First Report of Diaporthe novem Causing Postharvest Rot of Kiwifruit During Controlled Atmosphere Storage in Chile.

Chile is considered the third major exporter of kiwifruits (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson) worldwide after Italy and New Zealand (1). The genus Diaporthe Nitschke (anamorph: genus Phomopsis) has been reported as causing postharvest rot in kiwifruit (4). During the current study, 1,400 fruits arbitrarily collected from seven controlled atmosphere (CA) rooms after 90 days of storage conditions (2% O2, 5% CO2) determined that 21.5% of the fruit were affected by decay and 0.86% developed symptoms different than those caused by Botrytis cinerea, the main postharvest pathogen associated to kiwifruit. Symptoms were soft rot with brown skin that started at the stem-end and in severe cases affected the entire fruit. Internally, affected fruit showed browning and watery tissues. Twelve affected fruits were surface disinfested (75% ethanol) and small pieces of internal rotten tissues were placed on acidified potato dextrose agar (APDA) for 7 days at 20°C. Twelve isolates were obtained, and four of them were identified morphologically and molecularly as Diaporthe ambigua, a species that has been previously described causing rot in stored kiwifruits in Chile (2). However, eight other flat, white to grayish colonies with sparse dirty-white aerial mycelium at the edge of the dish were obtained (3). Black pycnidia contained unicellular, hyaline, biguttulate, oval to cylindrical alpha conidia, with obtuse ends of (7.9) 6.7 (5.3) × (2.9) 2.5 (2.1) µm (n = 30). These isolates were tentatively identified as a Diaporthe sp. The species identification was determined by sequencing comparison of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA (GenBank Accession Nos. KJ210020 to 24, KJ210027, and KJ210033) and a portion of beta-tubulin (BT) (KJ210034 to 38, KJ210041, and KJ210047) using primers ITS4-ITS5 and Bt2a-Bt2b, respectively. BLAST analyses showed 99 to 100% identity with D. novem J.M. Santos, Vrandecic & A.J.L Phillips reference ex-type (KC343156 and KC344124 for ITS and BT, respectively) (3). Eighteen mature kiwifruits cv. Hayward were inoculated using a sterile cork borer on the surface of the fruit and placing 5-mm agar plugs with mycelial of D. novem (DN-1-KF). An equal number of fruits treated with sterile agar plugs were used as negative controls. After 30 days at 0°C under CA, all inoculated fruit showed rot symptoms with lesions 7.8 to 16.4 mm in diameter. The same D. novem isolate was inoculated with 30 µl of a conidial suspension (106 conidia/ml) on the surface of 18 ripe kiwifruits that were previously wounded and non-wounded as described above. An equal number of wounded and non-wounded fruits, treated with 30 µl sterile water, were used as negative controls. All inoculated wounded fruits developed rot symptoms with necrotic lesions of 14.1 to 20.2 mm of diameter after 14 days at 25°C. Inoculated non-wounded and negative control fruits remained symptomless. Koch's postulates were fulfilled by re-isolating D. novem only from the symptomatic fruits. To our knowledge, this is the first report of rot caused by D. novem on kiwifruit during cold storage in Chile and worldwide. Therefore, both Diaporthe species appears to be associated to Diaporthe rot of kiwifruit in Chile. References: (1) Belrose, Inc. World Kiwifruit Review. Belrose, Inc. Publishers, Pullman, WA, 2012. (2) J. Auger et al. Plant Dis. 97:843, 2013. (3) R. Gomes et al. Persoonia 31:1, 2013. (4) L. Luongo et al. J. Plant Pathol. 93:205, 2011.

First Report of Blossom Blight Caused by Sclerotinia sclerotiorum on Japanese Plum, Nectarine, and Sweet Cherry Orchards in Chile.

