SYMBIOTIC NITROGEN FIXATION AND ESTABLISHMENT OF

SIX MONTANA NATIVE LEGUMES SPECIES

by

Sarah Jean Metcalf

A thesis submitted in partial fulfillment of the requirements for the degree

of

Masters of Science

in

Land Rehabilitation

MONTANA STATE UNIVERSITY

Bozeman, Montana

May 2005

© COPYRIGHT

By

Sarah Jean Metcalf

2005

All Rights Reserved

ii

APPROVAL

Of a thesis submitted by

Sarah Jean Metcalf

This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency and is ready for submission to the College of Graduate Studies.

Jeff Jacobsen

Approved for the Department of Land Resources and Environmental Sciences

Jon Wraith

Approved for the Department of College of Graduate Studies

Bruce McLeod

iii

STATEMENT OF PERMISSION TO USE

In presenting this thesis in partial fulfillment of the requirements for a master

degree at Montana State University-Bozeman, I agree that the Library shall make it

available to borrowers under the rules of the Library. If I have indicated my intention

to copyright this thesis by including a copyright notice page, copying is allowable only

for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright

Law. Requests for permission for extended quotation from or reproduction of this

thesis in whole or in part may be granted only by the copyright holder.

Sarah Metcalf

May 05, 2005

iv

ACKNOWLEDGEMENTS

First of all, I would like to acknowledge the Natural Resources Conservation

Service, the MSU Foundation Seed Program as well as the Department of Land

Resources and Environmental Sciences at MSU for funding this project. The

following people have helped to make this project a valuable experience for me. Jeff

Jacobsen helped me find this project; he has turned my life path in a whole new

direction, for which I will always be grateful. Clain Jones, my primary advisor,

worked very closely with me and was always around to help me solve problems. Tim

McDermott has been a wonderful mentor and provided me with opportunities too

numerous to list and Lina Butero-Griffiths, from his lab, who was very patient in

teaching protocols. Bill Grey has also given so much of his time and laboratory space

to help me with the pathogen aspects of this project. His enthusiasm for research and

discovery kept me going numerous times. I also want to thank Mark Majerus and

Larry Holzworth; both have given me great advice, encouragement and assistance over

the last two years. Dave Wichman and Bernie Schaff were appreciated for their

assistance in maintaining my field sites. I would also like to thank Aaron Mugaas,

Deirdre Willie, Ann McCauley, Teresa Warne, Ryan Weikel and Cole Germann for

their assistance in the laboratory and field. Special thanks to my family, especially my

parents who have been very supportive both financially and spiritually throughout this

project. Finally, I am thankful for my son Shea who inspired me to obtain this degree

though without whom completion would have been much easier.

v

TABLE OF CONTENTS

1. INTRODUCTION ...........................................................................................................1 Objectives ........................................................................................................................1 Background......................................................................................................................1 Materials and Methods.....................................................................................................1 Factors Effecting Nodulation and Legume Growth.........................................................4 Nodules ............................................................................................................................8 N2 Fixation in the Field....................................................................................................9 2. GREENHOUSE STUDIES: SOIL INOCULATION AND FUNGICIDE EFFECTS ON NODULATION AND BIOMASS.........................................................................12 Introduction....................................................................................................................12 Materials and Methods...................................................................................................12 Trial I: Effects of soil origin and N content on nodulation and biomass.......................12 Materials and Methods Trial 1...................................................................................13 Introduction................................................................................................................13 Seeding.......................................................................................................................15 Data Collection ..........................................................................................................15 Trial I Results..................................................................................................................16 Germination and emergence .....................................................................................16 Biomass.....................................................................................................................16 Nodulation.................................................................................................................19 Pathogenic Observations and Isolations ...................................................................20 Trial II: Effects of soil moisture content on nodulation and biomass..............................22 Materials and Methods..............................................................................................22 Introduction...............................................................................................................22 Seeding......................................................................................................................24 Data Collection .........................................................................................................24 Trial II Results .................................................................................................................24 Biomass.....................................................................................................................24 Nodulation.................................................................................................................25 Trial III: Effects of fungicide coating on nodulation, biomass and plant levels..............29 Materials and Methods..............................................................................................29 Introduction...............................................................................................................29 Seeding......................................................................................................................29 Data Collection .........................................................................................................30 Trial III Results ................................................................................................................30 Emergence and Plant Survival ..................................................................................30 Nodulation and Biomass ...........................................................................................32 Trial IV: Effects of fungicide drench on nodulation and biomass...................................36

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TABLE OF CONTENTS – Continued

Materials and Methods...........................................................................................36

Introduction............................................................................................................36 Seeding...................................................................................................................36

Data Collection ......................................................................................................37 Trail IV Results.............................................................................................................38 Biomass..................................................................................................................38 Root Structure, Nodules and Fungicide .................................................................39 N-content................................................................................................................41 Trial V: Effects of inoculation and fungicide application on nodulation and biomass ..............................................................................................................................42

Materials and Methods...........................................................................................42 Introduction............................................................................................................42 Seeding...................................................................................................................43

Trial V Results ..............................................................................................................44 Introduction............................................................................................................44 A. canadensis Combined Treatments....................................................................44

A. canadensis Nodulation ......................................................................................45 Inoculation .............................................................................................................46 Greenhouse Studies Summary ......................................................................................48 3. FIELD RESEARCH .....................................................................................................50 Introduction...........................................................................................................50 Materials and Methods..........................................................................................51 Site Preparation and Design..................................................................................51 Seeding..................................................................................................................52 Data Collection .....................................................................................................52 Field Research Results..................................................................................................55 Site Comparisons ..........................................................................................................55 Bridger ..........................................................................................................................56 Legumes...............................................................................................................56 Combined Biomass ..............................................................................................59 Grasses .................................................................................................................60 Summary ..............................................................................................................62 Moccasin .......................................................................................................................63 Legumes...............................................................................................................64 Combined Biomass ..............................................................................................65 Grasses .................................................................................................................66 Summary ..............................................................................................................67 Post Farm .....................................................................................................................67 Legumes...............................................................................................................68 Combined Biomass ..............................................................................................68 Grasses .................................................................................................................69

vii

TABLE OF CONTENTS – Continued

Summary ......................................................................................................................71 N Content .....................................................................................................................71 N2 fixation and Mass Balance......................................................................................76 Field Study Summary ..................................................................................................79 4. LEGUME LAND MANAGEMENT...........................................................................82 Introduction............................................................................................................82 Species Specific Information .................................................................................83

A. canadensis ....................................................................................................85 Dalea spp ...........................................................................................................86 H. boreale ..........................................................................................................87 L. argenteus........................................................................................................90 V. Americana......................................................................................................91

Technical Management Practices for Field Establishment of Native Legumes ....93 5. SUMMARY AND CONCLUSIONS ............................................................................99 REFERENCES CITED....................................................................................................102 APPENDICES .................................................................................................................111 APPENDIX A: PRELIMINARY GROWTH CHAMBER EXPERIMENTS ................112 APPENDIX B: INOCULATED AND NON-INOCULATED RESULTS FROM GREENHOUSE TRIAL V ..............................................................................................127

viii

LIST OF TABLES Table Page 1. Plant species seed stock and germination information ..................................................13 2. Field soil collection areas and characteristics................................................................14 3. Fungicide information and dilutions..............................................................................29 4. Rhizobia and legume inocula information .....................................................................43 5. Grass species and seed information ...............................................................................50 6. Field site information.....................................................................................................51 7. Native legume characteristics ........................................................................................84 A1. Six Legume and Ten Rhizobia Species.....................................................................102 A2. Plant survival in pouches AI .....................................................................................114 A3. Treatments and surviving plants in Trial AIII ..........................................................117 A4. Analysis of plant tissues and nutrient solution from growth pouches ......................121

ix

LIST OF FIGURES Figure Page 1. Emergence of all six native legumes in Geyser soil ......................................................17 2. Emergence of all six native legumes in Red Bluff soil..................................................18 3. Biomass from Trial I in Geyser and Red Bluff soil .......................................................18 4. Nodule mass from Trial I in Geyser and Red Bluff soil ................................................21 5. Nodule number from Trial I in Geyser and Red Bluff soil............................................22 6. Biomass from Trial II in Bridger and Post Farm soil ....................................................25 7. Biomass from Trial II in Bridger soil 60-80% FC soil moisture ...................................26 8. Biomass from Trial II in Post Farm soil 60-80% FC soil moisture ...............................26 9. Nodule mass from Trial II in Bridger soil 60-80% FC soil moisture ............................27 10. Nodule mass in Post Farm soil 60-80% FC soil moisture ...........................................28 11. Emergence of D. candida with fungicide seed coats in Geyser and Red Bluff soil ....31 12. Final plant number of D. candida with fungicide seed coats in Geyser and Red Bluff soil ............................................................................................................................31 13. Nodule mass of D. candida with fungicide seed coats in Geyser and Red Bluff soil .33 14. Photo-Nodules from D. candida metalaxyl vs. no fungicide.....................................34 15. Nodule number of D. candida with fungicide seed coats in Geyser and Red Bluff soil......................................................................................................................................34 16. Biomass of D. candida with fungicide seed coats in Geyser and Red Bluff soil ..............................................................................................................35 17. First harvest biomass of A. canadensis and L. argenteus with fungicide drench ........39 18. Second harvest biomass of A. canadensis and L. argenteus with fungicide drench....40 19. Photo- L. argenteus root condition when grown in Post Farm soil, fungicide vs. no fungicide treatment ............................................................................................................40

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LIST OF FIGURES-Continued Figure Page 20. Second harvest N concentration of biomass from A. canadensisand L. argenteus with fungicide drench ........................................................................................................42 21. Biomass from A. canadensis with fungicide drench and inoculation..........................45 22. Nodule number of A. canadensis with fungicide drench and inoculation ...................46 23. Nodule mass from A. canadensis with fungicide drench and inoculation...................47 24. Total N concentration of A. canadensis with fungicide drench and inoculation .........48 27. Field emergence of legumes in 2003 season................................................................57 28. Combined grasses and legume biomass at all three field sites ....................................58 29. Bridger site: legume dry shoot biomass.......................................................................60 30. Bridger site: combined grass and legume dry shoot biomass ......................................61 31. Bridger site: grasses dry shoot biomass .......................................................................61 32. Photo: Moccasin site: fall 2003 defoliation by grasshoppers, Spring 2004 site regrowth .............................................................................................................................63 33. Moccasin site: legume dry shoot biomass ...................................................................65 34. Moccasin site: combined grasses and legume dry shoot biomass ...............................66 35. Moccasin site: grasses dry shoot biomass....................................................................67 36. Post Farm site: legume dry shoot biomass...................................................................70 37. Post Farm site: combined grasses and legume dry shoot biomass...............................70 38. Post Farm site: grasses dry shoot biomass...................................................................71 39. N concentration of legume shoot biomass at all three field sites.................................74 40. Average shoot tissue N content at all three field sites .................................................75 41. Average shoot tissue N content of legumes vs. grasses at all three field sites ...........75

xi

LIST OF FIGURES-Continued Figure Page 42. Total N tissue mass balance for all three field sites.....................................................77 43. Bridger site: nitrogen mass balance .............................................................................78 44. Moccasin site: nitrogen mass balance..........................................................................78 45. Post Farm site: nitrogen mass balance .........................................................................79 46. Photo: A. canadensis at Post Farm site summer 2004. Seedpod and flowering ..........85 47. Photo: D. purpurea and D. candida H. boreale from Post Farm field site. Flowering and seedpod ......................................................................................................87 48. Photo: H. boreale from the Post Farm summer of 2004..............................................88 49. Photo: V. americana from the Post Farm field sit .......................................................90 50. Photo: L. argenteus at Post Farm site. Seedpods, root nodules and full row..............92 A1. Photo: native legumes vs. soybeans in growth chamber Trial AIII ..........................122 A2. Legume dry biomass from growth chamber Trial AIII............................................123 A3. Legume NO3-N from pouch solution in the growth chamber Trial AIII ..................124 A4. Legume NH4-N dry from pouch solution in growth chamber Trial AIII..................125 A5. Total N content of legumes biomass from growth chamber Trial AIII ....................125 A6. Total N per legume species from growth chamber Trial AIII ..................................126 B1. Trial V: biomass from Dalea spp. and H. boreale with fungicide drench and inoculation.....................................................................................................129 B2. Trial V: nodule mass from Dalea spp. and H. boreale with fungicide drench and inoculation.....................................................................................................130

xii

ABSTRACT

Legume species have been known to increase soil N content and are incorporated into land restoration seed mixes in hopes of improving degraded soils and plant communities. The goals of this project were to determine effects of soil type, soil moisture, inocula and fungicide treatments on legume establishment, N2 fixation capacity and plant biomass for six individual native legume species. In potted greenhouse studies, legumes were grown in five field soils to verify nodulation without inocula. In a second phase, plant biomass was measured in two soils at two moisture contents (60 and 80% of field capacity). The efficacies of Rhizobia inocula and fungicide treatments were also tested. The greenhouse studies were used to supplement data gathered at three field sites around Montana. Native soils contained Rhizobia that were specific to many of the plant species tested and induced nodulation. Soil moisture content, inoculation and fungicide treatments had less effect on nodulation and biomass than the soil characteristics. Fungicide treatments benefited establishment of Lupinus argenteus, and had mixed effects on Astragalus canadensis and Dalea candida. Benefits were dependent on soil nutrient content, microbial biota and other characteristics. Shoot biomass at each field site also varied significantly; in general A. canadensis had the highest nodulation and biomass, while Dalea purpurea and D. candida typically had the lowest biomasses and nodulation. This research should assist land managers in selecting species to suit a wide variety of ecological conditions and land restoration scenarios.

1

CHAPTER 1

INTRODUCTION

Objectives

The purpose of this project was to determine the amount of nitrogen gas (N2)

that can be fixed each by of six native legume species. Establishment and biomass

production of these species, in different field soils, was also examined. Specifically,

the goals of the project were to determine the effect of different field soils on

nodulation, total biomass production and N levels. Soil moisture content was

examined in relation to effects on nodulation, total biomass production and N content

for the selected legumes in two soils. The extent of nodulation, total yield, total N

uptake, and changes in available soil N (NO3-N + NH4-N) in different field soils for

the selected legume species was determined. Finally the effect of seed fungicide

treatments and inoculation on emergence and plant biomass was examined.

Background

Natural and disturbed ecosystems are often nitrogen (N) limited. Leguminous

plants have the potential to increase the levels of available nitrogen (N) in soils

(Seastedt and Knapp 1993; Towne and Knapp 1996; Tlusty et al. 2004; Unkovich and

Pate 2000). This project evaluated six native legume species that have been released

or are under consideration for release by the Natural Resources Conservation Service

(NRCS) Plant Materials Program to be used as additions to current prairie restoration

2

seed mixes. It attempted to determine the ability of each species to fix nitrogen gas

(N2) and measure the change in levels of bioavailable N in field soils. Another goal

was to establish field guidelines for the use of native legumes in land restoration,

because very little is known about the physiology and ecology of Montana native

legumes.

Many factors affect a grass-legume system and there are many levels on which

grasses and legumes compete and benefit one another in a prairie system, but these

systems can be difficult to establish and maintain (Frame and Newbould 1984, 1986;

Ross et. al. 1972; Thornley et al. 1995; Schwinning and Parsons 1996a, b, 1999).

Developing field protocols will hopefully help researchers and land managers achieve

more successful land restoration and lasting plant community establishment. These

protocols can also serve to improve information about species selection and

management in order to best utilize the N2 fixation potential of these species based on

growth habit and ecological niches. This research seeks to improve the knowledge

base of legume systems in order to determine and eliminate some barriers to successful

plant establishment. Current barriers are drought and salinity sensitivity, soil borne

pathogens, pests and a lack of knowledge on native species-specific rhizobia and the

quality of inocula currently in use (Schwinning and Parsons 1996 a, b, 1999; Thornley

et al. 1995; McDermott et al. 1991; Tlusty et al. 2004).

Humans have used legumes in agriculture for thousands of years either as a

food crop for humans and livestock, or as a rotational crop to supply N to non-legume

crops. The shift from biological to industrial sources of N fertilizer in agricultural

systems did not occur until the 20th century (Smil 2001; Crews and Peoples 2004). In

3

more recent times, legumes have been incorporated back into rotational farming,

especially in organic farming systems (Crews and Peoples 2004, Stockdale et al. 2000;

Smil 2001). It has been demonstrated that symbiotic N2 fixation enhances soil fertility

and productivity as well as increases carbon sequestration and nutrient conservation

(Cromwell and Winpenny 1993; Snapp et al. 2002). Some less developed nations with

low fertility soils cannot afford synthetic fertilizers and have never stopped the practice

of utilizing symbiotic N2 fixation (Crews and Peoples 2004; Snapp et al. 2002).

Symbiotic N2 fixation can actually be so effective that some researchers have noted

that many of the same problems that arise with poor usage of commercial fertilizers

can arise with improper management of legume crops (Scott et al. 2000; William and

Donald 1957). For example, legume crops can lead to soil acidification, N leaching

and excess N runoff in surface waters at levels just as high and detrimental to human

health and the environment as synthetic fertilizers (Fillery 2001; Heltar and Porter

1989; Peoples and Baldock 2001; Ridley et al. 2004; Scott et al. 2000). Ridley et al.

(2004) also found that N build up from legumes often exceeds 100 kg N ha-1 and with

high moisture levels, losses of N through leaching can be 15-35 kg N ha-1 yr-1. It is

unlikely however, in semi-arid regions utilizing native legume species, that leaching of

N build up would be a problem.

Native legume effects on yield of grasses and other crops, nodulation N2

fixation potential and soil fertility are highly variable (Crews and Peoples 2004; Jha et

al. 1995; Zahran 1998; 2001). Depending on the environmental conditions and the

species selected, legumes can be a great source of natural N, although there is always a

need for good management practices adapted to the region and species of interest.

