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Mature fruit cluster
Aflatoxin and its Control in Pistachios
Themis J. Michailides
University of California Davis
Kearney Agric. Research and Extension
Pistachio Short Course – 16 November 2017
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Tree nuts
• Low water activity &
• High in oilResistant to
spoilage
undesired damage (insects, drought
stress, etc.)
M O L D SToxic
secondary metabolites
“Mycotoxins”
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Mycotoxins = toxic compounds
illnesses
deaths
Aspergillus Penicillium Fusarium
Toxic effects of mycotoxins• Carcinogenic
• Mutagenic
• Teratogenic
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Certain species of Aspergillus produce
mycotoxins called aflatoxins?
AFs are secondary metabolites produced by:
•Aspergillus flavus and A. parasiticus
Aflatoxins B1, B2, G1, G2, M1
H
O
O
OO
O
OCH3
H
16
15
13
12
3 4
56
7
8
9
10
11
12
14
Aflatoxin B1
The most potent can cause liver cancer}
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Commodities contaminated with aflatoxins:
Highest risk of aflatoxin contamination:
Occasionally contaminated:
Corn
Peanuts
Cottonseed
Tree nuts (almonds, pistachios, walnuts)
Figs
Sorghum
Spices
Others
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Frequency of aflatoxin contamination
in California pistachio orchards
1 nut in 5,000 nuts (off years)
to
1 nut in 20,000 nuts (on years)
• USA
Aflatoxin B110 ppb
Total aflatoxins 15 ppb
• European Union
Aflatoxin B1 8 ppb
Total aflatoxins10 ppb
(in pistachios for direct consumption)
Regulatory limits for aflatoxins
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OUTLINE:
• Biology and fungal variability of fungi
producing aflatoxin
• Risk factors for aflatoxin contamination
• Biocontrol of aflatoxin
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OUTLINE:
• Biology and fungal variability of fungi
producing aflatoxin
• Risk factors for aflatoxin contamination
• Biocontrol of aflatoxins
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Molds that can produce aflatoxin in pistachio orchards
in California
Aspergillus flavus Aspergillus parasiticus
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most toxigenic
L - strain
Strains of Aspergillus flavus
M - strain S - strain
?about 50:50
toxigenic: atoxigenic
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Aflatoxin production by isolates of Aspergillus sect. Flavi
in liquid culture
Source: Dr. P. Cotty, USDA & Univ. of Arizona
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Density of A. flavus/A. parasiticus in soil
Orchard cfu/g
A 1.7
F 4.4
D 11.3
C 12.7
G 12.7
H 13.1
J 15.2
I 23.1
E 27.0
B 35.5
Selective medium Total fungal count = 58,000 cfu/g
propagules
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Sporulation on debris
Infection
of nuts
on trees
Survival on
orchard debris
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OUTLINE:
• Biology and fungal variability of fungi
producing aflatoxin
• Risk factors for aflatoxin contamination
• Biocontrol of aflatoxin
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Risk factors:
1) Navel orangeworm (NOW)
2) Harvest date
3) Location
4) Year (on /off)
5) Cultural practices
6) Rootstock
7) Various nut defects
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1. Navel orangeworm (NOW):
A moth on an early split pistachio
UC IPM photo
Adult of NOW
Places for
laying eggs
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Effect of feeding sites (wounds) on levels of aflatoxin
contamination
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Navel orangeworm (%)
0-0.5 0.5-1.0 1.0-2.0 >2.0
Sam
ple
s w
ith
afl
ato
xin
(%
)
0
10
20
30
40
> 0.0 ppb
> 10 ppb
> 100 ppb
Relationship of navel orangeworm infestation and
aflatoxin levels
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Si10
Sticky traps with NOW moths
Association of NOW with aflatoxigenic fungi
A. flavus
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Aspergillus sect. Flavi on NOW moths from
pistachio blocks (Madera Co.; 2012)
0
5
10
15
20
25
30
35
40
45
50
23-M
ar
6-A
pr
20-A
pr
4-M
ay18-M
ay
1-J
un
15-J
un
29-J
un
13-J
ul
27-J
ul
10-A
ug
24-A
ug
7-S
ep
Inc
ide
nc
e (
%)
Block 21
Block 22
AW S
AW North
McK
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Frequency estimate of aflatoxin-producing isolates
carried by NOW
Isolates/strains from NOW Orchard 1- Madera Orchard 2 - Madera
Isolates tested 41 24
S-strain recovered 2 3
L-strain positive for aflatoxin 7 7
Total toxigenic strains 9 10
Toxigenic strains (%) 22 % 42 %
Aspergillus sect. Flavi carried
by NOW at harvest (%)10 % 10 %
Toxigenic strains carried by
NOW at harvest (%)2.2 % 4.2 %
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2. Harvest date: Delaying harvest increases NOW
infestation
Year 1 Year 2Harvest date NOW(%) Harvest date NOW(%)
10 Sept. 1.8 14 Sept. 1.8
20 Sept. 5.2 20 Sept. 3.1
30 Sept. 12.1 28 Sept. 6.1
4 Oct. 9.1
12 Oct. 14.2
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y = 0.005e0.0489x
R² = 0.94867
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
7 14 21 28 35 42 49 56
Kings County 2016: Regression of Average Weekly NOW
damage over time (Day 7 = August 28)
Days from August 21
Pe
rce
nt N
OW
Da
ma
ge
Source: Dr. Joel Siegel, ARS-USDA
In general, the damage of NOW you have after the first
week of harvest will be doubled following the third week
of harvest (according to Dr. Siegel)
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Earlier 14 Sept.
