Natural products chemistry

and its applications to real-

world agricultural problems
John J. Beck
August 19, 2012
 Outline
Agricultural
Natural Products
Topics
Styrene from a
fungus
Volatiles for
detection of fungi
Host plant volatiles
to attract a major
insect pest
Natural Products
and Agriculture
 Natural
Products
Sustainability
Fungi as chemical
manufacturer?

Moldy almond
emissions
 off air flow to the RBF for 3 min, removing the air flow line, sampling
MS. A total of five Tenax columns were used for VOC collection:
with SPME (P, 3 min; E, 1 min; S, 30 s; T, 15 min), and analyzing by
mn 1, days 0-3 (69.7 h); column 2, days 3-7 (97.3 and 167.0 h);
GC-MS. A totalandof fiveh);Tenax columns
4 (2 were used for VOC collection:
Natural
mn 3, days 7-24 (405.9 572.9 column 10 g Tenax
columndays
mns in tandem), 1, days
24-45 0-3 (69.7
(509.6 andh); column
1082.5 2, days5, 3-7
h); column days (97.3 and 167.0 h);
66 (499.3column
and 1581.8 3, days 7-24 (405.9
h). Absorbed andwere
volatiles 572.9 h); column
desorbed from 4 (2 10 g Tenax
Tenax viacolumns in tandem),
protocol days(23). 24-45
Briefly, (509.6 andmedium
1082.5 h); column 5, days
Products
a published the Tenax
45-66
washed with (499.3 and
n-pentane, andthe 1581.8 h). Absorbed
n-pentane volatiles
was concentrated viawere desorbed from
ersion of the
the RBF
Tenax in avia
warm water bath and
a published distilled(23).
protocol until Briefly,
a mother the Tenax medium

Sustainability
r volumewas of ca. 100 mL.
washed with n-pentane, and the n-pentane was concentrated via
yrene Isolation, Semipreparative HPLC. Aliquots (100 L) of
immersion of the RBF in a warm water bath and distilled until a mother
mother liquor were separated via HPLC: 80:20, MeOH/H2O; flow,
mL/min; liquor
pressure, volume
70-72 of bar;ca. 100 mL.
detector, excitation at 285 nm and
sion at 385 Styrene Isolation,
nm. Commercial Semipreparative
styrene HPLC.the
was used to determine Aliquots (100 L) of
tion timethe mother
of the peak liquor were separated
for collection. via with
Pure styrene, HPLC: 80:20, MeOH/H2O; flow,
an elution
Noted large
of 20.8 1.5
min,mL/min;
gerator. Combined
was collected
styrene
pressure,
into a70-72
fractions
darkened
(ca. 10
bar;
vialdetector,
runs)
and stored
were
excitation
in a
transferred
emission at 385 nm. Commercial styrene was used to determine the
at 285 nm and

amounts of one
separatory funnel, and
retention timechilled
of thepentane
peak for (10collection.
mL) was added. The
Pure styrene, with an elution
ous methanol layer was partitioned and then washed twice
time of 20.8 min, was collected into a darkened vial and stored in amore
chilled pentane (2 10 mL). The combined organic layers were
refrigerator. Combined styrene fractions (ca. 10 runs) were transferred
compound
over anhydrous sodium sulfate and concentrated in Vacuo to a
to a separatory
me of approximately
aqueous
0.5 mL
methanol
funnel, and chilled
(composition
layer
pentane
checked
was partitioned
(10 mL) was added. The
by GC-MS
and then
tion, 1 L), and then a stream of nitrogen gas was passed over washed twice more
with
ial to near
E and GC-MS
Fungal bouquet
chilled
dryness
dried over
pentane
to afford
anhydrous
analysis,
pure(2styrene
10 (1.7
mL).mg),
sodiumallsulfate
which matched
Theconfirmed
data ofand
combined byorganic layers were
concentrated in Vacuo to a
the authentic
le. volume of approximately
Almond 0.5 mL (composition checked by GC-MS
COT Isolation, Semipreparative
injection, 1 L), and then a stream HPLC. Aliquotsof (900 L) of
nitrogen gas was passed over
mother liquor were separated PDA
via HPLC: 80:20, MeOH/H
the vial to near dryness to afford pure styrene (1.7 mg), confirmed by 2O
8.99 min; flow, 1.5 mL/min and then 99:1, MeOH/H2O 29-42
SPME and GC-MS analysis, which matched all data of the authentic
nm. SPME sample. Pure isolate on
flow, 2.0 mL/min; detector, excitation at 295 nm and emission at
headspace GC-MS was used to determine the retention
of the peakMCOT
8.5 min,the weremother
PDB Isolation,
for collection.
collected liquor
MCOT
into awere
Semipreparative
separated
darkened vial and
HPLC.
isomers, with an elution
viastored
HPLC:
time Aliquots (900 L) of
in a80:20,Figure 2. GC
MeOH/H 2O
traces of VOCs from (A) fungal bouquet on almonds, (B)
0-28.99 min;
gerator. Combined flow,
fractions (ca.1.5 mL/min
7 runs) wereand then 99:1,
transferred to aMeOH/H fungal bouquet on PDA, and (C) isolated fungus F. oxysporum strain on
2O 29-42
atory funnel,
min;and Fusarium
chilled
flow, 2.0 pentane
mL/min; (10detector,
mL) was added.
excitationThe aqueous
at 295 nm and PDA.
emission at
anol layer395
wasnm.partitioned and then washed twice more with chilled
oxysporum
SPME headspace GC-MS was used to determine the retention
ne (2 10 mL). The combined organic layers were dried over
time of the peak for collection. MCOT isomers, with anmeasured via SPME and GC-MS (Figure 2B). Styrene as the
elution time
drous sodium sulfate prior to concentration via gentle distillation GCsuccessfully
traces of VOCs from (A)with
fungal b
of 38.5 min, were collected into a darkened vial andprimary stored volatile
in a componentFigure 2.was duplicated
 nd (B) 13
Proposed Biosynthesis 13
C-labeled styrene with probable positions of C-labeling corresponding

g
r-
-
A)
+
)
is
y
d
e
);
B)
nt
n Figure 6. Proposed biosynthetic pathway of styrene by F. oxysporum
n showing probable positions of 13
C-labeling as asterisks. The pentose
Mass Fragmentation Pattern
 Where From
Here?
Optimize styrene
production
Growth medium
Growth conditions
Different strains
Biomass as feedstock
Green alternative?
90% styrene is industrial
Petroleum-based
1.3 x 1010 pounds

Beck et al. J. Agric. Food Chem.

2008, 56, 11392-11398
Volatile Natural Products
for Detecting Fungi?
 and identied. Several volatiles from the blanched almonds
of whole almonds. Several of these volatiles are considered fatty
Aflatoxin-
nal, nonanal, 3-octen-2-one, tetramethylpyrazine, and decanal.
harvest environment and illustrative of potential contamination
21 Samples of whole
ty acid decomposition were predominant in the samples that kernels and blanched
Contaminated
xanal, heptanal, octanal, and hexanoic acid increased 3-fold in
ween samples they could not be considered as indicator volatiles kernels
Almonds
d kernels contaminated with naturally occurring aspergilli and

sition, fungal contamination, volatile Colony Forming Units

(CFUs) of aspergilli and
non-aspergilli

Aflatoxin content

Volatile emissions of each

sample
Journal of Agricultural and Food Chemistry

Figure 1. Aatoxins B1, B2, G1, and G2 (AFB1, AFB2, AFG1, and
AFG2, respectively).

There are limited reports on the volatile output of agricultural

products specically contaminated with microbial bouquets
Beck et al. J. Agric. Food Chem.
containing A. avus and A. parasiticus. In two separate reports
Abramson2011, 59, demonstrated
et al.11,12 6180-6187 the existence of three indi-Figure 2. Pictures representative of whole (left) and blanched (right)
 went some form of blanching. The emission amounts of hexanal, heptanal, octanal, and hexanoic
es contaminated with aatoxin; however, due to variability between samples they could not be consid

Results
atoxin content. The emission prole of volatiles from almond kernels contaminated with naturally
ated fungi is heretofore unreported.
WORDS: almond, aatoxigenic aspergilli, fatty acid decomposition, fungal contamination, volatile

No direct correlation between

ODUCTION
aflatoxin content and volatile
09/2010 California orchards generated 1.41 billion
emission
(641 million profiles
kg) of almonds accounting for 80% of
roduction.1 Rigorous import limits set by the European
Large
or aatoxin increases
contamination in
in almonds some
have instances:
resulted
in rejected almond shipments.2 Aatoxins are toxic
in an

