Source: UNIV OF WISCONSIN submitted to
CHARACTERIZING SOYBEAN YIELD RESPONSE TO RHIZOBIAL INOCULANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0218132
Grant No.
(N/A)
Project No.
WIS01399
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2009
Project End Date
Mar 31, 2011
Grant Year
(N/A)
Project Director
Conley, S.
Recipient Organization
UNIV OF WISCONSIN
(N/A)
MADISON,WI 53706
Performing Department
Agronomy
Non Technical Summary
Soybean is a legume that requires more than 300 lbs of nitrogen to produce a 60 bu/a crop. Some of this nitrogen comes from soil organic matter, but the majority of the nitrogen is produced by a symbiotic relationship with rhizobia bacteria living on the plants roots. (Lindemann and Glover, 2003; Tien et al., 2002). This bacterium, Bradyrhizobium japonicum, fixes nitrogen from the air to produce ammonium which can be used by the plant (Balatti and Pueppke, 1992). Inoculating soybean seed with B. japonicum is widely used to ensure that adequate levels of healthy bacteria are present near the seed to facilitate N fixation. In fields where native rhizobia levels are low due to lack of soybean in the rotation or environmental stress, inoculation can increase yields by 50% or more. (Duong et al., 1984; Seneviratne et al., 2000). After an initial inoculation of a field, B. japonicum will survive for many years and future inoculation will not increase yields as much as the initial one. There is a mixture of results on the efficacy of successive inoculations after initial use of B. japonicum products. Some evidence that successive inoculation can increase yields is presented by Beuerlein, 2005 and Conley and Christmas, 2006. However a number of trials did not see yield increases (Abendroth and Elmore, 2006; Pedersen, 2003; Vitosh, 1997). In another study, use of soybean inoculant raised soybean yield in 6 of 14 site-years in fields that had been in soybean rotation in a Michigan study (Schulz and Thelen, 2008). Soybean inoculants are inexpensive and fairly convenient to use. The commercial inoculant industry in Wisconsin and the Midwest is vibrant and very competitive. Newer inoculants from several of these companies contain multiple improved strains of B. japonicum and claim better nitrogen fixation efficiency leading to higher yields. Recently, inoculant manufacturers have added lipochitooligosaccharide nod factors to their inoculant products. These products are meant to elicit responses which increase infection and nodule formation (Smith et al., 2004). Inoculation of soybean, while relatively inexpensive, can be very important to the profitability of a soybean crop. Understanding the environmental conditions when inoculation should be used would greatly benefit WI soybean growers and make crop input decisions easier. Previous research has not looked at a matrix of environmental factors that might affect the performance of inoculants.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20518201060100%
Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
Rhizobial inoculants have widely been promoted and used in Wisconsin due to our diverse rotations. It is widely known that if a field has never been in soybean or has been removed from soybean for > 4 years that inoculants are needed. In many areas of the state however, forages and small grains have been removed from the rotation and have been replaced with a soybean-corn or corn-corn-soybean rotations. Research is lacking regarding the annual need for inoculants in these "short" rotations. Our specific objectives are: 1.To develop a fast and reliable quantitative PCR assay based on real-time PCR technology and to compare its efficiency to the MPN-Plant Infection assay. 2.To determine if rhizobial inoculation is necessary the year after flooding events. 3.To quantify the effect of crop rotation and tillage on inoculant efficacy. 4.To quantify yield response of inoculants over various environmental conditions. Deliverables: The development of patentable molecular techniques to quantify soil rhizobia at the University of Wisconsin - Madison should represent a significant improvement for Wisconsin soybean growers. Growers will be then be able to make inoculation decisions rapidly (2 days vs. 5 weeks or more) and based on objective and reliable evidence. Using data collected from this series of experiments we hope to develop a prediction matrix from which growers can accurately assess the probability that an inoculant application will lead to increased yield and profitability. Lastly, we will also author a minimum of two research and two Extension publications (preliminary titles): 1.Rotation and tillage impact on soybean yield response to rhizobia 2.Flooding impact on soil rhizobia populations.
Project Methods
Objective 1. The most probable number (MPN) is the classical technique used to characterize rhizobia infection. MPN was developed by McCrady (1915) and later improved by other researchers (Cochran, 1950). In this technique axenic soybean plantlets are inoculated with diluted soil suspensions and grown for 4 weeks in a growth-chamber of a greenhouse. Plants are scored as positive or negative on the presence or absence of nodules and the MPN of rhizobia can be deduced from the classical McCrady table (Vincent, 1970). We propose to develop a quantitative PCR assay based on real-time PCR technology using the following procedure. DNA will be extracted from soil samples by lysing bacterial cells and purifying DNA from other cell and soil components of the crude extract using the SoilMasterTM DNA extraction kit (Epicenter Biotechnology). DNA will be quantified with the PicoGreen assay (Invitrogen) using a 96-well-plate fluorimeter. PCR Primers amplifying nodulation specificity genes such as nodS, nodU, nodZ, nodV, nodW and nolA will be designed using Primer3 and tested for their ability to detect specifically soybean rhizobia (Denarie et al., 1996). Once objective one is realized we will use this technique in objectives 2-4 below. For each objective soil samples will be taken at planting to quantify rhizobia populations. Rhizobia populations will be quantified according to the procedures described in Objective 1. Grain yield and end of season rhizobia populations will be quantified at R8 (physiological maturity) soybean. Objectives 2. We will identify a minimum of 3 flooded fields that were flooded for a minimum of 2 weeks in 2008. In each flooded zone we will conduct a randomized complete block design factorial experiment with 6 replications. The treatments will include an untreated check and liquid based inoculants. Objective 3. We will use the long-term rotation experiment located at Arlington WI to quantify Objective 3. This trial was established in 1983 and contains 14 different corn and soybean rotations. The experimental design is a randomized complete block split-split design with four replications. The main plot effect is tillage (conventional vs. no-till); the split-plot effect is rotation (Continuous soybean, soybean/corn rotation, 1st year soybean and corn, 2nd year soybean and corn, and third year soybean and corn); and the split-split-plot effect is inoculant treatment. Objective 4. We will use 9 sites from the Southern, Central, and North Central Region Roundup RR variety tests to field validate Objective 1 as well as quantify the effect of environment, soil type, and residual N levels on soybean yield. Across these locations, we have a variety of soil types ranging from sand to silty clay and irrigated and rainfed environments. We will select 3 high yielding soybean varieties to test at each region and each variety will receive one of three inoculant treatments: (untreated check, Optimize, or Excalibur). These inoculant treatments will be planted in a randomized complete block design with four replications using our standard plot planting and management practices.

