ISOLATION AND IDENTIFICATION OF VERTICILLIUM DAHLIAE CAUSING WILT ON POTATO IN PAKISTAN

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1 112 Pak. J. Phytopathol., Vol 24(2): , ISOLATION AND IDENTIFICATION OF VERTICILLIUM DAHLIAE CAUSING WILT ON POTATO IN PAKISTAN *Aasia Ashraf, *Abdul Rauf, **Muhammad Fahim Abbas and **Raheela Rehman, *Department of Plant Pathology PMAS Arid Agriculture University Rawalpindi, ** National Agricultural Research Centre, Park Road Islamabad-45500, Pakistan ABSTRACT During the year and , disease incidence of Verticillium dahliae in Rawalpindi (Taxila and Adyala) and Islamabad (National Agricultural Research Centre) was investigated in farmer s potato fields. During the year , maximum mean incidence was 24, 15 and 9% in Taxila, Adyala and NARC region respectively. Mean disease incidence increase 1% and 8% in Islamabad and Adyala while it was reduced by 3% in Taxila region respectively, during the second year of investigation. Over all mean disease incidence was increased by 2% comparison to disease incidence during second year. Maximum size of microsclerotia was recorded 63.9 µm in NARC while Taxila has smallest size of microsclerotia 35.7µm. Colony was dark brown to black and conidiophores were phialides, erect and septate. Ovate-oblong and elliptical conidia were singly associated to end of terminal branches before falling away. Taxila and Adyala isolate showed virulent behavior and Islamabad isolates were found the least virulent on Cardinal and Coroda. Most suitable temperature, ph and culture media were 26 C, 7 and verticillim specific media for the growth of V. dahliae respectively. Keywords: Verticillium dahliae, potato wilt, morphological characters. INTRODUCTION The genus name Verticillium derives from the morphological structure and the verticillate arrangement of conidiophores. The conidiospores are ovoid or ellipsoid and usually single celled, borne on long phialides which are specialized hyphae produced in a whorl around each conidiospore. The vegetative mycelium of Verticillium spp. is hyaline, septate and multinucleate and mostly haploid. V. dahliae can survive in the soil for nearly 15 years in the form of microsclerotia (Agrios, 2005). The cultivated potato (Solanum tuberosum L.) is ranked fourth in production after rice, wheat and maize (Rauscher et al., 2006) and this crop gives times more yield per hectare while calories production per unit area are higher than wheat, maize and rice. The potato tuber is an excellent source of carbohydrates, protein and vitamins (McGillivary, 1953). A significantly high number of pests and pathogens can be carried over from one generation to the next by propagated vegetative material. Verticillium wilt is a destructive fungal disease with a wide host range including vegetables (eggplant, pepper, potato, and tomato), field crops (soybean, sunflower, cotton and flax), trees (olive, ash and maple), shrubs (rose, lilac), fruits (strawberry), and weeds (Bhat and Subbarao, 1999). Most monocotyledonous plants are non-hosts of Verticillium species (Fradin and Thomma, 2006), the colonization of V. alboatrum and V. dahliae in some monocotyledonous plants such as barley, wheat and tulips has been reported (Mathre, 1989). In Pakistan, the annual production of potato is thousand tons which is cultivated over an area of thousand hectares (GOP 2011). In Pakistan, production of potato crop is low as compared to other potato growing countries of the world (Malik, 1995) due to biotic and abiotic factors. Among biotic factors, viral (Abbas et al., 2012) and soil-borne fungal diseases cause an economic loss of percent while two species, V. albo-atrum and V. dahliae, were observed for the first time in hilly areas of Pakistan (Turkensteen, 1986). Initial wilt symptoms are followed by yellowing and eventually browning and death of the leaves and stems. All the stems arising from the seed piece may not be affected. Wilting of the leaflets on one side of petiole often occurs and is an excellent diagnostic characteristic symptom of this pathological problem (Park et al., 1995). MATERIALS AND METHODS Sampling, isolation and multiplication of V. dahliae: Potato field of Taxila, Adyala and National Agricultural Research Centre (NARC) Islamabad region were investigated during the year 2010 and 2011as described by Bhat (2003). Samples suspected to Verticillium wilt were collected in labeled sterile polythene bags. Samples were cut into small pieces, dipped in 1% clorox solution and washed in sterile distilled water. Moisture was absorbed on sterile filter paper and samples were transferred on Potato Dextrose Agar (potato starch 20g, dextrose 20g, agar 20g and water 1L), Malt Extract Agar (malt extract 20g, agar 20g and water 1L) and cultured on Verticillim Selective Medium (VSM) (Ausher et al., 1975). All samples were incubated at 25 0 C for 7 days for multiplication of V. dahliae.

