Mycoflora associated with the seed samples of Cucurbit pepo L.collected from Pakistan

Abstract

Seventeen seed samples collected from Peshawar (and Mandibahuddin (1) areas of Pakistan were analyzed for the seed-borne mycoflora using standard blotter, agar plate and deep-freezing methods as suggested by ISTA. At least 100 fungal species belonging to 49 genera were isolated mutually from all the seed samples analyzed. Seed samples from Peshawar followed by Sukkur & Ghotki were highly infected with fungi. Agar plate method was found best for the isolation of fungi both qualitatively and quantitatively followed by standard blotter method. By using agar plate method, 79 species of 40 genera were isolated while 57 species of 29 fungal genera were isolated by the blotter method. Being frost sensitive, rot and decay of pumpkin seeds was observed in deep-freezing method. Species of Fusarium, Phoma and Macrophomina phaseolina were isolated by all three methods. However, the most dominant fungi were the species of Aspergillus followed by Rhizopus and Chaetomium. Good germination of seeds was observed in surface sterilized seeds treated with 1% Ca (OCl) 2, although surface sterilization was found less effective against fungal mycoflora. Atleast 95 species of 47 genera are newly reported from Pakistan.

Full text

Pak. J. Bot., 45(6): 2173-2179, 2013.
MYCOFLORA ASSOCIATED WITH THE SEED SAMPLES OF CUCURBITA PEPO L.
COLLECTED FROM PAKISTAN
SUMMIAYA RAHIM*, SHAHNAZ DAWAR AND M. JAVED ZAKI
Department of Botany, University of Karachi, Karachi-75270- Pakistan
*Corresponding author’s e-mail: sumy6@hotmail.com
Abstract
Seventeen seed samples collected from Peshawar (2), Swabi (1), Tordher (1), Fatu chack (1), Mardan (2), Karachi (4),
Islamabad (1), Murree (1), Abbottabad (1), Sukkur (1), Ghotki (1) and Mandibahuddin (1) areas of Pakistan were analyzed
for the seed-borne mycoflora using standard blotter, agar plate and deep-freezing methods as suggested by ISTA. At least
100 fungal species belonging to 49 genera were isolated mutually from all the seed samples analyzed. Seed samples from
Peshawar followed by Sukkur & Ghotki were highly infected with fungi. Agar plate method was found best for the isolation
of fungi both qualitatively and quantitatively followed by standard blotter method. By using agar plate method, 79 species of
40 genera were isolated while 57 species of 29 fungal genera were isolated by the blotter method. Being frost sensitive, rot
and decay of pumpkin seeds was observed in deep-freezing method. Species of Fusarium, Phoma and Macrophomina
phaseolina were isolated by all three methods. However, the most dominant fungi were the species of Aspergillus followed
by Rhizopus and Chaetomium. Good germination of seeds was observed in surface sterilized seeds treated with 1% Ca (OCl)
2, although surface sterilization was found less effective against fungal mycoflora. Atleast 95 species of 47 genera are newly
reported from Pakistan.
Introduction
Cucurbita pepo L., of the family Cucurbitaceae is
commonly known as zucchini, courgette or summer squash
when immature and pumpkin or winter squash when
mature. It is native of America but cultivated worldwide
with an annual production of 17.7 million tonnes from 1.4
million hectares (Anon., 2002). It is highly susceptible to
frost and cultivated mainly during may/June and harvested
around October. It is cultivated throughout Pakistan, as a
Kharif crop with an annual production of 45217 tonnes
