Phylogeny and taxonomy of the genus Gliocephalotrichum.

Species in the genus Gliocephalotrichum (= Leuconectria) (Hypocreales, Nectriaceae) are soilborne fungi, associated with post-harvest fruit spoilage of several important tropical fruit crops. Contemporary taxonomic studies of these fungi have relied on morphology and DNA sequence comparisons of the internal transcribed spacer region of the nuclear rDNA (ITS) and the ß-tubulin gene regions. Employing DNA sequence data from four loci (ß-tubulin, histone H3, ITS, and translation elongation factor 1-alpha) and morphological comparisons, the taxonomic status of the genus Gliocephalotrichum was re-evaluated. As a result five species are newly described, namely G. humicola (Taiwan, soil), G. mexicanum (rambutan fruit from Mexico), G. nephelii (rambutan fruit from Guatemala), G. queenslandicum (Australia, endophytic isolations) and G. simmonsii (rambutan fruit from Guatemala). Although species of Gliocephalotrichum are generally not regarded as important plant pathogens, their ability to cause post-harvest fruit rot could have an impact on fruit export and storage.

First Report of Neofusicoccum mangiferae Causing Rachis Necrosis and Inflorescence Blight of Mango (Mangifera indica) in Puerto Rico.

Inflorescence blight is a major disease in mango production (2,3). During a disease survey of mango in Puerto Rico conducted from February to April in 2009, 20% of the inflorescences were affected with inflorescence blight showing rachis and flower necrosis. Symptoms were observed in 70% of samples at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz. Blighted inflorescence tissue (necrotic and the interface between necrotic and healthy tissue) from mango cultivars 'Haden' and 'Irwin' were disinfested with 70% ethanol, rinsed with sterile water and transferred to acidified potato dextrose agar (APDA). Isolations (40%) produced fungi in the Botryosphaeriaceae. Isolates 90LY, 94LY, and 89LY were purified and identified morphologically using taxonomic keys (1,4) and by DNA sequence analyses as Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips. On APDA, colonies were gray with aerial mycelia that turned dark gray with age. Pycnidia were globose to pyriform and dark brown to black. Conidia (n = 50) were hyaline, ovoid, one-celled, and averaged 13.2 × 6.3 µm in size. PCR amplifications of the internal transcribed spacer region of rDNA using ITS5-ITS4 primers, and fragments of both ß-tubulin and translation elongation factor 1-alpha (EF1-α) genes using Bt2a-Bt2b and EF1728F-EF1986R primers, respectively, were sequenced and analyzed using BLASTn query. Accession numbers of gene sequences submitted to GenBank were KF479465 to 67 for ITS region; KF479468 to 70 for ß-tubulin; and KF479471 to 73 for EF1-α. All sequences were 99 to 100% identical to reference isolate CMW7024 (4) of N. mangiferae (GenBank Accession Nos. AY615185, AY615172, and DQ093221). For each fungal isolate, pathogenicity tests were conducted on mango trees using six randomly selected, healthy mango inflorescences at full bloom using two trees per cultivar. Both needle-wounded and unwounded inflorescences were inoculated with 5-mm diameter mycelial disks from 8-day-old cultures grown on APDA. Inflorescences were incubated in clear plastic bags for 8 days under field conditions. Controls were treated with APDA disks only. Inflorescences on 'Irwin' turned brown with necrosis extending from the rachis to flowers. Mycelial growth and inflorescence blight was observed with lesions ranging from 2 to 5 cm in length. On 'Haden,' the rachis tissues turned brown and necrotic with lesions ranging from 1.5 to 2 cm long and without mycelial growth. N. mangiferae was re-isolated from all diseased inflorescences, and no symptoms developed on controls, which fulfilled Koch's postulates. The test was repeated once. N. mangiferae was associated with blossom blight in Australia and South Africa (2,3). This is the first report of N. mangiferae causing rachis necrosis and inflorescence blight on mango in Puerto Rico. N. mangiferae belongs to a complex of pathogens causing inflorescence blight and rachis necrosis and, therefore, effective management of this important disease complex must involve control of this pathogen. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) G. I. Johnson et al. Ann. Appl. Biol. 119:465, 1991. (3) J. H. Lonsdale and J. M. Kotzé. Acta Hortic. 341:345, 1993. (4) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Last retrieved 5 February 2014 from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm .

First Report of Lasiodiplodia theobromae Causing Inflorescence Blight and Fruit Rot of Longan (Dimocarpus longan L.) in Puerto Rico.

