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J Phytopathol

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First Report of Fusarium proliferatum Infecting Carnation (Dianthus caryophyllus L.) in China Junxiang Zhang1,*, Xingxing Wu1,*, Yunqing Bi2, Yixin Wu3, Guanhui lin4, Yueqiu He3 and Zichao Mao3 1 2 3 4

College of Plant Protection, Yunnan Agricultural University, Kunming, 650201, China Institute of Agricultural Environmental and Resource, Yunnan Agricultural Academy, Kunming, 650205, China College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, 650201, China College of Energy and Environmental Science, Yunnan Normal University, Kunming, 650092, China

Keywords carnation, Dianthus caryophyllus, fungal plant pathogen, Fusarium proliferatum Correspondence Z. Mao, College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming, China. E-mail: [email protected] *Those authors contribute equally to this paper. Received: October 6, 2012; accepted: April 23, 2013.

Abstract In 2011, a wilt disease has been detected on carnation (Dianthus caryophyllus L.) cultivar ‘Light Pink Barbara’ in Kunming, Yunnan, China. A Fusarium sp. was consistently recovered from pieces of symptomatic tissues on Petri dishes containing potato dextrose agar (PDA). On the basis of morphological characteristics and molecular identification by DNA sequencing of ribosomal DNA internal transcribed spacer (rDNA ITS) and partial translation elongation factor-1a (TEF) gene region, following their phylogenetic trees construction, the putative causal agent was identified as Fusarium proliferatum (Matsushima) Nirenberg, and its pathogenicity was finally confirmed by Koch’s postulates. To our knowledge, this is the first report of a wilt disease caused by F. proliferatum on carnation in China.

doi: 10.1111/jph.12128

Introduction Carnation (Dianthus caryophyllus L.), which is traditionally prescribed as herbal medicine for treatment of fever, coronary and nervous disorders (Ali et al. 2008), is famous for being used as a cut flower in the florist trade and bedding plant in the garden as well. With wonderful aromatic and showy colours, the carnation flower has been used in food and cosmetic fields, for example, it is used as garnish in fruit salads, and the dried flower heads are used in cosmetic and stuffing of sachets. It was very popular and widely cultivated in China for their wonderful petals, which are usually double-fringed, with showy colour and pleasing fragrance. During a plant survey conducted in the horticultural farm of Yingmao Flower industry Co., Ltd., in Kunming City, Yunnan, China, in 2011. A severely wilt disease represented on carnation cultivar ‘Light Pink Barbara’ was first observed. Disease symptoms consisted of initially wilted, finally leading to wilting

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and death of the infected plants. The wilt disease resulted in great loss on the production of carnation cultivar ‘Light Pink Barbara’. The aim of this study was to identify the causal agent of the wilt disease for proper managing in future. Materials and Methods To isolate putative pathogens, the segments of infected stem, which were surface-sterilized by successive submersion in 70% ethanol for 30 s, and 1% sodium hypochloride (NaOCl) for 5 min, then three times washing in sterilized water for 2 min per time, were transferred onto a potato dextrose agar (PDA) plate. After 5-day incubation at 25°C, all colonies appeared on PDA looks like Fusarium spp. Those suspected fungal colonies were purified by subculturing of single conidia separated by micromanipulation (Leslie and Summerell 2006). The successive subculturing of single conidia resulted in a pure culture was initially named YM1. The isolated YM1 strain is

J Phytopathol 161 (2013) 850–854 Ó 2013 Blackwell Verlag GmbH

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deposited in Laboratory of Agro-diversity and Pests and Diseases Control, Ministry of Education of China, Yunnan Agricultural University. For further species identification of the isolate, carnation leaf agar (CLA) and PDA were used for morphological characteristics observation. The morphology of macroconidia, microconidia, conidiogenous cells and the chlamydospores was assessed from cultures grown on CLA and PDA. Morphological identifications of isolate YM1 were based on the description of Nelson et al. (1983) and Leslie and Summerell (2006). To confirm the putative causal fungus, total DNA of isolated YM1 was extracted and purified from mycelium using of a modified method (He 2000). The extracted DNA was used as template in PCR to amplify