14-22 Sept.
Later 22 Sept.
Reshakes
Sa
mp
les
wit
h a
fla
tox
in (
%)
0
10
20
30
40
50
> 0.0 ppb
> 10 ppb
> 100 ppb
Harvest dates, incidence, and amounts of
aflatoxin contamination
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Recommendations:
Follow an aggressive
insecticide program (spray
according to recommendations
given by UC entomologists).
Decrease number of nuts left
after harvest.
Winter sanitation should be
an annual practice.
Do not delay harvest.
Reduce damage by NOW
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Sweep the berms and grind nuts in the middles
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Samples with
County >100.0 ppb (%)
Merced 12.5 (2nd)
Tulare 3.6
Madera 5.8 (3rd)
Fresno 15.4 (1st)
Kern 1.4
Kings 0.0
Northern Calif. 0.0
3. Location/region: Incidence of aflatoxin contamination by county
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4. Effect of year (on or off): Results from
aflatoxin evaluations of library samples for 2001-
2005
Percentage of samples
Year >0.0 ppb >10.0 ppb >100.0 ppb
2001 (off) 27.4 13.9 3.8
2002 (on) 9.8 5.6 0.7
2003 (off) 34.7 19.7 2.7
2004 (on) 12.2 3.5 0.0
2005 (off) 16.0 4.8 0.9
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4. Effect of year (on and off): Frequency
of aflatoxin contamination in pistachio
orchards
1 nut in 5,000 nuts (off years)
to
1 nut in 20,000 nuts (on years)
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5. Cultural practices
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Infection by Aspergillus
fungi and infestation by
NOW
Early splits
Early split nuts are one of the
main sources of aflatoxin
contamination in pistachio
nuts
In addition to NOW damage:
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Incidence of early splits (ES) in 10 pistachio
orchards
Orchard Early split nuts (%)
I 0.6
J 0.6
C 0.8
G 2.0
H 2.7
F 3.4
D 3.4
A 4.3
B 5.7
E 6.5
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rough, shriveled hull smooth hull
Normal
Early splits
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Percentage of aflatoxins
0 20 40 60 80 100
Normal hulls
Smooth hulls
Rough hulls
1991 1992
Incidence of total aflatoxins detected in ES
and normal pistachio nuts
Characteristics of
nuts
ES
Year 1
Year 2
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Characteristics
of early split fruit
positive
samples
(%)
aflatoxins
per nut
(ppb)
% of total
aflatoxins
Rough hull;NOW 60 2998 83.7
Rough hull; no NOW 20 141 16.2
Smooth hull; NOW 20 2 0.1
Smooth hull; no NOW 0 0 0.0
Relationship of the hull appearance and NOW
infestation with kernels of early split pistachios
The normal nuts had no aflatoxins.
99.9%
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Early split nuts (%)
0 2 4 6 8 10 12
a
b
b
a
Skip one in May
Normal
Skip one in May
Normal
Recommendation: Apply sufficient irrigation during spring to
avoid tree stress and reduce ES.
Orchard A
Orchard B
IRRIGATION:
Effect of irrigation on incidence of early splits (ES)
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6. Rootstock: Four major rootstocks are used in pistachio in
California
Pistacia atlantica
UCB1 (hybrid)
PG I (Pioneer Gold)
PGII (Pioneer Gold)P. integerrima
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Effect of rootstock on the early split nuts in four
rootstock trials
Fresno County
Rootstock KAC Westside Kern Co. Madera Co.
Atlantica 4.7 a 0.8 a 0.4 b 16.6 a
PGII 4.0 ab 1.8 a 0.9 a 10.3 b
PGI 4.0 ab 0.1 b 0.3 bc 9.3 b
UCB-1 2.1 b 0.1 b 0.1 c 7.2 b
Recommendation: Use a rootstock that minimizes
early split nuts.
------------- Early splits (%) ---------------
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High in aflatoxins!
