'.., 110,328—335 (1984)
l.ig P.uil Parey, Berlin und Ha
M81 / lntcrGode: PHYZA3
Natural Pesticide Laboratory, Botany Department,
Gorakhpur Umversity, Gorakhpur—273001 (India)
Toxicity of Some Terpenoids
Against Fungi Infesting Fruits and Seeds
of Capsicum annuum L. During Storage
By
N. N. TRIPATHI, A. ASTHANA and S. N. DixiT
Received March 17, 1983
Ahstract
On mycoflora analysis of dried fruits and seeds of chilli stored for six months, Asprj^iKiis fliivus Jind /l. }ii'f^er were the more rrec^ucnt out of eighteen iungi isoia.ted. Some
terpenoids viz. Gitral, Citronellal, Gitronellol, Eugenol, Farnesol and Nerol showed absolute toxicity against A.flavus and A. niger at 0.5 per cent concentration. The miiiimura
against both these fungi were determined. Citral, Eugenol and Nerol exhibited a broad
rjini^c ot toxicity a.nd were found to be more efnca.cious than some synthetic tungiciucs.
Toxizitat einiger Terpenoide gegeniiber Pilzen,
die Friichte und Saatgut von Capsicum annuum wahrend der Lagerung befallen
Bei der Mykofloraanalyse von getrockncten Frtichten und Saatgut von Paprika, der
isolierten Pilze''n vor. Einige Terpenoide, namlich Citral, Citronellal, Citronetlol, Eugenol,
Earnesol und Nerol zeigten bei einer Konzentration von 0,5 % absolute Toxizitat gegenuber
A.flavHS und A. niger. Die minimale Hemmkonzemration (MIC) und Beschaffenheit (fungistatisch/fungizid) aller dieser Terpenoide gegenuber beiden Pilzen wurde bestimmt. Citral
Eugenol und Nerol wiesen einen breiten Toxizitatsbereich auf und waren wirksamer als
einige synthetische Eungizide.
Fungi are important destroyers of food during storage and transit. Their
m d carcinogenic metabolites have added a new dimension already
• :; -.ioragc problems. Fruits and seeds of chilli are important commodities
.
. ,r .Jorance Center Code St.temenn 003 1-948 l/84/1004-0328$02.50/0
ity of Somc Terpenoids Against Fungi Infes
which deteriorate badly following attacks by Aspergillus
flavus
during storage ( R O U T and R A T H 1972, MANOHARACHARY and
and A. niger
PADMAVATHY
1976, DHAWALE and KODMELWAR 1978, SEENAPPA et al. 1979, 1980). Although
many synthetic chemicals have been recommended for the control of
fungi
causing deterioration of food commodities during storage, the risk of residual
effects with respect to human health cannot easily be excluded (MATSUMURA
1972). However, in recent years secondary plant constituents have been found
to be promising as fungicides (FAWCETT and SPENCER 1969, 1970, BEYE 1978,
KuBO and NAKANISHI 1979, D I X I T and TRIPATHI 1982, D I X I T et al. 1983).
Materials and Methods
1. Storage of fruits and seeds of Capsicum annuum L.
SIX months at room temperatures of 19—32 C and at relative riumidities between 45 anci
95 %. The seeds obtained from mature fruits were also sun dried and stored similarly.
2. Mycoflora analysis
The usual agar plate (MUSKETT 1948) and standard blotter (DE TEMPE 1953) techniques were employed in the isolation, of fungi (borne externally or ititernally) from fruits
and seeds of stored samples.
For the agar plate tedinique, 10 fruits each from different containers (Jute bags and
tin bo.xes) were placed in Petri plates (2 fruits per Petri plate) containing malt agar medium. 50 seeds (10 seeds per Petri plate) were inoculated similarly. The plates were incubated at 2 4 ± 2 ° C for a week and pure cultures of all the fungi appearing were obtained
on a similar medium by the dilution plate method. Plates were examined daily for the
growth of fungi from the third day after the commencement of incubation.
Eor the standard blotter technique fruus and seeds were incubated on three layers of
filter papers whidi had been soaked in sterile distilled water, in Petri dishes at 2 4 ± 2 ° C .
For the isolation of internally borne fungi seeds were surface sterilized with 0.1 % mercuric chloride for upto 5 min.
