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Research Article
Screening of Chickpea Germplasm Against Fusarium Wilt in Pot Soil, Water Culture and Cultural Filtrate

Muhammad Sibtain, M.B. llyas , Iftikhar A. Khan and S.Sarwar Alam

Out of 31 lines evaluated against chickpea wilt caused by Fusarium oxysporum f.sp. ciceris, only three lines 99109, 99112 and 99115 were found to be highly resistant, two lines 1040/99 and 99104 were moderate resistant where as 26 lines were susceptible to highly susceptible. A very good correlation was observed among the three screening methods. i.e. screening of the germplasm against the pathogen using pot soil, water culture and spore free culture filtrate of the fungus.

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  How to cite this article:

Muhammad Sibtain, M.B. llyas , Iftikhar A. Khan and S.Sarwar Alam , 2001. Screening of Chickpea Germplasm Against Fusarium Wilt in Pot Soil, Water Culture and Cultural Filtrate. Journal of Biological Sciences, 1: 229-231.

DOI: 10.3923/jbs.2001.229.231



Chickpea (Cicer arietinum L.) is an important legume in the south East and Western Asia, Northern and Eastern Africa and Central and Southern America. Pakistan is the third major producer of chickpea after India and Turkey (Anonymous, 1998). In Pakistan, chickpea is grown over an area of 1118 thousand hectares with production of 607 kg/ha which is lower than that in India producing 813 kg/ha (Anonymous, 1996). This low yield is due to the lack of crop management and diseases affecting the crop. Among the fungal diseases, chickpea will caused by Fusarium oxysporum f.sp. ciceris is a serious problem in Pakistan (Khan, 1980), especially in thal area the districts of Jhang Lia, Khoshab, Bhukar and Mianwali. Wilt is a serious disease in Pakistan causing annual losses of about 12 million rupees (Sattar et al., 1953). Akhtar (1956) reported about 75 percent damage to the crop in severe cases.

The most ideal and economical way of controlling this disease is the use of host resistance which can be determined by different germplasm screening techniques viz., screening of germplasm in pot soil, water culture and cultural filtrate containing the phytotoxins. Phytotoxins are reported to be helpful not only in understanding the mechanism of pathogens but also in identifying resistant cells in tissue culture or in screening and breeding for disease resistance (Song et al., 1994) reports different levels of resistance/susceptibility in chickpea germplasm.

Materials and Methods

Screening in pot soil: A pure culture of Fusarium oxysporum f.sp.ciceris was obtained from phytopathological lab of NIAB. A sand maize meal medium was prepared in each of the several 250 ml Erlenmeyer flasks. This medium was autoclaved in the flasks at 15 lb psi for 20 minutes. Each flask was inoculated with a bit of fungus from a pure culture and incubated at 25°C for 15 days. A fungus soil mixture was prepared by hand mixing contents of each flask with 2 kg of non-autoclaved field soil. This soil was taken from a chickpea field where wilt normally occurred. Large (30 cm dia.) earthen pots were filled with inoculated soil approximately 10 kg of soil was required to fill each pot. The pots were watered and kept for 4 days at 25± before proceeding to the next step.

Forty to fifty seeds of highly susceptible cultivar (Aug 424) were sown in pot at 2-3 cm depth. Most plants exhibited wilting after 10 days, which were chopped and incorporated into the soil. This procedure was repeated until over 90% wilt was observed. These pots were than ready for screening. Ten seeds of each of the 31 test lines were planted in a pot. A highly susceptible line (Aug 424) served as control and a resistant line (Paidar 91) were grown as checks. The data on the number of wilted seedling in each pot for each test line were recorded and wilt incidence was calculated for each test line by using the formula.

The data on the mortality due to wilt were taken add the resistance/susceptibility of each line was determined by using a rating scale where 0-10% mortality = highly resistant. 11-20% = resistant 21-30% moderately resistant, 31-50% susceptible and 51-100 % highly susceptible (Iqbal et al., 1993).

Screening in water culture: Fifteen surface sterilized (5 min in 2.5% sodium hypochlorite) seeds of each of the test lines as well as susceptible checkline (Aug-424) were sown in autoclaved riverbed sand placed in 15 cm diameter pot. One pot containing 1 kg sand was used for each test line. Patato dextrose broth was prepared in one 250 ml flask, 100 ml of broth was placed and autoclaved at 15 lb psi for 20 minutes. This broth was then inoculated with a bit of the fungus (Fusarium oxysporum f.sp. ciceris) growth from a fresh culture tube. The inoculated plates were incubated on a shaker (8 hr each days) at room temperature for 10 days. Entire contents of the flask were diluted with sterilized distilled water to get the final inoculum dilution of 2.5% (about 2.5 liters of water were needed to obtain the desired dilution of the content of one flask). This dilution contained approx. 6.5×105 spores/ml. Ten days old (from prior sowing) seedlings were removed from sand. There root system was washed in running water and then rinsed in sterilized distilled water. These plants were shifted to 50 ml conical flask containing 40 ml of the inoculum prediluted. These flasks were placed under florescent light at 25±2°C and the flasks were shaked for 8 hrs sowing and lines were rated for their extent of resistance/susceptibility.

