First Report in Chile of Mycocentrospora acerina, Causal Agent of Peony (Paeonialactiflora) Red Spot

A survey to identify pathogens causing peony diseases in southern Chile (37°95' to 40°58'S) was conducted from 2008 to 2011. A noticeable symptom consisted of small red spots on leaves, stems, sepals, and first petals of the bud. The central part of the lesions (1 cm) enlarged, darkened, and became necrotic, coincident with rainy and cold weather, including frosts. Lesions grew but never coalesced completely. At the edges of leaves, the infected tissues cracked and produced a twisted and corrugated appearance. Rhizome symptoms were also observed and consisted of black spots, which enlarged to elongated lesions. Small pieces (~1 × 1 mm) of symptomatic leaves, previously washed with 2% NaOCl for 2 min were cultured on potato dextrose agar (PDA) and V8 juice agar (20%) media. The cultures were incubated in growth chambers at 20°C (dark) and 5°C (dark)/20°C (light) for 12/12 h (dark/light). Sporulation was obtained only with V8 under the dark and light regime. Mycelia growth was superficial and immersed in the media; hyphae were septate with thick black walls. Conidiophores were flexuous, not branched, and produced a single spore. Spores were elongate, multiseptate, with a long, strongly curved beak. After one week, sporulation decreased and thick-walled, round black clamydospores formed in the media. Spores ranged from 125 to 235 μm long and most frequently were 170 to 220 μm. The number of septa ranged from 5 to 12. Morphological and cultural characteristics fit the description of Mycocentrospora acerina (Harting) Deighton (1,2). DNA was obtained from fungal culture. The internal transcribed spacer (ITS) region was amplified using ITS1/ITS4 primers (3), and part of the amplicon (502 of 550 pb) was sequenced. The sequence was deposited in GeneBank (Accession No. KF015599) and showed 100% identity values with sequences of similar regions from M. acerina (strain ATCC 16259, Accession No. KF278454). Healthy leaves and rhizome pieces were washed with sterile distilled water and placed on sterile moist paper towel. A small agar disc with mycelium was placed on the leaves and rhizomes with and without wounding before inoculation. The inoculated materials were kept in closed boxes with high humidity at 5 to 20°C. Tests were positive in leaves and rhizomes with and without wounds. Seven days after leaf inoculations, necrotic symptoms developed. A second inoculation test in peony leaves of plants bagged with plastic under field conditions corroborated the in vitro test. Inoculated rhizomes developed dark orange lesions initially, then turned black with a watery consistency, similarly to affected rhizomes in the field. In both tests, controls showed no symptoms. To our knowledge, this is the first report of M. acerina on P. lactiflora globally (4). In an evaluation at Carillanca (38°41'S, 72°25'W), using a scale from 0 (no symptoms) to 7 (necrotic lesions covering >60% of foliage, with death of stems), 11 out of 31 peony varieties scored 0-1. As a reference, a score of 3 is the maximum damage allowed on peonies for export. These varieties might help to keep Chilean peonies in the market without an economic impact. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI. Surrey, England, 1971. (2) B. C. Sutton and I. A. S Gibson. M. acerina. CMI. Descriptions of Pathogenic Fungi and Bacteria, No. 537.1977. (3) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved April 16, 2014.