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The Remote Citroid Fruit Tree Glycosmis Pentaphylla Is a Host of Citrus Leprosis Virus C and Exhibits Novel Leprosis Symptoms Fi

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The Remote Citroid Fruit Tree Glycosmis Pentaphylla Is a Host of Citrus Leprosis Virus C and Exhibits Novel Leprosis Symptoms Fi 031_TESTI_DN_499 25-06-2009 12:55 Pagina 499 Journal of Plant Pathology (2009), 91 (2), 499-505 Edizioni ETS Pisa, 2009 499 DISEASE NOTE DISEASE NOTE THE REMOTE CITROID FRUIT TREE FIRST REPORT OF PHOMOPSIS GLYCOSMIS PENTAPHYLLA IS A HOST ACTINIDIAE CAUSING CANKERS ON OF CITRUS LEPROSIS VIRUS C AND SHOOTS OF KIWIFRUIT IN GREECE EXHIBITS NOVEL LEPROSIS SYMPTOMS T. Thomidis J. Freitas-Astúa1,2, A.L. Fadel2, R. Antonioli-Luizon2, M. Bas- tianel2, V.M. Novelli2, E.W. Kitajima3 and M.A. Machado2 Alexander Technological Education Institute of Thessaloniki, School of Agricultural Technology, Department 1Embrapa Cassava and Tropical Fruits, s/n, Cruz das Almas, BA, of Crop Production, 57400 Sindos, Thessaloniki, Greece Brazil, 44380-000 2Centro APTA Citros Sylvio Moreira-IAC, CP 4, Cordeirópolis, SP, In June and through summer 2007, wilted and blighted Brazil,13490-970 shoots with distinct dark cankers were observed on ki- 3Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, wifruit plants of cvs Hayward and Tsechelidis in the ESALQ, CP9, Piracicaba, SP, Brazil, 13418-900 province of Imathia (Greece). Isolations made on acidified potato dextrose agar yielded a fungus which was identified Citrus leprosis virus C (CiLV-C), the causal agent of as Phomopsis actinidiae (Henn.) Died. on the basis of mor- one of the main virus disease of citrus in Brazil, is consid- phological and sporulation characters. Circular chalk ered to have a narrow host range. However, recent studies white-colored aerial mycelial mats developed in culture af- ter incubation at 25°C for 7 days. After additional 3-4 have shown that some weeds, hedgerow and windbreak weeks, black, spherical or bluntly conical pycnidia 230-500 plants can host CiLV-C, thus may play a role in the epi- µm in size bearing a- and b-conidia were formed all over demiology of the disease (Bastianel et al., 2006). We now the mycelial mats. The conidia, extruded as yellowish or report that CiLV-C can infect Glycosmis pentaphylla Retz. milky conidial masses, were hyaline, unicellular, fusiform, DC., a rutaceous shrub native to tropical Asia. G. penta- filiform to hamate. Koch’s postulates were fulfilled by in- phylla is grown for its edible ripe fruits in gardens that can oculating with agar plugs from fungal colonies, 20 seg- be near citrus groves. G. pentaphylla leaves were infested ments (6 cm in length and 1.5-2 cm in diameter) of 1-year- with presumably viruliferous Brevipalpus phoenicis mites, old woody shoots of kiwifruit cv. Hayward as described by the vector of CiLV-C, which had been reared on leprosis- Jeffers et al. (1981). Ten segments, used as controls, were infected plants for 72 h. Twenty-four days after infestation, wounded and inoculated with an agar disk without fungal localized symptoms appeared as conspicuous dark spots, mycelium. Shoot segments inoculated with the fungus de- with a darker center and irregular borders surrounded by a veloped cankers similar to those observed in the field, small chlorotic halo. These spots differed significantly from which the same pathogen as that used for inocula- from the well-defined chlorotic to necrotic lesions often tions was recovered. Controls remained symptomless. P. ac- seen in leprotic Citrus spp. tissues. The symptoms re- tinidiae has been reported as the causal agent of leaf blight mained unchanged for 110 days. CiLV-C was detected in and fruit rot of kiwifruit in other countries (Jeong et al., symptomatic leaves by RT-PCR using specific primers that 2008) but, to our knowledge, this is the first report of its amplify a region of its putative movement protein gene occurrence in Greece, on kiwifruit showing cankers on the (Locali et al., 2003). Amplified products had the expected shoots. 344 bp size. Thirty-three amplicons were cloned and se- quenced (GenBank accession No. EU257507). Their de- Jeffers S.N., Aldwinckle H.S., Burr T.J., Arneson P.A., 1981. Ex- duced amino acid sequence was 93% identical to that of a cised twig assay for the study of apple tree crown rot Brazilian isolate of CiLV-C (YP_654542.1), confirming the pathogens in vitro. Plant Disease 65: 823-825. identification of the virus transmitted. This is the first re- port of CiLV-C infecting a remote citroid fruit tree, and G. Jeong I.H., Lim M.T., Kim G.H., Han T.W., Kim H.C., Kim M.J., pentaphylla is the only so far known CiLV-C host exhibit- Park H.S., Shin S.H., Hur J.S., Shin J.S., Koh Y.J., 2008. Inci- ing such particular symptoms. dences of leaf spots and blights on kiwifruit in Korea. Plant Bastianel M., Freitas-Astúa J., Kitajima E.W., Machado M.A., 2006. The Pathology Journal 24: 125-30. citrus leprosis pathosystem. Summa Phytopathologica 32: 211-220. Locali E.C., Freitas-Astúa J., Souza A.A., Takita M.A., Astúa-Monge G., Antonioli R., Kitajima E.W., Machado M.A., 2003. Development of a molecular tool for the diagnosis of leprosis, a major threat to the citrus production in the Americas. Plant Disease 87: 1317-1321. Corresponding author: J. Freitas-Astúa Corresponding author: T. Thomidis Fax: +55. 