4

Integration, rotation and/or incorporation of legumes as green manure, are currently

being proposed as best management practices by many ecologists and agronomists in

the field (Ridley et al. 2004). There is extensive literature on the subject of N2 fixation

in legumes and their role as an N source for non-legumes; however, very little is

known or published on legumes native to the U.S., and specifically, Montana.

Factors Affecting Nodulation and Legume Growth

The extent of nodulation and amount of N2 fixed by legumes is largely related

to plant size and root mass and will vary depending on the species of plant and the

strain of rhizobia which it hosts (Abel and Erdman 1985; Berg et al. 1988; Ham et al.

1971a; McDermott et al. 1991; Provorov et al. 2001; Tlusty et al. 2004; ). Individual

rates of N2 fixation will vary according to the plant’s growth stage and its demands for

N at a given time.

N2 fixation is an energetically expensive process costing the plant 16 ATP for

every N2 molecule reduced (Bergersen 1982). The energetic expense of the symbiosis

can be detrimental both to the plant host, due to loss of photosynthates, and to the

rhizobia, which could otherwise use carbon (C) sources for growth and reproduction

(Denison and Kiers 2004b). Rhizobial strains can be symbiotic, non-symbiotic, unable

to infect a host, or even parasitic, nodulating the host plant but fixing little or no N2

(Denison and Kiers 2004b). Many strains of rhizobia, utilizing any of these strategies,

can be present in soil at the same time (Berg et al. 1988; McDermott et al. 1991;

Moawad and Schmidt 1987). The strains in this study and within a soil can belong to

species of Rhizobium and Bradyrhizobium and will be referred to as rhizobia

5

throughout this paper (Graham et al. 1999; Tlusty et al. 2004; Zakhia and de Lajudie

2001). A variety of molecular and genetic mechanisms have evolved between the

legume and the rhizobia to regulate the nodulation and N2 fixation processes (Lum and

Hirsch 2003; Provorov et al. 2002). For example, the host plant regulates the oxygen

(O2) supply to the interior of the nodule and has the ability to cut off the O2 supply to

nodules that are not fixing N2, allowing the plant to select for beneficial rhizobia

(Denison and Kiers 2004a).

Soil nutrient availability is a primary factor driving the occurrence and function

of this symbiosis. If there is adequate N for plant growth, the symbiosis will be less

likely to occur (Lum and Hirsch 2003; Streeter 1988; Unkovich and Pate 2000; Vance

2001). However, when bioavailable N is limited, the costs are outweighed by the

benefits, and symbiosis will be initiated. With low N levels, a biochemical dialogue is

initiated between the host plant and the rhizobia, causing nodulation to occur (Caetano-

Anolleseld 1988; Lum and Hirsch 2003). Phosphorus (P) is another key nutrient for

effective symbiosis; if P is limited, symbiosis will often fail (Holford 1997; Lum and

Hirsch 2003; Marschner 1995; Vance 2001). The importance of P in symbiotic N2

fixation is indicated by an accumulation of P in nodules, compared to other organs,

during P-limited conditions (Israel 1993; Schulze 2004). There is evidence suggesting

that some legumes may employ a dual symbiosis by utilizing arbuscular mycorrhizae

to facilitate the acquisition of P in low nutrient soils (Lum and Hirsch 2003; Provorov

et al. 2002; Requena et al. 2001; Zahran 2001).

Light levels and increased photosynthesis will also increase N2 fixation as

photosynthates provide energy to fuel the reaction and increase plant biomass, as well

6

as the demand for N (Crews and Peoples 2004; Unkovich and Pate 2000). Nodulation

is also affected by temperature and pH due to influences on the rhizobia survival

(Bushby 1990). Maintaining temperature and pH in optimal ranges will directly affect

the extent of nodulation in the host plant; the range of tolerance will vary according to

the strain and its origin of adaptation (Bushby 1990; Bergersen 1982; Jacot et al. 1999;

b). High or low pH can damage rhizobia as well as root hairs and hinder the infection

processes. After infection has occurred, N2 fixation will continue as long as the host

plant can tolerate the pH level (Bergersen 1982).

It has also been found that temperature and pH tolerance ranges often

correspond to the environmental conditions in which the plant-rhizobia relationship

evolved. For example, native and high altitude species such as lupine or prairie clovers

are better adapted to the extreme environmental conditions that exist at altitude and

therefore perform better at lower temperatures as compared to tropical legumes such as

soybeans (Graham 2004; Jacot et al. 1999; Kessler et al. 1990; Svenning et al. 1991).

Plants inoculated with rhizobia species adapted to high altitude have shown higher

tolerance to stress conditions such as low nutrients, extreme pH, low moisture and cold

temperatures (Jacot et al. 1999; Johnson and Rumbaugh 1986; Svenning et al. 1991).

Typically, these stresses limit root infection by reducing bacterial populations in the

root zone and therefore decreasing the ability of the rhizobia to initiate nodulation

(Kessler et al. 1990).

Soil moisture is important to rhizobia survival. Continuous water films in the

soil benefit membrane structure and function of free-living bacteria and aid in their

distribution and movement through the soil (Bergersen 1982). Infection occurs when a

7

root hair grows through a resident population of free-living bacteria. In low moisture

conditions, this infection process may be inhibited. Low soil water content has been

seen to reduce nodulation by as much as 50% as measured by acetylene reduction and

respiration rates (Sinclair et al. 1987). If the moisture content of the nodule drops

below 80% by weight, the nodules begin to suffer from water stress causing respiration

and N2 fixation to decrease (Abdel-Wahab et al. 2002). Continued water stress can

cause irreversible damage to nodules and bacteria, permanently stopping N2 fixation,

until new nodules can be formed (Abdel-Wahab et al. 2002). High soil moisture or

saturation can lead to anaerobic conditions and will also stop N2 fixation because

rhizobia are microaerophilic and need some O2 for respiration (Dension and Kiers

2004b; Yu and Rengel 1999). High moisture is not as detrimental as low moisture and

the organisms suffer no physiological damage; when moisture levels drop, N2 fixation

will recommence (Bergersen 1982). Symbiotic processes are more sensitive to

saturation in some legumes, such as M. sativa, than they are in tropical legumes, which

show little response (Ahmed and Quilt 1980; Ismail et al. 1983; Williams and

DeMallorca 1984).

Other stresses such as heavy metal toxicity (Broos et al. 2004), high salinity

and nutrient deficiency can also stop or hinder the infection process or N2 fixation

(Saxena 1991; Yu and Rengel 1999). Legumes with high tolerance to water and other

stresses have been found to have a strong ability to make osmotic adjustments (Ford

1984). Legumes that form indeterminate nodules may be better able to tolerate stresses

than legumes with determinate nodules. The indeterminate nodule has the ability to

continue growing and forming new tissue from the indeterminate end, while the

8

determinate type will have to reform new nodules in order to continue N2 fixation

(Abdel-Wahab et al. 2002). Nodule morphology has less affect on a legume’s ability

to tolerate environmental stresses that affect rhizobial survival in the soil, such as

heavy metal contamination or other toxins (Broos et al. 2004).

Nodules

The extent of nodulation is related to the combination of the legume species

and the strain or strains of rhizobia that infect it (Denison and Kiers 2004b). It is also

related to the plant’s physiology and ability to form lateral roots. Infection occurs at

the root tip where cortex cells are rapidly dividing and growing outward (Hill 1980).

No published literature has been located showing a firm correlation between nodule

size, number and potential to fix N2 in an individual plant. Hill (1980) suggested that

an inhibitory substance produced by the apex of nodules may prevent other nodules

from forming in the same region of the root. Furthermore, it is thought that larger

functioning nodules may reduce the number of nodules on the nearby root, while

ineffective nodules will not produce the inhibitory substance leading to clusters of

small nodules on regions of the root (Hill 1980).

An indicator of nodule activity is the presence of leghemoglobin (LHb), an iron

containing protein similar to human hemoglobin, which binds O2 (Becana et al. 1986;

Bergersen 1982). It facilitates O2 diffusion throughout the interior of the nodule. N2

fixation is a reduction reaction and is hindered by the presence of O2; however, the

bacteroids require O2 to maintain metabolic function (Becana et al. 1986). While

conducting studies on drought and high salinity, Abdel-Wahab et al. (2001) found LHb

9

to be indicative of N2 fixation activity, as decreases in this protein within the nodule

were correlated with decreases in acetylene reduction assays (Guerin et al. 1991;

Irigoyen et al. 1992; Nash and Schulman 1976).

N2 Fixation in the Field

The primary product of N2 fixation is NH3, which quickly complexes with H+

to form the ammonium ion (NH4+). The NH4

+ can become toxic at high levels and is

exported to the cytosol and is immediately incorporated into amino acids (Abdel-

Wahab et al. 2002). These amino acids are transported throughout the plant to be

incorporated into the tissue or used in metabolic processes; excess amino acids are also

excreted into the surrounding soil by plant roots. Field studies have shown that some

legumes take up less N from the soil after nodulation (Armstrong et al. 1994; Phillips

and DeJong 1984; Unkovich and Pate 2000). Some of the N2 fixed is excreted into the

surrounding soil as amino acids and becomes available to non-legume species (Crews

and Peoples 2004). In addition, nodules have a short life span and as they senesce,

available N is released back into the soil and becomes bioavailable or is absorbed by

the host plant (Crews and Peoples 2004). The main pathway for N transfer from

legumes to adjacent crops occurs through the decomposition of root material rather

than through root exudates or direct transfer from mycorrhizal associations (Crews and

Peoples 2004; Trannin et al. 2000).

Many studies have found that intercropping of a legume species with grasses or

other non-leguminous species can have a significant influence on biomass of the non-

legume plant (Haynes 1980; Ledgard 1991). Sengul (2003) found that incorporating

10

sainfoin or M. sativa with three grasses increased the biomass and N content of the

grasses grown in conjunction with the legumes. Grasses grown in conjunction with

sainfoin had the largest increase in biomass, N and crude protein content; however,

both intercropped groups had significantly higher biomasses than did any of the single

stands of legumes or grasses (Gökkus et al. 1999; Sengul 2003). Furthermore,

mixtures enhanced utilization of symbiotically fixed N2 as well as increasing allopathic

effects and light absorption (Pubman and Duke 1978).

Ridley et al. (2004) contend that farming systems that rely on legumes as the

sole N supply are unsustainable because of competition for nutrients and water, as well

as poor timing between the supply and demand for N between the crops. Thornley

(2001) proposed an adaptation of the Hurley Pasture Model, which is a single-species

grassland simulator. This new model for management attempts to optimize not only N

uptake by non-legume crops, but also utilizes the benefits that legumes offer in C

sequestration (Thornley 1998). This model incorporates factors of competition that

occur for grass-legume systems and attempts to aid land managers in species selection

and crop management to successfully utilize legumes as a green manure (Schwinning

and Parsons 1996a;b; 1999; Thornley et al. 1995; 2001). Ridley et al. (2004) also

contend that the benefits of legumes can be substantial when crops are properly

managed. Thornley (2001) contends that the ideal approach to harnessing the benefits

of legumes is to utilize a polyculture system. With multiple legume and grass species

there is a variety of spatial and temporal zones for nutrient and water uptake as well as

varying rates of uptake and demand for these necessities (Crews and Peoples 2004;

11

Ridley et al. 2004). The variety of legumes and their N2 fixation efficiencies as well as

species and rhizobia combinations requires further investigation.

This study looks at potential N2 fixation, establishment, individual growth

requirements and potential benefits of the selected native legume species in Montana

soils. The work was organized into greenhouse studies (Chapter 2), field research

(Chapter 3) and management implications (Chapter 4).

12

CHAPTER 2

GREENHOUSE STUDIES: SOIL INOCULATION AND FUNGICIDE EFFECTS

ON NODULATION AND BIOMASS

Background

The following greenhouse studies were designed to provide information on the

effects of soil type, moisture content, and inoculation and fungicide treatments on

seedling emergence, nodulation and biomass. In addition, data on root biomass and the

extent of nodulation were collected. These data were compared to total plant biomass

and later used to supplement the limited field data of belowground plant biomass,

which is difficult to sample. The greenhouse portion of the research consisted of five

separate trials, each targeted to test different characteristics.

Materials and Methods

Seed stock information for each species is listed in Table 1. All six species

were obtained from the Bridger Plant Materials Center (BPMC) in Bridger, Montana.

The soils used for the greenhouse studies were collected from five field sites around

Montana and the soil characteristics are listed in Table 2.

13

Materials and Methods Trial I Introduction

The overall objective of this trial was to determine the effect of soil N and

characteristics on nodulation and total biomass in all six plant species. Specifically,

this experiment tested the effects of available N levels and other factors on nodulation

and biomass in soils from Geyser, MT near Great Falls and Red Bluff Agricultural

Research Station near Norris, MT (Table 2).

Table 1. Plant species seed stock and germination information.

Plant Species Common Name Pure Seed Germination Total Viability PLS*

------------------------------------%------------------------------ Astragalus canadensis

(AC) Canada Milk Vetch 94 6 88 82

Dalea candida (DC)

Slender White Prairie Clover

98 77 99 97

Dalea purpurea (DP)

Purple Prairie Clover 100 75 91 91

Hedysarum boreale (HB)

Northern Sweet Vetch 98 51 51 50

Lupinus argenteus (LA)

Silvery Lupine 100 0 82 82

Medicago sativa (MS)

M. sativa- Excellent 10A

98 93 94 94

Vicia americana (VA)

American Vetch 100 63 87 87

*PLS- pure live seed

14

Geyser was an active grass-M. sativa pasture that had been treated with N

fertilizer leading to a high N content that was 49 mg kg-1 higher than at Red Bluff. Red

Bluff is a native rangeland soil that had never been fertilized or cultivated, aside from

periodic grazing by livestock and wildlife. L. argenteus was identified growing in the

vicinity of the soil collection area. The presence of native legumes on the site was a

good indication that the field soil contained indigenous rhizobia. All six native prairie

legumes species from Table 1 were selected. No rhizobia inocula were applied in this

trial; therefore any nodulation was the result of the indigenous rhizobia populations in

the soils.

Table 2. Field soil collection areas and characteristics.

Site Cropping History

Texture FC* EC** pH NH4-N NO3-N Olsen P K

g H2O g soil-1 dS m-1 ----------------------mg kg-1------------------

Geyser Agriculture Clay Loam

ND*** 0.45 8.0 ND 76.1 7.9 366

Red Bluff Native Range

Sandy Loam

ND 0.19 7.0 ND 27.1 8.4 450

Bridger Agricultural Research

Silty Clay Loam

0.19 0.15 8.6 3.6 20.1 32.7 564

Moccasin Agricultural Research

Silt Loam

0.23 0.13 7.3 3.4 4.1 32.8 410

Post Farm Agricultural Research

Clay Loam

0.27 0.26 8.0 3.7 48.7 61.1 508

*FC- field capacity (_Pa) **EC- electrical conductivity ***- not determined

15

Seeding

Rocks and large gravel were removed from each soil. The six species, two

soils, and three replicates led to thirty-six pots (20 cm diameter), which were washed,

dried and then filled with approximately 1.2 kg of air-dried homogenized soil. Twenty

pure live seed (PLS), untreated and unconditioned, of each species were planted 1 cm

deep in three pots of each soil type. Each pot was then watered with 50 mL of water

and placed in the greenhouse at 20-26 ºC, the optimal temperature for the Dalea spp.

seeds to germinate (McGraw et al. 2003). Sixty mL of water were applied every other

day for the first two weeks after which time watering was reduced to every 4 d with the

same volume. At 45 d the plants in each pot were thinned to 4 plants per pot. Plants

were allowed to grow for a five-month period in the greenhouse.

Data Collection

Emergence was recorded daily for 14 d beginning 5 d after planting, then

weekly for the next 1.5 months. At the initiation of flowering, plants and soil were

removed from the pots by hand, and the roots and nodules were separated from the soil

by washing through a 1 mm screen sieve with cool tap water. All live biomass was

removed from the sieve and the entire plant was washed clean and stored at 2 ºC in

airtight plastic bags.

The total wet biomass of each plant was weighed. Aboveground biomass was

separated from the belowground biomass, and each wet component was weighed

separately. The aboveground material was placed in a drying oven at 40 ˚C for 14 d;

the dry biomass was then removed and weighed. The root nodules were separated

16

from the root system using forceps and then counted and weighed. Statistical analysis

of all data was assessed using JMP 5.1 (SAS Institute Inc. Cary, NC) analysis of

variance (ANOVA).

Results and Discussion Trial I

Germination and emergence

Native species adapted to semi-arid regions often have a slow growth rate and

the seed has a high longevity in the seed bank (McGraw et al. 2003). Germination

rates in natives are often much lower than non-native species adapted to less harsh

environments. In this experiment, D. purpurea had the highest emergence rate,

followed by D. candida (Figure 1-2). The majority of viable Dalea spp. seeds

germinated within two weeks after planting. The other four species were slower to

germinate and the number of seeds germinating continued to increase for four weeks.

A. canadensis and L. argenteus had the lowest germination rates although the A.

canadensis showed more of an increase later in the study, particularly in the Geyser

soil. L. argenteus consistently had a low germination rate through most of the trials in

this study.

Biomass Two of the native species, A. canadensis and L. argenteus, had significantly

higher dry shoot biomasses in Red Bluff soil than in Geyser soil, whereas the other

four species were not significantly different between soils (Figure 3). A. canadensis

had significantly higher biomass than the other species when grown in the Red Bluff

soil, although its biomass in the Geyser soil showed less variation from the other

17

species. L. argenteus had a moderate biomass in the Red Bluff soil, but in the Geyser

soil the plants began to show symptoms of root and stem rot around the second month

of the trial and by the end of the study all of the plants had died. The Dalea spp, H.

boreale and V. americana biomasses did not differ significantly between the soils.

The Dalea spp. had the smallest biomass in both soils, not surprisingly since it was

very slow growing and had a slender morphology. Apparently, soil characteristics,

specifically nutrient content and endemic microbial communities, have major

influences on the growth responses of at least some of these species.

Figure 1. Plant emergence for six native legume species, grown in Geyser soil DAS in 2002. (DAS)=days after seeding A. canadensis (AC), D. candida (DC), D. purpurea (DP), H. boreale (HB), L. argenteus (LA), V. americana (VA).

