Sansevieria trifasciata, commonly known as mother-in-law’s tongue, is a popular ornamental plant that can be found throughout Malaysia. Besides its attractive dark and light green variegation, S. trifasciata is tolerant to a wide variety of habitats. Sansevieria trifasciata were reported to be infected by Colletotrichum sansevieriae (Nakamura et al. 2006) and Chaetomella sp. (Li et al. 2013) associated with anthracnose and leaf spot, respectively. In 2015, another disease symptom, leaf blight was observed on S. trifasciata in several states of Malaysia. Scytalidium-like fungal isolates were recovered during preliminary fungal isolation from the samples.

During September 2015 and March 2017, diseased S. trifasciata with symptoms of leaf blight were collected from Penang and three other Malaysian states, namely Perak, Negeri Sembilan and Sarawak. Symptomatic samples were cut into approximately 1.5 cm2 fragments and surface sterilised in 70% ethanol (C2H5OH) solution and 1% sodium hypochlorite (NaOCl) solution for 3 min each. Then, the samples were rinsed thrice in sterile distilled water for 1 min each before being plated on potato dextrose agar (PDA) and incubated at 25 ± 3 °C for 3 to 5 days. Pure cultures were obtained by transferring a single conidium from the colony grown from surface sterilised samples onto fresh PDA and incubated as described previously. A mycelial plug 6 mm in diameter taken from a 7-days-old culture was transferred onto a PDA plate. The growth rate was recorded by measuring the colony diameter daily until the mycelia fully covered the plate. The isolates were cultured on 2% water agar (WA) overlaid with sterilised horsetail twigs (Casuarina equisetifolia) and carnation leaves, then were incubated as described previously to induce pycnidia formation. Measurements of 50 randomly selected conidia from pycnidia and arthrospores were taken.

A total of nine Scytalidium-like fungal isolates were obtained from diseased S. trifasciata showing leaf blight symptoms in four different states (Penang, Perak, Negeri Sembilan and Sarawak). Morphologically, all the isolates were identified as N. dimidiatum (Crous et al. 2006; Chuang et al. 2012; Masratul Hawa et al. 2013). Colonies on PDA were greyish white, gradually became greenish dark grey, dense with arthrospores and flat aerial mycelia, with dark pigmentation (Fig. 1a and b). Mycelial growth rate was 4.3 ± 1.0 cm/day on PDA. Sizes of arthric conidia ranged from 6.2 × 3.3 μm to 10.2 × 5.5 μm, 0 to 1 septate, hyaline to brown, and circular, oval or cylindrical with round to truncate ends (Fig. 1c). Conidia exuded in milky white cirrhus from pycnidia were one-celled, aseptate, oblong and 11.4 ± 0.7 μm × 4.9 ± 0.3 μm (Fig. 1d). All nine isolates of N. dimidiatum were deposited in Culture Collection Unit, Department of Plant Pathology, School of Biological Sciences, Universiti Sains Malaysia. Both sterile carnation leaves and horsetail twigs were used to induce the formation of pycnidia (Fig. 1e and f). However, pycnidia on carnation leaves tended to be covered by mycelia and arthric conidia, thus the horsetail twig was more suitable for examination of single-cell conidium from the conidiomata.

Fig. 1
figure 1

Morphological characteristics of N. dimidiatum isolate, (a) colony of N. dimidiatum on PDA, (b) pigmentation of N. dimidiatum on PDA, (c) arthric conidia, (d) conidia from pycnidia, (e) pycnidia and mycelia on carnation leaf, (f) pycnidia with conidia exuded from cirrhus on horsetail twig. Bars: c = 20 μm, d = 20 μm, e = 1000 μm, f = 200 μm

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Invisorb Spin Plant Mini Kit (Stratec, Germany) was used to extract the DNA of the isolates. The extraction was done according to the manufacturer’s instructions. Amplification of the ITS region was conducted using primers ITS1 and ITS4 (White et al. 1990).

PCRs were prepared in 50 μl containing 8 μl 5× GoTaq® Green Buffer (Promega, USA), 8 μl MgCl2 (25 mM) (Promega, USA), 1 μl dNTP Mix (10 mM) (Promega, USA), 0.8 μM of each primer (ITS1 and ITS4), 0.3 μl Taq polymerase (Promega, USA) and 0.6 μl genomic DNA. PCRs were run in MyCycler™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) that was programmed for an initial denaturation at 95 °C for 5 min, followed by 35 cycles (each 30 s at 95 °C, 30 s at 54 °C and 1 min at 72 °C) prior to a final extension of 5 min at 72 °C. PCR products were sent to a service provider for DNA sequencing.