Progress 04/01/09 to 03/31/11

Outputs
OUTPUTS: Branden Furseth my M.S. graduate student published 3 referred journal articles and 3 scientific abstracts on this work. He graduated in May 2011. PARTICIPANTS: Wisconsin Soybean Association and Marketing Board TARGET AUDIENCES: The target audience is Midwest certified crop consultants and growers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The symbiotic relationship between soybean [Glycine max (L.) Merr.] and rhizobial bacteria such as Bradyrhizobium japonicum, B. elkanii, Sinorhizobium fredii and Rhizobium etli allows biological nitrogen fixation (BNF), which along with residual soil N, can meet the seasonal needs of a soybean crop. Inoculants containing Bradyrhizobium japonicum are available for soybean [Glycine max (L.) Merr.] production but may not be necessary in fields where soybean previously has been produced. The objective of this study was to create a new method for quantifying soybean-specific rhizobia in the soil using quantitative real time polymerase chain reaction (qPCR). The regression analysis found the relationship between MPN and qPCR to follow the equation ln(MPN)=33.43-0.43(CT). The data have an adjusted R2 of 0.88. These data allow for the prediction of soybean-associated rhizobia populations in the soil based on the qPCR analysis of the extracted DNA. The objective of this study was to quantify the effect of flooding on the performance of rhizobia inoculants and the seed yield and quality response to the initial soil rhizobia population. Seed yield, protein, and oil did not respond to seed treatment across all locations. Though the lack of yield response to seed treatment suggests that initial soil rhizobia populations were adequate for optimum crop growth, and the negative correlation between yield and initial rhizobia population was unexpected, other environmental factors could be allowing the crop to compensate for any lack of biological nitrogen fixation due to a lack of rhizobia. The goal of this study was to improve the predictability of a positive soybean seed yield and quality response to inoculation by (i) characterizing inoculant product response in multiple environments, and (ii) quantifying the response of soybean to indigenous soil rhizobia populations. Two inoculants and a control were tested on three soybean varieties at nine locations in Wisconsin during 2009 and 2010. Soil samples were collected from each plot for the quantification of soil rhizobia. Seed yield, protein, and oil did not respond to inoculation across all 18 environments (P = 0.15, 0.38, and 0.30, respectively). Three environments responded positively to inoculation (P < 0.05), while the remainder showed no response. Yield of the control treatment was positively correlated with the soil rhizobia population level across all environments (P = 0.005), while the inoculated treatments showed no response. These data indicate a yield advantage for inoculation at lower rhizobia populations; however, given the large range of response with only three of 18 environments showing a positive response, a rhizobia population threshold to aid in the decision to treat soybean seeds with a rhizobia inoculant cannot be determined.