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

0 7 14 21 28 35 42

DAS

Emer

genc

e R

ate

ACDCDPHBLAVA

18

Figure 3. Average total dry shoot biomass of the six species in Geyser and Red Bluff soils.

ed e

c db c

b

e e

cb c

b

a

0

1

2

3

4

5

6

A C D C D P H B L A V A

S p e c ie s

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

G e y s e rR e d B lu ff

L S D = 0 . 6

Figure 2. Plant emergence rate for six native legume species, grown in Red Bluff soil DAS in 2002.

0%5%

10%15%20%25%30%35%40%45%

0 7 14 21 28 35 42DAS

Emer

genc

e R

ate

ACDC

DP

HB

LA

VA

19

Nodulation Soil nutrient and texture characteristics had less of an effect on plant biomass

than on nodulation and most likely N2-fixation. There was much higher nodulation in

the Red Bluff soil than in the Geyser soil. All the nodules collected had pink

pigmentation, indicating the presence of LHb, a sign that N2 fixation is probably taking

place (Abdel-Wahab et al. 2002). Many studies find a good correlation between

nodule parameters and plant production (Tlusty et al. 2004). Fresh weight may be a

better indicator of nodule functionality compared to nodule number, as active nodules

are typically heavier (Hill 1980). For example, H. boreale had a similar number of

nodules in both soils; however, the nodule biomass was much larger in Red Bluff as

was the total biomass, indicating perhaps a difference in N2 fixation amounts (Figure

4-5). However, H. boreale and L. argenteus both had much higher average nodule

weights than the other species (unpublished data). Both species are nodulated by

Bradyrhizobium, and express different nodule morphology from the other species as

well as one another.

Not only were there lower levels of nodulation in the Geyser soil, but two

species, L. argenteus and V. americana, did not form nodules at all in soil from this

site. The reduction and lack of nodulation in the Geyser soil is probably due in part to

the high N level of the soil, which is known to suppress nodulation (Schulze 2004).

This soil also had very low P content; the lack of P can drastically reduce the level of

nodulation and N2 fixation and may be a factor in the lower nodule mass in this soil

(Lum and Hirsch 2003). All the other legume species did nodulate, so it is unlikely

that the lack of nodulation in the L. argenteus and V. americana is due to soil

20

characteristics such as nutrients or pH, but rather has more to do with rhizobial

populations in that soil. There were no native legume species observed at the soil

collection site, which may be indicative of a lack of adequate species-specific rhizobia

in this soil. The Red Bluff site had native legumes present and showed much higher

nodulation in all species. Dalea spp., H. boreale and V. americana all had very low

nodulation in the Geyser soil compared to the Red Bluff soil, yet they had similar total

biomasses in both soils (Figure 3-4). This response raises the question of whether or

not the additional N provided by N2 fixation actually benefits biomass production, or

does it actually hinder it due to the additional energetic expense of the symbiosis? It

would serve to reason that biomass production would be slightly inhibited because a

portion of the energy that the plant produces would be directed toward the symbiosis,

whereas passive uptake of nutrients through the root system has a much lower

energetic cost. However, if a longer-term study had been conducted, the more

nodulated plants in the Red Bluff soil may have eventually out-competed the plants in

the Geyser soil, especially after the available soil N was depleted.

Pathogenic Observations and Isolations

The L. argenteus germinated in the Geyser soil, but the plants died during the

third month of the study, showing symptoms of disease from root and stem rot shortly

after their secondary leaves emerged. There was no sign of nodulation in this species

at any stage of plant growth in the Geyser soil. Because of the high N content in this

soil, it is unlikely that the lack of nodules was the cause of the plants’ deaths. It does

indicate, however, that the Geyser soil is probably lacking the species-specific rhizobia

21

required to establish effective symbiosis. Nodulation of the other five species was

verified within the first month of the trial. The diseased L. argenteus tissue was

isolated and several species of Fusarium spp. fungus were isolated. Although they

were not identified to the species level, this family of fungus is known to be a major

fungal pathogen leading to stem and root decay and can eventually kill the plant.

Schulze (2004) examines many mechanisms within the plant genome, as well

as the rhizobia, that regulate the symbiosis in response to physiological and

environmental stresses. Indigenous strains of rhizobia can have a large effect on plant

productivity.

Figure 4. Average dry nodule mass of all six species in Geyser and Red Bluff soils. No nodules were detected on L. argenteus and V. americana.

LS D -0 .05

e ee

cdc

decd

a

b

b

0.00

0.05

0 .10

0 .15

0 .20

0 .25

0 .30

A C D C D P HB LA V A

S p e c ie s

Nod

ule

Mas

s (g

pot

-1)

GeyserRed B luff

22

Trial II: Effects of soil moisture content on nodulation and biomass.

Materials and Methods

Introduction

In this experiment, the primary objective was to determine the effect of soil

moisture content on nodulation and total biomass for two legume species in two

different field soils. Two agricultural soils were selected from the Bridger Plant

Materials Center (BMPC) and the A.H. Post Farm, (Table 2). The four treatment

groups consisted of each of the six species grown in the two soils at moisture levels of

approximately 60% and 80% of each soil’s field capacity (FC). The soils were

submitted to the MSU Soil, Plant and Water Analytical Laboratory (SPWAL) to

measure the water holding capacity of each soil using a pressure plate at -33 kPa to

LSD-69

de ed

bc cdcd

b

a

d cd

0

100

200

300

400

500

600

AC DC DP HB LA VA

Species

Nod

ule

Den

sity

(No.

pot

-1

)

GeyserRed Bluff

Figure 5. Average nodule number from greenhouse Trial I in Geyser and Red Bluff soils.

23

approximate FC (Table 2). These values were then used to obtain the weights of each

pot at 60% and 80% of FC depending on the soil.

Seeding

Seeding followed the same protocol as listed in Trial I with the following

exceptions. An additional replica was added leading to four replicas for each

treatment. Each individual pot was weighed to make sure it was filled with exactly 1.2

kg of soil, and then water was added to bring the soil moisture to the FC of each soil,

by weight. Each pot was watered every other day to maintain FC moisture for 14 d to

allow the seedlings to properly germinate. At day 15, watering was reduced to keep

the treatments near 60% and 80% of FC by weight. Each pot was weighed every 3 d

and the appropriate volume of water was added to maintain the moisture level of each

treatment group. The plants were allowed to grow in the greenhouse for five months

during which time the soil moisture was kept between 55-65% or 75-85% FC.

Data collection

Harvest and data collection followed the same protocol as in Trial I. The data

was graphed in two different ways to better show differences between the moisture

treatments and the soil origins.

Trial II Results

Biomass

There was a high degree of variation between the species biomass between both

soil type and soil moisture treatments (Figures 6-8). There was more variation in

24

biomasses when the species were compared by soil origin (Figure 6) rather than by the

moisture content treatments; however, significant variation within species still existed

(Figure 7-8). All species but D. candida and L. argenteus had higher biomasses in the

Post Farm soil than the plants grown in the Bridger soil (Figure 6). The slower growth

in the Bridger soil could be due to several characteristics of that soil. The texture and

shrink-swell characteristics of the Bridger soil led to structure and drainage problems

in the greenhouse pots. By the end of the study much of the silt had washed out of the

pots leaving high clay content in the Bridger pots. This could have affected nutrient

uptake and benefited fungal pathogens. This soil also had a higher pH than the Post

Farm soil, which could have affected plant growth and rhizobial populations, although

endemic populations would probably be adapted.

There was some plant biomass variation within species at the different soil

moisture levels (Figure 7). Two of the six species, H. boreale and L. argenteus, had

significantly higher biomasses at the lower moisture content in the Bridger soil. There

was more variation within plant species between the different soil moisture treatments

when the plants were grown in the Post Farm soil (Figure 8). Four of the species had

larger biomasses at the upper moisture content, specifically, A. canadensis, H. boreale,

L. argenteus and V. americana.

L. argenteus germinated in the Bridger soil at both moisture level treatments,

but the seedlings in the upper moisture level showed symptoms of root decay and died

early in the study. Tissues from these seedlings were plated and species of Fusarium

spp. and Alternaria spp. were isolated. The higher moisture content may have enabled

the fungal pathogens to establish in these plants. V. americana did not survive the

25

duration of the study in either moisture treatment of the Bridger soil. There were no

pathogens isolated from this tissue, but it is possible that the pH 8 of the Bridger soil

was too high for this specie. The NRCS National Plant Database lists this specie as

having pH tolerance of 6.5-7.2. The Dalea spp. showed no significant plant biomass

differences in either soil.

Nodulation

Overall, there was little significant variation in nodule mass with soil moisture

(Figure 9-10). However, significant differences in nodule mass between species grown

in the different soils were fairly large.

d

c

d

cd

LSD-0.20bb

aba

a

cd

c

0

0.2

0.4

0.6

0.8

1

1.2

AC DC DP HB LA VA

Species

Dry

Sho

ot B

iom

ass

(g p

ot -1

) BridgerPost Farm

Figure 6. Average dry shoot biomass of all six species in Bridger and Post Farm soils. Species biomass averaged from both 60 and 80% FC treatments. A. canadensis (AC), D. candida (DC), D. purpurea (DP), H. boreale (HB), L. argenteus (LA), V. americana (VA).

26

Figure 7. Average dry shoot biomass of six species in Bridger soil at 60-80% field capacity. The L. argenteus 80% FC and V. americana of both treatments did not survive long enough in this soil to yield biomass data.

LSD-0.12

b

bc

a

bcbc

b

bc

bcbc

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

AC DC DP HB LA VASpecies

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

60% Field Capacity80% Field Capacity

Figure 8. Average dry shoot biomass of six species in Post Farm soils at 60- 80% field capacity.

d

bbb c

c

d

a

a

a

b

c d

d

0

0 .2

0 .4

0 .6

0 .8

1

1 .2

1 .4

A C D C D P H B L A V A

S p e c ie s

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

6 0 % F ie ld C a p a c ity8 0 % F ie ld C a p a c ity

L S D - 0 .1 5

27

L. argenteus and V. americana had substantial nodule mass in the Post Farm

soil, but showed no signs of nodulation in the Bridger soil prior to the plant

senescence. The H. boreale nodule mass showed very little variation between soils or

water contents. D. candida had significantly higher nodule mass at the upper moisture

level of the Bridger soil and it was the only nodule mass to have significant differences

between moisture treatments (Figure 9-10). There were, however, large variations

between the nodule mass within the species when grown in the different soils.

Typically, the Post Farm soil produced larger nodule mass; however, the Dalea spp.

had higher nodule masses in the Bridger soil (Figure 9-10). This may be due to the

fact that D. candida has been sown for seed production at BPMC and although it is not

planted on the study site specifically, it is possible that soil in the area would contain

substantial populations of rhizobia specific to the Dalea spp. A. canadensis nodule

mass, which did not vary with moisture, was almost ten-fold lower in the Bridger soil.

b

b

c

b

c

b

b

a

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

AC DC DP HB LA VA

Species

Nod

ule

Mas

s (g

pot

-1)

60% Field Capacity80% Field Capacity

LSD-0.07

Figure 9. Average nodule mass of all six species in Bridger soils at 60 and 80% field capacity.

28

Drought stress can have a large effect on nodulation and N2 fixation in many

legumes; it is possible that the greenhouse pots retained more water than expected or

perhaps lower soil moisture content is needed to see drought effects. During drought

stress, N2 fixation is often halted before other physiological processes such as

photosynthesis, transpiration, and even new tissue growth, possibly due to plant

restriction on O2 diffusion in the nodule (Sinclair et al. 1987; Durand et al. 1987). So

although the nodule number and mass did not show a large response to drought

conditions in this study, it is still possible that there was a reduction in N2 fixation,

which was not directly measured. It is also possible that because the rhizobia in the

test soils were endemic they could be better adapted to drought conditions than the

species used in other studies. This trial indicated that specific characteristics of each

soil, such as microbial community, shrink-swell potentially and nutrient content, had

d

a

bc

d

cdcd

d d

b

a

ccd

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

AC DC DP HB LA VA

Species

Nod

ule

Mas

s (g

pot

-1)

60% Field Capacity

80% Field Capacity

LSD-0.07

Figure 10. Average nodule mass of all six species in Post Farm soils at 60-80% field capacity.

29

more effect overall on the response of individual species than actual moisture

treatments.

Trial III-Effects of fungicide coating on nodulation, biomass and plant N levels

Materials and Methods

Introduction

The objective of this trial was to assess the effect of fungicide seed coating on

the emergence and survival of a native legume. A simple experiment was designed

with five treatments. The seeds of D. candida were treated with two fungicides

(fludioxonol and metalaxyl) accompanied by a control with no fungicide application.

The germination and emergence of the treated and untreated seeds were recorded in

two soils (Geyser and Red Bluff -Table 2).

Seeding

The D. candida seeds were shaken with each diluted fungicide in a plastic bag

at the label rate (Table 3). Once all the seeds were evenly coated, they were spread flat

on a metal sheet to air dry. The soils and pots were prepared according to the protocol

listed in the Trial I seeding procedures, with the following exceptions. The seeding

Table 3. Fungicide information and dilutions.

Active Ingredient

AI* Dilution H2O Volume Applied

% mL g-seed-1

Fludioxonol 40.3 1: 499 1.20 Metalaxyl 32.3 1: 99 1.04

*Active ingredient

30

rate was increased to 50 PLS per pot, which were planted in two parallel rows 1 cm

deep. Each pot was watered with 50 mL of water and covered with plastic wrap. The

pots were then placed in a growth chamber at 20 ºC with 16 h of light and 8 h of

darkness. Plants were watered every other day with 50 mL of water for one month,

after which watering was reduced to every four days with the same volume. Harvest

again took place five months after seeding.

Data Collection

Germination and emergence were recorded every other day for 14 d and then

every 7 d until 42 days after seeding (DAS). Harvest and data analysis again followed

the protocol described in the greenhouse Trial I.

Trial III Results

Emergence and Plant Survival In the Geyser soil, emergence and survival of plants were higher for both

fungicide and control treatments when compared to the Red Bluff soil (Figure 11). Of

the fungicide treatments, metalaxyl had the largest beneficial effect on survival when

compared to the fludioxonol treatment and the control group in the number of

surviving plants (Figure 12). Both soils had a higher emergence rate when seeds were

treated with the fungicide metalaxyl, a fungicide typically used to stave off slime

molds and other pathogens that have a negative effect on seed germination and young

seedlings. Plant numbers in the fludioxonol were not significantly different from the

31

control group. Overall plant emergence and survival was higher in the Geyser soil

than in the Red Bluff soil.

Figure 11. Emergence rates of D. candida, 42 DAS in Geyser (G) and Red Bluff (RB) soils. Fungicide treatments: fludioxonol (F), metalaxyl (M), and control (C).

0

10

20

30

40

50

60

70

0 7 14 21 28 35 42

DAS

Emer

genc

e (%

)

CGCRBFGFRBMGMRB

Figure 12. Average plant number, five months after seeding D. candida in Geyser and Red Bluff soils treated with fludioxonol and metalaxyl fungicides.

c

b

c

b

a

b

0

10

20

30

40

50

60

70

Control Fludioxonol Metalaxyl

Species

Plan

t Sur

viva

l (%

)

GeyserRed BluffLSD= 12

32

Nodulation and Biomass Nodule mass of D. candida in the Red Bluff soil was less with fungicide than in

the control (Figure 13). Nodule masses from the fungicide treatments in the Geyser soil

were not different from those in the control group. The nodule mass of the Red Bluff

control group was 0.03 g, ten-fold higher than the fungicide treatments, which had

masses between 0.003-0.006 g. However, in the Geyser soil, the control nodules were

just two times the mass in the fungicide treatments, with a mass of 0.004 g for the

control compared to 0.002 g in the treatments. Perhaps because legumes in the Geyser

soil already have difficulty forming nodules the decrease in mass due to fungicide

treatment is less substantial. Nodule numbers exhibited nearly identical results as

nodule mass (Figure 14).

Nodulation appeared to be negatively affected by fungicide treatments,

although the degree of the effect was dependent on soil characteristics. Several studies

have found that although chemical pesticides and fungicides may be effective to some

degree, they may have detrimental effects on symbiotic N2 fixation (Bagnasco et al.

1998). The nodules in the fungicide treatments were typically white in color lacking

the pronounced pink pigmentation often associated with LHb and active N2 fixation

machinery (Figure 15). It is possible that the process of symbiotic N2 fixation is

adversely affected by the two fungicide treatments; more testing should be done to

investigate other chemical and rate effects on legumes.

The proper development and function of nodules and N2 fixation appears to be

hindered by these fungicide treatments. There are several possibilities as to why these

effects are seen. All species of fungus in the soil would be killed by the fungicide

33

treatments. Requena et al. (2001) found that inoculation with indigenous rhizobia and

arbuscular mycorrhizal fungi enhanced legume establishment as well as soil fertility; it

is possible that there may be an indirect connection between mycorrhizal fungi and

symbiotic N2 fixation. Plants with mycorrhizal associations often display increased

resistance to pathogens meaning that it could be possible in some cases for a fungicide

to make a plant more susceptible to infection (Lum and Hirsch 2003). A second

possibility is that the dead fungal biomass following the fungicide treatments resulted

in a bloom of N in the soil as nutrients were released, hindering nodulation. Finally,

there could be a direct effect on rhizobial survival or behavior from the fungicide

chemicals.

Figure 13. Average nodule mass after five months, for D. candida in Geyser and Red Bluff soils treated with fludioxonol and metalaxyl fungicides.

bbb

a

bb

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Control F ludioxonol M etalaxy l

S pe cie s

Nod

ule

Mas

s (g

pot

-1)

GeyserRed B luff

LS D= 0.005

34

Figure 15. Nodules from D. candida, treated with metalaxyl (left) and without fungicide treatment (right).