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the partial translation elongation factor-1a (TEF) gene with the primers EF1 and EF2 (O’Donnell et al. 1998; Ali et al. 2008), the ribosomal DNA internal transcribed spacer (rDNA ITS) region with the universal primers pITS1/pITS4 (Lee et al. 2000). Amplification products were purified using the MinElute Gel Extraction Kit (Sangon Biotech Co. Ltd., Shanghai, China). The eluted fragment was ligated into pGEM-T easy vector (Promega product; Promega, Madison, WI, USA) following the supplier′s operating protocol. The amplified fragments on pGEM-T easy were sequenced by Sanger method in Sangon Biotech. The sequence alignment was conducted using the program CLUSTAL W multiple alignment in BIOEDIT (www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenic

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Fig. 1 Morphological characteristics of isolate YM1: (a) hyphens (arrow showing lightly constricted at the septa); (b) macroconidia formed on CLA; (c) microconidia; (d) chlamydospore; (e) chlamydospore; (f) solitary monophialides and branched conidiophores; (g) polyphialides and monophialides and branched conidiophores; (h) false head of microconidia; (i) microconidia chain on carnation leaf agar (CLA); (j) verticillately branched conidiophore with whorls of monophialides; (k) microconidia chain with ‘V’ shape on CLA; (l) anastomosis of sterile hyphens; (m) coiled hyphae. Scale bars represent 20 lm.


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trees were constructed using MEGA 5.03 with the maximum-likelihood (ML) method and Tamura–Nei model (Tamura et al. 2007) with bootstrap analysis (1000 repetitions). Pathogenicity test was conducted on the 2month-old healthy plants of carnation cultivar ‘Light Pink Barbara’. The root of the carnation was surfacewashed in sterilized water before inoculation. Inoculations were performed by dipping the roots with a conidial suspension (1 9 107 conidia/ml) for 30 min, while the control plants were immersed in sterile distilled water for same condition. The treated plants were transplanted into a 20 9 100 cm square plastic pot containing 20 kg of soil previously sterilized at pots in the glasshouse with temperature of 28 3°C. Each treatment was replicated on 10 plants, and the experiment was conducted three times duplicating treatments. Results and Discussion In morphological observation, Aerial mycelium was almost white and floccose in initial stage, and the reverse of colonies could become dull red in later stage on PDA. The conidiophores on aerial mycelium originated erect or prostrate from the substrate, sympodially branched, bearing slender and nearly cylindrical monophialides (Fig. 1a). Aerial conidiophores 43 18

branched verticillately with whorls of monophiales or sometimes polyphialides, normally with size of 23.4– 54.6 9 2.3–3.9 lm (av. 35.3 9 3.1 lm; Fig. 1f,g). Microconidia were mostly 0–1 septate and variable in shape, mostly obovoid to oval, and measured size of 5.2–18.2 9 2.6–5.2 lm (av.10.8 9 3.1 lm). Microconidia were produced in false head mostly on short monophialides but occasionally in chains (Fig. 1h,i). Macroconidia were fusoid, gradually becoming slender and pointed towards the ends, with an undifferentiated foot cell, usually 3–5 septate, and with measured size of 31.2–52.0 9 2.6–5.2 lm (av. 40.8 9 3.1 lm), mostly borne in false heads on monophialides (Fig. 1b). Chlamydospores and sterile coiled hyphae were observed in this isolated strain (Fig. 1d,e,m). According to description of Nelson et al. (1983) and Leslie and Summerell (2006), YM1, the isolated Fusarium sp., was morphologically identified as Fusarium proliferatum (Fig. 1). For further confirmation of YM1 identification by molecular methods, the amplified 528 bp of ITS rDNA sequence and 709 bp of partial TEF gene were sequenced and deposited in GenBank (accession no. HQ231757 and JX174033, respectively). A blast analysis for sequence similarity of rDNA ITS region and partial TEF gene confirmed the identity of the fungus, showing exact match with nucleotide sequences of published F. proliferatum. In the constructed phylo-

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Fig. 2 Phylogenetic tree constructed with sequences of internal transcribed spacer rDNA showing the closest known relatives of Fusarium proliferatum and using the maximum-likelihood (ML) method. Numbers above branches represent bootstrap support values higher than 83%. The Fusarium isolate infecting carnation (Dianthus caryophyllus L.) is shown in rectangle.



