Novel species of Pestalotiopsis fungi on Dracaena from Thailand

ABSTRACT A survey of the diversity and distribution of microfungi on Dracaena leaf litter in Songkhla Province (Thailand) yielded two collections of pestalotiopsis-like fungi. Analyses of a combined ITS, TEF1-α and TUB2 sequence data matrix were applied to infer the phylogenetic position of these new isolates in Pestalotiopsis. The phylogenies indicated that these two isolates were monophyletic and constituted a distinct lineage that perceived a taxonomic novelty in Pestalotiopsis. This clade shared a close phylogenetic affinity with P. adusta, P. krabiensis, P. pandanicola and P. papuana. The comparison of morphological features with the phylogenetically closely related taxa are given and the new species is introduced as Pestalotiopsis dracaenicola sp. nov. with comprehensive descriptions and illustrations herein.


Introduction
Dracaena is a monocotyledon belonging to the family Agavaceae that are used as ornamentals, herbs or medicinal plants (Pires et al. 2004). Dracaena consists of about 550-600 species in 18 genera including various shrubs and trees (Pires et al. 2004;Mabberley 2008). Species of Dracaena are widely distributed in the tropics and subtropical regions of the world. In Europe and Canada, most Dracaena plants are cultivated as ornamentals (Ilodibia et al. 2015). Dracaena marginata an important ornamental plant exported as a popular houseplant, has been shown to reduce the levels of formaldehyde in the air (Jaminson 2012). Robiansyah and Hajar (2017) have shown that there is a decline in the population of D. ombet throughout its native ranges due to overgrazing, disease by pathogens, human overexploitation, and climate change. The conservation actions for these species are hindered due to poor information about their natural enemies. The plant associated fungi which can be pathogens/opportunistic pathogens, may directly relevant with quarantine measures when the plant is exported as ornamentals to different regions. In contrast to the detailed studies on other hosts such as grasses, bamboo and palms in Thailand, information is still limited on Dracaena based fungi. Some taxa occurring on dead leaves of Dracaena are Colletotrichum gloeosporioides (D. sanderiana) (Stev enson 1975), Gloeosporium sp. (D. reflexa) (Giatgong 1980), Ophioceras chiangdaoense (D. loureiroi) (Thon gkantha et al. 2009), Parapallidocercospora thailandica (D. loureiroi)  and Phaeosphaeriopsis dracaenicola (Dracaena loureiroi) (Phookamsak et al. 2014). There have been two Pestalotiopsis species reported on Dracaena fragrans: P. affinis Y.X. Chen & G. Wei and P. dracaenea Yong Wang bis, Yu Song, K. Geng & K.D. Hyde. We are investigating the microfungi associated with monocotyledons in Thailand which has a high species diversity (Dai et al. 2017;. In this paper we introduce a novel species in Pestalotiopsis from Dracaena based on morphology coupled with multigene phylogeny.

Isolates and morphology
Dracaena leaf litter was collected from Songkhla Province in Thailand during May 2018. Collected samples were brought to the laboratory in plastic bags. Specimens were observed with a stereomicroscope (Motic SMZ-171). Mycelia or spore mass from specimens was directly isolated on potato dextrose agar (PDA) plates and incubated at 25-30°C. The culture was transferred to new PDA plates. Cultures were grown for 2-4 weeks and morphological characters, such as colour, colony and texture were recorded. The culture characteristics were photographed with a Canon EOS 600D digital camera fitted to a Nikon ECLIPSE Ni compound microscope. Measurements of morphological structures were taken from the widest and the longest parts of each structure. Whenever possible, more than 20 measurements were made. The lengths and widths were measured using the Tarosoft (R) Image Frame Work programme and images used for figures processed with Adobe Photoshop CS6 Extended v. 10.0 (Adobe Systems, USA).
The specimens were deposited in the Herbarium of Mae Fah Luang University (Herb. MFLU) and Culture Collection of Mae Fah Luang University (MFLUCC), Chiang Rai, Thailand. Facesoffungi and Index Fungorum numbers were submitted Index Fungorum 2020). New taxa were justified based on guidelines outlined by Jeewon and Hyde (2016).
The amplification reactions were performed in 25 μl volumes contained of 8.5 μl of sterilised H 2 O, 12.5 μl of Easy Taq PCR Super Mix [mixture of Easy Taq TM DNA Polymerase, dNTPs, and optimised buffer (Beijing Trans Gen Biotech Co., Chaoyang District, Beijing, PR China), 1 μl of each forward and reverse primers (10 pM) and 2 μl of DNA template (1.2 μg/ml)]. The PCR thermal cycle program for ITS and TEF1-α gene amplification was provided as initially 94°C for 3 mins, followed by 35 cycles of denaturation at 94°C for 30 secs, annealing at 55°C for 50 secs, elongation at 72°C for 90 secs, and final extension at 72°C for 10 mins. The PCR thermal cycle program for TUB2 gene amplification was provided as initially 94°C for 3 mins, followed by 35 cycles of denaturation at 95°C for 30 secs, annealing at 53°C for 30 secs, elongation at 72°C for 45 secs, and a final extension at 72°C for 90 secs. The PCR products were sent for sequencing at Sangon Biotech, Shanghai, China.

