Curvularia microspora sp. nov. associated with leaf diseases of Hippeastrum striatum in China

Abstract An undescribed Curvularia sp. was isolated from the leaf spot disease of Barbados Lily (Hippeastrum striatum (Lam.) Moore). Phylogenetic analyses of combined ITS, 28S, GPD1 and TEF1 sequence data place nine strains of this species in the trifolii-clade, but they clustered together as an independent lineage with strong support. This species was morphologically compared with related species in the trifolii-clade. Based on differences in morphology and phylogeny, it is concluded that this species is a new taxon, introduced as Curvularia microspora sp. nov. Pathogenicity testing determined the new species to be pathogenic on H. striatum.


Introduction
The genus Curvularia includes pathogens and saprobes of various plants, as well as opportunistic pathogens of humans and animals (Sivanesan 1987, Manamgoda et al. 2011, da Cunha et al. 2013) and has been well-studied in recent years. Identification of Curvularia spp. was previously mainly based on morphological descriptions and comparisons, however, the use of molecular taxonomy has solved many problems of resolving species (Valente et al. 1999, Mendoza et al. 2001. A multi-gene phylogenetic tree, based on the internal transcribed spacers including the 5.8S nuclear ribosomal DNA gene (ITS), the 5' end of the nuclear ribosomal large subunit (28S), fragments of the glycerol-3-phosphate dehydrogenase (GPD1) and translational elongation factor EF-1 alpha (TEF1) gene regions, was provided to identify fresh collections of Curvularia from various hosts and geographic locations worldwide (Manamgoda et al. 2015).
In this study, DNA sequences of ITS, 28S, GPD1 and TEF1 gene regions were used for phylogenetic analyses to identify a new Curvularia species. This was concluded based on the combined morphology and phylogeny. Curvularia microspora sp. nov., is introduced here, associated with leaf diseases of Hippeastrum striatum.

Isolation and morphological studies
All diseased samples were collected from the Medical Plants Herb Garden, in Chongqing City, Nanchuan County, China. This garden is located in a region of subtropical humid monsoon climate and has conserved more than 3000 kinds of medicinal plants.
In this study, all fungal strains were isolated by the single-spore technique in order to obtain pure cultures following the method of Chomnunti et al. (2014). Single spores were transferred to potato-dextrose agar (PDA) and incubated at room temperature (28 °C). After several weeks of incubation, the morphological characters were recorded following the methods of Manamgoda et al. (2011Manamgoda et al. ( , 2012. Conidia and conidiophores were observed using a compound microscope (Nikon Eclipse E600 DIC microscope and a Nikon DS-U2 camera or a Nikon 80i compound microscope fitted with a Canon 450D digital camera). The holotype specimen was deposited in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Ex-type cultures were also deposited in the culture collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC).

DNA extraction and sequencing
Fungal cultures were grown on PDA until nearly covering the whole Petri-dish (90 mm) at 28 °C. Fresh fungal mycelia were scraped with sterilised scalpels. A BI-OMIGA Fungus Genomic DNA Extraction Kit (GD2416) was used to extract fungal genome DNA. DNA Amplification was performed in a 25 μL reaction volume which contained 2.5 μL 10 × PCR buffer, 1 μL of each primer (10 μM), 1 μL template DNA and 0.25 μL Taq DNA polymerase (Promega, Madison, WI, USA). Primers ITS4 and ITS5 (White et al. 1990) were used to amplify the ITS region. The thermal cycling programme was: 3 min initial denaturation at 95 °C, followed by 30 cycles of 30 s denaturation at 94 °C, 30 s primers annealing at 52 °C, 1 min extension at 72 °C and a total 10 min extension at 72 °C. To amplify the GPD1 gene, the primers gpd1 and gpd2 were used (Berbee et al. 1999). The amplification programme included an initial denaturation step at 96 °C for 2 min, followed by 35 PCR cycles with 1 min at 96 °C, 1 min at 52 °C and 45 s at 72 °C with a final 10 min extension at 72 °C. The TEF1 and 28S regions were amplified using EF-526F/1567R and LR5/LROR primers respectively (Schoch et al. 2009). The 28S amplification programme included an initial denaturation step at 95 °C for 3 min followed by 30 cycles of 40 s denaturation at 94 °C, 50 s primer annealing at 52 °C, 1 min extension at 72 °C. The same PCR reaction was used to amplify TEF1 with the only change being the annealing temperature at 54 °C.