3. Evaluation of terpenoids for their fungitoxicity
Six terpenoids were evaluated for their toxicity against A. flavus and A. niger at
0.5% concentration following the technique of GROVER and MOORE (1962) using malt
extract agar medium (malt extract •""• 20.0 g^ dextrose ••""*• 20.0 g, agar •"•"" 20.0 g, peptone " ^
I.Og and distilled water 1000 ml). The requisite amount of each terpenoid was added to
pre-sterilized Petri dishes containing the medium using acetone (0.5 ml acetone/9.5 ml medium) as a solvent of terpenoids in order to get the desired concentration. Dishes containing
from the periphery of 7 day-old culture of the test fungi were aseptically placed on the
S-gar medium separately. Fungitoxicity was recorded in terms of percentage mycehal inhibition calculated by means of a standard tedinique (DIXIT et al. 1978).
Phytopith. Z., Bd. no, Heft 4
22
• 30
TRIPATHI, ASTHANA and DJXIT
4. Fungitoxic investigations with the terpenoids
11K- minimum
inhibitory
concentration
(MIC)
of
each terpenoid
was determined
ii.iiurc OT c'ACn tcrpenoiG \\'.'is di^tcrmincd tollowing the procedure or OARBER and HOUSTON
(1^39). The spectra ol Citral, Eu^cnol and Nerol, the three strongly fungitoxic terpenoids,
wore studied .it their respective hypotoxic, toxic and hypertoxic concentration (with respect
to their respective MlCs) against all the eighteen fungi isolated from the stored fruits and
seed.s. Further, the fungitoxic efficacy of these was compared with five prevalent synthetic
fungicides viz., organomercurial dust (Agrosan GN), carbendazim (Bavistin), copper oxychloride (Blitox-50), mancozeb (Dithane M-45) and zineb (Dithane Z-78). All the exper-
Results
1. Fungi associated externally with the fruits
Nine fungi, Aspergillus flavipes, A. flavus, A. fumigatus, A. niger, A.sydowi, CoUetotricbum sp., Rbizopus arrhizus, R. nigricans and Syncephalastrum
racemosum, were recorded as externally associated with fruits of Capsicum
annuum stored in Jute bags as well as in tin boxes by both techniques.
2. Fungi associated externally with the seeds
Sixteen fungi were found associated externally with seeds stored in jute
bags. Out of these Aspergillus awamori, A. flavipes, A. flavus, A. fumigatus,
A. nidulans, A. niger, A. sydowi, Cladosporium sp., Penicillium sp,, Rhizopus
arrbizus, R. nigricans and Syncephalastrum racemosum were obtained in both
the isolation techniques used. Alternaria alternata, Aspergillus ruber, Curvularia htnata, Fusarium oxysporum were, however, isolated by the standard
blotter paper technique only.
However, seeds stored in tin boxes were infested with 15 fungi. All these
fungi were the same found associated with seeds stored in jute bags. It is
noteworthy that seven out of 15 fungi were common to both the isolation
techniques. The other seven fungi viz., Aspergillus flavipes, A. niger, A. oryzae,
A. ruber, Rhizopus arrhizus, R. nigricans and Syncephalastrum racemosum
were isolated on agar plates only. Fusarium oxysporum was observed by the
standard blotter paper technique only.
3. Fungi associated internally with the seeds
A total of only ten fungi were recorded as being internally borne in seeds.
Seven of these viz., A. flavus, A. niger, A. sydowi, Cladosporium sp., Colletotrichum sp., Fusarium oxysporum and Penicillium sp. could be isolated by
'loth the techniques while Alternaria alternata, Aspergillus oryzae as well as
'•n-vtilaria limata appeared in the standard blotter paper technique in the
Tpplci Stored in jute bags.
of Sov
;i Infos
oids Ag;
The samples stored in tin boxes showed, however, nine internal funi
Aspergillus flavus, A. niger, A. oryzae, A. sydowi, Colletotridntm sp. we
isolated by both techniques. A. awamori and Penicillium sp. only occurred (
agar plates while Alternaria alternata and Curvularia lunata were specific
the standard blotter paper technique.
4. Fungitoxic propertie ; of terpenoids
AU the terpenoids showed absolute toxi :itymhibitmg the mycelial growth
of the test fungi completely (Table 1). Citral was fungicidal to both test fungi,
while Eugenol was fungicidal to A. flavus only. The rest of the terpenoids
were fungistatic to both fungi. Citral, Eugenol and Nerol exhibited a broad
range of toxicity at their respective toxic and hypertoxic (with respect to their
respective MICs) concentrations (Table 2). Citral inhibited 8, 12, 16 and
16 fungi while Eugenol inhibited 7, 14, 18 and 18 at .02, .03, .04 and .05
per cent concentrations respectively out of 18 fungi tested. Further, Nerol
showed complete mycelial inhibition of 9, 14, 18 and 18 fungi at .04, .05,
.06 and .07 per cent concentrations respectively out of 18 fungi tested. The
terpenoids were found to be more inhibitory than a.11 the synthetic rungicides
(Table 3) in the present investigation.
