Prevalence of Phytophthora Spp. in Citrus Nurseries of Vidarbha Region
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Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 3548-3555 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 8 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.908.409 Prevalence of Phytophthora spp. In Citrus Nurseries of Vidarbha Region R. S. Lad1*, R. M. Gade2 and Mina D. Koche3 1Department of Plant Pathology, Dr. P.D.K.V., Akola, Maharashtra, India 2College of Agriculture, Gadchiroli, Maharashtra, India 3Shri. Shivaji Agriculture College, Amravati, Maharashtra, India *Corresponding author ABSTRACT Phytophthora disease has been identified as one of the major causes of decline in citrus. It K e yw or ds causes the most serious and economically important soil borne diseases to citrus crops. In year 2016 and 2017 soil samples were collected from Thirty eight (38) surveyed nurseries Phytophthora, during August to November. Three species of Phytophthora viz. P. parasitica, P. Nursery, Survey, citrophthora and P. palmivora found associated to cause damping off, collar rot and root Morphology, Citrus rot in citrus. In the present studies these three of Phytophthora were isolated by using CMA-PARPH medium from collected soil samples. The occurrence of Phytophthora spp. Article Info was recorded in 35 out of 38 surveyed locations, with a range from 3.10 to 24.33 cfu / g of soil in the year 2016 and 2.33 to 24.99 cfu / g of soil in year 2017. Pooled data of infected Accepted: nurseries indicate propagules in the range of 2.94 to 24.66 cfu/g of soil. The morphological 26 July 2020 characters like mycelium and its appearance, sporangial papillation and their shapes and Available Online: presence of chlaymadospores of different isolated species were recorded. Presence of 10 August 2020 chlamydospores was observed only in P. nicotianae var. parasitica which were present as intercalary with oval shape and thick wall. Introduction Phytophthora disease has been identified as one of the major causes of decline in citrus. It Citrus occupies the position of most causes the most serious and economically remunerative fruit crops of India, having it important soil borne diseases to citrus crops. own niche in the international trade and world Three species of Phytophthora viz. P. finance. Nagpur mandarin, Sweet orange and parasitica, P. citrophthora and P. palmivora Acid lime are the three important species of found associated to cause damping off, collar citrus having international acceptance. This is rot and root rot in citrus (Naqvi, 1988; Gade, the second largest industry in India and third 2012). largest in production. Citrus is primarily grown in Andhra Pradesh, Maharashtra, The pathogen attack citrus plants right from Punjab, Karnataka, Uttaranchal, Bihar, Orissa, nursery causing damping off of seedlings, Assam and Gujarat (Lakshmi et al, 2014). decay of fibrous roots, crown rot, collar rot, foot rot and gummosis in mature orchards. 3548 Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 3548-3555 Nagpur mandarins face short life span and Materials and Methods low productivity because of these three species of Phytophthora (Das et al., 2011). Survey of citrus nursery and collection of Damping off in citrus seedlings occurred up soil samples to 24 per cent due to Phytophthora, thereafter, 5-15 per cent mortality occurred in secondary An extensive survey of citrus nurseries of beds (Naqvi, 2006). Amravati district has been made in year 2016 and 2017 and soil samples were collected Survey have been undertaken to see the from Thirty eight (38) surveyed nurseries association of Phytophthora spp. in Vidarbha during August to November. Samples from region where nearly 80L grafts are being particular nursery were collected from raised for sale every year (Gade, 2012). It is rhizosphere and combined to make a very well known that infected nursery plants composite sample (Naqvi, 1994; Timmer et are one of the major ways of movement of al., 1989). Phytophthora spp. in orchards. Isolation purification of pathogen culture The soil borne pathogen ‘Phytophthora’ causes tremendous losses in nurseries and Ten gram soil from each sample was diluted orchards of Nagpur mandarin in Vidarbha in 90 ml water having 0.25% agar. One ml region of Maharashtra and all over India is aliquot was spread on each of 10 plates of well. Unfortunately, since last 10 years or so, Corn meal agar (CMA-PARPH) selective growing Nagpur Mandarin has no longer medium (Kannwischer and Mitchell, 1978). remained a profitable proposition, as it is The plates were incubated at 280C for 2-3 being increasingly realized by citrus growers, days and number of colonies of Phytophthora beside with one or other problems. was counted. Soil in the second core was Nevertheless, the area under Nagpur flooded with water, baited with pieces of mandarin has considerably increased mainly citrus leaves and placed in the incubator for due to implementation of the different 48hr (Grimm and Alexander, 1973). The missions of the central