International Journal of Entomology and Nematology

Vol. 5(1), pp. 115-120, March, 2019. © www.premierpublishers.org. ISSN: 2326-7262

Research Article
Secondary Metabolites of the Entomopathogenic Fungus,
Cladosporium cladosporioides and its Relation to Toxicity
of Cotton Aphid, Aphis gossypii (Glov.)
1Nihal
Omar Shaker, 2Gehad Mohamed Mousa Ahmed, 3Heba Youssif El-Sayed Ibrahim*, 4Maha
Mohamed El-Sawy, 5Mohamed El-Hoseiny Mostafa, 6Heba Nagi Abd El-Rahman Ismail
1,4Applied Organic Chemistry Department, Faculty of Science (Girls), Al-Azhar University, Egypt
2,3,5,6Plant Protection Research Institute, Agriculture Research Center (ARC), Dokki, Giza, 12618, Egypt

Cladosporium cladosporioides is one of the promising entomopathogenic fungi acting as insect-

pathogenic microorganism or can be used as a source of toxins against insect pests. Ethyl
acetate extract of the secondary metabolites of C. cladosporioide was obtained, and its volatile
constituents were characterized using GC/MS technique. Also, two major compounds were
isolated and identified as 3-phenyl propanoic acid (6) and 3-(4β-hydroxy-6-pyranonyl)-5-
isopropylpyrrolidin-2-one (7). It's worthy to mention that this isolated compound (7), is reported
from C. cladosporioides for the first time. Also, the toxicity of the ethyl acetate extract of the
secondary metabolites of C. cladosporioides against both adults and nymphs of cotton aphid, A.
gossypii was determined. Data showed that C. cladosporioides ethyl acetate extract was most
effective against nymphs showing LC50 of 24.5827 ppm, LC90 of 128.7385 ppm and toxicity index
of 100%, while, it showed LC50 of 36.6959 ppm, LC90 of 154.4394 ppm and toxicity index of 76.69%
against adults.

Keywords: Cladosporium cladosporioides, secondary metabolites, toxicity, A. gossypii.

INTRODUCTION

Entomopathogenic fungi were among the first organisms et al. (2013) studied the extracts of C.
used for microbial control of insect pests. A number of cladosporioides (Fresen.) and isolated four compounds
fungal species has been recognized for this purpose including cladosporin; isocladosporin; 5′
(Gottel et al., 1990; Ferron et al., 1991). Many species of hydroxyasperentin; and cladosporin-8-methyl ether. Some
Cladosporium were used in the microbial control of plant- of these metabolites exhibit bioactive or insecticidal
insect pests like aphids and whiteflies which showed properties which enable us to use them for insect pest
resistance to chemical insecticides. C. cladosporioide was control.
recorded as natural pathogen of cowpea Aphis crassivora
(Ibrahim, 2012) and cabbage aphid, Brevicoryne brassicae Cotton aphid, Aphis gossypii is a serious pest damaging
L. (Ibrahim, 2017) revealing high virulence against aphid cultivated crops either directly by sucking plant sap or
populations. Many bioactive compounds were isolated indirectly by transmission of many plant viruses leading to
from C. cladosporiodes. Kobayashi et al. (1989) isolated great loss of crop yield. It effectively transmits polyviruses,
the calphostin family of natural products from fermented such as cucumber mosaic virus, watermelon mosaic virus
broths of C. cladosporioides. Also, Sakagami et al. (1995) 2 and zucchini yellow mosaic virus (Capinera, 2005).
isolated Cladosporol from the culture filtrate of C.
cladosporioide, which showed antitumor activity in mice. *Corresponding Author: Heba Youssif El-Sayed Ibrahim,
Some pentacyclic composts of cytokines and tyrosine Plant Protection Research Institute, Agriculture Research
kinase inhibitory properties, were isolated from C. Center (ARC), Dokki, Giza, 12618, Egypt. Email:
cladosporioides (Wrigley et al., 2001). In addition, Wang hyei_youssif@yahoo.com

Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladosporioides and its Relation to Toxicity of Cotton Aphid, Aphis gossypii (Glov.)
Shaker et al. 116
The extensive use of the traditional insecticides for
controlling this pest led to many problems in environment,
human health and non-target organisms. So, the bio-
insecticides which depend on microorganisms or their
toxins can be used as more secure alternatives of the
traditional insecticides.
The aim of this study is characterization and identification
of the bioactive compounds of C. cladosporioides
secondary metabolites using spectral analysis and
recognition of its relation to cotton aphid, A. gossypii
toxicity.
MATERIALS and METHODS
Entomopathogenic Fungi
The culture of C. cladosporioides was isolated from A.
craccivora (Ibrahim, 2012) and was identified by Assiut
Univ. Mycological Center (AUMC), Egypt.
The Tested Insect Pest
Cotton aphid, A. gossypii strain was obtained from the farm
of Faculty of Agriculture, Mansoura University, and has
been known to be free from any insecticides. Aphid strain
was reared on okra seedlings (2-3 weeks old) planted in
small pots (15 cm3) and kept under plastic greenhouse
conditions of 25± 5˚C, 70±7 RH and 10:14 hrs Light: Dark.
Cultivation of the Tested Fungus
The fungal strain of C. cladosporioides was cultivated on
autoclaved Sabouraud Dextrose yeast extract Agar
(SDYA) [10 g/L peptone, 40 g/L dextrose, 10 g/L yeast
extract and 20 g/L agar[ and incubated at 25± 2˚C and
80±5% RH. Then, small pieces of mycelial mats of 7-days
old culture were transferred to 1000 ml bottles for
autoclave containing 700 ml of (SDY) medium] i.e. the
same medium components without agar], and were
incubated under 25± 2˚C, and 12:12 hrs Light: Dark for 21
days. The culture fluid filtrated through two layers of
Whatman No. 1 filter paper.
Extraction of the Fungal Metabolites
Filtrate broth of C. cladosporioides was extracted three
times with methylene chloride followed by ethyl acetate
(three times also) using separating funnel. Ethyl acetate
fraction was filtered over anhydrous Na2SO4 and was
evaporated to dryness to obtain the final residues. A
sample of ethyl acetate residue was analyzed by GC/MS
Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladosporioides and its Relation to Toxicity of Cotton Aphid, Aphis gossypii (Glov.)
technique for characterization and identification of its
volatile components.
The residue of ethyl acetate fraction was
chromatographed on a silica gel column with CH2Cl2
containing increasing amounts of MeOH. The collected
similar sub-fractions were subjected to thin layer
chromatography (TLC) using different solvent systems.
Bands on (TLC) sheets were marked under ultra violet
(UV) light 254 and 365 nm and/or detected by spraying
with p-anisaldhyde– sulfuric acid reagent (Wagner et al.,
1984). The retardation factor or retention factor (Rf) was
calculated for each solvent system.
Efficiency of the fungal metabolite extracts against A.
gossypii
Ethyl acetate extract of the fungal secondary metabolites
was formulated as emulsion in distilled water containing
0.3% Tween 80. Four diluted series were prepared and
were tested immediately after preparation. Each ten
individuals of the same age were transferred to a Petri-dish
containing okra leaf to be considered as one replicate.
Each concentration had 3 replicates and another three
replicates sprayed only with water and 0.3% aqueous
Tween 80 to be considered as control. In case of testing
the susceptibility of nymphal stage, the adults were
allowed to lay nymphs on the surface of the host leaves for
a period of 24 hours, then the parents were removed.
When nymphs reaching the age of two days, they were
treated with the extract emulsion. All treatments were
maintained under laboratory conditions of 25 ± 2oC and
75± 5 % RH. Mortality percentages were recorded daily for
a week and were corrected by Abbott’s formula (1925).
Determination of 50 & 90% mortalities and slope values
were estimated according to Finney (1971). In addition, the
toxicity indexes were calculated by using Sun’s equation
(1950).
RESULTS AND DISCUSSION
The GC/MS chromatogram showed five peaks
corresponding to five compounds which were
characterized by comparing their mass spectra with those
of their analogous reported by NIST, Wiley & Pfleger
libraries. The obtained results were reported in Table 1.
Oleic acid, (Compound 2) showed insecticidal activity and
it was reported as a potent source of mosquito larvicidal
property (Gurunathan et al. 2016).

