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IDENTIFICATION of FUSARIUM OXYSPORUM F. Sp. OPUNTIARUM on NEW HOSTS of the CACTACEAE and EUPHORBIACEAE FAMILIES

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IDENTIFICATION of FUSARIUM OXYSPORUM F. Sp. OPUNTIARUM on NEW HOSTS of the CACTACEAE and EUPHORBIACEAE FAMILIES Journal of Plant Pathology (2017), 99 (2), 347-354 Edizioni ETS Pisa, 2017 347 IDENTIFICATION OF FUSARIUM OXYSPORUM f. sp. OPUNTIARUM ON NEW HOSTS OF THE CACTACEAE AND EUPHORBIACEAE FAMILIES D. Bertetti, G. Ortu, M.L. Gullino and A. Garibaldi Centre of Competence for the Innovation in the Agro-Environmental Sector (AGROINNOVA), University of Turin, Largo Braccini 2, 10095 Grugliasco, Italy. SUMMARY was more than 4 billion dollars in 2014 (USDA, 2015); in Canada, the import and export value of ornamental plant Fusarium oxysporum has recently been detected in com- products was about 418 and 312 million Canadian dollars, mercial nurseries in the Liguria region (northern Italy) on respectively, in 2013 (Statistical Overview of the Canadian new succulent plants belonging to the Cactaceae family Ornamental Industry, 2013); in Europe, the importing of (Astrophytum myriostigma, Cereus marginatus var. cristata, live plants and floriculture products reached more than C. peruvianus monstruosus and C. peruvianus florida) and 1,500 million euros in 2014 (European Commission, Ag- to the Euphorbiaceae family (Euphorbia mammillaris). The riculture and rural development, 2015). pathogen has been identified, for all the new hosts, from The ornamental industry in Italy is important in the morphological characteristics observed in vitro. The iden- agricultural sector because of its favourable climatic con- tifications have been confirmed by means of internal tran- ditions and specific economic situations that positively in- scribed spacer (ITS) analysis and/or by translation elonga- fluence the economic returns, with a production of about tion factor 1α (TEF) analysis. The aim of this work was 2,670 million euros in 2011 (Schimmenti et al., 2013). In to identify the forma specialis of the F. oxysporum isolates 2013, the production of ornamental and flowering plants obtained from new succulent plants. This has been investi- represented 5.4% of the total production in the agricul- gated by means of phylogenetic analysis based on the TEF tural sector in Italy (INEA, 2014). In 2010, a total of 4,271 gene and intergenic spacer (IGS), carried out on single- farms producing ornamental and flowering plants were spore isolates, together with pathogenicity assays. The located in the Liguria region, over an area of about 2,672 results of this research led to include four new succulent hectares (ISTAT, 2010). Various new genera and species hosts from Cactaceae as additional hosts to F. oxysporum are exploited in this region, because of their commercial f. sp. opuntiarum. This forma specialis has been identified importance. A particular fragment of the ornamental in- for the first time on a new host Euphorbia( mammillaris) dustry is that of succulent plants, which currently show a not belonging to the Cactaceae family. good market potential. The diversity of the crops and varieties, the effects of Keywords: ornamentals, succulent plants, soil-borne globalisation and of the intensive productions all lead pathogens, Fusarium wilt. to a multiplication of the number of potential pests and diseases that are able to infect new hosts. More than 120 different formae speciales of F. oxysporum have been de- scribed (Armstrong and Armstrong, 1981; O’Donnell and Cigelnik, 1999; Baayen et al., 2000; O’Donnell et al., 2009; Leslie, 2012). The detection and identification of formae speciales, which are classically based on pathogenicity as- INTRODUCTION says (Recorbet et al., 2003), are at present supported by molecular diagnostic tools (Lievens et al., 2012). Several The ornamental industry is economically important markers have been developed, on the basis of DNA se- throughout the world, and it also represents an interesting quences, in order to identify different formae speciales growth opportunity for developing countries. In industri- (Baayen et al., 2000; Groenewald et al., 2006). Genomic alized countries ornamental plants are purchased through- regions, such as the intergenic spacer region (IGS) or the out the year by a significant portion of the population, and translation elongation factor 1α (TEF), are useful but not moving the production of ornamental plants to develop- enough for correct identification (O’Donnell et al., 2009). ing countries would provide a remarkable source of in- Over the last few years, Fusarium rot or wilt symptoms come: in the USA, the wholesale value of floriculture crops have appeared on five new succulent hosts grown as pot- ted plants in commercial nurseries located in the Imperia Corresponding author: D. Bertetti Fax: + 39.011.6709307 province (Liguria region, northern Italy). The