PAGE, September - 2013; Volume 1(4) ISSN No. 2320 8694 IN-VITRO AND GREENHOUSE EVALUATION OF PHYTOPATHOGENIC POTENTIAL OF FUNGAL ISOLATES Alternaria tenuissima IN IRAN Mirkarimi HR *1, 2, Abasi moghadam A 2, Mozafari J 2 and Taheri Ardestani S 2 1 Department of Plant breeding, Faculty of Agriculture, science and research branch, Islamic Azad University, Tehran, Iran. 2 Department of Genetics & National Plant Gene- Bank of Iran, Seed and Plant Improvement Institute, Mahdasht RD, Karaj. Iran. Received: July 28, 2013; Revision: August 18, 2013; Accepted: August 28, 2013 Available Online September 20, 2013. KEYWORDS Early blight Greenhouse Agria Inoculation ABSTRACT Early blight of solanaceae is the most common reported disease in Iran. The disease caused by Alternaria species. Various isolates of Alternaria tenuissima were collected from various locations of the country. The collected Isolates were tested against tomato (Lycopersicon lycopersicum L.) crops in fields of Khansar Esfehan and National Plant Gene Bank of Iran. For determining the pathogenicity, these strains were tested against the genetic samples of Agria (susceptible), Delta (resistant control) and Cosmos. Symptoms were recorded in both in vitro and greenhouse conditions. Under in vitro condition genetic samples of Agria and Cosmos shows the pathogenic symptoms second day while the Delta is showing symptoms fifth day after inoculation. The reported symptoms were necrosis, chlorosis and damping. Under greenhouse evaluation, the symptoms became apparent in the genetic Agria fourth day, while in Cosmos at the eighth day and in Delta fourteenth day after inoculation. Finally, morphology and pathogenicity tests, asserted the isolation of pathogenic A. tenuissima. * Corresponding author E-mail: rezamirkarimi21@gmail.com (Hamid Reza Mirkarimi) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by Horizon Publisher (www.my-vision.webs.com/horizon.html). All rights reserved.
PAGE 237 Mirkarimi et al. 1 Introduction Early blight is one of the most destructive plant diseases, especially in the members of solanaceae family. The disease is ubiquitous disease in many countries around the world and caused by Alternaria solani, (Shahbazi et al., 2011). Hot and humid climatic conditions favor the Developments of the disease. Iran has a similar climatic condition that s why early blight of Solanaceae becomes most destructive disease in the Country. The initial symptoms of the disease appeared on the older and lower leaves; slowly it will cover the complete plant system (Ellis and Martin, 1882; Pscheidt, 1985). In Iran first exposure of A. alternata (Syn A. tenuis), A. brassicae and A. solani from was reported from the wheat, tomato and cabbage respectively (Esfandiari, 1948). The first written evidence of early blight in Iran was reported in 1977. (Taheri Ardestani et al., 2009). The identification and classification of the various isolates of Alterneria are depends on the spore morphology and the types of chain formation (Simmons, 1992). Over the past 30 years, some progress has been made in the taxonomy of this genus and emphasis on applying specific and standards methods. The specific procedures, including the use of low nutrient media, such as PCA, temperature 25-20 C under a light / dark 8/16 h using a white fluorescent lamp (Roberts, 2001; Andersen et al., 2001& 2002; Dugan & Peever, 2002). Recently six species of Alternaria responsible for causing spot disease in potato was isolated from the Iran (Taheri et al., 2007). A. tenuissima and A. interrupta were isolated from the infected potato crops, amongst these two A. tenuissima cultured on PCA medium. This fungus shows the dark brown appearance and concentric growth rings. Lamb of spores that grow directly from hyphae present in the culture medium, they were usually simple and at the end of the bend elbows 1-3 (Qusta, 1983; Taheri et al., 2007). The present study had been formulated for the finding out the pathogenicity of A. tenuissima under in-vitro and green house conditions. 2 Materials and Methods 2.1 Preparation of fungal suspension and cultural filtrate Fungal suspension was prepared from in vitro cultured fungal colonies and in double distilled sterilized water. The initial spore load of fungal suspension was maintained 10 6 which was calculated by haemocytometer. For obtaining cultural filtrate, fungal suspension maintained on PDB for 8-10 days, were used. The fungal suspension containing with spores passes through the four layers of cloth (Tiffany). The obtained filtrate was centrifuged for 15-20 minutes are 1000 rpm. Supernatant was passed through the bacterial filtrate and collected in sterilized vessels. 2.2 Morphological Identification To evaluate the morphological characteristics, pure isolates were taken from growing cultures, and there transferred to a flask containing PCA. The flasks were maintained on 20 ±2 C for a photoperiod of 6 hours of light and 18 hours of darkness for 7 days (Simmons, 1992). Finally, the samples were examined under microscope. 