2 Inoculums Preparation and Pathogenicity Test: Purified fungal cultures were used to produce inocula. Conidial suspension of isolates was prepared by adding 10 ml distilled water and the conidial density was adjusted to 10 7 conidia/ml by using haemocytometer. Potato tubers of Coroda and Cardinal were planted with four replications (one control) into earthen pots containing potting mixture of sand, common soil and farm yard manure (1:1:1) sterilized with 2% formalin. The roots of 3 to 4 weeks old seedlings were dipped in each inoculum for 30 min. and inoculated seedlings were replanted in pots. The remaining inocula were poured on the soil surface at the rate of 10ml/plant (Ebihara et al., 2003). Disease Rating Scale: Symptoms development was recorded after 3 weeks of inoculation. After 4 weeks, inoculated potato plants exhibited stunting, chlorosis and defoliation of lower leaves. The results were found positive. Disease observations were evaluated by the following scale in two sections i.e. foliar damage and vascular discoloration. Table 1: Disease rating scale of V. dahliae. Score Disease Reaction Incidence (%) 0 No Infection Immune Resistant Moderately Susceptible Highly Susceptible Characterization of V. dahliae: Fungal culture media were analyzed for physiological characterization as described by Fang et al., (1990) and colony morphology, microsclerotia and conidiophores were examined with assistance of compound microscope (Hall and Jackson, 1985). Morphological Characterization Identification: Isolates were identified on PDA medium based on published descriptions of the fungus (Hawksworth and Talboys, 1970). Morphological characteristics like microsclerotia, conidiophores, conidia and colony morphology were observed by using stereoscope and light microscope. Measurement of microsclerotia: V. dahliae was observed under microscope and microsclerotia size of each isolate was taken three times with the help of ocular micrometer after calibration. Morphology of conidiophores: Inocula from the margin of 14 days old culture of the isolates was examined microscopically observation being based on characters described by Domsch et al., Physiological Characterization Effect of Culture Media: Three artificial media were used to find out the most suitable medium for the mycelial growth. Each culture media was prepared in one liter of double distilled water and autoclaved at C at 15psi for 20 minutes. Media were cooled to 45 0 C and poured in 9cm Petri dishes for solidification. 113 Effect of Temperature: The experiment was conducted to compare the response of V. dahlia to temperature. Test cultures were initiated with a 4 mm diameter mycelial plug taken from the leading edge of a colony previously grown for two weeks in the dark at 25 C. Each pathogenic isolate of V. dahlia was evaluated at different temperature in dark. The diameters of colonies were measured after two weeks of incubation. Effect of Different ph Level: The test fungus was inoculated on PDA medium whose ph was adjusted to 5,6,7,8 and 9 by adding 10% phosphoric acid. RESULTS AND DISCUSSION Incidence of V. dahliae: From the tested samples, it was observed that lowest disease incidence was found in Islamabad region with mean incidence value of 9% and 10 % in first and second year respectively. Adyala fields mean incidence value increased 8% compared to the first year incidence. The highest incidence in 2010 was observed in Taxila fields (24%) which decreased 3% in the second year. This decline in wilt incidence in Taxila fields may be due to use of resistant seed tubers, crop rotation and unfavorable environmental condition for fungal which helped to reduce inoculums density in the soil for the next season. Mean incidence of V. dahliae was 16% and 18 % during the year 2011 and 2012 respectively. It was concluded that overall mean incidence of wilt disease is increasing 2% each year. Isolation and Identification of