Phytochemzstry,Vol 29, No 6, pp 1789 1791, 1990 w-9422/90 $3 oo+o 00

Prmted m Great Brltam Pergamon Press plc

STRESS EFFECTS ON COTTON LEAF PHYTOALEXINS ELICITED BY

CELL-FREE-MYCELIA EXTRACTS OF ASPERGILLUS FLAWS

H. J. ZERINGUE, JR

ARS/USDA, Southern Regional Research Center P 0. Box 19687 New Orleans, LA 70179, US A.

(Recetued 21 September 1989)

Key Word Index-Gossypwm jaous,aflatoxin.

hrrsutum; Malvaceae, cotton; phytoalexins, Aspergtllus

Abstract-The concentrattons of five induced phytoalexins, lacimlene C, lacinilene C 7-methyl ether, scopoletin,
2-hydroxy-7-methoxycadalene, and 2,7_dihydroxycadalene, were determined in cotton leaves treated wtth cell-free
mycehal extracts of Aspergillusflauus under a light regime of 14 hr light/l0 hr dark and temperature cycles of 25/20,
30/25, 35/30 and 40/35. Maximum concentrations were present in plants maintained at a post-inoculation
temperature regime of 30/25. In a separate experiment, the concentrations of the same elicited phytoalexms were
measured m cotton leaves treated with the fungal elicitor and maintained at different levels of plant moisture stress.
Leaf moisture stress values less than - 11.7 bars resulted in decreased production of the five cotton leaf phytoalexms
determined two days after the fungal elicitor was applied.

INTRODUCTION than 30/25 resulted in an overall 35% reduction in

induced phytoalexins while the highest temperature cycle
Phytoalexms are natural defence compounds produced
tested (40/35) resulted in an overall 43% reduction of
by the host plant in response to fungal, bacterial, and
induced phytoalexins
viral infections [l]. We have recently described the accu-
Plant moisture stress values less than - 11.7 bars re-
mulations of cotton (Gossypium hrsutum L.) sesquiter-
sulted in decreased amounts of the five induced
penoid phytoalexins 2,7,-dihydroxycadalene (DHC),
phytoalexins (Table 2). Even slight leaf wilt appearances
2-hydroxy-7-methoxycadalene (DHMC), their oxtdatton
products lacimlene C (LAC), lacinilene C 7-methyl ether (- 15.5 bars) at the time of fungal ehcitor treatment after
two days ( - 24.3 bars) resulted m an overall 45-58%
(LACME) and the coumarin scopoletin (SCOP), induced
reduction of the five phytoalexms produced when com-
in both cotton leaves and developing cotton boll carpel
pared to the induced phytoalexm production of non-wilt
surfaces when elictted by a cell-free extract of Aspergillus
flavus Link ex Fnes [2, 31. leaf appearance with PMS values of - 8.1 to - 11.7 bars.
Toxrgenrc strains of A. flavus in cottonseed can result
From these findings, tt is apparent that an ideal set of
environmental condittons of temperature and moisture
in pre- and post-harvest aflatoxin contamination of this
are necessary for the maxtmum induction of these cotton
feed and food crop. Aflatoxin B,, the most toxic of the
leaf phytoalexins. Cotton plants grown m the hot, dry,
aflatoxins is both teratogenic and carcinogenic [4].
desert regions of the southwest U.S.A. may be growing in
Aflatoxm contamination of cottonseed is a problem m
environmental conditions (temperature and moisture)
cotton grown in the hot, dry, low-elevation areas of
which do not favour the maximum productton of these
southwestern U.S. Our mvestigations are directed to-
phytoalexms. The inabthty of the host plant to produce
ward the elimmatton of the aflatoxm contamination of
defensive chemtcals quickly when attacked by A. _ilavus
cottonseed through the enhancement of natural defence
may be correlated with the incidence of aflatoxm con-
systems present in the cotton plant. The purpose of the
tammatton of cottonseed m the desert areas of the south-
current mvestigatton was to determine the effects of tem-
west U.S.A.
perature and plant moisture stress on the induction of
cotton leaf phytoalexms after treatment with the A.jaous
elicitor. EXPERIMENTAL

Fungi culture condrtlons, preparatton of fungal elicitor

RESULTS AND DISCUSSION
Both temperature and moisture stress had an effect on
phytoalexin productton when elicited by 10 pg amounts
of a cell-free mycelia extract of A. flaws containing 47%
protein and 29% carbohydrate. The highest concentra-
tions of the sesquiterpenoid and coumarin phytoalexins
were produced at temperature cycles of 30/25 measure-
ments being determined two days after treatment with
the fungal elicitor (Table 1). Temperature regimes lower
A toxlgenlc of A.Jaous SRRC 1OOOAwas maintained on
strain
potato dextrose agar plates Fungal spores were maculated into
the defined medmm of Adye and Mateles [S] contamed m 2.8 1
Femback culture flasks; the culture were maculated at 29 for 18
days without shakmg. The extraction of the fungal myceha and
the preparation of the fungal ehcttor has been described [6].
Plants, condzttons and leaf treatments. Four-month-old Delta
Pine 61 cotton plants were mamtamed in four separate envtron-
mental growth chambers, which were regulated at a hght cycle

