PERSISTENCE OF GLIOCEPHALOTRICHUM SPP. CAUSING FRUIT ROT OF RAMBUTAN (Nephelium lappaceum L.) IN PUERTO RICO Luz M.Serrato-Diaz 1, Elena I. Latoni-Brailowsky 2, Lydia I. Rivera-Vargas 2, Ricardo Goenaga 3 and Ronald D. French-Monar 1 ABSTRACT Characterize and identify Gliocephalotrichum spp. associated with fruit rot of rambutan in Puerto Rico based on morphology. Conduct pathogenicity tests on fruits of rambutan with isolates of Gliocephalotrichum. Characterize Gliocephalotrichum isolates associated with fruit rot of rambutan using molecular techniques. Rambutan is a native tropical fruit of South East Asia being the major producers: Thailand, Malaysia and Philippines. It is also produced in South and Central America (2,6). In United States rambutan’s production is concentrated in Hawaii and Puerto Rico. Hawaii has an estimated area of 100 acres planted with the highest rambutan yields in the world (2). In Puerto Rico, rambutan trees were introduced in 1927, but it was not until 1998 that commercials orchards were established in Mayaguez producing fruits for exportation mainly to U.S mainland. Worldwide, rambutan fruit rot is an important post-harvest disease and it has been associated with a complex of several pathogens. Six different Gliocephalotrichum species have been reported causing rambutan fruit rot and 30% post-harvest losses (3,4,7,8). In 2008, a pathogenic isolate of Gliocephalotrichum was obtained in a survey conducted at the Tropical Agriculture Research Station (TARS) (USDA- ARS), Mayaguez, Puerto Rico. Subsequential surveys were conducted in 2011 to determine if Gliocephalotrichum is a concurrent pathogen causing fruit rot through of the years. 1 Department of Plant Pathology and Microbiology, Texas AgriLife Extension Service-Texas A&M System, Amarillo, TX Department of Crops and Agroenvironmental Sciences, University of Puerto Rico-Mayaguez Campus, Mayaguez PR 00680, 3 USDA-ARS, Tropical Agriculture Research Station, Mayaguez PR Ellis, J.J., y C.W. Hesseltine A new genus of moniliales having penicilli subtenden by sterile hairs. Bulletin of the Torrey Botanical Club. 89 (1): FAO,2009.FAO Statistical Yearbook. Vol 4. last access November 14, Farungsang, U. S. Sangchote y N. Farungsang Appearance of quiescent fruit rot fungi on rambutan stored at 13°C and 25°C. ISHS Acta Horticulturae 321: Frontier in Tropical Fruit Research. Thailand. 4.Nishijima, K. A. y P. A. Follett First report of Lasmenia sp. and two species of Gliocephalotrichum on rambutan in Hawaii. Plant Disease. 86 (1): Rossman, A.Y., y G.J. Sammuels Leuconectria clusiae gen. nov. and its anamorph Gliocephalotrichum bulbilium with notes on Pseudonectria. Mycologia. 85(4): Salakpetch, S Rambutan production in Thailand, p In: N. Chomchalow and N. Sukhvibul (eds.). Acta Hort 665: Proceedings of the Second International Symposium on Lychee, Longan, Rambutan and other Sapindaceae Plants. ISHS, Leuven, Belgium. 7. Sangchote, S., U. Farungsang, y N. Farungsang Pre- and Postharvest infection of rambutan by pathogens and effects of post harvest treatments. Australian Center for International Agricultural Research. Cranberra. Pages pp. 8.Sivakumar, D., R.S.W. Wijeratnam, R.L.C. Wijesundera y M. Abeysekera Post-harvest diseases of rambutan (Nephelium lappaceum) in the western Province. Journal of the National Council of Sri Lanka. (25): White, T.J., T. Bruns, S. Lee y J. Taylor Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages In: PCR Protocols: a guide to methods and applications. M.A. Innis, D.H. Gelfand, J.J. Snisky and T.J White, eds. Academic Press, San Diego, CA. Worldwide, fruit rot of rambutan is an important problem that limits the storage, marketing and long-distance transportation of the fruit. A complex of pathogens has been reported to cause fruit rot of rambutan and significant post-harvest economic losses. During 2009 and 2011, rambutan fruit rot was observed on trees at the USDA-ARS Tropical Agricultural Station in Mayaguez, Puerto Rico. Infected fruit sections (1mm 2 ) were surface sterilized, rinsed with sterile deionized-distilled water and transferred to acidified potato dextrose agar (APDA). Using light and scanning electron microscopy, a total of 27 isolates of Gliocephalotrichum spp. were obtained and preliminarily identified morphologically into two species (Gsp1 and Gsp2). Pathogenicity tests were conducted on healthy superficially sterilized fruits that were inoculated with 5-mm mycelial disks of 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Fruits were kept in a humid chamber for eight days. Five days after inoculation (DAI), white mycelial growth for Gsp1 and yellowish mycelial growth for Gsp2 were observed on rambutan fruits. Eight DAI, fruit rot symptoms were observed on both Gsp1 and Gsp2 isolates. Fruits changed in color from red to brown, and, on