12/20/2013
Julie Grossman, Sarah Seehaver,
Thanwalee Sooksa-Nguan and
Mary Parr
Department of Soil Science,
North Carolina State University,
U.S.A.
Effects of Vicia villosa cover crop inoculation
on rhizobia ecology in organic farming systems
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12/20/2013
Sarah
Seehaver
Mary
Parr
Thanwalee ‘JiJY’
Sooksa-nguan
Hairy vetch (Vicia villosa); winter
annual legume cover crop
Study 1: Rhizobia population
diversity and inoculant
effectiveness
Study 2: Growth chamber
evaluation of genetically-distinct
rhizobia accessions
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12/20/2013
Cover crop use in organics
§205.205 Crop Rotation Standard, which states
that a "producer must implement a crop rotation
including but not limited to sod, cover crops,
green manure crops, and catch crops.
NOP, 2007
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Legume based organic systems
N2
Crop residue
BNF
Mineral Nitrogen
Decomposition
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Parr et al., Agronomy Journal 2011
Legume Biomass N (kg ha-1)
250.0
200.0
Vicia villosa (Hairy vetch)
Trifolium incarnatum
(Crimson Clover)
150.0
Pisum sativum
(Austrian Winter Pea)
100.0
50.0
0.0
All cover crops terminated in mid-May except those denoted
by * terminated in mid April
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Who inoculates?
Organic systems are dependent on successful
nodulation and BNF, and many organic systems are
inoculated
 Recent survey of 180 growers in the states of North
and South Carolina, U.S.A. (O’Connell, 2013)
 46% use inoculants regularly
 32% do not use inoculants
 16% use inoculants only when introducing new legume
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12/20/2013
N15 Natural Abundance
measurements of V. villosa
show this species to
outperform other cover
crop legumes in BNF across
3 site locations (Parr, 2011)
Study objectives:
Characterize rhizobia
isolated from field
inoculated V. villosa
root nodules and from
uninoculated soils to
determine population
diversity
Evaluate a subset of
genetically-distinct
rhizobia accessions for
symbiotic effectiveness
and performance
Parr, M.; Grossman,J.M.; Reberg-Horton, S.C.; Brinton, C. and Crozier, C. 2011. Nitrogen fixation of
legume cover crops in no-till organic corn production, Agronomy Journal 103(6), 1578-1590.
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Five Vicia villosa ecotypes:
Study 1: Experimental Design
• Auburn Early Cover (AUE)
• Auburn Merit (AUM)
• Winter hardy Early Cover
(EAR)
• USDA Purple Prosperity
(PRO)
• USDA Purple Bounty
(BOU)
At each of 3 sites:
Nodules collected from
each inoculated
treatment
AUM
AU
E
EAR
PRO
BOU
Soil collected from noninoculated perimeter for
trapping of rhizobia
Study 1
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Methods: inoculated plant nodules
• Early spring three root systems excavated and one
nodule removed from each
• Total 142 nodules from field-inoculated plants
Study 1
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Methods: resident rhizobia isolation
V. villosa planted in sterile 1:1
sand/vermiculite mixture to trap
rhizobia
50d of growth
RCBD design
Inoculated with 1mL of 5-1
soil dilutions (Bala, 2000)
Dilutions of non-inoculated soil
collected from field perimeters
Five nodules randomly
selected from each plant =
240 nodules representing
resident strains
Study 1
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Methods: Rhizobia Isolation
Nodule
Study 1
Rhizobia
Colony
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Methods: Rep-PCR Strain Identification
Each lane = one Rhizobia strain
1: Strains screened for nifH (nitrogen
fixing) gene
Primers used: 5'- TAC GGN AAR GGS
GGN ATC GGC AA -3' and reverse
primer sequence: 5'- AGC ATG TCY
TCS AGY TCN TCC A -3’
2: Genomic fingerprinting analysis
using rep-BOX PCR primer
3: GelCompar Sofware
 Sorts strains by similarities in
fingerprint patterns
4. Partial gene sequencing (620-bp) of
nodC: nodCfor540 –
nodCrev1160primers (Sarita et al, 2005)
Study 1
Similar
genetically related
Different
genetically distinct
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12/20/2013
Cosine coefficient (Opt:1.00%) (Tol 5.0%-5.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
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
Study 1
Isolated from
commercial
inoculant
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
Field/Inoculated
.
