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Study on the pathogenic characteristics of Phytophthora tabaci to Pythium humicum

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https://www.eduzhai.net/ International Journal of A griculture and Forestry 2013, 3(4): 159-161 DOI: 10.5923/j.ijaf.20130304.06 Characterization of Phytophthora nicotianae Pathogenic to Chamaerops humilis in Iran Eisa Naze rian1,*, Mansure h Mirabolfathi2 1National Research Station of Ornamental Plants, M ahallat, Iran 2Plant Protection Research Institute, Tehran, Iran Abstract Phytophthora nicotianae was isolated and identified using standard taxonomic criteria fro m Chamaerops humilis var. argentea showing inner basal leaves rot in Markazi province of Iran. Disease symptoms appeared as discoloration, water soaking on interior basal leaves. Collapse of an affected plant occurs less than one month. Lesion expands from the inner basal leaf to the tip. A ll affected plants turned to pale gray or silvery in advanced stage of disease. In pathogenicity test with insertion of mycelial plug of respective isolates, similar sympto ms produced as naturally infection. This is the first report of P. nicotianae in C. humilis fro m Iran. Keywords Blue Mediterranean Fan Pa lm, Landscape Plant, Orna mental Plants, Disease 1. Introduction Ornamental p lants are one of the major econo mic p lant commodit ies in Iran, gro wn either in greenhouse or in the field ; ho wev er th e majo rit y o f o rn amental p lants are cultivated in northern and central of the country. Recently, many o rnamental p lants v ariet ies were int roduced and cult ivated across the country, subsequently; new d isease occurred and cause loss to growers. Susceptibility of many ornamental p lants to Phytophthora nicotianae was reported previously[11-8]. Phytophthora nicotianae strains are able to infect d ifferent hosts. To date more than 301 d ifferent hosts of th is pathogen includ ing Allium cepa , Dianthus caryophil lus, Lycop ersicu m escul entu m and Eupho rbia pulcherrima we re repo rted[4-17]. Phytopathogen Ph yto phth o ra ni co tian ae hav e b een first repo rted on Chamaerops humilis var. argentea fro m Italy in 2011[5]. Moist env iron mental cond it ion and unsuitab le irrigat ion methods led to spread propaguls (sporangia and zoospore) of P. nicotianae. The disease seems to be spread quickly and there is cause for concern if diseased plants are found among healthy . Field observ at ions o f d isease sp read and past cultural histories of different plantings provide evidence that the disease may be rapid ly spread by cultural practices and overhead irrigat ion water. Disease dev elop ment can be reduced by sanitation practices include, the re moval of plant debris, sterilizing pot and use of disease free plant material. M any g ro wers also t reat p lants wit h ant i-o o my cetes fungicides such as phenylamide, mefeno xam and metalaxyl * Corresponding author: eisa_yas@yahoo.com (Eisa Nazerian) Published online at https://www.eduzhai.net Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved [17]. This study report symptomatology and pathogenicity of the causal agent on C. humilis. 2. Material and Methods In the summer of 2012, 15% o f a nursery stock, approximately to 4000 potted blue Mediterranean fan palms growing in an ornamental nursery in Markazi province showed dieback. Diseased tissues showing advanced disease symptoms were collected and disinfected with 5% NaClO for 3 min. Small pieces (0.5-1 cm) were p laced onto corn meal agar (CMA) and potato dextrose agar (PDA) media. For better production of sporangia, mycelia tips were placed onto V-8ju ice agar supplemented with 200pp m amp icillin, 50pp m mycostatin and 10ppm pentachloronitrobenzen[9]. Seven pure cultures of fungi were obtained using mycelia tips culture after incubation at 25° C for 7 days[10]. The width and length of 12 sporangia were measured for each isolate. The growth speed at 36°C was measured during 5 days, on 3 replicates for each isolate, on V-8 agar mediu m in the dark. Oospore production and determination of mating types were done using V8 med iu m. All isolates were stored in test tube on 5% V-8 juice agar at 25°C. In pathogenicity test C. humilis were grown under greenhouse condition in plastic pots (30 x30 cm) at 25°C. The soil mixture was as grower used and sterilized prior to use at 120°C for 30min. For inoculu m preparation, all isolates were grown on V-8 juice agar at 25°C for 5 days. To stimulate sporangia formation, p ieces