) in this high acid soil (pH=3.4).

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When white clover was grown in less acidic soil (pH=3.8), the same base

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fertilizer and additions of 100 and 200 mg kg-1 Mg addition, yield increased

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insignificantly at 100 Mg addition (+7%) and similarly at 200 Mg addition (+9.42%)

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(See Table 1).


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Effects of magnesium fertilizer on forage quality

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Leaf [crude protein] decreased while concentrations of measured microelements

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measured showed either no change or a decrease in leaves with Mg addition (See

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Figure 2).

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Crude Protein Concentrations: Low Mg addition (20 mg kg-1) had a significant

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negative effect on [crude protein] in ryegrass, and higher levels of Mg addition

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showed further [crude protein] decreases, whereas, only 400 mg kg-1 Mg addition

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significantly decreased leaf [crude protein] in white clover (Fig.2) grown in acidic soil

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(pH=3.4).

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Microelement concentrations: The [P] in ryegrass leaves was also significantly

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decreased by Mg addition, and more Mg additions had further effects, whereas, Mg

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addition had no effects on [P] in white clover at soil pH 3.4 (Fig.2). Low Mg addition

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had no significant effects on [Ca] in either species, but ≥50 mg kg-1 Mg addition and

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400 mg kg-1 Mg showed significant negative effects on [Ca] in ryegrass and white

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clover leaves, respectively (Fig.2). The [Mn] of ryegrass leaves significantly

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decreased with Mg addition ≥ 20 mg kg-1 Mg as did white clover leaves with 50 mg

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kg-1 Mg addition. The [Zn] remained unchanged in ryegrass leaves, but in white

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clover was significantly decreased by ≥100 mg kg-1 Mg (Fig.2). The [Na] in both

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species was not significantly influenced by Mg addition except at levels of 400 mg

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kg-1 Mg addition, where [Na] was significantly decreased in the leaves of both species

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(Fig.2). By contrast, [Mg] in both species’ leaves was significantly increased at low

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levels of Mg addition, and further increased by higher Mg additions (Fig.2). There

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was no significant influence of Mg addition on [Fe] or [K] in leaves of either species.

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Element Ratios: Mg addition showed no significant effects on either N:P or



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K:(Ca+Mg) ratios in leaves, but did show significantly lower K:Mg and Ca:Mg ratios

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in both species as Mg additions increased (Fig.3).

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With improved red soil pH (3.8 vs 3.4), we found that 100 mg kg-1 Mg

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addition for white clover substantially and significantly increased number and weight

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of nodules as well as leaf [crude protein], and more than doubled shoot [crude protein]

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and whole plant crude protein content: Higher Mg addition (200 mg kg-1) did not

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show further increases in these parameters (Table 1).

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Effects of additional nitrogen fertilizer on ryegrass yield and [crude protein]

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under ample magnesium soil conditions

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The urea addition in ample Mg soil (200 mg kg-1) of 0.7 g kg-1, significantly increased

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chlorophyll in ryegrass leaves after first cut, but there were no further increases in

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chlorophyll with 1 g kg-1 urea fertilizer additions (Fig.4). The first cut forage yield of

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ryegrass was not influenced by N fertilizer. The second cut yield was significantly

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increased by 0.7 g kg-1 fertilizer, and, as with chlorophyll, 1 g kg-1 fertilizer had no

279

further effects (Fig.4). The total forage yield (cut 1 plus cut 2) was only significantly

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increased by 1 mg kg-1 urea fertilizer (Fig.4).

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Low urea addition (0.1 g kg-1) had no effect on [crude protein] in either first or

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second cut ryegrass shoots, but higher (0.7 and 1 g kg-1) urea fertilizer addition

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significantly increased [crude protein] in both cuts, and the increase was more

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substantial in the second cut even though biomass also increased (Fig.4).


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Discussion

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Responses of forage yield to magnesium fertilizer

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As per previous studies and our hypothesis, the forage yield of ryegrass was

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significantly increased by low levels of Mg fertilizer. However, yield increase for

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white clover with Mg soil application was much less. We attribute white clover’s low

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yield response to Mg addition to low available N caused by inhibition of symbiotic

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rhizobium in the high acid soil (pH=3.4) 24 and the lower base N fertilizer used in this

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experiment. Some studies have shown no significant effects of Mg fertilizer on forage

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yield of silage corn 4, or grain yield of maize 26, 27. However, other studies have found

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positive effects of Mg fertilizer on yield of maize or soybean 4, 17, 28. The variability of

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yield in response to Mg may likely be caused by variations in availability and

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accessibility of nitrogen in the soil

