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Morphological and phylogenetic characterisation of novel Cytospora species associated with mangroves
expand article infoChada Norphanphoun§, Olivier Raspé|, Rajesh Jeewon#, Ting-Chi Wen, Kevin D. Hyde§
‡ Guizhou University, Guiyang, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Botanic Garden Meise Nieuwelaan, Meise, Belgium
¶ Fédération Wallonie-Bruxelles, Brussels, Belgium
# University of Mauritius, Moka, Mauritius
Open Access

Abstract

Mangroves are relatively unexplored habitats and have been shown to harbour a number of novel species of fungi. In this study, samples of microfungi were collected from symptomatic branches, stem and leaves of the mangrove species Xylocarpus granatum, X. moluccensis and Lumnitzera racemosa and examined morphologically. The phylogeny recovered supports our morphological data to introduce three new species, Cytospora lumnitzericola, C. thailandica and C. xylocarpi. In addition, a combined multi-gene DNA sequence dataset (ITS, LSU, ACT and RPB2) was analysed to investigate phylogenetic relationships of isolates and help in a more reliable species identification.

Keywords

3 new species, Cytosporaceae, Lumnitzera racemosa, Mangroves, Phylogeny, Taxonomy, Xylocarpus granatum, Xylocarpus moluccensis

Introduction

Mangroves are forests established in tropical and subtropical backwaters, estuaries, deltas and lagoons. These forests play a major role in the ecology of coastal tropical/subtropical waters, as they serve as hatchery and nursery habitats for marine organisms and protect coastlines from catastrophic events such as storms and tidal surges (Hyde and Jones 1988, Fisher and Spalding 1993, Hyde and Lee 1995, Hyde et al. 1998). The greatest diversity of mangrove species occurs in the mangroves of Indonesia, Malaysia and Thailand (Alias and Jones 2009, Alias et al. 2010).

Reports of fungi associated with mangroves are relatively few and data on diseases of mangroves are uncommon (Cribb and Cribb 1955, Kohlmeyer and Kohlmeyer 1979, Hyde and Jones 1988). So far, a number of fungi collected from mangroves are either saprobes (e.g Swe et al. 2008a, b, Devadatha et al. 2018, Li et al. 2018) or endophytes (e.g Liu et al. 2012, Doilom et al. 2017). One early species documented from mangroves is that of Stevens (1920) who reported a species of Anthostomella that was found from a leaf spot in red mangroves (Rhizophora mangle) in Puerto Rico. Later, McMillan (1964) reported Cercospora which caused leaf spot on red mangroves in Florida and Kohlmeyer (1969) documented an undescribed Cytospora species on R. mangle in Hawaii. Cytospora rhizophorae has also been reported as a marine fungus from Rhizophora mangle in southwest Puerto Rico (Wier et al. 2000). Later, Shivas et al. (2009) reported a serious disease, caused by Pseudocercospora avicenniae, on leaves of Avicennia marina in Cape Tribulation, Queensland.

Cytospora was introduced by Ehrenberg (1818) and belongs to the family Cytosporaceae in Diaporthales (Wijayawardene et al. 2018). Cytospora species are phytopathogens or saprobes (Wehmeyer 1975, Barr 1978, Eriksson 2001, Castlebury et al. 2002, Wijayawardene et al. 2018). Cytospora has a worldwide distribution and is an important pathogenic genus, causing canker and dieback disease on branches of a wide range of plants (Adams et al. 2005, 2006, Hyde et al. 2017, Norphanphoun et al. 2017). Currently, there are 614 epithets for Cytospora (Index Fungorum 2018, 14 June 2018) with an estimated 110 species in Kirk et al. (2008). Recently, fourteen new species were introduced to this genus by Norphanphoun et al. (2017). In this study, we report on three novel species of Cytospora associated with mangroves in Thailand. Detailed descriptions and illustrations of all the species identified are provided in this paper.

Material and methods

Sample collection and examination of specimens

Samples collected were dead branches of Xylocarpus granatum K.D. Koenig, X. moluccensis (Lam.) M. Roem. and leaf spots of Lumnitzera racemosa Willd. from Phetchaburi and Ranong provinces, Thailand in 2016. Specimens were returned to the laboratory in paper bags, examined and described following Norphanphoun et al. (2017). Morphological characters of ascomata and conidiomata were examined using a Motic SMZ 168 dissecting microscope. Hand sections were mounted in water and examined for morphological details. Micro-morphology was studied using a Nikon Ni compound microscope and photographed with a Canon EOS 600D digital camera fitted to the microscope. Photo-plates were made using Adobe Photoshop CS6 Extended version 13.0 × 64 (Adobe Systems, USA), while Tarosoft (R) Image Frame Work programme v. 0.9.7 was used for measurements.

