First Report of Phyllosticta citricarpa Causing Citrus Black Spot on Citrus sinensis and C. reticulata in India

Abstract

Citrus is an economically important fruit crop in India. In December 2016, citrus black spot (CBS)-like symptoms were observed on Nagpur mandarin (Citrus reticulata), ‘Mosambi’ sweet orange, and Cutter Valencia (C. sinensis) trees in two orchards situated at Mohpa and Kachimet localities of Nagpur district, Maharashtra state (21.14 to 21.30°N, 78.82 to 79.02°E). Later symptoms were also noticed on Nagpur mandarin and Mosambi fruits in the fresh fruit market of Nagpur (also known as “Orange City”). Fruit symptoms included hard spot, red-brown spot, freckle spot, and virulent spot. To isolate the causal agent, fruit lesions were surface disinfested with 1% NaOCl and plated on potato dextrose agar (PDA) and oatmeal agar (OA). Fifteen single-spore cultures resembling those of the genus Phyllosticta were obtained. On PDA, the colonies had irregular margins, with numerous pycnidia, lined by a translucent zone of colorless submerged mycelium. On OA, a distinct yellow pigment was produced that diffused into the medium around the colony. Conidia (9.6 to 10.3 × 5.9 to 9.0 μm; average, 9.9 × 7.3 μm; n = 30) were hyaline, aseptate, ovoid with a flattened base surrounded by a hyaline matrix (0.8 to 1.1 μm; average, 0.9 μm; n = 30) and a hyaline appendage (6.6 to 13.6 μm; average, 8.8 μm; n = 30) on the rounded apex, corresponding to published descriptions of P. citricarpa (sexual morph Guignardia citricarpa) (Baayen et al. 2002). Leptodothiorella spermatial state formed in pure culture, and spermatia (4.8 to 7.9 × 0.8 to 1.3 μm; average, 7.0 × 1.0 μm; n = 30) were hyaline, dumbbell shaped to cylindrical, and both ends bluntly rounded. No sexual structures were observed. DNA was extracted from six representative isolates (CB1 to CB6) using a Qiagen DNeasy Plant Mini Kit. The nuclear ribosomal internal transcribed spacer (ITS) region (GenBank accession nos. MF000337 to MF000341 and MF062501) and the translation elongation factor 1-alpha gene (tef1) (MF000342 to MF000347) were amplified and sequenced using universal primer pairs ITS5/ITS4 and EF1-728F/EF2, respectively. These isolates shared 99 to 100% nucleotide sequence similarity (for both ITS and tef1) among themselves and had 99 to 100% homology with P. citricarpa ITS sequences from the United States (GenBank no. KC311457) and South Africa (KF709953) and with the P. citricarpa tef1 sequence of an Australian isolate (GenBank no. JF343602). The P. citricarpa-specific primers GCN and GCMR (Peres et al. 2007) produced a 300-bp band from the DNA extracts of fruit lesions and pure cultures but not from healthy citrus tissue DNA. To confirm pathogenicity, two isolates (CB1 and CB4) were inoculated into Mosambi sweet orange (six fruits for each isolate), as described by Rodrigues et al. (2013). After 30 days, all the inoculation sites showed early hard spot-like CBS symptoms. Control fruits remained symptomless. P. citricarpa was consistently reisolated from the fruit lesions and identified by morphology and ITS sequencing. Earlier, P. citricarpa was noted on Citrus medica leaves in Chennai, India (Kumaran et al. 2008), but there was no proof of identification and characterization of the species. To our knowledge, this is the first confirmed report of P. citricarpa causing CBS in two economically important Citrus species, C. sinensis and C. reticulata, in India. The disease is of great importance to the Indian citrus industry because it inflicts serious fruit blemishes and can cause severe yield reduction under heavy disease pressure.