Curvicladiellapaphiopedili sp. nov. (Hypocreales, Nectriaceae), a new species of orchid (Paphiopedilum sp.) from Guizhou, China

Abstract Background An asexual fungus, collected from diseased leaves of Paphiopedilum sp. from Guizhou Province, China, and based on the phylogenetic analyses and morphological characters, it was identified as a new species in Curvicladiella. The genus Curvicladiella are recorded for the first time for China. New information The morphology of Curvicladiellapaphiopedili sp. nov. is characterised by penicillate conidiophores with a stipe, dull, tapering towards the apex, the curved stipe extension and cylindrical conidia. In the phylogenetic analyses of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data, this taxon was clustered as sister to Curvicladiellacignea within Nectriaceae.


Introduction
Nectriaceae (order Hypocreales) includes many important plant and human pathogens and some species have been used as biodegrading 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, Chaverri et al. 2011, Hirooka et al. 2012, Lombard and Crous 2012a, Lombard and Crous 2012b, Lombard et al. 2014a, Lombard et al. 2014b). Lombard et al. (2015) provided a phylogenetic backbone tree for Nectriaceae, based on combined sequence data of 10 gene regions. Curvicladiella is one of the genera in the Nectriaceae. Decock and Crous (1998) established Curvicladium (as Curvicladiella) with C. cigneum (as Curvicladiella cignea) as the type species. The genus is distinct from morphologicallysimilar 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. is identified as a new species in Curvicladiella, the artificial infection test shows that it is a pathogen and the specific infection process has been described by Song et al. (Song et al. 2020). Paphiopedilum is known as"slipper orchids", has a high ornamental value and can be used as household bonsai and garden plants (Luan et al. 2019).

Sample collection and isolation
Diseased orchid leaves were collected from Guizhou Botanical Garden, Guizhou Province, China (in August 2019). The samples were brought to the laboratory in envelopes, photographed and identified. Pieces of leaves (5 × 5 mm), half of which were diseased and half healthy, were sterilised by 75% ethanol for 5-10 s, rinsed three times with sterilised distilled water, placed on potato dextrose agar (PDA) and incubated at 25°C for two days (Fang 2001). Mycelia were transferred to PDA and incubated for ten days at 25°C to obtain the pure cultures. The morphological characters of the fungi obtained from the diseased leaves collected in the field and cultured with PDA, and the fungi obtained from the diseased leaves after an artificial infection test were observed using a Nikon SMZ 745 stereomicroscope. Measurements were made using Image Frame Work.
Pure cultures were deposited in Guizhou Culture Collection (GZCC) Guizhou, China and Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand. Herbarium specimens were deposited in the Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China and the Herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand.
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 from amongst suboptimal trees from each replicate by comparing likelihood scores under the GTR+GAMMA substitution model. Bayesian analyses were carried out using MrBayes 3.2 (Huelsenbeck 2012). MrModeltest 2.2 was used to choose the best-fit evolutionary model (Nylander 2004

Etymology
Refers to the genus name Paphiopedilum.

Phylogenetic analyses
The final alignment consists of the new species and the fungus obtained from the diseased leaves after use of the new species to infect the heathy Paphiopedilum and other genera of the families Nectriaceae. Additionally, the alignment of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data comprised a total of 3877 characters with gaps (734bp for cmdA, 529bp for his3, 616bp for ITS, 840bp for LSU, 548bp for tef1 and 610bp for tub2). The RAxML tree, based on analysis of cmdA, his3, ITS, LSU, tef1 and tub2 sequences data.
Bootstrap support values for ML and Bayesian greater than 75% and 0.95 were given near nodes, respectively. The tree was rooted with Campylocarpon fasciculare and Campylocarpon pseudofasciculare. The new isolate are shown in bold.
The dataset comprised 39 taxa with Campylocarpon fasciculare and C. pseudofasciculare as the outgroup taxa. The best scoring RAxML tree is shown in Fig. 3, with the Bayesian tree (not shown) having a similar topology with the ML tree. Curvicladiella paphiopedili was clustered as sister taxon to C. cignea within Nectriaceae with high support (99/1.00) (Fig. 3).