Occurrence of Black Scurf Disease of Potato in Multan (Punjab) Alongwith Its in vitro Chemical and Biotic Elicitor Mediated Management

Black scurf disease of potato, caused by fungus Rhizoctonia solani, is the most common and one of the oldest diseases of potato affecting stem and stolons. In recent years, the disease is reportedly present in the fields of potato in Pakistan especially in Punjab. Survey of different locations viz. Narangaabad, Band Bosan, Kaian Pur, Kotla Abdul-Fateh and Dhillun was conducted to assess the prevalence of disease in Multan region. Maximum disease incidence and severity of 95.00% and 3.1 rating, was recorded in Kotla Abdul-Fateh respectively. Sclerotial pieces showing characteristic symptoms of black scurf were detached from tuber surface and the fungus was isolated on potato dextrose agar medium. For in-vitro chemical and biotic elicitor testing, three different fungicides viz., Monceren, Topsin-M, and Triton were evaluated using poisoned food technique and antagonistic effect of two accessions of Triochoderma spp. viz. Trichoderma harzianum and Trichoderma viridae was determined against the sensitivity of fungus. Triton was found to be superior at all concentrations in inhibiting the radial mycelial growth of the fungus followed by Topsin-M and Monceren. Satisfactory results were obtained by the antagonistic effect of Trichoderma harzinaum and Trichoderma viridae with 70.00% and 66.00% respectively under in-vitro conditions. Although the fungicide chemistries exclusively control the fungus yet the evaluation of bioagents also remained prolific towards antagonism against Rhizoctonia solani. These investigations provide fresh information on the current status of black scurf disease of potato in fields of Multan and regarding the biochemical management against Rhizoctonia solani under in-vitro conditions and serve as a guide for the future prospects against this holistic disease.


Introduction
Potato (Solanum tuberosum L.) is an annual, herbaceous and dicotyledonous plant belonging to genus Solanum and family Solanaceae, a major vegetable crop of Pakistan, grown on an area of 185.1 thousand hectares with an annual production of 4104.4 thousand tonnes (Anonymous, 2013). Potato belongs to one botanical specie Solanum tuberosum but it comprises of thousands of varieties that vary in their characteristics. Potato's origin is from South America Andes. Chile and Peru fight over its origin (Anonymous, 2008). Haq et al. (2008) reported that half of population of Pakistan is at threshold level of food insecurity because of shortage and surge in prices of food items according to World Food Program. Researchers at International Potato Center have recommended that the emergent dilemma of food supply due to rise in prices and production of crops intended for bio-fuel rather than foodstuff could be lessened by an increase in potato cultivation (CIP, 2008). Potato crop is having serious and major pathological threats in Pakistan and during recent years, soil-borne and seed-borne diseases have turned out to be a major threat to this crop in Pakistan . the skin) on tubers and stem canker are the result of Rhizoctonia disease complex in potato (Tsror, 2010). Black scurf disease increases gradually as level of inoculum increases and sclerotia may develop on tuber surface even under primary low inoculum level, resultantly the control through fungicidal chemistries is not always useful especially when levels of initial inoculum are high (Tsror & Peretz 2005;Kyritsis & Wale, 2002). Idrees et al. (2009) evaluated Topsin-M, Score and copper oxychloride as a dressing on potato tuber against black scurf of potato and found Topsin-M to be the best fungicide against the fungus. Rauf et al. (2007a) reported Monceren a good fungicide for management of black scurf disease of potato, which decreased stem girdling, sprout killing, stem canker, and black scurf prevalence among the three tested fungicides viz. Dithane M-45, Benlate and Monceren. Diseased tubers treatment with Monceren greatly abridged the sclerotial viability during in vitro experiments (Campion et al., 2003;Wicks et al., 1995). Keeping in view the economic potential of the crop, present investigations were aimed to determine the disease incidence, severity, chemical and biotic elicitor mediated management to tackle with the disease.

Study Area
The study was carried out in March-April (2012)    and Dhillun (30.265 N and 71.490 E). During survey, diagonal (X) method was adopted for collection of samples and potato tubers weighing 5-6 kg were taken at random from the harvesting plants in the field.

Disease Incidence
20 potatoes were randomly selected from each surveyed location to determine disease incidence using formula proposed by Ahmed et al. (1995) Disease Incidence = No. of tubers infected × 100 Total Tubers Observed

Disease Severity
Disease severity was determined by using 0-5 disease severity grades based on percent tuber surface showing disease symptoms (Table 1), (Ahmed et al., 1995).

Isolation, Purification and Identification of Rhizoctonia solani
The infected potato tubers showing sclerotial masses were washed under tap water. Five sclerotial pieces, each of 3 to 5 mm length, were then excised from tubers with sterile forceps. Disinfection was done in 1% sodium hypochlorite solution for two minutes. Sclerotial pieces were dried on sterilized blotting papers and placed on 9 cm Pyrex glass Petri dishes containing potato dextrose agar medium. The plates were completely randomized in incubator at 25 °C with the illumination of 600 Lux. The tested material culture (Rhizoctonia solani) was multiplied on PDA for experimentation. The pathogen was identified on basis of morphological characteristics under the compound microscope (Parmeter, 1970;Sneh et al., 1991;Bhuiyan, 1994;Tredway & Burpee, 2001).

