Curvicladiella paphiopedili sp. nov., a new species on orchid (Paphiopedilum sp.) from Guizhou, China

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Introduction
Nectriaceae (Hypocreales, class) includes many important plant and human pathogens, and some species were used as biodegraders and biocontrol agents in industrial and commercial applications (Lombard et al. 2015). Based on molecular studies, many sexual genera in Nectriaceae were placed in Nectria sensu lato Samuels 1995, Rossman et al. 1999). However, Nectria sensu stricto is restricted to the type species N. cinnabarina (Tode) Fr et al. with tubercularia-like asexual morphs (Rossman 2000, Hirooka et al. 2012. A number of studies have treated taxonomic concepts within Nectriaceae based on multi-gene phylogenetic inference (Lombard et al. 2010a, Lombard et al. 2010b, Lombard and Crous 2012a, Lombard et al. 2014a, Lombard et al. 2014b, Lombard and Crous 2012b, Chaverri et al. 2011, Hirooka et al. 2012. Lombard et al. (2015) provided a phylogenetic backbone tree for Nectriaceae based on a combined sequence data of 10 gene regions. Decock and Crous (1998) established Curvicladium (as Curvicladiella) with C. cigneum (as Curvicladiella cignea) as the type species. The genus is distinct from morphologically similar genera, such as Cylindrocladium Morgan, Cylindrocladiella Boesew, Gliocladiopsis Saksena, Falcocladium Silveira, Alfenas, Crous, Wingf and Xenocylindrocladium Decock, Hennebert, Crous by having cylindrical conidia and stipe extensions (Decock and Crous 1998). Curvicladiella cignea is the only species in the genus.
Based on the phylogenetic analyses and morphological characters, the fungus collected from diseased leaves of Paphiopedilum sp. was identified as a new species in Curvicladiella, which has been proved to be a plant pathogen (Song et al. 2020).

Sample collection and isolation
Diseased orchid leaves were collected from Guizhou botanical garden, Guizhou Province, China (in August 2019). The samples were brought to laboratory in envelopes, photographed and identified. Pieces of leaves (5 × 5 mm), each with half part diseased and half healthy, were sterilized by 75% ethanol for 5-10 s, rinsed three times with sterilized distilled water, placed on potato dextrose agar (PDA) and incubated at 25°C for two days ( Fang 2001). Mycelia were transferred to PDA, incubated for ten days at 25°C to get the pure cultures. Morphological characters were observed using Nikon SMZ 745 stereomicroscope. Measurements were made using Image Frame Work.

DNA extraction, PCR amplification and sequencing
The fungal mycelia were scraped from the pure culture growing on PDA for ten days at 25ºC. DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China). Gene sequences were determined for 28S large subunit (LSU) nrDNA, calmodulin (cmdA), histone H3 (his3), internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), translation elongation factor 1-alpha (tef1) and βtubulin (tub2). Primer pairs used for amplifying each gene region were listed in Table 1. Polymerase chain reaction (PCR) was carried out in 25 ul reaction volume containing 12.5 ul 2 × PCR Master Mix (Sangon Biotech, China), 9.5 ul ddH O, 1ul of each primer and 1ul DNA template. The PCR products were examined by using 1.2% agarose electrophoresis gel stained with ethidium bromide and were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd, China. The nucleotide sequences were submitted in GenBank.
Maximum likelihood (ML) analysis was performed using raxmlGUI 1.3.1 (Silvestro and Michalak 2012). The optimal raxML tree search was conducted with 1000 bootstrap replicates and the default algorithm was used from a random starting tree for each replicate. The final tree was selected among suboptimal trees from each replicate by comparing likelihood scores under the GTR+GAMMA substitution model.
Maximum parsimony (MP) analysis was performed with the heuristic search in PAUP v. 4.0b10 (Swofford 2002). All characters were equally weight and unordered. Gaps were treated as missing in the alignment. Maxtrees were unlimited. All multiple, equally parsimonious trees were saved. The zero length of branches were collapsed. Clade stability was assessed by using a bootstrap (BT) analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa (Hillis and Bull 1993).

Etymology
Refers to the host name Paphiopedilum sp.

Phylogenetic analyses
The alignment of combined LSU, cmdA, his3, ITS, tef1 and tub2 sequence data comprised a total of 3872 characters with gaps (840bp for LSU, 734bp for cmdA, 529bp for his3, 611bp for ITS, 548bp for tef1 and 610bp for tub2). The dataset composed 38 taxa with Campylocarpon fasciculare and C. pseudofasciculare as the outgroup taxa. The best scoring RAxML tree was shown in Fig. 3, the MP and bayesian tree (not shown) had a similar topology with the ML tree. Curvicladiella paphiopedili was clustered as sister taxon to C. cignea within Nectriaceae with high support (99/98/1.00) (Fig. 3).

Discussion
Morphologically, Curvicladiella paphiopedili is similar to species in Calonectria, Cylindrocladium and Xenocylindrocladium, but distinct in having ellipsoidal or sphaeropedunculate chlamydospores (Fig. 2k), dull, tapering towards the apex (Fig. 1d, e, Fig. 2e-g), and curved extension stipes (Fig. 1f, g). Without obpyriform, ovoid, ellipsoidal or sphaeropedunculate vesicles (Lombard et al. 2010a, Pham et al. 2019 or coiled stipes ( Decock et al. 1997). The morphology of Curvicladiella paphiopedili is different from the type species Curvicladiella cignea with in the size of extension stipes, conidia and chlamydospores, without swollen cell below the apical septum, on the other hand, the curved position of stipes are different. In the phylogenetic analyses, the two taxa of Curvicladiella formed a well-supported monoclade and Curvicladiella aphiopedili represented a distinct lineage (Fig. 3).
022) for supporting this study. Lian-chai Song thanks Jing Yang for guiding the experiments and modifing the articles.  The RAxML tree based on analysis of LSU, cmdA, his3, ITS, tef1 and tub2 sequnces data.
Bootstrap support values for ML, MP and Bayesian greater than 75%, 75% and 0.95 were given near nodes respectively. The tree was rooted with Campylocarpon fasciculare and Campylocarpon pseudofasciculare. The new isolate was marked in red.

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Author-formatted, not peer-reviewed document posted on 08/11/2021. DOI:  Isolated taxa used in this study and their GenBank accession numbers. The type species are superscripted T and the newly generated sequences are indicated in red.