1789
1790 H J. ZERINGUE. JR

Table 1 Temperature effects* on the ehcltatlont of cotton leaf phytoalexmsf

Dmrnal temperature Phytoalexmsli Induced (pg/g fr wt leaf discs)

regime
Llght/dark# DHMC LACME DHC SCOP LAC

25,!20 21 3 + 1.5 140+13 81 kO.3 56+08 231+25

(0)f + (0) (0 4) + (0) (0) * (0) (0 8) + (0 1) (tr)
30/2Y 276+26 178k2 132+21 125+ 13 385+17
(tr)** (0 6) + (0 3) (tr) (1 8) + (0 4) (1 6) + (04)
35130 22 7 + 2.9 17.6 + 16 86kO6 95+08 216+12
(tr) (0 8) + (0 2) (tr) (1.2) + (0 3) (1 4) 4 (0 8)
4n/lY t.cz+ 1-Q s. 1. _c I_z 86% 1-z X7&01. 1.X41 1-Q
(tr) (tr) (tr) (0 8) + (0 2) (tr)

*Means + s d of two experimental runs with each run contammg at least I5 leaf discs from different leaves of
slmular maturity and posltlon harvested from different plants
TWounded leaves ehclted by a cell-free myceha extract of A $avus
$Determmed 48 hr after ehcltatlon
#14 hr hght/lO hr dark cycles
l\DHMC, 2-hydroxy-7-methoxycadalene, LACME, lacmllene C 7-methyl ether, DHC, 2,7,-dlhydroxycalalene;
SCOP, scopoletm, LAC, lacmllene C
TWounded leaves treated with sterile, deionized water only.
**Trace < 0 4

Table 2 Plant moisture stress (PMS) effects* on the ehcltatlont of cotton leaf phytoalexmsf

Phytoalexms Induced (pg/g fr wt leaf discs)

Two-day range Leaf appearance
of PMS (bars) and degree of wdt DHMC LACME DHC SCOP LAC

-81 normal
- tt7 normal 262+27 183529 1!3?05 14.6 + !.7 4041!3
- 155 wdt f
- 24 3 W&-f + 152+ !~2 86?j04 53_+ 1.0 4,OR !8! +22
-
- 22 5 wilt + + +
- 3L5 wdL+ + -5 + ll_+_Q7 22kcll 4.0.t 0.2 3.8. If: 0.5 4.1. _c 0.3

*Means + s d of two experimental runs at 30/25 with each run contammg at least 15 leaf discs from 15 different leaves of slmular
maturity and posltlon harvested from different plants
twounded leaves were induced by a cell-free myceha extract of A fiavus
$Determmed 48 hr after ehcltatlon

re@me of I4 hr light/l0 hr dark with separate temperature con- Leajmolsture stress measurements Delta Pme 61 plants grown
trols of 25/20, 30/25, 35130 and 40135 For each of the two m environmental chambers were maintained at 30/25 with
experimental runs, 20 leaves of approximately the same size and light/dark cycles as above Water was wlthheld from these plants
maturity were wounded with 4 mm diameter wounds and until several levels of wdtmg occurred. At wlltmg, several leaves
treated with either 20 ~1 sterile, deiomzed water or wounded and located half-way up the plant were harvested during the fifth
treated with 10 ,ug dry cell-free myceha extract suspended m hour of artificial light cycle and the plant moisture stress was
20 pl of sterile, deionized H,O The wounds were produced on determined usmg a pressure chamber (PMS Instrument Co.,
the undersurface of each leaf by lightly scraping with a surgical Corvalhs, OR 97330) Leaves posltloned on the plant m the same
scalpel Two days after treatment the leaves were harvested areas as those leaves harvested for PMS measurements, were
Extraction, tdknt$catlon, and quanrltatlon ojphytoaiexms In wounded- and- treated- with the cellXee fhngaf elicitor or with
the excised leafdlscs 15 mm diameter leaf discs (usually 20 leaf sterile deionized water by the same method described m the
discs at a time) were excised from wounded and treated areas on leaf-treatment, section above Water was withheld two more
the leaves The leaf discs were vacuum mfdtrated with 40 ml days after leaf treatment, then non-treated leaves and treated
50% aq EtOH m 125 ml flasks and were placed on a reclprocat- leaves were harvested from the stressed plants PMS was deter-
mg shaker for 12 hr at room temp The discs were removed from mined on the non-treated leaves m order to obtain a 2-day range
the EtOH extract by filtration and the extract was coned to of continued water stress on the treated leaves 15 mm diameter
dryness by rotary evaporation under vacuum at 50 The residue leaf discs were excised from each wounded and treated leaf and
was dissolved m MeOH to produce a 5% (w/v) solution which the phytoalexms were extracted and quantltated as prevtously
was spotted on slhca gel TLC plates and developed m described [Z]
MeCN-CHCI, (1.4) (v/v) Identification and TLC fluoroden-
sitometrlc quantltatlon procedures of the lacmdenes, cadalenes, Acknowledgement-The author thanks Mr L Scharfenstem for
and scopoletm were performed as previously reported [Z] the PMS measurements
 EnvIronmental effects on phytoalexm production 1791

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Control and Imphcations. Academtc Press, New York.
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112.
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