Postharvest Anthracnose of Persimmon Fruit Caused by Colletotrichum gloeosporioides First Reported in Spain.

Commercial production area and yield of sweet persimmon (Diospyros kaki L.) in Spain has doubled in the last 10 years to more than 5,000 ha and 50,000 tons, respectively, mainly because of the high quality and consumer demand for the Valencian autochthonous cultivar 'Rojo Brillante' in European markets. In a recent survey of decay on 'Rojo Brillante' persimmons stored in commercial packinghouses, fruit were found with disease symptoms of firm brown to dark brown round spots scattered on the fruit cheeks. Isolation of the potential causal agent (isolate IVIA QCV-2) was performed by disinfecting the surface of symptomatic fruit with alcohol, aseptically cutting pieces of infected peel tissue, and then plating them onto potato dextrose agar (PDA). The fungus grew fast, covering the entire plate surface (9 mm diameter) after 7 to 10 days of incubation at 25°C with cottony grayish mycelium that darkened with time. Masses of salmon-colored conidia were apparent in the center of some colonies. Conidia were one-celled, hyaline, aseptate, ovoid to oblong with rounded or obtuse ends, and 11.5 to 15.5 × 3.0 to 6.5 µm (n = 50). The identification of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. [synonym: Vermicularia gloeosporioides Penz.; teleomorph: Glomerella cingulata (Stoneman) Spauld. & H. Schrenk] was performed at the Instituto Valenciano de Microbiología (IVAMI, Bétera, Valencia, Spain) by macro and micro morphological observations and confirmed with the amplification and subsequent sequencing of the ribosomal DNA regions 5.8S-ITS2-28S, using the primers ITS3 and ITS4 (4). A representative nucleotide sequence was deposited in GenBank (Accession No. KC113600) and a BLAST search showed 99% identity with the strain C1254.3 of C. gloeosporioides (JX010153) (3). To fulfill Koch's postulates, selected healthy 'Rojo Brillante' persimmons were surface sterilized by dipping them for 2 min in a 0.5% sodium hypochlorite aqueous solution and thoroughly rinsing with fresh water. Mycelial plugs (5-mm diameter) from the edge of 7-day old colonies of isolate IVIA QCV-2 grown on PDA at 25°C were aseptically transferred to skin wounds (one plug per fruit). Wounded but not inoculated fruit were used as controls. Persimmons were placed in three humid chambers that each contained four fruit and incubated at 20°C for up to 21 days. The experiment was repeated twice. While inoculated persimmons developed anthracnose disease in all cases and C. gloeosporioides was consistently reisolated from these fruit, no decay was observed on control fruit. To our knowledge, this is the first report of C. gloeosporioides causing postharvest persimmon fruit rot in Spain. Persimmon anthracnose caused by this pathogen is well known in Asian countries such as China and Korea (1). This disease was also reported in Brazil (2). References: (1) J. H. Lee et al. Plant Pathol. J. 20:247, 2004. (2) M. A. S. Mendes et al. Fungos em Plants no Brasil. Embrapa-SPI/Embrapa-Cenargen, Brasilia, Brazil, 1998. (3) B. S. Weir et al. Stud. Mycol. 73:115, 2012. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press Inc., San Diego, CA, 1990.

First Report of Alternaria alternata Causing Postharvest Black Spot of Fresh Date Palm Fruit in Spain.

Commercial production of date palm fruit (Phoenix dactylifera L.) for fresh consumption has increased in the grove of Elx (Alacant Province, southeast Spain) after the successful development of tissue culture technologies and induced ripening and cold storage protocols. In a survey of losses after harvest, disease symptoms consisting of superficial, small, and firm black spots irregularly distributed throughout the fruit skin were observed in commercially handled and cold-stored fruit. At room temperature, superficial lesions expanded and produced dark mycelium. The potential causal agent was transferred to potato dextrose agar (PDA), incubated at 25°C in darkness, and subcultured on PDA. The identification was performed at the Spanish Type Culture Collection (CECT, University of Valencia, Spain) using colony morphology on PDA and malt extract agar at 26 or 37°C. At 26°C, the fungus rapidly produced cottony white mycelium that turned olivaceous and dark brown to black. Conidiophores were simple, straight or bent, with plain walls. Conidia were brown, obpyriform to ellipsoid (average 22 to 39 × 8 to 15 µm; n = 50), with both transversal and longitudinal septa, often observed in branched chains with more than 5 conidia. Growth occurred at 37°C. The identification of Alternaria alternata (Fr.:Fr.) Keissler was confirmed by the amplification and subsequent sequencing with the primers NL1 and NL4 of the region D1/D2 in the 5' end of the 28S rRNA gene of the isolate IVIA DAA-4 (GenBank Accession No. JX987100). A BLAST search showed 100% identity with A. alternata strain DAOM 216376 (JN938894). Selected healthy 'Medjool' dates were surface disinfected by dipping them for 2 min in a 0.5% sodium hypochlorite solution and thoroughly rinsed with fresh water. To fulfill Koch's postulates, 20 µl of a spore suspension at 1 × 105 spores per ml prepared from 7-day-old colonies grown on PDA were placed in fresh skin wounds made in disinfected fruit using a sterile stainless steel rod with a probe tip 1 mm wide and 2 mm in length (one wound per fruit; three humid chambers with nine fruits each). Wounded but not inoculated fruit were used as controls (one humid chamber with nine fruit). While disease symptoms were observed on all fruit inoculated with A. alternata (average black spots of 3, 6, and 12 mm after 4, 7, and 10 days of incubation at 20°C), no decay was observed on any of the control fruit. Reisolation of the fungus was performed from 10 infected dates and it was positive in all cases. A. alternata has been reported to cause date palm fruit disease in Israel (1) and Egypt (2), whereas Alternaria spp. have been cited in California (3) and Iran (4). To our knowledge, this is the first report of A. alternata causing date palm fruit rot in Spain. References: (1) R. Barkai-Golan et al. Hassadeh 69:1446, 1989. (2) H. M. El-Deeb et al. Acta Hort. 736:421, 2007. (3) H. S. Fawcett and L. J. Klotz. University of California Bulletin 522, 1932. (4) F. Karampourland and H. Pejman. Acta Hort. 736:431, 2007.

