First Report of Pestalotiopsis neglecta Causing Black Spot Needle Blight of Pinus sylvestris var. mongolica in China

Abstract

From May to October 2018, a total of 38 diseased Pinus sylvestris were found among 120 samples, with a field incidence rate up to 30%. The disease was found on needles and twigs of P. sylvestris. Initially, most disease lesions appeared on the tips of the needles, which were chlorotic to brown, and then expanded to cover the entire needles, which turned taupe. Eventually, the whole plant died. Pycnidia were initially buried in the epidermis and then exposed or semi-buried in the epidermis, separated or aggregated, globose, and dark-brown to black, and then expanded into an inverted V-shaped cracking(=223-897×38-105μm n=50). Most pycnidia were on the curved inner surface of the needles and fewer were distributed on the back. The conidia were five-celled clavate spindles. The color of the middle three cells was generally grayish brown to dark brown, and the fourth cell was slightly lighter in color and slightly constricted at the septum, 19.8-25.5×5.8-7.5μm (=23.9×6.3µm, n=50, std error. 0.20, 0.07); the septa were darker than the cells. The apical cells were hyaline and triangular, and the apical appendages were 2-3 (usually 2) tubulars with an open angle. The length of the apical appendages was 18.8-28.0 μm (=21.5µm, n=50, std error. 0.31); the tail cells were triangular and hyaline and had one basal handle or, in rare cases two 3.8-6.3 μm (=4.9µm, n=50, std error. 0.11). The colonies were white, dense and round in 3-5 days (PDA). The regions of the internal transcribed spacer (ITS) (ITS1/ITS2) and partialβ-tubulin (TUB) (T1/Bt-2b) were amplified and sequenced for BlASTn analysis and phylogenetic tree construction. BLASTn analyses of ITS (MN174666, KY558648) and TUB (MN176187) sequences exhibited 99.66%, 99.78% similarity to the sequences of P. neglecta 11G027 (LC387821), YY1A (EF055212), and YY8A (EF055250) in GenBank.The fungus was identified as Pestalotiopsis neglecta. For the pathogenicity test, the suspension was sprayed onto the needles of annual seedlings. After 40 days, typical symptoms appeared while control plants remained symptomless. After 80 days, the plants were diseased. The pathogen re-isolation infection rate was 40%. It will be important to develop phytosanitary management strategies of P. sylvestris and black spot needle blight.