Evaluation of various fungicides for the control of gram wilt caused by Fusarium oxysporium f.sp. ciceris

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1 African Journal of Agricultural Research Vol. 6(19), pp , 19 September, 2011 Available online at ISSN X 2011 Academic Journals Full Length Research Paper Evaluation of various fungicides for the control of gram wilt caused by Fusarium oxysporium f.sp. ciceris Muhammad Nasir Subhani 1 *, Shahbaz Talib Sahi 2, Safdar Hussain 1, Anser Ali 1, Javaid Iqbal 1 and Kiran Hameed 3 1 Sub-Campus D. G. Khan, University of Agriculture, Faisalabad, Pakistan. 2 Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan. 3 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan. Accepted 22 July, 2011 Fungitoxic effects of six fungicides, namely, Benomyl, Derosal, Ridomil, Cabrio Top, Vitavax and Prevent at four concentrations, 5, 10, 20 and 50 ppm were tested through poisoned food technique. There was a significant decrease in mycelial growth of the fungus with an increase in fungicidal concentration. The most effective fungicides in inhibiting the growth of the fungus, in descending order were Derosal, Benomyl and Vitavax as they caused 100, 95.81, and 70.96% reduction in mycelial growth, respectively at 5 ppm concentration. Ridomil and Prevent were least effective as they reduced and 39.02% mycelial growth respectively. The fungicides which proved to be most effective in vitro were tested in green house grown plants. These fungicides were compared on the basis of mean number of wilted plants at all dosage rates at 39 th day of sowing. Derosal and Benomyl were the most effective in reducing number of wilted plants followed by Vitavax, while the Cabrio Top was the least effective. Derosal and Benomyl exhibited 100% reduction in disease incidence, while Vitavex and Cabrio Top exhibited and 88.37% in disease incidence, respectively. Key words: Chickpea, wilt, Fusarium oxysporum f.sp. ciceris, fungicides. INTRODUCTION Chickpea (Cicer arietinum L.) is a bushy annual plant of the pea family with short, hairy pods containing usually two seeds. Chickpea is the world s third most important pulse crop, after dry beans (Phaseolus vulgaris L.) and dry peas (Pisum sativum L.) (Vishwadhar and Gurha, 1998). Chickpea is particularly an important high protein crop in Pakistan as it constitutes a major source of protein in animal feed and human diet especially for low income group, by supplementing their cereal diets. In Pakistan, this crop is grown over an area of 1050 thousand hectares with an average yield of 571 thousand tons (Anonymous, 2010). Chickpea production in Pakistan has been uncertain, erratic and low. Among factors responsible for low yield in Pakistan are the *Corresponding author. nasirsubhani@hotmail.com or mnasirsubhani@yahoo.com. outbreaks of different diseases such as Fusarium wilt, which is a serious disease of chickpea in India, Iran, Pakistan, Nepal, Burma, Spain, Tunisia and Mexico. It has been observed in Morocco, Algeria, and Syria. Nema and Khare (1973) reported that damage caused by the wilt disease is up to 61% if the attack takes place at the seedling stage and 43% at the flowering stage. In Pakistan disease may induce 10 to 50% loss every year (Khan et al., 2002). Fusarium wilt caused by Fusarium oxysporium f.sp. ciceris is one of the major soil or seed borne disease of chickpea (C. arietinum L.). Early stage wilting causes more losses as compared to loss caused by late wilting. Seeds harvested from wilted plants were lighter and duller than those from healthy plants (Haware and Nene, 1980). It causes complete loss in grain yield if the disease occurs in the vegetative and reproductive stages of the crop (Navas et al., 2000). The fungus F. oxysporium f. sp. ciceris, is a primarily soil borne

