Final report for LNC17-389
Project Information
Corn is North America’s most productive and most grown cereal. But it has pollution problems due to the necessity for large amounts of nitrogen fertilizers and problems with a lack of nutritional density in its grain. This research project focused on testing new hybrids bred at the Mandaamin Institute which have potential for resolving both of these problems. These hybrids possess microbial partnerships that enable the corn to efficiently take up minerals and nitrogen without the use of fertilizers. The hybrids were studied in the context of different farming systems and soil fertility conditions. Studies with microscopy, field trials on different farms, and mineral and natural isotope analyses showed that 1) the plants exercise rhizophagic cycles with seed associated bacteria, leading to nitrogen efficiency in field trials, high levels of δ15N in tissues, and grain with high protein and mineral contents; 2) these partnerships result in comparable yields to manured commercial hybrids where no manure is added to the Mandaamin hybrids; 3) Hybrids with the Mandaamin inbred C4-6 as a parent particularly express these traits and also appear to fix N2. 4) The C4-6 based Mandaamin hybrids also respond negatively to fresh manure but positively to high organic N and to high soil protein levels resulting from cattle manure. 5) The negative effect of fresh manure applications on the C4-6 based hybrids extends to yield and mineral uptake and this problem is worse on soils with low organic matter content. 6) The Mandaamin hybrids have grain with a high nutritional value due to their high content of the essential amino acids methionine and lysine and their enhanced mineral content and this may offset a slightly lower yield. Future research should be directed towards testing and confirming concepts developed in the course of this project while extending the testing to more farms. Breeding should be fostered that focusses on stability for both microbial assisted processes of rhizophagy and N2 fixation coupled with adequate yield performance and enhanced nutritional value under variable soil conditions with reduced N inputs.
Corn is the major cereal crop for North America and the most productive cereal in the world. However, conventional corn production is largely dependent on the use of mineral nitrogen fertilizers. Organic farmers often depend on bought in chicken manure. Nitrate production in the soil under corn, coupled with the use of these N fertilizers results in surplus nitrate in the soil. This nitrate is denitrified by bacteria to produce nitrous oxide, a potent greenhouse gas. The excess nitrate not used by the crop leaches from the soil and causes massive pollution of wells, lakes, rivers, and also the Dead Zone off the Gulf of Mexico. Modern hybrids have been found to have an enhanced ability to foster microbes in their rhizospheres that increase nitrification in the soil and denitrification (Favela et al., 2021).
Furthermore, the-sided focus on increased yields may have reduced nutrient density of crops and thereby affect the health of end users.
This project was about evaluating the impacts and value of new, experimental hybrids which have been developed to reduce the need for nitrogen fertilizers while increasing nutrient density. These hybrids were developed at the Mandaamin Institute, mostly under organic farming conditions that were N limited. They resulted from a field based breeding program (Goldstein et al. 2012; 2019) that, up to now, has utilized 51 growing seasons in Wisconsin, Chile, Puerto Rico, and Hawaii. The program selected corn for the needs of organic farming including for better nutritional value and nitrogen efficiency. The hybrids that resulted may have potential for resolving some of the Midwest’s most intractable problems with corn and N pollution that go beyond organic farming. This possibility is due to partnerships with microbes (Goldstein, 2016).
During this project the impacts and value of the Mandaamin hybrids were evaluated using on-farm testing. Objectives included 1) testing several Mandaamin hybrids on multiple farms to determine their agronomic and quality characteristics and effects of soil and soil management; 2) examining nutrient uptake, nitrogen efficiency and nitrogen fixation in the hybrids; 3) testing whether inoculating seed with N2 fixing bacteria would improve crop performance and N efficiency.
We used replicated trials and soil analyses to estimate yield, quality, and N efficiency of the Mandaamin hybrids on different farms. Manure trials were utilized, and bacterial inoculation was added as an extra test factor because it seemed increasing relevant to the matters at hand. In the descriptions below, these trials are referred to as the SARE manure/inoculation trials.
In the course of the project, events caused changes from the initial plan. Extensive soil and plant sampling and analyses that had been planned for 2018 proved not to be possible due to illness contracted by one of the two Mandaamin Institute technicians and illness by the principal investigator at the end of that growing season. Furthermore, additional sites and plots that had not been planned in the initial proposal became available in 2019 due to funding of an OREI project together with the University of Illinois. This OREI project involved un-replicated strip plots comparing multiple Mandaamin hybrids with commercial checks on different farms. These experiments are referred to as the varietal trials. A decision was made together with regional SARE leadership to incorporate those extra sites, to use SARE funds to better monitor these sites with soil and tissue testing, to modify the approach to the research accordingly, and to include a conventional farm into the plan. As will be shown below, pursuance of these varietal trials with SARE funds proved to be highly rewarding from the standpoint of gaining information.
Furthermore, to compensate for the first year, efforts were carried out in 2021 that included limited trials to clarify questions raised by results in preceding years and to analyze data from the overall effort.
The data gathered by the project was substantial. More analyses of the data in this report and other data are planned to further harvest pertinent results into the form of publishable papers. So this report should be regarded by the reader as preliminary information which will probably be modified in the review process. In this report we focus on the most important findings and there are a few experiments not reported on because their results were not essential to the overall findings. Aside from that, the ample tables and graphs used to describe the results have resulted in a large report. So in order to focus the reader on the outcomes of the project, rather than burying them first under voluminous tables, we will present our summary and discussion section first, then lead into the context and background sections followed by methods, description of results, and citations.
To become more sustainable and climate friendly, humanity needs to find alternatives to the massive use of nitrogen fertilizer. That fertilizer is energetically and monetarily expensive and non-renewable. It's breakdown product, nitrate, is polluting our wells, rivers, and lakes, causing the Dead Zone in the Gulf of Mexico, and releasing nitrous oxide into the atmosphere which is a potent greenhouse gas. Our political/economic system is currently locked into a paradigm that causes this pollution problem. Based on conventional thought, there is no practical solution nor major regulation that can reverse this trend. 2) Corn is both the most productive cereal and number one user of N fertilizer. It has been selected for a century to depend on nitrate fertilizers. Corn is also the major field crop for organic farmers and they use inputs including chicken manure to provide it with the nitrate and ammonium it needs to produce high yields.
The corn hybrids presently used by most organic farmers are bred by large private companies to perform under conventional conditions with high levels of synthetic nitrogen fertilizers, pesticide applications and high density plantings (Mastrodomenico et al. 2018). Conventional inbreds often lack vigor when they are grown for hybrid production under organic conditions, appearing to be nutrient deficient and competing poorly against weeds (Block, 2021). The experience of the PI/PD is that they often seem to lack plasticity of response (‘flex’) to low stands or low fertility conditions needed on organic farms. The production of organic inbreds, hybrids and grain is challenged by 1) a legal loophole which allows organic farmers to use conventional seed; 2) contamination from genetically engineered (GM) corn; 3) soil nutrient limitations [especially nitrogen (N)], and 4) climatic instability that causes stress, diseases, pests, and weeds. Chicken manure is often used as a fertilizer by organic corn producers to address N needs of corn, but it can be expensive, difficult to handle, potentially polluting of water and air, and is often derived from non-organic confinement operations.
The organic consumer expects that organic products are higher nutritional value, but conventional corn is being used by organic farmers and they have been bred for yield and not for quality. However, organic farmers have legitimate need for higher quality corn that is competitive. The Mandaamin Institute identified and is improving corn for the following parameters:
Methionine: Organic livestock producers need high quality feed from corn grain and silage that has protein that is high in methionine, cysteine, and lysine. This is important both for feeding organic poultry (Goldstein et al. 2008; Goldstein et al. 2012) and for dairy production (Lee et al. 2012). Organic poultry especially needs high methionine corn because a) methionine is the main limiting amino acid for poultry, b) the use of synthetic methionine in organic rations may be phased out by USDA (McEvoy 2015, Fanatico and Ellis 2016), and c) corn is the major ingredient in most poultry feed in the country.
Carotenoids: The color of egg yolks is important to the consumer and relates to their perception of better nutritional value. Brightly colored yolks bring a higher price at market. Yellow corn is relatively rich in carotenoids lutein and zeaxanthin, which are critical for coloring egg yolks. Epidemiological studies have shown that consumption of lutein and zeaxanthin is associated with reductions in oxidative stress, eye degenerative and cardiovascular diseases, and cancer (Zaheer, 2017). The high lipid content of eggs helps the human digestive system to absorb carotenoids (Zaheer, 2017), of which β-carotene and β-cryptoxanthin are also important constituents of corn. High levels of β-cryptoxanthin are most effective for increasing the provitamin A content of eggs (Liu et al., 2012). Visual selection for color is effective at increasing carotenoid content in corn and in eggs produced by hens fed on that corn (Burt et al. 2013).
The Mandaamin Institute has bred corn that seems to grow well without manure or nitrogen fertilizer inputs. Initial studies indicate the corn is competitive in yield, has better nutritional value, and obtains more of its nitrogen from microbial biomass and organic matter than does conventional corn. Its performance depends on plant/microbial partnerships that we believe are seed-borne. Despite its obvious potential, efforts to implement use of such corn will not succeed unless more wide scale evidence is gained, allowing understanding about how such partnerships work, their yields, reliability, how they impact N dynamics, and how to manage them. Organic poultry farmers need high methionine, high carotenoid corn grain, while organic corn producers need high yielding high methionine corn. Organic farmers, processors, and consumers need such corn but it needs to yield competitively. This project attempts to increase research on these alternative corns and to increase understanding of them amongst farmers and end users.
Cooperators
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Research
This project became part of a dynamic, quickly evolving, and novel research paradigm with evolving hypotheses. Initially we hypothesized that: 1) Mandaamin corn will reliably produce similar yields to conventional corn but with 30% higher (0.28%) methionine than conventional corn; 2) Inoculation with N-fixing bacteria will increase yield under low N conditions. Mandaamin corn will allow farmers to fertilize less with organic manures and if so, identify where the N is coming from.
As we progressed in learning from our research other hypotheses became apparent.
First, that Mandaaminn hybrids could have enhanced mineral uptake, increased delta 15N values of the nitrogen in the tissues, increased N2 fixation from the air, and better grain protein quality.
Second, that seed-borne bacteria cause rhizophagy in our corn and thereby cause many of these differences, and link them in some way .
Third, that hybrids based on the inbred C4-6 had these properties more than other commercial or Mandaamin hybrids.
Fourth, that application of fresh manured reduced yields, delta 15N, and mineral uptake in C4-6 derived hybrids due to 'endobiome interference' which means microbial interactions that disrupt the rhizophagy/microbial/plant partnership, and that this did not occur for commerical hybrids or to other Mandaamin hybrids.
Fifth, that some of the Mandaamin hybrids when not fertilized, could produce similar yields to fertilized commercial hybrids.
Soon after the inception of the research we became involved in testing our inbreds for seed borne endophytes together with James White at Rutgers University. Dr. White has pioneered research on such endophytes which in most wild plants enhance mineral uptake, growth, and health through the rhizophagy cycle (White et al 2018; 2019a, 2019b). In that cycle seed borne microbes are multiplied in roots, the bacterial cell walls and a portion of total bacterial bodies are consumed, and the bacteria are subsequently excreted through root hairs into the rhizosphere. The bacteria replenish their cell walls in the soil but may be taken up again by growing roots where they are again partially consumed and excreted. The likely biochemical dialogue that underpins the cycle is described in Chang et al. 2021.
Dr. White responded to the publication of our earlier research (Goldstein et al, 2019) by contacting us. We began cooperative research and discovered that that Mandaamin varieties (but not conventional varieties) were carrying gangs of seed borne bacterial endophytes in their seed. These bacteria were excreted from root hairs into the surrounding rhizosphere in relatively large amounts through the root hairs of our plants (see photo collage 1). Photos presented here are from seedlings that were grown axenically in Petri dishes in sterile agar following a light surface disinfection of seed. All inbreds were grown in the same nursery in 2019. Photo collage 2 shows the formation of bacterial aggregates in putative N2 fixing inbred C4-6 and bacteria and spore formation (transparent spheres) in the rhizophagic inbred C2B2. Photo collage 3 shows root tissues of conventional inbreds LH206 and Novartis 942 which showed no signs of bacterial colonization of roots. However, roots of seedlings from Novartis 942 showed colonization with fungal hyphae.