new hosts E-mail: [email protected] were succulent plants belonging to the Cactaceae family, 348 Fusarium oxysporum f. sp. opuntiarum on succulent plants Journal of Plant Pathology (2017), 99 (2), 347-354 that is, Astrophytum myriostigma (Garibaldi et al., 2015b), MATERIALS AND METHODS Cereus peruvianus monstruosus (Garibaldi et al., 2011), C. peruvianus florida (Garibaldi et al., 2015a) and C. mar- Fungal isolates. The F. oxysporum isolates were ob- ginatus var. cristata (Garibaldi et al., 2014) as well as to tained from diseased plants grown in nurseries located in the Euphorbiaceae family, that is, Euphorbia mammillaris Bordighera, Vallecrosia and Ventimiglia (Imperia prov- (Garibaldi et al., 2015c). Fusarium oxysporum has been ince, Liguria region) (Table 1). The isolates were obtained isolated and identified as the causal agent of the diseases by taking small pieces from the margin of symptomatic on all the host plants by means of morphological and mo- internal tissues and placing them on potato dextrose agar lecular methods. (PDA) and/or Komada Fusarium selective medium (Ko- The aim of this work was to investigate the forma specia- mada, 1975). To obtain pure isolates, colonies were sub- lis of the isolates of F. oxysporum obtained from succulent cultured on PDA. About 10 isolates were obtained from plants. each host; among these, two strains isolated from differ- ent plants were chosen for this study. Furthermore, two F. oxysporum f. sp. opuntiarum strains obtained from differ- ent collections were used as reference isolates: one strain isolated from Echinocactus grusonii (Polizzi and Vitale, 2004), gently provided by Prof. G.C. Polizzi (University of Table 1. The Fusarium oxysporum isolates used in this study. Catania, Italy), and the CBS 743.79 from the CBS-KNAW Strain* Host plant Place: city, Fungal Biodiversity Centre (The Netherlands). To ob- province, state tain the single-spore isolates used in this work (Table 1), DB13GIU05-22M Cereus marginatus Ventimiglia, a fungal suspension of each isolate was prepared in Po- Imperia, Italy tato Dextrose Broth (PDB), shaking cultures (90 rpm) at DB13GIU06-26M Cereus marginatus Ventimiglia, Imperia, Italy 25°C for 10 days. Then, each suspension was diluted to −8 DB210211-18M Cereus peruvianus monstruosus Bordighera, 1 × 10 CFU/ml. A drop from more diluted concentra- Imperia, Italy tions was spread on Komada selective medium. Single ger- DB220211-21M Cereus peruvianus monstruosus Bordighera, minated microconidia were selected by using an optical Imperia, Italy DB14OTT05-M1 Astrophytum myriostigma Vallecrosia, microscope. Imperia, Italy DB14OTT07-M1 Astrophytum myriostigma Vallecrosia, Pathogenicity assays. The isolates listed in Table 2 were Imperia, Italy DB14NOV08-M1 Cereus peruvianus florida Vallecrosia, artificially inoculated on Schlumbergera truncata plants (3 Imperia, Italy plants for each isolate), which are notoriously susceptible DB14NOV09-M1 Cereus peruvianus florida Vallecrosia, to F. oxysporum f. sp. opuntiarum (Lops et al., 2013). S. Imperia, Italy truncata healthy plants used for artificial inoculation were DB14OTT16-M1 Euphorbia mammillaris Vallecrosia, Imperia, Italy raised in a nursery and inoculated by wounding the stems DB14OTT17-M1 Euphorbia mammillaris Vallecrosia, (3 lesions/plant) with a sterilized needle contaminated Imperia, Italy with spores and mycelium taken from pure PDA cultures * In addition, two Fusarium oxysporum f. sp. opuntiarum strains were of the isolates (Talgø and Stensvand, 2013). Control plants used as reference isolates: Polizzi-31M (isolated from Echinocactus gru- were wounded with sterilized needles without any inocu- sonii, gently provided by Prof. Polizzi, University of Catania, Italy) and CBS 743.79 (isolated from Zygocactus truncatus and provided by the CBS- lum. All the plants were maintained under greenhouse KNAW Fungal Biodiversity Centre, The Netherlands). conditions, at mean daily temperatures ranging from 23 to 25°C and at mean daily RH ranging from 50 to 56%. Table 2. Pathogenicity test carried out on Schlumbergera truncata plants artificially inoculated with Fusarium oxysporum isolates obtained from succulent plants. Fusarium oxysporum tested isolates Susceptibility of Schlumbergera truncata Controls R* DB13GIU05-22M (from Cereus marginatus) HS DB13GIU06-26M (from Cereus marginatus) HS DB210211-18M (from Cereus peruvianus monstruosus) HS DB220211-21M (from Cereus peruvianus monstruosus) HS DB14OTT07-M1 (from Astrophytum myriostigma) HS DB14NOV09-M1 (from Cereus peruvianus florida) HS DB14OTT16-M1 (from Euphorbia mammillaris) HS Fusarium oxysporum f. sp. opuntiarum Polizzi-31M (from Echinocactus grusonii) HS Fusarium oxysporum f. sp. opuntiarum CBS 743.79 (from Zygocactus truncatus) HS *R = Resistant (disease index 0-5); PR = Partially Resistant (disease index 6-20); MS = Moderately Susceptible (disease index 21-50); S = Susceptible (disease
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