Sequences were aligned and analysed using MEGA7 (Kumar et al. 2016). The resulting sequences were deposited in GenBank. Based on BLAST searches, eight sequences (Accession No. MF580792-MF580799) and one sequence (KX401435) showed 100% (579/579) and 98% (572/583) identity to the epitype of Neoscytalidium dimidiatum (CBS499.66), respectively.

Phylogenetic relationships of the isolates were analysed using MEGA7. The ITS sequence for the type specimen of N. dimidiatum was incorporated for analysis using maximum likelihood (ML) method after the best model of nucleotide substitution was selected (Tamura 3-parameter). Bootstrap values were determined from 1000 bootstrap replicates. Gaps and missing data were eliminated. Isolates used in the phylogenetic study, including the type specimen and outgroups are listed in Table 1. Of the 590 characters in the multiple alignments, 522 were conserved sites, 58 were variable sites and 31 were parsimony informative sites. ML analysis based on ITS sequences indicated that all isolates of N. dimidiatum in the present study were grouped in the same clade with N. dimidiatum CBS499.66, supported by bootstrap value of 71% (Fig. 2).

Table 1 Isolates and GenBank accession numbers used in phylogenetic analysis
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Fig. 2
figure 2

Phylogenetic tree based on maximum likelihood (ML) analysis of ITS sequence data. Bootstrap value from 1000 replicates was shown at the node. Neofusicoccum vitifusiforme STE-U5252 and Neofusicoccum mangiferae CMW7024 represented outgroups. Isolates from the present study were indicated in bold

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Pathogenicity of all isolates obtained was assessed on potted S. trifasciata using conidial suspension and mycelial plug. Fungal conidial suspensions (1 × 106 conidia/ml) and mycelial plugs (6 mm in diameter) were prepared from 7-days-old cultures on PDA plates. A total of 0.1 ml of conidial suspension was injected into the leaves whereas mycelial plugs were placed with their upper surface facing downward on wounds made by a sterile needle. Sterile distilled water and PDA plugs without mycelia were used as controls. All control and inoculated plants were maintained in a plant house, School of Biological Sciences, Universiti Sains Malaysia at 25 to 31 °C for 2 weeks. Fungal isolates were then re-isolated from the symptomatic inoculated plants and re-identified. The experiment was repeated twice and three replicates were prepared for each isolate.

Inoculation of both methods on potted plants resulted in lesions with darkened centres around the wounded sites that expanded as browning to chlorosis of the tissues (Fig. 3a and b). Under humid conditions, mycelia with arthric conidia were observed on the lesions. No symptoms were observed on the controls. The pathogen was consistently re-isolated from the inoculated leaves of S. trifasciata, and morphological examination showed that it was N. dimidiatum.

Fig. 3
figure 3

Artificial inoculation of N. dimidiatum isolate on S. trifasciata, (a) pathogenicity test using conidial suspension, (b) pathogenicity test using mycelial plug on wounded leaves

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Neoscytalidium dimidiatum is an opportunistic pathogen of various hosts causing different types of diseases (Padin et al. 2005). This pathogen was reported to cause stem canker (Masratul Hawa et al. 2013; Sanahuja et al. 2016) and brown rot of dragon fruit (Chuang et al. 2012), canker of English walnut (Chen et al. 2013), leaf blight of white spider lily (Nurul Nadiah et al. 2017), dieback of lesser yam (Lin et al. 2017), dieback, stem-end rot and canker of mango (Sakalidis et al. 2011; Marques et al. 2013), wood canker of grapevine (Rolshausen et al. 2013), shoot blight, canker, and gummosis of citrus (Polizzi et al. 2009), human toenail infection (da Silva et al. 2016) and rhinosinusitis (Bakhshizadeh et al. 2014). The occurrence of N. dimidiatum on S. trifasciata contributes to the knowledge of the host range of this plant pathogenic fungus. Etiological information of N. dimidiatum will help to develop strategies and efforts to control this important pathogen that can pose a threat to plants and humans. To our knowledge, this is the first report of N. dimidiatum causing leaf blight on S. trifasciata in Malaysia and worldwide.