Table 1: The GC/MS analysis of ethyl acetate fraction of C. cladosporioides:
S.N Compound No. R.T.
1 4-Hydroxy-2-hexenoic acid -lactone (1) 10.30
2 9-Octadecenoic acid 27.91
=Oleic acid.=Rapinic acid (2)
3 Pentadecylbenzene 28.15
=1Phenylpentadecane (3)
Aspidocarpine alkaloid
4 =Aspidospermidin17ol, 28.61
1acetyl16methoxy- (4)
1,2Benzenedicarboxyl
5 ic acid,bis(2ethylhexyl) ester 43.59
=Diethylhexyl phthalate.(5)
The ethyl acetate fraction was chromatographed over
silica gel column using mixture of methylene chloride and
methanol as eluent, with increasing polarities. The major
compounds were isolated from this fraction were:
Compound 6
It was obtained by the eluent methylene chloride/ methanol
(9.5: 0.5) as a very fine colorless crystal with Rf = 0.65. It
gave a violet color upon spraying with p-anisaldehyde-
sulphuric acid reagent.
The 1H-NMR (Table 2) indicated that there's a mono
substituted benzene ring due to δ 7.20 ppm (m, 5H), in
addition to AA'BB' system in the up-field region
characteristic to α, β sp3 methylene protons attached to a
carboxylic acid group which appeared at δ 2.45 ppm (2H,
t, J=8.3 Hz, H-2) and δ 2.88 ppm (2H, t, J=8.3 Hz, H-3).
Therefore, the compound was established as 3-
phenylpropanoic acid (6).
The mass spectrum (EI-MS) of compound (6) showed a
molecular ion peak at m/z 150 corresponding to C9H10O2.
The spectrum also showed an ion peak at m/z 105 (17.5%)
due to expulsion of the carboxylic group. The base ion
peak appeared at m/z 91 (100%) due to loss of [C2H3O2]+.
Ion peak appeared at m/z 77 (17%) is corresponding to
[C6H5]+. All the previous spectral data supported that
compound (6) is 3-phenylpropanoic acid.
Compound 6
Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladosporioides and its Relation to Toxicity of Cotton Aphid, Aphis gossypii (Glov.)
Int. J. Entomol. Nematol. 117
Area% M.F. Mol. Wt.
0.04 C6H8O2 112
0.27 C18H34O2 282
3.16 C21H36 288
0.07 C22H30N2O3 370
0.07 C24H38O4 390
Table 2: 1H-NMR of compound (6) (CD3OD)
H atom δ value, ppm Multiplicity (J, Hz)
2 2.45 2H, t, J= 8.3 Hz
3 2.88 2H, t, J= 8.3 Hz
Ar- 7.17-7.20 5H, m
Compound 7
It was obtained from the column chromatography by the
eluent methylene chloride/ methanol (93:7), and it was
purified using TLC by eluent methylene chloride/Methanol
(9.5:0.5). It was obtained as a pale-yellow crystal with Rf =
0.38. It gave a violet color upon spraying with p-
anisaldehyde-sulphuric acid reagent.
The IR spectrum showed absorption band at 3450 cm -1
due to a hydroxylic groups and at 3276 cm -1 indicating the
presence of –NH amidic group besides, a broad band for
two amidic and ester carbonyl groups at 1663 cm -1. The
molecular formula was determined as C12H19O4 on the
basis of a prominent ion peak at m/z 241.1 [M]- observed
in the ESIMS negative mode spectrum.
The 1H NMR spectrum of (7) (Table 3) revealed the
presence of two secondary gemeinal methyl groups at
0.96 (3H, d, J= 6.3 Hz) and 0.97 (3H, d, J= 6.3 Hz) in
addition, to two signals for the amidic proton of
pyrrolidinone ring at 4.18 (1H, m) and the other proton of
pyranone ring at 4.52 (ddd, J= 11.1, 6.4, 0.5 Hz).
The 13C NMR spectrum (Table 3) showed total 12
resonances assigned to two sp2 carbons [two carbonyl
groups at δC 169.03 (C2) and 173.06 (C10)], Two methyl
groups [δC 22.24 (C12) and 23.24 (C13)], eight aliphatic
sp3 carbons (including three methylene and five methines)
as supported by the HSQC.
Detailed analysis of the 1H–1H COSY spectrum (Table 3)
disclosed the presence of two continuous spin systems
firstly, from H-4 and sequentially extending until H-12 and
H-13. The second spin system extended from H-6 to H-9.
HMBC experiment permit the substitution pattern for these
functional groups to be established as indicated in
compound (7). The most significant features were the
observed long rang correlation HMBC of the amidic
Shaker et al. 118

carbonyl carbon atom with the protons H-4a, H-4b, H-5 All the previous spectral data supported that compound (7)
and H-11 which located the ketonic group at C-2, of chemical formula, C12H19NO4 which was isolated from
moreover, the carbonyl ester carbon atom with H-6, H-7ax, ethyl acetate fraction is 3-(4β-hydroxy-6-pyranonyl)-5-
H-8, H-9eq protons which positioned the carbonyl group at isopropylpyrrolidin-2-one (7). According our knowledge,
C-10 as lactonic group. Further long rang correlations this is the first time of isolating and reporting compound (7)
through HMBC between the carbon atoms and the protons as secondary metabolite of C. cladosporioides.
established the proposed structure of compound (7).
Table 3: 1H, 13C-NMR and long rang HMBC data for compound (7) (CD3OD).
1 13
Atom H C Long range HMBC protons
1 --------- -----
2 -------- 169.03 H-4a, H-4b, H-5, H-11
3 3.30, brs 48.06
4a 1.51, m 39.36 H-5, H-11, H-12, H-13
4b 1.92, (dd , J= 8.6, 4.8 Hz)
5 4.18, m 54.57 H-3, H-4a, H-4b, H-11, H-12, H-13
6 4.52 (ddd , J= 11.1, 6.4, 0.5 Hz) 58.70 H-7ax, H-8, H-9ax
7 ax 2.09 (ddd , J= 13.3, 11.1, 4.2 Hz) 38.14 H-6, H-9ax
7 eq 2.28 (dd, J= 13.3, 6.4 Hz)
8 4.47 (t, J= 4.2 Hz) 69.10 H-7eq, H-9ax
9 ax 3.44(brd, J= 4.2 Hz) 55.16 H-7eq, H-8
9 eq 3.66 (dd, J= 12.8, 4.2 Hz)
10 ------------ 173.06 H-6, H-7ax, H-8, H-9eq
11 1.89, m 25.77 H-4a, H-4b, H-5, H-12, H-13
12 0.96 (d, J= 6.3 Hz) 22.24 H-4a, H-11, H-13
13 0.97 (d, J= 6.3 Hz) 23.24 H-4a, H-4b, H-5, H-11, H-12