Cultures were obtained by single spore isolation method outlined in Chomnunti et al. (2014). Single germinating spores were observed and photographed using a Nikon Ni compound microscope fitted with Canon EOS 600D digital camera. Geminated spores were transferred aseptically to 2% malt extract agar (MEA, malt extract agar powder 32 g in 1000 ml water) and incubated at room temperature (18−25 °C). A tissue isolation method was used for isolation of taxa from leaf spots of Lumnitzera racemosa. Leaves with leaf spots were cut into small pieces (0.5 × 0.5 cm2) using a sterilised blade and surface was sterilised using 70% ethanol for 1 minute, followed by three rinses with sterile distilled water, 1 minute in 3% sodium hypochlorite (NaOCl) and rinsed with sterile water for 1–2 minutes and dried by blotting on sterile filter paper. Four to five segments including the edge of the leaf spot were placed on water agar (WA) plates, supplemented with 100 mg/ml streptomycin. The dishes were incubated at 27 °C ± 2 °C for 7–10 days. Fungal colonies were transferred using single hyphal tips on to potato dextrose agar (PDA) plates throughout a 2-week period. Pure cultures were maintained for further studies on PDA (Bharathidasan and Panneerselvam 2011). The specimens/dried cultures and living cultures are deposited in the Herbarium Mae Fah Luang University (MFLU) and culture collection Mae Fah Luang University (MFLUCC), Chiang Rai, Thailand and duplicated in the International Collection of Micro-organisms from Plants (ICMP). Facesoffungi numbers were registered as in Jayasiri et al. (2015). New taxa are established based on recommendations as outlined by Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fresh fungal mycelia growing on MEA at room temperature (18−25 °C) for three weeks using a E.Z.N.A.TM Fungal DNA MiniKit (Omega Biotech, CA, USA) following the manufacturer’s protocols. Polymerase chain reactions (PCR) were carried out using primer pairs of ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') to amplify the ITS region (White et al. 1990), primer pairs of NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') to amplify part of the large subunit rDNA (28S, LSU) (O’Donnell 1993), the partial ACT region was amplified using primers ACT512F (5'-ATGTGCAAGGCCGGTTTCGC-3') and ACT783R (5'-TACGAGTCCTTCTGGCCCAT-3') (Carbone and Kohn 1999) and the partial RPB2 region was amplified using primers bRPB2-6F (5'-TGGGGYATGGTNTGYCCYGC-3') and bRPB2-7.1R (5'-CCCATRGCYTGYTTMCCCATDGC-3') (Matheny 2005).

The amplification reactions were carried out with the following protocol: 50 μl reaction volume containing 2 µl of DNA template, 2 µl of each forward and reverse primers, 25 µl of 2 × Bench TopTMTaq Master Mix (mixture of Taq DNA Polymerase (recombinant): 0.05 units/µl, MgCl2: 4 mM and dNTPs (dATP, dCTP, dGTP, dTTP): 0.4 mM) and 19 µl of double-distilled water (ddH2O) (sterilised water) using the thermal cycle programme in Norphanphoun et al. (2017). Purification and sequencing of PCR products with the same primers mentioned above were carried out at Life Biotechnology Co., Shanghai, China.

Phylogenetic analysis

The sequences were assembled by GENEIOUS Pro v. 11.0.5 (Biomatters) and BLAST searches were made to retrieve the closest matches in GenBank and multiple alignment also included recently published sequences (Norphanphoun et al. 2017, Hyde et al. 2017, 2018). Combined analyses of ITS1, 5.8S, ITS2, LSU, RPB2 and ACT sequence data of 86 taxa were performed under different optimality criteria (MP, ML, BI). Diaporthe eres (AFTOL-ID 935) was used as the outgroup taxon. In order to obtain a better picture of the phylogenetic relationships amongst our strains and closely related strains, a separate ITS1+ITS2 phylogeny was inferred, because only ITS sequences were available for many strains in that group and because less ambiguously aligned (and excluded) positions are expected in a dataset with narrower taxonomic coverage. Nineteen strains were selected for this analysis based on preliminary analyses and results from the multigene phylogeny. All sequences were aligned separately using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/; Katoh and Standley 2013) and Gblocks v. 0.91b was used to exclude ambiguously aligned positions in the ITS and ACT alignments (Castresana 2000, Talavera and Castresana 2007). A partition homogeneity test (PHT) was performed with PAUP 4.0b10* (Swofford 2002) to determine whether the individual datasets were congruent and could be combined. The combined sequence alignments were obtained from MEGA7 version 7.0.14 (Kumar et al. 2015), missing data were coded as question marks (?) and further manual adjustments were made wherever necessary in BioEdit 7.2.3 (Hall 1999). The combined sequence alignment was converted to NEXUS file for maximum parsimony analysis using ClustalX v. 2 (Larkin et al. 2007). The NEXUS file was prepared for MrModeltest v. 2.2 (Nylander 2004) in PAUP v.4.0b10 (Swofford 2002).

Maximum Parsimony (MP) analysis was performed using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10* (Swofford 2002) with 1000 bootstrap replicates using a heuristic search with random stepwise addition and tree-bisection reconnection (TBR), as detailed by Jeewon et al. (2002) and Cai et al. (2005). Maxtrees was set to 1000, branches of zero length were collapsed. The following descriptive tree statistics were calculated: parsimony tree length [TL], consistency index [CI], retention index [RI], rescaled consistency index [RC] and homoplasy index [HI].

For both Maximum Likelihood and Bayesian analyses, a partitioned analysis was performed with the following six partitions: ITS1+ITS2, 5.8S, LSU, ACT-exons, ACT-introns and RPB2. Maximum-likelihood (ML) analysis was performed with RAxML (Stamatakis 2006) implemented in the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE; Miller et al. 2010), 25 categories, 1000 rapid bootstrap replicates were run with the GTRGAMMA model of nucleotide evolution. Maximum likelihood bootstrap values (MLBS) equal or greater than 50% are given above each node.