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role in nitrogen-fixation of rhizobium, and Mg addition increased the soybean yield

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via that enhanced nitrogen-fixation 3. This explanation is supported by a significant

299

positive response of soybean forage yield to Mg addition when symbiotic rhizobium

300

was also enhanced, with positive correlations shown between overall plant yield and

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number or weight of soybean nodules 4. In the present experiment 2, we found Mg

302

addition increased white clover yield from 7.00 % to 9.42 % when the symbiotic

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rhizobium was enabled via increased soil pH, although this was not a significant

304

increase of yield. Such weak response of biomass in spite of assured existence of

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symbiotic rhizobium was likely caused by a low Mg addition (200 mg kg-1) compare

22

. One study found that Mg plays an important



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to that in first experiment (400 mg kg-1) or other factors in soil that were not measured

307

here.

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Responses of forage quality to magnesium fertilizer

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As per our hypothesis, the [crude protein] in annual ryegrass leaves in low N soil was

310

significantly decreased at all levels of Mg addition that showed significantly increased

311

biomass. However, the ryegrass shoot [crude protein] suffered no such influences with

312

less than 100 mg kg-1 Mg addition, but significantly decreased with more than 100 mg

313

kg-1 Mg addition (Fig. S1). This suggests that areas of low Mg soil (i.e. about

314

0.0015%) can add 100 mg kg-1 Mg to improve yield of ryegrass forage, and have no

315

decreases in protein content as shoots are usually used as fodder rather than for

316

grazing (animals usually selectively graze leaves). In contrast with our hypothesis of

317

increased [crude protein] in legume with Mg addition, the protein in white clover

318

showed no significant change with Mg addition up to and including 200 mg kg-1, and

319

even was significantly decreased at 400 mg kg-1 Mg addition, the one level of Mg

320

addition showing increased biomass. We attribute this to the absence of adequate N

321

fixation due to the inadequate symbiotic rhizobium for white clover in the acidic soil

322

condition: Thus, the legume -- white clover showed a response similar to the grass--

323

ryegrass, i.e. leaf [crude protein] decreased with increased yield brought on by Mg

324

addition. Similar phenomena were shown in a study of soybean

325

increased biomass via Mg addition caused [crude protein] dilution in both species

326

under these conditions, a suggestion supported by our findings of no significant


23

. We suggest that

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change of total leaf protein content in either species’ forage with significant increases

328

of biomass (ryegrass, p=0.42 and white clover, p=0.44). These results suggest that Mg

329

addition may enhance N use efficiency 22. By contrast, in a previous study we found

330

that protein in soybean with Mg addition (≥50 mg kg-1) was double that in the 0 Mg

331

addition control 4, a result also found by other studies 28, 29. In our second experiment

332

here presented, Mg fertilizer application to white clover in less acidic soil

333

significantly increased both leaf and shoot [crude protein] as well as total shoot crude

334

protein, while showing increased symbiotic rhizobium simultaneously. These results

335

are thus primarily due to more effective symbiotic rhizobium enhanced by Mg

336

addition 3, 4. The [crude protein] in white clover when grown in soil with abundant Mg

337

fertilizer was comparable to that of white clover in other studies (17%~21% )25. We

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also found that Mg fertilizer can increase yield and [crude protein] simultaneously in

339

ryegrass when supplied amply with urea N fertilizer. The values of [crude protein] in

340

annual ryegrass with low N fertilizer were lower compared to that (from 12 to 26%)

341

in the study of Redfearn et al (2002)

342

[crude protein] were similar to that in study of Redfearn et al (2002)30. Our present

343

results and previous studies together show that the variability of forage protein in

344

response to Mg fertilizer largely depends on available N, either from biological

345

fixation or fertilizer application in soil

346

meta-analysis showing that the effects of increased of CO2 “fertilizer” on protein

347

depend on plant functional group (legumes or non-legumes) and available nitrogen 32,

348

33

30

, but with ample N fertilizer, the values of

11, 28, 31

. This phenomenon was also found in a

.