19.3546.1399 Fax: +30.2310791351 E-mail: [email protected] E-mail: [email protected] Received August 18, 2008 Received December 18, 2008 Accepted January 27, 2009 Accepted March 5, 2009 031_TESTI_DN_499 25-06-2009 12:55 Pagina 500 500 Journal of Plant Pathology (2009), 91 (2), 499-505 DISEASE NOTE DISEASE NOTE FIRST REPORT OF PHOMOPSIS DIOSPYRI FIRST REPORT OF A 16SrV-B GROUP CAUSING SHOOT BLIGHTS ON PHYTOPLASMA ASSOCIATED WITH A PERSIMMON TREES IN GREECE LEAFROLL-TYPE DISEASE OF APRICOTS IN NORTHERN CHINA T. Thomidis and E. Navrozidis H.N. Yue, R.H. Sun, T.Wei and Y.F. Wu Alexander Technological Education Institute of Thessaloniki, School of Agricultural Technology, Department of Crop Production, College of Life Science, Northwest A&F University, Yangling, 57400 Sindos, Thessaloniki, Greece Shaanxi, 712100, P.R. of China In July and through summer 2006, persimmon trees In July 2007, a phytoplasma-like disease was observed on (Diospyros lotus) showing wilted and blighted shoots were apricot plants (Prunus armeniaca L.) grown in the Chinese observed in commercial orchards of the Imathia prefecture province of Shaanxi. Affected trees were stunted and showed (northern Greece). Close examination of the cortical tis- yellow, small and upward curled leaves. Incidence of this dis- sues of affected shoots revealed the presence of dark- ease was less than 15%. From samples collected from 14 coloured cankers. Isolations from the lower margins of symptomatic and six symptomless plants in six different or- these cankers made by plating tissue fragments ca. 3 mm in chards, total DNA was extracted from ca. 0.5 g of leaf midrib size on acidified potato dextrose agar, yielded a fungus or stem phloem tissue using a modified cetyltrimethyl-ammo- which, based on morphological and sporulation character- nium bromide (CTAB) method (Qi et al., 2004). DNA ex- istics, was identified as Phomopsis diospyri (Sacc.) Trav. & tracts were analyzed by nested PCR, using the 16S rRNA Spessa. Fungal colonies in culture had a white-coloured gene universal primer pairs R16mF2/R16mR1 followed by mycelium on whose surface dark-pigmented, flask-shaped R16F2n/R16R2 (Gundersen et al., 1996), which amplified a pycnidia developed, oozing one-celled, ovoid to fusoid 1.4 kb and a 1.2 kb product, respectively, from symptomatic curved conidia as milky or yellowish mucilaginous drops. plants only. RFLP analysis of the 1.2 kb products with AluI, Koch’s postulates were fulfilled by inoculating with agar MseI, HhaI, HpaI, RsaI, BfaI, HinfI and TaqI endonuclease (Lee et al., 1998), showed that all symptomatic plants were in- plugs from fungal colonies 20 segments (6 cm in length fected by a phytoplasma belonging to the subgroup B of the and 1.5-2 cm in diameter) of 1-year-old woody persimmon elm yellows group (16SrV-B) (Candidatus Phytoplasma shoots as described by Jeffers et al. (1981). Ten segments, ziziphi). The nucleotide sequence of cloned 16SrDNA (Gen- used as controls, were wounded and inoculated with an Bank Accession No. FJ572660) showed the highest identity agar disk without fungal mycelium. Shoot segments inocu- (99%) with comparable sequences of subgroup 16SrV-B, lated with the fungus developed cankers similar to those thereby confirming the results of RFLP analysis. Phytoplas- observed in the field, from which the same pathogen as mas of 16SrV-B subclade have previously been detected in that used for inoculations was recovered. Controls re- cherry in China and in peach in India (Lee et al., 2004), and mained symptomless. P. diospyri has been reported as the in Poland in apricot imported from China (Cieslinska et al., causal agent of shoot cankers and fruit rots of persimmon 2004). However, to our knowledge, this is the first molecular in other countries (Horst, 2008). However, to our knowl- evidence of the presence of a phytoplasma of the 16SrV-B edge, this is the first report from Greece documenting at- subgroup associated with a leafroll-type disease of apricot in tacks of P. diospyri to persimmon. northern China. Qi Y.X., Xie Y.X., Zhang H.Q., Zhu S.F., Liao X.L., Xiao Q.M., 2004. A improved method for extracting phytoplasma DNA. Biotechnol Bul- Jeffers S.N., Aldwinckle H.S., Burr T.J., Arneson P.A., 1981. Ex- letin 4: 44–46. cised twig assay for the study of apple tree crown rot Gundersen, D.E., Lee I.-M., 1996. Ultrasensitive detection of phytoplas- pathogens in vitro. Plant Disease 65: 823-825. mas by nested-PCR assays using two universal primer pairs. Phy- Horst R.K., 2008.
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