from an area of 4027 hectares (Anon., 2009). Pumpkins
vary in size and colors. The nutrient profile of pumpkin
seeds showed that they are low in calories, however the
seeds are rich source of Vitamin A, vitamin B1, B2, B3,
B6, B12, vitamin C, vitamin D, vitamin E, vitamin K,
pantothenic acid; minerals like calcium, iron, manganese,
magnesium, phosphorous, potassium, selenium, sodium,
zinc etc., number of amino acids and many other nutrients
are present in trace amount. They also contain wide variety
of antioxidants phytonutrient. Seeds are found to have
some benefits against diabetes, anti microbial activities and
cancer etc. (Mateljan, 2006). Seeds of pumpkin are flat and
oval with slightly pointed tip; colour may vary from species
to species. They are commonly known as Pepita. Pepitas
raw or roasted is a rich source of nutrition. Oil is also
extracted from Pepitas which is used in folk medicines.
A survey of literature showed that very little work has
been done on the seed-borne mycoflora of Pumpkin. The
fungi reported on Pumpkin seeds include the species of
Alternaria, Aspergillus, Fusarium, Penicillium, Sclerotium
and Macrophomina phaseolina (Ahmed et al., 1993). Fungi
damaging the pumpkin fruit and seeds are mostly soil borne
and attack either before or after harvest. Pumpkins are
temperature sensitive, storing under direct sunlight or in
frost, both cause decay and rot of the fruit. Jamiolkowska et
al., (2011) isolated the species of Fusarium from the roots
of zucchini, responsible for damping-off, stunting and stem
and root- rot of the plants. The fungi attacking pumpkin
includes the species of Fusarium causing Fusarium rot,
Macrophomina phaseolina causing char coal rot,
Sclerotinia, Collectotrichum lagenarium causing
Anthracnose, species of Erysiphales and Sphaerotheca
(causing powdery mildew, Septoria spp. (septoria leaf
spot), Phytophthora spp. causing Phytophthora rot,
Didymella bryoniae causing black rot, Cladosporium
cucumerinum (responsible for scab) and Plectosporium
tabacinum causing Plectosporium blight (Zitter et al.,
1996; Mc Grath, 2011). Seed-borne fungi reported from
Pakistan on cucurbits include Alternaria spp., Aspergillus
spp., Fusarium spp., Myrothecium roridum, Penicillium
spp, and Rhizopus spp. (Sultana & Ghaffar, 2009).
Due to their nutritional values and medicinal
properties, pumpkin is gaining interest of researchers and
agriculturists. Very little work has been reported
previously from Pakistan on pumpkin. Therefore keeping
in view their emerging economical importance, a
relatively new research work has been done to find out the
mycoflora associated with pumpkin seeds from Pakistan.
Materials and Methods
For the detection of seed-borne mycoflora, ISTA
(Anon., 1993) techniques i.e. Standard blotter method,
Agar plate method and Deep-freezing methods were used.
About 400 seeds of each sample were tested.
Collection of seeds: Pumpkin seed samples (17 samples)
were collected from the local markets of various areas of
Pakistan viz., Peshawar (2), Swabi (1), Tordher (1), Fatu
chack (1), Mardan (2), Karachi (4), Islamabad (1), Murree
(1), Abbottabad (1), Sukkur (1), Ghotki (1) and
Mandibahuddin (1).
Standard blotter method: Untreated and seeds after
treatment with 1% Ca (OCl)2 for 2 minutes were placed
on three layers of moistened blotter paper, 10 seeds per
Petri dish. The dishes were incubated for 5-7 days at
28±2°C under 12h, alternating cycle of artificial day light
(ADL) and darkness (Anon., 1993).

