Gentian rhodantha Franch. ex Hemsl, a perennial herb belonging to the Gentianaceae, is mainly distributed at high altitudes (900–1800 m) in mountainous areas in southwest China. The aerial parts of this herb (including flower, leaf, and stem) are widely used in Tibetan and Miao traditional ethnomedicine for the treatment of cough, bronchitis, hepatitis, and dysentery (Xu et al. 2011).
In August 2018, leaf spots were widely observed in a survey of cultivated field of G. rhodantha in Liupanshui, Guizhou Province, China. The typical symptoms on leaves were small brown spots in center or edge of the leaves, which expand to circular or irregular spots with purple halo (Fig. 2a) and even completely wither. In this study, three Pestalotiopsis-like strains (LBB062904, LBB062905 and LBB062906) were isolated from leaf spots of G. rhodantha field by single-spore isolation (Chomnunti et al. 2011).
For molecular analysis, total genomic DNA of fresh pure cultures were extracted using the SDS-CTAB method (Suwannarach et al. 2010). The internal transcribed spacers (ITS) region of rDNA molecule was amplified using primer pairs ITS5 and ITS4, the β-tubulin gene (TUB) region with BT2A and BT2B primer pairs, and the translation elongation factor 1-alpha (tef1) gene using the EF1-728F/EF2 (Maharachchikumbura et al. 2012). PCR was performed with the 25 μl reaction system consisting of 19.75 μl of double distilled water, 2.5 μl of 10 × Taq buffer with MgCl2 , 0.5 μl of dNTP (10 mM each), 0.5 μl of each primer (10 μM), 0.25 μl Taq DNA polymerase (5 U/μl), and 1.0 μl of DNA template. PCR amplification protocols were performed as described by Maharachchikumbura et al. (2012). The DNA sequences obtained in this study have been deposited in the DDBJ/EMBL/GenBank database with accession numbers MT539135 to MT539137 and MT671942 to MT671947.
Phylogenetic tree was constructed by MEGA X (Kumar et al. 2018) for the combined data set of the ITS, tef1 and TUB genes for 31 Pestalotiopsis strains (Table 1), P. camelliae CBS 443.62 was used as outgroup. Bootstrap value ≥ 50% (1000 replications) by the maximum likelihood (ML) methods were shown on the respective branch. The aligned results comprised 1413 characters including partial gaps (ITS: 1–509, tef1: 510–985 and TUB: 986–1413). Of these characters, 1275 were constant, 54 were variable and 106 were parsimony-informative. 31 Pestalotiopsis strains formed a strong clade (100% bootstrap support). Three strains obtained were clustered to P. trachicarpicola with an 82% bootstrap value (Fig. 1).
Strains sporulation were observed on synthetic nutrient-poor agar (SNA) amended with double-autoclaved pine needles placed on the agar surface, and incubated at 25°C for 15 days (Liu et al. 2017). Double-autoclaved Taxus chinensis needle was used to induce sexual forms, and the strains were inoculated in the center of SNA plates that contained needles and incubated at 20°C for 8 weeks.
The colonies on potato dextrose agar (PDA) were 7.30±0.51 cm (n=5) diameter at 25°C after 5 days, with undulate and redial edge, convex with plat surface, white to faint yellow on front, back pale honey-colored (Fig. 2b, c). Conidiomata pycnidial at pine needles in culture on SNA, globose, scattered, black, teardropform, 100–500 μm diameter (Fig. 2d–e); Conidiophores short, subcylindrical, hyaline, smooth, (Fig. 2f). Conidiogenous cells ampulliform, hyaline, 7.3–14.5×2.3–3.8 μm (x±SD=8.23± 4.33×2.56±0.78 μm, n=15). Conidia fusoid, olivaceous to thin yellow, 4-septate, 20–26×5–9 μm (x±SD=22.9±1.3×6.5±0.8 μm, n=30), thin-walled and verruculose wall (Fig. 2g–j); Basal cell obconic, thin-walled, hyaline, 3.5–6.4 μm long; Three median cells dolioform, versicolor, 13–17 μm long (x±SD=15.1±0.7 μm, n=30), verruculose, pale-yellow to brown, septa darker; apical cell conical, hyaline, smooth wall, 2.8–5.5 μm long, with 1–4 concurrent tubular apical appendages (mostly 3), filiform, unbranched, arising from the apical crest, 8–21 μm long; single basal appendage is straight, 3–7 μm long.