Bayesian Inference (BI) analysis was performed using the Markov Chain Monte Carlo (MCMC) method with MrBayes 3.2.2 (Ronquist et al. 2012). The best-fit nucleotide substitution model for each dataset was separately determined using MrModeltest version 2.2 (Nylander 2004). GTR+I+G was selected as the best-fit model for the ITS1+ITS2, LSU, ACT (ACT-exons and ACT-introns) and RPB2 datasets and K80 for 5.8S. The MCMC analyses, with four chains starting from random tree topology, were run for 5,000,000 or 10,000,000 generations for the combined dataset or the ITS1+ITS2 dataset. Trees were sampled every 100 generations. Tracer v. 1.5.0 was used to check the effective sampling sizes (ESS) that should be above 200, the stable likelihood plateaus and burn-in value (Rambaut et al. 2013). The first 5000 samples were excluded as burn-in.

The phylogram was visualised in FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/; Rambaut 2014) and edited in Adobe Illustrator CC and Adobe Photoshop CS6 Extended version 13.1.2 × 64. Newly generated sequences in this study are deposited in GenBank. The finalised alignment and tree were deposited in TreeBASE, submission ID: 22942 (combined sequence alignment) (Reviewer access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S22942?x-access-code=f9115cf637b0e4171aab1c980eb15830&format=html) and (Reviewer access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S22943?x-access-code=92a782825ac069b3fd761aff21fa2bf4&format=html) 22943 (ITS sequence alignment) (http://www.treebase.org).

GenBank accession numbers of the sequences used in phylogenetic analyses.

