<I>Neofusicoccum Luteum</I> As a Pathogen on Tejocote (<I

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 4-2013 Neofusicoccum luteum as a Pathogen on Tejocote (Crataegus mexicana) Anthony O. Adesemoye University of California, Riverside and Adekunle Ajasin University, [email protected] Joey S. Mayorquin University of California, Riverside, [email protected] Akif Eskalen University of California, Riverside, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/plantpathpapers Part of the Other Plant Sciences Commons, Plant Biology Commons, and the Plant Pathology Commons Adesemoye, Anthony O.; Mayorquin, Joey S.; and Eskalen, Akif, "Neofusicoccum luteum as a Pathogen on Tejocote (Crataegus mexicana)" (2013). Papers in Plant Pathology. 559. http://digitalcommons.unl.edu/plantpathpapers/559 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Phytopathologia Mediterranea (2013) 52, 1, 123−129 NEW OR UNUSUAL DISEASE REPORTS Neofusicoccum luteum as a pathogen on Tejocote (Crataegus mexicana) 1, 2 1 1 ANTHONY O. ADESEMOYE , JOEY S. MAYORQUIN and AKIF ESKALEN 1 Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA 2 Current Address: Department of Microbiology, Adekunle Ajasin University, P.M.B. 001, Akungba-Akoko, Ondo State, Nigeria Summary. Tejocote (Crataegus mexicana), a small pome crab-apple-like fruit, is becoming economically important in California with increasing production, so consideration of diseases that hinder the yield is important. Diseased trees of tejocote were observed in four orchards of Riverside and San Diego Counties of California. Ten symptomatic/asymptomatic samples were studied from each of the orchards. Five most frequently isolated fungi were identified on the basis of morphological characters and sequence data of the internal transcribed spacer ITS1-5.8S- ITS2 and partial β-tubulin gene. Three isolates were identified as Neofusicoccum luteum and two as Phomopsis sp. Pathogenicity tests were conducted by inoculating detached shoots of healthy tejocote trees. Significant lesions were observed on all shoots inoculated with the three N. luteum isolates (designated UCR1190, UCR1191, and UCR1192), but not on the shoots inoculated with other isolates or the non-inoculated controls. Results indicated that all three N. luteum isolates are aggressive pathogens on tejocote. This pathosystem should be further studied with a goal of designing appropriate disease management strategies. Key words: Botryosphaeriaceae; branch canker; dieback; Mexican hawthorn. Introduction sionally red to dark purple. Fruits may be eaten raw, cooked, or used as raw material in the processing Tejocote, also known as Mexican hawthorn (Cra- of concentrated pulp, jam, jellies, and marmalades taegus mexicana, Family Rosaceae), (www.plants. (Vivar-Vera et al., 2007). Tejocote production in Cali- usda.gov; Phipps et al., 1990) was recorded as intro- fornia is becoming economically important; thereby duced to California in 1868 (Riedel, 1957), but was generating attention about the possible pathogens not cultivated for fruit production. In the 1990s, the that are hindering its yield. United States Department of Agriculture Smuggling, Many fungal genera have been associated with Interdiction and Trade Compliance Program confis- dieback, and trunk and branch cankers in many cated many tejocote fruits at the point of entry dur- woody hosts, including Phomopsis sp. and members ing frequent attempts to get them into the United of the Botryosphaeriaceae. Specifically, Neofusicoc- States from overseas (www.ers.usda.gov). The fruits cum luteum has been reported as a pathogen associ- of C. mexicana are each small (1–3 cm in diameter), ated with dieback and canker in many woody hosts, pome, and crab-apple-like, with three to five brown including kiwifruit (Pennycook and Samuels, 1985), seeds and a hard skin covering the fleshy pulp that grapevine (Philips et al., 2002), avocado, (McDonald becomes soft when ripe. Fruit ripens from October et al., 2009), olives (Sergeeva et al., 2009), and cit- to December in California when the colour is mostly rus (Adesemoye et al., 2011). Usually, openings and greenish-yellow with russet or black spots or occa- wounds in host plants make invasion easier for spe- cies in the Botryosphaeriaceae, including N. luteum, and specifically, pruning wounds are important Corresponding author: A. Eskalen Fax: +1 951 827 4294 points of entry (Rolshausen et al., 2010; Amponsah E-mail: [email protected] et al. 2012). www.fupress.com/pm ISSN (print): 0031-9465 123 © Firenze University Press ISSN (online): 1593-2095 A.O. Adesemoye et al. In 2010, canker and dieback (Figure 1A) of tejo- Preliminary identification of fungal isolates was cote trees were observed in Riverside and San Diego based on morphological characters (Alexopoulos Counties of California. Symptoms included grayish et al., 1996; Farr et al., 2002; Luque et al., 2005). Iso- to brown discoloration on the rind, bark cracking lates suspected to belong to the Botryosphaeriaceae (Figure 1B), and when a cut was made through the were grown on oatmeal agar, slightly modified from tree, canker was seen covering xylem advancing re- Gooding and Lucas (1959) or 2% water agar on which gions (Figure 1C). The objective of the present study autoclaved pine needles were placed and incubated was to identify the pathogens associated with branch at 25°C under a UV light with 12 h photoperiod for canker and dieback of tejocote in the two counties. 