2 4556 Afr. J. Agric. Res. pathogen, however, few reports indicated that it can be transmitted through seeds (Haware et al., 1978). This pathogen is a facultative saprophyte which can survive in soil up to six years in the absence of susceptible host (Haware et al., 1986). Management of Fusarium wilt of chickpea is difficult to achieve and no single control measure is fully effective. Considering the nature of damage and survival ability of the fungus, the use of resistant varieties was considered to be the only economical and practical solution. Most of the resistant varieties have been found to be susceptible after some years because of breakdown in their resistance and evolution of variability in the pathogen. There appears to be no apparent reason as to why these already tested wilt resistant materials showed such a variable wilt reaction and which creates a doubt about the possibility of existence of physiologic forms of the pathogen. The pathogen with its high saprophytic ability can survive in soil for a long period during which it may have to go through different environmental stresses and biological competition which may lead to the existence of physiologic races. The most effective and practical method of control worldwide is to use fungicides (Gupta et al., 1988) or resistant cultivars. However, the effectiveness of host resistances is curtailed by the occurrence of pathogenic races in F. oxysporium f.sp. ciceris (Jimenez-Gasco et al., 2004). Therefore, integrated management strategies are the only solution to maintain plant health. These strategies should includes minimum and efficient use of chemicals for checking the pathogen population, encouragement of beneficial biological agents to reduce pathogen inoculum, modification of cultural practices and use of resistant varieties (Bendre and Barhate, 1998). Thus, there is a need to explore the efficacy of different fungicides to manage this disease below economic threshold level in the absence of resistant cultivars. MATERIAL AND METHODS Roots of diseased chickpea plants showing characteristic symptoms of wilt disease were collected from chickpea infected field of Plant Pathology section Ayyub Agriculture Research Institute, Faisalabad for the isolation of F. oxysporum f.sp. ciceris. Roots of chickpea plants were cut into 8 to 10 mm long segments, washed with tap water and surface sterilized by dipping in 0.1% mercuric chloride solution for 60 s. These pieces were washed three times in sterilized distilled water and were placed on sterilized filter paper sheets for drying. These dried segments were then plated on chickpea seed meal agar (CSMA) in Petri plate and were incubated at 25 C for more than one week for recovery of pathogen. Colonies of F. oxysporum along with some other colonies, that is, air borne fungi were recorded. F. oxysporum was purified by single spore method and was identified with the help of relevant literature of the Wollenweber and Reinking (1935) and Synder and Hausen (1940). Sensitivity of mycelial growth of F. oxysporum against different fungicides: Benomyl, Derosal, Ridomil, Cabrio Top, Vitavax and Prevent were evaluated at different concentrations, that is, 5, 10, 20 and 50 µg/ml by using modified Borum and Sunclair s technique (1968). A weighed quantity of each of the fungicide was amended with sterilized CSMA to obtain required concentration of fungicides. Chickpea seed meal agar without fungicide served as control. 25 ml of amended and non amended medium was poured in each of the four 90 mm diameter Petri dishes. After solidification 5 mm agar plugs containing F. oxysporum mycelium were cut from 10 days old CSMA and were placed in the centre of each Petri dish with the help of sterilized inoculating needle. These inoculated Petri dishes were incubated at 25 C. Data on radial mycelial growth (mm) of F. oxysporum were recorded after 7 days of incubation and percent inhibition was calculated by the formula given by Jagtap and Sontakke (2007): h. = h h 100 Data of colony diameter and percentage of inhibition were analyzed statistically to observe the difference among various treatments. Fungicides found to be the most effective in inhibiting the mycelial growth in vitro were further evaluated in the green house for the control of chickpea wilt in pots by soil drenching at dosage rates of 50, 100, 200 and500 µg/g of soil. F. oxysporum f.sp. ciceris inoculum was prepared by soaking chick pea seeds in tap water for overnight and then boiled until the upper seed coat became soft. These boiled seeds were spread over paper towel, were surface dried in sunshine, filled in polyethylene bags, and then plugged with cotton plugs having one inch plastic rings cut from half inch plastic pipe around their open ends and were autoclaved at 15 psi for one hour. The field soil was used in the pots and these pots were autoclaved for one hour at 15 psi. Sterilized pots