Mary Parr, January 18, 2011
Overview
Cover crops overview
 Rhizobia and inoculation of hairy vetch
 Biological N fixation (BNF) by winter annual
cover crops in North Carolina

Biological N Fixation
NH4+
NH4+
Legume Cover Crops
Grown during fallow period
 Terminated before cash crop planting

 Incorporation
 Mowing
 Herbicide

N mineralized for
cash crop
BNF in Grain Crops
N2
Export as Yield
Rhizobia
Nodulation
Soil Nitrogen
BNF in cover crop systems
N2
Decomposition
Nutrient Uptake
Rhizobia
Nodulation
Soil Nitrogen
Site locations

Work conducted on 3 North Carolina sites: Center for Environmental
Farming Systems (CEFS), Tidewater, and Piedmont
Piedomont
Tidewater
CEFS
CEFS
Tidewater
Piedmont
Field design



RCBD with 4 blocks
of inoculated vetch
in each location
Soil samples from
uninoculated field
margin used to trap
native rhizobia
Commercial
inoculant rhizobia
isolated in lab
Un-inoculated soil sample region
Inoculated
AU Merit
Inoculated AU
Early Cover
Harvested nodules
Inoculated Purple
Prosperity
Inoculated
“Winter Hardy
Early Cover”
One block of hairy vetch field cultivars
Rhizobia characterization
1. Trapping native
Rhizobia: Sterile
seedlings grown in
sterile sand/native
soil substrates for
28d; RCBD 4 reps
2. Rhizobia removed from
nodules through surface
sterilization and culturing
3. Two molecular methods for Rhizobia assessment:
 nifH gene amplification and digestion with restriction
enzymes Hpa II and Hae III
 rep-PCR (Repetitive element Polymerase Chain
Reaction) using BOX A1R primers