Figure 14. Average nodule density per plant after five months for D. candida in Geyser and Red Bluff soils treated with fludioxonol and metalaxyl fungicide.

b

bb

a

b

b

0

5

10

15

20

25

30

35

40

45

Control Fludioxonol Metalaxyl

Spe cie s

Nod

ule

Den

sity

(No.

pla

nt -1

)

GeyserRed Bluff

LSD= 12

35

In the Red Bluff soil, there was no significant variation in shoot biomass

between the fungicide treatments, but the control group had significantly higher

biomass (Figure 16). There was no significant variation within the Geyser treatments.

It is possible that the detrimental effect of the fungicide was not apparent in the Geyser

soil because infection and nodulation mechanisms were already constrained in the

Geyser soil (Figure 13). The benefits of fungicide treatments must be weighed with the

cost of possible reduction in nodule function. However, if pathogens are a problem at

a given site, fungicides may prove beneficial to the establishment of these species. If a

site does not have a history of soil pathogen problems, then fungicide application could

be detrimental to nodulation and long-term plant survival. The following trials further

examined the effects of fungicide treatments on nodulation and biomass.

Figure 16. Average dry shoot biomass of D. candida in Geyser and Red Bluff soils treated with fludioxonol and metalaxyl fungicides.

c cc

a

bcb

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Control Fludioxonol Metalaxyl

Species

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

GeyserRed Bluff

LSD=0.3

36

Trial IV: Effects of fungicide drench on nodulation and biomass

Materials and Methods Introduction

Due to the results in Trial III, further tests were designed to assess the effects of

fungicide treatments on nodulation in two native legumes (A. canadensis and L.

argenteus). These species were selected in order to test both a Rhizobium sp., which

nodulates A. canadensis, and a Bradyrhizobia sp., which nodulates L. argenteus. Soils

were collected from Bridger, Post Farm and Moccasin (Table 2). The treatment was a

soil drench, which soaks all the soil in the entire pot with label strength fungicide,

versus the seed coat, which only contacts the seed surface. This drench contained a

wide spectrum fungicide with the active ingredients etridozole (15%) and thiophanate-

methyl (35%). An equal volume of water was used as a drench for the control pots.

There were four replications of each treatment and there were two harvest times over

the course of the experiment. The objective was to assess how the effects of fungicide

on biomass and nodulation changed over time as the plants matured and the fungicide

became systemically diluted within the plant.

Seeding

The basic protocol from Trial I was followed, with the following exceptions.

During the first week in the greenhouse, the first drench application of 200 mL of

fungicide was applied to each pot at a dilution rate of 1000:1. This application was

repeated twice at 7 d intervals for a total of three applications. The control group

37

received a 200 mL water drench at each application. Each pot was watered every 3 d;

a high moisture level was used in order to promote fungal growth. The first harvest

took place at eight weeks after seeding and the second at five months.

Data Collection

Harvest followed the protocol from Trial I, but some additional data were

collected as follows. After removing biomass from the drying oven, the aboveground

biomass of each plant from the second harvest was ground and delivered to the

SPWAL for total percent N analysis of biomass using LECO combustion analysis.

Tissue samples of each treatment were collected from the roots, stems and leaves of

the plants. These tissues were surface sterilized by covering in a 10% sodium

hypochlorite (NaOCl) solution and stirring for five minutes; they were then rinsed with

sterilized DIH2O. The sterilized tissues were then placed on potato dextrose agar

plates (PDA), labeled, and allowed to grow at room temperature for a period of about 7

d. Fungi arising on the plates were identified with the assistance of Dr. Bill Grey of

MSU. Fungi suspected of being pathogenic species were isolated. Isolation was

achieved by taking a small sample from the leading edge of each fungus with a flame-

sterilized wire. This sample was placed on a sterile PDA plate. Isolates were allowed

to culture again for about 7 d at room temperature and were then identified to the

species level via microscopic observation of cellular structure.

38

Trial IV Results Biomass

The biomass from the fungicide treatments appeared to have more substantial

differences from the control during the first harvest indicating that fungicide effects

may be reduced as the fungicide is systemically diluted within the plant (Figure 17 and

18). The A. canadensis plants treated with fungicide in the Post Farm soil had higher

biomass than the control during the first harvest (Figure 17). Conversely, the biomass

of both species was higher in the Moccasin soil when there was no fungicide

application. L. argenteus biomass in the Moccasin soil decreased with fungicide.

Fungicide did not significantly affect biomass in the Bridger soil.

By the second harvest, there was also no significant variation in A. canadensis

biomass between the treatment and control groups in the Bridger soil (Figure 18).

However, the A. canadensis biomasses in the Post Farm and Moccasin soils were

higher in the control compared with the fungicide group. The L. argenteus biomass

from the second harvest in the Bridger soil was larger in the fungicide treatment than

in the control. However, this species no longer had any differences in the Post Farm or

the Moccasin soils (Figure 18). In treatment groups there may have been detrimental

chemical or salt effects from the fungicide that decreased with time. Compared to the

control group, some of the treated plants did show symptoms of chemical toxicity. The

EC of the fungicide solution was not measured, a control with a similar EC or salt

content should be used if these experiments are repeated. Fungicide treatments can

have a positive effect on seedling emergence and plant survival when fungal pathogens

39

are present and virulent in a soil. The benefits of fungicide are most pronounced in

earlier growth stages presumably because this is the period when most species are

more susceptible to disease. However, if there are no pathogens present or if fungal

species are non-virulent, the fungicide may be detrimental to plant health.

Root Structure, Nodules and Fungicide

The root structure of the treated plants from both harvests were healthier, more

flexible, and showed less discoloration and more flexibility, than the control group

which had large patches of pink and brown discoloration (Figure 19). The roots in the

control group were also very brittle and broke easily during harvest and washing.

However, the control plants were more extensively nodulated. The fungal pathogens

Fusarium spp. and Alternaria spp. were isolated from the plant tissues in both Bridger

and Post Farm soils. Specifically, F. solani and F. oxysporum were isolated from plant

tissue grown in the Post Farm soil. Both Fusarium spp. are known to be virulent

fungal pathogens that cause root and stem decay.

dd

d

cd

a b

b c

a

cd cd

b cb

cd

0

0 .1

0 .2

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A C C A C F L A C L A F

S p e c ie s a n d T re a tm e n t

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

B r id g e rM o cca s inP o s t F a rm

L S D - 0 .2 2

Figure 17. Effects of a water (C) drench vs. fungicide (F) drench on average shoot biomass of A. canadensis (AC) and L. argenteus (LA) 8 weeks after seeding.

40

f

de

f f

b

ede

cd de de

bc

a

0.00.51.01.52.02.53.03.54.04.5

AC C AC F LA C LA F

Spe cie s and Tre atme nt

Tota

l Dry

Bio

mas

s (g

pot

-1)

BridgerMoccasinPost Farm

LSD- 0.65

Figure 18. Effects of a water (C) drench vs. fungicide (F) drench on average dry shoot biomass of A. canadensis (AC) and L. argenteus (LA) 5 months after seeding.

Figure 19. L. argenteus grown in Post Farm soil fungicide treatment (left) shows healthier, more robust, root system while untreated control roots had more discoloration and were brittle.

41

N Content There was little significant variation in the percentage of N within the plant

tissue in the second harvest (data not shown); however, when total N content of the

plant was calculated by multiplying the N fraction by the total biomass there were

some significant differences (Figure 20). N content of plants in the Bridger soil were

significantly lower than the levels of the plants grown in the Post Farm and Moccasin

soils.

A. canadensis showed no significant variation in N content between treatments.

However, there were significant differences in total N content of plants grown in the

different soils. Plants from the Bridger soil had the lowest N content and those from

the Post Farm soil had the highest N content. The L. argenteus plants showed similar

significant variation in N content between sites but there was more variation between

treatments in this species. Plants from the Bridger soil showed no variation in either

species. However, L. argenteus from the Moccasin and Post Farm soils had

significantly higher N content in the control treatments. These results indicate that

fungicide may indeed have a detrimental effect on nodulation and/or N2 fixation.

Fungicides have an overall beneficial effect on the condition of root systems but there

was some evidence that fungicides may be harmful to rhizobia or interfere with the

establishment of fully functioning N2 fixation mechanisms.

42

Trial V: Effects of inoculation and fungicide application on nodulation and

biomass

Materials and Methods

Introduction

The final trial of the greenhouse research was designed to test the effects of

fungicide as well as the effects of inoculation on all six species. The primary focus

was to compare the ability of rhizobial strains from a commercial inocula versus the

ability of the endemic rhizobia species to nodulate the legumes. The effect of

fungicide on nodulation and plant growth response, and the combination of both

treatments, was also evaluated.

Figure 20. Effects of a water (C) drench vs. fungicide treatment (T) drench on average shoot biomass total N content of A. canadensis and L. argenteus from the second harvest.

de dee e

b

c

d d

a

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cc

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AC C AC T LA C LA T

Treatm ent

Shoo

t N C

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nt (g

pot

-1)

Bridge r

Moccas in

Pos t Fa rmLSD -0 .02

43

Seeding

In this trial, 5 cm diameter conetainers were used to add soil depth and increase

the stability of the field soils. Soils were collected from Moccasin and Post Farm

(Table 2) and available commercial and experimental inocula were obtained (Table 4).

There were a total of four treatments for each soil type with four replications. All six

species were tested with and without inocula (Table 4). A. canadensis, D. candida,

and L. argenteus were further tested with and without a fludioxonol fungicide seed

coating treatment as well as combining the fungicide with and without inocula. The

additional treatments were excluded to limit the size of the experiment.

Table 4. Rhizobia and legume inocula information.*

Plant Species Rhizobia Name/Type ID Label**

Astragalus canadensis Astragalus Spec. 1 LX1893 Dalea candida Type “M” Crownvetch/Dalea M-4 Dalea purpurea Type “M” Crownvetch/Dalea M-4 Hedysarum boreale Hedysarum spec. 2 LX1897 Vicia americana Vicia Spec. 5 LX1866 Lupinus argenteus Type “H” Lupine H-5 *Inocula obtained from Nitragin Worldwide, Brookfield, WI. **The LX ID labels are experimental and not available for commercial release.

Conetainers were washed and dried prior to filling with the pre-moistened soils.

Eight seeds were planted in each conetainer. Seedlings were thinned to one plant two

weeks after germination. Seeds in the fungicide treatment were coated as described in

Trial III. Following planting, 1 g of peat inocula (Table 4) was mixed with 1 mL of

water and the solution was pipetted over the top of each seed in the conetainer. The

seeds were then covered with 1 cm soil. Each conetainer was watered with 30 mL of

water and placed in the greenhouse to grow for 5 months. The plants were watered

44

with approximately 30 mL of water every 3 d during the growth period. Harvest

followed the procedure listed in Trial IV.

Results Trial V

Introduction

There were major differences in plant responses between soils. A. canadensis

had a much higher biomass than the D. candida, so the following discussion and

figures focus on A. canadensis only, in order to reduce variation and further investigate

the effects of the treatments. The results and a brief discussion on the inoculated vs.

non-inoculated treatments of Dalea spp., H. boreale and V. americana can be found in

Appendix B since there were not enough surviving plants to draw any significant

conclusions. The L. argenteus plants had very few survivors and were removed from

the statistical data analysis and left unpublished.

A. canadensis Biomass

The A. canadensis plants grown in the Moccasin soil generally had higher

biomass production than the plants in the Post Farm soil, although the inoculated only

treatments (CI) were similar in both soils (Figure 21). The combined fungicide and

inocula treatment in the Post Farm soil had the smallest biomass of all, and was

significantly lower compared to the fungicide treatment and the no treatment group.

The inoculation only treatment had the lowest biomass in the Moccasin soil and was

different from the non-inoculated control and fungicide treatments.

45

A. canadensis Nodulation

Nodule numbers in the Moccasin soils showed no differences (Figure 22). In

Post Farm soil, the largest nodule numbers were in the non-inoculated fungicide

treatment. The combined inocula-fungicide treatment had the fewest nodules and of

all the treatments.

Nodule masses showed more differences than the other dependent variables in

this study (Figure 23). All four treatments differed in the Post Farm soil, with the

largest nodule mass in the non-inoculated, fungicide treatment, followed by the non-

inoculated, no fungicide treatment. The combined inocula-fungicide treatment had the

lowest nodule mass; apparently there are some interactions between these treatments.

In the Moccasin soil, the non-inoculated, no fungicide treatment had a higher nodule

mass than all other treatments.

Figure 21. Average total dry biomass of A. canadensis with fungicide (F) and control (C) treatments; both inoculated (I) and non-inoculated (U), in Post Farm and Moccasin soils.

aa

ab

bc

b b

bc

c

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0.30.40.50.6

0.70.8

CU CI FU FI

Tre a tm e nt

Dry

Sho

ot B

iom

ass

(g p

ot-1

)

Moccas inPost FarmLSD-0.2

46

Inoculation

The indigenous rhizobia in the field soils evolved with these legume species

and could have a greater ability to survive and nodulate the plants in this experiment.

Wild legumes form effective symbioses with rhizobia strains isolated from field soils

and many wild legume species can be inoculated and put into crop production (Zahran

1998, 2001; Zahran et al. 1999). The indigenous and commercial strains probably have

differing abilities to form nodules and to fix N2. Some may be more beneficial to the

plants and soil than others. The introduction of the commercial and experimental

rhizobial strains may present competition and selection pressure hindering all strains’

ability to effectively nodulate the plants. Tlusty et al. (2004) found major differences

in plant response to inoculation with various species-specific strains isolated from field

soils. It has been found that inoculation with strains of B. japonicum was more

bc

ab

b

bc

bc

a

bc

d

0

20

40

60

80

100

120

140

160

CU CI FU FI

Treatment

Nod

ule

Den

sity

(No.

pot

-1)

Moccasin

Post FarmLSD-42

Figure 22. Average nodule number per pot of A. canadensis with fungicide and inoculated treatments in Post Farm and Moccasin soils.

47

effective on soybeans in soils that had not been previously cropped to soybeans (Abdel

and Erdman 1964; Weaver and Fredrick 1974; McDermott et al. 1999). The

competitiveness of rhizobial populations is related to the soil environment in which the

strain evolved (McDermott et al. 1991; Lum and Hirsch 2003; Jacot et al. 1999). In

order to get effective inocula for these native legumes it may be necessary to test a

variety of strains isolated from field soils to find effective inocula quality strains.

The total N content of the A. canadensis total biomass varied somewhat (Figure

24). In the Post Farm soil, the combined fungicide-inocula treatment had the lowest N

content. The other three treatments were similar to one another. Inoculation generally

decreased N content. With the combined fungicide-inocula treatment there was less of

an effect on plant N content in the Moccasin soil compared to the Post Farm soil.

Figure 23. Average nodule mass with fungicide and inocula in Post Farm and Moccasin soils.

a

b

b

bc

b

a

c

d

0.000.050.100.150.200.250.300.350.400.450.50

CU CI F U F I

Tre atme n t

Nod

ule

Mas

s (g

pot

-1)

MoccasinPost F armLSD-0.11

48

Greenhouse Studies Summary

Soils from different origins varied in texture, nutrient content, pH and perhaps

more importantly, they apparently contained different microbial communities.

Microbial populations evolve very rapidly and because bacteria, in particular, are

limited in their movement and dispersal, it is not unreasonable to expect the genotypes

of rhizobial strains to vary significantly according to geographic location and soil

environment. These soil characteristics or origins generally had more of an effect on

biomass, nodulation and overall emergence and survival of the legume species than did

most of the other treatments in this research project. A. canadensis often had the

largest amount of biomass, as well as the highest level of nodulation, both in weight

Figure 24. Average total N content of entire A. canadensis biomass from greenhouse Trial V with fungicide and inoculation treatments in Post Farm and Moccasin soils.

a

a

ab

c

bc bcbc

d

0.000

0.005

0.010

0.015

0.020

0.025

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0.035

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C U C I FU FI

T re atme nt

Tota

l N C

onte

nt (g

N p

lant

-1)

Moccas inPos t Farm

LSD -0.011

49

and number, throughout all the greenhouse trials. L. argenteus, H. boreale and V.

americana followed with interchanging, but not significantly different biomass levels.

H. boreale and L. argenteus had a moderate level of nodulation, followed by V.

americana, although these species all varied depending on the field soil tested. The

Dalea spp. generally had the lowest biomass and nodulation level of the six species.

Fungicide treatments, as well as inocula treatments, had varying effects on

plant biomass and the extent of nodulation. These plant responses were highly

dependent on the soil type and microbial populations. Fungicides had inconsistent

effects on emergence and survival, but in general, they appeared to negatively affect

nodulation. Inoculation also produced some mixed responses and generally decreased

shoot biomass, nodule number and mass, again depending upon the field soil used.

Host specificity, competition, and/or soil environment outside of the species’

adaptation ranges are all factors that can affect plant response and should be

considered when using introduced or commercial strains of inocula. It is likely that the

indigenous microbial communities in a soil are of primary importance to legumes.

Symbiosis of rhizobia and fungi, both beneficial and pathogenic, influence plant

survival and production. Isolation and research on endemic strains of rhizobia from

native legume species grown in field soils could improve inocula quality and

effectiveness and may benefit plant establishment.

50

CHAPTER 3

FIELD RESEARCH

Introduction

Legume-grass ecosystems have been found to improve soil fertility, habitat

potential and forage quality; however these systems can be difficult to establish and

maintain (Ridley et al. 2004). In this field study, the establishment, biomass and N2

fixation estimate of six native legume species when seeded with native grasses (Table

5) were evaluated at three field sites. In addition, changes in the total N level of the

soil and effects of legumes on grass biomass and N content were measured. The

selected field sites included the Bridger Plant Materials Center in Bridger (BPMC), the

MSU Post Farm Agricultural Research Center in Bozeman and the MSU Agricultural

Research Station in Moccasin; all three locations are in the state of Montana (Table 6).