SUMMIAYA RAHIM ET AL.,
2174
Agar plate method: Untreated and seeds after treatment
with 1% Ca (OCl)2 for 2 minutes were placed on Potato
dextrose agar (PDA), 10 seeds per Petri dish. The dishes
were incubated for 5-7 days at 28±2°C under 12h,
alternating cycle of artificial day light (ADL) and
darkness (Anon., 1993).
Deep-freezing method: Untreated and seeds after
treatment with 1% Ca (OCl)2 for 2 minutes were placed
on three layers of moistened blotter paper, 10 seeds per
Petri dish were incubated for 24h, each at 28±2°C and -
2°C followed by 5 days incubation at 28±2°C under 12h,
alternating cycle of artificial day light (ADL) and
darkness (Anon., 1993).
Identification of fungi: Mycoflora observed on seeds
were identified after reference to Barnett & Hunter
(1998), Domsch et al., (1980), Ellis (1971), Gilman
(1950), Hanlin (1989), Nelson et al., (1983), Raper &
Fennell (1965).
Analysis of data: Data was subjected to analysis of
variance (ANOVA) following the procedures as
suggested by Gomez & Gomez (1984).
Results
Atleast 100 species belonging to 49 genera viz.,
Absidia corymbifera
(Cohn) Sacc. & Trotter,
A.
cylindrospora
Hagem,
A. glauca
Hagem,
Acremonium
cerealis
(Karst).W.Gams.
, A. furcatum
F. & V. Moreau
ex W.Gams,
A. kiliense
Grutz,
A. murorum
(Corda)
W.Gams.
Acremonium
species Link ex Fr.,
Alternaria
(Fr.) Keissler,
A. cucumerina
(Ellis & Everh.) Elliott.
A.
dianthicola
Neergaard,
A. longipes
(Ellis & Everh.)
Mason.,
A
.
raphani
Groves & Skolko.,
A.tenuissima
(Kunze ex pers.) Wiltshire.,
Alternaria
species Nees ex Fr.
Nees
., Aspergillus flavus
Link ex Gray.,
A.fumigatus
Fres.,
A. glaucus
. Mich ex Fr.,
A.niger
Van Tieghem.,
A
.
oryzae
(Ahlburg) Cohn.,
A. parasiticus
Speare,
A.
sulphureus
Thom & Church
, A. terreus
Thom,
A. ustus
(Bain.) Thom & Church,
A
.
versicolor
(Vuill.)Tiraboschi,
Aspergillus
spp. Mich. ex Fr.,
Bahusakala olivaceonigra
(Berk. & Br.) Subram.,
Botrytis
cinerea
Pers. ex Nocca &
Balb.,
Brachysporium
obovatum
(Berk.) Sacc.,
Cephaliophora
irregularis
Thaxter.,
Chaetomium
bostrychodes
Zopf.,
C.cochliodes
Pall.,
C
.
crispatum
(Fuckel)Fuckel,
C
.
elatum
Kunze ex Steud.,
C
.
globosum
Kunze ex steud.,
C
.
indicum
Corda,
C
.
murorum
Corda,
C
.
spirale
Zopf,
Chaetomium
species Kunze ex Fr.,
Chuppia
sarcinifera
Deighton,
Cladosporium
cladosporioides
(Fres.) de Vries.,
C
.
cucumerinum
Ellis &
Arth.,
C
.
spaerospermum
Penz.,
Cochliobolus
nodulosus
Luttrell,
Coremiella
cubispora
Berk. & Curt.,
Curvularia
lunata
(wakker) Boedijn,
C. pallescens
Boedijn,
C
.
penniseti
(Mitra) Boedijn,
C
.
robusta
Kilpatrick &
Luttrell,
Drechslera
australiensis
(Bugnicourt) Subram. &
Jain ex M.B. Ellis,
D. bicolor
Paul & Parbery,
D.hawaiiensis
(Bugnicourt) Subram. & Jain,
Emericella
nidulans
(Eidam) Vuill.,
E
.
rugulosa
(Thom & Raper)
C.R. Benjamin,
Emericellopsis
terricola
van Beyma,
Epicoccum
purpurascens
Ehrenb. Ex Schlecht.,
Fusarium
oxysporum
Schlecht. emend. Sny. & Hans.,