No Taxon Straina Host Origin GenBank accession numbers References
ITS LSU RPB2 ACT
1 Cytospora abyssinica CMW 10181T Eucalyptus globulus Wondo Genet, Ethiopia AY347353 Adams et al. (2005)
2 C. acaciae CBS 468.69 Ceratonia siliqua Spain, Mallorca DQ243804 Adams et al. (2006)
3 C. ampulliformis MFLUCC 16-0583T Sorbus intermedia Russia KY417726 KY417760 KY417794 KY417692 Norphanphoun et al. (2017)
4 C. atrocirrhata HMBF156 KF225610 KF225624 KF498673 Fan et al. (2015a)
5 C. austromontana CMW 6735T Eucalyptus pauciflora Australia AY347361 Adams et al. (2005)
6 C. berberidis CFCC 89927T Berberis dasystachya China KR045620 KR045702 KU710948 KU710990 Liu et al. (2015)
7 C. berkeleyi StanfordT3T Eucalyptus globulus California, USA AY347350 Adams et al. (2005)
8 C. brevispora CBS 116829 Eucalyptus grandis Venezuela AF192321 Adams et al. (2005)
9 C. carbonacea CFCC 89947 Ulmus pumila Qinghai, China KR045622 KP310812 KU710950 KP310842 Yang et al. (2015)
10 C. centravillosa MFLUCC 16-1206T Sorbus domestica Italy MF190122 MF190068 MF377600 Senanayake et al. (2017)
11 C. ceratosperma MFLUCC 16-0625 Acer platanoides Russia KY563246 KY563248 KY563244 KY563242 Tibpromma et al. (2017)
12 C. chrysosperma HMBF151 KF225605 KF225619 KF498668 Fan et al. (2015a)
13 C. cinereostroma CMW 5700T Eucalyptus globulus Chile AY347377 Adams et al. (2005)
14 C. cotini MFLUCC 14-1050T Cotinus coggygria Russia KX430142 KX430143 KX430144 Norphanphoun et al. (2017)
15 C. curvata MFLUCC 15-0865 T Salix alba Russia KY417728 KY417694 Norphanphoun et al. (2017)
16 C. cypri CBS 201.42T Syringa sp. Switzerland DQ243801 Adams et al. (2006)
17 C. diatrypelloidea CMW 8549T Eucalyptus globulus Orbost, Australia AY347368 Adams et al. (2005)
18 C. disciformis CMW6509 AY347374 Adams et al. (2005)
19 C. donetzica MFLUCC 16-0574T Rosa sp. Russia KY417731 KY417765 KY417799 KY417697 Norphanphoun et al. (2017)
20 C. elaeagni CFCC 89632 Elaeagnus angustifolia Ningxia, China KR045626 KR045706 KU710955 KU710995 Fan et al. (2015b)
21 C. erumpens MFLUCC 16-0580T Salix × fragilis Russia KY417733 KY417767 KY417801 KY417699 Norphanphoun et al. (2017)
22 C. eriobotryae IMI136523T Eriobotrya japonica India AY347327 Adams et al. (2005)
23 C. eucalypti LSEQ Sequoia sempervirens California, USA AY347340 Adams et al. (2005)
24 C. eucalyptina CMW 5882 Eucalyptus grandis Cali, Columbia AY347375 Adams et al. (2005)
25 C. fabianae Dunnii Eucalyptus AY347360 Adams et al. (2005)
26 C. friesii CBS 113.81 Picea abies Norway AY347318 Adams et al. (2005)
27 C. gelida MFLUCC 16-0634 T Cotinus coggygria Russia KY563245 KY563247 KY563243 KY563241 Tibpromma et al. (2017)
28 C. germanica CXY1322 Elaeagnus oxycarpa China JQ086563 JX524617 Zhang et al. (2013)
29 C. gigalocus HMBF154 KF225608 KF225622 KF498671 Fan et al. (2015a)
30 C. gigaspora CFCC 89634T Salix psammophila China KF765671 KF765687 KU710960 KU711000 Fan et al. (2015b)
31 C. hippophaes CFCC 89636 KF76567878 KF765694 KF765710 Fan et al. (2015b)
32 C. japonica CBS375.29 Prunus persica Japan AF191185 Adams et al. (2002)
33 C. junipericola MFLUCC 17-0882T Juniperus communis Italy MF190125 MF190072 Senanayake et al. (2017)
34 C. kantschavelii 287-2 Populus deltoides Iran EF447367 Fotouhifar et al. (2010)
35 C. kunzei CBS 118556 Pinus radiata Eastern Cape, SA DQ243791 Adams et al. (2006)
36 C. leucostoma CFCC 50015 Sorbus pohuashanensis China KR045634 KR045714 KU711002 Yang et al. (2015)
37 C. longiostiolata MFLUCC 16-0628T Salix × fragilis Russia KY417734 KY417768 KY417802 KY417700 Norphanphoun et al. (2017)
38 C. lumnitzericola MFLUCC 17-0508 Lumnitzera racernosa Phetchaburi, Thailand MG975778 MH253461 MH253453 MH253457 In this study
39 C. mali CFCC 50044 Malus baccata Haidong, Qinghai KR045637 KR045717 KU710966 KU711005 Yang et al. (2015)
40 C. malicola 167 EF447414 Adams et al. (2002)
41 C. mali-sylvestris MFLUCC 16-0638 T Malus sylvestris Russia KY885017 KY885018 KY885020 KY885019 Hyde et al. (2017)
42 C. melnikii MFLUCC 15-0851T Malus domestica Russia KY417735 KY417769 KY417803 KY417701 Norphanphoun et al. (2017)
43 C. multicollis CBS 105.89T Quercus ilex subsp. rotundifolia Spain DQ243803 Adams et al. (2006)
44 C. myrtagena HiloTib1T Tibouchiina urvilleana Hilo, Hawaii AY347363 Adams et al. (2005)
45 C. nitschkii CMW10180T Eucalyptus globulus Wondo Genet, Ethiopia AY347356 Adams et al. (2005)
46 C. nivea MFLUCC 15-0860 Salix acutifolia Russia KY417737 KY417771 KY417805 KY417703 Norphanphoun et al. (2017)
47 C. palmae CXY1280T Cotinus coggygria Beijing, China JN411939 Zhang et al. (2014)
48 C. parakantschavelii MFLUCC 15-0857T Populus × sibirica Russia KY417738 KY417772 KY417806 KY417704 Norphanphoun et al. (2017)
49 C. parapersoonii T28.1T Prunus persicae Michigan, USA AF191181 Adams et al. (2002)
50 C. paratranslucens MFLUCC 16-0506T Populus alba var. bolleana Russia KY417741 KY417775 KY417809 KY417707 Norphanphoun et al. (2017)
51 C. parasitica MFLUCC 16-0507 Malus domestica Russia KY417740 KY417774 KY417808 KY417706 Norphanphoun et al. (2017)
52 C. pini CBS224.52T Pinus strobus New York AY347316 Adams (2005)
53 C. populina CFCC 89644 Salix psammophila Shaanxi, China KF765686 KF765702 KF765718 Fan et al. (2015b)
54 C. predappioensis MFLU 17-0327 Platanus hybrida Italy MH253451 MH253452 MH253450 MH253449 Hyde et al. (2018)
55 C. prunicola MFLU 17-0995 T Prunus sp. Italy MG742350 MG742351 MG742352 MG742353 Hyde et al. (2018)
56 C. pruinopsis CFCC 50034T Ulmus pumila Shaanxi, China KP281259 KP310806 KU710970 KP310836 Yang et al. (2015)
57 C. pruinosa CFCC 50036 Syzygium aromaticum Qinghai, China KP310800 KP310802 KP310832 Yang et al. (2015)
58 C. quercicola MFLUCC 14-0867T Quercus sp. Italy MF190129 MF190073 Senanayake et al. (2017)
59 C. rhizophorae ATCC38475 Rhizophora mangle LA, USA DQ996040 He et al. (2003)
60 C. rhizophorae ATCC66924 Haliclona caerulea HI, USA DQ092502 Unpublished
61 C. ribis CFCC 50026 Ulmus pumila Qinghai, China KP281267 KP310813 KU710972 KP310843 Yang et al. (2015)
62 C. rosae MFLUCC 14-0845T Rosa canina Italy MF190131 MF190075 Senanayake et al. (2017)
63 C. rosarum 218 EF447387 Fotouhifar et al. (2010)
64 C. rostrata CFCC 89909T Salix cupularis Gansu, China KR045643 KR045722 KU710974 KU711009 Unpublished
65 C. rusanovii MFLUCC 15-0854T Salix babylonica Russia KY417744 KY417778 KY417812 KY417710 Norphanphoun et al. (2017)
66 C. sacculus HMBF281 KF225615 KF225629 KF498678 Fan et al. (2015a)
67 C. salicacearum MFLUCC 16-0509T Salix alba Russia KY417746 KY417780 KY417814 KY417712 Norphanphoun et al. (2017)
68 C. salicicola MFLUCC 14-1052T Salix alba Russia KU982636 KU982635 KU982637 Li et al. (2016)
69 C. salicina MFLUCC 15-0862T Salix alba Russia KY417750 KY417784 KY417818 KY417716 Norphanphoun et al. (2017)
70 C. schulzeri CFCC 50040 Malus domestica Ningxia, China KR045649 KR045728 KU710980 KU711013 Unpublished
71 C. sibiraeae CFCC 50045T Sibiraea angustata Gansu, China KR045651 KR045730 KU710982 KU711015 Liu et al. (2015)
72 C. sorbi MFLUCC 16-0631T Sorbus aucuparia Russia KY417752 KY417786 KY417820 KY417718 Norphanphoun et al. (2017)
73 C. sorbicola MFLUCC 16-0584T Acer pseudoplatanus Russia KY417755 KY417789 KY417823 KY417721 Norphanphoun et al. (2017)
74 C. sordida HMBF159 KF225613 KF225627 KF498676 Fan et al. (2015a)
75 C. sophorae CFCC 50047 Styphnolobium japonicum Shanxi, China KR045653 KR045732 KU710984 KU711017 Fan et al. (2014)
76 C. sophoricola CFCC 89596 Styphnolobium japonicum Gansu, China KR045656 KR045735 KU710987 KU711020 Unpublished
77 C. tanaitica MFLUCC 14-1057T Betula pubescens Russia KT459411 KT459412 KT459413 Ariyawansa et al. (2015)
78 C. thailandica MFLUCC 17-0262 Xylocarpus moluccensis Ranong, Thailand MG975776 MH253463 MH253455 MH253459 In this study
79 C. thailandica MFLUCC 17-0263 Xylocarpus moluccensis Ranong, Thailand MG975777 MH253464 MH253456 MH253460 In this study
80 C. tibouchinae CPC 26333T Tibouchina semidecandra La Reunion, France KX228284 KX228335 Unpublished
81 C. translucens 35 EF447403 Fotouhifar et al. (2010)
82 C. ulmi MFLUCC 15-0863T Ulmus minor Russia KY417759 KY417793 KY417827 KY417725 Norphanphoun et al. (2017)
83 C. valsoidea CMW 4309T Eucalyptus grandis Sibisa, North Sumatra AF192312 Adams et al. (2005)
84 C. variostromatica CMW 6766T Eucalyptus globulus Australia AY347366 Adams et al. (2005)
85 C. vinacea CBS 141585T Vitis sp. New Hampshire, USA KX256256 Lawrence et al. (2017)
86 C. xylocarpi MFLUCC 17-0251 Xylocarpus granatum Ranong, Thailand MG975775 MH253462 MH253454 MH253458 In this study
87 Diaporthe eres AFTOL-ID 935 DQ491514 DQ470919 Spatafora et al. (2006)
88 C.rhizophorae A761 Morinda officinalis China KU529867 Unpublished
89 C.rhizophorae HAB16R13 Cinnamomum porrectum Malaysia HQ336045 Harun et al. (2011)
90 C.rhizophorae M225 Rhizophora mucronata Philippines KR056292 Unpublished
91 C.rhizophorae MUCC302 Eucalyptus grandis Australia EU301057 Unpublished