Blossom blight of Japanese plum (Prunus salicina), nectarine (P. persica var. nectarina), and sweet cherry (P. avium) was observed in commercial orchards in central Chile in 2012. Disease prevalence of 8% and 1% were estimated in 2012 and 2013, respectively. Early symptoms appeared as small pale-brown necrotic lesions on the petals that eventually affected the entire flowers. White and cottony fungal colonies were consistently isolated on potato dextrose agar acidified with 0.5 ml/liter of 92% lactic acid (APDA), incubated for 5 days at 20°C. Black spherical to elongated sclerotia of 2.5 to 4.2 × 2.8 to 5.3 mm (n = 60) were formed on APDA. This fungus was tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary. The identity of the fungus was confirmed by BLAST analysis of the internal transcribed spacer (ITS) region (GenBank Accession Nos. KF148604 to KF148609) of rDNA, amplified with PCR primers ITS1/ITS4 (3), demonstrating a 99 to 100% similarity with the reference S. sclerotiorum strains (EU082466 and JX307092). The pathogenicity was studied in detached flowers of 'Larry Ann' Japanese plum, 'Summer Bright' nectarine, and 'Bing' sweet cherry that were inoculated with a mycelial suspension (106 fragments/ml) of six isolates of S. sclerotiorum and incubated for 5 days at 20°C in humid chambers (>80% relative humidity). Inoculated flowers developed a light brown petal necrosis that eventually comprised the entire flower. The same S. sclerotiorum isolates were inoculated in mature fruits of 'Larry Ann' Japanese plum, 'Summer Bright' nectarine, and 'Staccato' sweet cherry. Surface disinfected (1% NaOCl for 1 min) fruits were inoculated by placing a mycelium plug (4 mm in diameter) into a wound made with a sterile scalpel and incubated for 3 days at 20°C in humid chambers. Symptoms consisted on light brown soft lesions that varied from 8.7 to 46.5 mm in diameter. A superficial white and cottony septated mycelium was also obtained. An equal number of non-inoculated flowers and wounded but non-inoculated fruits remained healthy. S. sclerotiorum was re-isolated from 100% of the artificially inoculated flowers and fruits, completing Koch's postulates. S. sclerotiorum was reported causing shoot blight on apricot (P. armeniaca), lemon tree (Citrus limon), and table grapes (Vitis vinifera) in Chile (1,2), and to our knowledge, this is the first report of S. sclerotiorum associated with blossom blight in Japanese plum, nectarine, and sweet cherry in Chile. References: (1) R. Acuña. Compendio de Bacterias y Hongos de Frutales y Vides en Chile. Servicio Agrícola y Ganadero, Santiago, Chile, 2010. (2) B. A. Latorre and M. J. Guerrero. Plant Dis. 85:1122, 2001. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Monilinia fructicola Causing Brown Rot on Stored Japanese Plum Fruit in Chile.

In autumn 2013, fruit of Japanese plum (Prunus salicina) cvs. Angelino and Black Kat developed an unusual brown and soft rot after 2 months in cold storage (0°C) on nearly 1% of the fruit. Fruit showed small, circular, light brown spots that eventually destroyed the entire fruit. Small sporodochia appeared on the fruit surface. Fruit was harvested from orchards located near San Francisco de Mostazal (33°59' S, 70°41' W), Chile. Small pieces of diseased tissue were selected from margins of lesions of surface disinfected (96% ethanol) fruit (n = 7) and placed on acidified potato dextrose agar (PDA) plates for 5 days at 20°C. Light brown colonies with even margins and concentric rings of spores were obtained. The conidia of five isolates were one-celled, hyaline, lemon-shaped, (min. 10.7) 14.9 ± 1.5 (max. 18.6) × (min. 8.1) 9.4 ± 0.8 (max. 10.8) µm (n = 30), and borne in branched monilioid chains. This fungus was identified as Monilinia fructicola (G. Winter) Honey (1). Identification was confirmed by amplifying and sequencing the ribosomal ITS1-5.8S-ITS2 region using ITS1 and ITS4 primers (3). BLAST analysis of Chilean plum isolates (GenBank Accession Nos. KF148610 and KF148611) were 99 to 100% identical to isolates of M. fructicola originating from the United States (DQ314727 and HQ846966, respectively) and 100% identical to the first Chilean isolate (JN001480) found in nectarines originating from California at the supermarkets in Santiago in June 2009. Koch's postulates were fulfilled by reproducing brown rot symptoms on mature wounded Japanese plums cv. Angelino (n = 8) inoculated with 10 µl of a conidial suspension (105 conidia/ml) or with a mycelium plug (5-mm diameter). After 2 days in humid chambers (>80% relative humidity) at 25°C, all inoculated fruit developed brown rot symptoms with necrotic lesion means of 15.8 and 21.5 mm in diameter in fruit inoculated with conidia and mycelium, respectively. Non-inoculated control fruit remained healthy. Re-isolations were performed on PDA and the presence of M. fructicola was morphologically confirmed in 100% of the symptomatic fruits. To our knowledge, this is the first report demonstrating the presence of M. fructicola causing brown rot in stored Japanese plums in Chile after its first interception in 2009 in Chile, suggesting that this pathogen has been established in the field. Currently, M. fructicola is a quarantine organism under official control, restricted to Prunus orchards between Santiago and Nancagua in central Chile (2). References: (1) EPPO. EPPO Bull. 39:337, 2009. (2) Servicio Agrícola y Ganadero, SAG, Ministerio de Agricultura, Gobierno de Chile. www.sag.cl , accessed 15 November 2013. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, NY, 1990.

