First report of Truncatella angustata causing leaf blight on Thuja plicata Donn ex D. Don in Canada

Abstract

Western redcedar (Thuja plicata Donn ex D. Don) is one of the most important commercial tree species in British Columbia, generates more than $1 billion in economic activity annually and about 8-10 million trees are planted in reforestation efforts (Gregory et al. 2018). It has been selected as the provincial tree of British Columbia (BC) because of its tremendous economic, ecoogical and cultural value. However, foliar diseases such as leaf blights have serious impact on redcedar growth and may cause significant loss of tree volume (Russell, 2007). Our 2014 - 2015 surveys of western redcedar forests in coastal areas of BC indicated high incidence of a distinctive type of blight. We observed the incidence of this disease on more than 80% of western redcedar (approximately 493) trees from late May to early December. Early symptoms appeared as circular to oval, brownish to black spots (2–3 mm), 1–5 spots per branch tip, scattered at the tip margins. Sequentially, the spots enlarged and developed into necrotic lesions on both young and old leaves. More than 50 symptomatic leaves from 10 different trees were collected and rinsed in distilled water then surface-sterilized with three times washing in Tween 20 (%5 solution) for 2 minutes (each time) and %70 ethanol for 30 second (3 times repeat). Tissues from under lesions were placed on MEA (Malt Extract Agar; Phyto Tech® labs-Product ID: M498) and PDA (Potato Dextrose Agar; Phyto Tech® Labs-Product ID: P772). The plates were incubated at 21°C in the dark. They developed distinct dull white to brown, cottony colonies with each black acervuli approximately 450-500µm. The isolates produced fusiform conidia with four cells. They didn’t have any distinct color. The conidiophore size was approximately 23-24 x 2-3 µm with mostly hyaline to light brown color, branched and conidiogenous was hyaline and not branched and simple. The spore size was approximately 15-20μm by 7-10μm with three transverse septa and endogenous papillae with hyaline apical appendages. Next, we collected spores and replated them on fresh MEA media culture and placed back in the incubator to produce pure cultures. We studied conidia from leaves of trees mentioned above using light and electron microscopy using Hitachi S-3500N Scanning Electron Microscope (Noshad et al. 2023). After morphological study, further identification to the species level conducted using Zambounis and Wenneker’s approach (Zambounis 2019; Wenneker,2017). Genomic DNA from two single-spore isolates were isolated and sequenced. Sequences of ITS (Internal Transcribed Spacer) region amplified using primers ITS1/ITS4 and sequenced. Final sequences were deposited in Genbank and published (accession numbers OP086244 and OP086251). Blast analysis of these sequences showed 99% and 99% resemblances with T. angustata sequence (Sutton 1980). To verify its pathogenicity, we performed a comprehensive pathogenicity test to fulfill Koch’s postulates. We collected their distinctive spores in an aseptic environment and standardized them (5000/ml) using a haemocytometer. Then we inoculated 100 western redcedar seedlings (three years old) by injecting standardized spore suspension solution (inoculum) using ultra-fine 0.3ml, 31G, 8mm syringes (approximately 0.1ml per inoculation site). Ten positive control seedlings were inoculated with distilled water and ten negative control seedlings were not inoculated at all. All inoculated (experimental) seedlings demonstrated same symptoms (black spots and characteristic spores) after eight weeks. None of the control seedlings showed any similar symptoms. In the next stage, we isolated and cultured spores from inoculated seedlings and studied them. The identity of reisolates confirmed using DNA sequencing. We used these spores for our next set of disease screening which was successful again. We identified Truncatella angustata (Pers.) Hughes as the causal agent for shoot-tip blight (STB) on western redcedar by examining morphological and molecular characteristics of the pathogen. This is the first report of T. angustata as a primary pathogen on western redcedar in British Columbia, Canada.