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First Report of Eutypa lata Causing Branch Dieback and Cankers on Cape Willow in South Africa

Moyo, P., Mostert, L., Dedekind, R., van Jaarsveld, W. J., Pierron, R., Halleen, F.
Plant disease 2018 v.102 no.10 pp. 2033
DNA primers, Eutypa lata, Salix, asci, ascospores, branches, chloramphenicol, conidia, culture media, dead wood, demonstration farms, dieback, discoloration, ethanol, fruit crops, fruit trees, fungi, genes, girdling, inoculum, internal transcribed spacers, mycelium, necrosis, orchards, pathogenicity, pathogens, perithecia, polymerase chain reaction, ribosomal DNA, shoots, sodium hypochlorite, streams, tissues, tubulin, vineyards, wood, South Africa
The Cape willow (Salix mucronata Thun.), a member of the Salicaceae, is a widespread and evergreen tree that grows along streams and river banks in South Africa (Burtt Davy 1922). Between April 2014 and December 2015, several willow trees in close proximity to vineyards in the Western Cape Province of South Africa were observed to be affected by twig and whole branch dieback as well as girdling cankers, often extending from branches to the trunks. Stromata containing fruiting bodies (perithecia) were also observed on dead wood of several trees. Internally, irregularly shaped light-brown to dark-brown lesions, reddish-brown lesions, brown-black streaks, and sectorial necrosis were observed. Wood tissues collected from the border between symptomatic and healthy tissue were surface sterilized (70% ethanol for 30 s, 2.5% sodium hypochlorite for 2 min, and 70% ethanol for 30 s) and plated on potato dextrose agar plates containing chloromycetin (250 mg/liter) (PDA-C). Isolations from fruiting bodies were also conducted (Moyo et al. 2018). Cultures were incubated at 24°C, and colonies were cottony white initially, turning gray with age. Conidia produced on PDA-C were filiform measuring (17 to) 21 to 39 (to 42) × 1 to 2 μm (n = 60). Perithecia were 300 to 550 µm in diameter, and asci were spindle shaped, eight spored, and 28 to 65 × 5 to 8 μm. Ascospores were subhyaline, allantoid, and 7 to 12 × 2 μm; all fit the description of Eutypa lata (Pers.) Tul. & C. Tul. (Munkvold 2001). The rDNA internal transcribed spacer (ITS) region and β-tubulin (TUB2) gene of two isolates (STEU 8521 and STEU 8522) were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Moyo et al. 2018). The sequences obtained were deposited in GenBank (accession nos. MG978310 and MG978311 for ITS, MG978306 and MG978307 for TUB2). Nucleotide BLAST analysis of ITS sequences showed a 100% identity matching with E. lata strain ONCC1 (KF453557) and strain LATA1 (KR909218) for STEU 8521 and STEU 8522, respectively. TUB2 sequences for both isolates were 100% similar to E. lata strain CBS 622.84 (DQ006964). Pathogenicity of the two isolates was tested on shoots of mature willow trees at the ARC experimental farm Nietvoorbij, Stellenbosch, Western Cape, between March and June 2017. Inoculations were done by placing a mycelial plug into 4-mm wounds. Sterile PDA plugs were used to inoculate control shoots, and all inoculated wounds were sealed with Parafilm. Each isolate or control treatment was inoculated onto five different healthy shoots on one tree. The experiment was repeated on a second tree. After 12 weeks, inoculated shoots showed brown to black lesions extending in an upward and downward direction from the inoculation point. No discoloration was observed on control shoots. Lesion lengths ranged from 19.3 to 25.4 mm. The inoculated fungus was isolated from all the inoculated shoots but not from the control shoots. To our knowledge, this is the first report of E. lata causing cankers on the Cape willow in South Africa. This finding is important because willow could potentially serve as an inoculum source of E. lata to vineyards and other fruit tree orchards and, therefore, needs to be taken into consideration when managing trunk disease pathogens on fruit crops in South Africa.