Transcript AMCOE REPORT Understanding Biological Control of Aflatoxin

Understanding Biological Control of Aflatoxin
Contamination of Corn
K. Damann, LSU; B. Bluhm, U of A; T. Allen, MSU
Report to the National Corn Growers Association
February 25, 2014
San Antonio, TX
Outline
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Introduction & rationale
Field experiments
Spore trapping results
Hybrids for reducing aflatoxin contamination
Future work
RATIONALE FOR UNDERSTANDING BIOLOGICAL CONTROL:
Touching or physical interaction is necessary for intraspecific aflatoxin inhibition.
There is specificity in the touch inhibition of aflatoxin synthesis, i.e. just because
a nontoxigenic strain inhibits toxigenic strain A doesn’t mean that it can also
inhibit toxigenic strain B. (PLoS ONE 6(8): e2347. doi.1371/journal.pone.0023470)
That means that each nontoxigenic biocontrol strain has a spectrum of toxigenic
strains it can and cannot inhibit.
In order to be able to protect corn from contamination a mixture of nontoxigenic
strains which together have the ability to complement each other’s lack of
inhibitory ability appears to be the best approach.
There also appears to be specificity in which toxigenic strains in the soil can infect
or not infect corn. (Phytopathology 101:952-959)
Knowing the “infectious aggressive” strains in the soil (those which infect kernels)
or characteristics of those strains gives us a target against which to select the
most effective biocontrol strains.
In order to determine the “infectious aggressive” strains we are spore trapping to
determine which strains “fly” and when and how our biocontrol strains “fly”.
Suspended Disk Culture
Screened ~ 50 tox- kernel isolates vs 53 tox+.
Af 51 completely inhibited AFB1 of Af 53.
FILTER INSERT-PLATE WELL: Af 51- /53+
In insert:
toxigenic isolate 53 + GS Medium
Filter membrane
Toxin
In well:
atoxigenic isolate 51 + GS Medium
In insert:
½ 53 + ½ 51 + GS Medium
No Toxin
Filter membrane
In well:
½ 53 + ½ 51 + GS Medium
20
0
40
80
60
Atox 21 10 lbs. + Aflaguard 10
lbs.
Atox 21 10 lbs.
Atox 19 10 lbs. + Aflaguard 10
lbs.
Atox 19 10 lbs.
Atox 17 10 lbs. + Aflaguard 10
lbs.
Atox 17 10 lbs.
Aflaguard 20 lbs.
Aflaguard 10 lbs.
Control
Aflatoxin B1 in PPB
2010 St. Joseph Biocontrol Test
120
100
2013
FIELD EXPERIMENTS
BARLEY INOCULUM BIOCONTROL
Louisiana
Mississippi
Arkansas
SPRAY INOCULUM BIOCONTROL
Louisiana
1
0
2
4
Tox 4 + 19 + AG
5
3
Tox 4 + 51 + AG
Tox 4 + 17 + AG
Tox 4 + AG
Tox 4 + 51
6
Tox 4 + 19
Tox 4 + 17
Tox 4
Aflatoxin B1 in ng/ml
7
Damann Barley Dispersal Test
Toxigenic 4 with Atoxigenic A. flavus
2
1
3
Tox 4 + 51 + AG
Tox 4 + 19 + AG
Tox 4 + 17 + AG
4
Tox 4 + AG
5
Tox 4 + 51
8
Tox 4 + 19
9
Tox 4 + 17
Tox 4
0
Nontreated
Aflatoxin B1 in ng/ml
10
Allen Barley Dispersal Field Test
Toxigenic 4 with Atoxigenic A. flavus
7
6
Bluhm field test in Arkansas was flooded
out and no data is available.
Conclusion: Louisiana and Mississippi
showed negligible contamination.
The environmental conditions were not
conducive to aflatoxin contamination
Aflatoxin B1 in ng/ml
40
20
0
TOX 4 + 51 + AG
TOX 4 + 19 + AG
TOX 4 + 17 + AG
TOX 4 + AG
TOX 4 + 51
TOX 4 + 19
80
TOX 4 + 17
100
TOX 4
120
No Treatment
140
Ben Hur Spray Test
Toxigenic 4 with Atoxigenic
A. flavus
60
SPORE TRAPPING EXPERIMENTS
Ionic spore trap
Stub is vortexed in 300ul .001% SDS to remove attached
conidia and liquid suspension spread on an AFPA selective
medium plate.