This classical demonstration of rhizophagy has been shown in other plants to increase mineral uptake and to induce root branching, root hair production, and incorporation of nitrogen from the air (White et al., 2018; 2019a, 2019b). More recent work by the White lab, with numerous plant species, has revealed the nature of the biochemical dialogue between microbes fostered in growing shoots and reproductive organs (Chang et al., 2019; Micci et al., 2022). These endophytic bacteria are fostered and fed by plants in the area around plant hairs (trichomes). Biochemical studies with various stains have confirmed parts of the hypothesis that these microbes, working in the shoots and reproductive organs of different non-leguminous species, are engaged in N2 fixation. The quantity of N2 that can be fixed by such endophytes is presently unknown, but initial studies suggest more N is taken from the air in shoots than in roots (Micci et al., 2022).
The results from the White lab have informed our thinking because they could help explain many of the phenomena discovered in the course of this project described below relating to mineral uptake and N acquisition. For example, the putative N2 fixing inbred C4-6 developed at the Mandaamin Institute (Photo collage 4) and used in the trials below, and other inbreds as well, have relatively hairy leaves and stalks as well as highly branched, relatively disease free rooting systems (photo collage 5 and 6).
Our ideas about nitrogen fixation have also evolved in the course of this project in consonance with the results of our studies. Unequivocal confirmation of nitrogen fixation in the field is difficult to achieve. The δ15N value assesses the ratio between the two natural isotopes 14N and 15N. The higher the value, the greater the concentration of 15N. Soil microbial biomass and extractable organic N mostly have higher 15N content than bulk soil and air and hence high δ15N values (Craine et al., 2015). This is because bacteria consume soil organic matter and through isotopic partitioning they release low δ15N nitrate and ammonium into the soil while retaining more 15N in their bodies. They also selectively release more 14N than 15N into the atmosphere during denitrification.
As rhizophagy involves the oxidative destruction of microbes to obtain nutrients it can be assumed that N uptake in plants that have active rhizophagy will be skewed in favor of 15N. Our previous studies (Goldstein, et al., 2019) showed that the grain resulting from many of our inbreds and populations had higher δ15N values than conventional inbreds and hybrids, suggesting that they obtain a portion of their N from microbial biomass and easily extractable organic N. But the grain of very high chlorophyll, putative N fixing populations had much lower δ15N values indicating that they were getting up to half of their N from the air.
An alternate explanation to explain difference in plant δ15N values might be fractionation of nitrogen isotopes in the plant. In some cases fractionation might occur due to the enzymes nitrate reductase, nitrite reductase, and glutamate and glutamine synthase (Craine et al., 2015). But this kind of fractionation, if it does occur in vivo, would discriminate against 15N and would therefore be expected to reduce the 15N content of grain, not increase it. Furthermore, according to Craine et al (2015) “If the major N source for plants in soils is inorganic N, the δ15N of plants should more closely correlate with the δ15N of that source than total N. Although a more comprehensive survey and broader sampling are required, published values of foliar δ15N largely reflect the signatures of inorganic N available in soil. The vicinity of most plots to the identity line indicates that, in most non-boreal sites, plants mainly acquire NH4 + and NO3 − from soil and this uptake occurs without any large isotopic fractionation.” Figure 3 in Craine et al (2015) shows a compilation of data from multiple authors that indicates a tight relationship between the 15N composition of inorganic N and the 15N content of foliage. Hence it seems most likely if the Mandaamin corn has a higher 15N content in its body than neighboring check varieties then it probably relies at least partially on a source of N nutrition that has a higher 15N content. In soils, this 15N enriched source is probably microbial biomass or easily extractable organic matter composed of decomposing microbes.
Based on the evidence and inferences above it is further hypothesized that 1) the Mandaamin varieties have active rhizophagy cycles. 2) Rhizophagy causes higher δ15N by increasing N uptake from microbial biomass and easily extractable organic N. 3) As roots are probably the most active site for microbial turnover, a high δ15N should be especially apparent in roots. 4) In the cases where N fixation is also fostered by foliar endophytes, δ15N is lower in the sink organs such as grain due to a ready supply of depleted δ15N in the form of nitrate or ammonium. The 15N enriched N taken up from through rhizophagy would be progressively diluted with 15N depleted N from atmospheric fixation, leading to lowered δ15N values due to greater 14N enrichment from fixation. This lowered δ15N might be especially apparent in grain.
Hence in the best N efficient hybrids the δ15N levels might be simultaneously increased and decreased in the plant parts by these competing plant/microbial activities. It seems probable that the overall balance may shift from growing phase to growing phase and from year to year depending on growing conditions and plant allocation of resources to partnerships, and that one process may obscure the other from the standpoint of δ15N assessments. Furthermore, it is expected that δ15N values would shift depending on whether the microbes are predominantly engaged in N2 fixation and supplying nitrate or ammonium to the plant with depleted δ15N or they are being consumed by the plant providing materials enriched in δ15N.
Due to the volume of data and limitations of the online system, all Tables and Graphics referenced below are in a pdf.
Here is a link to the tables, photos, and diagrams associated with this report: (Document Containing Tables, Photos, and Diagrams)
The experiments carried out between 2018 and 2021 during this grant comprised mainly two types of trials. The first were the SARE funded trials to examine the effect of manure and inoculation with nitrogen fixing bacteria on the yield and quality of single N efficient hybrids relative to conventional hybrids. The second major emphasis was on examining the performance of multiple hybrids on different organic and conventional farm sites, with and without manuring. The latter effort was jointly funded with OREI and SARE.
The sites chosen were on twelve working farms located from Southeastern to Northwestern Wisconsin. Dr. Micheal Travis (UW Extension-Pepin County) and Chase Cummings (Pepin County Conservationist) took part in guiding and conducting the research in the Durand/Arkansaw area. Farmers performed the research by tilling, planting, weed control. Wisconsin farmers Anibas (Arkansaw), Beiler (Rewey), Clark (Spring Green), Doudlah (Evansville), Esch (Fennimore), Adsit (East Troy), Egre (Cambridge), Michael Fields Agricultural Institute (Troy), Anon (Darlington), Weiss/Bauer (Durand), and Zinniker (Elkhorn) participated in the research, in a gradient stretching from the Southeast to the Northwest portions of the state. Research was also carried out on the Lengacher farm in NE Ohio and on the Michael Fields Agricultural Institute farm in Elkhorn, WI. Farm Weiss/Bauer was conventionally managed monoculture corn with reduced chemical inputs but with a history of large inputs of dairy manure. The other sites were organically managed. The rotations and the inputs for 2019 are shown for several of the farms in Table 1.
Inclusion of these farms allowed us to examine and contrast the performance of the Mandaamin hybrids under farm conditions with different kinds of management histories. The Adsit, Egre, Beiler, and Doudlah farms are organic operations that are largely based on arable cropping. The Adsit and Doudlah farms rely on bought in chicken or other livestock manure. The Anibas, Zinniker, and Anon farms are longer term organic cattle based farms with some arable cropping, perennial forage, and ample cattle manure applications.
Research was carried out in strip plots planted on the farms, with various amount of replication. Strip plots ranged from 40 feet to several hundred feet in length and were two rows wide on the Beiler, Zinniker, and Anon sites, respectively, and three rows wide for Anibas and Clark. Spacing between rows was 2.5 feet for farms Adsit, Beiler, Clark, Egre, Stoltzfus, Michael Fields Agricultural Institute, and Weiss/Bauer, 3.167 feet at Zinniker, and 0.125 feet at Doudlah.
Varietal trials consisted in plantings of a set of Mandaamin hybrids and commercial hybrid checks. Row numbers per plot generally varied generally from two to four rows. Distance between rows was generally 30 inches and planting density was generally 30,000 plants/acre. Various hybrids were compared on these farms. Though the hybrid identity varied from year to year, 17.461 (105 day) and 17.2B24 (108 day) were used as constant Mandaamin checks each year alongside commercial hybrid checks. The commercial checks consisted of Foundation Organic hybrid FOS8507 (110 day maturity) in 2018 or FOS8500 (105 day maturity) in 2019, 2020, and 2021. In some of the studies, varieties were planted across manured and non-manured strips. With few exceptions, strip plots were generally unreplicated on each site but a randomized version of the same design was planted on all farms within a given year.
2019 varietal trials: In 2019 hybrid comparisons were made on 7 farms, but study also focused effort on five farms to study N cycles on a subset of four hybrids across three different farming systems. The four hybrids chosen were the FOS8500 control, 17.461, 17.2B24, and C2B2-1.C46. The latter three Mandaamin experimental hybrids were essentially derived by crossing three parents: 17, C4-6-1, and C2B2 (17.461=17 x C4-6-1; 172B24=17 x C2B2;. C2B2-1.46=C2B2-1 x C4-6-1).
Table 1 describes sites used for varietal trials in 2019.
In preceding years the C2B2-1.C46 routinely appeared to show the greatest signs of being N efficient by its robust early growth coupled with very dark foliage and high chlorophyll scores, and Mandaamin Institute breeders assumed it to be the most likely to be fixing N2 (see photo 2 below). 17.461 appeared quite similar, but not as extreme in its coloration and early vigor. Of the three, 17.2B24 seemed to have more normal early growth and chlorophyll scores and it was rated as being efficient at obtaining N from organic matter but not necessarily N2 fixing.
2020 varietal trials: In 2020, nine different hybrids were grown in similar strip trials on multiple sites. The assortment consisted of the FOS8500 check 17.2B24, 17.461, and six mostly early Mandaamin hybrids, four of which had received only minimal testing in the past. The hybrids ranged from 92 to 108 day relative maturity. The trials took place on two adjacent, manured and non-manured sites on the Beiler farm, on a non-manured site on the Clark and Egre farms, on a manured site on the Esch and Weiss/Bauer farms, and on a not manured site on the Lengacher Farm. The Egre farm site was largely decimated by deer. The Clark farm had too much variation in soil moisture gradients from subsoil seepage to give reliable results. The Lengacher farm produced valid grain yields, but because the farm was located in Ohio we were unable to get full data on plant populations and stover yields. We gained full sets of data from the two sites at Beiler, and single sites at Esch, Goldstein, and Weiss/Bauer farms where corn followed after pumpkins strawberries, alfalfa, and corn, respectively. Grain and stalk samples were sent to USDA in Morris Minnesota for determination of mineral contents in grain and to UC Davis for isotope analyses. Grain samples were sent to the Grain Testing Lab at Iowa State University for determination of grain nutritional value, density, and ethanol yield.
Table 2 describes sites used for varietal trials in 2020 and provides notes on weeds and animal damage.
Manure/inoculation trials. The experimental design generally used in manure/inoculation trials from 2018 to 2020 was a split block design with manuring being the first split applied to half of the experimental area. Manure application was targeted at 10 t/acre of livestock manure. Superimposed across this in the form of strip plots was a 2x2 factorial design consisting of two hybrids which were either inoculated or not inoculated with a proprietary mixture of nitrogen fixing bacteria which originated from the Mandaamin Institute in conjunction with the inoculate company TerraMax. These treatments were laid out as strip plots across manured and non-manured treatments, and they were replicated three times. In several cases the experiment was modified by not including the manure application. In other cases the entire field was manured.
In the SARE funded inoculation trials, two hybrids were always compared, a Mandaamin hybrid and a check. In 2018 the two hybrids were a commercial hybrid from Foundation Organic Seed (FOS8507) with a relative maturity of 110 days and an experimental Mandaamin Institute hybrid (C461.C2B2) of the same maturity with putative N efficiency. During 2019 the experimental Mandaamin hybrid 17.461 was compared with the commercial FOS hybrid FOS8500. These hybrids both possess a relative maturity rating of 105 days. During 2020 the experimental Mandaamin hybrid 15.461 was compared with the commercial FOS hybrid FOS8500. These hybrids both possess a relative maturity rating of 105 days. The FOS8500 and 8507 represented the highest yielding hybrids available from the Foundation Organic Seed company. They were based on seed available from reputable commercial seed brokerage companies and have tested at the top of yield trials on multiple sites (personal communication, 2018, Steve Mohr, owner Foundation Direct Seed company.