Table 5. Grass seed and species information Plant Species Common Name Pure Seed Germination Total Viability PLS*

------------------------------------%--------------------------- Poa secunda Sandberg Bluegrass 93 4 89 83 Elymus trachycaulus Slender Wheatgrass

98 93 93 91

Pascopyrum smithii Western Wheatgrass 93 92 92 86 Elymus lanceolatus Streambank Wheatgrass 98 99 99 97

* PLS- pure live seed

51

Table 6. Field Site Information Bridger Moccasin Post Farm

Past Crop Fallow Grasses Barley Fallow Grasses

Last legume + 10 yrs + 10 yrs 3 yrs Fertilizer No No No Pesticide Applied:

June 2004 Mustang

Applied: July and August 2004 Metathyon No

Irrigation Monthly: May-Sept 2003/2004

Sprinkled 38 mm No

1 time August apx. 2 hours

flooded Yearly Precipitation

Average (mm) 292 390 414

2003 Precipitation (mm) 169 371 345 2004 Precipitation (mm) 215 294 384 Temperature Min-Max

(ºC)

May (ºC) 0.55-16.7 3.3-16.7 1.1-18.3 June (ºC) 11.6-31.6 10-26.7 10-27.8

Materials and Methods

Site Preparation and Design

Soil characteristics were analyzed and basic soil properties at each site were

listed in Table 2. Information on the monthly temperature and precipitation amounts,

as well as site history, fertilizer and irrigation levels for each field site are included in

Table 6. Bridger and Post Farm were tilled and rolled to pack the soil prior to planting.

Moccasin was directly seeded perpendicular to stubble rows.

The treatments at all three sites consisted of alternating rows of a native grass

seed mixture (Table 5) and each legume species listed in Table 1. In addition, two

comparison treatments were used; M. sativa as a crop legume specie was alternated

with the native grass mix, and four native grass plots were seeded without legumes.

Treatments were replicated four times at each of the three field sites, in a complete

52

randomized design. Each site had 42 plots, 1.5 m by 3 m each, consisting of 35

treatments and 7 demonstration blocks in which the grasses were inter-seeded in the

same row as each of the legumes. The total area of each site was approximately 512

m2. Seeds were planted at a rate of 20 pure live seeds per linear foot totaling 200 PLS

per row in each treatment block. Grasses were seeded at 480 PLS per row; they were

inadvertently seeded at twice the normal rate.

Seeding

A four-row drill seeder was used to plant four rows spaced at 0.3 m and seeded

1.3 cm deep. The entire research block was surrounded with the grass seed mix in

order to reduce edge effects. Maintenance of the sites was minimal. Each site was

weeded several times the first season depending on the need for weed control at each

site. Weed removal took place one time at the beginning of the second season. Other

maintenance operations, such as insecticide treatment and irrigation, are listed in Table

6.

Data Collection

Following seeding, soil cores were taken with a 4 cm diameter soil auger 60 cm

deep at the Post Farm and Bridger. At Moccasin, the core depth was decreased to 30

cm due to high gravel content. Each core was placed in a 20 L plastic bucket,

homogenized, placed in a soil sample bag and stored in a cooler. The soil core and

bucket were cleaned using a bottlebrush and paper towels to remove any remaining

53

soil and prevent contamination between samples. Wet weight of each sample was

collected and the samples were dried in a soil dryer at 40˚ C for 14 d and weighed

again to calculate soil moisture content. The samples were then submitted to the MSU

SPWAL for analysis of NO3-N. NH4-N was not measured in 2004 because

concentrations in 2003 were generally very low (1 mg kg-1). Composite samples from

each site were used for routine soil analysis, consisting of texture and selected nutrient

content (Table 2).

Immediately following harvest, soil samples were again collected from each

plot as described earlier. At Moccasin the depth was again reduced to 30 cm. Each

soil sample then went through the prior procedure.

During the first season, germination numbers for both grasses and legumes

were recorded. Harvest began at the Post Farm during the third week of July, 2004.

Moccasin was harvested the fourth week of July and the Bridger site was harvested the

first week of August. Plant biomass was collected during the second season by placing

a 0.6 m by 1.5 m wooden frame over the center of the two interior rows of each plot,

again to reduce edge effects. The grasses and legumes were then clipped separately at

the ground level and the fresh biomass was placed in a 20 L plastic bucket and

weighed in the field. Each row was sub-sampled and weighed again; these sub

samples were then placed in a plant-drying oven at 40º C for 7 d. At this point, the

samples were reweighed and moisture content for each plot was calculated. Each

sample was then ground and submitted to the MSU SPWAL for analysis of total N via

LECO combustion.

54

To estimate the difference in N2 fixation between species, a N mass balance

was calculated by the following equation: N mass balance = N uptake + (soil N 2004-

soil N 2003) in the upper 60 cm (30 cm at Moccasin) of the soil profile. This formula

assumes that leaching, denitrification, mineralization, immobilization and volatilization

were fairly constant between plots. By assuming that, differences in the mass balance

were equated directly with differences in N2 fixation amounts between plots within

each site. although N uptake for below the soil sampling depth would overestimate N

fixation. N content of tissues was multiplied by biomass production to determine N

uptake. Total dry biomass N was estimated to be 1.82 x shoot N content, based on

shoot N: root N ratios from greenhouse studies. The grass-grass plots were used as a

comparison because N2 fixed in these plots should have been essentially zero in each

plot.

Nodulation was verified in the field for each species by collecting a portion of

the root mass and inspecting it for nodulation. Nodule presence and pigmentation were

recorded. Plant tissue was also collected to isolate and identify plant pathogens as

described in Trial IV of the greenhouse studies. Statistical analysis was performed

using a natural log transformation of the data means.

In order to test the accuracy of the data collection as well as the laboratory

analysis, quality control procedures were taken by splitting two field duplicates of two

plant and soil samples every sixteenth sample for each site (n=6) (Dollhopf 2000).

These duplicate samples were labeled as part of the sample sequence and submitted

with the remainder of the samples for data analysis. These splits were used to calculate

the % relative standard deviation (% RSD) for our data samples. The % RSD levels

55

for N content of the soil, dry grass shoot biomass, and dry legume shoot biomass were

18%, 11% and 8% respectively. These are fairly substantial error margins, however, it

is not uncommon to see larger errors when studying natural systems. As will be seen,

the differences between biomasses are substantial; therefore, these errors likely do not

affect the conclusions of the study.

Field Research Results

Site Comparisons Native legumes can have a fairly low germination rate; therefore, emergence

was measured two months after seeding (Figure 27). Germination at the Moccasin site

was the highest, followed by the Post Farm and finally Bridger. Moisture levels at the

time of seeding followed the same pattern with Moccasin having the highest moisture

and Bridger the lowest levels; this probably in part affected the emergence rates at each

site. A. canadensis had low rates at all three sites, although it is slightly outside of its

historical range. M. sativa typically had a higher rate of emergence than the native

legumes, although H. boreale had similar rates at the Moccasin location. According to

McGraw et al. (2003) the optimal temperature for emergence of Dalea spp. is around

25 °C, much higher than the temperatures at the time of seeding. With lower

temperatures, the time to germinate is increased and the total number of seeds to

germinate is slightly decreased; however, the difference in emergence rates for these

species showed no marked difference between 15-30 °C (McGraw et al. 2003). Being

able to germinate well across a wide variety of temperatures is an advantage that native

plant species often have (McGraw et al. 2003).

56

The Post Farm location had the most favorable environmental conditions and

was the highest producer in total biomass of all sites; both the grasses and the legumes

out-performed those at the other two sites (Figure 28). Moccasin and Bridger both had

lower moisture levels as well as insect and wildlife grazing. Plant biomass at the

Moccasin location varied the least between plots and the native legumes at this site

typically produced larger biomasses than those at Bridger, with the exception of A.

canadensis. The M. sativa was very prolific at Bridger and biomass production was

comparable to that of the Post Farm.

Bridger

Bridger had the lowest precipitation of the three sites and the annual rainfall

during the study period was well below the long-term average (Table 6). The plots

were irrigated monthly with sprinkle irrigation to deliver approximately 38 mm across

the site during each irrigation cycle.

Legumes

At two months after planting, less than 20% of the native legumes seeded at

this site had emerged (Figure 27). The emergence rates of the native species at this site

did not vary much and M. sativa had twice the average emergence (44%). M. sativa

had the highest biomass by far (Figure 29), which could be due to the fact that M.

sativa was so prolific at this site and had been previously grown in the vicinity.

Specifically, some M. sativa plants were removed from the other legume and grass

rows during the weeding sessions. M. sativa also has a deeper root system than native

57

legumes and may have been able to access nutrients and deep soil moisture (USDA-

NRCS 2004). Accumulation of N below the root zone is common in irrigated systems

due to leaching. Deeper rooted species, such as M. sativa, can take advantage of N

fixed by more shallow rooted legumes (Ridley et al. 2004).

A. canadensis had the second highest biomass, and was similar to the

biomasses produced by the grass-grass plots (Figure 29). Of the native species this

was typically the most successful during this study. D. candida, L. argenteus and H.

boreale had the next highest biomasses of the native legumes. D. candida is currently

being grown and produced in other plots at BPMC. Two species of insects were found

parasitizing the root system of this species. Many of the legume plots showed signs of

drought stress and grazing damage from insects, antelope and rabbits at the time of

harvest, which would negatively affect biomass results.

Figure 27. Legume emergence in 2003 field season 2 months after planting. A. canadensis (AC), D. candida (DC), D. purpurea (DP), H. boreale (HB), L. argenteus (LA), Grass Mixture (GR), M. sativa (MS), V. americana (VA).

ab

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The H. boreale showed the highest level of water stress and plants in several of

the plots were wilted at the time of harvest. This specie was quick to recover when soil

moisture was restored, but it was also the first specie to show symptoms of wilting. It

was observed in the greenhouse that this specie was able to regenerate from

belowground biomass after extreme drought and loss of most of the original

aboveground biomass.

Nodulation was verified in five of the seven species at this site; L. argenteus

and V. americana showed no signs of nodulation although removal of an intact root

system was not possible. The NRCS lists these two plants as being adapted to pH

ranges slightly lower than that of the soil at Bridger (Table 6), which could affect the

rhizobia as well as plant growth. These are the same species that performed poorly in

Figure 28. Average total combined grasses and legume dry aboveground biomass from field sites.

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this soil during the greenhouse phases. M. sativa was also verified to have nodulation.

It is possible that rhizobia were introduced to the soil by seed or soil at some point. It

is not likely that indigenous rhizobia in Montana soils would be specific to M. sativa.

All of the species except H. boreale flowered at Bridger, although not to the

extent seen at the other two sites. Several of the Dalea spp. plants showed signs of

seed formation at the time of harvest; however, none of the other plants were close to

setting seed, possibly because harvest took place well before most of these species

typically set seed, usually in late August or early September. H. boreale was probably

unable to produce flowers due to the stress from drought and grazing. This specie is

one of the most palatable of the native species and was grazed heavily, which would

effect total biomass data collection.

Combined Biomass The biomass of the grasses plus the native legumes at Bridger showed the most

variation of the three sites (Figure 28 and 30). M. sativa plots had the highest biomass.

A. canadensis and the grass only plots had the next largest biomasses. D. candida and

L. argenteus had the third highest biomasses but were only slightly higher than the

other natives. The results at Bridger were the opposite of the results at the other two

sites, where the grass-native legume plots typically produced the largest grass biomass,

which somewhat reduced the yield in total biomass between the plots. The relative

biomass order of the combined grass-legumes treatments at the Bridger site was similar

to results of the legume biomass (Figure 29 and 30).

60

Grasses

The grasses in the A. canadensis plots had the largest biomass and were similar

to the biomass of the grasses in the grass-grass plots (Figure 31). This suggests that

this native specie had little or no influence on grass biomass production at this site. D.

candida and L. argenteus plots had the next highest grass biomass. H. boreale, D.

purpurea, V. americana and L. argenteus grasses had similar biomasses. This site was

surface irrigated and it is possible that the legume root systems remained close to the

surface in order to obtain moisture, whereas at the other sites, with no irrigation,

moisture was probably obtained deeper in the soil profile This may have resulted in

more competition between the grasses and the smaller native legumes because they

would have obtained moisture and nutrients from the same root zone.

Figure 29. Average legume dry shoot biomass from the Bridger field site. Note log scale.

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Figure 31. Average dry shoot biomass of grasses grown between legume rows from the Bridger field site.

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The largest grass biomass grew between M. sativa rows. This was the only site

where M. sativa plots produced the largest grass biomass; at the other two sites, the

grass biomass in the native legume plots was often larger. It is possible that the deep-

rooted system of the M. sativa plants reduced competition and/or helped maintain soil

moisture and nutrient availability in the rhizosphere, which could benefit the grasses.

Summary The soil at Bridger is fine in texture and has a fairly high salinity and pH

compared to the other sites. It also had the lowest rainfall of the three sites during the

study, yet it was irrigated periodically. The combination of these environmental factors

could have led to difficulties in plant establishment. Insect damage was apparent on all

of the species at the site to varying degrees. Scat from antelope and rabbits was found

in the M. sativa, Dalea spp. and H. boreale plots and there were signs of grazing. M.

sativa was the most prolific species across the site and produced the highest legume

and grass biomasses. The natives often had smaller biomasses at this site and showed

signs of water stress and insect damage, which probably reduced biomass. The

conditions at this site were very poor; however, the survival of plants over the duration

of the study yielded valuable information on how these species respond to adverse

conditions. Many studies have noted the importance of native legume species in N-

limited, high stress conditions because their adaptive strategies and associated rhizobia

can be a primary source of N (Jacot et al. 2000; McDermott et al. 1991; Tlusty et al.

2004; Abdel-Wahab et al. 2002).

63

Moccasin

The annual precipitation at Moccasin during the 2003 season was substantially

higher than during the 2004 season. In July of 2003, biomass production appeared to

be high, however, in August, grasshoppers defoliated the site. Plants recovered fairly

well in 2004 and all seven legume species were able to persist and grow, although the

growth appeared to be reduced from the previous season (Figure 32). In the

greenhouse studies, it was found that approximately 50-60% of the N in legumes was

stored in the root material. It is possible that these plants were able to over winter by

utilizing stored materials in the root system and regenerating new aboveground

biomass. This regenerative ability may prove useful to land managers, as these

species seem to be able to tolerate fairly substantial grazing.

Figure 32. The Moccasin field site in the fall of 2003 following the defoliation of legumes and grasses by grasshoppers (left) and in the spring of 2004 following, regrowth of aboveground biomass at the site (right).

64

At Moccasin, the native legumes had the highest emergence rates of the three

sites (Figure 27). The H. boreale had over 40% emergence, which is higher than

expected for a native species in the field (McGraw et al. 2003). Emergence of the

other natives was between 20-30% with A. canadensis having the lowest rate of

emergence at just over 10%. M. sativa again had the highest level of emergence at

50%, but was not different from the H. boreale. The overall higher emergence at this

site may be due to two primary factors. First, Moccasin had fairly high soil moisture at

seeding, which should have benefited seed germination and emergence. Also, at this

site, seeds were sown into stubble rows, which offer some protection from

environmental elements to seeds and seedlings.

Legumes

A. canadensis did poorly compared to its growth at the other two sites and may

not respond as well as other species to heavy grazing (Figure 33). Astragalus spp. are

considered to be toxic, potentially causing illness and starvation in livestock, wildlife

and humans. These traits may indicate that the plant has adapted to grazing by

producing toxic substances to deter herbivores, rather than responding with aggressive

regrowth. This species is also considered to be adapted to fine textured soils (NRCS,

2004); perhaps the rocky soil texture at this site negatively affected its growth.

Some of the species that did poorly at the Post Farm and Bridger did very well

at this site. H. boreale had the largest biomass of the natives; it also flowered

extensively and some plants were beginning to form seed in July 2004. This specie

recovered very well from the defoliation, and sent out new shoots in response to

65

grazing. The morphology of the H. boreale at this site was very similar to that of M.

sativa. The D. candida and L. argenteus had the next highest biomass and both species

flowered and set some seed. D. purpurea, A. canadensis and V. americana had the

smallest biomass and only the D. purpurea flowered.

Combined Biomass

This site showed the least variation between species. Specifically, when total

biomass was combined, none of the native legume plots varied from one another. Only

the biomasses of the L. argenteus and V. americana were significantly lower than the

M. sativa (Figure 34). Even this response was minimal when compared to the growth

at the other two sites.

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66

Grasses

The grasses in the D. purpurea and grass-grass plots had the highest biomass

production (Figure 35). A. canadensis, H. boreale and D. candida grass biomass were

similar to one another but smaller than the D. purpurea. Grasses grown with V.

americana and L. argenteus had the smallest biomass of the native legume plots. The

grasses in the L. argenteus plots were not different than the grasses from M. sativa

plots. M. sativa had a more aggressive growth rate and probably created more

competition for sunlight, water and nutrients than the native species did, resulting in

reduced grass biomass in the M. sativa plots.

Figure 34. Average of combined grasses and legume dry shoot biomass from the Moccasin field site.

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Summary

Overall biomass production at Moccasin was fairly low for all the species

except H. boreale, which did fairly well at this site. Many of the plants, including the

grasses, were negatively impacted by the defoliation that occurred in August 2003.

This probably resulted in physiological stress within the plants, as well as permanently

removing the N contained in the shoot biomass from that season. The recovery of the

site was impressive. All the species were able to re-grow the following season,

although visually, biomass appeared to be substantially decreased. Again, adaptive

strategies of these native species lead to some interesting responses to a variety of

environmental stresses, and may warrant further study.

Figure 35. Average dry shoot biomass of grasses grown between legume rows from the Moccasin field site.

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Post Farm

Post Farm had the best growing conditions of the three sites over the course of

the study. Precipitation was adequate and there was very little grazing pressure at this

site. M. sativa again had the largest rate of emergence at about 40% while most of the

native species had approximately 20% emergence (Figure 27). A. canadensis had the

lowest emergence of all species. Less than 10% of the seeds emerged, which was less

than the emergence at the other two sites. This was the first site to be seeded; perhaps

the earlier time and lower nighttime temperatures decreased the seedling emergence.

Temperature can have a major effect on seed emergence, and lower temperatures

typically decrease the days to emergence, maximum total emergence and emergence

rates (McGraw et al. 2003).