2.3 In vitro evaluation of A. tenuissima pathogenicity Plantlets raised by tissue culture were used for in vitro evaluation. These plantlets were exposed to the Culture filtrate, this evaluation was done in two ways: a. The plantlets were exposed to the filtrate by spraying the filtrate on the leaves of plantlets. The plantlets were maintained in the incubator at 23 ± 2 C temperature and 8/16 hours light and dark periods respectively (Rodriguez et al., 2007). b. In second method all three types of plantlets were uprooted from the test tubes and transferred in to the five micro liter filtrate containing test tubes. The plantlets were maintained in the incubator at 23 ± 2 C temperature and 8/16 hours light and dark periods respectively (Rodriguez et al. 2007). 2.4 Greenhouse evaluation of A. tenuissima pathogenicity Plantlets were raised in the laboratory at 4-5 leaves stages these were transferred in to the pots containing peat moss: perlite: soil, leaf in ratio of 2: 1: 1 under green house conditions. When the plants were 25 to 30 days old, the fungal spore suspension was sprayed on the plants by using content geysers. 2.5 Pathogenicity test In the end of each experiment infected leaves were washed by tap and double distilled sterile water 5-6 times. The infected leaves were surface sterilized by sodium hypochlorite solution (0.5%) for 35 seconds and again washed 2-3 times by double distilled sterilized water. Finally, the pieces are placed on sterile filter paper (for drying) and then transferred to the PCA culture medium (Taheri Ardestani et al., 2009). 3 Results and Discussion 3.1 Morphological characteristics The kh6 isolate colonies were often a milky white color rarely it was showing olive brown color. The hyphae produced by the colonies are in abundant in number and aerial in position. The fungal conidia are elliptical with 2-7 transverse and 1-3 longitudinal walls. Several bunches of conidiophores were also reported (Figure 1). The findings are in conformity with the finding of Taheri Ardestani et al. (2009).
PAGE In-vitro and greenhouse evaluation of phytopathogenic potential of fungal isolates Alternaria tenuissima in Iran. 238 20 X Magnification 100 X Magnification Figure 1 Microscopic images of isolated fungal spores A. tenuissima. Figure 2 In vitro evaluation of genetic samples with the fungal isolates, the both method. 3.2 In vitro evaluation A. tenuissima pathogenicity The isolates of A. tenuissima were showing positive signs of disease development on Delta, Cosmos and Agria isolates. These isolates start showing disease symptoms between two to five days of fungal filtrates applications (Figure 2). It was reported that Agria, was the most susceptible genotypes under disease progress curve It was noteworthy that genotype data (resistant control) are also shown symptoms of the disease, and the isolates proved to be pathogenic. 3.3 Greenhouse evaluation of A. tenuissima pathogenicity Under green house conditions, the appearance of symptoms are delayed then the in vitro application. Under green house condition, on Agria first disease symptoms appeared only after the four days fungal filtrate spraying while in the case of Cosmos and Delta first symptoms appeared eighth and fourteenth day after spraying of filtrate (Figure 3). Like in-vitro evaluation here also Agria was found the most susceptible isolates on the progression of disease curve. Rodriguez et al. (2006) reported that disease first appeared on the middle leaves and from here it can spread to top and bottom leaves of the tree and under green house condition maximum symptoms were appeared between four to eight days. In this manner the findings of the present study are in conformity with the findings of Rodriguez et al. (2006).
PAGE 239 Mirkarimi et al. Figure 3 Greenhouse evaluation, showing spots and small necrotic. Figure 4 Disease testing. Cultured leaf spots after inoculation, the cultures PCA and a growing colony of leaf spots after inoculation. 3.4 Pathogenicity test he infected leaves were harvested and used for the pathogenicity test (Figure 4). The structure of mycelium, conidia and conidiophores shows the similarity with the findings of Taheri Ardestani et al. (2009). Therefore, its proved that the leaves are infected by fungal isolates A. tenuissima kh6. References Anderson B, Kroger E, Roberts RG (2001) Chemical and morphological segregation of Alternaria alternata, A. gaisen and A. longipes. Mycological Research 105: 291-299. Andersen B, Kroger E, Roberts RG )2002( Chemical and morphological segregation of Alternaria arborescense, A. infectoria and A. tenuissima species groups. Mycological Research 106: 170-182. Dugan FM, Peever TL )2002( Morphological and cultural differentiation of described species of Alternaria from poaceae. Mycotaxon 83: 229-264. Ellis JB, Martin GB )1882( Macrosporium solani E&M. American Naturalist 16: 1003. Esfandiari E )1948( Troixieme liste des fungi ramasses en Iran. Entomologie et Phytopathologie Appliquees 8: 1-12. Pscheidt JW )1985( Epidemiology and control of potato early blight, caused by Alternaria solani. PhD thesis, Review of early blight of potato 101 University of Wisconsin-Madison.
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