of each inoculu m derived fro m V-8 ju ice agar was cu ltured in to 100 ml of sterile 1% potassiumn itrate solution in five petridishes[7]. These cultures were g rown under uv light at 25°C for 5 days. Pathogenicity tests were 160 Eisa Nazerian et al.: Characterization of Phytophthora nicotianae Pathogenic to Chamaerops humilis in Iran performed by wound-inoculation with a cork borer of 8 three-year-old potted fan palms. After disinfect ion of the inoculation surface with 70% ethyl alcohol, a mycelial plug of seven-day-old colonies grown on V-8 ju ice agar was inserted into the basal stem and the hole was covered with the removed tissue and sealed with Parafilm®[5]. To compare the isolates behavior in pathogenicity and aggressiveness, an experiment was conducted in a completely randomized design with 3 replications per isolate and 8 plants per replication. Disease severity readings were made at 10 and 20 days after inoculation on each plant of every pot[15]. The experiment was carried out at the National Research Station of Ornamental Plants in Iran during 2012. Pots were irrigated daily with tap water. The greenhouse condition was conducted at 25 ± 2°C and 43% RH. Classification to species was confirmed by ribosomal DNA sequencing. After DNA extraction fro m pure culture, amp lification of DNA in PCR was carried out using ITS4 and ITS6 prime rs[16]. The PCR consisted of 1 cycle of 95°C for 2 min; 30 cyc les of 95°C for 20 s, 55°C for 25 s, 72° C for 50 s; and a fina l cyc le of 72° C for 10 min[3]. A 853bp band was obtained fro m amp lification of each ITS-PCR product in 1.5 % agarose gel w/v run in TG buffer (3gr/li Tris-Base MW = 121.10, 28.8 gr/ li g lycine MW = 75.07), stained with 1.0 % ethidium bro mide, visualized under uv light and photographed. 3. Results and Discussion A dark bro wn rot on the petiole base and blight of the unopened spear leaves were the main d isease symptoms (Figure1). isolates were A1 mating type and formed oogonia with smooth walls on V-8 juice[2]. The sporangia width/length ratio, were almost 1.3[1]. All isolates were also produced abundant chlamydospors on V-8 ju ice agar. The above characteristics were agreed with other studies[13]. Using of the ITS6/ITS4 primers wh ich caused the amp lification of the expected bands (853bp) in isolates (Figure 2), was in concord with its result of classification based on the morphological features in this research. BLAST analysis of the 853-bp frag ment showed 99% identity with the sequences of P. nicotianae isolates exists in NCBI data b as es . Figure 2. IT S-PCR banding pattern of six isolates (lane 1-6) of P. nicotianae isolated from blue Mediterranean fan palm. M; Molecular marker 1 kb, lane 7; Control without DNA In pathogenicity test P. nicotianae caused severe disease reaction on C. humilis and was practically identical. Normally, the outer leaves remained healthy, so the disease symptoms could be seen only at the advanced stage of disease development (because the nature of the plant). Bro wnish to black lesion on inner basal leaves enlarged rapidly toward the leaves tips were characteristic symptoms on C. humilis. The lesion caused bud to die. Merg ing lesion or quickly e xpanding rot, developed around the basal interior leaves. When the basal of the emerg ing leaf became infected, the rot rapidly spread into the termina l bud and kill the plant. P. nicotianae was re-isolated fro m p lants which inoculated in pathogenicity test. The pathogenic behaviors of the isolates showed sever inner basal leaf rot 10 days after inoculation with all seven isolates. The symptoms were similar to naturally infection (Figure 3). Figure 1. Natural infection of blue Mediterranean fan palm caused by Phytophthora nicotianae, (a: unopened spear leaves, b: browning of basal st em) Characteristics of seven isolates obtained fro m affected samples were studied based on morphological characteristics and PCR amp lification. Morphological characteristics of the myceliu m clearly showed that P. nicotianae Breda de Haan was pathogen affecting C. humilis. Obpyriform, monopapillate sporangia, stoloniferous myceliu m and amphygenous antheridium, and g rew between 8°C and 36°C on V-8 ju ice agar were the main characteristics. None of isolates produced oogonium on CMA or PDA med ia. A ll Figure 3. Cross section of bud basal rot caused by Phytophthora nicotianae in pathogenicity test, photographed after 10 days On the 20th day, plants inoculated with P. nicotianae were completely collapsed and died. In spite of the low