Ascomatas on Taxus chinensis needle were 130–320 μm diam, black, gregarious, immersed in leaves epidermis and raised slightly (Fig. 2k). Asci 8-spored, bitunicate, cylindrical, 67–94×6–10 μm (x±SD=84.8±7.6×9.4 ±1.3 μm, n=30) (Fig. 2 l–m), mature ascus without wall, indistinct J+ (amyloid ring) in apical apparatus (Fig. 2n). Ascospores 13.5–15.2×5.1–7.5 μm (x±SD=14.3±0.9 ×6.5±0.4 μm, n=30), uniseriate or interlaced, 2–4 cells (mostly 3) ascospore concolorous, light yellow to brown, oblong to fusiform, smooth or verrucose, brown septate and slightly constricted at the septa(Fig. 2o–r). Based on molecular analysis and unsexual and sexual characters, three strains were identified as P. trachicarpicola Y.M. Zhang & K.D. Hyde (Zhang et al. 2012), To the best of my knowledge, this is the first report of P. trachicarpicola causing leaf spot of G. rhodantha.
P. trachicarpicola have many asexual features similar to P. neglecta, P. kenyana and P. oryzae, which have thin and olivaceous walled conidia, concolourous medial cells and branches of apical appendages. But there are some significant differences between them (Table 2). The conidia of P. trachicarpicola (x=22.9×6.5 μm) are shorter than those of P. kenyana (x=25.5 μm) and P. oryzae (x=26.9 μm) and the apical appendages of P. trachicarpicola (8–21 μm) are shorter than P. neglecta and P. oryzae (Maharachchikumbura et al. 2014; Steyaert, 1953; Zhang et al. 2012). The asci and ascospores of P. trachycarpicola is morphologically mostly similar to P. neglecta and P. accidenta, but the asci of P. trachycarpicola are thinner than P. neglecta, and ends of the ascospores of P. trachicarpicola are more rounded and some are verrucose compare with P. neglecta and P. accidenta (Kobayashi et al.2001; Zhu et al, 1991).
In pathogenicity tests, detached leaves of G. rhodantha were surface-disinfected with 75% EtOH for 30 s and washed five times with sterilized water. Leaves were slightly wounded with a sterile needle and then inoculated a 2 mm diam PDA mycelial disk and inoculated sterilized PDA as controls and placed in Petri dishes at 25°C. There were 10 leaves for each treatment and performed three times. The inoculated leaves had observed symptoms consisted of circular, brown to black spots and highly similar to pathology characteristics of the specimen at 7th days after inoculation (Fig. 2s–t). None of the control leaves had symptoms. The same fungus was re-isolated from symptomatic lesion.
To determine the optimal temperature and pH on spore germination and mycelial growth of P. trachicarpicola, eight temperature gradients (5–40°C) and ten pH values (3.0–12.0) of PDA medium were designed as a randomized single factor experiment. All pates for pH tests were cultured at 25°C. Experiments were conducted 3 times in the dark. Germination rate were calculated we counted the number of total and germinated spore in 10 visions under low-power microscopic (10 × 10) at 12 h, and measured the diameters of colony after 7 days. The results showed that the mycelia grew at a temperature range of 10 to 35°C, with optimum growth at 20 to 25°C. The conidia sprout had a temperature range of 15–40°C, and the optimum temperature were 25–30°C (Fig. 3a). After treatment of medium with different pH values, mycelia could grow at pH 4 to 12, with optimum growth rate and conidia germination rate at 8 and 6–9, respectively (Fig. 3b).
Brown leaf spot and leaf blight caused by Mycochaetophora gentianae (Nekoduka et al. 2013) and Septoria gentianae (Verkley et al. 2013) were two serious disease on overground parts of Gentiana plants and widely occurred in fields, but they symptoms were significantly different with leaf spot in size, color and edge type in focuses. There were without any records of Pestalotiopsis diseases on wild and cultivated G. rhodantha yet. Therefore, we propose that leaf spot caused by P. trachicarpicola be added as novel disease on G. rhodantha.