V. dahliae: A total of 30 isolates of V. dahliae were isolated from wilted potato. All the isolates in vitro were examined based on the size of conidia, formation of resting structures and color of colonies. Their mycelium was septate with phialides in a characteristic arrangement on the conidiophores. Microsclerotia were also observed on all the cultures as globose to oblongate dark melanised structures throughout the colonies with no presence of dark mycelia which showed that all the tested isolates were morphologically similar and identified as V. dalhiae. These characteristics were compared with the description of Hawksworth and Talboys, Pathogenicity of Isolates: Variation in disease rating in respect of V. dahliae isolates towards both the tested exhibited variability. Cardinal showed symptoms involving dropping of leaves, vascular discoloration, wilting and plant mortality and showed susceptible behavior. Reaction of potato cultivar to 9 isolates of the fungus indicated that Coroda showed resistant reaction to TAX-1, TAX-5, NARC-10, NARC-2 and AD-8 isolates. Whereas, TAX-7, NARC-6, AD-3 and AD-4 isolates were moderately susceptible to Coroda. Cardinal showed highly susceptible response to TAX-7, NARC-2 and AD-3. Among 9 isolates none was found immune on two potato cultivars in green house. Out of 54 replications only 21 were resistant and just showed foliar damage

3 while 30 showed moderately susceptible response exhibiting both foliar and vascular discoloration and 3 isolates were highly susceptible and showed 100 % yellowing and stunting of the plant. V. dahliae isolates TAX-7 and AD-3 were the most virulent among all isolates and TAX-1 and NARC-10 were least virulent. Coroda was more resistant as compared to Cardinal (Table 2). Table 2: Pathogenicity of V. dahliae Isolates on two Potato Cultivars. Sr. No Isolates TAX-1 TAX-5 TAX-7 NARC-6 NARC-10 NARC-2 AD-8 AD-3 AD-4 V1(Cardinal) R1 R2 R3 Mean DI (%) + _ + 66 V2 (Coroda) R1 R2 R3 Mean DI (%) Grand Mean Morphological Characterization Identification of the Pathogen: Wilt isolates of V. dahliae were examined based on the size of conidia, formation of resting structures and color of colonies. Brown to black microsclerotia arising from single hyphae by repeated budding and consisting of swollen, an almost globular cell was observed (Hawksworth and Talboys, 1970). Morphological and Cultural Characteristics of V. dahliae: Infected tissues produced microsclerotium after two weeks of cultivation. In case of V. dahliae color of colony was dark brown to black microsclerotia arising from single hyphae by repeated budding and consisting of swollen, almost globular cells (Hawksworth and Talboys, 1970). Measurement of Microsclerotia: Data on size of microsclerotia revealed that NARC-2 has the maximum size of microsclerotia (63.9 µm) with mean length (27.3 µm) of thrice replicates of V. dahlia (Table 3). The isolate TAX-7 has least length of microsclerotia (35.7µm) while maximum width was 42.2 µm and the mean microsclerotia size of three replicates of TAX-7 was 30.2 µm. The microsclerotia width of isolate AD-3 was almost similar to isolate TAX-7 and length of isolates AD-3, AD-4 and AD-8 were intermediate to the isolates of NARC and Taxila (Table 3). Morphology of Conidiophores: Morphological characteristics of isolate TAX-7 were considered for morphology of the fungus. Conidiophores of the fungus were erect, septate and branched. The short branched being arranged in whorls, with the terminal branches (phialides) usually quite pointed at the apices. Conidia were formed singly, terminally and for short time associated in spore balls at the end of branches before falling away. Conidia were elliptical, ovate-oblong or spherical, unicellular and hyaline or very slightly colored. Similar characters were described by Isaac (1697) and later by Anderson (2008) based on conidiophores morphology. Physiological Characterization Effect of Culture