average, mycelia of Gsp1 and Gsp2 covered 50 and 60% of the fruit, respectively. Conidiophores were observed on spintems (hair-likes-appendages on fruit surface). Both species were re-isolated from diseased plant tissue, thus fulfilling Koch’s postulates. To identify the species of the isolates obtained from the surveys on both years, PCR amplifications of the ITS rDNA region are being conducted. Gliocephalotrichum spp. have already been associated with rambutan fruit rot in Costa Rica, Hawaii, Sri Lanka and Thailand. INTRODUCTION OBJECTIVES MATERIALS AND METHODS Recollection of plant material In 2008 and 2011, fruits showing rot were collected at the TARS (USDA-ARS), Mayaguez, Puerto Rico. Samples were processed at the Plant Pathology Laboratory, Department of Crops and Agroenvironmental Sciences, UPR-Mayaguez Campus. Fruit rotting sections (1mm 2 ) were surface sterilized. Tissue was transferred to Petri dishes containing acidified potato dextrose agar (APDA). Fungal isolates were identified to genus using taxonomic keys (1,5). Isolation of Gliocephalotrichum spp. Molecular characterization of Gliocephalotrichum isolates Pathogenicity Tests Pathogenicity tests were conducted under laboratory conditions on five superficially sterilized fruits. Unwounded healthy tissues were inoculated with 5mm mycelial disks from each fungal isolate grown in APDA. Untreated controls were inoculated with APDA disks only. Koch’s postulates were fulfilled by re-isolation of inoculated fungi from diseased tissue. Experiment was repeated twice. Data was taken at 5 and 8 DAI. RESULTS A total of 28 isolates of Gliocephalotrichum spp. were obtained from survey conducted during 2008 and In 2008, an isolate of Gliocephalotrichum bulbilium J.J. Ellis & Hesselt. was identified using taxonomic keys (2,3,9) and amplification of the rDNA ITS region (Accesion No. GU299862). This isolate had a sequence identity of 95% with the strains of G. bulbilium listed in the GenBank sequences data and caused fruit rot of rambutan at 8 DAI (Fig. 1) REFERENCES Figure 1. Gliocephalotrichum bulbilium A. Conidiophore with conidiogenous penicilli, conidia and stipes. B. Bulbilloid aggregate. C. Mycelia covering 100% of the fruit at 8 DAI. DNA was extracted using DNAeasy Plant Kit (Qiagen, MD) from mycelium and it was amplified with primers ITS1 and ITS4 (9). Thermocycler conditions were: 94°for 4 min, followed by 35 cycles of 94° for 1 min, 55° for 30 sec, 72° for 1 min, and with a final extension at 72° for 4 min (9). PCR product was cleaned with QIAquick PCR purification Kit (Qiagen, MD). PCR product was sequence at the SGF UPR-Río Piedras and nucleotide sequence obtained was analyzed using Sequencher® (v. 4.9, Gene Codes Corporation, MI). Figure 3. Gliocephalotrichum sp. (Gsp2) A. Conidiophore with conidiogenous penicilli and stipes using light microscopy. B. Chlamydospore. C. Conidiophore using a stereomicroscope. D. Conidiophore and stipe using SEM. In 2011, 27 isolates of Gliocephalotrichum were identified, and distributed in two species (Gsp1 and Gsp2) (Fig 2 and 3). Five DAI, white mycelial growth was observed with Gsp1 and golden mycelial growth with Gsp2 on fruits of rambutan. Mycelia of Gsp1 and Gsp2 covered 50 and 60% of the fruit, respectively (Fig 4). Eight DAI, fruit rot symptoms were observed on both Gsp1 and Gsp2 isolates. A BC D E Figure 4. Pathogenicity test on fruits of rambutan with Gsp1 and Gsp2 isolates at 5DAI. A. Controls. B and C. Inoculated fruits with Gsp1 isolates. D and E. Inoculated fruits with Gsp2 CONCLUSIONS Both Gliocephalotrichum species (Gsp1 and Gsp2) were pathogenic to rambutan fruits causing fruit rot. Gsp2 was more pathogenic than Gsp1 with 60% and 50% of area covered, respectively. Five DAI, white mycelial growth was observed with Gsp1 and golden mycelial growth for Gsp2 on inoculated fruits. Eight DAI, both species produced conidiophores on spintems (hair-likes-appendages on fruit surface). For isolates of Gsp1, conidiophores had stipe extensions mostly directly subtending the conidiogenous penicilli with aggregates bulbilloid. For isolates of Gsp2, conidiophores had stipe extensions rising at some distance of the conidiogenous penicilli. Chlamydospores were unicellular, brown, smooth and thick-walled. Evidently, two species were found causing fruit rot in Puerto Rico. To confirm morphological parameters observed, current studies are being conducted to molecularly characterize isolates obtained in survey conducted during A BC 20 µm 50 µm Figure 2. Gliocephalotrichum sp. (Gsp1) A. Conidiophore with conidiogenous penicilli, conidia and stipes using light microscopy. B. Bulbilloid aggregates. C. Conidiophore and stipes using a stereomicroscope. D. Conidiophore and stipe using Scanning Electron microscopy (SEM). A B C D 25 µm 50 µm10 µm D B CA 20 µm 15 µm