Table 1: Chickpea varieties/lines showing different levels of resistance/susceptibility against Fusarium wilt
*Lines raised by Plant Genetics and Breeding Department, University of Agriculture, Faisalabad

Results and Discussion

The green-house screening method is relatively less time consuming cheaper and more reliable as compared to field screening which often has the chances of some escape as the wilt disease is temperature dependent and the level of inoculum may vary at different places. The greening house potscreening of chickpea germplasm revealed that out of 31 testlines evaluated for their level of resistance/susceptibility of Fusarium wilt, four lines 99107, 99109, 99106 and 1040/99 were found to be resistant, five lines 1009/99, found to be resistant, five lines 1009/99 588, 1001/99 99108 and 99115 were moderate resistant while all the other lines were susceptible to highly susceptible which could not withstand against wilt. The source of resistance to Fusarium wilt in chickpea germplasm are not uncommon and a number of workers have reported the occurrence of high level of resistance to Fusarium wilt (Kaushal and Singh, 1990; Yu and Su, 1997).

Chickpea lines that showed upto 20 per cent mortality in pots found resistant to water culture and cultural filtrate of Fusarium oxysporum f. sp. ciceris but with more than 30% mortality was susceptible (Table 1). However 5 lines 99109, 99112, 99105 and 99104, out of 31 lines having zero percent mortality were found to be highly resistant during water culture and culture filtrate screening. Only one line 1040/99 was moderately resistant, all other lines were highly susceptible. There are several other reports where culture filtrate and water culture were used for the selection of disease resistant plant (Behnke, 1979; Nene et al., 1981; Frane et al., 1991; Alam, 1994). Chickpea varieties/lines showing different levels of resistance/susceptibility against Fusarium wilt.

Akhtar, M., 1956. Further studies on the wilt disease of gram in Punjab. M.Sc. Thesis, University of the Punjab, Lahore, Pakistan.

Alam, S.S., 1994. The toxins of ascochyta rabiei and Fusarium oxysporum f. sp. ciceris, their potential use in selection for blight and wilt resistance in chickpea. Proceedings of 9th IUPAC Symposium on Chemistry Natural Product, January 16-20, 1994, Karachi, Pakistan -.

Anonymous, 1996. Agriucltural statistics of Pakistan. Ministry of Food and Agriculture and Livestock Division, Islamabad, Pakistan.

Anonymous, 1998. FAO Year Book. Food Organization of the United Nations, Rome, Italy.

Behnke, M., 1979. Selection of potato callus for resistance to culture filtrates of Phytophthora infestans and regeneration of resistant plants. Theor. Applied Genet., 55: 69-71.
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Frane, B., K.F. Yu, B.R. Christie and K.P. Pauls, 1991. In vitro selection for resistance to verticillium wilt in alfalfa (Medicago sativa L.) using a fungal culture filtrate. Physiol. Mol. Plant Pathol., 38: 325-340.
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Iqbal, M.J., K. Iftikhar and M.B. Ilyas, 1993. Evaluation of chickpea germplasm for resistance against wilt disease (Fusarium oxysporum). J. Agric. Res., 31: 449-453.
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Kaushal, R.P. and B.M. Singh, 1990. Pot screening of chickpea germplasm against chickpea wilt. Int. Chickpea Newslet., 23: 20-21.

Khan, I.U., 1980. Chickpea pathology in Pakistan. Proceedings of the International Workshop on Chickpea Improvement, February 28-March 2, 1979, ICRISAT., Hyderabad, India, pp: 257-.

Nene, Y.I., M.P. Hawar and M.P. Reddy, 1981. Chickpea diseases: Resistant screening technique. ICRIST Inform. Bull., 10: 195-201.

Sattar, A., A.G. Arif and M. Mohy-ud-Din, 1953. Effect of soil temperature and moisture on the incidence of gram blight. Pak. J. Sci. Res., 5: 16-21.

Song, H.S., S.M. Lim and J.M. Widholm, 1994. Selection and regeneration of soybean resistant to phytotoxic culture filtrate of septoria glycinea. Phytopathology, 84: 948-951.
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Yu, K.H. and T. Su, 1997. Pot screening of chickpea germplasm lines against wilt. Int. Chickpea Newslett., 4: 19-20.

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