Ta ble
of terpenoids
T »rpe . o i d .
C tral
C
ellal
c tron ellol
Et gen ol
Fa
ol
N
1
aga,nsttest
fungi
MIC ('/, ) a n d n a
A. flavus
0.03=O.OS
0.25
0.030.08
0.06
^j'.niger
0.04-0.09
0.07
0.04
0.10
0.05
Discussion
For the mycoflora analysis the agar plate technique has been found more
useful than the standard blotter technique (SURYANARAINA and BHOMBE 1961,
ToMKiNGS 1929). Fiowever, NEERGAARD and SAAD (1962) and MISRA (1978)
reported both methods equally good and considered them to be supplementary
to each other. The present investigation showed that for isolation of all fungi
involved both methods should be employed as some fungi were isolated by the
blotter method while others were isolated only by the agar plate method.
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Tahle 3
: Aspergilius flavus and A. t
Common name of
fungicides/terpenoids
Trad e name
offu igicides
Organo mercurial dust
Carbendazim
Copper oxychloride
Mancozeb
Zineb
Citral
Eugenol
Nerol
1
1
MIC (%)
A. flavus
A. mger
Agro anGN
Bavis tin
Blitox-50
Ditha ne M-45
Dkha ne Z-7S
0.4
0.5
4.5
4.0
0.2
0.5
0.3
5.0
3.0
0.3
—
—
0.03
0.03
0.06
0.04
0.04
0.05
—
Of the fungi isolated Aspergillusflavusand A. niger occurred more frequently on the fruits and seeds. Therefore, these were selected as the test fungi
in the present investigation.
Although terpenoids constitute a group of biologically active compounds
(ToMiYAMA et al. 1968, SHATACHER and YOELKASHAMAN 1970, BONDARENKO
et al. 1972, HEROUT 1970), little attention has been paid to their fungitoxic
properties.
According to AGARWAL et al. (1979), o-pinene, /?-pinene, p-cymene,
geraniol and k-terpineol were low to moderately active while car-3-ene, camphene and 1,8 cineol were either inactive or possessed very low activity against
A. flavus and A. niger. Thymol was moderately active while its isomer carvacrol possessed strong activity. It is interesting to note that in the present study
all the terpenoids exhibited strong toxicity against both test fungi.
FRENCH et al. (1978) found citral stimulated the conidial germination of
P. digitatum and P. italicum, surprisingly it showed strong fungitoxicity in
the present investigation. The compound was further found to be fungicidal,
similar to the findings of SINGH (1977). Citronellal, Citroneliol and Eugenol,
however, were found to be fungistatic, as earlier observed by KURITA (1982).
It is noteworthy that Citral, Eugenol and Nerol possessed stronger fungitoxic
activity than the synthetic fungicides tested.
Thus the terpenoids, due to their strong fungitoxicity, broad spectrum
and superiority over synthetic fungicides, could be exploited as a safe postharvest protectant against fungal infestation for chilli fruits and seeds during
storage.
The authors are thankful to the Head of the Deptt, of Botany, Gorakhpur University
for providing laboratory facilities; to the Director, C. M. L, Kew, England for the identification of isolates and to Miss H. CHANDRA for a critical review of the manuscript. A
Post Doctoral Fellowship awarded by the Council of Scientific and Industrial Research,
New Delhi to N.N.T. is gratefully at^nowledged.
Literature
\
\i\s\i
I
C
S AiAiHiLA
inuS
u u u n o i d s i^inist AspLr^ilii
Ui M 1
SINUA
I V / 9 Studies on the antifungal activity of
I n d n n Phytopath
32,104—105.
I '"S lnscuiudcs Ironi d u \ct,(.t^bk kl^^dunl. PI. Res. Dev. 7, 13—31.
SKt)Kti(u)if \i Ki,>
<linlUL,B^^
DHWWII
iiid
\i
L
csscntiiloil
S I)
md R
MisffrNKO\A
1972: Antibiotic properties of Psoralea
Micrubiol Zit ^KY\ JV) 34, 612—616.