Image analysis using Bionumerics software
(Applied Maths)
Example of gel derived from repPCR analysis
Nodule numbers on non-inoculated vetch (native rhizobia)
varied by site, but not by hairy vetch variety
80
70
b
a
a
60
Auburn Merit
50
Nodule number
Vetch Varieties
Auburn Early Cover
40
Early Cover - Winter
Hardy
Experimental
Population - MEL
30
20
10
Parr, unpublished
0
Tidewater
CEFS
Piedmont
IV V
II
4% 4%
2%
Tidewater
V
3%
I
90%
Native rhizobia nifH genes
varied across sites
Piedmont
I
II
III
IV
IV
25%
III V
4%3%
III
6%
CEFS
II
93%
I
66%
Five distinct banding
patterns were identified
V
100
95
90
85
80
75
70
65
60
55
50
45
40
BOX A1R
35
30
BOX A1R
.
Inoc
17
Inoculant
.
Inoc
18
Inoculant
.
Inoc
24
Inoculant
.
Inoc
11
Inoculant
.
Inoc
13
Inoculant
.
Inoc
10
Inoculant
.
Inoc
14
Inoculant
.
Inoc
9
Inoculant
.
Inoc
12
Inoculant
.
Inoc
4
Inoculant
.
Inoc
5
Inoculant
.
Inoc
8
Inoculant
.
Inoc
6
Inoculant
.
Inoc
7
Inoculant rhizobia
Inoculant
.
149
Piedmont
AU Merit
.
163
CEFS/C2
Purple Prosperi.Native
.
102
Tidewater/O2
.
108
CEFS/C2
EAR
Native
.
211
CEFS/C2
Early Cover
Native
.
158
Tidewater/O2
AU Merit
.
159
Tidewater/O2
AU Merit
.
140
Piedmont
Purple Prosperi.Native
.
113
Tidewater/O2
Purple Prosperi.Native
.
142
Piedmont
Purple Prosperi.Native
.
120
Piedmont
EAR
Native
.
203
Piedmont
EAR
Native
.
134
Piedmont
Early Cover
Native
.
266
Tidewater/O2
Early Cover
Native
.
171
Piedmont
Early Cover
Native
.
265
Tidewater/O2
Early Cover
Native
.
263
Tidewater/O2
Early Cover
Native
.
256
Piedmont
Purple Prosperi.Native
.
257
Piedmont
Purple Prosperi.Native
.
135
Piedmont
Early Cover
Native
.
195
Tidewater/O2
Early Cover
Native
.
196
Tidewater/O2
Early Cover
Native
.
231
Piedmont
EAR
Native
.
199
Tidewater/O2
Early Cover
Native
.
150
Piedmont
AU Merit
Native
.
151
Piedmont
AU Merit
Native
.
254
Piedmont
Purple Prosperi.Native
.
186
Tidewater/O2
AU Merit
.
187
Tidewater/O2
AU Merit
.
160
Tidewater/O2
AU Merit
.
229
Piedmont
EAR
Native
.
335
Tidewater/O2
AU Merit
Native
.
239
CEFS/C2
Purple Prosperi.Native
.
338
Tidewater/O2
Purple Prosperi.Native
.
340
Tidewater/O2
Purple Prosperi.Native
.
341
Tidewater/O2
Purple Prosperi.Native
.
339
Tidewater/O2
Purple Prosperi.Native
.
337
Tidewater/O2
Purple Prosperi.Native
.
278
Tidewater/O2
Purple Prosperi.Native
.
197
Tidewater/O2
Early Cover
Native
.
206
Tidewater/O2
EAR
Native
.
144
Tidewater/O2
EAR
Native
.
117
Piedmont
EAR
Native
.
204
Piedmont
EAR
Native
.
245
Tidewater/O2
AU Merit
Native
.
280
Piedmont
AU Merit
Native
.
121
Piedmont
EAR
Native
.
200
Piedmont
EAR
Native
.
249
Tidewater/O2
EAR
Native
.
227
Piedmont
EAR
Native
.
264
Tidewater/O2
Early Cover
Native
.
244
Tidewater/O2
AU Merit
Native
.
99
Tidewater/O2
Field/Inoculated
.
105
Tidewater/O2
Field/Inoculated
.
079
Tidewater/O2
Field/Inoculated
.
087
Tidewater/O2
Field/Inoculated
.
094
Tidewater/O2
Field/Inoculated
.
090
Tidewater/O2
Field/Inoculated
.
091
Tidewater/O2
Field/Inoculated
.
092
Tidewater/O2
Field/Inoculated
.
097
Tidewater/O2
Field/Inoculated
.
095
Tidewater/O2
.
088
Tidewater/O2
.
72
Tidewater/O2
.
73
Tidewater/O2
.
190
Piedmont
AU Merit
Native
.
191
Piedmont
AU Merit
Native
.
192
Piedmont
AU Merit
Native
.
357
Piedmont
.
347
Piedmont
.
342
Piedmont
EAR
Native
.
344
Piedmont
EAR
Native
.
133
Piedmont
Early Cover
Native
.
222
Piedmont
Purple Prosperi.Native
.
328
Piedmont
Purple Prosperi.Native
.
138
Piedmont
Purple Prosperi.Native
.
282
Piedmont
AU Merit
.
292
Tidewater inoculated-plant rhizobia were 90%
similar to each other, but only 60% similar to
native Tidewater/Piedmont rhizobia
Field/Inoculated
Field/Inoculated
Field/Inoculated
Field/Inoculated
Field/Inoculated
Field/Inoculated
Native
AU Merit
Early Cover
EAR
Native
Field/Inoculated
Piedmont
Piedmont
EAR
EAR
Native
Native
Piedmont
EAR
Native
CEFS/C2
AU Merit
Native
.
202
.
273
CEFS/C2
AU Merit
.Inoc
Inoc
22
.
Native
Piedmont native and a few inoculated-plant
rhizobia
Piedmont
Piedmont
.202
201
.
Phylogenetic tree
comparing all
rhizobia isolates
Most Piedmont and Tidewater native rhizobia
shown to be similar (85% similarity)
Piedmont
Piedmont
.350
201
.
.
273
Field/Inoculated
Piedmont
.
350
.
174
Native
Field/Inoculated
Native
Native
Inoculant
Inoculant
. 3
Inoc
Inoculant
.
358
Piedmont
.
168
CEFS/C2
Purple Prosperi.Native
.
238
CEFS/C2
Purple Prosperi.Native
.
180
CEFS/C2
EAR
Field/Inoculated
Native
.
181
CEFS/C2
EAR
Native
.
235
CEFS/C2
EAR
Native
.
236
CEFS/C2
EAR
Native
.
240
CEFS/C2
Purple Prosperi.Native
.
038
CEFS/C2
Field/Inoculated
.
045
CEFS/C2
Field/Inoculated
.
040
CEFS/C2
Field/Inoculated
.
051
CEFS/C2
.
035
CEFS/C2
.
270
CEFS/C2
AU Merit
Native
.
304
CEFS/C2
EAR
Native
.
306
CEFS/C2
EAR
Native
.
182
CEFS/C2
EAR
Native
.
169
CEFS/C2
Purple Prosperi.Native
.
109
CEFS/C2
EAR
.
129
CEFS/C2
AU Merit
.
130
CEFS/C2
AU Merit
.
128
CEFS/C2
AU Merit
.
215
CEFS/C2
Early Cover
Native
.
216
CEFS/C2
Early Cover
Native
.
110
CEFS/C2
EAR
Native
.
314
CEFS/C2
Purple Prosperi.Native
.
312
CEFS/C2
Purple Prosperi.Native
.
322
CEFS/C2
Early Cover
Field/Inoculated
Field/Inoculated
Native
Native
CEFS native and inoculated-plant
rhizobia only 30% similar to any other
cluster
Objectives
Determine amount of biomass produced
by 14 different legumes
 Quantify total N accumulated
 Determine %N derived from the
atmosphere (%Ndfa)
 Investigate how values change with kill
dates