number of International Journal of A griculture and Forestry 2013, 3(4): 159-161 161 isolates examined here; it is concluded that no significant variations observed in aggressiveness among isolates in severity index and bud rot parameters in this study. However, variation in aggressiveness among isolates of Phytophthora spp. and within isolates of P. nicotianahas been recognized before[14]. Th is result is important because it shows that particular hosts that display the disease symptoms only in advanced stage of disease development can serve as a powerful repository of inoculum for Phytophthora nicotianae. This disease happened at nurseries with high mo istures. No application of preventive fungicide, overhead irrigation caused excessive moisture, favor for infection and disease development. It is clear that asexual reproduction is a dominant character in Phytophthora spp. Controlling the spread of Phytophthora within and among production sites seems difficult. In many production facilities, plants do not show apparent symptoms before well establishment of the infection or even plants which treated with protective fungicides may seem healthy until the fungicides lose efficacy and pathogen population increase[6-12] Overall, use of water free fro m fungal inoculum using absorption mats below pots to filter out recirculating inoculum, adding some minerals to irrigation water, drench irrigation instead of over heading and use of preventive fungicide are reco mmended for disease management. report of Phytophthora nicotianae as pathogen of blue M editerranean fan palm. New Disease Report, 23, 3. [6] Ferrin, D. M ., Rohde, R. G., 1992, Population dynamics of Phytophthora parasitica the cause of root and crown rot of Cathranthus roseus, in relation to fungicide use. Plant Disease, 76, 60-63. [7] Harada, G., and Kondo, N., 2009, Adzuki bean leaf infection by Phytophthora vignae f. sp. adzukicola and resistance evaluation using detached leaves inoculated with zoospores. Journal of General Plant Pathology, 75, 52-55. [8] Hine, R. B., and Aragaki, H., 1952, A new wilt disease of carnations in Hawaii caused by Phytophthora parasitica. Hawaii Farm Science, 11, 6. [9] Ko, W. H., Chang, H. S., Su, H. J. 1978, Isolates of Phytophthora cinamomi from Taiwan as evidence for an Asian origin of the species. Transactions of the British Mycological Society, 72, 496-499. [10] Ko, W. H., 1981, Reversible change of Phytophthora parasitica. Journal of General M icrobiology, 125, 451-454. [11] Klotz, L. J., De Wolfe, T.A. Wong, P.P., 1958, Decay of fibrous roots of citrus. Phytopathology, 48, 616-622. [12] Lamour, K. h., Daughtrey, M . L. Benson, D.M ., Hawang, Y., Hausbeck, M .K., 2003, Etiology of Phytophthora dreshleri and P. nicotiana (P. parasitica) diseases affecting floriculture crops. Plant Disease, 87, 854-858. [13] Stamps, D. J., Waterhouse, G. M ., Newhook, F. J., Hall, G. S., 1990, revised tabular key to the species of Phytophthora. CABI. Mycology Institute Mycology Paper No. 162, 28 pp. REFERENCES [1] Allagui, M . B., Lepoivre, P., 1996, Comparaison de différentes techniques d´inoculation du piment par Phytophthora nicotianae var parasitica. Agronomie, 16, 433-440. [2] Chang, T. T., and Ko, W. H., 1990, Effect of metalaxyl on mating type of Phytophthora infestans and P. parasitica. Annal Phytopathological Society of Japan, 56(2), 194-198. [3] Cooke, D. E. L., Drenth, A., Duncan, J. M ., Wagels, G., Brasier C. M ., 2000, A molecular phylogeny of Phytophthora and related Oomycetes. Fungal Genetic Biology, 30, 17-32. [4] Erwin, D. C., and Ribeiro, O. K., 1996, Phytop hthora diseases worldwide. APS Press, St Paul, USA. [5] Faedda, R., Pane, A., Granata, G., San Lio, G. D., 2011, First [14] Sullivan, M . J., M elton, T. A., Shew, H. D., 2005, Fitness of races 0 and 1 of Phytophthora parasitica var. nicotianae. Plant Disease, 89, 1220-1228. [15] Townsend, G. R., Heuberger, J. W., 1943, M ethods for estimating losses caused by diseases in fungicide experiments. Plant Disease Report, 27(17), 340-343. [16] White, T. J., Bruns, T., Lee, S., Taylor, J., 1990, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to M ethods and Applications (M . A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds.), pp. 315–322. Academic Press, San Diego. [17] Yoshimura, M . A., Uchida, J. Y., Aragaki, A., 1985, Etiology and control of poinesstia blight caus ed by Phytophthoranicot iane var. parasitica and P. derechsleri. Plant Disease, 69, 511-513.

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