Dimocarpus longan L., commonly known as longan, is a tropical fruit tree of the Sapindaceae family. From 2008 to 2010, a disease survey for longan was conducted in March and October in Puerto Rico. Fruit rot and inflorescence blight (rotting of the rachis, rachilla, and flowers) were observed in fields of longan at the USDA-ARS Research Farm in Isabela, and two commercial orchards in Puerto Rico. Tissue sections (1 mm2) of diseased inflorescences and surface of the fruit were disinfested with 70% ethanol, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Three isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Lt) were isolated from symptomatic tissue and identified morpho-molecularly using a taxonomic key for the Botryosphaeriaceae and DNA sequence analysis (1). In APDA, colonies of Lt had initial greenish-gray aerial mycelia that turned dark brown with age. Pycnidia were dark brown to black. Immature conidia were sub-ovoid to ellipsoid, apex rounded, truncate at the base, thick-walled, hyaline, and one-celled, becoming dark brown, two-celled, and with irregular longitudinal striations when mature. Conidia (n = 50) for all the isolates averaged 26.9 µm long by 13 µm wide. For molecular identification, the ITS1-5.8S-ITS2 region and fragments of the ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and BLASTn searches done in GenBank. Accession numbers of gene sequences of Lt submitted to GenBank were KC964546, KC964547, and KC964548 for ITS region; KC964549, KC964550, and KC964551 for ß-tubulin; and KC964552, KC964553, and KC964554 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of Lt reported in GenBank (accessions AY640255, EU673110, and AY640258). Pathogenicity tests were conducted on six random healthy non-detached inflorescences of longan and six healthy detached fruits per isolate. Unwounded inflorescences and fruit were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Inflorescences were enclosed in plastic bags for 5 days under field conditions while fruits were kept in a humid chamber using plastic boxes for 5 days under laboratory conditions of 25°C and 12 h of fluorescent light. Untreated controls were inoculated with APDA disks only. The experiment was repeated once. Five days after inoculation, isolates of Lt caused inflorescence blight, fruit rot, and aril (flesh) rot. Inflorescences turned brown and flower mummification was observed on the inflorescences. The exocarp (peel) and endocarp (aril) turned dark brown and mycelial growth and pycnidia of Lt were observed on fruits. Untreated controls did not show any symptoms and no fungi were re-isolated from tissue. In diseased inflorescences and fruits, Lt was re-isolated from diseased tissue and identified using morphological and molecular parameters, thus fulfilling Koch's postulates. Lt has been reported to cause dieback, stem end rot, and fruit rot on a wide range of plants host (2,4). In longan, Lt has been reported causing fruit rot in Thailand (3). To our knowledge, this is the first time that Lt has been reported causing inflorescence blight in longan and the first report of Lt causing fruit rot in Puerto Rico. References: (1) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 26 November 2013. (2) B. Slippers et al. Mycologia 97:99, 2005. (3) P. Suwanakood et al. Asian J. Biol. Ed. 3:47, 2007. (4) A. F. Wright and P. F. Harmon. Plant Dis. 93:962, 2009.

First Report of Diaporthe pseudomangiferae Causing Inflorescence Rot, Rachis Canker, and Flower Abortion of Mango.

