First Report of Canker Disease Caused by Neofusicoccum parvum and N. australe on Blueberry Bushes in Spain.

A field survey conducted in September 2009 at five plantations of six different cultivars of southern highbush blueberries (Vaccinium spp.) in Huelva, Spain, yielded 35 diseased plants. Diseased plants exhibited red-brown cankers and stem dieback. Blueberry cultivation in Huelva rose from 290 ha in 2007 to 777 ha in 2012, and the increase of these symptoms is of concern to producers. Stem pieces cut from the edge of lesions on infected plants were surface-disinfected with 5% sodium hypochlorite and cultured on potato dextrose agar (PDA). Based on colony characteristics on PDA, 18 colonies (one each from 18 different plants) were identified as Botryosphaeria spp. Species identities were confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS), rDNA, and elongation factor 1-alpha (EF1-α) sequences, using ITS1-ITS4 (3) and EF728f-EF986r (2) as primer pairs, respectively. BLAST searches of GenBank showed a high similarity of the isolate sequences to the reference sequences. Molecular results confirmed these species as Neofusicoccum parvum, N. australe, and B. dothidea. N. parvum was the most prevalent (on 34% of the plants analyzed), followed by N. australe and B. dothidea (9% each). In phylogenetic analyses, isolates that clustered in the same group belonged to the same species with a high homogeneity index (>99%). One representative isolate of each species was selected for a pathogenicity assay. Amplified sequences from each selected isolate were deposited in GenBank with the following accession numbers: N. parvum, KC556958 (ITS) and KC556961 (EF); N. australe, KC556959 (ITS) and KC556962 (EF); and B. dothidea, KC556960 (ITS) and KC556963 (EF). The pathogenicity assay of these three isolates was conducted using two cultivars of southern highbush blueberry, 'Misty' and 'Star.' The isolates were cultured on acidified PDA at 25°C for 5 days. Stems of the plants were wounded at a height of 10 cm with a drill (5 mm diameter and ~4 mm deep). Six replicates per cultivar were inoculated per isolate by placing a colonized agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identically with sterile agar plugs were used as controls. The plants were then maintained at 100% relative humidity for 2 h. This trial was conducted in a growth chamber at 28°C (night) and 30°C (day) with a 14-h photoperiod for 3 months. Disease was measured on a six-point scale: 0 = healthy plant; 1 = plant with a canker smaller than 3.5 cm; 2 = plant with a canker bigger than 3.5 cm; 3 = plant with one dry shoot; 4 = plant with some dry shoots; 5 = dead plant. At the end of the trial, disease was expressed as area under the disease progress curve. The results showed the N. parvum isolate to be the most aggressive, followed by the N. australe isolate. Espinoza et al. (1) also found that N. parvum showed more aggressiveness than N. australe on blueberries in Chile. B. dothidea was not pathogenic and behaved similarly to the controls (P < 0.05). Each pathogen was reisolated from all the inoculated plants, fulfilling Koch's postulates. To our knowledge, this is the first report of isolates of these pathogens, N. parvum and N. australe, causing stem canker and dieback on blueberry bushes in Spain. References: (1) J. G. Espinoza et al. Plant Dis. 93:1187, 2009. (2) A. J. L. Phillips et al. Mycol. 97:513, 2005. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: a Guide to Methods and Amplifications. M. A. Innis et al., eds. Academic Press, San Diego, CA. 1990.