J Phytopathol 159:579–581 (2011)  2011 Blackwell Verlag GmbH doi: 10.1111/j.1439-0434.2011.01798.x Short Communication Department of Plant Protection, Faculty of Agriculture, University of Ankara, Turkey Involvement of Phytophthora citrophthora in Kiwifruit Decline in Turkey Sec eç_il l Akilli killi1, Ç_igdem lubaş Serce erçe2, Yakup akup Zeka eka_i Katircioglu atircioğlu3, Az z_iz z Karakaya arakaya3 and Sal al_ih h Maden aden3 ğdem Ulubas Authors addresses: 1Faculty of Science, Department of Biology, Çankırı,Çankırı Karatekin University, Turkey; 2Faculty of Agriculture, Department of Plant Protection, Hatay, Mustafa Kemal University, Turkey; 3Faculty of Agriculture, Department of Plant Protection, University of Ankara, 06110, Dışkapı, Ankara, Turkey (correspondence to A. Karakaya. E-mail: karakaya@agri.ankara.edu.tr) Received October 7, 2010; accepted February 18, 2011 Keywords: Phytophthora citrophthora, kiwifruit, Turkey Abstract Materials and Methods Vine decline of kiwifruit was found in an orchard in the Rize province in Turkey. About half of the vines showed poor growth, leaf discoloration and dieback symptoms. From the necrotic feeder roots of the diseased vines, a Phytophthora sp. was isolated. The causal agent of the disease was identified as Phytophthora citrophthora by morphological characteristics and comparing sequences of Internal Transcribed Spacer (ITS) region. Phytophthora citrophthora caused bark necroses averaging 4.2 ± 1.4 cm in length when inoculated on the rooted canes. Study site and sampling Introduction Kiwifruit (Actinidia deliciosa) is native to Southeast Asia. Today it is grown in many countries including New Zealand, Italy, Chile, China and Korea (Sale 1990). It has been grown on 1629 ha in Turkey, mainly in the coastal areas of the Black Sea region with production of 23 000 tons (Anonymous 2009). Rize, which is located in Eastern Black Sea region, is an important kiwifruit growing area with production of 5360 tons on 310 ha in 2009. Several Phytophthora species affecting kiwifruit have been reported (Erwin and Ribeiro 2005). Phytophthora citrophthora, P. cryptogea, P. megasperma, P. cactorum, P. cinnamomi, P. citricola P. drechsleri, P. gonapodyides, P. nicotiana, P. lateralis and Phytophthora sp. were recovered from kiwifruit in USA, Chile, Italy, New Zealand, Korea, France and China (Conn et al. 1991, Latorre et al. 1991; Stewart and McCarrison 1991; Huang and Qi 1998; Lee et al. 2001; Miller 1998; Ciccarese et al. 1992). In an orchard in Ardeşen, Rize, Turkey, a Phytophthora sp. was consistently isolated from diseased roots of kiwifruit. This study was conducted to find out the causal organism responsible for the poor growth and death of kiwifruit plants. Decline symptoms were reported from a kiwi plantation treated by farm manure in the Ardeşen district of Rize province, Turkey. Almost half of the vines in the 0.25 ha study area showed decline symptoms. Three vines, 3–5 years old, were uprooted; roots and lower stem sections were removed and brought to the laboratory. Phytophthora isolation Necrotic bark, feeder roots and inner root and stem tissues showing discoloration were plated onto modified selective PARPNH medium, in which Grated Carrot Agar (GCA) (40 g thinly grated carrot and 18 g agar per liter) was a base medium (Jung et al. 2002) and incubated at 20C in dark. Colonies growing from plated tissues were transferred to GCA amended with b-sitosterol 30 lg⁄ml, thiamine hydrochloride 1 lg⁄ml, and tryptophane 20 lg⁄ml (Wilcox and Ellis 1989). Pathogenicity test Pathogenicity of the isolate was assessed using the method of Lee et al. (2001). Stem inoculation tests were carried out on 1-year-old rooted cuttings of 10 kiwifruit cuttings (cultivar Hayward). The wound was covered with wet, autoclaved cotton wool and sealed with Parafilm. Ten control cuttings were treated similarly with sterile GCA disks. Inoculated plants were kept in a growth chamber at 25 ± 1C. Morphological and physiological characteristics Cultural and morphological characteristics of the isolates were studied on cultures grown on malt extract agar (MEA), corn meal agar (CMA), GCA and potato dextrose agar (PDA). Identification of Phytophthora sp. was done using the features cited by Stamps et al. 580 Akilli et al. (1990), Erwin and Ribeiro (2005) and Gallegly and Hong (2008). PCR protocol for ITS region amplification and sequence analysis A fragment from the ITS region of the isolate was amplified using the universal primer pairs ITS-6 (5¢ GAA GGT GAA GTC GTA ACA AGG 3¢) (13) and ITS-4 (5¢ TCC TCC GCT TAT TGA TATGC 3¢). The PCR conditions and cycling program were performed according to Camele et al. (2005). PCR products were separated in 1% agarose gels stained with ethidium bromide, and visualized under UV light. Sequence analysis was done by REFGEN (Gen Araştırmaları ve Biyoteknoloji Ltd. Şti., Gümüş Bloklar 5–6 ODTÜTeknokent, Ankara, Turkey). The nucleotide sequence was submitted to Genbank with the accession number of HQ676131. Results and Discussion Disease symptoms were observed on kiwi vines treated by farm manure. Symptoms were observed especially (a) (b) (c) (d) (e) (f) (g) (h) (i) on the slopes. A Phytophthora sp. was consistently isolated from diseased feeder roots on modified selective medium PARPNH. Phytophthora sp. was not isolated from other plant tissues. The Phytophthora species grown on CMA and GCA produced sporangia profusely in sterile and nonsterile soil extracts. Morphological aspects of the sporangia and cultural aspects of the isolate used (Fig. 1a–f) were in accordance with the descriptions cited by Erwin and Riberio (2005) and Gallegly and Hong (2008) except Stamps et al. (1990) who reported the sporangia as caducous. We also observed some caducous sporangia. We assume that caducity is not a distinguishing character for P. citropthora. Identification of P. citrophthora based on this feature may mislead researchers since sporangial morphology of the species resemble those of P. capsici. Nucleotide sequence of the kiwifruit isolate of P. citrophthora (Acc. No HQ676131) had 99–100% homology with other P. citrophthora isolates in Genbank (e.g. Acc. No: EU000063, EF193228, GU259198, GU259190, GU259189, GU259187, GU259003, GU133068). Fig. 1 Morphological and pathological characteristics of Phytophthora citrophthora isolated from kiwifruit. Sporangia having swellings on the pedicel and lateral attachments (a and b), bizarre shaped sporangia with lateral pedicel attachment (c), colonial growth on MEA, PDA and corn meal agar (d, e, and f, respectively), bark cankers (g and h) formed by the isolate on 1 year old rooted plants (i) non inoculated control plant Involvement of Phytophthora citrophthora on Kiwifruit Pathogenicity of the P. citrophthora isolate on kiwi fruit stems was proved. Thirty days after inoculation bark lesions formed on the stems measured 2.6–7.0 (average 4.2 ± 1.4) cm in length (Fig. 1g,h). Control plants showed only small necrotic areas girdled by new callus (Fig. 1i). Phytophthora citrophthora was reisolated from the diseased area. Pathogenicity of P. citrophthora on kiwifruit was also reported by other researchers (Latorre et al. 1991). 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