ndian Journal of Experimental Biology

vol. 38, January 2000, pp. 84-87

Fungal metabolites from Aspergillus niger AN27 related to

plant growth promotion
Goutam Mondal* 1, Prem Dureja2 & Bi neeta Se n**

Division of Plant Pathology and 2Divi sion of Agricultural Chemicals, Indian Agricult ural Researc h ln sti lulc,
New Delhi 110 012, India

Received I Apri/ 1999; revised 20 September /99 9

Two met abolites ha ve been isolated from AsJJergillus niger AN27, a biocontrol agent, and identilicd as 2-carhmymet hyl
3-n-hexyl maleic acid (co mpound I) and 2-mcthylene-3-hexylbutanedioic acid (co mpou nd 2). Their biological activities
related to crop growth promotion have been assayed. Both the compounds increased germination and improved crop vigour.
Compound I was more effective for increase in germination and shoot length, whereas compound 2 had relati vely greater
role in increasing the root length and biomass of caul iflower seedlings .

Pl ant vigour is an important factor in resi stance of

crop to several diseases' . Introduced biocontrol
agents, mostly bacteri a on ly have been reported to
26
increase plant vigour as we ll as yie ld - . Among the
fungal bi ocontro l agents, Trichoderma spp. and some
unidentified sporu lating and non sporulating fungal
isolates have been reported to promote plant growth
7" 1?
"
an d suppress sot"lb orne d tseases
-_
Funga l metabolites re lated to plant growth
13 15
promotion have been iden tified in a few cases -
16
Almass i et al.
have reported some secondary
metabolites viz. 2-carboxyme thyl 3-n-hexyl ma le ic
acid anh ydride, 2-methylene-3-hexylbutaned io ic acid
and 2- methylene-3-(6-hydroxy hexy l)-butanedioic
acid which they isolated from an unspec ific isolate of
A. niger. But none of these fungi has been reported to
have biocontrol ability.
Efficacy of A. niger strain AN27 has been tested
against different soi l bome funga l plant pathogens in
17 1
laboratory and in field - ~. The principal antifunga l
compou nd has been iso lated from this strain and
ide ntified as trans and cis-4(3acetoxy-6-met hoxy-2hydroxyphenyl)-2-methoxy-butanolide20.
Bes ides
controlling disease, the isolate a lso promotes growth
18
and crop yie ld . The present pape r re ports the
iso lation of two compou nd s from the biocontrol
agent, A. niger strain AN27 and their plant growt h
promoting act ivity.
*Present address-Pulses and Oil sccds
Berhampore, Murshidabad-742 I0 I Indi a
** Correspondent author

Research

Stat ion,

Test crop-Cau lifl ower cv. Pusa :mow ball K I

obtained from the Division of Vegetabl e C rops, of th e
Institute was used as the test crop for bi oassay.

Isolation

of

secondarv tnetuh(l/i /es-G rowth

promoting compounds were is o lated as described
16
earlier Aspergillus niger AN27 was in oc ulated in
Sabouraud broth med ium (40g, dex trose; I Og,
peptone in I litre H 20 , pH 5.6)@ 2ml of 2.5x lOR cfu
mr' in eac h of 500ml med ium lots . The flasks were
incubated in a shaker BOD at I SO rpm for 7days at
30C. T he culture broth was filtered through a
Whatman filter paper (No.3) and th e filtrate was
extracted with ethylacetate. The organ ic layer was
separated, dried over anhydrous sodium su lph ate and
concentrated on a rotary vacuum evaporator. The dark
brown oi ly residue obtained showed th e presence of
three metabo lites o n thin layer chromatograph y usin g
hexane:ethylacetate:aceti c acid (60 :40:0 .1 ). These
metabolites were separated by co lumn c hroma tography on silica ge l e lutin g with dich loromethane to
ethylacetate. The separat ed
metabo lites were
c haracterised by nuclear m<:~ g n e ti c resonance (NM R )
and mass spectroscopy. Quantific<:~ti o n was ca rried
out on a thermo-separation product model spectra
sys tem P2000, hi g h perfo rmance I iqu id c hromatography (HPLC), equipped with variabll: wavel e ngth
UV- 150, UV-V IS d<:!tect o r and a Rheody ne inj ector
1
(20 J..ll loop). H-NMR spectra were reco rded o n
Variati EM 360L (60 MHz) and Bruker 400 AC (400
MHz) instruments . Deute ro-chlorofo rm (CDCI 1 ) and
pyridine were used as so lvents and tetra met hylsila ne