First Report of Diplodia seriata Causing Loquat Fruit Rot in Spain.

Spain is the second largest loquat (Eriobotrya japonica Lindl.) producer in the world, with about 40,000 t per year. 'Algerie' is the main cultivar planted in Alicante province (SE of Spain; Lat. 38.40° N, Long. 0.08° W), where more than 80% of Spanish commercial loquat plantations are located. In a survey of fruit losses at harvest, irregular brownish superficial dry spots (5 to 15 mm) located mainly near the stem end were observed on fruits from different orchards. After incubation at 20°C for 14 days, the spots on fruit expanded rapidly and turned to dark brown or black, producing black, unilocular, ostiolate, and thick-walled pycnidia. Isolation was performed by disinfecting the surface of symptomatic fruits with alcohol and aseptically cutting pieces of infected peel tissue and plating them in potato dextrose agar (PDA) dishes. The potential causal agent (isolate IVIA GCA-5) was identified in the Spanish Type Culture Collection (CECT, University of Valencia, Valencia, Spain). The fungus grew rapidly on both PDA and malt extract agar (MEA) at 26°C, covering the entire plate surface with dark gray mycelium within 4 days. The plate reverse was dark gray to black. The conidia were brown and aseptate, with the apex broadly rounded and the base rounded or truncate, and 23 × 11 µm (n = 50). The identification of Diplodia seriata De Not. was molecularly confirmed with the amplification with the primers ITS1 and ITS4 and subsequent sequencing of the internal transcribed spacer ITS1-5.8S-ITS2 region of the rDNA extracted from the isolate IVIA GCA-5 (GenBank Accession No. JX987099). Furthermore, the region D1/D2 in the 5' end of the 28S rDNA gene was amplified with the primers NL1 and NL4 and sequenced (JX997743). A nucleotide BLAST analysis showed in both cases 100% identity with D. seriata [EF127892 (3) and AY928050, respectively]. To fulfill Koch's postulates, 5-mm diameter mycelial plugs from 7-day-old colonies of isolate IVIA GCA-5 grown on PDA at 25°C were aseptically transferred to skin wounds on superficially disinfected 'Algerie' loquats (one plug per fruit; n = 9). Wounded but not inoculated fruit were used as controls. The experiment was repeated three times. Inoculated fruit developed lesions of 18 to 100 mm after 7 to 21 days of incubation at 20°C. No lesion was observed on controls. The fungus was consistently reisolated from inoculated fruit. D. seriata is a broadly spread pathogen causing cankers, blight, dieback, and fruit rots in vines and many fruit trees. In Spain, it has been reported to cause fruit rot of olive (1) and branch dieback in olive (2) and grapevine (4). To our knowledge, this is the first report worldwide of D. seriata causing loquat fruit rot. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007. (4) J. R. Úrbez-Torres et al. Plant Dis. 90:835, 2006.

First Report of Penicillium expansum Causing Postharvest Blue Mold of Fresh Date Palm Fruit (Phoenix dactylifera) in Spain.