F
.
semitectum
Berk. & Rav.,
Fusarium
species Link ex Fr.,
Glomerella
cingulata
Spauld. & v. Schrenk.,
Gonitrichum
macrocladium
(Sacc.) Hughes.,
Helminthosphaerica
clavariarum
(Tul.) Fuckel,
Humicola
fuscoatra
Traaen,
Macrophomina
phaseolina
(Tassi) Goid,
Melanospora
sp.
Corda,
Memnoniella
echinata
(Riv.) Galloway,
M
.
subsimplex
(Cooke) Deighton,
Monilia
sp. Pers. ex Fr.,
Monodictys
levis
(Wiltshire) Hughes,
Mucor
hiemalis
Wehmer,
M
.
mucedo
Mich. Ex St. Am.,
Myrothecium
cinctum
(Corda) Sacc.,
M
.
roridum
Tode ex Steudel,
Nectria
inventa
Pethybr,
N
.
ventricosa
C. Booth,
Neosartorya
fischeri
(Wehmer) Malloch & Cain,
Nigrospora
oryzae
Hudson,
N
.
sphaerica
(Sacc.) Mason,
Nigrospora
species Zimmermann,
Paecilomyces
species
Bain.,
Papulaspora
irregularis
Hotson ,
Penicillium
Link
ex Fr.,
Phoma
eupyrena
Sacc.,
P
.
exigua
Desm.,
P
.
medicaginis
Malbr. & Roum.,
Rhizopus
arrhizus
Fischer,
R
.
oryzae
Went & Prinsen Geerligs,
R
.
stolonifer
(Ehrenb.
Ex Link) Lind,
Scytilidium
lignicola
Pisante,
Septotrullula
bacilligera
Höhnel,
Stachybotrys
cylindrospora
C.W.
Jensen,
Staphylotrichum
coccosporum
J. Meyer & Nicot ,
Taeniolella
exillis
(Karst.) Hughes,
Trichocladium
opacum
(Corda) Hughes,
Trichoderma
hamatum
(Bonord.) Bain,
T
.
harzianum
Rifai,
T
.
viride
Pers. ex
Gray were isolated and identified from the seed samples
collected from various localities of Pakistan by ISTA
techniques. Out of 100 species isolated, except for
Alternaria
,
Aspergillus
,
Fusarium
,
M
.
phaseolina
and
Penicillium
(Ahmed
et al
., 1993), all other fungi are
newly reported from Pakistan. Agar plate method was
found to be more suitable for the isolation of fungi
followed by blotter method. Agar plate method yielded 79
fungal species belonging to 40 genera where as blotter
method yielded 57 species belonging to 29 genera.
Pumpkins as well as its seeds are highly frost sensitive,
deep-freezing method yielded around 26 species
belonging to 15 genera (Table 1). Pathogenic fungi like
M
.
phaseolina
, species of
Fusarium
and
Phoma
were
observed on seeds causing char-coal rot, damping off, rot
and decay of seeds and seedlings. Very heavy infection of
seeds was observed by the species of
Aspergillus flavus
(p<0.001) and
A. niger
(p<0.001),
Rhizopus
and
Chaetomium
. These fungi were responsible for the
complete rottening of seeds and seedlings. Mites were
also observed on the seed samples infested with
Chaetomium
species. Seeds surface sterilized with 1% Ca
(OCl)
2
has not produced any significant effect on
mycoflora of seeds however good germination was
observed during incubation of surface sterilized seeds.
Being frost sensitive, rot and decay of seeds subjected to
deep-freezing method was observed. Most of the fungi
isolated from seed samples (both pathogenic and storage)
are known to produce mycotoxins. Seed samples collected
from Peshawar, Ghotki and Sukkur were found to be
highly infected with fungi.