NOTES

(TMS) as internal standard. A HRGC-MEGA2 series

gas chromatograph coupled to a FISONS-TRIO 1000
ion trap mass spectrometer and co nnected with a
Panasonic KX-Pll50 multi-mode printer was used.
The ioni sation potential was 70 ev. Gas
chromatograph was equipped with a SE -54 capill ary
column ( 15m x 0.25 mm i.d., film thi ck ness 0. 1 - 0.15
11m). GC condition s were - initial temperature 70 C
for I min. ; and then heated up @ 10C min .-1 to 250C
for 15 min .
Bioassay- In blotter test. 2m! of compound was
used at different concentrations in sterili sed deionised
water. Twenty caulifl ower seeds were soaked for I hr
at the same dilution of th e compound, placed on filter
paper and incubated for 4days in a growth chamber at
27C, 78-85 % RH , and 500 11E/ m2 for IOhr Iight
intensity (white flu orescent tubes). Three replication s
were kept for each diluti on .
In vial test, 7days old cauliflower seed lings grown
on steri li sed sand were suspended in vials (9 ml )
filled with dilutions of th e compounds in sterili sed
MS solution 2 1 (wi thout sucrose and vitamins). Vial s
containing one seedling each , were kept in growth
chamber for 15days. Ten replication s we re kept for
each treatment.
Ethylacetate ex tract of culture fi ltrate yielded three
secondary metabolites (Rf values 0.77, 0.67 and
COMPOUND 1
NMR ( CDCh)
0.88 (3H, t )
1.3 - 1.6 ( 8H )
2.51 ( 2H , dd )
3.51 ( 2H, s)
10.52 ( lH, s, OH)

MASS SPECTRUM
m/z = 222 ( w--18)

85

0.57). These compounds were quantified by HPLC as

19.84 mg of compound I, 14.56 mg of compoun d 2
and 0.46 mg of compoun d 3 per litre of culture
filtrate. Compound I separated out as an oil and was
further purified by preparati ve TLC. It s NMR spectra
showed a tripl et at 8 0. 88 (CH,). a multip let at 8 1 .~1.6 (4 x CH 2), a tripl et at 8 2.51 (C H2 co nnec ted to a
double bond) and a singlet at 8 :u I (C H2 ) . Its IR
spectrum showed a broad abs orption ba nd from ~6002300 cm-1 for OH group and absorpti on band at 1720
cm 1 for carbony l group. Its mass spect rum showed a
molecular ion peak at m/z 240 (ve ry weak) wit h base
peak at m/z 222 (M+- 18) . NMR spectrum of
compo und 2 (a white crystalline so lid) sh owed a
triplet at 8 0.83 (C H1), a multi plet at 8 1.25 0-lH. -1- x
CH 2), a multiplet at 8 1.8 (C H2 ) . a trip let at 8 :us
(CH) , and two singlets at 8 5.80 and 6.40 for =CH ~
protons respectively and at 8 9.60. 2 proton for OH
group. Its IR spectrum showed the presence or a
broad band at 3600-2300 cm 1 for hyd roxy l group and
a carbonyl abso rption band at 1720 cm- It s mas.
spectrum showed a molecular ion peak at m/z 2 14
(M+) with fragment ion peaks at m/z 197 (M +-OHl.
180 (M+-2 x OH). On the basis of above spec tral d<Jta.
compound s I and 2 we re identi fied as 2-ca rhoxymethyl 3-n-hexy l mal eic acid and 2- mcth ylc nc -~
hexy lbutanedioic acid (hexy l itaconi c ac id) respec1

COMPOUND 2
NMR ( Pyridine)
0.83 ( 3H, t)
1.25 ( 8H , bs )
1.80 (2H, m)
3.35 ( lH' t)
5.80 ( lH, s)
9.60 ( 2H , brs, 2 x COOH )
MASS SPECTRlJ M
m/z = 214 ( M + )

