First Report of Clonostachys rosea Causing Root Rot of Lonicera japonica Thunb. in China.

Abstract

Lonicera japonica Thunb., belonging to Caprifoliaceae, is a widely cultivated traditional Chinese medicinal plant with high economic value. In May of 2024, symptoms of a root rot disease were observed on L. japonica in commercial plantings in Longju Town, Baitu Town, and Gaoliang Town in Wanzhou District (30°23'50″N, 107°52'22″E), Chongqing City (southwestern China). Aboveground parts of infected plants were entirely necrotic and displayed weakened lignification of roots with incidence rate of 45 to 70%. To identify the pathogen, root tissues from 17 diseased plants were collected, surface disinfected in 75% ethanol for 30 seconds, immersed in 5% NaClO for 1 minute, rinsed three times with sterile water, dried on sterile absorbent paper, placed on potato dextrose agar (PDA), and incubated at 28°C in the dark. After 5 days, 95% of the samples developed similar-appearing colonies. A single isolate, B1-V1, was selected for morphological and molecular characterization. After 15 days of incubation at 28℃ on PDA, colonies of B1-V1 reached a diameter of 80 mm; white mycelium grew slightly above the surface of the plate, was velvety in texture, and produced a yellow pigment. Hyaline, septate hyphae produced verticillate conidia that were branched like broomsticks, and conidiophores were transparent and elliptical with a single conidiophore measuring (2.31 - 4.75) × (5.33 - 8.75) μm (n = 32). The morphology was consistent with a previous description of Clonostachys rosea (Wang et al. 2024). The internal transcribed spacer region (ITS), β-tubulin gene (TUB2) and second subunit of RNA polymerase II gene (RPB2) were amplified by primers ITS1/ITS4 (Wang et al. 2024), T1/βt2b (Wang et al. 2024). and RPB2F/RPB2R (Long et al. 2021), respectively. The corresponding sequences from B1-V1 were submitted to GenBank under accession numbers PV368674 (ITS), PV390661 (TUB2), and PV390662 (RPB2). According to a BLAST search, the pathogen displayed the highest sequence similarity to strains GY12 (ITS: OR789466.1, identities: 545/545 bp), JABY1 (TUB2: OP868694.1, identities: 601/617bp) and HZL-6-8 (RPB2: PP486409.1, identities: 896/900bp). A phylogenetic tree was constructed based on concatenated ITS-TUB2-RPB2 sequences using Maximum Parsimony methods by MEGA7 software, revealing that the isolate was most closely related to C. rosea strain TMB (ITS: 491/498bp, TUB2: 311/455bp, RPB2:753/753bp). To confirm pathogenicity, both cuttings derived from branches of two-year-old plants of L. japonica Thunb. and one-year-old plants (n = 6 per group) were surface-sterilized with 75% ethanol. Roots were wounded with a sterile needle and soaked in a 4 × 10⁷ conidia/mL suspension of isolate B1-V1 for 20 days. After inoculation, symptoms including leaf wilting, yellowing, and rot browning and rot were observed, and the number of new roots significantly decreased. Colonies re-isolated from symptomatic roots were morphologically consistent with the original inoculum, thus fulfilling Koch's postulates (Wang et al, 2024). To our knowledge, this is the first report of C. rosea causing root rot in L. japonica Thunb. and provides an essential basis for root rot disease management on this crop in the future.