Whole
tes produced almonds
by Aspergillus avus(3-fold
and A. increase)
parasiticus,
us fungi of tree nut orchards, and represent a grave food
oblem dueto AFB1 > 20 ppb
their carcinogenic and teratogenic attri-
Hexanal,
A. parasiticus is capable heptanal,
of producing octanal,
aatoxins B1, B2,acetic
G1, and
AFB1, AFB2, AFG1, hexanoic
and AFG2,acid
respectively) (Figure 1),
atoxigenic strains of A. avus produce primarily AFB1
B2. AFB1 isthe AFG1 > 10 ppb aatoxin and con-
most predominant
4
Hexanal,
to be the most toxic. heptanal,
It should be notedoctanal, hexanoicFigure
that AFB2, acid 1. Aatoxins B1, B2, G1, and G
nd AFG2 are typically not present if AFB1 is absent, AFG2, respectively).
 Conclusion

Significant differences between

blanched and whole almond emissions
This study of established fungi on
almonds inconclusive
no statistically significant correlation
between aflatoxin and volatiles

Beck et al. J. Agric. Food Chem. 2011, 59, 6180-6187

 Conclusion

Significant differences between blanched

and whole almond emissions
This study of established fungi on
almonds inconclusive
no statistically significant correlation
between aflatoxin and volatiles

but dont touch that dialimminent

manuscript
 f 23 compounds were consistently detected and identied. Several volatiles from
Volatile Natural Products
nt increases when compared to the emissions of whole almonds. Several of these volat
oducts and included hexanal, heptanal, octanal, nonanal, 3-octen-2-one, tetramethy
for Monitoring Insect Pests?
nvestigated were characteristic of a typical postharvest environment and illustrative of p
ransport container. Volatiles indicative of fatty acid decomposition were predomin
of blanching. The emission amounts of hexanal, heptanal, octanal, and hexanoic a
with aatoxin; however, due to variability between samples they could not be consider
The emission prole of volatiles from almond kernels contaminated with naturally
etofore unreported.
d, aatoxigenic aspergilli, fatty acid decomposition, fungal contamination, volatile

rnia orchards generated 1.41 billion

) of almonds accounting for 80% of
rous import limits set by the European
mination in almonds have resulted in an
mond shipments.2 Aatoxins are toxic
y Aspergillus avus and A. parasiticus,
 Host Plant
Volatiles to
Attract?
Ex situ
damaged
undamaged
almonds
pistachios
In situ
Ambient
Premise: navel orangeworm moths
Fungal attracted to damaged hosts
 Volatiles
From Natural
Damage?
Hull split

Damage
other insects
mechanical
Blend of Volatiles from
Hull Split Collection
O O O

OH O O O

OH O
12 4 4 1 1

Pared down from

larger blend
1-octen-3-ol from
damaged almonds
EtOAc as solvent
 Results

Moths Captured
Orchard Treatment NOW Total Female Male
Almond Blend 155 59 96
Meal 20 19 1
Blank 2 1 1
Pistachio Blend 32 20 12
Meal 2 2 0
Blank 0 0 0

Beck, J. J.; Higbee, B. S.; Light, D. M.; Gee, W. S.; Merrill, G. B.; Hayashi, J. M.
Hull split and damaged almond volatiles attract male and female navel
orangeworm moths. J. Agric. Food Chem. 2012 ASAP, DOI: 10.1021/jf302658v
Summary

Styrene produced
from a fungus found
in almond orchards

Alternative source of
polystyrene?
Summary

Volatile emissions for O O O

early warning detection

of contaminated tree O O

nuts? OH OH

Food safety
 Summary
O O O O

OH OH OO
O O

OH O
Volatile natural 12 4
12 4 4 1 1
products as attractants O O O

for an agricultural OH O O

insect pest OH
12 4 4 1

Food safety
 Acknowledgements
USDA-ARS Paramount Farming Co.
Wai S. Gee Bradley S. Higbee
Noreen E. Mahoney Johnny Magana
Douglas M. Light Ashlee Pedro
CRADA 58-3K95-7-1198
Gloria B. Merrill
Jennifer M. Hayashi California Pistachio Research Board
Jeffrey D. Palumbo TFCA 58-5325-9-365
Teresa L. OKeeffe
Almond Board of California
Daniel Cook
TFCA 58-5325-8-419
Klaus Dragull
James Baker California Department of Food and
Inna Ovchinnikova Agriculture 208-5325-163