2–4 weeks (McDonald and Eskalen, 2011) until fertile pycnidia were formed. Digital images of 20 conidia Materials and methods per isolate were taken with a Leica DFC420 camera mounted on a compound microscope (Olympus Sample collection, isolation, and morphology of BX40). The length and width of the conidia were de- fungal isolates termined using the SPOT Imaging Software v4.7.0.35 In 2010, symptomatic branches of dying tejocote (Diagnostic Instruments, Inc., MI, USA). Conidial trees, most of which showed severe canker and die- characteristics were compared with those in previ- back, were sampled from four orchards in Riverside ous reports (Phillips et al., 2002; Luque et al., 2005; and San Diego Counties, California. Ten branch sam- Úrbez-Torres et al., 2006). ples were collected from each orchard and transport- ed in a cooler to the laboratory at the University of DNA extraction, PCR amplification, and sequencing California, Riverside (UCR). Samples were washed with deionized water and cankers were surface- Total genomic DNA was extracted from fungal disinfested with 95% ethanol and flamed. Pieces of isolates by shaking in FastPrep24 (MP Biomedicals symptomatic tissues, 1–2 mm2 in size, were plated LLC, Solon, OH, USA) at a speed of 4.0 for 20 s, and onto nutrient agar (NA) and incubated for about 36 h using the methods of Cenis (1992). Polymerase chain at 27°C to isolate bacteria. Similar tissues were plat- reaction (PCR) was performed in a MyCycler (Bio- ed onto potato dextrose agar amended with 0.01% Rad Laboratories, Inc., Hercules, CA, USA) using tetracycline (PDA-tet) and incubated in the dark for primers Bt2a/2b for β-tubulin (BT) and/or ITS4/5 4 d at 25°C to isolate fungi. Pure cultures of fungal for 18S rRNA, internal transcribed spacer ITS1-5.8S- isolates were obtained by transferring hyphal tips to ITS2, and 28S rRNA regions (White et al., 1990; Farr et fresh PDA-tet plates while single colonies of bacte- al. 2002; Slippers et al., 2004). Each PCR reaction con- rial isolates were streaked on fresh NA. tained 12.5 μL of GoTaq Green Master Mix (Prome- Figure 1. (a) A tejocote tree showing dieback, (b) canker on the trunk, and (c) cross section of a branch canker. 124 Phytopathologia Mediterranea Neofusicoccum luteum on Tejocote ga, Madison, WI), 9.3 μL of PCR-grade water, 0.6 μL old detached shoots using a 3 mm diam. cork borer, of 10 μM of each primer, and 2 μL of DNA template. and the freshly wounded surfaces were inoculated The reaction protocol included an initial preheat at with 3 mm diam. mycelial plugs of 5 d old cultures 94°C for 2 min, followed by 35 cycles of denatura- of each isolate growing on PDA-tet (Adesemoye and tion at 94°C for 15 s, annealing at 58°C for 15 s, and Eskalen, 2011). Inoculated wounds and shoot ends extension at 72°C for 45 s, and a final extension at were covered with petroleum jelly and wrapped with 72°C for 5 min. Parafilm to prevent desiccation. Control shoots were The PCR products were verified in 1% agarose inoculated with sterile agar plugs and incubated sim- gel and the gels were documented with Dark Reader ilarly. There were ten replicate shoots for each isolate DR88X transilluminator (Clare Chemical Research, and the control treatment. Shoots were incubated at Dolores, CO, USA). The PCR products were purified 25°C in moist chambers for 4 weeks after which time using Isopure PCR purification kit (catalog # CM- lesion lengths were recorded. The experiment was re- 0100-100, Denville Scientific Inc, Metuchen, NJ). The peated. Re-isolation and identification of fungi from quality of the PCR products was estimated with a the inoculated and control shoots were carried out NanoDrop Spectrophotometer ND-1000 (Thermal using similar methods as described above. Scientific, Wilmington, DE). Sequencing was done at the Institute for Integrative Genome Biology of Data analyses the University of California, Riverside. Sequences were edited and aligned with Sequencher software Lesion lengths were analyzed using GLM proce- 4.6 (Gene Codes, Corporation, Ann Arbor, MI, USA). dure of Statistical Analysis System 9.3 (SAS Institute, The sequences have been deposited in GenBank un- Cary, NC), and Fisher’s protected LSD was used to der Accession numbers JF921867 to JF921871 for ITS separate means with P=0.05.

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