Although mango (Mangifera indica L.) is a very important tropical fruit crop, limited studies have been conducted on fungal pathogens affecting the inflorescences. During a disease survey conducted from 2008 to 2010, 50% of the inflorescences were affected with inflorescence rot, rachis canker, and flower abortion characterized by blackening of plant tissue with soft rot lesions and suken lesions on the rachis, respectively. Symptoms were observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz, Puerto Rico. Five diseased pieces of 350 inflorescences from cvs. Haden and Irwin were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Among several typical or common fungi, three isolates of Diaporthe pseudomangiferae (Dp) R.R. Gomes, C. Glienke & Crous were obtained from symptomatic tissue and identified morphologically using taxonomic keys and DNA sequence comparisons (1,2). On APDA, colonies of Dp initially had white-gray moderate aerial mycelia. Pycnidia were black and superficial on cultures with a central ostiole that exuded beige to light orange conidial droplets. Alpha conidia (n = 50) were aseptate, hyaline, smooth, fusiform, apex rounded and base truncate, averaged 7.34 µm long by 2.60 µm wide. Beta conidia (n = 50) were spindle-shaped, aseptate, hyaline and smooth, averaged 22.03 µm long by 1.53 µm wide. DNA analysis of the ITS1-5.8S-ITS2 region using primers ITS5 and ITS4, and fragments of both ß-tubulin and translation elongation factor 1 alpha (EF1-α) genes using primers T1 and Bt2b, and EF1-728F and EF1-986R, respectively, were sequenced and compared using BLASTn with sequences available in the GenBank. Accession numbers of gene sequences of Dp submitted to GenBank were KF616498 to KF616500 for ITS region, KF616501 to KF616503 for ß-tubulin, and KF616504 to KF616506 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS 388.89 of Dp in GenBank. For each fungal isolate, pathogenicity tests were conducted on six random healthy non-detached mango inflorescences for both cvs. Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. On cv. Haden, isolates of Dp caused rachis canker (sunken lesion on the rachis) at 8 days post inoculation (dpi). On cv. Irwin, isolates of Dp caused inflorescence rot. Initially, white mycelia was observed on inflorescences but eventually inflorescences turned brown and flower abortion was observed at 8 dpi. Untreated controls did not show any of the above symptoms and no fungi were re-isolated from tissue. From diseased inflorescences, Dp was re-isolated, thus fulfilling Koch's postulates. Diaporthe spp. have been associated with fruit rots, stem cankers, decay, and wilt on a wide range of plant hosts (3,4). Recently, Dp was associated with fruit peel of mango in Mexico and the Dominican Republic (2). To our knowledge, this is the first report of Dp causing inflorescence rot, rachis canker, and flower abortion in mango. References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. APS Press. St. Paul, MN, 1998. (2) R. R. Gomes et al. Persoonia. 31:1, 2013. (3) J. M. Santos et al. Persoonia 27:9, 2011. (4) S. M. Thompson et al. Persoonia 27:80, 2011.

First Report of Calonectria hongkongensis Causing Fruit Rot of Rambutan (Nephelium lappaceum).

Fruit rot of rambutan is a pre- and post-harvest disease problem of rambutan orchards. In 2011, fruit rot was observed at USDA-ARS orchards in Mayaguez, Puerto Rico. Infected fruit were collected and 1 mm2 tissue sections were surface disinfested with 70% ethanol followed by 0.5% sodium hypochlorite. Infected fruit were rinsed with sterile, deionized, double-distilled water and transferred to acidified potato dextrose agar (APDA). Plates were incubated at 25 ± 1°C for 6 days. Three isolates of Calonectria hongkongensis (Cah), CBS134083, CBS134084, and CBS134085, were identified morphologically using taxonomic keys (2,3). In APDA, colonies of Cah produced raw sienna to rust-colored aerial mycelial growth. Conidiophores of Cah had a penicillate arrangement of primary to quaternary branches of 2 to 6 phialides. Conidia (n = 50) were cylindrical, hyaline, 1-septate, rounded at both ends, and 44 to 52 µm × 3.5 to 4.5 µm. Conidiophores produced terminal and lateral stipe extensions with terminal sphaeropedunculate vesicles that were 8 to 12 µm wide. Subglobose to ovoid perithecia, 300 to 500 µm × 200 to 350 µm and orange to red-brown, were produced in groups of 3. Asci were clavate and contained 8 ascospores aggregated at the top of the ascus. Ascospores (n = 50) were hyaline, guttulate, fusoid with rounded ends, straight to curved, 1-septate with constriction at the septum, and 28 to 36 µm × 4 to 7 µm. For molecular identification, the ITS rDNA, fragments of ß-tubulin (BT), histone H3 (HIS3), and elongation factor (EF1-α) genes were amplified by PCR, sequenced, and compared using BLASTn with Calonectria spp. submitted to the NCBI GenBank. The sequences of Cah submitted to GenBank include accessions KC342208, KC342206, and KC342207 for ITS; KC342217, KC342215, and KC342216 for BT; KC342211, KC342209, and KC342210 for HIS3; and KC342214, KC342212, and KC342213 for EF1α. The sequences were >99% or identical with the ex-type specimen of Cah CBS 114828 for all genes used. Pathogenicity tests were conducted on 5 healthy superficially sterilized fruits per isolate. Both scalpel-wounded and unwounded fruit tissues were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C under 12 h of fluorescent light. The test was repeated once. Three days after inoculation (DAI), white mycelial growth was observed on the fruit. Five DAI, the fruit changed color from red to brown and yellowish mycelia colonized 50 to 62% of the fruit surface. Eight DAI, all the fruit turned brown, the mycelium growth covered the entire fruit, and conidiophores were produced on spinterns (hairlike appendages). Fruit rot of spinterns, exocarp (skin), endocarp (aril), and light brown discoloration were observed inside the fruit. Untreated controls showed no symptoms of fruit rot and no fungi were reisolated from tissue. Cah was reisolated from diseased tissue, fulfilling Koch's postulates. Calonectria spp. (or their Cylindrocladium asexual states) have been associated with lychee decline syndrome in North Vietnam (1). Both fruits belong to the Sapindaceae family. To our knowledge, this is the first report of Cah causing fruit rot of rambutan. References: (1) L. M. Coates et al. Diseases of Longan, Lychee and Rambutan. Pages 307-325 in: Diseases of Tropical Fruit Crops. R. C. Ploetz, ed. CABI Publishing, Cambridge, MA, 2003. (2) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. APS Press, St Paul, MN, 2002. (3) P. W. Crous, et al. Stud. Mycol. 50:415, 2004.

