First Report of Root Collar Canker Caused by Neoscytalidium dimidiatum on Jacaranda mimosifolia in China.

Jacaranda mimosifolia is widely cultivated as a garden ornamental tree. In July 2023, an unknown root collar canker of J. mimosifolia was discovered in green belts of Qingxiu District, Nanning, China, with a 8% incidence rate. Crowns of affected trees ranged from reddish brown leaves to deciduous or dead. Root collar tissue became necrotic matched by underbark dark brown lesions with irregular margins, and rotted at last. Six diseased plants distributed within 3000 m2 were choosed, and 24 root collar tissues were surface sterilized and placed on potato dextrose agar (PDA) plates to incubate at 28℃ for 3 to 5 days. Same colonies were consistently isolated from 18 tissues, and three isolates (M3-B1-1, M3-B1-2 and M3-B1-3) were purified for morphological and molecular determination. These isolates formed colonies with lush aerial mycelia rapidly, which covered a 90 mm plate in 72h. The colonies were initially white, then grayish-green to black. Arthrospores were colourless to light brown, short columnar, aseptate, truncate base, averaging 12.1±2.5 µm × 3.4±0.7 µm, sometimes formed arthric chains. Chlamydospores were dark brown, round or oval, aseptate, averaging 8.7±1.6 µm × 5.0±0.9 µm. Mature pycnidia and conidia produced for about 50 days on oatmeal agar medium (OMA), and conidia were colorless, oblong, aseptate, averaging 11.2±1.2 µm × 6.0±1.4 µm. These morphological characteristics were consistent with the description of Neoscytalidium dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Genomic DNA was extracted from three isolates. The partial ITS region, TUB2 and TEF1-α genes were amplified (White et al., 1990; Glass and Donaldson 1995; Carbone and Kohn 1999). The sequences were deposited in GenBank (ITS: PP939650-PP939652; TUB2: PP942728-PP942730; TEF1-α: PP942731-PP942733). Blastn analysis revealed that ITS sequences of three isolates showed 99.8%, 100%, 100% identity (506 bp out of 507 bp, 507 bp out of 507 bp, 507 bp out of 507 bp) to N. dimidiatum C21 (KX447539), the TUB2 sequences showed 100% identity (436 bp out of 436 bp, 437 bp out of 437 bp, 437 bp out of 437 bp) to N.dimidiatum LNeo (ON099066), and the TEF1-α sequences showed 99.64% identity (276 bp out of 277 bp) to N.dimidiatum ARM230 (MK495384), respectively. Phylogenetic analysis based on concatenated ITS, TUB2 and TEF1-α sequences showed that three isolates were clustered into the same clade as N. dimidiatum. To fulfill Koch's postulates, pathogenicity of these isolates was tested on healthy two-year-old J. mimosifolia trees. Stem and root collar were wounded and placed mycelial plugs (8mm), and the inoculation sites were wrapped with parafilm or covered with nursery substrate to maintain the humidity. Four plants were inoculated with each isolate. As a control, four plants were inoculated with noncolonized PDA plugs. All treated plants were kept in a greenhouse at 28 ± 3°C and 70% relative humidity. Foliar blight and necrotic lesions around inoculation points were observed about 65 days after inoculation, and 50% of inoculated trees exhibited symptoms, whereas the control trees remained symptomless. Neoscytalidium dimidiatum was successfully reisolated from symptomatic tissue via morphological analysis. To our knowledge, this is the first report of root collar canker caused by N. dimidiatum on J. mimosifolia. Neoscytalidium dimidiatum has a wide range of hosts, including pitaya, pine, mulberry, pear, grape, locust tree (Luo et al. 2024). This finding will help in controlling of the disease epidemic.