First report of Curvularia muehlenbeckiae, the causal agent of Curvularia leaf spot on pecan (Carya illinoinensis) in China.

Abstract

The pecan (Carya illinoinensis) industry is largely affected by the increased incidence of diseases (Xiao et al 2021). Leaf spot symptoms were identified in an orchard of cultivar Pawnee pecan trees at Zhejiang A&F University, Zhejiang, China in August 2020. Small black spots occurred on the veins and edges of the leaves and nearby tissues turned yellow and slightly deformed (May to July). The spots (0.5-1.5cm) spread to all leaves with 25% to 40% leaf drop occurring in almost all trees between August and October. The causal pathogen was isolated from leaves with target symptoms using the following method: surface sterilized with 70% alcohol (2×, 30 s), rinsed with sterilized water (3×), leaf spots excised and placed on PCA media, and left to incubate at 28℃ in the dark for 3-d. Mycelium on the edge of each clone was excised and incubated on fresh oatmeal agar medium with a 12-hour light/dark cycle for 7-d to obtain conidia. Single spore isolates were germinated on PDA medium under the same conditions as previously described, one 5-mm hyphal plug was transferred to fresh PCA media to obtain the pure cultures. The pathogenicity of the isolates were verified using Koch's postulates. Non-wounded healthy leaves (disinfected with 1% NaClO) of cv. Pawnee (disease susceptible) were obtained from seedlings grown in green-house at 26 ℃ and inoculated with 5-mm hyphal plugs and a conidia-hyphae suspension (~106/mL) containing one-week old purified cultures. After 3-15 days post-inoculation, small black spots appeared on the leaves inoculated with isolate P-6 (the only pathogenic isolate from the leaf spots in the orchard) and grew larger until the whole leaf wilted while the control leaves remained asymptomatic. The experiment was repeated two times with two bio-replicates each run. Finally, the pathogen was re-isolated from infected leaves, which showed the same symptoms as the previous isolate. Aerial mycelia of P-6 turned from white to gray and substrate mycelia from brown to black. Colonies had a fimbriate margin before mycelia filled the medium. Hyphae were septate, branched, brown or black, smooth wall and 1.4-10 µm in width. Conidiophore single-branch, dark brown, curved or straight, 1.93-5.51×44.12-104.41 μm width, conidiogenous cells 6.66-16.67 µm (terminal) and 8.82-23.33 µm (intercalary) length, mono- to polytretic, proliferating sympodially. Conidia (n=20) four cells, 15.7 - 25.7 μm × 7.1 - 11.4 μm wdth, swelling and curving from the basal cell to the third. The bending angle was 5° to 80°. The middle two cells were brown and usually verruculose, the basal and apical cells paler and less ornamented. No sexual morph observed. The morphological characteristics matched previous descriptions of Curvularia species (Madrid et al 2014). The identity of P-6 was confirmed by phylogenetic reconstruction using the concatenated sequences of ITS rDNA, partial GAPDH, LSU, TEF-1α and RPB2 regions (Raza et al 2019). The genomic DNA of P-6 was extracted by the M5 hipermix-MF859 (Mei5 Biotechnology). The sequences of P-6 were used for nucleotide BLAST against the Standard databases and model strains were selected to construct the concatenated sequences of GAPDH-ITS-LSU-TEF-1α-RPB2 for building a phylogenetic tree. This analysis identified P-6 as a strain of C. muehlenbeckiae, a species with few reports other than in gramineous crops (Raza et al 2019; Chen et al 2021; Cui et al 2020; Ni et al 2016). This is the first report of C. muehlenbeckiae on pecan in China and worldwide.