First Report of Pseudonectria buxi causing leaf and stem blight on boxwood in Santa Catarina, Brazil

Abstract

Buxus sempervirens is an ornamental plant used commonly for hedges and topiaries all over the world (Henricot and Culham 2002). Pseudonectria buxi (DC.) Seifert, Grafenhan & Schroers or P. rousseliana (Mont.) Seaver is a fungal pathogen that causes leaf and stem blight on boxwood (Buxus sp.). This disease has been reported in Europe and North America, as well as China (Garibaldi et al. 2016; Shi and Hsiang 2014a,b). Plants of B. sempervirens exhibiting bronzed, yellow, and blighted leaves, stem dieback symptoms, and orange sporodochia on leaves and stems were observed in January 2016 in Passo de Torres District, Bellatorres, Santa Catarina, Brazil. Twenty diseased leaves were collected, surface disinfested in 1% NaOCl for 1 min, placed on potato dextrose agar (PDA), and incubated for 5 to 7 days at room temperature (25°C) with a 12-h photoperiod to assess macroscopic morphological characteristics. Light microscopy and scanning electron microscopy were used to assess the micromorphological characteristics. Isolated colonies consisted of white fluffy mycelium with an orange colored center. Conidia were hyaline, single celled, elliptical, and formed on single and multiple branched phialides. Mean conidia size was 7.70 × 3.20 µm and ranged from 6.10 to 10.80 × 2.50 to 3.70 μm. Mean chlamydospore size was 7.10 × 4.60 μm and ranged from 4.80 to 11.00 × 3.40 to 6.70 μm. For molecular confirmation of identification, total genomic DNA was extracted from conidia and mycelium of the fungus and a portion of the beta-tubulin gene was amplified using primers btub_F750: and btub_R1400: according to the protocol of Shi and Hsiang (2014a). Sequences with around 592 bp were subjected to BLAST search in NCBI. Results showed 99 and 97% sequence identify with two isolates of P. buxi (syn. P. rousseliana), DQ522522 (612/613 bp) and KC819609 (574/592 bp), respectively. The DNA sequence was deposited in GenBank as accession no. KX254345 and the fungal strain was deposited in the Mycological Collection of the Federal University of Lavras with the CML 3515 code. Pathogenicity experiments were conducted by placing six detached leaves (1 to 2.5 cm in length) of B. sempervirens with three leaves, either the abaxial or adaxial surface facing upward in the same 9-cm diameter plastic Petri dish lined with filter paper moistened with 2 ml of sterile deionized water. Three detached leaves were placed in each Petri dish and wounded using sterile dissecting needle. Nonwounded leaves were also included as a control. Leaves were inoculated by spraying each leaf with 1 ml of a 1 × 106 conidia suspension. Wounded or nonwounded leaves of control plants were sprayed with 1 ml of sterilize water. Plates were incubated at 25°C with 12-h photoperiod for 11 days and leaves were examined for disease symptoms. Wounded leaves inoculated with conidia on the abaxial surface turned brown. No signs or symptoms were observed on either nonwounded inoculated leaves or on leaves sprayed with water. The fungus was reisolated the diseased wounded leaves and identified using morphological and molecular method as described above for the original field samples as established in Koch’s postulates. To our knowledge, this is first report of P. buxi causing leaf blight on boxwood in Santa Catarina, Brazil. This disease can occur at all stages of plant production where there is wounding, and it could spread in nurseries of B. sempervirens and cause serious economic losses to ornamental producers in Brazil.