Soil samples Our intent was to capture any different effects soil quality might have on the different hybrids and their N efficiency/N2 fixation, and the effects the hybrids might be having on soils. Towards that ends, samples were taken before planting in some of the varietal trials and manure/inoculation trials. After planting soil samples from the organic farms were gathered from the varietal trials and analyzed at the University of Illinois by as part of the OREI funded project there. Furthermore, with SARE funding in 2019 we sampled OREI and SARE sites in September before harvest under selected hybrids to examine soil quality and relationship to crop performance. Soil samples for these trials were taken to a depth of 8 inches under four hybrids in the OREI trials and under two hybrids in the SARE trials. Samples were sent to Woods End Soil Testing Lab in Maine for soil quality measurement and to Cornell University for determination of soil protein content.
Plant samples. To determine population density and grain yield, ears and population data were gathered on three 8.75 foot samples that were taken from each plot at regularly spaced sampling sites of approximately 30 feet apart. On the Doudlah farm, two samples of that length were taken on each of three sites sampled. Three stalks from each of the sample sites were pooled across plots, weighed, ground and subsequently tested for moisture content in order to determine the dry matter yield of stalks. A 12 x 12 x 8 inch deep soil/root monolith was extracted from a centered crown of one plant on each sample site. Root samples were soaked, spray washed and dried to determine dry matter content in roots. Grain yield was calculated assuming 56 pounds of grain per bushel, and results were standardized for grain moisture of 15.5%. Grain, stalk and root samples were shred, mixed and subsamples were ground with a Wiley mill. Samples were sent to the USDA-ARS North Central Soil Conservation Research Lab in Morris Minnesota (Abdullah Jaradat, Jane Johnson, Chris Wente) for analysis of mineral content using ICP and for N and C content using a Leco elemental analyzer. Nutrient values were analyzed both in terms of tissue concentration and total plant uptake. Samples were further ground, mixed, and packed by USDA into capsules and sent to the UC Davis isotope facility for analysis of 14N and 15N isotopes and for determination of the δ15N ratio.
Initial studies with isotopes in 2009 and 2010 showed that the grain resulting from inbreds and populations from the breeding program had higher δ15N values than conventional inbreds, but that the grain of N fixing populations had much lower δ15N values. We assume that rhizophagy causes higher δ15N by increasing N uptake from microbial biomass and from easily decomposable organic matter (Craine et al. 2015). We also assume that in some cases, where N fixation is fostered by endophytes, the grain δ15N is lower than for conventional inbreds because the N taken up from the soil is progressively diluted with N from the air, leading to greater 14N enrichment. Hence in the best N efficient hybrids the δ15N levels are being increased and decreased in the plant parts by these competing plant/microbial activities.
Based on these suppositions, nitrogen derived from air (NDFA) for the grain of the different hybrids was determined in two different ways; internally, contrasting δ15N (delta 15N) values for the parts of a given hybrid between the high values found in roots or stalks and the low values found in grain; or by subtracting the δ15N value for a given Mandaamin hybrid from the check, dividing the product by the δ15N value for the check, and multiplying that result times 100. Application of these formulas for determining nitrogen acquired from soil/microbial biomass/organic matter or from the air by the Mandaamin hybrids relative to the FOS hybrid are explained in the results section.
Data analysis Data was analyzed with Excel spread-sheets and JMP software. Response factor data was explored utilizing linear models, analysis of variance, means generation, and means separation. Where appropriate an analysis of variance included relevant covariates for partitioning out contributing variation. In several cases, population density was utilized when necessary as a covariate to compensate for differences in plant stands, but other correlative factors were used where appropriate to clarify sources of variation for δ15N scores. In such cases adjusted least square means were generated and various models were compared for their effects on explaining total variation and estimates of fixation. Analyses of variance were often partitioned for the % of the total SS (sums of squares) attributed to a given factor. Analyses were made of roots, stalks plus leaves (stover), and grain. Harvest index values were calculated as yield in grain/total yield of grain and stover) x 100. Indexes were calculated for grain yield in 2019 and for grain yield and uptake of minerals in 2020 varietal trials.
The strip plot design used for the variety trials was un-replicated within sites but the farms served as replicates. This basic design does not allow testing for farm differences in the farm x hybrid interaction as that latter interaction is used as an error term to test both farms and hybrids. However, the types of farms we were testing fell into three very different groups. These were organic cattle-based farms which utilized their cattle manure and forages in their rotations alongside production of corn and small grains (Anibas, Anon, Zinniker). The organic arable group consisted in farms that mainly utilized chicken manure to sustain mostly arable production (Adsit, Beiler, Doudlah), though forage crops were also grown occasionally at Beiler’s. The third group consisted of two conventionally managed fields (Weiss and Bauer) with monoculture corn that utilized inputs of starter fertilizer and herbicides and had a history of high inputs with cattle slurry from a dairy operation. In 2019 we tested data from two sites in each of these three types (arable organic=Beiler, Doudlah), (organic cattle = Anon and Zinniker), and conventional manured corn = two fields at Weiss/Bauer). On the Doudlah site chicken manure was not applied in 2019. On the Weiss/Bauer fields, one received only starter fertilizer; the other received starter and manure. Analyzing data by type of farm enabled us to test for significant effects due to type of farm, hybrid, and type x hybrid. Due to the levels of variation inherent in the on-farm research we did, we considered p values to be worthy of discussion if they were approximately 10% or less.
Due to the volume of data and limitations of the online system, all Tables and Graphics referenced below are in a pdf.
Here is a link to the tables, photos, and diagrams associated with this report: (Document Containing Tables, Photos, and Diagrams)
Over all yields.
Throughout the experiments the hybrid 17.461 characterized itself as probably the best adapted of the Mandaamin hybrids that possessed the same relative maturity (105 days) as the commercial hybrid check FOS8500. Table 3 summarizes overall yield data from 14 strip trials on different farms where 17.461 was compared with FOS8500. More information on these two hybrids is presented in sections below from specific trials. Here we are comparing yields across two years and many different kinds of farms, farming systems, and climatic factors. There were 4 sites where comparisons were made in the context of an arable organic cropping system that was manured, and 3 sites where it was not manured. There were five sites where comparisons were made in the context of a cattle farming operation with manure applied but two sites where manure was not applied. Due to this structure, and the need to make relevant comparisons in the context of different conditions on the different farm sites, the 17.461 yields were converted to a percentage of the FOS8500 yield on the same site. These percentages were then analyzed using an analysis of variance with system, manuring, hybrid and all the interactions of these factors. Doing this standardized the results so the focus was on the relative difference between the two hybrids irrespective of the general yield level.
The analysis of variance of the data converted into relative values shows no significant factors for analysis of the raw data. However, when the data was adjusted as % of the FOS8500 control there were significant effects for all factors except hybrid . In the latter analysis there were identical sum of squares for manuring and manuring x hybrid as well as for system x manuring and manuring x hybrid x system. Highlights of the yield trials were that:
- The 17.461 averaged 139 bu/acre and the FOS averaged 144 bu/acre.
- the cattle system yielded 38 bu/acre more than the arable systems.
- The manured fields yielded 22 bu/acre more than the not manured.
- The 17.461 yielded 28 bu/a more than the FOS8500 in the arable system without manure which was 37% more for the standardized comparisons.
- The 17.461 yielded 24 bu/a less than the FOS8500 in the arable system with manure which was 15% less for the standardized comparisons.
- The 17.461 yielded 5 bu/a less than the FOS8500 in the cattle system without manure which was 4% less for the standardized comparisons.
- The 17.461 yielded 16 bu/a less than the FOS8500 in the cattle system with manure which was 8% less for the standardized comparisons.
The yield data from strip trials are portrayed graphically in Diagram 1. Simple inspection suggests that system is generally a major factor with the cattle system out-yielding the arable system. Furthermore, manuring appears to be a major factor for the 17.461 with manured corn especially yielding lower in the arable system.
Yield trials, paired manured and manured strip trials.
A second set of experiments was carried out for 2019 to 2021 on five sites where different Mandaamin hybrids were planted across manured and not manured plots. The hybrids grown on the plots included FOS8500, five hybrids with a C4-6 parent, and one (2019) or two hybrids (2020 and 2021) with NG10 as one of the parents. Because identity of the hybrids varied from year to year we averaged the yields of the C4-6 group of hybrids on each site and the NG10 group of hybrids on each site and compared them with the FOS8500 in an analysis of variance.
The analysis of variance is shown in Table 6. The only significant effect was of system.
Irrespective of lack of statistical significance, the averages showed the same tendency as the experiment previously described. The arable system (Beiler Farm) yielded 94 bushels/acre while the cattle systems yielded 122 bu/acre. The manured plots yielded 114 bu/acre while the not manured yielded 107. Fos 8500 yielded 125 bu/axre while the C46 hybrids yielded 109 and the NG10 hybrids yielded 97 bu/acre. Diagram 2 shows the interaction between manuring and hybrids. Again manure appeared to reduce yields of the C4-6 hybrids while increasing yields of the FOS8500 and the NG10 hybrids.
Why does manure have a negative effect on hybrids that have C4-6 (461) as a parent?
An open enigma is why the negative effect on C4-6 hybrids, and why is it seemingly worse in its effect on lower yielding sites? We analyzed the mineral contents of the Beiler unreplicated strip trial which was on a lower organic matter upland soil. Table 7 shows that manuring decreased grain yields 20%, nitrogen uptake 32%, uptake of macronutrients 20-22%, and especially total uptake of micronutrients 46%) across the 461 hybrids. Hybrids that respond positively to manuring had 38% more yield, 3% more N uptake, 8-16% more uptake of macronutrients, but 19% less micronutrient uptake. They had a positive response to manure or they had a lesser negative response. Most hybrids responded to manuring with some degree of decrease in uptake of micronutrients but it was much larger for the 461 hybrids. We assume this extreme reaction of the hybrids with C4-6 as a parent has to do with disruption of rhizophagy by the manure.
On the Weiss farm, which had a high fertility soils with a very high soil protein content, also sponsored hybrid trials with and without manure in 2019. Results shown in Table 8 are that 17.461 did respond negatively to manuring with a 19% reduction in grain yield and a 6% reduction in N uptake. However the effect of manure was positive for uptake of other nutrients. The differences between the response on the two sites could relate to the necessity for hybrids like 17.461 to exercise rhizophagy on soils with lower nutrient availability, and that they are prevented from doing that when manure is applied. This is not as large an issue where nutrients are more available such as they were on the Weiss farm in 2019. See section below on varietal trials in 2019.
Results Varietal Trials 2019.
Soil test results. Table 9 shows soil test results from samples taken in early June from recently planted corn plots on all the farms except for the Weiss/Bauer fields, which was not analyzed at that time. Nutrient levels varied with high levels of calcium for the Adsit, Beiler, Anon 1 and 2 farms. Inspection of the two columns on the right hand side of the table reveals large difference between the two management approaches. On average, the farms with arable cash cropping based on inputs of chicken manure had greater levels of phosphorus and potassium. There was little difference in amounts of available N in June for the corn plants. However, the cattle-based operations had greater stocks of organic matter, carbon, and nitrogen and a higher C/N ratio. This included more particulate organic matter and greater stocks of potentially mineralizable nitrogen.
To test significance of difference the three arable organic farms were compared with values for the three organic farms with cattle (Table 10). There was no difference in the amount of total inorganic N available in the spring. The arable organic had 157% more Bray P, 63% more K, 12% more Ca,and 25% more Mg than did the organic cattle sites. However, they also had only 73% as much Total N, 64% as much total C, 89% as high a level of C/N and 54% as much particulate organic matter C.
We analyzed crop performance parameters with system, hybrid, hybrid x system, and total organic N and C/N as covariates. The percentages of the total variance for performance that were accounted for by soil organic N plus + C/N were 36% for grain yield, 57% for stalks/acre, 51% for roots/acre, 45% for N uptake/acre, 66% for C uptake/acre, 45% for tissue %N, 87% for δ15N in grain and 27% for δ15N in roots.