were filled 2/3 with the soil and infested with the pathogen, that is, F. oxysporum by mixing autoclaved soil with mass culture of the fungus prepared in polyethylene bags. Eight healthy surface sterilized seeds of chickpea were sown in each pot with 2 to 3 cm deep. Aqueous suspension of fungicides, namely, Benomy, Derosal, Carbendazim, Ridomil, Score and Vitavax were drenched into the pot soil, with each of the four concentrations, that is, 50, 100, 200 and 500 µg/g in three replications. There were three pots for each dosage rate and represent a replication. Fungicides concentrations, that is, 50, 100, 200 and 500 µg/g suspended in water were used for drenching. Application of fungicides was repeated seven days after seedling emergence. Three pots of each of the infested non drenched and non infested but drenched treatment served as control. The pots were irrigated when needed with the distilled water. The disease incidence was recorded 39th day of sowing. The data were recorded and analyzed statistically to visualize the difference between the fungicidal treatments by a given formula as under: Mortality Reduction Percentage = 100

3 Subhani et al Table 1. Mean mycelial growth in vitro at various concentrations of fungicides. Mean colony mycelial growth (mm) at various concentrations 5 ppm 10 ppm 20 ppm 50 ppm Benomyl 5.24 l 5.37 l 4.45 l 0.00 o Ridomil e f g h Derosal 0.00 n 0.00 n 0.00 n 0.00 n Cabrio Top i j k 5.58 l Prevent b c d g Vitavax 3.54 lm 1.64 mn 0.84 mn 0.44 mn Control a a a a CV= Table 2. Mean colony mycelial growth inhibition at various concentrations. Mean colony mycelial growth inhibition (%) 5 ppm 10 ppm 20 ppm 50 ppm Benomyl f f e a Ridomil m l k j Derosal a a a a Cabrio Top i h g f Prevent p o n k Vitavax d c b a CV= RESULTS AND DISCUSSION Fungitoxic effects of six fungicides, namely, Benomyl 50SC, Ridomil, Derosal 60 WP, Cabrio Top, Prevent and Vitavax at four concentrations 5, 10, 20 and 50 ppm were tested in vitro through poisoned food technique. Results obtained on the fungitoxicity of fungicides against F. oxysporum f.sp. ciceris in vitro are presented in Table 1. In general, there was a significant decrease in mycellial growth of the fungus with an increase in fungicidal concentration. However, when growth response of fungus to fungicidal concentrations was compared on the basis of mean comparison after an incubation period of 7 days at 25 C (Table 2), Derosal, Shinkar, Vitavax, Cabrio Top, Ridomil and prevent caused 100.0, 95.93, 88.09, 79.20, and 21.49% reduction in mycelial growth respectively (Table 2). Thus, the most effective fungicides in inhibiting the growth of the fungus, in descending order were Derosal, Benomyl and Vitavex as they impacted 100, and 88.09% reduction in mycelial growth of fungus respectively. Prevent and Ridomil were the least effective in inhibiting the mycelial growth, that is, and 39.02% respectively (Table 2). Analysis of variance shows significant interaction between concentration and reduction of mycellial growth of F. oxysporum f.sp. ciceris as an increase in fungicide concentration decreases the mycellial growth of the fungus. However, for Derosal at all tested concentrations and Benomyl 50 ppm were statistically equally effective (Table 2). Preliminary evaluation of the comparative effect of fungicides on the mycelial growth of F. oxysporum revealed that the effectiveness of fungicides in inhibiting the mycelial growth of the pathogen varied in general and a significantly increased in the inhibition of mycelial growth with an increased fungicide concentration. The effectiveness of fungicides in inhibiting the growth of fungus, in descending order were Derosal, Shinkar, Vitavax, Cabrio Top, Ridomil and Prevent The effectiveeness of Derosal and Benlate in controlling chickpea wilt has been reported by other worker as well (Ghosh and Sinha, 1981; Mani and Sethi, 1984; Jimenez-Diaz and Troperoeasas, 1989; Kotasthane et al., 1987). The comparative effectiveness of Derosal on the inhibition of mycelial growth of F. oxysporum has been reported by Khalid (1993) who tested 10 fungicides against mycelial growth of F. oxysporum f.sp. ciceris by poisoned food technique. Benlate and Derosal completely controlled the growth of the fungus. Similarly these fungicides were also the most effective as soil drenches at 200 and 500 ug/g dosage rates respectively. Similarly the effectiveness of Benlate, Derosal, Tecto-60 and Topsin-M against the mycelial growth of the fungus have also been reported (Iqbal et al., 1996). Jagtap and Sontakke (2007) reported that chemical seed treatment with Thiram (0.15%) + Carbendazim (0.1%) proved to be