349

Leaf [K] in both species remained stable in the face of increased biomass

350

with Mg addition. But in disagreement with our expectations and structure-function

351

chain 12, there were negative influences of Mg addition on [P] in ryegrass and [Ca] in

352

both species. In both disagreement and alignment with our expectation,

353

microelements showed no significant decrease for [Fe], a decreased [Zn] only in

354

white clover, and decreased [Mn] and [Na] in both species. This negative effect of Mg

355

fertilizer on [Mn] has also been found in maize in a study which also found that Mg

356

addition decreased [K] 17,. Surprisingly, Klein et al (1982) found that [Ca] and [Mn]

357

increased in tuber of potatoes with Mg addition 19. Such inconsistent results may be

358

caused by differences in organ of study, available elements in soil and/or additive

359

amounts of Mg fertilizer. Such negative influences of Mg addition on quality are quite

360

probably largely due to a yield dilution effect. The other micromineral elements

361

measured in this study suffering no significant influences from increased yield caused

362

by Mg addition suggest possible positive effects of Mg addition on these elements’

363

uptake by roots and a resulting positive effect of such increased microelements on

364

yield. Our results suggest that the “dilution effect” of Mg addition yield depends on

365

species, microelement and organ. Excessive Mg addition may disturb the balance

366

between K or Ca and Mg as reflected in significant decreases of K:Mg and Ca:Mg

367

ratios with rising Mg addition.

368

Implications for fertilization in grassland agro-ecosystem

369

Our results suggest that Mg fertilizer alone may play a more important role in

370

leguminous crops and forage quality compared to that of grass: Mg not only plays a


Page 16 of 30

Page 17 of 30


22

371

role in increased nitrogen utilization efficiency

, but it also enhances biological

372

nitrogen fixation in appropriate soil pH 3. As suggested by Gerendás and Führs (2013)

373

11

374

positive effects as it has very important roles in rhizobium nitrogen-fixation. In acid

375

soils, alkaline Mg fertilizer, such as MgO or alkaline Mg fertilizer with lime, should

376

be used in order to increase soil pH, benefiting rhizobium nodule formation and

377

activity 24. For grass, our results show sufficient N fertilizer along with Mg fertilizer

378

(200 mg kg-1 Mg fertilizer with 0.7 g kg-1 N fertilizer) can avoid the dilution effects on

379

crude protein shown with Mg fertilization alone.

, Mg fertilizer should be applied before flowering or earlier to maximize these

380

In this study, we only measured crude protein, rather than the different

381

forms of protein. Further studies should consider if Mg addition causes any change of

382

protein form in forage or seed19, 25. Excessive Mg fertilizer not only has negative

383

influences on [Ca], but may also affect the balance between K or Ca and Mg (See

384

Figure 3). Therefore, appropriate Mg fertilizer should be applied according to soil Mg

385

availability. Our results suggest that microelement dilution effects caused by Mg

386

fertilizer were also significant for some of our considered elements, but depend on

387

species. Therefore, attention needs to be paid to microelements, such as Zn in white

388

clover, to avoid “hidden” deficiency of elements and assure high nutritional quality

389

along with high yields.

390

In this study, we found that the forage crop protein response to Mg fertilizer

391

depends in large part on the availability of N in soil, which for legume also depends

392

on soil pH. These different responses to Mg addition between legume and grass are

393

primarily caused by different functions of their symbiotic microorganisms. Significant

394

dilution effects of [crude protein] with Mg addition in grass was mimicked in legume



395

with acidic soil that precluded rhizobium growth and presumably minimized N2

396

fixation. Added N fertilizer in the grass or maximizing N2 fixation in the legume via

397

less acidic soil eliminated and even reversed this [crude protein] dilution effect in

398

both species. The Mg addition also decreased [Ca] in both species, which may result

399

in imbalance between Mg and Ca for consumers, but the appropriate increase of [Mg]

400

caused by Mg fertilizer may decrease the risk of grass tetany via decreasing K:Mg

401

ratio. The other nutritionally relevant influences of “dilution effects” caused by Mg

402

fertilizer on microelements depend on species. These results suggest that we should

403

fertilize with adequate N or assure biological fixation of N when fertilizing with Mg.

404

‘A forgotten element in crop production,’ Mg deserves high importance for

405

maintaining crop yield and high-quality food.

406 407

Acknowledgments

408

We thank Shuijing Hu for helpful suggestions on the experiment, and staffs in Baima

409

base for assistance in the greenhouse. This work was financially supported by

410

National Natural Science Foundation of China (NSFC 600030), and National Basic

411

Research Program of China (also called 973 Program, 2014CB441001).

412

Supporting Information Available: [Results of response of nutrient concentrations in

413

ryegrass shoot to Mg addition (Fig. S1), and the effects of N addition under ample

414

magnesium soil conditions on mineral element concentrations in ryegrass shoots (Fig.

415

S2)].

416


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508 509



510

Figure captions

511

Figure 1 Effect of Mg fertilizer application in soil on leaf chlorophyll concentration

512

(expressed as SPAD values) (a) and forage yield (aboveground biomass) (b).

513

Different capital letters indicate statistically significant differences (Tukey HSD,

514

p

Effects of Magnesium Fertilizer on the Forage Crude Protein Content

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