Relationship between spring tests before planting and soil quality tests before harvest, varietal trials, 2019:
The relationship between the three farming systems and the soil test values are shown in Table 11. Obviously the use of cattle slurry on the conventional farm had boosted the soil protein levels beyond those in the other systems. The lower aggregate stability and higher bulk density on this farm was probably related to a greater content of sand in the soil composition. The percentage relationship between the arable and cattle based organic systems for the different fall measured parameters paralleled the results for organic matter related parameters in in the spring samples.
Table 11. Relationship between farming systems and September soil test values in 2019.
Relationships between the most pertinent parameters for the spring and fall sampling are shown in Table 12. These linear model analyses used total organic nitrogen and carbon (TON and TOC), C/N ratio, particulate organic matter carbon (POM)-C., and potentially mineralizable N (PMN) as determinant factors and protein, protein score, labile amino N (SLAN), nitrate, aggregate stability, bulk density, and CO2 respiration as respondent factors. Using the former factors as x factors explained the majority of the variation in the nitrogen related soil quality tests. In fact, the vast majority of the variation for the nitrogen related parameters could be explained by total carbon and nitrogen and the relationship between them (99.6% for soil protein, 99.7% for protein score; 96% for SLAN; 75% for nitrate, and even 85% for CO2 respiration). Soil C, N, and C/N attributed less to the variation in aggregate stability (4.1%) and bulk density (12.1%) but there, the young fractions of particulate organic matter C and potentially mineralizable N were important.
Table 12. Relationships between spring and fall soil tests in 2019.
Following analysis of the data that had been condensed into types it became clear that there were significant differences between the arable organic, cattle based organic and conventional manured monoculture for every parameter measured. There were significant effects of farming systems on soil protein, protein core, CO2 respiration, SLAN, Aggregate stability, and bulk density (<0.0001) (Table 13). Regarding effects of systems and hybrids on the soil, contrasts showed that soil under 17.461 and 17.2B24 had higher aggregate stability but lower SLAN and nitrate production than FOS8500.
Table 13. Effects of hybrids on soil characteristics in 2019 varietal trials.
Hybrid performance: The average yields of the different varieties are shown in Tables 14 and 15.
Table 14. Overall yields from varietal trials in 2019
Initial observations were 1) the commercial check gave the highest overall yield; 2) that the cattle-based systems had higher grain yields; 3) manure application at the Weiss farm did not appear to affect overall yields; 4) some varieties, especially the check, gave lower yields where they were not manured.
Table 15. Yield results for manured and not manured trials and for arable cash and cattle based trials in 2019.
On average, the best producer on the cash system farms or under unmanured conditions was 17.2B24. . In the cases where manuring was omitted (Doudlah and Weiss notM) the yields of FOS8500 dropped strongly relative to the Mandaamin hybrids and in the case of Weiss, relative to the side by side manured plots.
Detailed research, varietal trials, 2019: As was referred to above, in order to elucidate relationships between farms, soils, and nitrogen efficiency/fixation, we focused research on five farms (Beiler, Doudlah, Anon, Weiss/Bauer, and Zinniker). Weiss/Bauer had two sites one with starter fertilizer, another with starter fertilizer and manure. That site had a history of manure slurry applications from the Weiss dairy operation. Farms were grouped according to farm type as described above. Anon had two replicated sites with different planting dates which were averaged. We studied plant performance, nutrient and N relationships and soil on a subset of four hybrids. The four hybrids were described above as the FOS8500 control, 17.461, 17.2B24, and C2B2-1.C46. Data was analyzed using type of farm, hybrid, and type x hybrid as sources of variation in an analysis of variance.
Before describing detailed statistics, the general patterns in relationships between the different parameters in magnitude are shown in Table 16. Here, results for the different hybrids are presented as a percentage of the FOS8500 checks in each system. Values in red indicate values that are 5% or more below the check value. Values for accumulated elements were calculated as an average % difference for the ratio of Mandaamin hybrid/FOS8500 across measurements of Al, Cu, Fe, Mn, Si, Sr, Ti, Zn for the microelements and of C, N, Ca, Mg, K, P, and S for the macro-elements.
Averaged across the systems, Mandaamin hybrids had lower (10-11% grain yields), greater N accumulation (3-9%), greater accumulation of macroelements (14 to 20%); greater accumulation of microelements (5-19%), greater accumulation of dry matter and N in stalks, and in part in roots. The hybrid C2B2-1.C46 differed from the other Mandaamin hybrids by having lower stalk production and macroelement uptake.
Table 16. Grain yield and mineral uptake as affected by hybrid and farming systems in 2019.
For each parameter analysis of variance revealed significant differences due to plant part, but not to hybrid, pedigree, farm x hybrid, or part x hybrid[1]. Yields of carbon, nitrogen, and dry matter are shown in Table 17 including information on significance of differences. The arable farming systems had the lowest total dry matter yield and lowest yield of grain, stalks, and roots. The highest grain yields and the highest partition of N into grain were achieved in the organic cattle based systems, but the monoculture corn system achieved the highest stalk and N yields.
The FOS8500 had significantly higher harvest index which paralleled higher dry matter accumulation in grain and percent of total plant N in grain than did the two hybrids with 17 as a parent (17.2B24 and 17.461). However the latter two hybrids had greater total N uptake, dry matter in stalks, and % N in grain. 17.2B24 had the most C and N uptake. However, the two hybrids with C2B2 as a parent also had more total C, N, and dry matter in their roots than did 17.461 and FOS8500.
Table 17. Grain, stalk (stover) and root yields, harvest index (HI), total N, and the percentage of total N in grain for hybrid variety trials in 2019.
An analysis was made to model to which extent uptake of minerals was influenced by covariates consisting of the δ15N values for grain, stalks, and roots. Only δ15N for grain showed significant effects and those relationships were all positive relationships (Table 18 and Diagram 3).
Table 18 shows results of analysis of variance examining effects of covariates on nutrient uptake per acre.
Diagram 3. Relationship between δN15 and uptake of nutrients. Positive relationship between δ15N (x axis) and mineral uptake (y axis) for the same trial on six different farms composed of 3 farming systems, 3 Mandaamin hybrids and FOS8500, with anova factors system, hybrids, systems x hybrid, and δ15N as a covariate. Positive relationships were significant at p<1% for S, Si, Ti; p<5% for Al, Cu, Mn, N, Sr; p< 10% for C, Mg; p<15% for P, K; p <20% for yield.
Soil test values generally correlated poorly with both %N and δ15N in tissues. On the other hand, both of these parameters seemed to correlate well with plant production and nutrient uptake parameters. The %N seemed to have a central place as a high level of correlation was found between %N and total C (R2=62%), total N (R2=78%), total dry matter (R2=22%), and P (R2-62%), S (R2=69%), Cu (R2=12%), and Zn (R2=34%) uptake by plant parts. The δ15N had similar significant correlation values with most of these parameters, but the R2 values were lower than for %N. This indicates that the two kinds of measurements are linked. Approximately 60 to 69% of the N that was accumulated in the plant was found in the grain. But correlations between the δ15N in the grain and the same performance and uptake factors showed little relationship.
[1] Throughout this report there is a significant difference between means at p=5% if the means within a parameter do not share the same letter after the mean values.
An analysis was made to model to which extent uptake of minerals was influenced by covariates consisting of the δ15N values for grain, stalks, and roots. Only δ15N for grain showed significant effects and those relationships were all positive relationships (Table 18 and Diagram 3).
Table 18 shows results of analysis of variance examining effects of covariates on nutrient uptake per acre.
Diagram 3. Relationship between δN15 and uptake of nutrients. Positive relationship between δ15N (x axis) and mineral uptake (y axis) for the same trial on six different farms composed of 3 farming systems, 3 Mandaamin hybrids and FOS8500, with anova factors system, hybrids, systems x hybrid, and δ15N as a covariate. Positive relationships were significant at p<1% for S, Si, Ti; p<5% for Al, Cu, Mn, N, Sr; p< 10% for C, Mg; p<15% for P, K; p <20% for yield
Soil test values generally correlated poorly with both %N and δ15N in tissues. On the other hand, both of these parameters seemed to correlate well with plant production and nutrient uptake parameters. The %N seemed to have a central place as a high level of correlation was found between %N and total C (R2=62%), total N (R2=78%), total dry matter (R2=22%), and P (R2-62%), S (R2=69%), Cu (R2=12%), and Zn (R2=34%) uptake by plant parts. The δ15N had similar significant correlation values with most of these parameters, but the R2 values were lower than for %N. This indicates that the two kinds of measurements are linked. Approximately 60 to 69% of the N that was accumulated in the plant was found in the grain. But correlations between the δ15N in the grain and the same performance and uptake factors showed little relationship.
Soil tests as explanatory factors for crop performance: Regression studies tested a model that combined soil protein, protein core, CO2 respiration, SLAN, aggregate stability, and bulk density for its relationship to crop performance parameters. Soil protein content, protein score, aggregate stability and bulk density proved significantly correlated with many crop performance parameters. The combination of these factors accounted for the vast majority of total sum of squares variation. There was little significant little or no significant statistical relationship between the other soil tests and crop performance parameters. Stalk yield related to bulk density and nitrate. The model tested accounted for less of the variation for %N and δ15N. There was no statistical significance for any of the individual tests for %N; and only aggregate stability (p=0.01) and bulk density (p=0.07) showed some level of significance in relationship to δ15N.
Table 19. Relationship between soil tests and crop performance for variety trials in 2019.
Nitrogen efficiency and fixation in varietal trials, 2019:
Two isotopes are found in nature: 15N and 14N. Little 15N is found, but for what there is it tends to accumulate more in soil organic matter than in air. The relative amount of 15N is expressed by the δ15N value which also indicates the ratio between the two natural isotopes. As the values increase, there is more 15N. Air is richer in 14N than soil. Nitrogen fixation from the air involves the accumulation of N2 from the atmosphere which dilutes the relative amount of 15N and depresses the δ15N.
Diagram 4 shows that the Mandaamin hybrids had higher values of δ15N in their roots and lower values in their grain than the commercial check. Increase in 15N in roots is probably associated with uptake of 15N rich easily extractable soil organic matter or microbial biomass. These results fit with our hypothesis that the Mandaamin hybrids increase uptake of N from microbial sources or their decomposition products and also fix N2. To fit the data with appropriate calculations we made the following assumptions:
- The 15N content in the roots represented the isotope composition of N derived from the soil coupled with microbial biomass.
- The difference in root δ15N between the FOS8500 and the Mandaamin hybrids was due to the roots of the Mandaamin hybrids appropriating more N from microbial biomass or easily extratable organic matter.
- The difference between the highest δ15N achieved in the root or the stalk and the δ15N found in the grain indicated the dilution effect due to N2 fixation and the amount of N derived from the air (NDFA) in the grain.
Diagram 4. The δ15N values for hybrids and their plant parts.
Diagram 5 shows the interaction between farming systems and δ15N levels for different organs of the four hybrids. There was little difference between the roots, stalks and grain δ15N levels for 17.461 on the arable organic sites, but substantial difference between it and the FOS8500 control. In that case the difference in the grain δ15N levels between that hybrid and the control were used to calculate NDFA.
Table 20. Data for %N and δ15N levels of hybrids grown in 2019.
Diagram 5. The δ15N values for hybrids and their plant parts grown in different farming systems.
Table 21. Estimates of nitrogen derived from air (NDFA) and microbial biomass in 2019.
The relationship between N uptake by the crop and the δ15N ratios in grain also seemed to differ according to farming system (Diagram 6). The arable organic system had the highest δ15N values which is congruent with the fact that the arable soils had the lowest C/N ratios. Soils with low C/N ratios have have microbial biomass with high δ15N values (Craine et al., 2015). According to our calculations, the arable organic system had the lowest nitrogen derived from air values (17%), but medium estimates for N from microbial biomass (20%). This is assumed to have resulted in predominance of rhizophagy contributing loading N in the grain and a positive and significant linear relationship between δ15N and %N in grain (R2=0.836, p = 0.0001 for intercept and 0.0015 for the δ15N effect). On the other hand, corn grown in the conventional monoculture system averaged the highest N derived from air values (32%) but the lowest N derived from microbial/plant rhizophagy (14%). It showed a negative relationship between δ15N and %N in grain (R2=0.3334, p = 0.0001 for intercept and 0.1339 for the δ15N effect), suggesting indeed that N2 fixation was contributing more to grain loading with N. On the other hand, the corn grown in the cattle based organic system had the second highest estimates for N derived from the air (26%) and the highest values for N derived from rhizophagy (31%). It showed no clear relationship between δ15N and %N in grain (R2=0.0545, p = 0.0001 for intercept and 0.578 for the δ15N effect). This implies that rhizophagy and N2 fixation were both contributing to grain loading with N.