Results

Phylogenetic analysis of combined ITS, LSU, ACT and RPB2 sequences

The combined alignment of ITS, LSU, ACT and RPB2 sequences comprised 86 taxa, including our strains, with Diaporthe eres (CBS 183.5) as the outgroup taxon. The total length of the dataset was 2037 characters including alignment gaps (1–199, 200–357, 358–518, 519–1056, 1057–1296 and 1297–2037 corresponding to ITS1, 5.8S, ITS2, LSU, ACT and RPB2, respectively). The combined dataset contained 1426 constant, 144 parsimony uninformative and 467 parsimony informative characters. The result from the partition homogeneity test (PHT) was not significant (level 95%), indicating that the individual datasets were congruent and could be combined. The combined dataset was analysed using MP, ML and Bayesian analyses. The trees generated under different optimality criteria were essentially similar in topology and did not differ significantly (data not shown). The descriptive statistics of the phylogram generated from MP analysis based on the combined dataset of ITS, LSU, ACT and RPB2 (Fig. 1) were TL = 2418, CI = 0.375, RI = 0.650, RC = 0.244, HI = 0.625. The best scoring likelihood tree selected with a final value for the combined dataset = -14466.797686. The aligned sequence matrix of the ITS1+ITS2 dataset comprising 19 taxa had 279 constant, 23 parsimony uninformative and 57 parsimony informative characters. The descriptive statistics of the most parsimonious tree (Fig. 2) were TL = 2418, CI = 0.375, RI = 0.650, RC = 0.244, HI = 0.625. The best scoring likelihood tree obtained for the ITS1+ITS2 dataset had a log-likelihood of= -1276.782916.

Figure 1. 

Phylogram generated from maximum parsimony analyses based on analysis of combined ITS, LSU, ACT and RPB2 sequence data. The tree is rooted to Diaporthe eres (AFTOL-ID 935). Maximum parsimony and maximum likelihood bootstrap values ≥50%, Bayesian posterior probabilities ≥0.90 (MPBS/MLBS/PP) are given at the nodes. The species obtained in this study are in blue font. Ex-type taxa from other studies are in black bold.

Figure 2. 

Maximum parsimony phylogenetic tree inferred from ITS1 and ITS2 sequence data. Maximum parsimony and maximum likelihood bootstrap values ≥50%, Bayesian posterior probabilities ≥0.90 (MPBS/MLBS/BIPP) are given at the nodes. The species obtained in this study are in blue font. Ex-type taxa from other studies are in black bold.

Taxonomy

Cytospora lumnitzericola Norphanphoun, T.C. Wen & K.D. Hyde, sp. nov.

Index Fungorum number: IF554778; Facesoffungi number: FoF 04603
Figure 3

Etymology

refers to the host where the fungus was isolated.