First Report of Lasiodiplodia theobromae Causing Inflorescence Blight of Mango.

Mango (Mangifera indica L.) is an important tropical fruit crop in Puerto Rico. During a disease survey from 2008 to 2010, inflorescence blight was observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz as a rotting of the rachis (main axis of the inflorescence), rachilla (lateral axis), and flowers. Diseased inflorescences from cultivars 'Haden' and 'Irwin' were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Two isolates of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. were isolated from symptomatic tissue and identified morphologically using a Botryosphaeriaceae taxonomic key (3). In APDA, colonies of L. theobromae had initial greenish gray aerial mycelia that turned dark brown with age. Pycnidia were uniloculate and dark brown to black in color. Conidiogenous cells were hyaline, cylindrical, and holoblastic. Immature conidia were subovoid to ellipsoid, apex rounded, truncate at the base, thick walled, hyaline and one-celled, becoming dark brown, two-celled with irregular longitudinal striations when mature. Conidia (n = 50) averaged 26.88 µm long by 12.98 µm wide. Genomic DNA was extracted from pure cultures using a Qiagen DNeasy Plant Mini Kit. PCR amplification of three genes was used to support morphological identification. DNA analysis of the ITS1-5.8S-ITS2 region, and fragments of both ß-tubulin and elongation factor 1 alpha (EF1-α) genes were sequenced and compared using BLASTN with sequences available in GenBank. Accession numbers of gene sequences of L. theobromae from Puerto Rico submitted to GenBank were: KC631659 and KC631660 for ITS region; KC631651 and KC631652 for ß-tubulin; and KC631655 and KC631656 for EF1α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS164.96 of L. theobromae reported in GenBank. Pathogenicity tests were conducted on six random healthy non-detached mango inflorescences from cultivars Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. For both cultivars, isolates of L. theobromae caused inflorescence (rachis, rachilla, and flowers) blight, 8 days after inoculation. Inflorescences turned brown and profuse mycelial growth was observed on the inflorescences. Untreated controls were disease-free and no fungi were reisolated from tissue. L. theobromae was reisolated from diseased inflorescences, fulfilling Koch's postulates. Fungi in the family Botryosphaeriaceae have been associated with stem-end rot, fruit rot, branch dieback, blossom blight, and cankers on mango (1,2,4). Worldwide, L. theobromae has only been reported causing dieback, stem end rot and fruit rot in mango (1,2). To our knowledge, this is the first report of L. theobromae causing inflorescence blight in mango. References: (1) N. I. Hui-Fang et al. Botanical Stud. 53:467, 2012. (2) A. M. Ismail et al. Australas. Plant Pathol. 41:649, 2012. (3) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 6 August 2013. (4) B. Slippers et al. Mycologia 97:99, 2005.

First Report of Neofusicoccum parvum Causing Rachis Necrosis of Mango (Mangifera indica) in Puerto Rico.