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genetic trees of ITS (Fig. 2) and TEF (Fig. 3), the isolate YM1 were placed within a clade composed of reported F. proliferatum isolates. In pathogenicity test, the isolate YM1 was inoculated on roots of carnation as described above. Wilt symptoms first appeared on carnation after 5–7 days after inoculation (Fig. 4). Initially the plants get wilted, and later the older leaves converted into greyish colour but unaffecting healthy leaves remained green; eventually, the stem shrivelled and turned grey. The control plants remained healthy. Koch’s Postulate further confirms the F. proliferatum as the novel causal agent of wilt disease of carnation. Fusarium oxysporum has commonly been reported to cause a vascular wilt disease of carnation and regarded as the greatest threat to the production worldwide.

Moreover, Fusarium stem rot is generally associated with the three species F. avenaceum, F. graminearum and F. culmorum (Nelson et al. 1981; Wright et al.1997). F. proliferatum is a prevalent pathogen of agriculturally important crops with wide host range (Leslie and Summerell 2006). F. proliferatum is the pathogen of some economically importance plants including citrus fruit, banana, orchids, sorghum, asparagus (Leslie and Summerell 2006), Asparagus officinalis (von Bargen et al. 2009), onion (Bayraktar and Dolar 2011) and garlic bulbs (Tonti et al. 2012). Recently, in Yunnan and Sichuan provinces, China, it has been known as the causal agent of malformation disease of mango (Zhan et al. 2010). However, to the best of our knowledge, this is the first report of F. proliferatum infecting carnation (Dianthus caryophyllus L.) in China. 52 61

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Fig. 3 Phylogenetic tree constructed with sequences of partial translation elongation factor1a (TEF) gene sequences showing the closest known relatives of Fusarium proliferatum and using the maximum-likelihood (ML) method. Numbers above branches represent bootstrap support values higher than 99%. The Fusarium isolate infecting carnation (Dianthus caryophyllus L.) is shown in rectangle.

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Fig. 4 Symptoms induced by root infections of Fusarium proliferatum on carnation cultivar ‘Light Pink Barbara’: (a) healthy plant; (b) wilting plant; (c) dying plant with greyish; (d) dead plant.


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Acknowledgements The research was supported by the National Natural Science Foundation of China (NO.40861019), Panchun Yang, the general manger of Yingmao Flower industry Co., Ltd., provided great convenience on samples collection and paper discussing. References Ali A, Afrasiab H, Naz A, Rauf M, Iqbal J. (2008) An efficient protocol for in vitro propagation of carnation (Dianthus caryophyllus). Pak J Bot 40:111–121. von Bargen S, Martinez O, Schadock I, Eisold A-M, Gossmann M, B€ uttner C. (2009) Genetic variability of phytopathogenic Fusarium proliferatum associated with crown rot in Asparagus officinalis. J Phytopathol 157: 446–456. Bayraktar H, Dolar FS. (2011) Molecular identification and genetic diversity of Fusarium species associated with onion fields in Turkey. J Phytopathol 159:28–34. He Y-Q. (2000) An improved protocol for fungal DNA preparation. Mycosystema 19:434. Lee Y-M, Choi Y-K, Min B-R. (2000) PCR-RFLP and sequence analysis of the rDNA ITS region in the Fusarium spp. J Microbiol 38:66–73. Leslie JF, Summerell BA. (2006) The Fusarium Laboratory Manual. Ames, IA, USA, Blackwell Publishing Professional.

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Nelson PE, Horst RK, Woltz SS. (1981) Fusarium diseases of ornamental plants. In: Nelson PE, Toussoun TA, Cook RJ. (eds) Fusarium: Diseases, Biology and Taxonomy. University Park, PA, USA, The Pennsylvania State University, pp 121–128. Nelson PE, Toussoun TA, Marasas WFO. (1983) Fusarium Species: An Illustrated Manual for Identification, 1st edn. University Park, PA, USA, Pennsylvania State University Press. O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC. (1998) Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci USA 95:2044–2049. Tamura K, Dudley J, Nei M, Kumar S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. Tonti S, Pra MD, Nipoti P, Prodi A, Alberti I. (2012) First report of Fusarium proliferatum causing rot of stored garlic bulbs (Allium sativum L.) in Italy. J Phytopathol 160:761–763. Wright GEK, Say M, Pascoe LG, Guest DL. (1997) Incidence and symptoms of Fusarium diseases of carnations in Victoria. Australas Plant Pathol 26:44–53. Zhan R-L, Yang S-J, Ho H-H, Liu F, Zhao Y-L, Chang J-M, He Y-B. (2010) Mango malformation disease in South China caused by Fusarium proliferatum. J Phytopathol 158:721–725.