Holotype

MFLU 18-1227

Isolated from leaf spot of Lumnitzera racemosa. Culture characteristic: Colonies on MEA reaching 5–6 cm diameter after 2 days at room temperature, colonies circular to irregular, medium dense, flat or effuse, slightly raised, with edge fimbriate, fluffy to fairly fluffy, white to grey from above, light yellow to green from below; not producing pigments in agar. Asexual morph: Conidiogenous cells (8–)8.5–14 × 0.6–1.4(–1.6) μm (x‒ = 8.4 × 1.4, n = 15), blastic, enteroblastic, flask-shaped, phialidic, hyaline and smooth-walled. Conidia (3.7–)4–4.5 × 1–1.3(–1.5) µm (x‒ = 4 × 1.2 µm, n = 30), unicellular, subcylindrical, hyaline, smooth-walled.

Material examined

THAILAND, Phetchaburi Province, the Sirindhorn International Environmental Park, on leaf spot of Lumnitzera racemosa, 30 November 2016, Norphanphoun Chada NNS23-2a (MFLU 18-1227 dried culture, holotype; PDD, isotype); ex-type-living culture, MFLUCC 17-0508, ICMP.

Notes

Based on the multigene phylogeny, Cytospora lumnitzericola is closely related to Cytospora thailandica (Fig. 1). Although conidial sizes of both species are similar, they have significant differences in nucleotides: ITS (26 nt), ACT (22 nt), and RPB2 (53 nt) (Table 5). The phylogeny derived from the ITS regions depicts C. lumnitzericola as an independent lineage close to C. brevispora CBS 116829 and C. eucalyptina CMW5882 (Fig. 2). In future, more collections are needed to confirm whether C. lumnitzericola can exist as a saprobe or endophyte as well as performing tests to confirm its pathogenicity.

Figure 3. 

Cytospora lumnitzericola (MFLUCC 17-0508, from culture). a Mangrove collecting site b, c Lumnitzera racemosa in mangroves forest d, e Colonies on MEA after 6 days (left) and 30 days (right) (d-from above, e-from below) f, g Conidiomata produced on MEA h, l Transverse sections of conidioma i, j, n Conidiogenous cells with attached conidia k, m Conidia. Scale bars: f = 1000 µm, g, h = 500 µm, i, j = 10 µm, k = 5 µm.

Cytospora thailandica Norphanphoun, T.C. Wen & K.D. Hyde, sp. nov.

Index Fungorum number: IF554779; Facesoffungi number: FoF 04605
Figure 4

Etymology

refers to the country where the fungus was collected.

Holotype

MFLU 17-0709

Associated with twigs and branches of Xylocarpus moluccensis. Sexual morph: Stromata immersed in bark. Ascostromata 400–1000 × 70–250 µm diameter, semi-immersed in host tissue, scattered, erumpent, uni- or multi-loculate, with ostiolar neck. Ostiole 70–150 µm diameter, numerous, dark brown to black, at the same level as the disc, occasionally area below disc a lighter entostroma. Peridium comprising several layers of cell of textura angularis, with innermost layer thick, brown, outer layer dark brown. Hamathecium comprising long cylindrical, cellular, anastomosed paraphyses. Asci (21–)23–25 × 4.1–4.7(–5) μm (x‒ = 22 × 4.3 μm, n = 15), 6–8-spored, unitunicate, clavate to elongate obovoid, with a J-, refractive apical ring. Ascospores (5.6–)6–6.8 × 1.3–1.5(–2) μm (x‒ = 6.6 × 1.5 μm, n = 20), biseriate, elongate-allantoid, unicellular, hyaline, smooth-walled. Asexual morph: Conidiomata 400–1200 × 180–380 µm diameter, semi-immersed in host tissue, solitary, erumpent, scattered, discoid, circular to ovoid, with multi-loculate, pycnidial, embedded in stromatic tissue, with ostiole. Ostioles 230–300 µm long, with an ostiolar neck. Peridium comprising few layers of cells of textura angularis, with innermost layer thin, pale brown, outer layer brown to dark brown. Conidiophores unbranched or occasionally branched at the bases, formed from the innermost layer of pycnidial wall, with conidiogenous cells. Conidiogenous cells (3.3–)6–9.1 × 1–1.3(–1.7) μm (x‒ = 6 × 1.3 μm, n = 15), blastic, enteroblastic, flask-shaped, phialidic, hyaline and smooth-walled. Conidia (3.3–)3.8–4 × 1–1.3(–1.5) µm (x‒ = 3.8 × 1.3 µm, n = 30), unicellular, subcylindrical, hyaline, smooth-walled.

Material examined

THAILAND, Ranong Province, Ngao Mangrove Forest, on branches of Xylocarpus moluccensis, 6 December 2016, Norphanphoun Chada NG02a (MFLU 17-0709, holotype; PDD, isotype); ex-type-living cultures, MFLUCC 17-0262, MFLUCC 17-0263, ICMP.

Notes

Cytospora thailandica was collected from branches of Xylocarpus moluccensis. The new species resembles some other Cytospora species, but is characterised by uni- or multi-loculate ascomata/conidiomata with unicellular, subcylindrical and hyaline spores in both morphs. Cytospora species associated with Xylocarpus granatum is also reported in this study as C. xylocarpi (MFLUCC 17-0251, Fig. 5). Cytospora xylocarpi is similar to C. thailandica in its conidiomata being multi-loculate and in the length of conidia in the asexual morph (C. xylocarpi: conidia 3 × 1.1 µm versus 3.8 × 1.3 µm in C. thailandica). However, C. thailandica differs from C. xylocarpi in having shorter ostiolar necks and larger asci and ascospores (Table 2). Phylogenetic analysis of our combined gene also reveals C. thailandica is closely related to C. lumnitzericola (Fig. 1), but there are nucleotide differences as mentioned in notes of C. lumnitzericola. The individual ITS1+ITS2 phylogenetic tree also indicates that C. thailandica is distinct with good support (Fig. 2).

