“Cladosporium rot” on “Bosc” pear fruit during cold storage causes significant economic losses and has been reported in recent years in the productive valleys of Río Negro and Neuquén. The species involved were not determined. During 2016-2017, “Bosc” pears (Pyrus communis) in cold storage chambers exhibited external brownish black circular spots caused by Cladosporium spp. The objective of this work was to determine the Cladosporium species that caused the above mentioned symptoms. The morphological and molecular analyses of the partial sequence of the actin gene (ACT) supported the identification. Cladosporium macrocarpum, Cladosporium subtilissimum and Cladosporium floccosum were determined as the species involved in the disease. Although Cladosporium has been reported to cause pear rot, this is the first report to identify these species as causal agents of this fruit disease.
En los valles productivos de Río Negro y Neuquén, se ha reportado en los últimos años la presencia de podredumbre de peras «Bosc» causada por Cladosporium, lo que generó significativas pérdidas económicas. Las especies involucradas no fueron determinadas. Se detectó la aparición de manchas circulares negras parduzcas en peras de dicha variedad en cámaras de almacenamiento en frío durante 2016-2017. El objetivo del presente trabajo fue determinar las especies de Cladosporium causantes de los síntomas mencionados. La identificación fue llevada a cabo por caracterización morfológica y el análisis molecular de la secuencia parcial del gen de actina (ACT). Se pudo determinar que Cladosporium macrocarpum, Cladosporium subtilissimum y Cladosporium floccosum fueron las especies implicadas. Si bien la podredumbre en peras causada por Cladosporium ha sido previamente reportada, este es el primer informe que identifica a estas especies entre los agentes causales de la enfermedad.
Cladosporium species can cause lesions in healthy pears according to in vitro studies5,7,14. Cladosporium herbarum was reported as a causal agent of Cladosporium rot on Bosc pear cultivars in the USA9. Moreover, the species C. herbarum and Cladosporium sp. were reported as postharvest phytopathogens of pears in the Netherlands15. Postharvest Cladosporium rots were also reported in Argentina on Beurré Bosc and Golden Russet Bosc pears in Northern Patagonia5,12. A correct and accurate identification of the species is necessary because the name of the species involves a set of characteristics such as growth features, pathogenicity or production of mycotoxins, which allow to predict their behavior1.
“Bosc” pears were affected by rot spots during cold storage (2016-2017) in the High Valley of Río Negro, a fruit producing region of Northern Patagonia in Argentina. The symptoms consisted of one or more brownish black circular spots that extended over the rind, light brown on the edges and dark brown to black in the center (Fig. 1). Therefore, the objective of this work was to determine the Cladosporium species that were involved in this fruit disease.
Fifteen symptomatic “Bosc” pears, stored unprocessed during approximately 2 months in bins inside conventional cold storage chambers at -0.5°C, were obtained from three commercial establishments (5 pieces from each). Fruits were superficially disinfected with a solution of sodium hypochlorite (1:10) for 5minutes and rinsed by immersion twice in sterile distilled water. Infected internal tissue fragments were aseptically extracted from the spots and placed on potato dextrose agar supplemented with 0.1% chloramphenicol (PDA+C). Plates were incubated for 7 days at 25°C. The pathogens were identified at the genus level according to Pitt and Hocking8 as Cladosporium species. Characterization based on macroscopic features of the colonies clustered the isolates into nine different morphological groups (designated as G1 to G9). The microscopic characteristics of the isolates were determined in SNA (synthetic nutrient-poor agar) medium after 14 days of incubation at 25°C under close UV light3. DNA extraction was performed using the DNeasy Plant Mini Kit following the manufacturer's instructions (Qiagen, Intl) and genomic DNA was quantified with the Qubit 2.0 fluorometer (Life Technologies, Intl.). The partial sequence of the actin gene (ACT) was amplified using the primers ACT-512F: ATGTGCAAGGCCGGTTTCGC and ACT-783R: TACGAGTCCTTCTGGCCCAT to obtain resolution at the species level3,14. Sequencing of the fragments was done by Macrogen Inc. (Seoul, Korea). Pathogenicity tests were performed using the toothpick technique2 and verified according to Koch's postulates.
After the molecular analysis, the nine different morphological groups were reduced to 3 species as causal agents of the disease. BLAST analysis of 200bp fragments from the isolates with Cladosporium strain reference sequences obtained from the GenBank showed 100% identity to Cladosporium subtilissimum (isolate 53ACT GenBank Accession No. MG680545.1), Cladosporium macrocarpum (isolate 12ACT GenBank Accession No. MG680533.1) and Cladosporium floccosum (culture CPC 17802 GenBank Accession No. MF473823.1). As a result of the pathogenicity tests, the C. subtilissimum isolates produced a 1.5cm lesion on the surface of the fruits with internal necrosis 1.1cm deep (dry tissue that emerges like a plug). C. macrocarpum isolates produced an average lesion of 1.5cm on the surface of the fruit with wet internal necrosis averaging 1.7cm. C. floccosum isolates caused an external average lesion of 1cm surrounded by a brown halo with dark brown internal necrosis extending 1.5cm deep (dry tissue that emerges like a plug).
Cladosporium species are predominant in indoor and outdoor environments4,11,13. C. macrocarpun and C. subtilissimum have been reported in a previous study conducted in rural environments of the High Valley of Río Negro productive region in Northern Patagonia in which eleven species were determined. Pathogenicity tests revealed that C. macrocarpun and C. subtilissimum, among other Cladosporium species, caused disease on pears14. The presence of these and other potentially phytopathogenic species in the air warns about the potential risk of infections by these causal agents during cold storage and/or growing seasons in the field. Emerging diseases can be expected in the context of climate change, such as that which has been occurring in Northern Patagonia (Argentina)6,10. These findings contribute to implementing appropriate preventive measures to reduce losses in pear production due to Cladosporium rot.
GenBank Accession numbers are MK410437-MK410445 (under examination and processing by the GenBank annotation staff).
Conflict of interestNone
To María del Valle Leiva, Soledad Ramírez and María de los Ángeles Pérez. This research was financially supported by Universidad Nacional de Río Negro and CONICET.