Mango is an important tropical fruit crop in Puerto Rico that has been grown in the island for centuries. One of the major disease issues in mango production is rotting of the rachis (main axis stem of the inflorescence). During a disease survey from 2008 to 2010, rachis and flower necrosis were observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz. Diseased inflorescences from cultivars Haden and Irwin were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile, deionized, double-distilled water, and transferred to acidified potato dextrose agar (APDA). Two isolates, 91LY and K15C, of Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. were purified and identified morphologically using taxonomic keys (1,4) and DNA sequence comparisons. In APDA, colonies of N. parvum were whitish grey with aerial mycelia turning dark gray with age. Pycnidia were uni- or multilocular and dark brown to black in color. Conidiogenous cells were hyaline and holoblastic. Conidia were hyaline, ellipsoid, smooth, and one-celled with sub-obtuse apex and truncate base. Conidia (n = 50) were 16.75 µm long by 5.5 µm wide. PCR amplification of three genes was used to support morphological identification. DNA analysis of ITS1-5.8S-ITS2 region, and fragments of both ß-tubulin and elongation factor 1-alpha (EF1-α) genes were sequenced and compared using BLASTn with other sequences of N. parvum submitted to the NCBI GenBank. Accession numbers of gene sequences of N. parvum submitted to GenBank were: KC631661 and KC631662 for ITS region; KC631653 and KC631654 for ß-tubulin; and KC631657 and KC631658 for EF1-α. For all genes used, sequences were 99 to 100% identical to ex-type specimen CMW9081 of N. parvum reported in GenBank. Pathogenicity tests were conducted on mango trees using six random healthy non-detached mango inflorescences for both Haden and Irwin cultivars and for both isolates. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA and kept in a humid chamber using plastic bags for 8 days under field temperature, light, and other environmental conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. For both cultivars, at 8 days after inoculation, isolates of N. parvum caused rachis necrosis ranging from 20 to 35 mm in rachis length. On cultivar Irwin, inflorescences turned brown and the necrosis was extended from the rachis to the flowers. On cultivar Haden, inflorescences turned brown and only rachis necrosis was observed. Untreated controls showed no symptoms and no fungi were reisolated from tissue. N. parvum was reisolated from diseased inflorescences, fulfilling Koch's postulates. Worldwide, N. parvum has been associated with stem-end rot, branch dieback, blossom blight, and cankers on mango (2,3). To our knowledge, this is the first report of N. parvum causing rachis necrosis on mango in Puerto Rico. References: (1) A. J. L. Phillips. Key to the various lineages in "Botryosphaeria" Version 01 2007. Retrieved from http://www.crem.fct.unl.pt/botryosphaeria_site/key.htm , 6 August 2013. (2) G. I. Johnson et al. Ann. Appl. Biol. 120:225, 1992. (3) B. Slippers et al. Mycologia 97:99, 2005. (4) P. W. Crous et al. Stud. Mycol. 55:235, 2006.

First Report of Gliocephalotrichum bulbilium and G. simplex Causing Fruit Rot of Rambutan in Puerto Rico.

Post-harvest disease losses of rambutan (Nephelium lappaceum L.) have been reported worldwide and several pathogens have been associated with fruit rot (3,4). In 2011, fruit rot of rambutan was observed on 11-year-old trees at the USDA-ARS Tropical Agriculture Research Station in Mayaguez, Puerto Rico. Infected fruit sections (1 mm2) were surface-sterilized, rinsed with sterile deionized-distilled water, and transferred to acidified potato dextrose agar (APDA). Gliocephalotrichum bulbilium J.J. Ellis & Hesseltine (Gb) and G. simplex (J.A. Meyer) B. Wiley & E. Simmons (Gs) were identified using a taxonomic key (1). In corn meal agar (CMA), five isolates of Gb were light yellow-to-light brown. Conidiophores had sterile stipe extensions ranging from 120 to 150 µm long and were produced contiguous to the erect conidiogenous penicilli. Conidia were unicellular, smooth, oblong to elliptical, and 5.5 to 7.5 µm long by 2.0 to 2.5 µm wide. Bulbilloid aggregates were observed and averaged 70 µm long. In CMA, five isolates of Gs were light brown-to-chestnut brown. Conidiophores had sterile stipe extensions 130 to 180 µm long that were produced approximately 15 to 30 µm away from the conidiogenous penicilli. Conidia were unicellular, smooth, cylindrical to elliptical, and with slightly curved ends ranging from 6.5 to 8.5 µm long by 2.0 to 2.5 µm wide. Chlamydospores were unicellular, brown, smooth and thick-walled, averaging 35 µm long. Pathogenicity tests were conducted on five detached fruits per isolate. Five isolates of each Gliocephalotrichum spp. were inoculated on fruits using 5-mm mycelial disks of 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Inoculated fruit was kept in a humid chamber for 8 days at 25°C under 12 hours of fluorescent light. Test was repeated once. Five days after inoculation (DAI), white mycelial growth for Gb and golden mycelial growth for Gs were observed on rambutan fruits. Eight DAI, fruit rot, and aril (flesh) rot symptoms were observed on fruits inoculated with isolates of Gb and Gs. Infected fruit changed in color from red to brown, and, on average, mycelia of Gb and Gs covered 50 and 60% of the fruit, respectively. Conidiophores were observed on spintems (hair-like appendages). Control fruit did not rot. Both species were reisolated from diseased plant tissue, thus fulfilling Koch's postulates. For molecular identification of these species of Gliocephalotrichum, the ITS1-5.8S-ITS2 region of the rDNA and a fragment of the ß-tubulin gene were amplified by PCR and aligned with other Gb and Gs sequences in NCBI GenBank for comparison. The sequences submitted to GenBank included Gs Accession Nos. JQ688045 and JQ688046 and Gb Accession Nos. JQ688044 and JQ68847 for the ITS sequences. For the ß-tubulin gene, Gs Accession Nos. JQ688049 and JQ688050 and Gb Accession Nos. JQ688048 and JQ688051. Both DNA regions had 99.9 to 100% sequence identity to other isolates of Gb and Gs reported in GenBank (1). Gliocephalotrichum spp. have been associated with rambutan fruit rot in Hawaii, Sri Lanka and Thailand (2,4). To our knowledge, this is the first report of G. bulbilium and G. simplex causing fruit rot of rambutan in Puerto Rico. References: (1) C. Decock et al. Mycologia 98:488, 2006. (2) K. A. Nishijima and P. A. Follett. Plant Dis. 86:71, 2002. (3) L. M. Serrato et al. Phytopathology 100:S176, 2010. (4) D. Sivakumar et al. J. Natn. Sci. Coun. Sri Lanka 25:225, 1997.