Manejo con onabotulinumtoxin A del dolor y distonía focal en paciente tetrapléjico pediátrico / Pain and focal dystonia in a pediatric quadriplegic patient: management with onabotulinumtoxin A: a case report

Introduction: Focal dystonia can cause pain and interfere with rehabilitation goals and affect quality of life and family, in a pediatric patient with recent organic brain damage. Objective: This study examined the effects of onabotulinumtoxin A on focal dystonia, posture malalignment, and pain during the sub-acute phase. Method: At four mounths of progress, a 10 years old girl, with mixed quadriparesis secondary to tuberculous meningoencephalitis is presented. The patient was semi-conscious, malnourished, gastrostomized, and exhibited pain at upper right limb mobilization. Physical exam evidenced abnormal scapular posture, and fixed cephalic rotation. Initial treatmnt consisted of neurodevelopmental techniques added to oral antiespatic medication, with no clinical results. Subsequent treatment consisted of onabotulinumtoxin A infiltration, guide with electromyographic technique to those muscles that showed greater dystonic activity, mapped during previous evaluations. Pain was measured according to the Visual Pediatric Analogue Scale, and passive axial alignment and spasticity according to a modified version of the Ashworth Scale, pre and post treatment. Results: Two weeks after infiltration, the patient showed an objective decrease in pain during mobilization, and achieved aligned axial posture both while standing and sitting. At three months follows-up, patient maintained a decline in the dystonic pattern, which allowed adequate positioning to enable the integration of both upper limbs during activities, without pain interference. Discussion: The use of onabotulinumtoxin A could play a role in the management of focal dystonia and a secondary pain, during the sub-acute phase of organic brain damage.

Introducción: En un paciente pediátrico con daño orgánico cerebral reciente, la distonía focal puede ser causa de dolor que interfiera con los objetivos de rehabilitación y repercuta en la calidad de vida del paciente y su familia. Objetivo: Mostrar el efecto de la onabotulinumtoxin A sobre cuadro de distonía focal, con alteración postural y dolor en pacientes en etapa subaguda. Método: Presentamos a una niña de 10 años, portadora de tetraplejia mixta secundaria a meningoencefalitis tuberculosa, estado de mínima conciencia, desnutrición, gastrostomizada, de cuatro meses de evolución, quien presentaba dolor a la movilización de la extremidad superior derecha y al examen físico; se encontró compromiso postural de escápula y giro mantenido cefálico. Se inició manejo con técnicas de neurodesarrollo y antiespásticos orales sin respuesta clínica. Se efectuó infiltración de onabotulinumtoxin A con técnica de guía electromiográfica en los músculos que presentaron mayor descarga distónica mapeados en evaluación previa. Se objetivó el dolor según Escala visual análoga pediátrica, alineamiento pasivo axial y espasticidad según Escala modificada de Ashworth, pre y post procedimiento. Resultados: Tras dos semanas post infiltración, se objetivó paciente con disminución del dolor frente a la movilización, con postura alineada axial tanto en decúbito como sedente. A los tres meses mantenía disminución en hipertonía, posicionamiento adecuado que permitía actividades de integración de ambas extremidades superiores sin interferencia por dolor. Discusión: El uso de onabotulinumtoxin A puede cumplir función en el control de la hipertonía y en el control del dolor secundario, en un paciente de evolución subaguda posterior a daño cerebral adquirido.