E. E. Ferrada

Identification and Characterization of Botrytis Blossom Blight of Japanese Plums Caused by Botrytis cinerea and B. prunorum sp. nov. in Chile

Blossom blight is a destructive disease of plums (Prunus salicina) when humid and temperate weather conditions occur in Chile. Disease incidence ranging from 4 to 53% has been observed. Symptoms include light brown petal necrosis, starting as light brown mottles or V-shaped necrosis at the margins of the petals, progressing to the stamen and pistils. In this study, the etiology of blossom blight of plums was determined. High- and low-sporulating isolates of Botrytis were obtained consistently from blighted blossoms and apparently healthy flowers of plums. Based on colony morphology, conidial production and molecular phylogenetic analysis, these high- and low-sporulating isolates were identified as B. cinerea and B. prunorum sp. nov., respectively. Phylogenetic analysis of the genes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) grouped B. prunorum isolates in a single cluster, distantly from B. cinerea and other Botrytis species. The phylogenetic analysis of necrosis and ethylene-inducing protein (NEP1 and NEP2) genes corroborated these results. Analysis of the internal transcribed spacer region and large-subunit (26S) ribosomal DNA and detection of Boty and Flipper transposable elements, were not useful to differentiate between these Botrytis species. Both species were pathogenic on plum flowers and the fruit of plums, apples, and kiwifruits. However, B. prunorum was less virulent than B. cinerea. These pathogens were re-isolated from inoculated and diseased tissues; thus, Kochs postulates were fulfilled, confirming its role in blossom blight of plums. B. cinerea was predominant, suggesting that B. prunorum may play a secondary role in the epidemiology of blossom blight in plums in Chile. This study clearly demonstrated that the etiology of blossom blight of plums is caused by B. cinerea and B. prunorum, which constitute a species complex living in sympatry on plums and possibly on other stone fruit trees.

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. Kochs 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.

Gray mold caused by Botrytis cinerea limits grape production in Chile

Gray mold (GM) caused by Botrytis cinerea is a major disease of grapes ( Vitis vinifera ) that substantially reduces the yield and quality of grape production in temperate and humid regions of the world. B. cinerea is a necrotrophic fungus that attacks the non-lignified aerial organs of grapes; in particular, berries are highly susceptible during ripening. The polycyclic nature and exponential progress exhibited by GM at the beginning of the its epidemic, as well as the abundant inoculum production, the high dissemination efficiency, the wide host range and the high genetic variability of B. cinerea , explain the difficulties encountered in attempting to control GM. At present, integrated disease management, including cultural and chemical control, is the main control strategy. These control measures can be used to reduce the initial inoculum or to lower the disease infection rate. However, control measures that reduce the infection rate are the most effective means of controlling GM. Important progress toward understanding the complexity of the biology and epidemiology of this pathogen has occurred in recent decades. This has allowed the improvement and development of more effective and sustainable control strategies against B. cinerea . This review article provides a recent update regarding grape GM, with special emphasis on Chilean production conditions. La pudricion gris (PG) causada por Botrytis cinerea, es una de las principales enfermedades de la vid ( Vitis vinifera ) que limita la produccion y reduce los rendimientos y la calidad de la fruta en zonas templadas y humedas a nivel mundial. B. cinerea es un hongo necrotrofo que ataca organos aereos no lignificados de la vid, siendo las bayas altamente susceptibles durante la maduracion. La naturaleza policiclica y el desarrollo exponencial de las epidemias de PG, junto con la abundancia de inoculo, la eficiente dispersion mas el amplio rango de hospederos y gran variabilidad genetica que presenta B. cinerea, explican las dificultades para lograr un control satisfactorio. Ante lo cual se hace necesario realizar una estrategia de control integrado que incluya medias de control cultural y quimico. Estas medidas pueden estar orientadas a reducir el inoculo inicial o la tasa de progreso de la enfermedad, siendo las medidas de control destinadas a reducir la tasa de progreso las que mas aporta al control de PG. En las ultimas decadas se han producido importantes progresos en el conocimiento de la compleja biologia de este patogeno y de los aspectos epidemiologicos de la PG. Esto ha permitido mejorar las estrategias de control logrando alternativas mas efectivas y sustentables. En este articulo se revisan los aportes cientificos recientes realizados en relacion con la PG de la vid, teniendo especial enfasis en la situacion del vinedo chileno.

Identification and Characterization of Diaporthe ambigua, D. australafricana, D. novem, and D. rudis Causing a Postharvest Fruit Rot in Kiwifruit

Diaporthe spp. are important plant pathogens causing wood cankers, blight, dieback, and fruit rot in a wide range of hosts. During surveys conducted during the 2013 and 2014 seasons, a postharvest rot in Hayward kiwifruit (Actinidia deliciosa) was observed in Chile. In order to identify the species of Diaporthe associated with this fruit rot, symptomatic fruit were collected from seven kiwifruit packinghouses located between San Francisco de Mostazal and Curicó (central Chile). Twenty-four isolates of Diaporthe spp. were identified from infected fruit based on morphological and cultural characters and analyses of nucleotides sequences of three loci, including the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequences of the β-tubulin, and translation elongation factor 1-α genes. The Diaporthe spp. identified were Diaporthe ambigua, D. australafricana, D. novem, and D. rudis. Multilocus phylogenetic analysis revealed that Chilean isolates were grouped in separate clades with their correspondent ex-types species. All species of Diaporthe were pathogenic on wounded kiwifruit after 30 days at 0°C under normal and controlled-atmosphere (2% O2 and 5% CO2) storage and they were sensitive to benomyl, pyraclostrobin, and tebuconazole fungicides. D. ambigua isolates were the most virulent based on the lesion length measured in inoculated Hayward and Jintao kiwifruit. These findings confirm D. ambigua, D. australafricana, D. novem, and D. rudis as the causal agents of kiwifruit rot during cold storage in Chile. The specie D. actinidiae, a common of Diaporthe sp. found associated with kiwifruit rot, was not identified in the present study.