RESUMO
Pseudomonas syringae pv. actinidiae (Psa), the agent causing bacterial canker of kiwifruit, has been present in the Principality of Asturias (PA), Northern Spain, since 2013, although with restricted distribution. In this study, 53 strains collected in kiwifruit orchards in PA during the period 2014-2020 were characterized by a polyphasic approach including biochemical and phylogenetic analysis. Thirty-three strains, previously identified by PCR as Psa, have been found to be a homogeneous group in phylogenetic analysis, which seems to indicate that there have been few introductions of the pathogen into the region. Two strains were confirmed as P. syringae pv. actinidifoliorum (Pfm), so this is the first report of Pfm in the PA. The remaining 18 strains were found to be close to P. avellanae and P. syringae pv. antirrhini or to strains described as Pfm look-alikes. Pathogenicity tests carried out on peppers with a selection of strains have shown that both Psa and Pfm caused clear damage, while the 18 atypical strains caused variable lesions. It would be necessary to carry out pathogenicity testing of atypical strains on kiwifruit plants to study the role of these strains in the kiwifruit pathosystem to evaluate their pathogenic potential in this crop.
RESUMO
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.
RESUMO
BACKGROUND: The Asian chestnut gall wasp (ACGW) Dryocosmus kuriphilus Yasumatsu is the most severe pest of the genus Castanea worldwide. After its arrival in Europe, the insect dispersed rapidly from Italy to other European countries, as well as spreading over long distances within each country because of the unintentional transport of infested chestnut material owing to the fact that infestation inside the dormant buds is visually undetectable. Hot water treatment (HWT) has been tested in terms of pest and diseases control in propagation processes with a wide range of plant material and species, including the Asian chestnut Castanea mollissima cv 'Qing'. Taking into account the different thermal tolerances of plant species, an experiment to disinfect Castanea sativa scions from ACGW by HWT was performed. The effects of the immersion of dormant scions for 10 min at two water temperatures (49 and 52 °C), with and without a previous period of water hydration, were tested. RESULTS: A 10 min soak at 49 °C was effective in killing ACGW larvae while retaining an excellent percentage of successful grafts. The pre-HWT hydration stage did not influence the success of the treatment. Positive shoot growth was also recorded after HWT. CONCLUSIONS: We report for the first time the effectiveness of a simple and environmentally friendly protocol based on hot water for the disinfection of European chestnut (C. sativa) scions against D. kuriphilus. This will enable nurseries to supply plants free from this pest, preventing the insect dispersion, which will have a positive socio-economic impact to the chestnut tree sector. Also positive environmental impact will be favoured as gall wasp proliferation will decrease. © 2019 Society of Chemical Industry.
