INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
Physiological Studies of Fusarium oxysporum F. Sp. Ciceri
SAJID FAROOQ, SH. MUHAMMAD IQBAL1† AND CH. ABDUL RAUF
Department of Plant Pathology, University of Arid Agriculture, Rawalpindi
†Pulses Programme, National Agricultural Research Centre, Islamabad
1Corresponding author’s e-mail: firstname.lastname@example.org
In vitro studies were conducted on the effect of culture, media, carbon and nitrogen sources, temperature and pH levels on
mycelial growth of F. oxysporum f.sp. ciceri. The fungus grew the best on Czapek dox agar and chickpea seed-meal agar
media among eight culture media that were tried. Glucose was found to be the best source of carbon whereas peptone was the
best source of nitrogen. Growth of F. oxysporum was maximum at 300C after seven days of inoculation, which was reduced
drastically below 150C and above 350C. The most suitable pH level for growth of fungus was 7.0 and 6.0.
Key Words: Fusarium oxysporum; In vitro; Culture media; pH; Carbon; Nitrogen; Mycelial growth
Chickpea (Cicer arietinum L.) is one of the most
important grain legumes and constitutes about 70% of the
pulse crops grown in Pakistan. The national average yield
(550 kg ha-1) of chickpea is very low as compared to the
other chickpea producing countries of the world
(Anonymous, 2003). Although a number of biotic and
abiotic factors contribute for low chickpea production but
endemic occurrence of wilt disease caused by Fusarium
oxysporum f.sp. ciceri is of significant importance.
Chickpea wilt is worldwide in occurrence and has been
reported from many countries of the world (Nene et al.,
Since the creation of Pakistan Fusariun wilt had
always been remained a serious problem in chickpea crop.
Although, no precise information on the losses caused by
the disease is available, but the yield loss may vary from 10
to 100% depending on the environmental conditions
(Grewal & Pal, 1970). Likewise, an estimated annual loss of
rupees 12 million was reported due to wilt disease in
Pakistan (Sattar et al., 1953). In 1956, the disease appeared
in epidemic form and more than 75% crop losses were
reported (Akhtar, 1956).
The wilt pathogen is soil-borne and survives through
chlamydospores in seed and dead plant debris in soil
(Haware et al, 1978). Since, the fungus can survive in the
soil for several years, it is not possible to control the disease
through normal crop rotations. Although a number of
chickpea lines have been reported as resistant to wilt from
different countries of the world (Nene et al., 1981), but their
success has been highly localized due to location-specific
races of the pathogen (Singh & Reddy, 1991). The seed-
borne inoculum can be eradicated by seed-dressing
fungicides: Benlate-T: Benomyl 30% + Thiram 30% @
1.5% (Haware et al., 1978).
Resistance to vascular wilt may be expressed before
the pathogen gains entry into the xylem of plants or even
after that. The very fact that the vascular wilt fungi are
confined to the xylem until the later phases of the disease is
itself a manifestation of resistance on the part of extra
vascular tissues. Since vascular fungi are facultative
parasites, they obviously find the xylem environment
relatively free of severe host reactions. These peculiar
features have to be born in mind to identify the factors of
resistance to wilt disease. The present investigation was
conducted to study the effect of physiological factors on the
mycelial growth of the fungus.
MATERIALS AND METHODS
Studies of the following physiological aspects of F.
oxysporum f.sp ciceri were conducted in vitro.
Effect of Culture Media. Following eight culture media
were used to find out the most suitable one for the mycelial
growth of the fungus. Each culture medium was prepared in
1 liter of water and autoclaved at 1200C at 15 psi for 20 min.
These were cooled to 450C and then poured in 9 cm petri
dishes for solidification. Chickpea Seed meal Extract Agar
(CSMA) Medium (Chickpea seed meal extract 20 g,
Dextrose 20 g, Agar 20 g), Potato Dextrose Agar (PDA)
Medium (Potato starch 20 g, Dextrose 20 g, Agar 20 g),
Cornmeal Agar Medium (Cornmeal 20 g, Dextrose 20 g,
Agar 20 g), Malt Extract Agar Medium (Malt extract 20 g,
Agar 25 g, Peptone 2 g), Cape dox Agar Medium (Sodium
nitrate 2 g, Potassium nitrate 1 g, Magnesium sulphate 0.5 g,
Potassium chloride 0.5 g, Ferrous sulphate 3 g, Sucrose 30
g, Agar 20 g), Sabouroud’s Agar Medium (Dextrose 40 g,
Peptone 10 g, Agar 20 g), Waksman‘s Agar Medium (Agar
26 g, Glucose 10 g, Peptone 5 g, Potassium dihydrogen
phosphate 1 g, Magnesium sulphate 0.5 g), Richard’s Agar
Medium (Agar 20 g, Sucrose 50 g, Potassium nitrate 10 g,
Potassium dihydrogen phosphate 5 g, Magnesium sulphate
FAROOQ et al. / Int. J. Agri. Biol., Vol. 7, No. 2, 2005
Effect of Different Carbon and Nitrogen Sources.