Colonies counted (>1000) and 10 / plate isolated and single
spored resulting in 295 isolates over ~ 30 days.
DNA extracted, quantified, and SSR PCR performed with
selected primers.
Gel patterns analyzed to determine the putative identity of
the isolates. This was done in the Ben Hur fields where we
knew the identity of the applied nontoxigenic biocontrol
strains and toxigenic strain.
51
51
?
?
4
8
8
4
2013/07 to 2013/08
Ionic Spore Trap Sampling from Ben Hur
For each spore trap stub placed in a 15 ml tube, add 300 µl .001% SDS, vortex for 10 seconds and spread on a single AFPA plate. Try to isolate 10 colonies from each plate.
Isolate # and Isolate Designation
W:/KDamann Lab/Cathy/Data/2013-07 to 2013-08 Ionic Spore Trap Sampling from Ben Hur
# Hours
Sampler
Collected
Number
Day
Date
Time
# Times
Sampler
Collected
Total # of
Colonies
Detected
Total # of
Colonies
Isolated
Average Wind Direction ˚
1
2
3
4
5
6
7
8
1
Wednesday
7/10/2013
3PM-9AM
18
12
0
0
279
2
Thursday
7/11/2013
10AM-8AM
23
15
55
10
314
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
3
Friday
7/12/2013
9AM-8AM
24
16
20
10
11
VCG4
51
VCG4
VCG?
VCG4
VCG4
VCG4
VCG4
4*
Monday
7/15/2013
9AM-8AM
72
48
18
10
50,140,131
VCG4
VCG4
VCG4
VCG4
VCG4
5
Tuesday
7/16/2013
9AM-9AM
17
46
10
106
VCG4
VCG4
VCG4
VCG4
6
Wednesday
7/17/2013
10AM-7AM
22
14
23
10
120
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
7
Thursday
7/18/2013
8AM-9AM
26
18
28
10
34
VCG4
VCG4
VCG4
VCG4
8
Friday
7/19/2013
10AM-8AM
23
15
38
10
154
AG
VCG4
VCG4
VCG4
VCG4
9
Monday
7/22/2013
9AM-10AM
74
50
34
10
243,228,248
VCG4
AG
10*
Tuesday
7/23/2013
11AM-8AM
22
14
19
10
275
VCG?
VCG4
VCG4
11
Wednesday
7/24/2013
9AM-7AM
23
15
25
10
293
VCG4
AG
VCG4
12
Thursday
7/25/2013
8AM-8AM
25
17
33
10
340
13
Friday
7/26/2013
9AM-8AM
24
16
35
10
241
14*
Monday
7/29/2013
9AM-7AM
71
47
100's*
0
259,0.5,286
15
Tuesday
7/30/2013
8AM-8AM
25
17
5
5
280
16
Wednesday
7/31/2013
9AM-8AM
24
16
100's*
0
296
17
Thursday
8/1/2013
9AM-8AM
24
16
36
10
316
VCG4
VCG4
VCG4
18
Friday
8/2/2013
9AM-8AM
24
16
22
10
280
51
VCG4
51
19
Monday
8/5/2013
9AM-8AM
72
48
4
4
302+327+290
AG
20
Tuesday
8/6/2013
9AM-8AM
24
16
36
10
288
51
VCG4
AG
21
Wednesday
8/7/2013
9AM-9AM
25
17
18
10
284
51
VCG4
VCG4
22
Thursday
8/8/2013
10AM-8AM
23
15
13
10
244
VCG4
23
Friday
8/9/2013
9AM-7AM
23
15
53
10
119
24
Monday
8/12/2013
8AM-9AM
74
50
7
7
158,154,168
VCG4
25
Tuesday
8/13/2013
10AM-8AM
23
15
10
10
243
26
Wednesday
8/14/2013
9AM-8AM
24
16
10
10
282
27
Thursday
8/15/2013
9AM-8AM
24
16
13
10
27
AG
28
Friday
8/16/2013
9AM-9AM
25
17
54
10
20
29
Monday
8/19/2013
10AM-9AM
72
48
47
10
104,67,126
30
Thursday
8/22/2013
10AM-9AM
72
48
17
10
145,141,111
31
Wednesday
8/26/2013
10AM-8AM
95
63
55
10
104,122,130,124
32
33
Thursday
Monday
8/29/2013
9/9/2013
9AM-8AM
9AM-9AM
72
265
48
177
72
161
10
10
10AM-9AM
24
25
Tuesday
9/10/2013
16
Total:
9
9
1016
295
VCG4
AG
VCG4
51
VCG4
AG
VCG4
VCG4
AG
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
51
VCG4
VCG4
VCG4
VCG4
VCG4
10
VCG4
51
VCG?