088
Tidewater/O2
Field/Inoculated
.
72
Tidewater/O2
Field/Inoculated
.
73
Tidewater/O2
Field/Inoculated
.
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
Native
.
292
Piedmont
AU Merit
Native
.
174
Piedmont
Early Cover
Native
.
350
Piedmont
.
201
Piedmont
EAR
Native
.
202
Piedmont
EAR
Native
.
273
CEFS/C2
AU Merit
Native
.
Inoc
2
Native
Field/Inoculated
Native strains
Isolated from field
inoculated plants
Field/Inoculated
Field/Inoculated
Mixed native and
inoculated strains
Phylogenetic tree
of rhizobia strains
Commercial
inoculants showed
differences in
fingerprint banding
patterns from
strains isolated
from nodules
Rhizobia from
inoculated soils
generally clustered
with rhizobia from
un-inoculated soils
From phylogenetic
tree 25 genetically
distinct rhizobia
were selected for
further analysis
Field/Inoculated
Inoculant
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Sequencing of
NodC revealed
lack of match with
inoculant strains
Rhizobia from
Inoculated
field-grown
plants
Commercial
inoculant
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12/20/2013
Study 2: Strain evaluation
Experimental Design
 Hairy vetch seeds surface
sterilized and planted in sterile
leonard jars in a growth
chamber.
 Bottom watered using N-free
Hoagland nutrient solution
(Broughton and Dilworth, 1971)
 The 25 selected strains grown in
TY broth and cell culture used as
inoculant.
 Design: RCBD with four replicate
units and 10 replicates of uninoculated plants
 50d growth then assessed for
nodulation and growth
parameters
Study 2
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12/20/2013
Nodule mass per
unit plant biomass (mg)
Nodule number per
unit plant biomass
Plant nitrogen (%)
HV 342
HV 342
HV 341
HV 341
HV 341
HV 338
HV 338
HV 338
HV 337
HV 337
HV 337
HV 332
HV 332
HV 304
HV 304
HV 278
HV 278
HV 263
HV 263
HV 257
HV 257
HV 256
HV 256
HV 229
HV 229
HV 227
HV 227
HV 227
HV 202
HV 202
HV 202
HV 201
HV 201
HV 201
HV 133
HV 133
HV 133
HV 120
HV 120
HV 120
HV 357
HV 357
HV 347
HV 347
HV 099
HV 099
HV 079
HV 079
HV 072
HV 072
C 13
C 13
C 10
C 10
C 10
C 05
C 05
C 05
C 03
C 03
C 03
Native rhizobia strains
HV 342
HV 332
HV 304
HV 278
HV 263
HV 257
HV 256
HV 229
Field inoculated
HV 357
HV 347
HV 099
HV 079
HV 072
C 13
0
20
40
60
80
0
100
200
300
400
500
Surrogate
for
nitrogen
fixation
0
2
4
6
Study 2
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Plants inoculated
with strains from
commercial
sources
(inoculant or
inoculatedplants) had higher
N concentrations
Effect of rhizobia source on V. villosa
nitrogen concentration (%N)
*
*
% N of plants
inoculated
with rhizobia
from each
source
Study 2
Commercial
inoculant
Inoculated
plants
Native soil
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12/20/2013
Conclusions
• Inoculated hairy vetch (Vicia villosa) contain nodule rhizobia
that are genetically dissimilar to those in the original source
inoculant
• Sequencing of the nodC region of a selection of 25 strains
show all strains to be Rhizobium leguminosarum
• Assessment of strain performance show resident strains to be
both poor and strong performers in terms of plant growth.
• Visit poster number 1900 (Seehaver) for the sister study
evaluating inoculant effectiveness and genetic diversity
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12/20/2013
A lab isn’t a team without its students…..
Thanks!
Projects supported by the Fulbright and NIFA Integrated Organics Programs
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