Sequence alignment and phylogenetic analyses
Separate ITS, TEF1-α and TUB2 DNA sequences were subjected to BLAST search engine tool of NCBI for verification and selection of taxa for subsequent phylogenetic analyses. Taxa used in the analyses were obtained from sequence data of Pestalotiopsis and related taxa (Table 1) were downloaded from GenBank. Sequence alignments were performed in MAFFT v. 7.220 (mafft.cbrc.jp/alignment/server, Katoh et al. 2017) for each gene locus. Phylogenetic analyses were conducted on a combined dataset of ITS, TEF1-α and TUB2 sequence data. The sequence datasets were combined using BioEdit v.7.2.3 (Hall 1999). Phylogenetic analyses of both individual and combined aligned data were performed under maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference analyses (BI) criteria. Parsimony analysis was carried with the heuristic search option in PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 with the following parameter settings: characters unordered with equal weight, random taxon addition, branch swapping with tree bisection-reconnection (TBR) algorithm, branches collapsing if the maximum branch length was zero. Alignment gaps were treated as missing characters in the analysis of the combined data set, where they occurred in relatively conserved regions. Trees were inferred using the heuristic search option with 1000 random sequence additions, with maxtrees set at 1000. Descriptive tree statistics for parsimony; Tree Length (TL), Consistency Index (CI), Retention Index (RI), Relative Consistency Index (RC) and Homoplasy Index (HI) were calculated for trees generated under different optimality criteria. The Kishino-Hasegawa tests (Kishino and Hasegawa 1989) were performed in order to determine whether trees were significantly different. Maximum parsimony bootstrap values (MP) equal or greater than 60% are given above each node (Figure 1) (Rannala and Yang 1996) by Markov Chain Monte Carlo sampling (BMCMC). GTR+I + G was used in the command. Six simultaneous Markov chains were run for 10,000,000 generations and trees were sampled every 200th generation. The distribution of log-likelihood scores was examined to determine stationary phase for each search and to decide if extra runs were required to achieve convergence, using the program Tracer 1.5 (Rambaut and Drummond 2007). First 20% of generated trees were discarded and remaining 80% of trees were used to calculate posterior probabilities of the majority rule consensus tree. BYPP greater than 0.95 are given above each node (Figure 1). Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTR+I + G model of evolution. Maximum likelihood bootstrap values (ML) equal or greater than 60% are given above each node (Figure 1). The phylogenetic trees were shown in FigTree v. 1.4 (Rambaut 2012) and edited using Microsoft Office Power Point 2007 and Adobe illustrator CS3 (Adobe Systems Inc., USA). Sequences derived in this study were deposited in GenBank (Table 1). The finalised alignment and tree were deposited in TreeBASE, submission ID: 26152.

Culture characteristics.
Conidia germinating on PDA within 12 hours reaching 6 cm diameter after 6 days at 25-30°C, circular, floccose to fluffy; white mycelium with aerial on the surface, producing black spore masses.
Material examined. THAILAND (Table 2). Meanwhile, Pestalotiopsis adusta was reported on leaves of Prunus cerasus in USA, from a PVC gasket of a refrigerator door and from Syzygium species in Thailand . Pestalotiopsis krabiensis and P. pandanicola were found on Pandanus sp. in Thailand . Pestalotiopsis dracaenea (HGUP4037) and Pestalotiopsis affinis (Hsp2000 II-6600) also found on Dracaena (D. fragrans) from China (Chen et al. 2002;Ariyawansa et al. 2015).
Pestalotiopsis affinis (Hsp2000 II-6600) only known from its morphological descriptions and there are no DNA based sequence data to compare the phylogenetic relationship with our new species. P. dracaenea (HGUP4037) is not monophyletic with Pestalotiopsis dracaenicola (Figure 1).

Acknowledgements
We are grateful to the Thailand Research Fund (TRF) grant no PHD60K0147, and Kunming Institute of Botany for the help with molecular work. Shaun Pennycook is thanked for nomenclatural advice. K.D. Hyde would like to thank the Thailand Research Fund project entitled 'The future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (No. DBG6080013)' and 'Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion ( -0913)  T  -G  -T  C  G  C  T  T  A  G  C  C  C  C  T  C  G  P. dracaenicola (18-0914)  T  -G  -T  C  G  C  T  T  A  G  C  C  C  C  T  C  G  P. dracaenea (HGUP4037)  C  T  T  G  C  A  A  G  A  A  G  A  G  T  -G  -T  -Doilom, Yong Wang, Dhandevi Pem and Deping Wei, for their precious help during this research.

Disclosure statement
No potential conflict of interest was reported by the authors.