4 4558 Afr. J. Agric. Res. Table 3. Effect of fungicidal drenches at each of the four fungicides dosage rates on the number of wilted plants after 39 days of sowing. Dosage rates (µg/g of soil) 50 ppm 100 ppm 200 ppm 500 ppm Cabrio top 7.33 a 4.00 c 2.67 de 2.00 ef Vitavax 5.33 b 3.00 d 1.00 gh 1.00 gh Derosal 3.00 d 2.67 de 1.00 gh 0.66 ghi Benomyl 3.33 cd 3.00 d 2.66 de 1.00 gh Control inoculated 8.00 a 8.00 a 8.00 a 8.00 a Control un inoculated 0.00 i 0.00 i 0.00 i 0.00 i CV= Table 4. Effect of fungicidal drenches at each of the four dosage rates on the number of wilted plants after 39 days of sowing. Dosage Rates (µg/g of soil) 50 ppm 100 ppm 200 ppm 500 ppm Cabrio top k j f d Vitavax i e c b Derosal g d b 100 a Shinkar h f 100 a 100 a Control inoculated 0.00 l 0.00 l 0.00 l 0.00 l Control un inoculated 100 a 100 a 100 a 100 a CV= the most effective against F. oxysporium f. sp. ciceri. Greenhouse evaluation of fungicides for the control of chickpea wilts by drenching technique The data regarding the fungicidal drenches at each of 50, 100, 200 and 500 µg/g dosage rates on number of wilted plants recorded at 39th day of sowing is presented in Table 3 and seedling mortality reduction over untreated control is given in Table 4. The data revealed that the effect of fungicides in reducing number of wilted plants varied with the individual fungicides and the effect increased with the increased dosage rate (Tables 3 and 4). The four test fungicides when compared on the basis of mean number of wilted plants at all dosage rates recorded at 39th day of sowing, Benomyl and Derosal were found to be the most effective fungicides followed by Vitavex, and Cabrio Top (Tables 3 and 4). Benomyl reduced the disease incidence 100% at 200 and 500 µg/g while Derosal exhibited 96.41% at 200 µg/g and 100% reduction at 500 µg/g (Table 4). On the other hand Vitavax and Cabrio Top exhibited and 88.37% reduction of seedling mortality at 500 µg/g concentration (Table 4). In this study a continuous trend was seen that seedling mortality was reducing with the increase in the concentration from 50 to 500 µg/g, which shows the effectiveness of all these fungicides but in lesser or higher effects. As Benomyl shows maximum effectiveness and Cabrio Top with least effectiveness but still the trend was the same, in which an increased concentration the percentage of survival percentage of the seedlings was increased. Fusarium wilt caused by F. oxysporum f.sp. ciceris (Padwick) Matuo & K Sato (Foc) is a major constraint to chickpea (C. arietinum L.) production throughout the world and particularly in the Indian subcontinent and the Mediterranean basin (Nene et al., 1989). The integrated management strategies should includes minimum use of chemicals for reducing the pathogen population, encouragement of beneficial biological agents to reduce pathogen inoculum, modification of cultural practices and use of resistant varieties (Bendre and Barhate, 1998). In the greenhouse evaluation, which used fungicides as drench Derosal was found to be the most effective in controlling the disease in potted plants and in reducing the number of wilt-effected plants, Derosal was followed by Shinkar, Vitavax and Cabrio. The effectiveness of Benomyl, Rhizolax and Topsin-M (Thiophanate methyl) has been reported by Ilyas et al. (1992) as soil drench while Benlate and Derosal have also been reported to reduce the number of linseed wilted plants in greenhouse (Ilyas et al., 1996). Ilyas et al. (1992) tested 10 fungicides against the mycelial growth of F. oxysporum f.sp. ciceris