Compound 7
Bioassay
Toxicity of ethyl acetate extract of C. cladosporioides
metabolite against A. gossypii
Data in Table 4, showed that cumulative mortality
percentages of both adults and nymphs increased with
increasing concentrations of the tested extract. Also, a fast
activity against aphids was cleared after 24 hours of
application.
Aphid nymphs showed more susceptibility than adults.
Data in Fig. 2 showed that C. cladosporioides ethyl acetate
extract revealed cumulative mortality of cotton aphid
nymphs ranged between 20.00-53.33% at first day post
treatment. The mortality percentages increased to range
between 40.00-96.67% at the 3rd day post treatment. The
maximum mortality percentages were recorded at 5th day
post treatment to range between 46.67-96.67% at different
tested concentrations.

C. cladosporioides ethyl acetate extract showed
cumulative mortality of cotton aphid adults ranged
between 20.00- 46.67% at first day post treatment (Fig. 1).
The maximum mortality percentages were obtained at 5th
day post treatment ranged between 40-96.67% at different
tested concentrations. Cotton aphid treated with C.
cladosporioides ethyl acetate extract shrunk, lost its weight
and turned to reddish brown color.
Also, data showed that C. cladosporioides ethyl acetate
extract was most effective against nymphs showing LC50
of 24.5827 ppm, LC90 of 128.7385 ppm and toxicity index
of 100%, while, it showed LC50 of 36.6959 ppm, LC90 of
154.4394 ppm and toxicity index of 66.99% against adults.
Our data agreed with Ibrahim (2012) who determined the
virulence of C. cladosporioides against cowpea aphid,
Aphis craccivora. Her results showed that the ethyl acetate

Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladosporioides and its Relation to Toxicity of Cotton Aphid, Aphis gossypii (Glov.)
 Int. J. Entomol. Nematol. 119

extract of C. cladosporioides showed high virulence
against all developmental stages of aphids and nymphs
showed more susceptibility than adults.
We suggested that the volatile compounds may be
contributed toxicity to this extract. Also, presence of
compounds like the alkaloidal compound (7) may be the
responsible for the toxicity and insecticidal properties of
the ethyl acetate extract of C. cladosporioides secondary
metabolites.

Table 4: Efficiency of different solvents extracts of entomopathogenic fungi, C. cladosporioides against cotton
aphid, A. gossypii under laboratory conditions of 25 ± 20 C, 75 ± 5% RH.
Mortality %at indicated day
Treatment Conc. after treatment. LC50 and LC90 and Slope X2 Toxicity
(ppm) 1st 3rd 5th 7th confidence limits confidence limits ±SE Index
day day day day at 95% at 95%
Adults 25 20.00 33.33 40.00 40.00
50 30.00 53.33 56.67 56.67 36.6959 154.4394 2.0534
100 33.33 73.33 80.00 80.00 ± 0.5749 66.99
200 46.67 96.67 96.67 96.67 23.313 48.921 109.026 294.5629 0.3904
Nymphs 25 20.00 40.00 46.67 46.67
50 26.67 63.33 73.33 73.33 24.5827 128.7385 1.7823
100 36.67 76.67 83.33 83.33 10.8511 36.3283 88.4876 275.5471 ± 0.3403 100.00
200 53.33 96.67 96.67 96.67 0.4038

CONCLUSION

The present study illustrated the importance of the

entomopathogenic fungi as great source of natural
products that having insecticidal activities. New compound
was isolated from ethyl acetate extract of C.
cladosporioides secondary metabolites. This compound
was identified as 3-(4β-hydroxy-6-pyranonyl)-5-
isopropylpyrrolidin-2-one.

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Fig. 2: The cumulative mortality % of ethyl acetate extract
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Secondary Metabolites of the Entomopathogenic Fungus, Cladosporium cladosporioides and its Relation to Toxicity of Cotton Aphid, Aphis gossypii (Glov.)