Similarly, a positive linear relationship was found between δ15N and total N uptake for the arable organic system (R2 = 0.573, p = 0.067 for intercept and 0.0296 for the δ15N effect. However there was no significant correlation for the conventional (R2 = 0.158) or the organic cattle system (R2 = 0.044).
Diagram 6. Relationship between %N and δ15N in grain for different farming systems.
The hybrid 17.461 showed a strong negative linear relationship between δ15N and %N (R2=0.60, p = 0.0019 for intercept and 0.07 for the δ15N effect) and a similar relationship between δ15N and lbs of N/acre (R2=0.534, p = 0.102 for intercept and 0.099 for the δ15N effect). None of the other hybrids showed such relationships (R2 values lower than 0.16).
Concentration of nutrients in varietal trials of 2019. Mineral analyses on the 2019 varietal trial revealed statistically significant effects of systems and plant parts on most of the minerals tested. The factors tested in the analysis that were associated with pedigree (pedigree, system x plant part, system x pedigree, system x plant part x pedigree) accounted for significant portions of the total variation for macronutrients P, S, and for micronutrients Al, Cu, Fe, Mn, Ti, and Zn. For macronutrients, 5 to 28% of the total sum of squares accounted for by the model was associated with pedigree. Pedigree affected variation more for micronutrients where 2 to 83% of the total variation was associated with pedigree.
Table 22. Analysis of variance for different macronutrients in plant tissues in 2019 hybrid trials.
The mean values for the plant part x pedigree interaction for macronutrients and microminerals are shown in Table 23. Also shown is the value averaged across all macronutrients or microminerals for the Mandaamin hybrids relative to the FOS8500 check.
On average the 17.461 had 14% and 13% higher macronutrient content in its grain and stalks, respectively, but 6% less in its roots than did the FOS8500. It had also 30% more and 6% less micronutrients in its grain and stalks, respectively, but 37% higher micronutrient content in its roots than did the FOS8500.
The C2B2.C46 had 12% higher and 3% lower macronutrient content in its grain and stalks relative to FOS8500. But it had equal amount of macronutrients in its roots as did the FOS8500. It had also 6% higher and 7% less micronutrients in its grain and stalks, respectively, but 67% higher micronutrient content in its roots than did the FOS8500.
The 17.2B24 had 6% and 12% higher macronutrient content in its grain and stalks relative to FOS8500. But it had 9% lower content of macronutrients in its roots as did the FOS8500. It had also 6% higher and 4% more micronutrients in its grain and stalks, respectively, but only a 1% difference in micronutrient content in its roots than did the FOS8500.
To summarize, the two hybrids with C4-6 in their parentage showed enhanced ability to accumulate macronutrients and micronutrients in their grain. This was especially the case for 17.461 which had 14% more macronutrients and 30% higher micronutrient content than the FOS8500. They also showed enhanced ability to accumulate micronutrients in their roots relative to the FOS8500 (37% and 67% more for the 17.461 and C2B2.C46, respectively).
Table 23. Concentration of minerals in hybrid tissue parts, hybrid trials 2019.
For macronutrient uptake, the differences between the average for the Mandaamin hybrids and the FOS8500 in nutrient concentration were greatest (8%) in the conventional system, intermediate (6%) in the organic cattle system, and least (2%) in the organic arable system. For micronutrient uptake, the differences between the Mandaamin hybrids and the FOS8500 were least (-5%) in the conventional system, and about the same (17% and 18%, respectively) in the organic cattle and the organic arable system. Hence enhanced micronutrient concentration for the Mandaamin hybrids was associated with the organic sites while enhanced macronutrient concentration was associated with the conventional sites.
Table 24. Concentration of mineral macronutrients in hybrids grown in variety trials in 2019.
Total nutrient uptake per acre, 2019 Varietal trials. The nutrient uptake by plants (total of root, stalk and grain) in the experiment is shown in Tables 25 and 26. Statistical analysis revealed few significant differences between hybrids for individual nutrients. But in the aggregate, the Mandaamin hybrids averaged the same C production per acre, but 13% more macronutrient uptake than the FOS8500. This was 7% more N, 44% more Ca, 6% more K, 15% more Mg, 2% more P, and 6% more S. The 17.461, 17.2B24, and C2B2.C46 averaged 19%, 16% and 5 % greater uptake of macronutrients per acre, respectively than did the FOS8500. Relative to the check the highest average uptake by the Mandaamin hybrids (21%) was in the conventional system, while the organic arable and organic cattle systems averaged 8% and 11% more than the check, respectively.
Table 25. Total uptake of macro nutrients in hybrid corn grown in different farming systems in 2019.
Table 26. Total uptake of nutrients relative to the commercial check FOS8500 when grown in different farming systems in 2019.
The Mandaamin hybrids averaged 16% more uptake of microelements. On average they had 31% more Al, 4% less Cu, 19% more Fe, 5% more Mn, 19% more Si, 37% more Sr, 32% more Ti, and 8% less Zn than the FOS8500. The Mandaamin hybrids grown in the organic livestock system averaged 27% more uptake than the FOS8500 but the other two systems both averaged 11% more uptake.
Table 27. Total uptake of micromineras in hybrid corn grown in different farming systems in 2019.
Table 28. Total uptake of microelements relative to commercial check when grown in different systems in 2019.
Analysis of variance showed significant differences between hybrids (p <0.0001) and minerals (p<0.01).
Table 29. Analysis of variance for total mineral uptake in the varietal trials of 2019 that evaluated the pedigree x mineral interaction.
The values presented in tab les x and y were based on ratios calculated from LS means. The data was reanalyzed by taking ratios between raw data then analyzing it. Based on least square means derived from that analysis of variance, on average the three hybrids 17.461, 17.2B24, and C2B2.C46 had 24%, 20%, and 12% greater mineral uptake relative to FOS8500. According to t tests the 17.461 did not differ significantly only from 17.2B24, which did not differ significantly from C2B2.C46. All of the Mandaamin hybrids differed significantly from FOS8500. These estimates are slightly higher than previous estimates probably due to not using LS means for calculating the initial ratios with the FOS8500 standard values.
A summary of the differences between hybrids and elements is shown in Table 30. the highest % increase was associated with the macroelements Ca and Mg and with microelements Al, Fe, Sr, Ti, and Si. The 17.461 and 17.2B24 hybrids had only 6 and 8% more C production than the FOS8500. However, these two hybrids respectively took up 62% and 63% more Ca, 36% and 25% more Al, 25 and 28% more Si, 45% and 55% more Sr, and 6% and 35% more Ti, than did the FOS8500. The high uptake of these minerals by the Mandaamin hybrids suggests enhanced mobilization of minerals from the solid portions of the soil including alumino-silicate clay particles, from parent mineral particles, and from limestone rather than from passive reliance on the soil solution.
Table 30. LS mean values for the relative uptake of nutrients by hybrids based on FOS8500 as a standard for the varietal trials in 2019.
Varietal trials in 2020. These trials tested nine different experimental hybrids ranging from 92 day to 108 day relative maturity. For the eight sites tested, we managed to obtain valid and complete data for five sites. Data analysis used an analysis of variance with hybrid as the main factor and plant population density and δ15N as covariates. Utilization of covariates appeared necessary primarily because of deviations in plant population results and secondarily as a research tool to clarify the effects of δ15N on variance. Results were that all three factors significantly affected grain production but only hybrid and δ15N significantly affected protein, oil, starch, ethanol yield, lysine methionine, and cysteine. Population density did not affect grain quality. The δ15N covariate had positive effects on grain yield, harvest index, grain protein, density, lysine, methionine, and cysteine, but negative effects on stover yield, grain oil, starch, and ethanol yield. Hence δ15N was positively associated with protein accumulation and quality but negatively associated with non protein components. The portion of variance associated with δ15N was significantly approximately half or more of the total sum of squares (SS) that were accounted by the model for harvest index, grain protein and amino acids, starch, ethanol yield, and density.
Yields and quality results are shown in Table 31 and 32. Grain yields and harvest index values were lower in 2020 than they had been in 2019 FOS8500, 17.2B24, and 17.461 averaged 177, 156, and 158 bu/acre in 2019 but they averaged 130, 96, and 128 bu/acre, respectively, in 2020. Harvest index for FOS8500, 17.2B24, and 17.461 averaged 64%, 58%, 57%, and 64% in 2019. But in 2020 they averaged 47%, 40%, and 37%, respectively. This implies that growing conditions in 2020 were stressful and this is corroborated by visual evidence of greater weed problems on the Goldstein (severe thistle infestation) and Beiler sites in 2020. For hybrids grown in 2020, yields were generally lower for the earlier hybrids with lower relative maturity (RM) ratings (see Table 31). An exception to this was the earliest hybrid (K5N.NG10) which yielded 109 bu/acre. As in 2019, the 17.461 had the highest stover yield which was 40% higher than for the check. Though hybrid effects on harvest index were not statistically significant, the FOS8500 again had a higher value (15% more, p=0.153) than the other hybrids. The FOS8500 also had statistically significantly higher starch and ethanol yield than the Mandaamin hybrids but the magnitude of the difference was only +3%. The Mandaamin hybrids differed in their yields and quality characteristics between themselves and there were significant differences between them and the FOS8500. On average the FOS8500 had 17% less protein, 8% less oil, 15% less lysine, 23% less methionine, and 14% less cysteine than the average value for the Mandaamin hybrids.
Table 31. Yield and grain quality of nine hybrids grown on five sites in the varietal trials of 2020.
Table 32. Yield and grain quality of nine hybrids grown on five sites relative to commercial check.
Table 33. Analysis of variance for characteristics of hybrids grown in varietal trials in 2020.
Variances associated with agronomic characteristics including covariant information on plant population density, δ15N, %N content of grain, N uptake by grain, and plant population are shown in Table 32. There was no significant effect of hybrid pedigree if the anova model included only pedigree as a factor for testing δ15N, %N content of grain, and pounds of N taken up by grain. If %N and %C in grain were taken as covariates in addition to pedigree the analysis of variance produced F values with p = 0.0001 for %N, p=0.0002 for %C, and p=0.0957 for pedigree. When δ15N itself was analyzed with that same model, 80.2% of the total sum of squares was associated with %N. Conversely, Table 34 shows results obtained by analyzing %N in grain by pedigree, δ15N, and plant population density. δ15N accounted for large amounts of the total sum of squares. This again shows the tight relationship between δ15N and %N or % protein in grain and the quixotic nature of these two parameters which describe source and magnitude of supply of nitrogen to the grain. They correlate with many parameters but are plastic factors that synthesize many processes and they have to be mutually considered when analyzing hybrid effects.
The relationship between δ15N and the %N in the grain was tested for the individual hybrids using regression analysis despite the low sample sizes. 17.461 stood out in the trials for having similar yields to FOS8500 but the highest levels of δ15N, %N in grain, and N uptake in grain (see least square means in Table 34). Regression coefficients showed a much higher intercept value and a lower slope coefficient than for the other hybrids. Though the slope coefficient was not significant nevertheless, the graphic representation of this data in a scatter-gram (Diagram 7) suggests that 17.461 has a higher and more stable content of N in grain that was maintained at higher δ15N values.
Table 34. Agronomic characteristics and linear equations for hybrid trials in 2020.
Diagram 7. Relationships between δ15N and %N for hybrids.
Stover production and composition in the 2020 variety trials:
Stover production and composition is shown in Table 35 and 36. The analysis of variance showed that though pedigree did not significantly affect δ15N it affected stover yield, stover N, and %N. The δ15N values significantly affected stover yield, stover N and %N as well. Though it accounted for little of the total variation in stover yield and stover N, it accounted for about half of the variation found in %N.