Czapeck Dox agar medium (in one liter of water) was used
as the medium for studying the effect of carbon and nitrogen
Nitrogen sources. Three nitrogen compounds viz;
Potassium nitrates 10 g, Sodium nitrate 8.5 g and Peptone
2.5 g were amended in Czapeck Dox agar medium.
Carbon sources. Three carbon compounds viz; glucose
13.5 g, sucrose 12.5 g and starch 12.5 g were tried
individually as a constitute of carbon source in Czapeck
Dox agar medium.
Effect of Temperature. The fungus F. oxysporum was
inoculated in Czapeck Dox medium using five petri dishes
for each temperature, which was applied at 5, 10, 15, 20, 25,
30 and 350C.
Effec t of Different pH Levels. The test fungus was
inoculated on Czapeck Dox agar medium whose pH was
adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0, respectively.
All these experiments were conducted in five
replicates. Plates were inoculated by placing 4 mm agar
medium plugs containing active mycelium of the fungus
and were placed in the centre of the petri dishes. Plates were
incubated at 300C (except for the study of temperatures).
Observations on linear growth were recorded after seven
days of inoculation.
RESULTS AND DISCUSSION
Effect of culture media. The results of the experiment
revealed that the Czapeck Dox agar and CSMA media were
the best for the radial growth of F. oxysporum as this fungus
gave maximum growth of 85 and 80 mm, respectively, after
seven days of inoculation followed by Cornmeal agar and
Malt extract agar media which showed growth of 70 and 65
mm, respectively (Fig. 1). Khanzada et al. (2003) also
reported similar results with M. phaseolina. Different
synthetic and non synthetic cultural media have profound
influence on cultural and morphological characteristics of
fungus (Shaikh, 1974). Haware et al. (1986) had modified
Cape do agar medium by adding PCNB, streptomycin and
malachite green. This medium is highly effective for the
growth of F. oxysporum.
Effect of different carbon and nitrogen sources. The
results of this experiment indicated that all the carbon
sources were suitable for the fungus growth. However,
glucose was found to be the best carbon source for this
purpose (Fig. 2). The fungus may convert certain forms of
complex carbon compounds into simple form, which may
be readily metabolized (Bais et al., 1970).
As is evident from Fig. 3, Peptone was found to be
best source of nitrogen for F. oxysporum and the maximum
growth of fungus 90 mm was attained. It was followed by
Potassium nitrate. On potassium nitrate (KNO3), the growth
of fungus was 80 mm after seven days of inoculation.
Similar observations were made by Hussain et al. (2003) for
the mycelial study of Sclerotium rolfsii Sacc. Tariq et al.
(1993) obtained the maximum growth of Botrytis
glaiolorum when glucose and potassium nitrate were used
as carbon and nitrogen sources, respectively.
Fig. 1. Effect of different culture media on the
mycelial growth of F. oxysporum f.sp. cicri
CSMA Cornmeal Malt extract WaksmanRichardsd PDA Czpekdox Sabroud
Fig. 2. Effect of carbon sources on the mycelial
growth of. F. oxysporum f.sp. cicri
Fig. 3. Effect of nitrogen sources on the mycelial
growth of F. oxysporum f.sp. cicri
PHYSIOLOGICAL STUDIES OF Fusarium oxysporum F. SP. Ciceri / Int. J. Agri. Biol., Vol. 7, No. 2, 2005
Results of our study indicated that the role of C: N
ratio is very important. The fungus readily colonizes organic
substances in the soil. Increased inoculum potential and
disease severity are positively correlated with the food base
of organic substances. Crop debris that serves as a food base
can also serve as an infection bridge. The fungus becomes
active primarily near the soil surface and hyphea of the
fungus penetrate into the roots and cause severe blockage in
the xylem vessels which results in the wilting of plants.