Research Sites
2009: Piedmont and Tidewater Stations
 2010: Caswell and Piedmont Stations

Tidewater
Piedmont
Caswell
Soil Types

2009
 Tidewater: Portsmouth (Fine-loamy over
sandy or sandy-skeletal, mixed, semiactive,
thermic Typic Umbraquults)
 Piedmont: Lloyd (Fine, kaolinitic, thermic
Rhodic Kanhapludults )

2010
 Caswell: Pocalla loamy sand (Loamy, siliceous,
subactive, thermic Arenic Plinthic Paleudults)
 Piedmont: Mecklenburg Loam (Fine, mixed,
active, thermic Ultic Hapludalfs)
Species
Cultivars
Abbreviation
Vicia villosa (Hairy Vetch)
AU Merit
AUM
AU Early Cover
AUE
Winter Hardy Early Cover
EAR
Purple Prosperity
PRO
Variety Unstated
VET
AU Sunrise
AUS
AU Robin
AUR
Dixie
DIX
Tibbee
TIB
Trifolium mischelanium (Balansa Clover) (2010)
Frontier
FRO
Trifolium alexandrinum (Berseem Clover)
Bigbee
BIG
Trifolium subterraneum (Subterranean Clover)
Denmark
DEN
Pisum sativum sp. Arvense (Austrian Winter
Pea)
Whistler
WHI
Variety Unstated
PEA
TifBlue 78
TIF
Vicia sativa (Common Vetch)
Trifolium incarnatum (Crimson Clover)
Lupinus angustifolius (Lupin)
Berseem Clover
Balansa Clover
Crimson Clover
Austrian Winter Pea
Hairy Vetch
Subterranean Clover
Lupine
Common Vetch
Field Experiment Design
Main Plot – CRBD: Cover Crop + Controls
randomly distributed


Sub Plot – Strip
plot, termination
Date/Plant Date
R1 R3 R2 R1 R3 R2 R1 R3 R2
R1 R3 R2 R1 R3 R2 R1 R3 R2
R1 R3 R2 R1 R3 R2 R1 R3 R2
Legume biomass production
Rye Mixes
Crimson Clover
Hairy Vetch
Pea
* Denotes data from 2010 only
Nitrogen content of Legumes
Rye Mixes
Hairy Vetch
Pea
Crimson Clover
* Denotes data from 2010 only
Peak Biomass N groups
Vetch has higher
N content later in
spring
Berseem clover
gains N until late
May
Crimson clover
peaks in N
content in mid –
late April
Vetches have lowest C:N Ratios
Crimson Clover
Hairy Vetch
Mixes
* Denotes data from 2010 only
Nitrogen fixed 2010
Mixes
HV
Pea
HV
CC
Pea
Mixes
CC
Conclusions of N-Fixation

Peak N content varies by species and date

Vetch and pea lower C:N than clovers and
mixes

Tested cover crops acquired a great deal of N
from atmosphere.

Between 50 (Caswell) and >150 (Piedmont)
kg N ha-1 entering system from atmosphere.
Acknowledgements
Committee Members: Julie Grossman, Chris Reberg-Horton, Carl Crozier
Technicians: Sarah Seehaver, Carrie Brinton
Lab Members
Objectives



Determine rate cover
crop N mineralizes
Determine N
availability to corn
roots
How cover crop mulch
treatments affect corn
yields
Treatments

Treatments:
 AU Early Cover Hairy
Vetch (HV)
 AU Sunrise Crimson
Clover (CC)
 AU Early Cover +Rye
(HV+RYE)

Controls
 168 Kg N ha-1
 0 kg N ha-1
Soil N Monitoring
Mineral N Pool: Soil
Samples
Mineralization Flux: PRS
Probes
Ion exchange
resin
membranes
collect NO3 and
NH4
20 cm composite soil
samples taken every 2 wks
Extracted with 1M KCl and
analyzed for NO3 and NH4
PVC
collars to
exclude
root
competitio
n
Grain Yields Tidewater 2009
Grain Yields Piedmont 2010
Organic Agriculture in the US