Publications

  • Furseth, B. J., Conley, S. P., and Ane, J.. 2012. Soybean Response to Soil Rhizobia and Seed Applied Rhizobia Inoculants in Wisconsin. Crop Sci: 52:339-344.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Branden Furseth, my M.S. graduate student, published 2 referred journal articles and 2 scientific abstracts on this work. He expects to graduate in February of 2011. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: researchers, growers, certified crop consultants PROJECT MODIFICATIONS: none

Impacts
The symbiotic relationship between soybean [Glycine max (L.) Merr.] and rhizobial bacteria such as Bradyrhizobium japonicum, B. elkanii, Sinorhizobium fredii and Rhizobium etli allows biological nitrogen fixation (BNF), which along with residual soil N, can meet the seasonal needs of a soybean crop. Inoculants containing Bradyrhizobium japonicum are available for soybean [Glycine max (L.) Merr.] production but may not be necessary in fields where soybean previously has been produced. The objective of this study was to create a new method for quantifying soybean-specific rhizobia in the soil using quantitative real time polymerase chain reaction (qPCR). The regression analysis found the relationship between MPN and qPCR to follow the equation ln(MPN)=33.43-0.43(CT). The data have an adjusted R2 of 0.88. These data allow for the prediction of soybean-associated rhizobia populations in the soil based on the qPCR analysis of the extracted DNA. The objective of this study was to quantify the effect of flooding on the performance of rhizobia inoculants and the seed yield and quality response to the initial soil rhizobia population. Seed yield, protein, and oil did not respond to seed treatment across all locations. Though the lack of yield response to seed treatment suggests that initial soil rhizobia populations were adequate for optimum crop growth, and the negative correlation between yield and initial rhizobia population was unexpected, other environmental factors could be allowing the crop to compensate for any lack of biological nitrogen fixation due to a lack of rhizobia.

Publications

  • Furseth, B. J., Conley, S. P., and Ane, J.. 2010. Soybean Seed Yield and Quality Response to Rhizobia Population and Rhizobia Inoculants on Previously Flooded Sites in Southern Wisconsin. Agronomy Journal. Accepted for publication 12/18/10.
  • Furseth, B. J., Conley, S. P., and Ane, J.. 2010. Enumeration of Soybean-Associated Rhizobia with Quantitative Real Time Polymerase Chain Reaction. Crop Sci. 50: 2591-2596.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The 2009 growing season was the first year of research for this Hatch project, therefore I have no outcomes/impacts to report as of now. This data will be presented extensively during the 2010 season. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Objective 1. To develop a fast and reliable quantitative PCR assay based on real-time PCR technology and to compare its efficiency to the MPN-Plant Infection assay.Two primer sets were designed for each of the nodZ and noeI specificity genes of Bradyrhizobium. Initial PCR testing indicated one high quality primer set from each gene (nodZ-A and noeI-B). The efficiency of these two primer sets was evaluated using a qPCR primer optimization procedure. The efficiencies were 88.9% and 98.5% respectively for the nodZ-A and noeI-B primers. Efficiency scores of 90% or greater are desirable.Objective 2. To determine if rhizobial inoculation is necessary after flooding events. Across flooded and non-flooded sites and inoculants, there was no significant yield response at any location. Corresponding soil rhizobia data will be analyzed this winter and any conclusions or recommendations can be made with this data.Objective 3. To quantify the effect of crop rotation and tillage on inoculant efficacy.In 2009, we initiated a comparison of several soybean inoculants in the long-term rotation experiment located at Arlington WI. Soil samples were taken at planting and harvest to quantify rhizobia populations. Rhizobia populations will be quantified according to the procedures described in Objective 1.Plots were planted on 18-May and harvested on 12-October. Yield was significantly different for rotation, but not for tillage or inoculant treatment. Highest yields were obtained from the 1st year soybean (52.9 bu/a) and lowest from the 4th year soybean (45.2 bu/a). A rotation x inoculant interaction for yield was significant. Highest overall yields were obtained in the 1st year soybean and corn/soybean rotations. The soybean inoculant Optimize, significantly increased yields in the 5th and 3rd year rotations compared to the control. Once the qPCR method from objective 1 is confirmed and soil rhizobia levels are determined we hope to better explain the rotation by inoculant yield interactions. Objective 4. To quantify yield response of inoculants over various environmental conditions.In 2009, we used 9 sites from the Southern, Central, and North Central Region Roundup variety tests to field validate Objective 1 as well as quantify the effect of environment, soil type, and residual N levels on soybean yield. Across the three regions, there was significant yield response to seed treatments. Overall, the CruiserMaxx treatment produced the highest yield. Both Optimize and Excalibre (our inoculant treatments) out-yielded the UTC. We cannot make recommendations based on this single year of data, but propose to continue this work and will attempt to correlate these yield results with the soil rhizobia analysis as that is completed.

Publications

  • No publications reported this period