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
AG
AG
VCG4
VCG4
VCG4
VCG4
VCG4
AG
VCG4
AG
VCG4
VCG4
VCG4
VCG4
AG
VCG4
VCG4
VCG4
VCG?
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
51
VCG4
AG
VCG4
51
51
VCG4
VCG4
AG
AG
VCG4
51
VCG4
VCG4
51
VCG4
VCG4
AG
VCG4
VCG4
51
VCG4
VCG4
VCG4
VCG4
51
51
VCG4
VCG4
VCG4
51
VCG4
VCG4
AG
AG
VCG4
VCG4
VCG4
VCG4
VCG4
AG
VCG4
VCG4
VCG4
VCG4
VCG4
VCG4
AG
AG
VCG4
VCG4
VCG4
51
72,81,247
AG
AG
AG
VCG4
VCG4
VCG?
269,267,268,273,289,142,
120,138,132,128,158
VCG4
34**
51
9
141
VCG4
VCG4
51
51
VCG4
51
AG
26/295
8.8%
VCG4
133/295
45.1%
VCG8
0/295
0.0%
VCG?
5/295
1.7%
22/295
7.5%
186/295
63.1%
VCG1
17
19
51
Identified Isolates
HYBRIDS FOR REDUCING
CONTAMINATION
Agrisure 3000GT
Agrisure Viptera 3111
120
Hybrid Comparison –St. Joseph, LA
3000GT (1, 7) vs Viptera 3111(others)
100
Aflatoxin B1 in ng/ml
80
60
40
20
9
0
1
2
3
4
5
6
7
8
10
11
12
20
Amount of Fumonisin
Found in Specially Treated Syngenta Plots 2013
18
16
14
12
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10
11
12
FUTURE WORK
Development of a high throughput industrial strength assay
for aflatoxin production which can be used to screen for
mutants affected in ability to “touch inhibit” or to be “touch
inhibited”.
Development of an Agrobacterium insertional mutagenesis
and selection system for Aspergillus flavus to be used to
generate mutants impaired in touch inhibition.
Harold Lambert A. flavus isolates in coconut cream with aflatoxin levels (ng/ml)
(10µl 5x10⁵ spores/ml in 500µl coconut cream:H₂O incubated for 5 days at 30˚C)
2 A13
22 A6
2 A5
2 A3
2A2
2 A1
27,144
38,360
25,589
0
0
B13
B12
B9
B5
B4
A17
3,675
9,102
6,976
0
37,374
C19
C3
C2
C1
B19
B17
14,269
46,226
52,832
0
8,387
3,342
E2
D10
D9
D4
D3
D2
41,117
44,162
0
45,344
40,038
31,201
59,902
35,633
A flavus toxigenic 53 paired with biocontrol strains
10µl 5x10⁵ spores/ml in 500µl coconut cream:H₂O incubated 5 days at 30˚C.
53
51 + 53
AG + 53
17 + 53
19 + 53 Af36 + 53
USES OF THE “YELLOW” ASSAY
Quick qualitative screen for aflatoxin production.
Quick qualitative assay for detecting isolates having
intraspecific aflatoxin inhibitory ability (touch inhibition)
when paired (putative biocontrol strains).
Quick assay for atoxigenic mutants impaired in intraspecific aflatoxin inhibition. (yellow when paired)
Quick assay for toxigenic mutants impaired in ability
to be inhibited. (yellow when paired)
Aspergillus flavus
disease/ biocontrol cycle
on corn
Parasitic biocontrol =
“competitive inclusion” or
“competitive phenotype
conversion” by “touch
inhibition” or intraspecific
aflatoxin inhibition by
thigmo-down regulation of
aflatoxin synthesis which is
strain specific.
Saprophytic biocontrol =
“competitive exclusion” by
lowering inoculum potential
is epidemiology based.
This provides sustainability.
Photo: Gary Payne, N. C. State Univ.
QUESTIONS?