First Report of Goss's Bacterial Wilt and Leaf Blight (Clavibacter michiganensis subsp. nebraskensis) of Corn in Texas.

In September 2009, the University of Nebraska-Lincoln Plant and Pest Diagnostic Clinic received leaf samples of hybrid corn (Zea mays L.) displaying long, necrotic lesions with wavy margins. The lesions had discontinuous water-soaked spots that are indicative of Goss's bacterial wilt and leaf blight. The symptomatic leaves were submitted from Dallam County, located in the Texas Panhandle (northwest Texas). According to the USDA Farm Service Agency and the National Agricultural Statistics Service, in 2009 Dallam County had 54,025 ha planted to corn. This is approximately 19% of the total corn planted in the 26 counties in the Texas Panhandle and 6% of the total corn planted in the state of Texas. Extracts from the infected leaf tissue tested positive for Clavibacter michiganensis subsp. nebraskensis with a commercially available ELISA test (Neogen Inc., Scotland, UK). Isolation from the infected tissue onto CNS selective media (1) resulted in round, dark orange, mucoid colonies that tested gram positive with the Gram-stain test. BLAST nucleotide sequence alignments of the amplified 500-bp 16S rRNA region of the suspect culture's genome (2) revealed a 96% similarity for C. michiganensis subsp. nebraskensis (NCBI BLAST Accession No. U09381.1). To fulfill Koch's postulates, three sweet corn plants (Golden Cross Bantam) at growth stage V3 to V4 were inoculated in the greenhouse with a suspension of approximately 1 × 109 CFU/ml from suspect cultures grown on CNS for 5 days. Wounds approximately 6.5 cm long were created with sterile scissors on the fifth leaf from the bottom running parallel to the veins on either side of the midrib at the leaf apex. The leaf apex was dipped into 150 ml of the inoculum suspension for 5 s. Approximately 6 days after inoculation, discontinuous, water-soaked spots consistent with the symptoms on the original symptomatic leaves appeared on all the inoculated leaves near the site of infection. Colonies consistent with C. michiganensis subsp. nebraskensis (dark orange, mucoid) were reisolated onto CNS, completing Koch's postulates. To our knowledge, this is the first report of Goss's bacterial wilt and leaf blight on corn in Texas and because it is a residue-borne pathogen, the probability of it becoming a resident disease is relatively high. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) X. Li and S. H. De Boer. 1995. Phytopathology 85:837, 1995.

First Report of a Lasmenia sp. Causing Rachis Necrosis, Flower Abortion, Fruit Rot, and Leaf Spots on Rambutan in Puerto Rico.