government. leaves were transferred to petri dishes and examined for the presence of papillate The purpose of current study is to estimate sporangia under compound microscope. prevalence of Phytophthora spp. in selective Subculture of Phytophthora from infected leaf citrus nurseries and orchards. An intensive pieces was easily accomplished by rapid roving survey of nurseries in contributes submerging infected leaf pieces in PARPH to assess the present status, distribution of medium and transferring hyphal tips as they plant pathogens prevalent in particular areas. grew in the medium. As soon as the growth of Such surveys are helpful to characterize local fungus was observed, small portion was isolates of pathogen. transferred to the slants. The survey and current status about Composition of CMA-PARPH selective availability of disease free planting material media (Mitchell et al., 1986) in study area is also one of the initial views of this research work. It is generally agreed that Corn meal agar- 17 g/ lit, Pimaricin- 10 mg/ the environment is the driving force in the lit, Ampicilin- 250 mg/ lit, Refampicin- 10 development of epidemics (Hardwick, 1998). mg/ lit, PCNB- 100 mg/ lit, Hymexazole- 50 mg/ lit, Distilled water-1000 ml. 3549 Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 3548-3555 Production of sporangia Phytophthora spp. viz., Phytophthora nicotianae var. parasitica, P. citropthora and Sporangia are the cells or vessels in which P. palmivora were isolated from collected zoospore are formed. Production of sporangia samples. P. nicotianae var. parasitica was was carried out by the water culture method. found prominently involved in causing A thin layer of sterile distilled water is added damping off, collar rot and root rot in to the surface level of discs in Petri plates surveyed citrus nurseries. containing discs of fungus growth removed from culture. Plates are incubated under cool The isolation of Phytophthora was done by white fluorescent light for 1-3 days and using leaf bait technique (Grimm and observed daily under the dissecting scope for Alexander, 1973) and soil spreading sporangia. (Kannwisher and Mitchell, 1978) method. Phytophthora was isolated on CMA-PARPH Morphological Identification and medium (Michell et al., 1986). The use of characterization of the pathogen selective medium was found effective in the detection of Phytophthora when low Phytophthora spp. was observed under 40X inoculum was present in the soil (Zitko et al., magnification on compound microscope and 1987; Gade, 2009). identified on the basis of their morphological characteristics like mycelium structure, shape For propagule count soil spreading method of sporangia, presence of chlamydospore etc. was used accordingly 35 out of 38 soil and confirmed with the help of CMI samples showed presence of Phytophthora in publications. citrus nurseries (Table 1). Presence of the pathogen may be because of raising of Results and Discussion nurseries on same piece of land and to the roadside to attract the customer and free Survey of citrus nurseries access to enter in to the nursery. Pathogen population can be build up even phytosanitary Citrus nurseries were surveyed in Amravati measures are taken. Ridings et al., (1977) district of Vidarbha region of Maharashtra. showed that even with strict sanitary Citrus nurseries in this district were selected practices, recontamination of disinfected areas during 2016 and 2017 to record prevalence of occurred when it was present near to the Phytophthora spp. Soil samples from the nursery. nurseries were collected during prevalence of the disease in citrus nursery. Collected Gade (2009) and Gade (2012) reported that samples were subjected for isolation of Phytophthora parasitica is associated with Phytophthora spp. on selective medium disease in citrus nurseries of Vidarbha in the (CMA-PARPH). range of 28 to 46 cfu/g soil. Present results of association of Phytophthora with citrus The occurrence of Phytophthora spp. was rhizosphere confirms the findings of Das et recorded in 35 out of 38 surveyed locations, al., (2016) who reported similar results and with a range from 3.10 to 24.33 cfu / g of soil isolated Phytophthora palmivora, P. in the year 2016 and 2.33 to 24.99 cfu / g of parasitica and P. citrophthora from the soil soil in year 2017. Pooled data of infected samples collected from citrus growing areas nurseries indicate propagules in the range of of Nagpur, Wardha and Amravati districts. 2.94 to 24.66 cfu/g of soil (Table 1). Three 3550 Int.J.Curr.Microbiol.App.Sci (2020) 9(8): 3548-3555 Table.1 Prevalence of Phytophthora spp. in citrus nurseries of Amravati district Sr. Location GPS Average cfu / g soil No. (Latitude/Longitude) 2016 2017 Pooled 1 Badnera 20.844719/77.721848 10.44 14.21 12.33 2 Shivaji Agri. College 20.950424/77.775459 8.88 13.10 11.00 3 Govt. Nursery 20.948980/77.781731 6.77 9.0 7.89 4 Jarud (Hole Nursery) 21.457016/78.228668 12.22 21.77 17.00 5 Tembhurkheda 21.504112/78.215836 11.77 14.77 13.27 6 Warud 21.496664/78.266365 10.11 6.88 8.50 7 Wai mauje (S.