Figure 4. 

Cytospora thailandica (MFLU 17-0709, holotype). a Xylocarpus moluccensis b Branch of Xylocarpus moluccensis c Ascostromata on host substrate d, e Surface of ascomata f Transverse sections through ascostroma to show distribution of locules g–h Longitudinal sections through ascostroma to show distribution of locules i Peridium j Ostiolar neck kakd, n Asci l, m Apical ring oa–of Ascospores p Surface of conidioma q Transverse sections through conidioma to show distribution of locules r, s Longitudinal sections through conidioma to show distribution of locules t Peridium u Ostiolar neck va–vc, w Conidiogenous cells with attached conidia x, y Conidia za, zb Colonies on MEA (za-from above, zb-from below). Scale bars: d = 1000 µm, e–g = 400 µm, h, j, p–s = 200 µm, i, u = 100 µm, ka–kd, n = 10 µm, l, m = 2 µm, oa–of, va–vc, w = 5 µm, t = 50 µm, x, y = 4 µm.

Synopsis of species of Cytospora discussed in the paper.

Taxon Sexual morph Asexual morph References
Ascostoma Ostiolar neck Asci Ascospores Conidiomata Ostiolar neck Conidiogenous cell Conidia
C. lumnitzericola 8.4 × 1.4 4 × 1.2 In this study
C. rhizophorae 370–500 × 100–310 30 × 10–25 13–20 × 1–1.8 3–6 × 1.1–1.5 Kohlm. and Kohlm. (1971)
C. thailandica 400–1000 × 70–250 70–150 22 × 4.3 6.6 × 1.5 400–1200 × 180–380 230–300 6 × 1.3 3.8 × 1.3 In this study
C. xylocarpi 230–600 × 90–250 160–200 26 × 4 5.7 × 1.8 700–1200 × 400–480 200–250 8.5× 1.4 3 × 1 In this study

Cytospora xylocarpi Norphanphoun, T.C. Wen & K.D. Hyde, sp. nov.

Index Fungorum number: IF554810; Facesoffungi number: FoF 04604
Figure 5

Etymology

refers to the host genus that fungus was collected.

Holotype

MFLU 17-0708

Associated with Xylocarpus granatum branches. Sexual morph: Stromata immersed in bark. Ascostromata 230–600 × 90–250 µm diameter, semi-immersed in host tissue, scattered, erumpent, multi-loculate, with ostiolar neck. Ostiole 160–200 µm diameter, numerous, dark brown to black, at the same level as the disc, occasionally area surrounded with white hyphae. Peridium comprising several layers of cells of textura angularis, with innermost layer thick, pale brown, outer layer dark brown to black. Hamathecium comprising long cylindrical, cellular, anastomosed paraphyses. Asci (22–)24–28.8 × 3.6–4.8(–5.1) μm (x‒ = 26 × 4 μm, n = 15), 6–8-spored, unitunicate, clavate to elongate obovoid, with a refractive, J-, apical ring. Ascospores (5.5–)6–6.5 × 1.7–1.8(–2) μm (x‒ = 5.7 × 1.8 μm, n = 20), biseriate, elongate-allantoid, unicellular hyaline, smooth-walled. Asexual morph: Conidiomata 700–1200 × 400–480 µm diameter, semi-immersed in host tissue, solitary, erumpent, scattered, multi-loculate, with ostiole. Ostioles 200–250 µm long, with 1–2 ostiolar necks. Peridium comprising several layers of cells of textura angularis, with innermost layer brown, outer layer dark brown to black. Conidiophores unbranched or occasionally branched at the bases, formed from the innermost layer of pycnidial wall, with conidiogenous cells. Conidiogenous cells (6.3–)7.9–10 × 0.9–1.4(–1.6) μm (x‒ = 8.5× 1.4 μm, n = 15), blastic, enteroblastic, flask-shaped, phialidic, hyaline and smooth-walled. Conidia (2.4–)3–3.1 × 0.8–1(–1.2) µm (x‒ = 3 × 1 µm, n = 30), unicellular, subcylindrical, hyaline, smooth-walled.

Material examined

THAILAND, Ranong Province, Ngao Mangrove Forest, on branches of Xylocarpus granatum, 6 December 2016, Norphanphoun Chada NG09b (MFLU 17-0708, holotype; PDD); ex-type-living cultures, MFLUCC 17-0251, ICMP.

Notes

The asexual morph of C. xylocarpi, studied here, is most similar to C. rhizophorae from dead roots of Rhizophora mangle L. in Guatemala, in having multi-loculate conidiomata and allantoid, slightly curved, hyaline and 3–6 × 1.1–1.5 μm conidia (Kohlmeyer and Kohlmeyer 1971). However, the phylogenies, generated herein, show that C. xylocarpi is distinct from C. rhizophorae (ATCC 38475), a strain from Rhizophora mangle that was identified by Kohlmeyer, the author of the species (Fig. 2). The two species also differ by 25 substitutions in ITS1+ITS2 and were collected from different hosts. Therefore, the collection in the present study is designated as a new species.