Rambutan (Nephelium lappaceum L.) is a tropical fruit tree that has increased in importance for fruit growers in Puerto Rico. In 2008 and 2009, fruit rot and lesions on leaves and inflorescences were observed. A total of 276 diseased samples were collected from commercial orchards, orchards at the University of Puerto Rico, and the USDA-ARS in Mayaguez. Plant tissue was disinfested and plated on acidified potato dextrose agar (APDA). Besides other typical fungi associated with these tissue samples (2,3), 130 unknown isolates were identified as a Lasmenia sp. at the Fungal Biodiversity Centre (CBS), the Netherlands and the University of Puerto Rico using taxonomic keys (1,4). Sequencing of the rDNA with primers ITS 1 and ITS 4 and Lr5 and LR0R corresponding to the (internal transcribed spacer) ITS1-5.8S-ITS2 region and the partial region of the large ribosomal subunit (LSU), respectively, was completed. Five isolates (CBS 124122 to 124126) were deposited at the CBS. In APDA, colonies of a Lasmenia sp. were cream-colored with dark brown concentric rings and immersed, hyaline, branched, and septate mycelium. Acervuli were produced on APDA and plant tissue that was sampled from field and clean tissue that was inoculated with a Lasmenia sp. Conidia were 10 to 12 × 4 to 5 µm, light brown, thick walled, obclavate, aseptate, and the apex was obtuse with a scar at the base. Conidiophores were hyaline, septate, cylindrical, and sparingly branched. The conidiogenous cells were hyaline, cylindrical, and holoblastic. Pathogenicity tests were done on 12 healthy, superficially sterilized fruits under laboratory conditions, on four random leaves in each of six 6-month-old rambutan seedlings under greenhouse conditions, and on four flowers in six random inflorescences for each of six mature trees from an orchard. Tests were repeated. Either wounded or unwounded tissues were inoculated with a conidial suspension (2 to 4.5 × 106 conidia/ml) and 5-mm mycelial disks from each fungal isolate grown in APDA. After 5 days, a Lasmenia sp. produced necrotic spots on leaves, rachis necrosis and flower abortion, fruit rot, and water-soaked lesions on the fruit surface that spread to cause an aril (flesh) rot. Acervuli were produced on fruit spintems (hair-like appendages). Koch's postulates were fulfilled by reisolation of inoculated fungi from diseased tissue. A complete sequence for the ITS region for four isolates of a Lasmenia sp. was submitted to NCBI GenBank (Accession Nos. GU797405, GU797406, GU797407, and JF838336). Complete sequences of the LSU region for all five isolates were submitted to GenBank (Accession Nos. JF838337, JF838338, JF838339, JF838340, and JF838341). For both types of sequences, the identity was 100% between isolates. Although there is no DNA sequence data for the genus Lasmenia, a BLASTN search indicates a closer affinity to the Cryphonectriaceae (Diaporthales) (1). A Lasmenia sp. has been reported from Hawaii as causing fruit rot in rambutan (2). To our knowledge, this is the first report of a Lasmenia sp. causing rachis necrosis and flower abortion worldwide, and the first report of fruit rot and necrotic spots on leaves of rambutan in Puerto Rico. References: (1) M. N. Kamat et al. Rev. Mycol. 38:19, 1973. (2) K. A. Nishijima and P. A. Follett. Plant Dis. 86:71, 2002. (3) L. M. Serrato et al. Phytopathology (Abstr.) 100(suppl):S176, 2010. (4) B. C. Sutton. The Coelomycetes: Fungi Imperfecti with Pycnidia Acervuli and Stromata. CMI. Kew, Surrey, England, 1980.

First Report of "Candidatus Liberibacter solanacearum" on Field Tomatoes in the United States.

In August 2008, 30% of tomato (Solanum lycopersicum) plants in plots in Lubbock County, Texas showed yellowing, lateral stem dieback, upward leaf curling, enlargement of stems, adventitious roots, and swollen nodes. Yellowing in leaves was similar to that seen with zebra chip disease (ZC) of potato that was confirmed in a potato field 112 km away in July 2008 and was associated with a 'Candidatus Liberibacter' species (1), similar to findings earlier in 2008 in New Zealand and California (2,3). Tissue from four symptomatic plants of cv. Spitfire and two of cv. Celebrity were collected and DNA was extracted from midribs and petioles with a FastDNA Spin Kit (Qbiogene, Inc., Carlsbad, CA,). PCR amplification was done with 16S rRNA gene primers OA2 and OI2c, which are specific for "Ca. Liberibacter solanacearum" from potato and tomato and amplify a 1.1-kb fragment of the 16S rRNA gene of this new species (1,3). Amplicons of 1.1 kb were obtained from all samples and these were sequenced in both orientations (McLab, San Francisco, CA). Sequences of the 16S rRNA gene were identical for both Spitfire and Celebrity and were submitted to the NCBI as GenBank Accession Nos. FJ939136 and FJ939137, respectively. On the basis of a BLAST search, sequence alignments revealed 99.9% identity with a new species of 'Ca. Liberibacter' from potato (EU884128 and EU884129) in Texas (1); 99.7% identity with the new species "Ca. Liberibacter solanacearum" described from potato and tomato (3) in New Zealand (EU849020 and EU834130, respectively) and from the potato psyllid Bactericera cockerelli in California (2) (EU812559, EU812556); 97% identity with 'Ca L. asiaticus' from citrus in Malaysia (EU224393) and 94% identity with both 'Ca. L. africanus' and 'Ca. L. americanus' from citrus (EU921620 and AY742824, respectively). A neighbor-joining cladogram constructed using the 16S rRNA gene fragments delineated four clusters corresponding to each species, and these sequences clustered with "Ca. L. solanacearum". A second PCR analysis was conducted with the CL514F/CL514R primer pair, which amplifies a sequence from the rplJ and rplL ribosomal protein genes of "Ca. L. solanacearum". The resulting 669-bp products were 100% identical to a sequence reported from tomato in Mexico (FJ498807). This sequence was submitted to NCBI (GU169328). ZC, a disease causing losses to the potato industry, is associated with a 'Candidatus Liberibacter' species (1-3) and was reported in Central America and Mexico in the 1990s, in Texas in 2000, and more recently in other states in the United States (4). In 2008, a "Ca. Liberibacter solanacearum" was detected on Capsicum annuum, S. betaceum, and Physalis peruviana in New Zealand (3). Several studies have shown that the potato psyllid, B. cockerelli, is a potential vector for this pathogen (2,4). To our knowledge, this is the first report of "Ca. Liberibacter solanacearum" in field tomatoes showing ZC-like foliar disease symptoms in the United States. References: (1). J. A. Abad et al. Plant Dis. 93:108, 2009 (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) G. A. Secor et al. Plant Dis. 93:574, 2009.