A survey of postharvest losses of commercially handled and cold-stored fruit of fresh date palm (Phoenix dactylifera L.), cvs. Medjool and Hayani, was conducted in the 2009 and 2010 seasons in the grove of Elx (Alacant Province, Southeast Spain). Disease symptoms consisting of circular, light brown, soft spots located in any part of the fruit skin were observed in 2 to 5% of the fruit. At room temperature, the lesions expanded rapidly and blue mold symptoms were apparent. The potential causal agent (isolate IVIA NiAA-2) was transferred to PDA and incubated at 25°C. The identification was performed at the Spanish Type Culture Collection (CECT, University of Valencia, Spain) based on colony morphology of the isolate grown on Czapeck yeast extract agar (CYA) and malt extract agar (MEA) at 26°C. Colonies were circular (average diameter of 40 mm at 7 days), radially sulcate, with dense velvety white mycelium, and very abundant, bluish green conidia. The underside of the plates showed light brown and pale green colonies on CYA and MEA, respectively. On CYA, but not on MEA, a light yellow exudate was produced and a brownish pigment diffused into the medium. At 5 and 37°C on CYA, white microcolonies and no colonies were observed, respectively. Conidia were ellipsoidal to subglobose, smooth and thin walled, measuring 3.0 to 3.5 × 2.5 to 3.0 µm (n = 50) (4). Based on these morphological characteristics, the isolate IVIA NiAA-2 was tentatively identified as Penicillium expansum L. To confirm the identity, we amplified and sequenced the rDNA internal transcribed spacer (ITS) region with primers ITS1 and ITS4 (GenBank Accession No. KC169942). A BLAST search showed 99% identity and 100% query coverage with P. expansum strain NRRL 6069 (DQ339562) (2). Selected healthy dates cv. Medjool were surface disinfected by dipping in 0.5% sodium hypochlorite for 2 min followed by thorough rinsing in deionized water. Pathogenicity was tested by pipetting 20 µl of a spore suspension (1 × 106 spores per ml), prepared from 7-day PDA cultures, onto fresh skin wounds, which were made on disinfected fruit using a sterile, stainless steel rod with a probe tip 1 mm in width × 2 mm in length (one wound on each of nine dates, incubated in one humid chamber). Disinfected, wounded, and non-inoculated dates were used as controls. The procedure was repeated three times. Disease symptoms were observed on all inoculated fruit (average lesion size of 6, 15, and 22 mm after 4, 7, and 10 days of incubation at 20°C, respectively) and P. expansum was consistently reisolated, thereby fulfilling Koch's postulates. No decay was observed on any of the non-inoculated fruit. Unidentified species of Penicillium have been reported to cause date palm fruit rot (1,3). To our knowledge, this is the first report of P. expansum causing postharvest decay of date palm fruit in Spain. References: (1) M. Djerbi. Diseases of the Date Palm. FAO Regional Project, Rome, 1983. (2) M. A. Dombrink-Kurtzman. Antonie Van Leeuwenhoek 91:179, 2007. (3) S. Ibrahim and M. A. Rahma. Bayero J. Pure Appl. Sci. 2:127, 2009. (4) R. A. Samson et al. Introduction to Food-Borne Fungi. Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, 1995.

Immunocytochemical investigation of normal and chronic lymphocytic leukaemia lymphocytes reveals unexpectedly frequent reactivity with some myelomonocytic associated antibodies.

Information about the expression of some myelomonocytic markers in lymphocytes of patients with B-CLL is scarce. We studied the CD13, CD14, CD11c and CD68 surface antigens in 42 controls and in 38 patients with B-CLL to detect their possible reactivity. Eighty-nine percent of B-CLL expressed very strongly the CD14 antigen; on the contrary, the other myelomonocytic antigens tested were very weakly expressed. Forty-one of 42 controls showed a few CD14-positive lymphocytes with a statistical difference between normal and CLL lymphocytes. No statistical difference was recorded either between CD14 expression and Rai's staging system or Binet's stages, nor between CD14 and bone marrow involvement and doubling time or between CD14 and heavy or light chain expression. A minor B lymphocytic subset in humans coexpresses the CD14 and CD5 antigens, it being increasingly speculated that B chronic lymphocyte leukaemias originate precisely from this B CD5- and CD14-positive cells. Just as the CD5 antigen is regarded as an excellent B-CLL marker, it seems to us that a strong expression of the CD14 antigen might have the same diagnostic relevance.

Isochromosome 14q in myeloid dysplastic disorder.

A case of refractory anemia (RA) with an isochromosome 14q is described. This finding is compared with other hematologic disorders with trisomy 14 as the sole abnormality.

Circulating erythroid and megakaryocytic progenitors in polycythaemia vera and essential thrombocythaemia.

We studied the behaviour in culture of erythroid and megakaryocyte progenitor cells (BFU-E, CFU-MK) obtained from peripheral blood (PB) in 38 patients: 15 with essential thrombocythaemia, 3 with reactive thrombocytosis, 16 with polycythaemia vera and 4 with secondary polyglobulia. Clonal erythroid growth without added erythropoietin was observed in all patients with polycythaemia vera and in 5 out of 15 with essential thrombocythaemia, but in none of the patients with reactive thrombocytosis or secondary polyglobulia or in controls. When the CFU-MK were cultured without phytohaemagglutinin-stimulated medium (PHA-LCM), all patients with essential thrombocythaemia and 7 out of 16 with polycythaemia vera showed circulating CFU-MK but none of those with reactive thrombocytosis or secondary polyglobulia or controls did so. This study indicates that the growth in vitro of megakaryocytic and erythroid progenitors from such a readily available source as peripheral blood can be valuable in the diagnosis of certain borderline cases of thrombocytosis or erythrocytosis.