Table 35. Analysis of variance for stover parameters of the 2020 varietal trials, showing p values for significance and the % of the total SS associated with the different sources of variation.
The hybrids that produced the most stover were 17.461 and K5N.NG10. These two hybrids appeared to pursue a very different investment strategy than did the FOS8500 in terms of growth allocation. They had very high δ15N contents in their stalks, produced a lot of stover with relatively large amounts of N (see Table 36) as well as Ca in the stover. They possessed average %N in the stover but produced grain with high protein contents. The plants seemed to be investing in rhizophagy, vegetative growth, and loading N, Ca, and other minerals into grain. The 17.461 had 53% higher δ15N, 36% higher stover yield, 37% higher stover N, but the same %N in its stover as did the FOS8500. The K5N.NG10 had 72% higher δ15N, 17% higher stover yield, 58% higher stover N, and 45% higher %N in its stover as did the FOS8500 Similar data with respect to stover production were obtained in 2019 with the Mandaamin hybrids, especially with 17.461.
Table 36. Effects of hybrids on stover yield and N content; varietal trials 2020.
Nutrient composition of grain in 2020 variety trials: The nutrient concentrations of grain and stalks were also analyzed using the factor hybrid pedigree and the covariates δ15N and plant population density. Values showed substantial variation was accounted for by all three factors. Pedigree accounted for an average of 54% of the total sum of squares while δ15N accounted for an average of 34% of the total sum of squares.
Table 37. Results of an analysis of variance of grain composition analyzing pedigree, plants/acre, δ15N, and plant density for varietal trials in 2020.
The concentration of nutrients in grain for different hybrids are shown in Table 38 . Their value in percent, relative to FOS8500 are shown in Table 39. The Mandaamin hybrids averaged 27% more minerals in their grain. This consisted mainly in 63% more Ca, 20% more Cu, 22% more Mn, 10% more P, 14% more S, 24% more Zn, and 9% more N. However, the Mandaamin hybrids had 25% less Fe and 5% less K than the check. As had been the case in 2019, 17.461 led the other hybrids in terms of concentration of nutrients in its grain. On average it averaged 61% higher nutrient values in its grain than the check. This included 270% more Ca, 99% more Cu, 10% more Mg, 41% more Mn, 9% more P, 19% more S, and 36% more Zn. High relative levels were distributed amongst different kinds of minerals. As yields of 17.461 were very similar to FOS8500 these very large differences are clearly not due to yield reduction and some kind of nutrient concentration effect.
Table 39. Relationships between Mandaamin hybrids and a hybrid check for mineral composition of grain; varietal trials 2020.
These values in the table above represent ratios of least square means generated by the anova model with covariates. The ratios based on raw values relative to FOS8500 were re-analyzed for significance using an analysis of variance with hybrids, minerals and hybrids x minerals as factors and plant population and δ15N values as covariates. Additional ICP data were included for P and S at two different wavelengths. Contrasts were used to compare the different kinds of hybrids. The analysis of variance in Table 40 shows that approximately half of the sum of squares (SS) variance was attributed to the pedigree x mineral interaction, and most of the rest to pedigree and mineral.
Table 40. Analysis of variance of relative values for mineral composition of grain with FOS8500 as the standard for varietal trials in 2020.
The table 41 showing the interaction between nutrient and pedigree shows the same major kinds of differences as were shown in Table 39 , with minor differences.
Table 41 shows relative values for nutrient composition of grain based on an analysis of variance with pedigree, mineral, pedigree x mineral as main factors and δ15N, and plant population density as covariates.
The hybrids with 461 as a parent averaged 122% while the FOS8500 averaged 99%. The contrast was significantly different at p=0.0043. The hybrids with NG10 as a parent averaged 114%. The contrast with FOS8500 was p=0.0636. The 17.461 averaged 158% while the 172B24 averaged 102 and the difference was significant at p = 0.0001. Hybrids with 461 as a parent did not differ significantly from hybrids with NG10 as a parent (p = 0.195).
Nutrient composition of stalks in 2020 variety trials: The analysis of variance for nutrient composition of stover is shown in Table 42 based on Pedigree as main factor and δ15N and plants/acre as covariates. The mean values and relative values are shown in Tables 43 and 44. On average 52% of the total SS associated with variation for the various components was associated with pedigree and 26% was associated with δ15N. However, statistically significant effects of pedigree were found only for Mg while significant effects of δ15N and plants/acre were found for most of the parameters tested.
Table 42. Analysis of variance table for stover composition results from 2020 variety trials.
Table 43. LS mean values for stover composition results from 2020 variety trials.
Table 44. LS mean values for stover composition results from 2020 variety trials relative to the check.
The results from stover mineral analysis suggested that there was much less difference between the Mandaamin hybrids and the check for stover than had been present for grain. On average the Mandaamin hybrids had only 3% more minerals than did the check. The major differences were again for that the Mandaamin hybrids There were 14-18% increases for Ca, Mg, P, and S but 13 and 20% less Cu and Fe.
A fuller analysis of variance took into account the pedigree x mineral interaction, more minerals, and the usual covariates (see Table 45). The analysis showed significant effects for pedigree, minerals, δ15N, and plants/acre. Most variation was found for mineral and mineral x pedigree.
Table 45. Analysis of variance considering minerals as a factor. Hybrid trials 2020.
The effects of pedigree as shown in Table 46 indicates that UR65.461, another 461 hybrid with 105 day RM, had the highest concentrations of minerals in its stover. However, this did not seem to be conveyed to its grain (see Table 38). On the other hand, the two Mandaamin hybrids with the highest stover and grain yields (17.461 and K5N.NG10) had also high relative concentrations of minerals in their stover and grain. There was a positive correlation between relative concentration of minerals in grain and the stover yield (R2 = 0.48; p = 0.056) as well as for the grain yield (R2 = 0.56; p = 0.032).
Table 46 shows least square values for the concentration of minerals in stover and grain relative to FOS8500 and the actual stover and grain yields.
The full interaction between hybrid pedigree and mineral type is shown in Table 47. The pattern shown is similar to that shown in Table 46. The differences between Mandaamin hybrids and FOS8500 ranged from 1 to 16%. On average the Mandaamin hybrids had 6% more minerals than the FOS8500 in their stover whereas the grain of the same hybrids averaged 17% more minerals than the FOS8500 in their grain.
Table 47. Total mineral uptake by stover for different hybrids as a % of the FOS8500 check. 2020 variety trials.
Total uptake of minerals in 2020 varietal trials.
An analysis of variance took into account all the minerals in the analysis. The models utilized hybrid pedigree, mineral, pedigree x mineral, with δ15N in grain, δ15N in stover, and plants/acre as covariates. The analysis of variance for total uptake of nutrient (Table 48) showed significant effects of pedigree, mineral, and of all the covariates (plants/acre, and δ15N in stover and grain. Pedigree accounted for 49% of the total SS.
Table 48. Analysis of variance for total mineral uptake in lbs/acre. Varietal trials, 2020.
The effect of hybrids is shown in the form of least square means in Table 49. The later maturing hybrids tended to have higher nutrient uptake values. 17.461 had 132% which was 29% higher than for the check FOS8500 which had 103%. LS means for the hybrid pedigree x mineral interaction are shown in Table 50.
Table 49. Relative uptake of minerals for different hybrids using mineral as a factor In analysis.
Table 50. LS mean values for the hybrid x mineral interaction for uptake of minerals in grain and stover and grain + stover for varieties grown in 2020.
The data for the interaction between hybrids and minerals is shown in Table 49 relative to the FOS8500 check. On average the Mandaamin hybrids showed especially higher uptake of Calcium.
Table 51. Total uptake of minerals by hybrids in 2020 as % of FOS8500.
Harvest index for minerals; varietal trials 2020.
The higher yielding Mandaamin hybrids appeared to have allocated more nutrients into grain than the check. To test whether this had to do with partitioning of the mineral from stover to grain we calculated the harvest index values for the minerals taken up by the corn. We analyzed harvest index for each mineral. In the anova the main factor was hybrid pedigree and the covariates were plants/acre and the δ15N in stover and grain. Table 52 shows that though pedigree accounted for 26% (range 16 to 75%) of the total variation, it was not a statistically significant effect. Plants/acre accounted for an average of 6% of the variation and the effect was not significant for any minerals. However the δ15N in stover and grain accounted on average for 58% of the total SS variation and in very many cases, especially for the δ15N in grain, the effects were statistically significant. Thus on the individual mineral basis, δ15N was playing the predominant role in determining harvest index. The effect of δ15N in grain on overall harvest index was positive for most minerals. The effect of δ15N in stover on overall harvest index was negative for most minerals. Though effects of both kinds of δ15N were not statistically significant for calcium and effects of δ15N in stover were not statistically significant for Cu and Fe, these minerals still had the same pattern of positive responses of HI to δ15N in grain and negative responses to δ15N in stover. This may be due to a source-sink relationships where stover supplies grain with δ15N and minerals.
Table 52. Probabilities and percent allocation of sums of squares for analysis of variance of harvest indices with pedigree as a main factor and covariates.
Results Manure/Inoculation trials, 2018: In 2018 two hybrids were compared on three farms. These farms are coded as Beiler, Clark, and Anon, and they are located near the towns of Rewey, Spring Green, and Fayette, WI. Towards the end of the season the experimental hybrid C461.C2B2 showed it was susceptible to an infestation of the fungal disease Grey Leaf Spot (caused by Cercospora zeae-maydis) which was present in pandemic proportions on all sites. This disease decimated foliage on all sites during grain fill and undoubtedly contributed to premature dry down and lower yields.
Analysis of variance for grain yield showed significant treatment and interaction effects only for farm, hybrids, and farm x hybrid interactions (all at p < 0.0001). The population density covariate was significant at p < 0.0096. There was no significant effect of manure or of inoculation on the sites.
Yield results for the significant farm x hybrid interaction are shown in Table 53. On average, the FOS8507 yielded 123.2 bu/acre. On average, the C46.C2B4 yielded 92.4 bu/acre. Undoubtedly a portion of this decrease was due to disease susceptibility as previous trials had shown competitive yields under more normal yield conditions. These results caused us to switch Mandaamin hybrids in subsequent years.
Table 53. Results of manure and inoculation trials on three farms in 2018.
Anibas Farm inoculation study 2019. The same design was applied in 2019 on the Anibas farm. However, all plots were manured with dairy manure and there were only the hybrid and inoculation treatments. Grain was harvested as for 2018. However, roots and stalks were harvested from the same areas as yield shredded ground, and submitted to USDA in Morris MN for mineral analysis with ICP and C and N analysis with a Leco analyzer. Results from the analysis of variance for grain yield are shown in Table 54.
Analysis of yield results showed that inoculation had no effect on yield but hybrids differed significantly. The FOS8500 yielded 152.3 bu/acre and the 17.461 yielded 125.7 bu/acre. Results from the mineral analysis are shown in Table 55. There was no direct effect of inoculation on mineral nutrient accumulation. The main significant factors in analyses of variance, when they occurred, were or the hybrid, the part of the plant sampled, and the interaction between the hybrid and the part of the plant. These present for some elements and not for other (see Table 54).
Table 54. P level of significance or the effect of factors hybrid, parts (grain, stalk, roots) and the hybrid x part interaction for farm A in 2019.
The results indicate profound differences in uptake and mobilization of certain nutrients, and their accumulation in grain. The hybrid 17.461 mobilized higher concentrations of nitrogen, carbon, phosphorus, and sulfur into its grain than did FOS8500 (see Table x). FOS 8500 tended to accumulate higher levels of potassium, magnesium, and sulfur in its roots than 17.461. Conversely, 17.461 accumulated higher levels of carbon, aluminum, copper and zinc in its roots and silica in its stalks.
Table 55. The relationship between hybrid and plant part on Anibas farm in 2019. Significant differences between hybrids for the same plant part are shown in bold print.
Fertiization and inoculation study on Weiss/Bauer farm in 2019.
