Differential Responses of Soybean and Sorghum Growth, Nitrogen

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Differential responses of soybean and sorghum growth, nitrogen uptake and microbial metabolism in the rhizosphere to cattle manure application: A rhizobox study Qingnan Chu, Zhimin Sha, Takuji Nakamura, Norikuni Oka, Mitsuru Osaki, and Toshihiro Watanabe J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03046 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

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Journal of Agricultural and Food Chemistry

Differential responses of soybean and sorghum growth, nitrogen uptake and microbial metabolism in the rhizosphere to cattle manure application: A rhizobox study Qingnan Chu,† Zhimin Sha,‡ Takuji Nakamura,§Norikuni Oka,§Mitsuru Osaki,† Toshihiro Watanabe*,† Author information †

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

‡

School of Agriculture and Biology, Shanghai Jiaotong University, 200240, Shanghai,

China §

NARO Hokkaido Agricultural Research Center, Sapporo 062-8555, Japan

Information for corresponding author *Toshihiro Watanabe, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Tel: +81 11 706 2498; Fax: +81 11 706 2498 E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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In this study, we determined the capacity of soybean (Glycine max L. Merr. cv.

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Hoyoharuka) and sorghum (Sorghum bicolor L. Moench. cv. Hybrid Sorgo) to utilize

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different forms of nitrogen (N) in a rhizobox system. Seedlings were grown for 35

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days without N, or with 130 mg N kg-1 soil as ammonium sulfate or farmyard cattle

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manure. The soil fractions at different distances from the root were sliced millimeter

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by millimeter in the rhizobox system. We assessed the distribution of different forms

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of N and microbial metabolism in different soil fractions in the rhizosphere. There are

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no treatment-dependent changes in biomass production in the roots and shoots of

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soybeans, however, the ammonium and manure treatment yielded 1.30 and 1.40 times

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higher shoot biomass of sorghum than the control. Moreover, the depletion of

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inorganic N and total amino acids (TAA) in the rhizosphere was largely undetectable

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at various distances from the soybean roots, regardless of the treatments employed.

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The addition of ammonium sulfate resulted in a decrease in the nitrate concentration

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gradient as the distance decreased from the sorghum roots. The addition of manure to

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the soil increased the N content in the sorghum shoots, 1.57 times higher than the

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control; this increase was negatively correlated with the concentrations of TAA in the

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soil of the root compartment. In addition, the application of manure simultaneously

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induced TAA depletion (i.e. the TAA concentration in root compartment was 1.48

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times higher than that in bulk soil) and greater microbial activity and diversity in the

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sorghum rhizosphere, where higher microbial consumption of asparagine, glutamic

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acid, and phenylalanine were also observed near the roots. Our results are first to

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present the evidence that sorghum may possess a high capacity for taking up amino

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acids as a consequence of organic matter application and microbial metabolism.

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Keywords: amino acid, microbial metabolism, nitrogen, rhizobox, rhizosphere,

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sorghum

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Introduction

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N is indispensible for sustaining human activities through its role in the production of

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food, animal feed, and synthetic chemicals.1 Depending on food demand, the

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projected consumption of N fertilizers is likely to increase from 105 Mt in 2010 to

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180 Mt by 2050.2 Thus, the effective utilization of organic materials as N source for

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crop production is important for the development of sustainable agriculture. The

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actual pool sizes of organic forms of N can be large in agricultural soils,3 but higher

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plants generally cannot efficiently assimilate the abundant sources of N bound up in

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the soil.4 Fully comprehending the different capacities of crops to assimilate organic

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N is important for advancing crop productivity and sustainable agriculture.

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It has been well documented that the responses to organic matter in terms of growth

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and N uptake differ among plant species.5-9 Some plants possess the ability to acquire

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and metabolize organic N (ON) efficiently in the form of amino acids,10-11

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peptides,12-13 and proteins.6 Once applied to the soil, organic matters undergo stepwise

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mineralization, including proteolysis, ammonification, and nitrification, where

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proteinaceous compounds are converted into amino acids, ammonium (NH4+), and

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nitrate (NO3–) by soil bacteria and fungi. Among the primary microbial extracellular

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proteases in soils, alkaline metalloprotease, neutral metalloprotease, and serine

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protease are encoded by the apr, npr and sub genes, respectively.14-15 The interactions

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between plant roots and microbes in the rhizosphere with applied organic fertilizer

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have been investigated extensively because of their importance for nutrient

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management and soil carbon (C) dynamics.16-20 Microorganisms are less limited by

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the ON pool than plants because they are able to release enzymes to utilize

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macromolecular soil organic N.21 Most plant roots provide soil microbes with C

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nutrition, while the microorganisms can decompose soil ON and make N available to

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the roots.18,

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increase the rates of N mineralization and bacterial numbers, as well as change the

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microbial-root interactions to accelerate the flux of N from organic sources into the

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plant-available N pool.18-19 However, the microbial factors that affect its capability to

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use ON, as well as the forms utilized have not been well elucidated.

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Among the cereal crops, the sorghum has been demonstrated to have the capacity to

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utilize more ON than maize and pearl millet in soil or hydroponic growing

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conditions.5,

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capability to absorb proteinaceous compounds but that amino acids may be a more

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readily utilizable source of N.5 In a previous study, we showed that soybean do not

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efficiently utilize ON,8 and the N uptake by soybean did not increase significantly

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The microbial responses to the addition of organic matter likely

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Okamoto and Okada 2004 suggested that sorghum have the

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with the farmyard cattle manure application.25 Consequently, soybeans can be used as

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a comparative plant in studies of ON utilization compared with sorghum. Moreover,

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the capacity of symbiotic nitrogen fixation by soybean and the difference of sink N

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demand between C3 plant (soybean) and C4 plant (sorghum) may cause contrasting N

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utilization. Therefore, in the current study, we aim to investigate the difference of N

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uptake and utilization between soybean and sorghum receiving ON application, which

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is quite important for nitrogen management in the agricultural production.