5 Subhani et al and reported that Benomyl, Topsin-M, (Thiophenatemethyel), Tilt (propeconazole) and Rhizolax were the most effective in inhibiting the fungus. In soil drench test on infected potted plants in the green house, Banomyl was the most effective, followed by Rhizolax and thiophenate-methyl. In these studies possibility of any other factor affecting or increasing the incidence of seedling mortality was also tested by sowing these seedlings without adding the F. oxysporum f.sp. ciceris (Padwick) Matuo & K Sato (Foc) inoculum in the pots. Results of the pots having 100% healthy seedlings showed that the death of the seedlings were only due to the presence or attack of the fungus rather than any other factor. This was also obvious from the reduction of seedling mortality in fungicide application and 100% mortality in case of fungus inoculated control pots. From the above in vitro and green house results indicated that in the absence of resistant varieties we can reduce the incidence gram wilt by soil drenching with any of the Benomyl, Derosal, Vitavax and Cabrio Top fungicides. REFERENCES Anonymous (2010). Agricultural statistics of Food, Agriculture and Live Stock. Economic wing, Government of Pakistan, Islamabad. Bendre NJ, Barhate BG (1998). A souvenir on Disease Management in Chickpea. M.P.K.V., Rahuri during 10th Dec Borum DF, Sinclair JB (1968). Evidence for systemic protection against Rhizoctonia solani with Vitavax in cotton seedlings. Phytopathol., 58: Ghosh MK, Sinha AK (1981). Laboratary evaluation of some systemic fungicides against Fusarium Wilt of pigeon pea. Pesticides, 15(1): Gupta RP, Katiyar RP, Singh DP (1988). Seed treatment with Bavistin and Rhizobium and its effect on wilt incidence, nodulation and yield of chickpea. Pesticides, 22: Haware MP, Nene YL (1980). Influence of wilt at different stages on the yield loss in chickpea. Trop. Grain Legume Bull., 19: Haware MP, Nene YL, Mathur SB (1986). Seed borne diseases of chickpea. Techn. Bull. No.1, ICRISAT, p. 32. Ilyas MB, Iqbal MJ, Iftikhar K (1992). Evaluation of some fungicides against Fusarium oxysporum f.sp. ciceris and chickpea wilt. Pak. J. Phaytopahtol., 4 (1-2): 5-8. Ilyas MB, Khan MW, Mohy-ud-Din A (1996). Evaluation of some fungicides against Fusarium oxysporum f.sp. lini and linseed wilt Pak. J. Phytopathol., 8(1): Iqbal SM, Bashir M, Rauf CA, Malik BA (1996). Efficacy of fungicides against soil-borne pathogens of chickpea. Pak. J. Phytopathol., 8(1): Jimenez-Diaz RM, Trapero-Casas A, Cabrera-de-La-Colina J (1989). Races of Fusarium oxysporum f.sp. ciceris infecting chickpea in southern Spain. In: Tjamos E.C. and Beckman C.H. (eds.). Vascular Wilt Diseases of Plants, pp Springer-Verlag, Berlin, Germany. Jimenez-Gasco MM, Navas-Cortes JA, Jimenez-Diaz RM (2004). The Fusarium oxysporum f.sp. ciceris/cicer arietinum pathosystem: a case study of the evolution of plant-pathogenic fungi into races and pathotypes. Int. Microbiol., 7: Mani A, Sethi CL (1984). Influence of seed treatment on seedling emergence of chickpea in presence of Meloidogyne incognita, Fusarium oxysporum f.sp. ciceris and F. solani. Indian J. Nematol., 14(1): Jagtap GP, Sontakke PL (2007). Management of chickpea wilt caused by Fusarium oxysporium f. sp. ciceri Afr. J. of Agric. Res., 2(12): Khalid T (1993). Screening of chickpea Germplasm and fungitoxicant against chickpea wilt disease. M.Sc. Thesis, Dept. of Pl. Path. Univ. Agri. Faisalabad. Khan IA, Alam SS, Haq A, Jabbar A (2002). Selection for resistant to wilt in relation with phenols in Chickpea. Int. Chickpea Pigeonpea Newslett., 9: Kotasthane SR, Gupta OM, Khare MN (1987). Influence of fungicidal treatment and soil amendment on the development of Fusarium nudum propagules in soil and pigeon pea wilt. Indian J. Phytopaht., 40(2): Navas-Cortes JA, Hau B, Jimenez-Diaz RM (2000). Yield loss in chickpea in relation to development to Fusarium wilt epidemics. Phytopatholology, 90: Nema KG, Khare MN (1973). A conspectus of wilt Bengal gram in Madhya Pradesh. Symposium on wilt problem and breeding for wilt resistance in Bengal gram. September, 1973 at Indian Agricultural Research Institute, New Dehli, India, p. 4(Abst). Nene YL, Haware MP, Reddy MV, Phillips JC, Castro EL, Kotashthane SR, Gupta O, Singh G, Shukla P (1989). Identification of broad-based and stable resistance to wilt and root-rots in chickpea. Indian Phytopathol., 42: Synder WC, Hausen HN (1940). The species concept in Fusarium. American J. Bot., 27: Vishwadhar, Gurha SN (1998). Integrated Management of Chickipea Diseases. Integrated Pest and disease management. Rajeev K, Upadhyay KG, Mukerji BP, Chamola and Dubey OP (eds.) APH Publishing Co., New Delhi (India), p Wollenwebber HW, Reinking OA (1935). Die verbreitung Fusaritm in der Hature. (The Distribution of Fusarium in nature), Berlin, R. fried land und, sohm, p. 80.