Identification and Characterization of Diaporthe citri as the Causal Agent of Melanose in Lemon in China

Abstract

Lemon, widely used in food, medicine, cosmetics, and other industries, has considerable value as a commodity and horticultural product. Previous research has shown that the fungus Diaporthe citri infects several citrus species, including mandarin, lemon, sweet orange, pomelo, and grapefruit, in China. Although D. citri has been reported to cause melanose disease in lemons in China, key pathological evidence, such as Koch's postulates fulfillment on lemon fruits and detailed morphological characterization, is still lacking. In May 2018, fruits, leaves, and twigs were observed to be infected with melanose disease in lemon orchards in Chongqing municipality in China. The symptoms appeared as small black discrete spots on the surface of fruits, leaves, and twigs without obvious prominent and convex pustules. D. citri was isolated consistently from symptomatic organs and identified provisionally based on the morphological characteristics. The identification was confirmed using sequencing and multigene phylogenetic analysis of ITS, TUB, TEF, HIS, and CAL regions. Pathogenicity tests were performed using a conidium suspension, and melanose symptoms similar to those observed in the field were reproduced. To our knowledge, this study provides the first comprehensive evidence for D. citri as a causal agent of melanose disease in lemons in China, including morphological characterization and pathogenicity assays on lemon fruits. This report broadens the spectrum of hosts of D. citri in China and provides useful information for the management of melanose in lemons.

Keywords: Citrus limon; Diaporthe citri; melanose disease; molecular identification; pathogenicity test.

Figure 1

Morphological characteristics of D. citri. (A–C) Colony morphology on PDA medium after 5-day incubation at 25 °C. (D–F) Pycnidial conidiomata produced on PDA. (G,I) Ellipsoidal α-conidia. (H) Both α- and β-conidia (black arrows indicate β-conidia). Scale bars: 20 μm (G), 100 μm (H,I), 300 μm (B,C,E,F), 500 μm (D).

Figure 2

Phylogenetic tree of the Diaporthe species based on concatenated ITS, TUB, TEF, HIS, and CAL sequences. The majority rule consensus tree from maximum likelihood (ML) analysis was shown for the phylogenetic relationships of Diaporthe species, the tested isolates (CQTN-1, CQTN-2, and CQTN-3) grouped with D. citri strain CBS134239, AR3405, ZJUE0413, ZJUE0254, and CPC34235 in the same clade. The tree was rooted with Diaporthella corylina (CBS 121124). ML bootstrap values > 50% are shown at the branch nodes. Culture collection numbers are indicated behind the species names. Phylogram was generated with ML analysis based on the K2+G model.

Figure 3

Symptomatology and pathogenicity assessment of citrus melanose caused by D. citri. (A,B) Field symptoms on fruits with characteristic melanized punctate lesions (0.3–1 mm diam). (C,D) Leaf lesions under natural infection. (E,F) Twigs symptoms observed in orchard conditions. (G) Artificially inoculated detached leaves showing enhanced lesion density due to controlled spore concentration (1 × 10⁶ conidia/mL). (H) Potted plant inoculation reproducing field-identical fruit symptoms. (I) Detached leaf assay results.