Phylogenetic analysis
DNA sequences from these isolates and reference sequences were downloaded from GenBank and analysed by maximum parsimony (MP) and maximum likelihood (ML) ( Table 1). Sequences were optimised manually to allow maximum alignment and maximum sequence similarity, as detailed in Manamgoda et al. (2012). The alignment document of four phylogenetic markers has been submitted to TreeBase (https:// treebase.org/; Accession number: 21970). A partition homogeneity test (PHT) was performed with 1000 replicates via PAUP v. 4.0b10 (Swofford 2003) to evaluate statistical congruence amongst sequence data of 28S, ITS, GPD1 and TEF1 gene regions. MP analyses were performed in PAUP v. 4.0b10 (Swofford 2003), using the heuristic search option with 1,000 random taxa addition and tree bisection and reconnection (TBR) as the branch swapping algorithm. Maxtrees were set to 10,000. The characters in the alignment document were ordered accordingly: 28S+ITS+GPD1+TEF1, with equal weight and gaps were treated as missing data. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. Maximum likelihood (ML) trees of DNA sequences were obtained by a heuristic search using the TrN + I + G model, which was deduced as the best fit for the data by the likelihood ratio test using the MODELTEST wer3.7 and MrMTgui version 1.01 (Posada and Crandall 1998).

Pathogenicity test
Pathogenicity of this species was determined by inoculating healthy leaves of Hippeastrum striatum and Canna indica L. with 5 mm diameter mycelial plugs, cut from the margins of 10-day-old actively growing cultures; the control was treated with sterile agar plugs. Both inoculated and control plants were kept in a moist chamber at 25 °C for 7 days and observed for disease symptom development. Infected leaves were collected and the fungus was re-isolated in PDA medium and compared against the original strains. Control plants were sprayed with sterilised distilled water. Table 1. GenBank accession numbers of isolates include in this study.

Habitat and distribution. Isolated from leaf diseases of H. striatum and Canna indica in China
Etymology. microspora, referring to this species producing obviously smaller conidia.

Pathogenicity test
Test plants (Hippeastrum striatum) were inoculated with 5 mm diam mycelial plugs of Curvularia microspora with two replicates of each plants and the inoculation experiment was repeated two times (with different sporulation generations). Hippeastrum striatum leaves both exhibited brown to dark brown necrotic spots (Figure 3a, b) after 7 days, which were very similar to those of natural infection (Figure 2a, b). The DNA sequencing result (ITS region), after re-isolation, identified this as C. microspora. The successful re-isolation of C. microspora from the inoculated leaves of H, striatum established a credible proof of pathogenicity. All test plants were covered with polyethylene bags for 7 days. However, on Canna indica, disease symptoms did not appear again.
Curvularia species can cause severe or opportunistic diseases of different plant taxa and are often a threat to agricultural production by reducing yield and quality. In the trifolii-clade, all species except for C. borreriae, have been reported as causing plant disease. This is especially true of C. trifolii and C. pallescens, which cause serious diseases of Agrostis stolonifera and Gloriosa superba respectively (Table 2). Koch's postulates were performed to show that C. microspora causes leaf spot disease of Hippeastrum striatum (Figure 3), but on Canna indica might only be saprobic or endophytic. Hippeastrum striatum as an economic ornamental plant is grown in some areas of China, thus there is a need to continue investigation on the biology of this species in order to determine whether it can cause serious disease outbreaks.