COOH

COOH
H

2 - carboxymethyl - 3 - hexyl - maleic anhydride

2- Methylene- 3- hexyl- butanedillic acid

( hexyl itaconic acid )

Fig. !- Spectral analysis of two crystallised growth promoting compounds isol ated fro m A.I'J )('rgillus nign AN27

86

I DI AN J EXP BI OL . JANUARY 2000

16
lettuce and ri ce . Impact on seed ge rmin al ion and
crop vigour has not bee n stu d ied. Moreover
metabolite producti on is iso late s pec i fic 1 r'~ 221 . Fu nga l
metabolites related to grow th promot ion have bee n
identified in A. nidu/an .1 14 . and Neocosll wsporo
11
tecta ISs
va s uz
. assa et a /. I1ave .t ~ o IatecI a growtI1
promotin g co mpound cont aining rad ic loni c acid from
an un identified fun gus. These fun gi have not bee n
reported for bi oco ntrol acti vit y. In thi s co ntext. A.
Higer AN27 has proved itself as a myco parasite of
seve ral important so il borne fun gal pl ant pa th oge ns
viz ., Fusarium oxy sp o ntlll c iceri , Macropho tll ina
phaseolilla ,
Py thiw11
o p ha n idemwt un1
and
14
Rhizoctonia so /a ni in vit m . It reduces d isease
incidence of Fusa rium wilt of musk melon 171'J and

Fig. 2-Cau li ll ower seedli ngs raised from seeds: C-untreated; !treated with compouund I ( I 0 ppm): 2-t reated with compou nd 2
(25 ppm).

ti vely (Fig. I ). Co mpound 3 could not be ide ntified as

th e isolated amount was inadequ ate.
On th e bas is of bioassays, co mpound s I and 2 were
found to enh ance ge rmin ati on and growth of
cauliflower seedling (Fig. 2). The co mpounds I and 2
at I00 ppm increased ge rmin ati on up to 25 and 13 per
cent and bi omass up to 59.0 and 66.3 per cent
respec tively, over co ntrol. Increase in shoot length
was up to 46.6 per ce nt at I 0 ppm of co mpound I
wh ereas up to 38 per cent at 50 ppm by co mpound 2.
Compounds I and 2 increased 27.8 and 34.5 per cent
root length at 10 and 25 ppm respec ti ve ly. Howeve r,
hi gher concentrati ons of both the co mpound s retarded
plant growth .
On the bas is of bioassay, it was co ncluded th at
compound I was more effecti ve in increasing
germin ati on and shoot length whereas co mpound 2
was more res ponsible for promoting bi omass and root
length. Though both the co mpound s had been iso lated
. Iates 16---
?? ? 1
, on Iy 2f rom un spect. f.tc A . ru.ger tso
meth ylene-3-hexylbutanedi oic ac id has been quoted
responsible for shoot and root length promoti on in

charcoa I rot of potato I 'J -' 4 over 87 and 93 per cent

respecti ve ly. It also increases potat o yield ove r I ~.-ttl
per cent bes "d
t es crop v1.gour I X I 'J .-'4 . A mong ,.un ga I
bi ocontrol agents, T richode m 1o spp . h;~ ve been
studi ed and fo un d to enhance plant vigo ur 7x 2' .
However, till now, spec ifi c compounds res ponsible
for growth pro moti on have not been identifi ed in any
fun gus acting simultaneously as a btOco ntro l age nt. It
can be presumed that th e co mpounds iso lated from A.
n ige r A 27 may have a ro le in promotin g yie ld by
improv ing crop vigo ur. T he strain can be used for
crop protecti on and enh anceme nt or prod ucti on.
Tox ic effects of th e compound s at hi gher
concentrati on inhibiting germinat i:)l1 an d reta rd ing
growth of caulifl ower is a ge neral c h ;~rac t e r i s ti c of
,(,
any growth regul ator th at pro m o t e~ plant growt h- .
The work was support ed from a grant from
Department of Bi otec hn ology, M inistry o r Sc ience
and Tec hn ology, Government of Ind ia. Since re th anks
are clue to Shri Sanj eev Kumar an d Mi ss Shahana
Majumcler for in valu able help.

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