Legumes

Biomass of M. sativa was much higher than that of the native legumes (Figure

36). A. canadensis, L. argenteus and H. boreale had the largest biomasses of the

native legumes, followed by V. americana and D. candida. D. purpurea had the

smallest biomass, perhaps due to the large grass biomass in these plots, which

appeared to cause a shade effect on the smaller, slower growing legumes. The growth

form of the plants at this site was slightly different than at the other two sites; the

plants at this site were much larger and fuller in most cases.

Nodules were difficult to locate at this site and the plants appeared to be much

more deeply rooted compared to the other sites. Only L. argenteus and A. canadensis

were verified to have nodulation. The soil surface was dry and hard during harvest and

69

it was difficult to obtain an intact root system. However, since all the native species

nodulated in Post Farm soil during the greenhouse studies, it is suspected that there

was nodulation in the field as well. The plots here were only irrigated once, late in the

first season. It is possible that the legumes sent roots deep into the soil in order to

access water. All seven legume species flowered at this site and were beginning to set

seed at harvest.

Combined biomass

A. canadensis, L. argenteus, M. sativa and the grass-grass rows had the highest

combined biomass of grasses and legumes at the Post Farm followed by D. candida, H.

boreale and V. americana (Figure 37). Interestingly, the native legume-grass plots

competed well with M. sativa-grass plots. The D. purpurea combined biomass at this

site was significantly lower than any of the other species. The combined biomass of

the native plots at this site were much larger than those of the other sites, indicating

that under good environmental conditions the native legumes are capable of producing

high amounts of biomass.

Grasses The grasses within the native legume rows produced close to 1000 g m-2 of dry

biomass, which was the highest grass biomass of the three sites (Figure 38). Grass

biomasses were similar when grown between native legume species and were higher

than the grass-grass and M. sativa plots. The grass-grass plots had an average biomass

of about 700 g m-2, while the M. sativa plots produced only half of the grass biomass

70

of the native legume plots. Due to the high biomass production at this site,

competition for resources was probably higher in the grass-grass and M. sativa plots.

Figure 36. Average dry above ground biomass of legumes from the Post Farm field site. Note log scale.

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Summary

Overall, the Post Farm had the highest biomass production of all sites. It also

had the best environmental conditions for the 2003 and 2004 growing seasons. Under

good growing conditions, the native legumes were apparently capable of producing

high biomasses; they were not as productive as the non-natives, although they were

very similar at the Post Farm. Three of the species produced approximately 500 g dry

biomass m-2, which is about half the production of M. sativa. The other three species

had production under 100 g dry biomass m-2. Grasses had the largest biomasses when

grown between native legume rows, probably due to lower levels of competition.

Figure 38. Average dry shoot biomass of grasses grown between legume rows from the Post Farm field site.

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Nitrogen Content

The quality of a plant species as a source of N in a habitat community, as

forage or as green manure can be partially assessed by measuring the N content of the

plant tissue. From these measurements the total amount of N stored in the plant tissue

can be calculated, this information can be useful in selecting plant species for a given

environment. The N and total crude protein content of grasses has been found to be

higher in a grass-legume mixture than in single stand crops (Gökkus et al. 1999;

Sengul 2003; Tasun 1968; Whitehead 1999). There was some variation in the tissue N

content in the legumes at each site in this study (Figure 39). The legumes at Bridger

often had similar and occasionally higher N content than those at the Post Farm and

Moccasin.

At Bridger, D. purpurea had the highest tissue N content of all the species, but

was similar to M. sativa (Figure 39). L. argenteus and V. americana had the lowest

tissue N content as was expected due to lack of nodulation. The N concentrations from

the grass-grass plots at Bridger were significantly lower than the levels in the legumes

(Figure 39-40). The grasses from the M. sativa plots had the highest N concentration

while grasses grown with D. candida had the lowest (Figure 40). There were no

differences in the grasses from the other plots. The high N content of the grasses from

the M. sativa plots is likely due to the extensive root system of the species at the site.

Uptake of mineral N from soils fluctuates as factors such as plant demand, root

dispersal, surface area, volume and growth rates change; it also varies in the midst of

plant genotype and genera (Armstrong et al. 1994; Clements et al. 1993; Gregory

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1988). N availability in the soil was high enough that the N needs of the plant could be

met without the need for N2 fixation.

The plants at Moccasin were highly variable in N content, perhaps because

there was less available N at the Moccasin site to begin with causing the plants to be

more dependent on N2 fixation (Figure 39). A. canadensis, D. purpurea and L.

argenteus had the highest N contents at this site, although the N content of M. sativa

and H. boreale were statistically similar to that of L. argenteus. D. candida and V.

americana had the lowest N contents, but were not statistically similar to one another.

The N content from the grass-grass plots at Moccasin was very low and similar to the

level at the Post Farm (Figure 39). Overall, the grasses at this site had the lowest N

content of all three sites and there was very little difference between plots (Figure 40).

The Dalea spp. at the Post Farm had significantly higher N content than the other

species, which did not differ (Figure 39). The grass-grass N content was very low, but

did not vary significantly from the grasses in the other plots (Figure 40).

The grass tissue N content varied between sites. Grasses from the M. sativa

plots at the Bridger site had the highest N content of all the grasses at each site. The

grasses had a much lower N content than the legumes and the combined average N

content of grasses from this study did not differ between legume plots (Figure 41).

This suggests that two seasons of legume growth are not sufficient to alter the N

content of the soil. The average N content of the native legumes showed no large

differences.

The N content of the grasses and soils showed no increase after two seasons of

growth, however, the N content of the legume biomass could still be beneficial. As the

74

legume plant tissue senesces it would release the assimilated N and thru mineralization

processes in the soil it could likely become bioavailable, thus potentially increasing

soil N. Transfer of N between legumes and adjacent crops occurs primarily through

the decomposition of root material rather than root exudates, so N from the biomass

can be released over winter and become available for plant uptake in the spring (Jha et

al. 1995; Zahran 1998 and 2001). The higher N content of the legumes also makes the

palatable species beneficial to herbivores because the high N content typically results

in higher quality forage (Sengul 2003; Wilkins et al 2001). Grazing is another means

that N in legume crops can become bioavailable within an ecosystem.

Figure 39. Average shoot tissue N content of legumes at three field sites.

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Figure 40. Average shoot tissue N content of grasses at three field sites.

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N2 fixation and N Mass Balance

N mass balances allow analysis of N changes over a season as plants grow and

senesce. Differences in the N mass balance may suggest differences in N2 fixed by the

different legumes, especially if they are higher than the grass-grass plots.

Bridger had the largest differences in N balances between species, with M.

sativa having the only positive N balance (Figure 42 and 43). This could possibly be

due to its aggressive growth at this site. With a larger root mass than the natives it

could have increased the extent of nodulation and improved this specie’s ability to fix

N2. A. canadensis had the least negative N balance at the site, indicating that it may fix

more N2 than the other native species; however, it was similar to the grass-grass plots

suggesting that N differences were more likely due to processes other than N2 fixation

(Figure 43). The Dalea spp., H. boreale and V. americana were all similar while L.

argenteus was the lowest N producer on the site but was not statistically lower than V.

americana.

The N mass balance at the Moccasin site yielded positive results for all species,

suggesting that more N2 fixation was probably occurring here than at the other sites,

although it could also be due to mineralization (Figure 42). The soil at Moccasin had

the lowest initial N concentration of the three sites; this may have increased the N2

fixation in the legumes at this site. M. sativa had a significantly larger N balance than

the native legumes. The N balances for D. purpurea and H. boreale were higher than

those of the other native species at Moccasin (Figure 44). The A. canadensis and D.

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candida N balances were smaller than, but not statistically different from, that of H.

boreale. They also were not different than any of the other native legume species.

The M. sativa N balance was higher than for the native species at the Post Farm

(Figure 45). A. canadensis had the highest N balance of the native species at 100 kg N

ha-1. This specie also had the largest N balance of the natives at all three sites and may

be the most effective N2 fixer (Figure 42). It is realized that the differences in the N

balance could also reflect differences in N uptake from the soil below 60 cm. Deeper-

rooted species, such as M. sativa, may be capable of accessing N at a depth that the

other species cannot, resulting in higher N balances, which could indicate higher rates

of N2 fixation that are actually a result of increased deep N uptake. V. americana had

the lowest N balance at –41 kg N ha-1, although it was not different from the H.

boreale, which had an N balance near zero.

Figure 42. Total N mass balance for each plot at each sites.

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Figure 43. Bridger N mass balance.

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Figure 44. Moccasin nitrogen mass balance.

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Field Study Summary

Striking variations can be found in total N accumulation and plant production

between individual species in any given season (Beck 1992; Schulz et al. 1999;

Unkovich and Pate 2000). This makes it difficult, if not impossible, to make direct

estimates of N2 fixation and yield in a given year. In this field study there were large

biomass variations between sites (Figure 28). Within the legumes, M. sativa had the

highest biomass at all three sites followed by A. canadensis. Grasses were better able

to compete with native legumes, whereas M. sativa was more efficient at competing

for resources. Often the biomass of the grasses was higher when they were grown in

combination with a native legume versus the M. sativa-grass plots (Figures 31, 35 and

38). At Moccasin the grass biomass from the all grass plots were similar to the grass in

Figure 45. Post Farm nitrogen mass balance.

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the native legume plots. At the Post Farm, the grass-grass and the M. sativa-grass plots

had the lowest grass biomass whereas at Bridger the M. sativa-grass and grass only

plots had the highest grass biomass. Biomass at the Post Farm was significantly higher

than the other two sites (Figure 46), likely due to more precipitation (Table 6).

There was no evidence of grazing by wildlife and little evidence of damage

from insects at the Post Farm, but there was some grazing by wildlife and heavy insect

damage at both Bridger and Moccasin. At Bridger and the Post Farm, A. canadensis

had the highest biomass and the highest relative estimate of N2 fixation at all three

sites. V. americana, L. argenteus and the Dalea spp. often had the lower biomasses

and lower N2 fixation estimates. V. americana and H. boreale are two of the most

palatable species; they appeared to be favored among herbivores and may have

endured heavy grazing at some sites. Any tissue that was grazed was not available for

N analysis; however, in a natural habitat any N from the legume tissue not utilized by

the herbivores is later released and becomes bioavailable in the ecosystem. H. boreale

had the highest production of the palatable natives and may prove to be important to

rangeland managers.

N uptake from the field soils was substantial over the short course of this study.

When N mass balances of the legumes were compared to those of the grass-grass plots,

it did not appear that native legumes were substantially increasing N availability. The

introduced specie, M. sativa, produced the most positive N balance at all three sites.

This species has a larger and deeper root system than the native legumes and likely has

a higher N uptake rate and demand than the native species. Due to this large root

system, M. sativa can likely take up N from deeper in the soil profile than 60 cm,

81

which was the soil sampling depth. Either of these possibilities could be expected due

to the aggressive growth of this species compared to the slower growing native species

and a low estimate of N uptake would result in a low estimate of N2 fixation.

N2 fixation estimates were largest at the Post Farm, which had higher initial N

concentration in the soil. The concentration of N within the legume tissue was much

higher than that of the grasses. However, due to the lower biomass yields, the overall

amount of N in the legumes was often lower than or comparable to the grasses. One

management strategy to harness the N content and N2 fixation potential of native

legumes is to allow the legume crop to over-winter, because small amounts of N2 are

fixed year round and as roots and nodules senesce, during the period of no growth, N

will become bioavailable for the next season’s growth (Zahran 2001). Legumes can

also be tilled into the soil and utilized as green-manure; however, a more aggressive

species such as M. sativa would be more beneficial and easier to establish for this role

versus the difficult native species (Banda et al 1994; Snapp et al. 2002). As forage,

grass-legume systems have an advantage, not only can N be transferred between

species, but these systems are often found to have increased yield and light utilization

(Brougham 1958; Haynes 1980; Ledgard 1991). The biomass production of these

systems is also very balanced for use as forage; the grasses have high production and

nutrient content in the spring whereas legumes have higher biomass production in the

summer and early fall (Brougham 1958; Mooso and Wedin 1990).

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CHAPTER 4

LEGUME LAND MANAMENT AND ESTABLISHMENT METHODS

Introduction

In Chapter 1, the history and use of legumes as a natural source of N for crops

and soil remediation was discussed. There is a wide range of uses for legumes in order

to improve soils and increase bioavailable N for plant use. This chapter is dedicated to

land uses of legumes, the individual requirements and parameters of each species, as

well as potential benefits and drawbacks (Table 7). This will provide a reference for

land managers in order to aid in species selection and more successful plant

establishment.

M. sativa yielded higher biomasses than the natives and there may be many

cases where the most beneficial legume would be an introduced variety. However, in

land rehabilitation and habitat restoration scenarios, establishment of non-native

species is often prohibited by environmental policies. For example, introduced species

are undesirable selections for situations like National Parks or fragile habitat

restoration projects. Introduced species are often more aggressive and have a higher

demand for nutrients and water than natives, which often require less maintenance, as

they have evolved regionally and are better adapted to a semi-arid mountain

environment. In this study, the native species often had a higher or similar N

concentration to the M. sativa, meaning that they could be integrated into various

environments and utilized to improve diversity, habitat and forage quality. Legumes

assume a variety of growth forms and are typically more prostrate than grasses which

83

tend to grow more erect; in a mixture, these varied growth forms can improve light

usage and root production and minimize species competition (Haynes 1980; Mooso

and Wedin 1990; Sengul 2003). The variety in legume growth and flower morphology

can also benefit and attract many species of insects, birds and mammals. During this

study, numerous species of birds, insects and mammals were observed to have visited

and utilized the study sites.

Native legumes further benefit wildlife as forage and not only do they have a

high N and protein content but they have been shown to increase the N and crude

protein content of adjacent grasses (Brougham 1958; Haynes 1980; Ledgard 1991).

Most of the native legume species in this study showed a strong ability to re-grow

following defoliation and grazing. In the fall of the 2003 field season, grasshoppers

defoliated the plants at the Moccasin site. There were no aboveground remains of any

of the native legumes in the study area, but by spring 2004, the legumes were growing

again on the site (Figure 32), having sent out aboveground biomass from the intact root

systems.

Species Specific Information

This section will look at the individual species and go over some of their

individual characteristics. Table 7 presents basic information on the species and is

followed by a detailed section on each individual native legume species in this study.

Unless otherwise stated, information on plant characteristics not obtained from this

study was from the USDA-NRCS National Plant Database (http://plant.usda.gov).

84

Table 7. Native legume characteristics

M. sativa

D. candida

D. purpurea

H. boreale

V. americana

L. argenteus

A. canadensis

Mature Height (cm)*

92 61 92 61 38 46 92

Drought Tolerance

**

Medium Medium Medium Medium Medium-Low Medium

Medium

pH Range * 6.1-8 6-8 6-8 5.3-8.1 5.9-7.2 7-8 6-8

Growth Form */**

Single Crown Erect

Multi-stem Decumbent

Multi-stem Erect

Single crown Semi-erect

Rhizomatous Decumbent

Multi-stem Erect

Multi-stem Erect can be Stoloniferous and prostrate

Flower Color */**

Purple White Purple Red Purple Blue White

Seed Vigor */**

High Medium Medium Medium Low Low Medium

Harvest/ Drought

Regrowth**

Rapid Moderate Slow Rapid Moderate-Rapid Moderate Moderate-

Rapid

Palatability * High High slight

toxicity High High High Low

Severe toxicity

Low Mild toxicity

Root Morphology

*/**

Tap root not

distinct

Central tap root

Central tap root

Viney root system

Viney root system

Tap root

not distinct, large lateral

roots

Viney root structure

Root Depth (cm) * 61 35 41 46 25 30 25.4

Soil type * All textures All textures All textures All textures

Coarse or Medium texture

All textures Medium texture

Nodule Morphology

*** I I I I I D I

* Data from USDA-NRCS National Plant Database ** From study data *** I- indeterminate D-determinate

85

A. canadensis This very hardy plant grew well and generally produced the highest biomass of

the natives throughout this study. The seed is very small like the Dalea spp. but it is

much more vigorous upon germination. Most seeds germinated within the first 14

days following planting and the seedling grew much more quickly than the other

natives in the study. The leaves have a typical pea shape, and multiple stems can reach

a height of almost one meter (Figure 46). It can be stoloniferous and prostrate, forming

new plants from underground. It has a web-like root system, which spreads throughout

the soil, and like H. boreale and V. americana, can probably be beneficial in reducing

erosion.

The flowers are large and white in color. They consist of cone shaped

flowerets that form the larger cone-like flower. The seedpods are large and upright, so

Figure 46. A. canadensis at Post Farm site Summer 2004, seedpod (left) flowering (right). Photos: S. Metcalf

86

seed production would probably not be difficult in this species. This species is

particularly attractive to birds and insects. However, it is only mildly palatable and

Astragalus spp. are actually known to be toxic to humans and animals although A.

canadensis has a lower toxicity than other Astragalus species. This species is

commonly known as “loco weed” and many livestock producers claim that after an

animal consumes it they will preferentially graze it over other feed. Many legumes

have been found to have high concentrations of certain amino acids, the type depends

on the plant species. Astragalus sp. has been used medicinally for thousands of years

as an immune system booster to be used only when the body is free of illness

(Ritchason, 1995). The toxicity of A. canadensis may have been an adaptation to

protect it from grazing.

At the Moccasin site, this species had the lowest biomass of all three sites in

2004 and was drastically reduced from the biomass it produced in 2003. This species

appears to be less capable of sending out new shoots in response to grazing compared

to the other legumes. Drought affects this species, but it is slower to show the signs of

wilting seen in the H. boreale and it recovers quickly after soil moisture is restored. It

should most likely not be planted in a drought prone areas or soils.

Dalea spp.

The prairie clovers send out a large central taproot, which can penetrate

deep into the soil and access moisture deep in the soil profile. As was seen during this

research project, the species have a fairly high drought tolerance and do well in dry

areas and well-drained soils.