The treatments on farm Weiss/Bauer differed from the trial at farm Anibas in that the rotation was corn after corn instead of alfalfa, a starter fertilizer with an unspecified amount of N was applied to all plots, and a slurry application was added to half of the plots. There were significant treatment effects due to hybrid (p <0.0001), and fertilization x hybrid x inoculate (p = 0.0257), and possible effects of fertilization x inoculate (p = 0.0846).
The fertilization x hybrid x inoculate interaction shown in Table 56 shows that FOS outyielded 17.461 and that inoculation had a negative or positive effect on yield, depending on the level of fertilization and on the hybrid. For FOS 8500 the inoculation had a negative effect at the high level of fertilization, but a positive effect at the low level of fertilization For 17.461 the inoculation effects were not significant.
Table 56. fertilization x hybrid x inoculate interaction grain yield in bu/a for the Weiss/Bauer farm in 2019.
Anibas Farm trials 2020.
The same design was applied in 2020 on the Anibas farm near Arkansaw, WI and on the Beiler farm near Rewey, WI in 2020. During this season we compared the experimental Mandaamin hybrid 15.461 with the commercial FOS hybrid FOS8500. These hybrids both possess a relative maturity rating of 105 days. Results from the analysis of variance were no significant effects of any of the treatments or interactions between them on the grain yield of the corn except for Inoculate x hybrid (p=0.037). The Mandaamin hybrid responded negatively to inoculation while the conventional hybrid responded positively.
- Here is a link to the final SARE report with tables, photos, and diagrams included: (Full Report with Figures, Photos, and Diagrams)
- Here is a link to the tables, photos, and diagrams associated with this report: (Document Containing Tables, Photos, and Diagrams)
- Microscopic inspection of seedlings of Mandaamin inbreds that were grown under axenic conditions at Rutgers University showed the presence of large numbers of seed-borne bacteria that were multiplying and living inside root cells. These bacteria were also visibly excreted from root hairs into the rhizosphere. Microscopic stains showed that the bacteria were surrounded by oxidative substance produced by the plants to degrade the bacteria. In addition to this, observation of the putative N2 fixing inbred C4-6 suggested a unique relationship as bacteria living in root cells appeared imbedded in some kind of gel-like, cloudy matrix.
- These bacteria are thought to enhance nutrient availability in the rhizosphere and provide the plant with nutrients associated with oxidized microbial biomass through rhizophagy cycles. Such cycles entail the excretion of mineral depleted bacteria from root hairs and the reabsorption of mineral enriched bacteria by young growing roots.
- Rhizophagy results in uptake of macro- and micronutrients both from turnover of microbial bodies in plant tissues and from N2 fixation (White et al., 2018; 2019a,b). N2 fixation involves a specific biochemical dialogue between bacteria and plant where the microbes produce nitric oxide and ammonium as antioxidants to protect themselves from superoxide produced by the plant (Chang et al., 2019; Micci et al., 2022).
- At question is whether hybrids developed from inbreds that are rhizophagic can yield competitively with conventional hybrids. Yields of Mandaamin hybrid 17.461 were compared with yields of the commercial check FOS8500 on 14 farm sites over two years. The 17.461 averaged 139 bu/acre and the FOS8500 averaged 144 bu/acre. Corn hybrids grown on cattle farms yielded 38 bu/acre more than the corn grown in arable organic systems which had lower contents of soil organic N. The 17.461 yielded 28 bu/acre more than the FOS8500 in the arable system without manure which was 37% more. The 17.461 yielded 24 bu/acre less than the FOS8500 in the arable system with manure which was 15% less for the standardized comparisons. The 17.461 also yielded 5 bu/acre less than the FOS8500 in the cattle system without manure which was 4% less for the standardized comparisons. The 17.461 yielded 16 bu/acre less than the FOS8500 in the cattle system with manure which was 8% less for the standardized comparisons. These results show that manure has a negative effect on 17.461 but a positive effect on FOS8500. The negative effect on yield due to application of manure is increased when the corn is grown on poorer soils in an arable system.
- Another set of field trials compared FOS8500 with various hybrids of varying maturities made with the putative N2 fixing inbred C4-6 and another set made with the Mandaamin inbred NG10. The corn yielded 28 bu/acre more in the cattle system. Though results were not significantly different, the interaction between manuring and hybrid type showed negative responses of the C4-6 hybrids to manuring but positive responses of the NG10 hybrids and FOS8500 to manuring.
- A major question arising from this research is why the C4-6 hybrids respond negatively to manure on the poorer soils in the arable cropping systems? A study on such a soil on the Beiler farm showed that manuring decreased grain yields 20%, nitrogen uptake 32%, uptake of macronutrients 20-22%, and especially total uptake of micronutrients 46% across the C4-6 based hybrids. The commercial hybrid and other kinds of Mandaamin hybrids responded positively to manuring with 38% more yield, 3% more N uptake, 8-16% more uptake of macronutrients, but 19% less micronutrient uptake. Strip trials with and without manure were also carried out on the Weiss/Bauer farm, which had a very high soil fertility due to repeated applications of dairy manure. The 17.461 did respond negatively to manuring with a 19% reduction in grain yield and a 6% reduction in N uptake. However the effect of manure was positive for uptake of other nutrients. The differences between the response on the Beiler and Weiss/Bauer sites could relate to the necessity for hybrids like 17.461 to exercise rhizophagy on soils with lower nutrient availability, and that they are prevented from doing that when manure is applied.
- Other field studies indicated that grain yields were 10-11% lower for the highest yielding Mandaamin hybrids with comparable maturity relative to conventional hybrids when grown under manured conditions. This confirms results from a multiyear OREI study in Illinois, Indiana, and Wisconsin which is otherwise not described here but which overlaps this study. The Mandaamin hybrids of comparable maturity to commercial checks had generally lower harvest index values but they had higher stover (stalks + leaves + husk) and root production.
- Analysis of data from OREI and SARE trials showed a higher methionine, lysine, carotenoid, and mineral content in the grain of the Mandaamin hybrids. This also confirms results from the multiyear OREI study. The commercial check had more starch but less oil and protein in the grain and their protein was of poorer nutritional value.
- Diet modelling with a cooperating poultry scientist (not shown in this report) that considered methionine contents showed the same value per acre for the organic poultry feeder of the Mandaamin crop as for the commercial organic hybrid, despite 11% less yield. This was because the higher content of methionine in the Mandaamin hybrids allowed for replacement of expensive organic soybean meal in the ration.
- On farm studies with the Mandaamin hybrids showed that they had increased acquisition of macronutrients and microelements from the soil relative to conventional hybrids and higher contents of minerals and other nutrients in the grain. The magnitude of this enhancement in mineral uptake and concentration in plant tissues varied with sites, hybrids and years. In 2019 the relative values were calculated by taking the ratio of least square mean values between Mandaamin hybrids and the commercial check. The concentration of nutrients in grain for three Mandaamin hybrids ranged from 6 to 14% higher for macronutrients and 6-30% higher for micronutrients than the check. Total uptake ranged between Mandaamin hybrids from 5-19% more for macronutrients and 14-20% more for micronutrients. Ratios calculated using raw data showed the Mandaamin hybrids ranging from 12-24% higher than the check across all nutrients. The highest value in almost every case was for the 17.461 hybrid. In 2020 ratios based on raw data showed that nutrient concentration in grain across 8 different Mandaamin hybrids averaged 20% higher than the check. The 17.461 again had the highest mineral contents.
- Natural isotope abundance studies of the Mandaamin hybrids indicated enhanced N2 This paralleled increased mobilization of nutrients from soil into grain. Both processes seemed to be linked.
- The Mandaamin corn hybrids differed from each other in their abilities to acquire N from microbial biomass/organic matter or to fix N2. In 2019 trials the hybrids that had C4-6 in their parentage appeared to extract the most N from air and microbial biomass/soil organic matter. They also took up the most minerals and partitioned more into grain. The 2020 trials showed the critical importance of the other half of a C4-6 hybrid in stimulating nutrient uptake, partitioning of nutrients into grain, and yield.
- The natural abundance method for assessing nitrogen fixation utilizes differences in relevant δ 15N values that indicate the relative amounts of natural 15N and 14N isotopes in the total N. In 2019 results supported the hypothesis that the active rhizophagy cycles in the Mandaamin hybrids cause higher δ15N (i.e. more 15N) in root and stover. This occurred again in 2020 but also in grain. Based on the scientific literature it is most probable that the enhanced 15N uptake is sourced from microbial biomass and easily extractable organic N made up of microbial necromass. In 2019 the 15N-rich N taken up from the soil appeared to be progressively diluted from stalks to grain, leading to greater 14N enrichment in the grain and strong decreases in δ15 This may have been associated with absorption of depleted N occurring as a result of N2 fixation in the Mandaamin hybrids, that possibly took place in foliar tissues (Micci et al., 2022). Hence, in the best N efficient hybrids the δ15N levels may be simultaneously increased and decreased in the plant parts by these competing plant/microbial activities.
- The percent N or protein in grain and δ15N in grain proved to be the two central and inter-related indicators of crop performance. Both δ15N and %N correlated strongly with each other and positively with uptake and concentration levels for numerous minerals as well as for grain quality parameters including essential amino acids, starch, oil, and density. In multi-site trials in 2019 and 2020 analyses showed the majority of variation in δ15N was associated with %N (or % protein) in grain, and the converse was also true.
- The balance of fixation and acquisition from soil microbial biomass/organic matter depended on hybrid, farm history, soil quality, and year of trials. The highest estimates for fixation level of N2 into grain (48%) and acquisition of N from microbial biomass/easily available organic matter (58%) was estimated for the C2B2.C46 hybrid when grown under organic arable systems in 2019. However, the other Mandaamin hybrids tested appeared to fix or extract more N from biomass or the air when they were grown under the organic or conventional cattle systems.
- In 2019, rhizophagy seemed to predominate for the Mandaamin hybrids as a source of N for grain in the arable organic system specifically on the organic-arable system’s N depleted soils. Similarly, in 2020, the δ15N results in 2020 showed that rhizophagy predominated in its effects on grain protein-N loading, possibly obscuring inputs from fixation. The growing conditions in 2020 were more stressful and resulted in lower yields and harvest index values for the varietal trials. In this case stover generally had a lower δ15N value than grain. A possible explanation for these differences from 2020 may be less fixation of N from the air in the foliage in 2020 than in 2019 due to stress conditions. Also, the C2B2.C46 hybrid, which had appeared to fix large amounts of N2 even on the poorer, arable organic soils in 2019, was not included in the 2020 trials.
- In the 2020 variety trials the uptake of minerals, C, and N into grain in 2020 was positively associated with δ15N level of the grain but negatively associated with the δ15N level of stover. Similarly, the mineral uptake in the stover was positively associated with the δ15N levels in the stover, but negatively associated with the δ15N levels in the grain. This reciprocal relationship can be explained as a source-sink relationship. The grain is a sink for N15 enriched N and minerals that are loaded into the grain from the stover source which depletes the stover.
- In both years the C4-6 based hybrids took up the most minerals, and also had high δ15N levels in their stover and higher %N in their grain. In 2019 when roots were analyzed, the C4-6 hybrids had exceptionally high microelement contents in their roots. In both 2019 and 2020 trials, 17.461 produced grain with high mineral contents. In both years it produced large quantities of stover and demonstrated the strongest ability to acquire minerals and to partition micronutrients into grain. In the 2020 varietal trials 17.461 averaged 61% higher mineral contents in its grain than the check. This difference was exceptionally high. It included 270% more Ca, 99% more Cu, 10% more Mg, 41% more Mn, 9% more P, 19% more S, and 36% more Zn. As yields of 17.461 were very similar to FOS8500 these very large differences are clearly not due to yield reduction coupled with some kind of nutrient concentration effect.
- To evaluate the partitioning of minerals into grain we calculated a harvest index value for each mineral (harvest index = (mineral in grain/mineral in grain + stover)x 100).In the 2020 varietal trials the δ15N in grain and stover was associated with a major part of the variation in the harvest index of individual minerals. As had been the case for mineral uptake into grain, harvest indices for minerals were positively associated with the the δ15N levels in the grain but negatively associated with the δ15N levels in the stover. This again makes sense as the outcome of a sink-source relationship with the assumption that the movement of N derived from rhizophagy into grain is coupled with enhanced mineral uptake derived from rhizophagy into grain. Hence the harvest index and mineral uptake are probably linked to the strength of exercise of the microbial partnership.
