First report of Neopestalotiopsis sp. causing trunk disease on European chestnut in Spain

In April 2021, a brownish-brown vascular lesion was observed in the stemwood of a five-year-old chestnut (Castanea sativa Mill.) rootstock when cutting for grafting in Villaviciosa (Asturias, Northern Spain). To identify the causal agent, a cross section of the steam was cut, surface-sterilized with 96% ethanol and dried, plated on potato dextrose agar (PDA) and incubated at 25ºC. Fungal colonies were consistently isolated and after 5 days developed abundant greyish-white mycelium. For molecular identification, the internal transcribed spacer (ITS) gene region of rDNA of the strain LPPAF-975 was amplified with the ITS1/ITS4 primers (White et al. 1990) using the TerraTM PCR Direct Polymerase Mix (Takara Bio Company, CA, USA). This sequence was deposited in GenBank (accession no: OR002144) and showed 99.80% identity over a 507 pb alignment with the Neopestalotiopsis isolate 328-16 (accession no: OK166668) isolated on blueberry in Serbia and Nespestalotiopsis australis strains LNZH0701 and LNZH0752 (accession nos: OM919511-12) both isolated on blueberry in China. To clarify the identification, beta-tubulin (tub2) and translation elongation factor alpha-1 (tef1-a) were amplified (Glass & Donaldson 1995, Walker et al. 2010, respectively). Beta-tubulin sequence (accession no: OR001747) was 99.52% of identity with sequences of several species of Neopestalotiopsis, and elongation factor (accession no: OR001748) was 99.57% identity with sequences previously deposited for N. clavispora (accession nos: OP684010-11, MZ097377-79). The phylogenetic tree was constructed with the three concatenated sequences using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993), and their topological robustness was evaluated by bootstrap analysis based on 1,000 replicates using Mega 11 (Tamura et al, 2021). However, strain LPPAF-975 clustered with N. javaensis, N. rosae and N. vacciniicola, therefore the species remains unidentified. Pathogenicity tests were carried out on ten five-year-old chestnut plants on which a half 5-mm-diameter PDA plug from the edge of an actively growing colony of the fungus was inoculated by a cut in the bark of one to three branches per plant and then wrapped with Parafilm©. Five plants inoculated as above but without the fungus were used as controls. Plants were cultivated in pots provided with drip irrigation in a tunnel under natural conditions. The assay was conducted twice. External cankers around the inoculated area were observed a month after inoculation, while control plants did not show any lesions. The fungus was re-isolated on all the inoculated plants, but not from controls. As all the re-isolated strains had the same morphology, one of them was randomly selected and identified by sequencing, thus fulfilling Koch's postulates. Cross-sections of plants showed lesions similar to that initially found, with 100% of the plants damaged at the point of inoculation, and 80 and 65% 1 cm above and below, respectively. Pathogen was newly re-isolated and identified from one of these cross-sections. To the best of our knowledge, this is the first report worldwide of Neopestalotiopsis sp. causing disease on Castanea sativa. This pathogen could represent a threat to the maintenance of the biodiversity of traditional chestnut varieties, multiplied by grafting on rootstocks in nurseries, which would incur considerable economic losses.