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Previously, Youssef and Chino (1988) established a rhizobox system to study the

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distribution of nutrients in the different soil fractions of the rhizosphere,26 and this

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rhizobox system has been used extensively to map the distribution of N in different

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rhizospheric soil fractions.18-19,

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distribution of N and microbial metabolism has not been determined in a rhizobox

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study. Therefore, we designed a rhizobox experiment to examine the distribution of

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inorganic N, amino acids, and the microbial metabolism in the rhizospheric soil of

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soybean and sorghum after the addition of inorganic and organic N fertilizers. We

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analyzed the interactions between plant N uptake, the microbial utilization of organic

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compounds, and the distribution of inorganic N and amino acids in different

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rhizospheric soil fractions.

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Materials and Methods

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Experimental soil Soil classified as Haplic Fluvisols according to the US Soil

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However, the spatial relationship between the

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Taxonomy Classification was collected from agricultural fields at Hokkaido

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University. The basic properties of the experimental soil were: pH 6.05, total nitrogen

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4.5 g kg–1, ammonium-N 4.05 mg kg–1, nitrate-N 13.4 mg kg–1, available P (Truog-P)

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194 mg kg–1, exchangeable K 0.36 cmol kg–1, cation exchange capacity 37.5 cmol kg-1,

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and base saturation 30%.

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Experimental design All experiments were performed in a greenhouse. Rhizobox

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culture chambers (20 × 10 × 15 cm, Fig. 1) were used to separate the root zone soil

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from the rhizospheric and bulk soils away from the root zone, according to the

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method described by Youssef and Chino (1988) and Li et al. (2007).26-27 The rhizobox

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contained two types of compartments: a root compartment (2 × 10 × 15 cm, Fig. 1),

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and two rhizosphere compartments on each side of the root compartment (Fig. 1). The

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root compartment was comprised of a perspex frame and nylon cloth (pore radius
14 mm

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(L) outward from the nylon mesh of root compartment (Fig. 1). The soil collected in

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the middle root compartment was defined as RC. Any soil at distances more than 14

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mm from the RC was regarded as the bulk soil (BS). Two soil slices obtained at the

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same distance from the root plane on either side were mixed together. After slicing the

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soil samples were sieved rapidly through a 2-mm sieve. Several fresh soil samples

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were then stored at 4 °C for Biolog EcoplateTM analysis, whereas the other samples

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were frozen rapidly with liquid nitrogen, stored at -80 °C, and then lyophilized before

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analyzing the TAA and inorganic N concentrations.

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Inorganic N and TAA determination in plant and soil samples To analyze the N

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contents of plant samples, the samples were digested with (H2SO4 [98%] -H2O2) and

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the N concentrations were determined using the Kjeldahl method (Page et al. 1982). 28

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The concentrations of inorganic N, TAA, and available mineral elements were

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analyzed in the soil samples. The inorganic N (NH4+-N and NO3–-N) was extracted

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from the soil by shaking with 2 M KCl. 9 The soil (1g) was shaken with the 2 M KCl

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(5 ml) in 10 ml volume polycarbonate tubes on an end-to-end shaker (200 rpm, 30

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min). The soil suspension was centrifuged (15,000g, 15min, 4°C) and the supernatant

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was collected. The inorganic N (NH4+-N and NO3–-N) in the supernatant was

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determined by colorimetric assays at 630 nm and 538 nm, as described by Page

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(1982). The TAA was extracted from soil by shaking with 6 M HCl and the

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supernatant collection was the same as inorganic N. The concentrations of TAA were

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determined in 1:5 (w/v) soil (1 g) /HCl extracts with a colorimetric assay at 570 nm

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using the ninhydrin method, as described by Jones (2002).29

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Community level physiological profile (CLPP) analysis CLPPs were determined

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using Biolog EcoplatesTM (Biolog Inc., CA, USA). Each EcoplateTM consisted of

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triplicate combinations of 32 wells containing 31 different carbon compounds

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(carbohydrates, carboxylic acids, amino acids, amines, polymers, and miscellaneous),

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and a control (water) in each replicate. Tetrazolium dye was added to each substrate

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and liberated via the microbial breakdown of individual carbon compounds. Soil

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suspensions (1 g soil in 10 mL of 0.85% sterile saline solution) were shaken and then

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pre-inoculated for 24 h before inoculation to allow microbial utilization of any soluble

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organic compounds present in the soil. Hundred-fold dilutions were prepared and

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150-µL aliquots of the dilutions were added to each well of a Biolog Ecoplate. The

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plates were incubated at 25°C and the color development in each well was recorded as

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the optical density at 595 nm using a plate reader at regular 12-h intervals (from 24 to

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120 h). The plates were incubated in the dark at 25°C between measurements.

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Normalization was performed against the blank well (control) for each replicate and

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the average well color development (AWCD) was determined by calculating the mean

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of each well’s absorbance value (Abs) at 595 nm for every reading time. The Shannon

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diversity index (DQ = – Σpilog2pi, where pi is the ratio of the Abs of a particular well

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relative to the sum of the Abs of all the microplate wells, and DQ represents the

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microbial diversity) was calculated by treating the Abs for each well as equivalent to

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the abundance of individuals in each species.30

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Statistical analysis All statistical analyses were performed using SPSS version 18.0

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(SPSS Inc. Chicago, IL, USA). One-way analysis of variance (ANOVA) with was

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used to evaluate the results at P

Differential Responses of Soybean and Sorghum Growth, Nitrogen

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