7. Various nut defects: Damaged by other means
(DBOM)
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DBOM (or C-nuts) from a processing plant (after surface sterilization)
No stain Yellow stain
A.
fla
vu
s (
%)
0
20
40
60
80
100
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suture
staining
7. Various defects: Early splits and stained nuts
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7. Nuts with various defects
DBOM
OILY
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Apply sufficient irrigation during spring to
avoid tree stress.
Use a rootstock that minimizes early
split nuts (i.e. UCB1, PGI).
Recommendations to reduce aflatoxin:
Control navel orangeworm (NOW). ***
Reduce early splits.**
Do not delay harvest. ***
Sort out damaged and defected nuts.**
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Control of Aflatoxins (biocontrol):
Use of atoxigenic strains of Aspergillus
flavus as biopesticides to reduce aflatoxins
(Atoxigenic strains = those strains that do not produce aflatoxins)
Rationale: The atoxigenic strains when applied in the field, increase in numbers, and displace the aflatoxigenic strains.
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most toxigenic
L - strain
Strains of Aspergillus flavus
M - strain S - strain
undescribedabout 50:50
toxigenic: atoxigenic
AF36
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Natural occurrence of the selected atoxigenic strain
AF36 among A. flavus isolates from orchards in
California
AF36 (%)
County Pistachio Almond Fig
Butte … 6.5 …
Colusa … 3.0 …
Fresno 3.1 … 6.1
Glenn … 4.4 …
Kern 12.7 8.5 …
Madera 7.2 5.0 7.2
Merced 15.0 … 5.8
Tulare 2.9 … …
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Flood-irrigated orchard initially (2001); and since
(2002):
Micro-sprinkler irrigated orchard (AF36) (2003)
AF36
Micro-sprinkler
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As applied
After growth
of AF36
AF36 inoculum Sporulation
Irrigation is needed for
spore production
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Inoculum of atoxigenic strain AF36 stored and
shipped to the growers
50 lbs
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Delivery of AF36 inoculum for pistachio treatment
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AF36
Inoculum
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Application rate: 10 lbs. per acre
(=11.2 kg per ha)
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After irrigation, the wet wheat seeds will produce
spores of AF36
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Year
AF
36
(%
)
0
20
40
60
80
100
AF36 product
Untreated
2008 2009 2010 2011
ns
a
b
a
b
a
b
a
b
a
b
Percentage of A. flavus isolates from soil belonging to AF36
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Non-treated
Orchard
Fungal content is the same in treated and untreated orchardsTreated Orchard
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Label has orchard location, date, and grower.
Library samples for aflatoxin analyses
Doster et al. (2014), Plant
Disease 98:948-956
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Reduction in contaminated samples with aflatoxin
– second harvest
2008 2009 2010 2011 2008-2011
Re
ductio
n o
f co
nta
min
ate
d s
am
ple
s (
%)
0
10
20
30
40
50
20.4%
38.6%
44.9%
36.7%39.9%
(4 years average)
Reduction in aflatoxin-contaminated pistachio
samples (1st and 2nd harvests)
(Doster et al. (2014), Plant Disease 98:948-956)
P value =0.0033
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2009 2010 2011 2009-2011
Reduction o
f conta
min
ate
d s
am
ple
s (
%)
0
20
40
60
80
100
23.6%
85.4%
58.1%54.6%
(3 years average)
Reduction in aflatoxin-contaminated pistachio
samples (2nd harvest)
2008
-No
sam
ple
sAbout
55%
reduction
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About 73,000 acres of pistachios have
been treated in 2012
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Treated pistachio acreage with AF36
Year Acreage treated (ha)
2012 73,000 acres (29,500 ha)
2013 150,000 acres (60,700 ha)
2014 200,000 acres (80,900 ha)
2015 200,000 acres (80,900 ha)
2016 200,000 acres (80,900 ha)
2017 200,000 acres (81,000 ha)
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Substrates (carriers) of biocontrol strain AF36
wheat sorghum
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AFLATOXIN
CONTAMINATION RISK
IN TREE NUTS
(Agent)
Aspergillus
(toxigenic)
(Host)
pistachio
(Environment)- Moisture
- Temperature
- Soil
AF36 (atoxigenic)
We cannot change;
except:
a) chose the right
rootstock;
b) avoid tree stress
c) we can reduce
NOW damage
We can change!
We cannot change
Navel
orangeworm
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Acknowledgments:
• P. Cotty
• J. Siegel
• M. Doster
• D. Morgan
• M. Donner
• R. Puckett
• D. Felts
• H. Reyes
• Paramount Farming. Co. (now
Wonderful Orchards Co.)
• Setton Farms
• Nichols Farms & Keenan Farms
• & many pistachio growers
Thank you
California Pistachio Research
Board, USDA, and IR-4
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Nut length, dark stain, and navel orangeworm
(NOW) infestation
Nut length (mm)
NO
W (
%)
0
5
10
15
20
25
10-15 mm
16-17 mm
18-19 mm
>19 mm
No dark stain Dark stain 1-10%