V KnuwrL'* AR
197S Studies on mycoflora of chilli seeds. Seed
Rcsi-irdi ( N L \ \ Uilhi) 6, 21 — ^0
Di TiMM
1
1^'S^
Ihc bkutcr method of seed health testing. Proc. Int. Seed test Assocn
2S, 1.13—LSI
l)i\ir
S N
N
N
iRUMHi
-ind b C iRiiArHi
197S: Fungitoxicity of some seed ex-
t r a u s N i t Acid Su L u t t r s 1, 2S7—2SS
iiid R n lRir».rHi i'l\2 N n u i i l h OLcurrinj, H-Naphthoquinones. Promising fungilo.i.xm,
P h i t i studies on Inm^ xnd fossil p h n t s , Pant Comm. Vol. 77—85.
N. K. UuuFY, and N. N. TRIPATHI, 1983; Fungitoxic essential oils vis-a-vis disease
n nrrul In HUSAIN
\
B R SINOH K SINGH, and V. P. AGNIHOTRI (eds.),
RLLUU \ d \ i n i c s m P h n t Pitholo..,> 248—2S1. Prof. H . K. Saksena Festschrift,
Print House ( I n d n ) L U C K N O W
D C (cd ) run.,iudcs
m t d ^ n c e d treuise Vol. II, 637—663. Acadetiiic Press,'
INC Ui New \ o r k i^ London
\nd
, 1970: Plant chemotherapy with natural products
Ann Rev Phytopath
8 403-41S
hRi\(H
K
C
R
K
l.KC
I
M
LMLFRHL
I iniii ttaii dl itxutm xnd P iiiluum
C
1. GRAH.AM, J. J. SMOOT, and P. E.
Ph) topuhology 68, S77—882.
<.7\MiR K H
md B R HOUSTON 1 9 I 9 An inhibitor of Verticillium
totton seed Ph\ti>p-ithoKi,v 49, 449—4=^0
i'r.,inism Sih oti
)?iutuoh
albo-atrum in
ind S" / ; \ ; Ph) topathology 52, 876—880.
lif ROL 1 V iV/0 Stinic rclitions bLt\\i_Ln pliiits insects and their lsoprenoids. In: R-ElN"
HLLD L m d \
LixxsCHiTz (eds) Progress in Phytochemistry, Vol. 2, 143-202.
\ \ i l e \ Intersotnei I ondon
K L L O I ind k NAKAMSHI 1979 Sunie teipeiioid insect antifeedants from tropical plants.
Press O i t o r d m d >-\fl ^ o r k
kuRiTA
N 19S2 Reteiit fandinf,s on the intimieiobial effect of essential oil components.
F Antibiet Antitunf; iptnts 10, 301 - 3 0 b
AlAfOHARACHAR-i C l n d K P\DMA\ATH'i 1976 run.;i associated with chiUi Seeds. Geobios
3, 99—100.
MATSIVHRA F 19^2 Current pcstieide situ Uion in the United States. In: MATSUMURA, F.,
G. M. BousH, and T. MISATO (eds.), Environmental toxicology of Pesticides, 33—60.
Aeidemie Press Ne\^ \ ork iiid London
MisR^ N
197S Stud) on lungi eiusing deterioration of foodstuffs in storage. Ph. D
Thesi Gorikhpur Uni%ersits I n d n
\liskfTT \ h
194S leehnique ioi the e x n i i n i t i o n of seeds for the presence of seedb )rne fun.;i Trins Brit M}col Soc 30, 74—8S
1
WKi) P
ind SAAD 1962 Seed hcilth testmi, of rice, A contribution to developin nt nl hbor-itor\ routine testin., method Indian Phytopath. 15, 85—111.
K
ind G G RATH 1972 Note on seed borne disease of chilli. Indian Phyto-
Toxicity ot Some Terpenoids A^^in.st I'lingi Iriic.stin|^ I'ruiis tind Seeds
335
SEENAPPA, M., L. W . STOBBS, and A. G. KEMPTON, 1979: The role of insects in the biodeterioration of Indian red peppers by fungi. Int. Biod. Bull. 15, 96—102.
Phytopathology 70, 218—222.
1221—1223.
SINGH, I •, \/77\ Antirun^jl tictivity in underground ptirts or nigner pJtints. Pn. L). Thesis,
Gorakhpur University, India.
seeds. Indian Phytopath. 14, 30—41.
infected by pathogens. Phytopathology 58, 115—116.
ToMKiNGS, R. G., 1929: Study on the growth of moulds. Proc. Roy. Soc. (London) B. 105,
375—401.
Authors' address: N. N. TRIPATHI, A. ASTHANA and S. N. DIXIT, Natural Pesticide