First Report of the Detection of 'Candidatus Liberibacter' Species in Zebra Chip Disease-Infected Potato Plants in the United States.

Zebra chip (ZC), an emerging disease causing economic losses to the potato chip industry, has been reported since the early 1990s in Central America and Mexico and in Texas during 2000 (4). ZC was subsequently found in Nebraska, Colorado, New Mexico, Arizona, Nevada, California, and Kansas (3). Severe losses to potato crops were reported in the last few years in Mexico, Guatemala, and Texas (4). Foliar symptoms include purple top, shortened internodes, small leaves, enlargement of the stems, swollen axillary buds, and aerial tubers. Chips made from infected tubers exhibit dark stripes that become markedly more visible upon frying, and hence, are unacceptable to manufacturers. Infected tubers may or may not produce plants when planted. The causal agent of ZC is not known and has been the subject of increased investigation. The pathogen is believed to be transmitted by the potato psyllid, Bactericera cockerelli, and the association of the vector with the disease is well documented (3). Following the report of a potential new liberibacter species in solanaceous crops in New Zealand, we sought to identify this liberibacter species in plants with symptoms of the ZC disease. Six potato plants (cv. Russet Norkota) exhibiting typical ZC symptoms were collected in Olton, TX in June of 2008. DNA was extracted from roots, stems, midribs, and petioles of the infected plants using a FastDNA Spin Kit and the FastPrep Instrument (Qbiogene, Inc., Carlsbad, CA). Negative controls from known healthy potato plants were included. PCR amplification was carried out with 'Candidatus L. asiaticus' omp primers (1), 16S rDNA primers specific for 'Ca. L. asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' (1), and 16S rDNA primers OA2 (GenBank Accession No. EU834130) and OI2c (2). Amplicons from 12 samples were directly sequenced in both orientations (McLab, San Francisco CA). PCR amplifications using species-specific primers for the citrus huanglongbing liberibacter were negative. However, 1.1- and 1.8-kb amplicons were obtained with the OA2/OI2C and omp primers, respectively. The sequences for the rDNA were submitted to NCBI GenBank (Accession Nos. EU884128 and EU884129). BLASTN alignment of the 16S rDNA sequences obtained with primers OA2 and OI2c revealed 99.7% identity with a new species of 'Ca. Liberibacter' identified in New Zealand affecting potato (GenBank Accession No. EU849020) and tomato (GenBank Accession No. EU834130), 97% identity with 'Ca. L. asiaticus', and 94% with 'Ca. L. africanus' and 'Ca. L. americanus'. The neighbor-joining phylogenetic tree constructed using the 16S rDNA fragments delineated four clusters corresponding to each of the liberibacter species. These results confirm that 'Ca. Liberibacter' spp. DNA sequences were obtained from potatoes showing ZC-like symptoms, suggesting that a new species of this genus may be involved in causing ZC disease. To our knowledge, this is the first report of the detection of 'Ca. Liberibacter' spp. in potatoes showing ZC disease in the United States. References: (1) C. Bastianel et al. Appl. Environ. Microbiol. 71:6473, 2005. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) G. A. Secor and V. V. Rivera-Varas. Rev. Latinoamericana de la Papa (suppl.)1:1, 2004.