First Report of Neofusicoccum parvum Causing Stem Blight and Dieback of Highbush Blueberry in the Czech Republic

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F62156489%3A43510%2F23%3A43924433" target="_blank" >RIV/62156489:43510/23:43924433 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1094/PDIS-03-23-0595-PDN" target="_blank" >https://doi.org/10.1094/PDIS-03-23-0595-PDN</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1094/PDIS-03-23-0595-PDN" target="_blank" >10.1094/PDIS-03-23-0595-PDN</a>

Jazyk výsledku

angličtina

Název v původním jazyce

First Report of Neofusicoccum parvum Causing Stem Blight and Dieback of Highbush Blueberry in the Czech Republic

Popis výsledku v původním jazyce

Neofusicoccum parvum (Pennycook &amp; Samuels) Crous, Slippers &amp; A.J.L. Phillips, is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, and apoplexy), and the plant usually declines within a few weeks (Slippers and Wingfield 2007). In August 2022, blueberry plants displaying the symptoms described above were observed in a production orchard located in Hovorany, Czech Republic. Around 3% of 1,000 observed plants were symptomatic. To identify the pathogen, leaves, stems, and roots of three diseased plants were collected, sectioned into small pieces (5 x 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% NaOCl), rinsed three times in sterile distilled water, plated on PDA supplemented with 0.5 g/liter of streptomycin sulfate (Biosynth, Staad, Switzerland), and incubated at 25oC for 2 weeks in the dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. Thirty-three fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 days at 25oC) were felty and white to iron gray in the center. Conidiomata were observed on sterile pine needles on 2% water agar at 25oC under near-UV light after 2 weeks (110 to 220 x 60 to 175 μm). Conidia (n = 30) were cylindrical to ellipsoidal, hyaline, 0 to 1 septate, and 3.8 to 8.1 x 2 to 3 μm. Two representative isolates (CBS 149846 and CBS 149847) were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. The partial ITS regions, tub2 gene, and tef gene were amplified from the genomic DNA of both isolates using primers and following protocols previously described by Eichmeier et al. (2020). Newly generated sequences were deposited in NCBI GenBank (accession nos. OQ376566 and OQ376567 for ITS, OQ401701 and OQ401702 for tub2, and OQ401699 and OQ401700 for tef), which were &gt;99% identical (ITS, 483 of 484 nt; tub2, 426 of 430 nt; tef, 230 of 232 nt) with the ex-type ITS (AY236943), tub2 (AY236888), and tef (AY236917) sequences of the N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, 1-year-old canes of ten 2-year-old V. corymbosum plants grown in pots were wounded by a 5-mm-diameter cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs as controls. The wounds were covered by sterile wet cotton and sealed with Parafilm, and the inoculated plants were maintained in a growth chamber at 20oC with a 12-h/12-h light/dark period. Within 2 weeks, inoculated shoots changed color from green to dark brown and exhibited dark necroses under the bark; after 1 month, the inoculated plants declined, whereas the controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100% reisolation rate, and its identity was confirmed by sequencing the ITS region. The experiment was repeated. N. parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal, and Uruguay (Farr and Rossman 2021). Pecenka et al. (2021) reported the presence of another pathogen, Lasiodiplodia theobromae (Pat.) Griffon &amp; Maubl., from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

Název v anglickém jazyce

First Report of Neofusicoccum parvum Causing Stem Blight and Dieback of Highbush Blueberry in the Czech Republic

Popis výsledku anglicky

Neofusicoccum parvum (Pennycook &amp; Samuels) Crous, Slippers &amp; A.J.L. Phillips, is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, and apoplexy), and the plant usually declines within a few weeks (Slippers and Wingfield 2007). In August 2022, blueberry plants displaying the symptoms described above were observed in a production orchard located in Hovorany, Czech Republic. Around 3% of 1,000 observed plants were symptomatic. To identify the pathogen, leaves, stems, and roots of three diseased plants were collected, sectioned into small pieces (5 x 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% NaOCl), rinsed three times in sterile distilled water, plated on PDA supplemented with 0.5 g/liter of streptomycin sulfate (Biosynth, Staad, Switzerland), and incubated at 25oC for 2 weeks in the dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. Thirty-three fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 days at 25oC) were felty and white to iron gray in the center. Conidiomata were observed on sterile pine needles on 2% water agar at 25oC under near-UV light after 2 weeks (110 to 220 x 60 to 175 μm). Conidia (n = 30) were cylindrical to ellipsoidal, hyaline, 0 to 1 septate, and 3.8 to 8.1 x 2 to 3 μm. Two representative isolates (CBS 149846 and CBS 149847) were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. The partial ITS regions, tub2 gene, and tef gene were amplified from the genomic DNA of both isolates using primers and following protocols previously described by Eichmeier et al. (2020). Newly generated sequences were deposited in NCBI GenBank (accession nos. OQ376566 and OQ376567 for ITS, OQ401701 and OQ401702 for tub2, and OQ401699 and OQ401700 for tef), which were &gt;99% identical (ITS, 483 of 484 nt; tub2, 426 of 430 nt; tef, 230 of 232 nt) with the ex-type ITS (AY236943), tub2 (AY236888), and tef (AY236917) sequences of the N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, 1-year-old canes of ten 2-year-old V. corymbosum plants grown in pots were wounded by a 5-mm-diameter cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs as controls. The wounds were covered by sterile wet cotton and sealed with Parafilm, and the inoculated plants were maintained in a growth chamber at 20oC with a 12-h/12-h light/dark period. Within 2 weeks, inoculated shoots changed color from green to dark brown and exhibited dark necroses under the bark; after 1 month, the inoculated plants declined, whereas the controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100% reisolation rate, and its identity was confirmed by sequencing the ITS region. The experiment was repeated. N. parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal, and Uruguay (Farr and Rossman 2021). Pecenka et al. (2021) reported the presence of another pathogen, Lasiodiplodia theobromae (Pat.) Griffon &amp; Maubl., from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

Druh

O - Ostatní výsledky

CEP obor

—

OECD FORD obor

40105 - Horticulture, viticulture

Projekt

<a href="/cs/project/EF16_017%2F0002334" target="_blank" >EF16_017/0002334: Výzkumná infrastruktura pro mladé vědce</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů