First Report of Leaf Spot on Paphiopedilum micranthum Caused by Neopestalotiopsis saprophytica in China

Abstract

As a high value ornamental plant, Paphiopedilum micranthum is planted in many provinces in China. In December 2018, a leaf spot disease on P. micranthum was observed in the gardens of Leye County (N 24.78°，E 106.55°), in Guangxi province. At the onset of the infection，the brown spots appeared on the leaves. Subsequently, the tissues around the spots gradually dried up, and the spots gradually expanded to form the necrotic areas. In severe cases, these areas connected to form a large dry rot area on the surface of the leaves, which destroyed the ornamental value of P. micranthum. This leaf spot disease was seen throughout whole year with the maximum incidence (about 30%) in July and August. Beside Leye County, this disease was also found in Nanning (N22.82°/span>, E108.37°) and Yulin(N22.63°, E110.17°), and has been spreading in other parts of Guangxi province. To isolate the pathogen, leaf sections (5 mm × 5 mm) were prepared, surface-sterilized with 75% ethanol (for 30 s) and 2% sodium hypochlorite (NaOCl) (for 1.5 min), rinsed five times with sterilized water. Then placed them on potato dextrose agar (PDA) with 300μg/mL Ticarcillin Sodium and Clavulanate Potassium, and incubated at 28°C for two days. The hyphae grown from the sections were transferred to new PDA media and incubated in the dark until the conidia produced. The single conidia was transferred to new media for pure culture. The isolated strains were cultured on PDA media for seven days at 28℃/span>. The colony attained 80–90 mm diam, with undulate edge, white-coloured, dense aerial mycelium on the surface; reverse similar in colour. After 10-15 days of culturing, the brown gregarious conidiomata were produced on the surface of the mycelium. Conidiomata were globose, confluent and erumpent, exuding globose conidial masses. Conidia (18 to 28 μm × 4 to 7 μm ) (n=10) were fusoid and straight to slightly curved, with 4 septa. The apical cell was hyaline and cylindrical to subcylindrical, with 2-3 filiform apical appendages arising from the apical crest. The 3 cells in the middle were brown, and the cell near the basal cell was lighter than the other two. The basal cell was also hyaline, with single appendage thawas easy to abscise. These morphological characteristics resembled Neopestalotiopsis spp. (Maharachchikumbura, S.S.N，et al. 2014). For further identification, the total genomic DNA of the pathogen was extracted with the CTAB method. For phylogenetic analyses, the first internal transcribed spacer region, the 5.8S nrRNA gene, the second internal transcribed spacer region and the 28S nrRNA gene (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes were amplified with primer pairs ITS5/ITS4, T1/Bt-2b and EF1-728F/EF-2 (Maharachchikumbura, S.S.N，et al. 2014). Finally, the pathogen was identified as Neopestalotiopsis saprophytica basing on phylogenetic tree by the Maximum Parsimony (MP) analyses. The most isolated strains (70%) were this species. The other isolated strains were some saprophytic fungi-such as Alternaria. The pathogenicity of these strains were verified by Koch's postulates. A 30 ml conidial suspension (106 spores/ml) of strains was used to inoculate the healthy plants of P. micranthum and with sterile distilled water as a control. The conidial suspension was smeared on five healthy leaves of each plant. There were five pots of P. micranthum being used in each inoculation treatment and control treatment. The experiment was repeated three times. These treated plants were cultured at room temperature (26-34℃) for appearance of symptoms. After two gap days, brownish spots were appeared on the surface of the leaves. All the treatments inoculated by N. saprophytica appeared the similar symptom as observed in field. The other strains treatments and control treatment did not appear the similar symptom. Similar pathogen was isolated from the necrotic areas again. The re-isolations were identified as N. saprophytica by morphological observation and phylogenetic analyses. According to the other researches, N. saprophytica not only could cause leaf spot on Calanthe sinica in China (Rang S F, 2017), but also on Elaeis guineensis in Malaysia (Ismail S I, et al, 2017). This pathogen can cause leaf spot on P. micranthum, which hasn’t been reported. To the best of our knowledge, this is the first report of Neopestalotiopsis saprophytica causing leaf spot on Paphiopedilum micranthum in China. Furthermore, It is necessary to develop effective management strategies for controlling this disease.