87

Both species send out several stems, all of which can flower if conditions are

conducive. The D. purpurea is typically more erect than the D. candida in growth

form (Figure 47). The stalks are thin with narrow leaves. The flower is cone-shaped

and formed by many small combined flowerets. This specie is palatable to wildlife

and livestock and the flowers attract a large variety of pollinators. The D. purpurea

leaves are slightly less robust than the D. candida, although there is very little other

difference between the two species aside from flower color. Both species come from

similar seeds, which are very small, and germinated in the greenhouse studies within 7-

14 days of sowing. Germination increased with higher moisture levels during the first

month after planting. Once these plants were established, they were fairly tolerant of

low moisture levels. They also appear to be quite tolerant of low nutrient levels based

on the slow growth rate, occasionally taking two seasons to reach a mature flowering

Figure 47. D. purpurea (Left) and D. candida (Right). Photo from USDA-NRCS National Plant Database http//:plants.usda.gov.

88

stage. These species could easily be used in any land management practice, from

landscaping to wildland restoration. They would also be a good selection for crop

rotation and incorporation into grazing or hay pasture.

Due to the small seed size and slow growth rate, they have difficulty

establishing when inter-seeded with grasses and other more vigorous species.

Broadcast seeding to create a patchy landscape would be most beneficial to getting a

lasting stand of the Dalea spp. The largest specimens of these species in the field

study were at the ends of rows and in gaps where they had ample room to grow and

little competitive pressure from other plants.

H. boreale

This species has a moderately sized seed for a native species and has a

rather long germination period. Seeds in the greenhouse studies often continued to

germinate for up to three months after the planting date.

Figure 48. H. boreale from Post Farm field site summer of 2004. Flowering (left) seedpod (right).

89

H. boreale has a single crown, semi-erect growth form (Figure 48). It sends many

small roots into the soil forming a web-like structure in the soil. The root system

seems to be fairly shallow and does not appear to penetrate as deeply into the soil

profile as the taproot species. This type of root system should be extremely beneficial

in stabilizing soil and reducing surface soil erosion. It has moderate to low drought

tolerance and the leaves and stems wilt at around 55% field capacity. In the

greenhouse, it recovered quickly once soil moisture was increased and was even

capable of regenerating new aboveground biomass from the root structure, even after

the original aboveground biomass had reached permanent wilting point. No data was

located on the exact point at which the root system is no longer capable of

regenerating, but this species does possess a strong ability to regenerate. H. boreale

had the highest biomass at the Moccasin site and showed extreme tolerance to the

grazing, sending many shoots out from the root system. This native species is the most

similar to M. sativa, which is currently the one of the most utilized legumes in the

United States.

The flowers of this species are a typical pea shape and are usually red to pink in

color although there are some different varieties. The seedpod has several seeds within

it that are separately encased; this growth form may make seed harvest difficult (Figure

48). H. boreale is extremely palatable and high in protein content. In the field study,

wildlife readily grazed this species. The flowers are also very beneficial to nectivores

and pollinators; several species were observed frequenting these flowers at both the

Post Farm and Moccasin field sites.

90

L. argenteus This species is very common in Montana and can be found growing from the

open valley floors to the top of the Beartooth Pass. It has a large hard seed and

scarification from winter freeze-thaw cycles or even mild fire can improve germination

rates (Tracy and McNaughton, 1997). It had a very low germination rate and it was

often a month before the seeds germinated and many were simply not viable. This

species is moderately palatable and toxic. It can cause birth defects, such as locked

joints in the fetus of cattle and sheep if it is consumed during the spring by pregnant

animals. Again, high amino acid concentrations are suspected of hindering certain

metabolic processes.

Figure 49. L. argenteus from the Post Farm site. Seedpods (upper left), root nodules (upper right) and full row (below).

91

L. argenteus typically performs better in medium to coarse soils with fairly low

soil moisture and good drainage. It is intolerant of saturation and in moist conditions

can be highly susceptible to fungal pathogens (Saxena 1991; Cakmak and Marschner

1992; Anon 1998; Yu and Rengel 1999). The flower is a large cone shape and blue to

purple in color (Figure 49). Like many legume species, it is broken down into

flowerets and attracts many nectivores and pollinators. The plant is multi-stemmed

and erect with a large leaf and the entire plant is covered in fine silver hairs.

Although L. argenteus is very difficult to cultivate in the greenhouse, it is

remarkably hardy in the wild. It does well over a variety of habitats and elevations and

seems to establish in many harsh areas where the soils are poor. It is often a primary

colonizer in disturbed areas and is also common in open areas of climax community

forests (Tracy and McNaughton, 1996). Overall, L. argenteus is a valuable addition to

restoration seed mixes, especially in high altitude environments where species

selection is limited by extreme environmental conditions and N2 fixation is thought to

be a primary source of N (Jacot et al. 1999). It could be beneficial to further

investigate the adaptation strategies of this species and its associated bradyrhizobia, as

well as assessing N2 fixation capacities of this specie across altitudinal gradients.

V. americana This species has a medium sized seed that is round and fairly hard shelled;

scarification can improve germination. The species had a high degree of variability in

germination with some seeds germinating within seven days, but germination

continued for a two-month period. The growth form is rhizomatous and decumbent; it

92

sends out many tendril like roots and shoots that can climb and weave around other

vegetation (Figure 50).

It does not appear to harm adjacent vegetation as some vine plants can via

strangulation. The root system is diffuse and runs throughout the soil. Roots can

protrude from the soil and convert into aboveground biomass. This species has much

less drought tolerance than the other species and wilts quickly, although it can also

regenerate to some degree from the root system. In the greenhouse and growth

chamber studies, it was apparent that this specie could tolerate a higher level of soil

moisture and longer periods of saturation than the other species in the study. It can be

grown in areas that have higher precipitation and slower drainage.

V. americana is another highly palatable species that is often sought out by

livestock and wildlife. The preferential grazing of this species may make estimates of

total biomass difficult to determine, as it has an extensive root system that will

Figure 50. V. americana from the Post Farm field site. Photo: S. Metcalf

93

continue to send out aboveground biomass in response to grazing pressure. The

flowers are typical pea flowers and are small and purple in color, and the rhizomatous

growth form of this species may make seed production difficult. Birds and insects

seeking nectar often utilize this species.

Technical Management Practices for Field Establishment of Native Legumes

Legume-grass systems have been shown to enhance soil productivity and

fertility through symbiotic N2 fixation, carbon sequestration and conservation of

minerals (Snapp et al. 2002, 2002). The capacity of legumes to fix N2 symbiotically

depends on many factors and can be highly variable depending on soil and

environmental condition as well as the plant species selected. The presence of species-

specific rhizobia is another primary factor affecting N2 fixation. Soil nutrients, N

demands of the plant, moisture levels, pH and a number of other environmental factors

will influence the amount of N2 actually fixed by a certain legume. Because of all

these factors it is difficult to generalize what kind of production can be expected

from a specific legume at a site for any given year, let alone predict what kind of yield

a polyculture will produce (Beck 1992; Schulz et al. 1999; Unkovich and Pate 2000).

Regardless of inconsistencies, it is clear that native legumes are capable of forming

nodules and participating in symbiotic N2 fixation and can benefit soils in multiple

ways. Legume biomass stores a high percentage of N and as the plants senesce or are

consumed, this N will become available to other biota (Zahran 2001).

Native species are slower growing and assimilate nutrients at a slower rate than

agricultural species. Conflicting evidence has been found on legumes’ ability to

94

scavenge N before and after nodulation (Unkovich and Pate 2000). Rennie and Debutz

(1986) showed that non-nodulated and nodulated chickpeas took up the same amount

of N from the soil. Jensen (1997) found nodulated soybeans to take up less N form the

soil after nodulation, but other studies have found that nodulated N2 fixing legumes can

scavenge more N than non-nodulated legumes (George et al. 1993; Sparrow et al

1995). No studies have been found looking at the N uptake rates of these native

species. The ability of plant species to access soil N has much to do with the plant

genotype and its root growth and morphology, as well as the plant demand for N at a

given time (Clements et al. 1993; Armstrong et all 1994; Gregory 1988).

To minimize competition for resources and better utilize available N the best

strategy is careful selection of the plant species within a community (Myers et al.

1997; Crews and Peoples 2004). Selection of diverse species that have different

growth and root morphologies will improve the structure and function of a plant

community. Diversity can reduce species competition and optimize resource

utilization; it can also reduce the risk of large population losses due to pests and

disease.

For successful plant community establishment, seeding should take place in a

mosaic pattern rather than in rows. Species with a faster more aggressive growth habit,

such as grasses and M. sativa should be seeded at a lower rate than the slower growing

native legumes. Diversity will decrease the initial competition with neighboring plants

and allow the natives more space and time to reach a mature state. Broadcast seeding

is a good method of sowing seeds. This should be followed by light mulching or

raking to incorporate the seeds into the soil. Often seeding and inoculation take place

95

in the fall and both seeds and rhizobia are expected to over-winter close to the soil

surface. This can greatly reduce seedling emergence and offers a long period for

rhiziobia to persist in the soil before nodulation can occur (Graham 2004). Sowing

legume and grass seed in the early spring can increase seedling survival and decrease

the time to nodulation. The method of sowing the seeds over stubble rows at Moccasin

was highly effective in reducing weed invasion. It also may have increased seedling

emergence by providing protection against elements such as intense sun, wind, rain or

low temperatures.

Treatments such as fungicides, inocula and fertilizers can be useful in getting

plants established depending on the environmental characteristics at a site. Each of

these treatments has been shown to be either beneficial or detrimental to native legume

survival, and the use of such treatments should be determined on a site by site basis.

Fungicide treatments had a positive effect on seedling emergence and plant survival

when fungal pathogens were present and virulent in a soil. The benefits of fungicide

were most pronounced in earlier growth stages, presumably because this is the time

period when most species are more susceptible to disease. If there are no pathogens

present or if fungal species are non-virulent, the fungicides’ detrimental effects on

plant health may be more than its benefits, resulting in reduced biomass and growth

rate of plants. Fungicides do have an overall beneficial effect on the condition of root

systems, but there is evidence that fungicides may be harmful to rhizobia or interfere

with the establishment of properly functioning N2 fixation mechanisms (Bagnasco

1998). The use of fungicides in the field should be assessed at the time of seeding, as

the threat of pathogens at a given site can change each season. Once plants have

96

become established the need for fungicide is reduced as plants are more susceptible

during early growth stages.

Inoculation can be beneficial in some cases; however, in most of the soils

tested, it was unnecessary. It has been found that inoculation with indigenous rhizobia

can enhance not only plant establishment, but increase soil fertility (Zahran 2001). It

has also been found that nodulation induced by inoculation with a specific strain can be

very low in soils where endemic rhizobia are present and legumes are already known

to nodulate (McDermott et al. 1991). Inocula for native species are limited in

availability and rhizobia for most of the species cannot be obtained commercially. In

addition, many strains that are available are still experimental and not fully understood.

There are many other strains that have not yet been studied or isolated. One method of

obtaining inocula for native legumes in the field is to incorporate a native top soil

known to contain rhizobia specific to the species of interest into the seedbed as an

inocula. This method will allow for the legumes to be inoculated by species that have

adapted to the environmental conditions of the area. If a soil is lacking in species

specific rhizobia then inoculation may increase the extent of nodulation. However, in

soil that contains indigenous rhizobia the addition of inocula may create a competition

between different strains potentially hindering nodulation. Another more intensive, yet

potentially highly effective, method of obtaining inocula is to isolate rhizobia from

native plants that are well nodulated and known to be effective at N2 fixation. These

isolates can be further tested and utilized as a specific inocula; this could, however, be

a long costly process.

97

Elevated levels of N in the soil have been shown to repress nodulation, but it

has also been shown that fertilizers can increase legume production (Sengul 2003;

Unkovich and Pate 2000). The first few seasons of growth are critical to lasting plant

community establishment. If amendments such as fertilizer and fungicide benefit the

seedlings in getting established then they should be considered even if initially

nodulation is hindered. Infection and nodulation occur at the root tips, and because

plants are always generating new roots, nodulation will eventually occur as soil N is

depleted and demand for N increases with plant growth.

Controlling weeds and invasive species may be one of the most important

aspects in the first couple seasons. Many of these native plants reach a substantial size

but are slower to grow and are often out-competed by invasive species with more

aggressive growth habits. If given time to establish properly, native species will have

a better chance at lasting establishment in an area. Larger plants are better able to

compete for space and nutrients within the community and therefore have a better

chance of persisting.

Once established, these native species are hardy, require little maintenance, and

are also capable of reseeding themselves and persisting in the plant community.

Selecting species based on specific site parameters is important to successful plant

establishment and long-term survival. For example, grasses and cereal roots are

usually concentrated near the surface of the soil while legume roots grow and contract

to follow the wetting front of the soil. These differences affect N uptake (Unkovich

and Pate 2000). In some cases, a shallower rooted legume may lend more N to

adjacent grasses. However, intermixture with deeper rooted species such as H. boreale

98

or M. sativa should be utilized to access N that can build up below the root zone. Deep

rooted species are particularly useful in buffer areas of wetlands to absorb N and

prevent excessive N leaching (Crews and Peoples 2004). Legumes have also been

found to be highly beneficial in conserving N when allowed to grow over the fallow

season (Crews and Peoples 2004). They continue to fix N2 as well as scavenging some

soil N, reducing N leaching and volatilization (Crews and Peoples 2004; George et al,

1993). At planting, N from these legumes can be utilized as green-manure or they can

be mowed and grasses or other plants can be seeded over them as with other stubble

rows. The availability of N from decaying legume biomass has been found to be better

synchronized with the demand for N uptake than single and multiple applications of N

fertilizers (Becker and Ladha 1997; Gliessman 1998; Zahran 2001).

Legumes can be a great addition to a number of situations from agricultural

production to a home garden or highway median. Native legumes offer special

benefits because they are suitable for use in the Conservation Reserve Program and

plant establishment in backcountry habitat restoration. The various parameters here

are meant to help land managers with species selection and sowing methods to most

effectively establish native legumes. As with most natural systems, however, there are

no set parameters or given growth responses. There is a large degree of variability

even within a species and each plant can respond differently to certain environmental

conditions.

99

CHAPTER 5

SUMMARY AND CONCLUSIONS Overall this project was very successful. It looked at the individual yields of

six native legume species. Throughout the course of greenhouse and field studies,

factors affecting plant growth and nodulation were analyzed. Rough estimates of N2

fixation capacity were measured at three field sites in Montana.

The primary factors influencing legume response in this study were soil

characteristics such as nutrient content and microbial populations. Significant

variation occurred within species grown in different soils. Components such as soil

nutrient content (primarily N and P) and microbial populations within the soil had a

large effect on how a legume nodulates and, therefore, the amount of N that can be

fixed. Soil nutrient content and endemic microbial communities had major influences

on the growth responses of these species. Germination, emergence, biomass and

nodulation all responded differently depending on soil characteristics.

Agricultural species such as M. sativa produced higher biomass and had higher

estimated levels of N2 fixation when compared to native legumes. Native legumes

were slower growing, but are well-adapted to the semi-arid conditions of Montana and

may be very useful in land rehabilitation projects.

Legumes are able to regulate the rhizobial symbiosis in response to

physiological and environmental conditions. When moisture content was varied, there

was less variation in species growth response between moisture treatments than there

was with the soil origin. A. canadensis, H. boreale, and V. americana often increased

100

in biomass at higher soil moisture levels. The Dalea spp. was less responsive to soil

moisture changes.

Nodule mass was not as dependent on soil moisture and responded more to the

field soil characteristics. There were differences in nodule mass between species grown

in the different soils. This is logical, as the endemic rhizobia in the soil were

responsible for most of the nodulation in this study, and the introduced species from

the inocula in Trial V typically showed a lower degree of nodulation. Whether there

was competition between the endemic rhizobia and the introduced inocula, or the

legumes were not specific to the inocula strains, is not clear. Once again the

parameters that affect successful nodulation are extensive and highly dependent on

plant and rhizobia genotype and environmental conditions.

Fungicide treatments increased seed emergence in some cases and may have

improved plant survival, but overall, there were also many detrimental treatment

effects. Fungicides negatively affected nodulation in general and there was a

pronounced decrease in nodule number and mass depending on soil characteristics. It

is possible that some aspect of the N2 fixation mechanism could be affected by

fungicide applications and an apparent decrease in LHb in the nodules. The cost and

benefits of fungicides must be weighed in order to determine if the threat of pathogens

is worse than the losses to biomass and nodulation that can occur. A combination of

factors probably influences plant response. It is possible that fungicides will benefit

plants when there is risk from plant pathogens. However, the chemical stress to the

plant may be detrimental when there is no threat from pathogens. Application of

101

fungicides, as well as inocula, should be assessed on a site-by-site basis taking into

account specific environmental conditions and soil characteristics.

M. sativa had the highest biomass at all three sites as was expected. A.

canadensis had the strongest overall performance of the natives and did fairly well at

all of the sites and in the greenhouse. Biomasses of the grasses were often highest

when they were grown in combination with a native legume, although at Bridger, the

M. sativa-grass plots had the highest biomass. Biomass at the Post Farm was higher

than the other two sites and the estimated N2 fixation amounts were above average. N

uptake from the field soils was substantial over the course of the study. The N mass

balance showed that native legumes were not substantially increasing N availability

within the soil, but that a fair amount of N was assimilated in the biomass. M. sativa

produced much higher N levels than the native legumes at all three sites. N2 fixation

estimates were higher at sites with lower initial N concentration in the soil; symbiotic

N2 fixation may become more valuable to plants when soil N is limited. Legume

tissues have higher N concentrations than the grasses; therefore, native legumes would

provide higher protein forage for wildlife when used in habitat restoration, as long as

non-toxic species are selected.

Native legumes offer a variety of benefits to any setting. With a variety of

flower colors and growth habits, they offer diversity to seed mixes and can replace M.

sativa as a N source in areas where non-native species may be undesirable. It is hoped

that land managers can use the results of this study to optimizing species selection for a

range of land uses.