Media: Three artificial culture media were used to check their efficiency for growth of the V. dahliae under laboratory conditions. TAX-7, NARC-2 and AD-3 isolates of V. dahliae after 10 days of incubation showed the maximum mean Table 3: Size of Microsclerotia of V. dahliae Isolates. Isolates Name Size of Microsclerotia (µm) Length (Mean length of width (Mean width of three replications) three replications) TAX (26.0) 36.0 (12.3) NARC (17.4) 41.5 (8.7) AD (19.6) 41.2 (14.4) TAX (24.5) 28.7 (9.8) NARC (27.3) 41.6 (17.5) AD (16.5) 36.0 (10.3) TAX (30.2) 42.2 (18.1) NARC (21.4) 30.1 (8.1) AD (21.1) 30.4 (9.0) 114

4 growth percentage on VSM (7.5%) followed by MEA (6.7 %) and PDA (6.2%) as shown in Table 4. These findings have much resemblance with the results of Naseem et al., According to these scientists, highest number of colonies was isolated on MEA than PDA. Effect of Temperature: Each pathogenic isolate of V. dahlia was evaluated at 18, 22, 24, 26, 30 and 32 C in dark. The fungus grew at temperature of o C. Colony growth was measured after two weeks of inoculation. Growth of the fungus was drastically reduced below 18 o C and above 28 o C (Fig 2). A temperature range from o C favored the mycelia growth of TAX-7. It was observed that at 22 o C the growth of fungus was maximum 3.5 cm after 7days, while at 24 o C growth fast 0.6 cm i.e. 4.1 cm and at 26 o C it was 4.4 cm after 7 days of inoculation. Isolate of V. dahliae failed to grow at 30 o C in culture. These results were consistent with Katazawa and Suzui s (1980) studies who reported that growth of fungus at different temperature ranging from o C. They found temperature 26 o C was best suited for growth of V. dahliae and at this temperature mycelia growth was maximum. Fig 1: Effect of ph on the Mycelial Growth of V. dahliae. Effect of ph: Isolates TAX-7 was observed at all the ph levels but it was maximum at ph 5 after 7 days of incubation and growth of was decreased by increasing ph level. This study has been supported by Susuri et al., 1999 who observed that growth occur over a wide range of ph 4.5-7, the optimum ph for Verticillium growth was 5 which is also similar to the findings of Katazawa and Suzui s (1980) for best suited ph of fungal growth. The pathogen grows well at ph 5 and gives 60 mm colony growth and it responded well up to ph 7 by giving 74 mm colony growth but it declines gradually as ph value increases and growth stop (Fig 1). The present status of incidence must be brought under economic threshold levels in order to protect the production of highly valued food crop in Pakistan. Use of resistant cultivars, crop rotation along with no host, alternation of sowing time, integrated disease management based upon best combination of various control measures should urgently be brought into practice to reduce the inoculums of V. dahliae in the field. Only this can help combating against extensively damaging disease of wilting in potato. Table 4: Colonies growth of V. dahliae on PDA, MEA and SVM. Fig 2: Growth of V. dahliae at different Temperatures. REFRENCES Abbas, M.F., S. Hammed, A. Rauf, Q. Nosheen, A. Ghani, A. Qadir and S. Zakia Incidence of six viruses in potato growing areas of Pakistan. Pak. J. Phytopath. 24 (1): Anderson, J.P., E. Badruzsaufari, P.M. Schenk, J.M. Manners, O.J. Desmond, C. Ehlert, D.J. Maclean, P.R. Ebert and K. Kazan Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis, The Plant Cell, Agrios, G. N Plant pathology, Fifth Edition, Academic Press New York, London, Ausher, R. Katan and S. Ovadia An improved selective medium for the isolation of Verticillium dahlia Phytoparasitica. 3: Bhat, R.G. and K.V. Subbarao Host range specificity in Verticillium dahliae. Phytopathol., 89: Bhat, R.G., R.F., Smith, S.T. Koike, B.M. Wu and K.V. Subbarao Characterization of Verticillium dahliae isolates and wilt epidemics in pepper. Plant Disease 87:

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