First Report of Diaporthe cynaroidis and D. australafricana Associated with Walnut Branch Canker in Chile

Abstract

In 2017, with more than 40,000 planted hectares, Chile became the second exporter of English walnut (Juglans regia) worldwide with a production of about 100,000 tons1. In 2017, low incidence of branch canker from old pruning wounds was observed in young walnut orchards cv. ‘Chandler’ in the Maule region of Chile. Fifty symptomatic wood samples were collected from ten trees in two orchards. Isolates were recovered from the wood necrotic lesions displaying a light to dark brown discoloration in the cortical and vascular tissue as previously described2. The isolates were identified as Diaporthe cynaroidis and Diaporthe australafricana based on morphological characteristics and DNA sequencing. Both species were grown on Potato Dextrose Agar medium and sterile grape leaves to induce sporulation but only D. cynaroidis produced alpha conidiospores. Alpha conidia were fusiform to ellipsoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown at maturity, with size of 6.1 µm ± 0.5 × 2.5 µm ± 0.1. Beta conidia were extremely rare. Fungal DNA was extracted from fungal colonies with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), elongation factor (EF) and s-tubulin (sT) loci were amplified using primer pairs (ITS1/ITS4, EF728F/EF987R, and Bt2a/Bt2b) and methods previously described3. One isolate from each species was sequenced and D. cynaroidis (isolate #2248) and D. australafricana (isolate #2249) showed 99.13% and 99.65% sequence homology with NCBI voucher specimens MB#506209 and MB#344439, respectively. Sequences were deposited in the GenBank database; D. australafricana accession numbers ITS, KC343038; EF, KC343764; BT, KC344006; and D. cynaroidis accession numbers: ITS, KC343058; EF, KC343784; BT, KC344026. Pathogenicity tests were performed by drilling a hole in stems of two-year old walnut trees cv. ‘Chandler’ (n=20) and inoculating with D. cynaroidis and D. australafricana isolates using 25 µl of inoculum at 1×104 spores/µl. Ten control plants were treated with 25 µl sterile water. The experiment was repeated two times on separate sets of trees. After 16 weeks of incubation in a shaded open house, the lesions were light to dark brown in both the cortical and vascular tissues. The lesion length produced by D. australafricana averaged 31.9 mm ± 8.8 mm, D. cynaroides averaged 26.5 mm ± 7.1 mm and both were significantly longer (P <0.0082 and P <0.0061, respectively) than the control, which averaged 1.3 mm ± 0.6 mm. Both Diaporthe species were re-isolated from all the inoculated plants and identified by colony morphology in order to fulfill Koch’s postulate. Previously, only D. neotheicola and D. rushicola were reported to be pathogenic to walnut in California2 and Spain4. D. australafricana was only reported as the causal agent of stem canker of grapevine (Vitis vinifera L.) in Australia and South Africa5 and of blueberry (Vaccinium corymbosum L.) in Chile6. Additionally, D. cynaroidis has been identified as a pathogen of king protea (Protea cynaroides L.) in South Africa6. Blueberry, grape, and king protea are all grown in Chile and these findings warrant further investigation in order to determine the host range, distribution and incidence of these pathogens in the area.