First report of charcoal rot, caused by macrophomina phaseolina, on Blueberry in Southwestern Spain

Abstract

In Europe, Huelva province in Southwestern Spain is the main berry production area. Blueberry(Vaccinium spp.) was introduced during the early 90?s as an addition to strawberry cultivation.From 2011 to 2018, blueberry acreage increased from 777 ha to 3,000 ha. In May 2015 andSeptember 2017, wilted southern highbush blueberry plants (cvs. "Star", "Ventura", and "Legacy")were collected from three orchards located in the Huelva production area (Gibraleón and Moguer).The diseased plants showed drying of foliage and brown discoloration of stems and roots. Root andstem of symptomatic plants were surface sterilized (2 min,1% sodium hypochlorite), rinsed, dried,and plated on potato dextrose agar (PDA). After 7 days at 30º C in the dark, fungal isolatesproduced numerous black, round to ovoid shaped sclerotia with an average diameter of 98 μm(range: 61 to 128 μm). Genomic DNA from a single sclerotium isolate (TOR-872) was extractedfollowing the technique described by Bekesiova et al. (1999). Four DNA regions were amplifiedand sequenced: the exon region of translation elongation factor 1- α (TEF-1 α), β-tubulin (β-TUB),calmodulin (CAL), and the ITS region. TEF-1 α was amplified with the EF1-728F and EF1-986Rprimers (Carbone and Kohn, 1999), the β-TUB with the T1 and T22 primers (O?Donnell andCigelnik, 1997), the CAL with the CAL-228F and CAL-737R primers (Carbone and Kohn, 1999)and the ITS with the ITS5 and ITS4 primers (White et al., 1990). After BLASTing the 4 sequencesagainst the GenBank database, the top hits corresponded to Macrophomina phaseolina with a 99-100% of sequence identity for all cases. Our sequences were submitted to GenBank underAccession numbers: MK447854 (TEF-1 α), MK447918 (β-TUB), MK447823 (CAL) andMK447886 (ITS). Morphological and molecular results confirmed this isolate as M. phaseolina(Holliday and Punithalingam, 1970). In Gibraleón, in 7.87% of nearly dead plants (cv. Ventura)only M. phaseolina was isolated, whereas in Moguer disease incidence was 30, 7, and 2.27% in cvs."Star", "Ventura", and "Legacy", respectively. Inoculum for pathogenicity testing was produced bygrowing isolates TOR-872 and TOR-862 (both from diseased blueberry plants) on PDA. Inaddition, pathogenicity of a M. phaseolina isolate (TOR-102), from a strawberry soil and confirmedas pathogenic to strawberry, was tested because blueberry is usually cultivated in soils wherestrawberry had grown. Six potted blueberry plants (cv. "Star") per isolate were inoculated bysubstrate irrigation with 50 ml of a sclerotia suspension (104 sclerotia/ml) of each isolate. Sixcontrol plants were irrigated with water. Plants were held at 28°C and 40/70% relative humidity(day/night) in a growth chamber with a 16-h photoperiod. Four months after inoculation, themortality of the inoculated plants was 33, 50 and 50% for isolates TOR-862, TOR-872, and TOR-102, respectively. M. phaseolina was reisolated from all dead plants. No symptoms were observedin control plants. Macrophomina phaseolina has been associated with a blight disease on blueberryin Serbia (Popović et al., 2018) but this is the first report of charcoal rot on blueberry in Spain. Theaggressiveness of the strawberry soil isolate was also confirmed on blueberry. In Spain, chemicalfumigation in soil is banned in blueberry production. Thus, blueberry may be grown on fields with a previous history of strawberry production and carry-over of M. phaseolina causing charcoal rot maysignificantly impact crop production.