Efficacy of Three Fungicides for Management of Rhizoctonia solani
Poisoned food technique (Dhingra & Sinclair, 1985) was used to determine the efficacy of the three fungicides www.ccsenet.org/jas Journal of Agricultural Science Vol. 6, No. 9;2014 viz. Monceren at the rate of 0.6, 0.3, and 0.9 ml (v/v), Topsin-M at the rate of 0.4, 0.2 and 0.6 g (w/v) and Triton at the rate of 1.2, 0.9, and 1.5 ml (v/v) at 1 kg seed treatment dose compared with the recommended and standard field doses ( Table 2). The tested concentrations were amended in Petri dishes containing potato dextrose agar medium. Four replicated plates were used for each concentration of every fungicide with completely randomized design (CRD) and four replicated plates received no fungicide served as control. Incubation was done at 28 C and data of mycelial radial growth was recorded after seven days when the growth of control plates completely covered the petri dishes. Percent inhibition of mycelial growth was determined based on colony diameter on control plate using the formula (Sunder et al., 1995).

Biological Control of Rhizoctonia solani Through Antagnostic Fungi
Antagnostic effect of Trichoderma species viz. Trichoderma harzianum and Trichoderma viridae was investigated against Rhizoctonia solani on potato dextrose agar medium by dual culture technique (Morton & Strouble, 1955;Skidmore & Dickinson, 1976). Each treatment was replicated four times in completely randomized design. The petri dishes received only mycelial discs of tested pathogen the Rhizoctonia solani served as control. The petri dishes were incubated at 25 °C temperature until the radial mycelial growth of the tested pathogen; Rhizoctonia solani covered the whole control plate. Percent inhibition of radial growth was calculated in relation to growth of control plate by the formula (Edington et al., 1971).

L = [(C -T) / C] × 100
Where, L = Inhibition of radial mycelial growth; C = Pathogen Radial growth measurement in control; T = Pathogen Radial growth in the presence of Trichoderma spp.

Statistical Analysis
The data of mycelial radial growth fungus was analyzed for analysis of variance (ANOVA) using SAS (statistical analysis system version 9.1). Treatment means were compared by least significant difference (LSD) test at (P  0.05).

Results
Surveyed results indicated the prevalence of disease at all visited areas. The aboveground parts of potato plants showed aerial tubers, rolling of leaves and chlorosis before harvesting while the most obvious symptoms of the black scurf were observed as sclerotial masses on tubers after harvesting of crop. Maximum disease incidence (95%) was recorded at Kotla Abdul Fateh and Dhillun followed by Narangabaad (65%) and minimum disease incidence (45%) was observed at Band Bosan.  The Rhizoctonia solani was initially identified by the presence of thread-like whitish mycelial growth, which gradually turned to dark brown followed by production of sclerotia on the culture that seemed to be submerged in the agar plates. Large aggregates of sclerotia were spherical and irregularly shaped. Anastomosis showed septate hyphae to be branched at 90 C to the main hypha with slight tapering at branch origin with no sexual

Discussion
Black scurf, caused by Rhizoctonia solani, is becoming more common and serious disease in the potato fields of Multan region; may inflict enormous losses to the production of potato crop with the occurrence of disease symptoms in the fields as black sclerotial masses on tuber surface and aerial tubers on foliar parts (Wharton et al., 2007), Rhizoctonia solani was isolated from the diseased potato tubers on potato dextrose agar medium and identified through microscopic characterization (Ritche et al., 2009). Disease incidence remained 70% whereas severity on 0-5 rating scale remained 1.8. Highest mean disease incidence and severity of 95% and 3.1 rating was found in Kotla Abdul-Fateh and Moaza Dhillun localities in Multan. Rauf et al. (2007b) reported that black surf disease of potato caused by Rhizoctonia solani was a common fungal disease in all the eight potato production agro-ecological zones of Pakistan with highest mean disease incidence and severity of 65% and 2.95 rating respectively in Punjab Province. A total of 80% of potato crop is produced in Punjab in Pakistan (Rauf et al., 2007b), potato production is rigorous in the areas near cold stores and bazaars in the towns (Geddes et al., 1985). High prevalence of black scurf of potato in Punjab is due to the favorable climatic condition for the inoculum, extensive monocropping of cultivar Desiree, which is the highly susceptible to black scurf of potato, poor cultural practices and lack of quarantine measures for disease (Hooker, 1981;Khan & Naumann, 1989). Low soil temperature, high moisture level together with zinc deficiency was the major factors favoring the development of disease in the surveyed locations (Ahmed et al., 1995;Thongbai et al., 1993). Potato crop cultivated without proper rotation tactics may lead to the encouragement of soil-borne diseases (Zanoni, 1991).
Management through fungicidal chemistries is only effective when the levels of initial inoculum were not high as the quantity of Rhizoctonia solani on tubers increases, the effectiveness of fungicides decreases (Tsror & Peretz, 2005 (Perello et al., 2003;Otadoh et al., 2011;Singh et al., 2008;Wilson et al., 2008).

Conclusion
In conclusion, the frightening situation of black scurf demands some multidimensional management tactics instantly to be taken for disease control and seed certification.

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