First report of Lasiodiplodia theobromae causing dieback of Citrus reticulata Blanco in India

Abstract

Citrus reticulata Blanco is an important plant grown in Darjeeling Hills, India, for its fruits. In December 2024, dieback symptoms with a disease incidence of 96% were observed among 200 plants aged 35-50 years within the survey area at Chhota Mangwa (27.0499° N, 88.4089° E), Darjeeling district, West Bengal, India. The trees declined over a period of three years. Progressive chlorosis was observed in upper canopy leaves, which developed to necrosis and finally extended downwards, resembling the dieback symptom of the twigs. The necrotic leaves were detached. In severe cases wilting of the branches, premature fruit drop, necrosis of vascular tissue, and eventually death of the trees were also observed. Infected twig tips from fifteen plants with similar symptoms were cut into 5mm pieces, surface sterilized (70% ethanol for 30s, 0.01% HgCl2 for 15s), washed and cultured on PDA plates for 2 days at 28°C. Five pure cultures with similar morphology were obtained by mycelial tip transfer on PDA plates. Isolates initially exhibited fast-growing grayish-white dense aerial mycelia that turned to grayish-black within 10 days. Conidiomata developed after 10 days and were black, globose to sub-globose; immature conidia were ovate, double walled, hyaline, aseptate, and on maturation after 17 days the conidial wall turned dark brown showing a striate appearance with a median septum and size ranging from 19.83-25.80 µm x 11.97-15.81 µm (n=50) morphologically identified the isolates as Lasiodiplodia sp. (Alves et al. 2008; Philips et al. 2013). Molecular identification of the representative isolate MY4 involved amplification of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (tef-1α), β-tubulin (tub2), and RNA polymerase subunit II (rpb2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b (Glass and Donaldson 1995) and rpb2-LasF/rpb2-LasR (Cruywagen et al. 2016) with GenBank accession Nos. PX447540, PX515998, PX515997, PX515996 respectively exhibited 100% identity (497/668 bp) with L. theobromae strain CBS 111530 (FJ150695) for ITS; 99.36% identity (316/466 bp) with strain ICMP 22643 (ON262790) for tef-1α; 100% identity (461/459 bp) with strain CBS 164.96 (EU673110) for tub2; and 99.54% identity (649/911 bp) with culture MFLUCC 23-0075 (OR148430) for rpb2. Phylogenetic analysis with the concatenated sequences of four genes using MEGA 11 confirmed MY4 as Lasiodiplodia theobromae. To confirm the pathogenicity of the isolate, twenty healthy 18-month-old saplings of C. reticulata were taken. Ten saplings were spray-inoculated with a 10 5 conidia/ml suspension of the isolate MY4. The other ten saplings sprayed with sterile distilled water served as controls. Both inoculated and control setups were kept under the greenhouse at 27 ± 2°C, 80% RH, 12-h light/12-h dark photoperiod. After 12 days, all the inoculated plants showed typical dieback symptoms characterized by yellowing of leaves near the shoot apex, followed by progressive browning and death of affected tissues advancing downward, accompanied by leaf abscission; while control plants remained symptom-free. L. theobromae was successfully reisolated from symptomatic twigs, and its identity was confirmed by morphology and ITS sequencing fulfilling Koch’s postulates. Previously, L. theobromae was reported to cause dieback in Citrus reticulata in China (Xiao et al. 2021), cupuaçu (Theobroma grandiflorum) trees in Brazil (de Souza et al. 2025), and nutmeg (Myristica fragrans Houtt.) in India (Biju et al. 2021); L. iraniensis in C. reticulata in Pakistan (Fayyaz et al. 2018). To our knowledge, this is the first report of L. theobromae causing dieback of Citrus reticulata in India. The disease caused severe damage to Chhota Mangwa orange production.