102

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APPENDICES

112

APPENDIX A

PRELIMINARY GROWTH CHAMBER EXPERIMENTS

113

APPENDIX A

GREENHOUSE STUDIES

Introduction

The overall goal in this phase was to determine a base rate for symbiotic N2

fixation levels as well as the extent of nodulation for each of the six native legume

species. Each species was placed in a growth pouch containing a liquid plant nutrient

solution containing nutrients essential to plant growth, excluding nitrogen (McDermott

and Graham 1990). This allows measurement of the amount of N the plant is capable

of fixing. The experimental treatments consisted of inoculated and non-inoculated

plants.

Materials and Methods

Rhizobia inocula, which are known to be specific to five of the plant species,

were obtained from Dr. Peter Graham at the University of Minnesota. Inocula for H.

boreale were provided by Dr. Peter van Berkum via Patrick Elia with the Agricultural

Research Service in Maryland. Seedstock for each species was obtained from the

BPMC.

Preparation: Rhizobia contamination check

Each inocula strain was removed from the refrigerator and placed under the

sterile hood. Using a sterile wire loop, each species was streaked across a yeast

manatol agar (YMA) plate (Graham 1963).

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Table A1: Six Legume and Ten Rhizobia Species Plant Species Inocula Type Dalea candida 6808, 6815, 7240, 7205 Rhizobia sp. Dalea purpurea 6808, 6815, 7240, 7205 Rhizobia sp. Hedysarum boreale 3174 Bradyrhizobia sp. Vicia americana 6005, 6101 Rhizobia sp. Lupinus argenteus 6003 Bradyrhizobia sp. Astragalus canadensis 6335, 6355 Rhizobia sp.

The inoculated plates were placed in an incubator at 27 ˚C for 5 d to allow the

colonies to grow. The plates were then removed from the incubator and the bacterial

growth patterns were analyzed. All rhizobia strains appeared to be free of

contamination based on ocular inspection of colony growth.

Seed sterilization methods

Seeds were all surface sterilized to remove any rhizobia or other microbes that

might interfere with the results of this experiment.

Method I

1. 95% EtOH for 30 seconds

2. 5% NaOCl Soln. 5 min

3. Distilled water, rinse 3 min, repeat 5 times

This method of sterilization was used for D. candida, D. purpurea, and A. canadensis.

H. boreale was sterilized following the above procedure except the NaOCl solution

was diluted to 3%, based on preliminary evidence of seed damage at 5%.

Method II

1. 95% EtOH for 30 seconds

2. Concentrated sulfuric acid 15 min

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3. Distilled water rinse quick

4. Distilled water, rinse for 3 min, repeat 5 times

This method was used for V. americana and L. argenteus, the harder seeded species

(Somasegaran et al. 1983). The L. argenteus seeds which still showed bacterial

contamination after method II sterilization were further sterilized using method I.

Following sterilization, seeds were plated on YMA in Petri dishes and allowed

to germinate for 7-10 d, at which point the seedlings were at least 1.5 cm in length.

Any plates that showed signs of bacterial or fungal contamination were discarded. The

uncontaminated seeds were inoculated and placed in growth pouches.

Inocula preparation 15 ml of sterile yeast manatol broth (YMB) solution was added to an

Erlenmeyer flask. Using a flame sterilized wire loop a small sample of each rhizobia

strain was collected from a pure colony on the previously streaked plates. This sample

was added to the YMB solution. These solutions were incubated and shaken in a water

bath at 27º C for 4 d.

Planting and Inoculation Trial A1 Growth pouches, forceps and a 60 mL glass cylinder were autoclaved for 0.5

h. Two L of N-free plant growth solution were autoclaved for 0.75 h. All autoclaving

was conducted at 120ºC.

The following was performed under the sterile hood using aseptic technique.

Each growth pouch was filled with 50 mL of the plant nutrient solution. Two healthy

seedlings were removed with forceps from an uncontaminated germination Petri dish

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and placed in the slit at the top of each growth pouch so that the root was in contact

with the moist paper within the pouch. 0.1 mL of each rhizobia culture was added to

10 mL of YMB solution. 1 mL of this dilution was added to the root tip of each

seedling. Each pouch received a total of 2 mL of inocula. Each legume species was

combined with each available rhizobia strain. Only one replication was used during

this initial trial. Following inoculation the pouches were placed in a growth chamber

set to cycle between 18 ºC and 30 ºC with a 16 h cycle of light and 8 h of darkness.

Nutrient solution and sterile DIH2O were added alternately to each pouch as needed to

maintain a moist contact between the growth pouch and the plant root system. The

plants were allowed to grow in the chamber for 4 months at which point they were

removed and harvested.

Data Collection The presence and quantity of nodules were recorded for each surviving pouch.

Plants were removed from the growth pouches and weighed, then placed in a drying

oven at 30˚ C for 7 d at which point they were removed and weighed again. The

minimum biomass required for laboratory analysis was not reached and the experiment

was terminated.

Results and Data Trial A1

Thirteen of the original forty plants survived. Nodulation occurred in the

Dalea spp. and the V. Americana. The other species did not show nodulation. The L.

argenteus does not tolerate saturation well and did not survive long enough to

nodulate. H. boreale showed root curling but did not form nodules indicating that the

117

rhizobia perhaps induced the initial biochemical signaling process and root curling

began but the infection process could not be completed. H. boreale did not nodulate in

either trial. There was only one experimental inocula for this plant specie and it may

not have been specific to H. boreale.

Trial AII

Introduction This experiment was designed to test a new protocol for growing native

legumes in a N-free setting. Native legumes are typically dryland species; it may be

that the saturated conditions within the growth pouches hindered the growth of these

Table A2. Plant survival in each pouch. Treatment Alive Nodules DC CONT + + + + DC6808 - - - - DC6815 - - - - DC7240 + - + - DC7205 + - + - DP CONT + + + + DP6808 - - - - DP6815 - - - - DP7240 - - - - DP7205 - - - - LA CONT - - - - LA6003 - - - - AC CONT - - - - AC 6335 + - - - AC 6355 - - - - VA CONT + + - - VA6005 + + - + VA6101 - - - - HB CONT + - - - HB3174 + - - - Each pouch has two healthy seedlings. ++: Signifies both seedlings either survived or nodulated, depending on the column. +-: One plant survived or nodulated --: Neither plant survived or nodulated.

118

species. Jars of sand were used in place of growth pouches in hopes that the reduced

moisture level would increase the growth rate of these native species. Preparation of

seeds and inocula followed the procedure outlined in Trial AI.

Planting and Inoculation Quart mason jars containing 480 g of sand and 50 ml of deionized water were

autoclaved for 2 h. Growth pouches, forceps and a 100 mL cylinder were autoclaved

for 0.5 h and 2 L of N-free plant growth solution was autoclaved for 0.75 h. All of the

following were performed under the sterile hood using aseptic technique. Four

seedlings were removed with forceps from the sterile Petri dish and were planted in the

sand in each jar. 0.1 mL of each rhizobia culture was added to 10 mL of YMB

solution. 1 ml of the diluted inocula was added to the sand at the base of each

seedling. Each jar received a total of 4 mL of inocula. Each jar received 40 mL of

growth solution and additional DIH2O was added so that the moisture level of the sand

in all jars was at FC by weight. Each jar was covered with autoclaved plastic and

ventilated with small perforations to allow for airflow, but limit contamination. Jars

were placed in a growth chamber set at 12 ºC to 24 ºC and having a 16-hour cycle of

light.

Maintenance Jars were weighed and nutrient solution was added to maintain FC until roots

reached the base of the jar at which point the soil moisture was maintained at 90% FC.

Plants were allowed to grow for four months attempting to reach the dry weight

119

required for N analysis (0.1g). Unfortunately this weight was not reached and the

experiment was terminated. No data were collected from this trial.

Trial AIII

Introduction This trial attempted to correct the problems that arose during the course of

Trial AI and AII. Native legume seeds are very small and slow growing compared to

the crop legumes that the original protocol was adapted from. Some of the plants also

had a yellowing burn patterns around the leaf edges as well as signs of stunted growth,

indicating that perhaps the nutrient solution was too concentrated for the slow nutrient

uptake of native plants. The changes made to the protocol follow.

Materials and Methods In Trial AII the growth solution evaporated rapidly in the growth chamber

allowing seedlings to dry out quickly. This time, pouches were sealed prior to

autoclaving, leaving only small openings for the seedlings. Seeds were allowed to

germinate in the Petri dishes for 14 d so that the roots were approximately 3 cm long.

The nutrient solution was diluted to one-quarter strength in Trial AII. Planting and

sterilization procedures followed those outlined in Trial AI. During inoculation the

rhizobia culture was not correctly diluted. 1ml of bacterial culture, instead of 0.1 ml,

was added to 10 ml of YMB solution. The pouches were placed in the same growth

chamber from Trial AI and allowed to grow for 3 months. After little growth increase,

0.05 mg L-1 of N was added to the main nutrient solution, in hopes of boosting the

growth rate of the seedlings. Each pouch received 10 mL of the N-free nutrient

120

solution as the liquid level reduced through evaporation. Again this experiment was

terminated due to insufficient biomass, however, the replicates were combined in order

to get N content of the plant tissues. The nutrient solutions from the pouches were

also analyzed for soluble NO3-N and NH4-N content.

Trial AIII Data Nine of the original 36 pouches survived and were submitted for N analysis

(Tables A3-A4). The individual biomass was again too small for analysis. The dry

weights for these plants ranged between 0.014 g and 0.077 g; 0.1 g are required for

testing so the replicates were combined. The N content for the four species that

survived, D. candida, D. purpurea, A. canadensis, and V. americana was measured

using Total Kjeldahl N analysis.

Trial AIII Results The L. argenteus in both the treatment and control group died shortly after

transfer to the growth pouch, apparently unable to survive in the saturated conditions

of the growth pouches. The other five species grew for the first few weeks, after which

the treatment group stopped growing. Nodulation occurred in both the treatment and

the control groups but after two months of little increase in growth, the leaves turned

yellow and began to fall off and all the plants in the treatment group died. The control

plants also formed nodules suggesting contamination may have occurred during the

daily watering process. Two nutrient solutions could be used in the future to reduce

risk of this occurring again. The improper dilution of the inocula may also have

increased contamination risk by increasing the number of rhizobia present by 10 fold.

121

Of the treatment group, only two V. americana plants survived, along with

sixteen plants from the control group. Many of the native species cannot tolerate

extended periods of saturation and therefore did not fare well in the growth pouches.

The native legumes were very slow growing in the growth chamber and H.

boreale and L. argenteus rarely survived the duration of the experiments. In fact, L.

argenteus usually died shortly after germination. This was probably because the

species has no tolerance for saturated conditions. The overall biomass and growth rate

Table A3. Treatments and surviving plants.

Treatment Alive NodulesDC2 + - 3 - DC3 + - 3 - DC4 + + 2 3 DP1 + + 2 2 DP4 + + 2 2 AC3 + + 3 4 AC4 + + 2 1 VA1 + + 2 2 VA2 + + 2 3 + +: 2 surviving plants +-: 1 surviving plant

Table A4. Laboratory analysis of plant tissues and nutrient solution from growth pouches. Plant Sp/Rx

Total Dry Weight (g)

Nodule Number

Plant Number

NO3-N (mg L-1)

NH4-N (mg L-1)

Tissue (%N)

ACC3 0.014 3 2 0.16 1.29 ACC 2.66 ACC4 0.041 3 2 0.22 0.38 DCC2 0.021 3 1 0.05 2.22 DCC3 0.021 4 2 1.09 0.49 DCC 3.23 DCC4 0.026 3 1 0.05 0.38 DPC1 0.033 4 2 0.06 0.26 DPC3 0.022 4 2 0.05 0.32 DPC 2.53 DPC4 0.014 4 2 0.05 0.45 VAC 0.077 2 2 0.14 1.46 VAO 2.20 VAT 0.101 3 2 0.06 0.46

122

of the native legumes were much less than that of the soybeans, an agricultural species

for which these protocols were originally designed. After three months in the growth

chamber soybeans were substantially larger than the native species (Figure A1).

Nitragin Inc. has a protocol set up for testing native species in the growth chamber. It

is similar to the sand jars in Trial II with slight modifications that reduce the moisture

level and alleviate the saturated conditions. If further research is performed on these

native species this method should be adapted for more successful completion of growth

chamber experiments.

Figure A1. Growth pouches containing N-free plant nutrient solution, native legumes (left) and soybeans (right) in the growth chamber. The native legume pouches were seeded 1 month prior to the soybeans and still had comparatively lower biomasses.

123

Biomass

The V. americana has the highest tolerance for saturated conditions. Once this

species germinated and established in the pouches it was quite hardy in the growth

chamber. The biomasses of the V. americana were the largest in most of the

greenhouse experiments. The other surviving species did not vary much and produced

approximately 0.03 g of dry biomass.

Nodulation and N Content

Typically the legumes in the growth chamber formed 2-4 nodules, which were

pink in color indicating LHb was present, and that N2 fixation was occurring. There

was little variation between species. Although the D. purpurea had the highest

average number of nodules, it was only slightly larger than the other species, which

0.000.010.020.030.040.050.060.070.080.090.10

AC DC DP VA

Species

Dry

Tot

al B

iom

ass

(g)

Figure A2. Average legume total dry biomass from growth chamber pouches.

124

averaged about 3 nodules per plant. N content was measured to analyze the extent to

which N2 fixation was occurring, although the extent of conclusions that can be drawn

is very limited due to the lack of adequate replicas.

D. candida solution contained more than twice the NO3 content of the other

four species (Figure A3). D. purpurea had the lowest level, but was fairly similar to

the remaining two species. There was less variation in the concentration of NH4 in the

pouches; all the species had about 1mg L-1 except for the D. purpurea, which only had

0.4 mg L-1. This species also had the highest number of nodules, the low N production

of this species may suggest that the nodule function of this legume-rhizobia

combination was not as effective as the other species. The N content of the plant tissue

did not vary much. V. americana had the largest biomass, so when the total N content

per plant was calculated it had the highest amount of N.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

AC DC DP VA

Species

Nod

ule

Den

sity

(No.

pou

ch -1)

Figure A3. Average nodule number in growth chamber pouches.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

AC DC DP VA

Species

NO

3-N

(mg

L-1)

Figure A3. NO3-N concentration in growth chamber pouches.

125

0.00

0.20

0.40

0.60

0.80

1.00

1.20

AC DC DP VA

Species

NH

4-N

(mg

L-1

)

Figure A4. NH4-N concentration in growth chamber pouches.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

AC DC DP VA

Spe cie s

Bio

mas

s N

Con

trat

ion

(%)

Figure A5. Total N concentration of legume biomass from growth chamber.

126

Summary

The growth chamber experiments were highly unsuccessful, but it was apparent

that the method of testing N2 fixation potential using N-free plant nutrient solution

could be highly effective. Native species are adapted to dry, low nutrient soil and

fairly harsh environmental conditions. The proper protocol must be set up to give

these native species growing conditions that are more similar to their specialized

adaptations. Alleviation of saturated conditions and dilution of the nutrient content in

the growth solution could help these species perform better in these types of

experiments. Under the proper conditions, a more accurate measure of the true N2

fixation potential of these native species could be accomplished.

Figure A6. Average total N per legume species of legume biomass from growth chamber.

0.0000.0010.0010.0020.0020.0030.0030.0040.0040.005

AC DC DP VA

Species

Bio

mas

s N

(g N

pla

nt

-1)

127

APPENDIX B

INOCULATION RESULTS FROM GREENHOUSE TRIAL V

128

APPENDIX B

INOCULATION RESULTS FROM GREENHOUSE TRIAL V

Inoculated and Non-inoculated Results From Greenhouse Trial V

The inoculated and non-inoculated section of this had very poor plant survival

so statistics were not performed on the following data due to unequal and/or

insufficient replicas. D. candida biomass showed no response to inoculation in the

Moccasin soil (Figure B1), whereas, in the Post Farm soil, inoculation apparently

increased the shoot biomass. Biomass of D. purpurea decreased with inocula in the

Moccasin soil but slightly increased with inoculants in the Post Farm soil. H. boreale

showed no response to inoculation in the Moccasin soil but increased in biomass

substantially with inoculation in the Post Farm soil.

D. candida showed little change in nodule mass in response to inoculation in

both soils (Figure B2). The D. purpurea failed to form nodules when inoculated in the

Moccasin soil although the non-inoculated plants formed fairly large nodules. In the

Post Farm, soil inoculation may have increased the mass of the nodules in this specie.

Inoculation apparently increased H. boreale nodule weight substantially in the Post

Farm soil; however, inoculation in the Moccasin soil had essentially no effect or

perhaps an adverse effect on nodulation. The various responses to inoculation could be

due in part to competition between the introduced and endemic rhizobia, as well as the

ability of the different strains to induce nodulation in the host plants. Apparently, the

soil origin and its characteristics are of primary importance when it comes to the plant

129

growth responses to these treatments; it is likely that a combination of factors are

involved.

It is possible that fungicides will benefit plants when there is a risk of virulent

plant pathogens. However, the chemical stress to the plant may be detrimental when

there is no threat of pathogens infecting a plant. The same holds true for inoculation. If

a soil is lacking in species-specific rhizobia, then inoculation can increase the extent of

nodulation. However, the addition of inocula may create competition between the

introduced strain of rhizobia and the indigenous strain.

Figure B1. Average dry shoot biomass response of Dalea spp. and H. boreale from Trial V inoculated (I) vs. un-inoculated (U). Statistics were not performed due to unequal and/or insufficient replicas, due to poor survival.

0

1

2

3

4

5

6

7

DCI DCU DPI DPU HBI HBU

Treatment

Dry

Sho

ot B

iom

ass

(g p

ot -1

)

MoccasinPost Farm

130

Figure B2. Average nodule mass in greenhouse Trial V with fungicide and inoculation treatments in Post Farm and Moccasin soils. Statistics were not performed due to unequal and/or insufficient replicas, due to poor survival.

0

0.05

0.1

0.15

0.2

0.25

DCI DCU DPI DPU HBI HBU

Treatment

Nod

ule

mas

s (g

pot

-1)

MoccasinPost Farm