MYCOFLORA OF CUCURBITA PEPO L. COLLECTED FROM PAKISTAN 2175

SUMMIAYA RAHIM ET AL.,
2176

MYCOFLORA OF CUCURBITA PEPO L. COLLECTED FROM PAKISTAN 2177

SUMMIAYA RAHIM ET AL.,
2178
Discussion
Of the 17 seed samples tested, samples collected from
Peshawar, Mardan, Abbottabad, Murree, Swabi,
Mandibahuddin, Ghotki and Sukkur were found to be
infected with pathogenic fungi like Fusarium spp,
M.phaseolina, and Phoma spp.
Quantitatively as well as qualitatively, agar plate
method was found to the best for the isolation of most the
fungi from Pumpkin seeds. Unlike Sultana & Ghaffar
(2009) who found blotter and deep-freezing methods most
suitable for the seeds of bottle gourd. High incidence of
Aspergillus species caused retarded growth and decay of
seeds and seedlings. Chaetomium species were also
observed in higher frequency on the seeds, as Chaetomium
is cellulose decomposing fungus (Domsch et al., 1980)
blotter method was found to be good for the isolation of
Chaetomium species. Various sizes of sclerotia of
M.phaseolina were observed on seeds causing char-coal rot
and decay. Similar results were also observed by Sultana &
Ghaffar (2009) on bottle gourd, where the fungi have
produced small sized sclerotia and black rot of seeds. Such
similar results were also reported by Maholay & Sohi,
(1982); Maholay, (1988, 1989), where M.phaseolina has
produced black rot on the seeds of muskmelon, bottle
gourd and squashes. Fusarium species are responsible for
the seed rot, seedling blight and wilt in number of
cucurbitaceous crops (Booth, 1971). Weidenborner (2001)
isolated 25 different species belonging to 17 genera from
the seeds of pumpkin using different media for isolation
which included the species of Absidia, Alternaria,
Aspergillus, Chaetomium, Cladosporium, Epicoccum,
Eurotium, Fusarium, Mucor, Phoma, Penicillium,
Rhizopus, scopulariopsis and Trichoderma etc. Due to
higher infection of fungi, seeds failed to germinate in agar
plate method and as fungal infection were low on blotter
method, the seeds germinated well on it. Such similar
results were also reported by Kaiser et al., (1989) on lentil
seeds where seeds failed to germinate due to high fungal
infection. Overall, good germination of seeds was observed
on surface sterilized seeds. Similar results were observed
by Odofin (2010) who reported that treatment with bleach
has enhanced the germination rate of okra seeds.
These saprophytic as well as pathogenic fungi attack
pumpkin before and after harvest causing rot, decay, scab,
blight etc of the pumpkin in the field as well as after
harvest during the storage of fruit. Mostly the fungi are
present as dormant mycelium in the tissues of fruits and
seeds and cause infection when the environment is suitable
for their germination. From the consumption point of view
presence of so many fungi both pathogenic and
saprophytic, is not a good sign. Nearly all the fungi isolated
hereby, are known to produce mycotoxins. Niaz et al.,
(2012) reported that out of 59 maize seed samples, 50 were
found to be contaminated with aflatoxins, while 43 seed
samples were contaminated with zearalenone. Aspergillus
species are responsible for the production of aflatoxins.
Aflatoxins are carcinogenic and responsible for the
production of aspergillosis and systemic infections in man,
animals and birds (Raper & Fennell, 1965). Storage and
pathogenic fungi are responsible for the loss of germination
and discoloration of seeds (Barton, 1961; Harrington, 1963;
Golumbic & Laudani, 1966; Naqvi et al., 2012). Alternaria
spp. produces mycotoxins such as alternariols, altenuens,
altertoxins and tenuazonic acid (King & Schade, 1984).
Most of the Chaetomium species are cellulose
decomposing fungi causing soft rot, decay and
decomposition of wide variety of hosts besides being food
for mites (Domsch et al., 1980). Fungi forming fruiting
bodies always have high mycotoxins production ability and
are more pathogenic. Presence of Melanospora sp. and
other ascomycetes showed that the seed samples were
highly infected with pathogenic fungi.
Studies on the pumpkin seeds showed that pumpkin
fruit is highly susceptible to fungal infestation before and
after harvest while the seeds are prone to pathogenic as
well as saprophytic fungi during storage. Care must be
taken while handling the seeds; they must be cleaned and
properly washed before drying the seeds for storage to
avoid any fungal infection, mites and insects attack. Being
agricultural state, proper steps must be taken to avoid
diseases and damage to the crop due to fungal mycoflora,
for saving economy of the country.
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(Received for publication 15 September 2012)