Effect of temperature. As evident from Fig. 4, the fungus
grew at the temperature range of 10–350C. However,
growth of the fungus was drastically reduced below 150C
and started to decline above 350C, as these temperatures did
not favour for growth of the fungus. It was observed that at
250C and 300C, the fungus attained the maximum growth
76.8 and 85.4 mm while at 250C, it was 59.3 mm after seven
days of inoculation. No growth was observed at 50C. Gupta
et al. (1986) reported similar findings regarding temperature
requirements to this fungus. Soil temperature relationship
indicated that suitable temperature for development of
chickpea wilt is 25-300C (Chauhan, 1965).
Effect of different pH levels. Growth of the fungus was
obtained at all the pH levels tested but it was maximum at
pH 7 where it was 80 mm after seven days of inoculation
(Fig. 5). pH 6 (74 mm) and pH 8 (65 mm) were also
favourable. Growth of the fungus decreased by increasing or
decreasing the pH level from the neutral level. The results of
the present study are in agreement with those achieved by
Hayes (1978). This fungus can tolerate a wide range of pH
5.0–6.5 (Shaikh, 1974).
Akhtar, M., 1956. Further studies on the wilt disease of gram in Punjab.
M.Sc. Thesis. University of Punjab. Lahore
Bais, B.S., S.B. Singh and D.V. Singh, 1970. Effect of different carbon and
nitrogen sources on the growth and sporulation of Curvularia
pallescens. Indian Phytopath., 23: 511–7
Chauhan, S.K., 1965. The interaction of certain soil conditions in relation to
the occurrence of Fusarium wilt of gram. Indian J. Agric. Sci., 35: 52
Grewal, J.S. and M. Pal, 1970. Fungal diseases of gram and arhar. Proc. IV
Annual Workshop of Pulse Crops, p. 168. PAU, Ludhiana
Gupta, O., M.N. Khare and S.R. Kotasthane, 1986. Variability among six
isolates of Fusarium oxysporum f.sp. ciceri causing wilt of chickpea.
Indian Phytopathol., 39: 279
Haware, M.P, Y.L. Nene and R. Rajeshware, 1978. Eradication of
Fusarium oxysporum f.sp. ciceri transmitted in chickpea seed.
Phytopathology, 68: 1364–7
Haware, M.P, Y.L. Nene and S.B. Mathur, 1986. Seed–borne diseases of
chickpea. Techn. Bullet. No.1, ICRISAT, p. 32. India
Hayes, W.A., 1978. Nutrition, substrates and principles of disease control.
In: The Biology and Cultivation of Edible Mushrooms, pp. 202–22.
Academic press. New York
Hussain, A., S.M. Iqbal, N. Ayub and A.M. Haqqani, 2003. Physiological
studies of Sclerotium rolfsii. Pakistan J. Plant Pathol., 2: 102–6
Iqbal, S.M., M.S.A. Khan and A. Bakhsh, 2003. Physiological studies on
Ascochyta rabiei (Pass.) Lab. MYCOPATH 1: 137–41
Khanzada, S.A., S.M. Iqbal and A.M. Haqqani, 2003. Physiological studies
on Macrophomina phaseolina. MYCOPATH, 1: 31–4
Nene, Y.L., M.P. Haware and M.V. Reddy, 1981. Chickpea disease
screening techniques. ICRISAT Information Bulletin, 3: 44
Nene, Y.L., V.K. Sheila and S.B. Sharma, 1984. A world list of chickpea
(Cicer aretinum L.) and pigeonpea (Cajanus cajan L.(Millsp)
pathogens. ICRISAT Pulses Pathology Progress Report, 32: 19
Sattar, A., A.G. Arif and M. Mohu–ud–Din, 1953. Effect of soil temperature
and moisture on the incidence of gram wilt. Pakistan J. Sci. Res., 5:
Singh, K.B. and M.V. Reddy, 1991. Advances in disease resistance
breeding in chickpea. Advances in Agron., 45: 191–222
Shaikh, M.H., 1974. Studies on wilt of gram (Cicer arietinum L.) caused by
Fusarium oxysporum f.sp. ciceri in Marathwada Region. M.Sc.
Thesis, Marathwada Krishi Vidyapeeth, Parbhani, India
Tariq, M., J.H. Mirza and A.B. Shakir, 1993. Physiological studies of
Botrytis gladiolorum and its in vitro sensitivity to fungicides.
Pakistan J. Phytopathol., 5: 89–92
(Received 20 November 2004; Accepted 18 December 2005)
Fig. 4. Effect of different pH levels on the mycelial
growth of F. oxysporum f.sp. cicri
pH 5pH 6pH 7pH 8pH 9
Fig. 5. Effect of temperature on the mycelial growth
of F. oxysporum f.sp. cicri