- Studies with different kinds of soil tests in 2019 showed that the Mandaamin hybrids had the highest yields and nutrient uptake when they were grown on cattle manured soils with high levels of total organic-N, high C/N ratios and high soil protein content. Hence management oriented towards building organic-N and soil protein may be determinative for enhancing stabile, high level agronomic performance of the Mandaamin hybrids.
- Uptake of specific mineral nutrients varied from year to year and site to site. The high uptake of certain minerals by the Mandaamin hybrids in 2019 studies suggests enhanced mobilization of minerals from the solid portions of the soil including alumino-silicate clay particles, from parent mineral particles, and from limestone rather than from passive reliance on the soil solution.
- Multiple experiments showed that inoculation with N2 fixing bacteria did not improve performance of Mandaamin hybrids but inoculation may have had some benefits on the performance of conventional hybrids.
- The inoculation and manure trial results suggest that the Mandaamin hybrids have established internal microbial cultures and that addition of bacteria to the seed or soil in the form of manure may interfere with rhizophagy and hence, crop performance.
- The negative response to direct manuring and the lower nitrate and available amino acids found before harvest under the Mandaamin hybrid roots suggest lower nitrification under their roots and lower reliance on mineral N nitrification from soil and manure.
- Future studies might be carried out to test whether the suppositions derived from this research remain valid with more data gained after larger-scale application of the Mandaamin hybrids on farms. We are at the beginning of learning about relationships between breeding, rhizophagy, seed-borne endophytes, soil fertility, soil microbial biomass and microbial necromass, N fertilizers and yield. It is important to gather more data comparing N need, utilization, and fertilization of soil under different farming systems and learning how to optimize hybrid partnerships with endophytes. And finally, it is important to continue breeding for hybrid/endophyte partnerships with greater stability for both microbial assisted processes of rhizophagy and N2 fixation coupled with adequate yield performance and enhanced nutritional value under conditions with reduced N inputs.
Education
A field day was held on one of the research sites (Graham Adsit) in September, 2018 with approximately 87 attendees. The project was advertised to local and organic farmers. Due to weather conditions (the first sunny day after a lot of rain) attendance was more limited than was expected. There was a lunch and a two hour session to discuss farmer input on corn and quality and research.
There was interest that came in the spring from UW-Extension (Dr. Micheal Travis), NRCS personnel and a local citizen-based group in Western Wisconsin for Mandaamin Institute’s N efficient/N2 fixing corn. This is due to problems with widespread nitrate contamination of well water. In 2018 I gave a seminar in Mid May on our work followed by discussion (about 20 people)and a field day on September 20th (40 people, mostly farmers). The latter event was filmed but I don't have a copy. The local paper wrote two articles, one after each event. One of them is attached 180927-Courier-Wedge-N-Field-Day. The group in Pepin County produced several on-farm field trials; one was replicated with Mandaamin hybrids and reduced N fertilizer rates with a conventional dairy farmer (Don Weiss). Trials are being planned again for 2019.
In addition, hour-long talks on our research were given at Agrienergy's farmer winter meetings on Jan. 23rd in Springfield, IL (ca. 100 attendees) and on Feb 6th in Shakopee, MN (ca. 40 attendees). Both of those were filmed and one of the films is attached. A third hour-long presentation on our work with N efficient corn was given to farmers at the eOrganic Grain Conference and Tradeshow in Champaign, organized by the Land Connection. That session had about 60 people attend. It was also filmed but I don't have a copy yet.
Presentations were given to two scientific congresses on organic agriculture in the Fall of 2021. One sponsored from Switzerland (Second International Biodynamic Conference) (40 people) and the other from France (International IFOAM, Scientific conference) (40 people). An oral presentation was given at the MOSES conference in 2022 in La Crosse to 50 farmers and seed company people.
All sessions included considerable feedback and discussion with farmers to understand the significance of our results.
In the second year two meetings were held with farmers in Durand, Wisconsin to discuss the project in conjunction with Mike Travis, UW-Extension. The area has sandy soils and nitrogen pollution of surface and well water is a big problem. The first was a spring meeting with farmers and others, but I have lost documentation on the date so I am not including it. The other was a field day on the Weiss Farm, held in October 3, 2019 where a group of approximately 25 farmers met to discuss results and to view test plots. The event was about nitrogen and corn and the flier is attached. Results were that several farmers were enthusiastic about the way the Mandaamin corn looked without fertilizer and they requested seed to try. Mike and his colleagues applied for a grant and got funding to pay for seed. Unfortunately, due to wet weather hybrid seed production failed in 2019 and we have to delay getting seed to farmers.
In addition to this, on March 12th, Walter Goldstein also met with a group of about 20 farmers and ag educators near Durand to show slides, discuss the research, and to enlist aid in carrying out trials in 2020.
Also, Walter Goldstein competed to present a poster at the scientific session of the MOSES organic conference in LaCrosse, Wisconsin in February 2019, and in February of 2022. The results presented show yields of Mandaamin hybrids relative to conventional hybrids on farms in three states and the quality differences found in 2018. Results presented in 2019 show somewhat lower yields for Mandaamin hybrids (the best yielded 11% less than hybrid checks on all states over 2 years. However nutritional quality was much higher (micronutrients, protein quality, carotenoids) for the Mandaamin cultivars. The paper was viewed by many farmers and leaders in the organic movement. The second poster showed similar yields but that manure could interfere in the yield of Mandaamin hybrids. Both posters are attached.
Farmers:
Farmers articulated a keen interest in obtaining seed and seeing our programs evolve to meet their needs. A network of organic farmers in WI tested the high methionine corn in the SARE/OREI trials. Corn appeared to produce competitive though slightly lower (10-11%) yields, but with considerably higher protein, methionine, lysine, oil, mineral, and carotenoid content. Farmers were interested in N efficiency to reduce manure use and where no manure was applied, the Mandaamin corn out-yielded the conventionally bred corn, at times producing equivalent yields to conventional corn that had been manured. Farmers also showed interest in cross incompatibility to pollen from GM plants because GM contamination of corn can cause milk processors to drop farms or shipped grain to be rejected, and restrictions have tightened. Two organic farmers (Mark Zinniker, Elkhorn, WI and Moses Beiler, Rewey, WI) are growing the high methionine corn and feeding it, without synthetic methionine, to small flocks of layers. Beiler (personal communication, M. Beiler, 2019) reported that egg production went up in comparison to feeding of a commercial organic diet that included commercial hybrid corn grain + synthetic methionine inputs. Both farmers have grown Mandaamin cultivars in 2019, 2020, and 2021 and Beiler is working to grow grain to feed his 10,000 layers based on high methionine hybrids.
End User Companies: Interaction has occurred with large and small companies, including those engaged in organic seed, feed, poultry production or grain marketing. Long-term dialogues have occurred with a consortium of organic poultry companies (Methionine Task Force) about the need for high methionine corn hybrids that can yield competitively with commercial hybrids. They are presently considering a medium scale feeding trial to test the corn. Since 2019 we have been advising one medium-sized poultry company, and one of our longer-term farm cooperators, in growing sufficient high methionine grain to do a feeding trial on 10,000 birds. Furthermore, Jack Algiere (Farm Director of Stone Barn Center in Pocantico Hills,NY), is planning a poultry feeding trial based on high methionine corn with advice from Klaas and Mary-Howell Martens of Lakeview Organic Grain (producers of organic grains and feeds with an active interest in natural forms of methionine for feeding poultry). A tortilla maker in Madison, WI and a chip maker in MN are testing the corn for product.
Extension/NRCS: Mandaamin Institute has been testing its N efficient/putatively N2 fixing corn in its SARE project together with organic and conventional farmers, UW-Extension, NRCS personnel and a local citizen-based, water quality group in Pepin County, Western Wisconsin. Interest is in reducing nitrate contamination of well water. Farmers were encouraged by yield results with Mandaamin hybrids when fertilizer inputs were reduced. Further larger scale trials with multiple farmers were hindered by a lack of seed in 2020.
Seed companies: Discussions and visits included the PI/PD with organic seedsmen Maury Johnson (Blue River Hybrids, Ames, IA); Jeff Block, (Gro Alliance, Cuba City, WI); Jesse Niggemann (Lakeland Organics, Colfax, WI); and Steve Mohr (Foundation Organic Seed, Onalaska, WI). They expressed a need for robust inbreds that compete with weeds and thrive under organic management. The effort to introduce alternative corn is hindered by lack of hybrid seed. The Foundation Organic/Direct Seed Company (FOS) in Onalaska, WI, has helped the Mandaamin Institute to multiply seed of high methionine inbreds and produced hybrid seed in winter nurseries. In 2020 FOS and Mandaamin Institute grew approximately 50 acres of inbred or hybrid production seed corn for farmer trials near Onalaska and East Troy, WI, and in 2021 they grew 30 acres with three different hybrids. In 2021 FOS marketed its first high methionine hybrid (17.C2B2) and in 2022 they are marketing three hybrids from the Mandaamin program including 17.C2B2 and 17.C46. Chris Cashen from Breathe Deep Farm in Hudson, NY has grown corn for seed and grain multiplication from high methionine corn to foster the effort.
Outreach Farmer/Academic: Dr. Goldstein presented results from the project both in academic papers (Goldstein, et al. 2019) and at academic and organic farmer conferences. In 2019 research findings of Mandaamin’s breeding for microbial traits were presented at Crop Science Society of America meetings. In 2021 Walter Goldstein virtually presented results from research at the International Federation of Organic Agriculture Meetings hosted in France and at the International Biodynamic Research Meeting hosted in Switzerland. In 2022 he presented at the virtual Organic Seed Alliance conference hosted in Corvallis, Oregon, and presented at the MOSES conference in La Crosse, WI.
Farmer-based NGOs. In 2020 Walter Goldstein presented information on the project to the Northern Plains Sustainable Agriculture Society (NPSAS) farmers in one seminar (40 people) and a Farmer Breeders Club gathering (20 people). Trials were run on five organic sites in MN, ND, and SD with early maturing hybrids from the Mandaamin program in conjunction with NPSAS farmers and North Dakota State University (NDSU). Members of the NPSAS Farmer Breeding Club and the NPSAS Board attended field events and met in a special retreat with Dr. Goldstein. The Mandaamin Institute continues breeding early corn for that bioregion.
Consultations. In addition, Walter Goldstein has had numerous visits/consultations with various seed or feed companies and particularly Amish farmers about the corn with approximately 50 people.
Project Activities
Educational & Outreach Activities
Participation Summary:
- Two newspaper articles were written about our work in Durand Co. by the local newspaper, The Leader-Telegram. (Article 1 and Article 2)
- We were highlighted twice in the NPSAS newsletter, The Germinator (Spring 2020 Issue and Spring 2021 Issue).
- The magazine Discover published an article on us and other researchers working on N2 fixing corn.
- Numerous meetings (26) including field days, seminars, consultations, poster presentations were given to farmers to talk about our work. Presentations were filmed.
- Organic Seed Alliance presentation video
- 2019 Organic Grain Conference presentation video
- A research paper documenting our research was published in a refereed journal called Open Agriculture. That paper is written by myself, Abdullah Jaradat, Linda Pollak, and Major Goodman, all established corn scientists.
- Multiple seminars, powerpoint presentations, and two posters were presented.
Learning Outcomes
- Nitrogen fixing corn
- Soil and root health and yield stability
- protein quality of corn and methionine
- corn breeding
- microbial partnerships with corn
Project Outcomes
learning how to reduce N fertilizer use.
Numerous farmers have indicated 1) that they want to try high methionine; 2) that they would like that corn to be cross incompatible with GM corn; 3) that they want to know how much fertilizer they can reduce if they grow the corn. Grain buyers/feed companies want a lot of grain to feed to chickens. Many are interested in how to manage manures to make the most of N efficient corn.
Corn is the major cereal crop in the Midwestern region for farmers. It is important to have long-term support for public breeding of climate friendly hybrids that provide services such as N efficiency, enhanced mineral uptake, and better nutritional value grain. These corn varieties need continued efforts to ensure that yields and disease resistance are competitive.