Our phylogeny also indicates a close relationship to unpublished sequences from GenBank (Figs 1, 2). Given that no morphological descriptions are available for these, the similarity in the ITS1 and ITS2 sequence between our strain and the sequences from GenBank (HAB16R13, M225, A761, MUCC302) are presented in Table 3. Those strains were collected from different hosts (Table 3) and, together with our strain, show substantial variation in ITS1 and ITS2 (Table 4). More collections are needed to further study morphological and genetic variation in this group.

Figure 5. 

Cytospora xylocarpi (MFLU 17-0708, holotype). a Xylocarpus granatum b Branch of Xylocarpus granatum c Ascostromata on host substrate d Surface of ascomata e Transverse sections through ascostroma to show distribution of locules f, g Longitudinal sections through ascostroma to show distribution of locules h Peridium il, n Asci m, o Ascospores p Germinating spore q, r Colonies on MEA (q-from above, r-below) s Transverse sections through conidioma to show distribution of locules t Longitudinal sections through conidioma to show distribution of locules u, v Conidiogenous cells with attached conidia w Mature conidia. Scale bars: c = 2000 µm, d–f = 500 µm, g = 200 µm, h = 20 µm, i, p = 10 µm, j–o, u–w = 5 µm, s, t = 400 µm.

GenBank BLAST search from ITS1 and ITS2 of Cytospora xylocarpi (MFLUCC 17-0251) with sequence from GenBank identified as Cytospora rhizophorae.

Toxon Strain Host Country Accessions ITS1 ITS2 ITS1+ITS2 Identities (I), Query cover (QC) References
C.rhizophorae HAB16R13 Cinnamomum porrectum Malaysia HQ336045 213/215 167/169 380/384 I=98.9%, QC=99% Harun et al. (2011)
C.rhizophorae M225 Rhizophora mucronata Philippines KR056292 213/217 167/169 380/386 I=98.4%, QC=100% Unpublished
C.rhizophorae A761 Morinda officinalis China KU529867 213/217 166/169 379/386 I=98.2%, QC=100% Unpublished
C.rhizophorae MUCC302 Eucalyptus grandis Australia EU301057 213/217 164/169 377/386 I=97.7%, QC=100% Unpublished
C. rhizophorae ATCC38475 Rhizophora mangle LA, USA DQ996040 187/202 156/166 343/368 I=93.2%, QC=100% He et al. (2003)

Nucleotide differences in the ITS1+ITS2 of Cytospora xylocarpi (MFLUCC 17-0251) with sequence from GenBank identified as Cytospora rhizophorae.

Taxon Strain ITS1
14 16 18 30 92 93 96 99 102 103 104 105 113 115 118 119 135 154
C. xylocarpi MFLUCC 17-0251 - G A C C C C G G G C G C T T C A G
C. rhizophorae ATCC38475 G A C T G A T A T T T A T - C T - T
C.rhizophorae HAB16R13 ? G A C C C C G G G C G C T T C A T
C.rhizophorae M225 ? G A T C C C G G G C G C T T C A T
C.rhizophorae A761 ? G A T C C C G G G C G C T T C A T
C.rhizophorae MUCC302 ? G A T C C C G G G C G C - T C A T
Taxon Strain ITS2
13 24 40 46 47 50 51 75 111 112 115 123
C. xylocarpi MFLUCC 17-0251 C C A T - T T C A A C T
C. rhizophorae ATCC38475 T T - T - - T C G T A T
C.rhizophorae HAB16R13 C T A T - T T C A A C C
C.rhizophorae M225 C T A T - T T C A A C T
C.rhizophorae A761 C T A T T - T C A A C T
C.rhizophorae MUCC302 C T A - - - - T A A C T

Nucleotides differences in the ITS, ACT and RPB2 sequences of Cytospora lumnitzericola, C. thailandica and C. xylocarpi.

Taxon Strain ITS
29 88 91 92 93 94 96 97 99 101 102 103 104 105 106 107 108 111
C. lumnitzericola MFLUCC 17-0508 T C T T T T C T C G G A C T A T A G
C. thailandica MFLUCC 17-0262 T - T - - - T C T C A G - - A C G C
C. thailandica MFLUCC 17-0263 T - T - - - T C T C A G - - A C G C
C. xylocarpi MFLUCC 17-0251 C C C - - C C C C G G G - - G C G G
Taxon Strain ITS
119 120 121 122 123 124 125 134 157 389 396 404 405 412 413 414 415 420
C. lumnitzericola MFLUCC 17-0508 T T C - - - - - T T A A - - - - T G
C. thailandica MFLUCC 17-0262 C T T C - G G - T T G T T - - - - A
C. thailandica MFLUCC 17-0263 C T T C - G G - T T G T T - - - - A
C. xylocarpi MFLUCC 17-0251 T C T C C G G A G C A A A C T T T G
Taxon Strain ITS ACT
439 468 485 487 488 74 78 80 92 95 96 97 107 122 125 129 136 137
C. lumnitzericola MFLUCC 17-0508 T T C T A G C A T T - - C T A G A A
C. thailandica MFLUCC 17-0262 T T T C T T G A A T - - T C T G A G
C. thailandica MFLUCC 17-0263 T T T C T T G A A T - - T C T G A G
C. xylocarpi MFLUCC 17-0251 C C C T T G C T A C C C T C A A G A
Taxon Strain ACT
139 146 147 148 149 150 152 159 165 198 209 210 212 215 216 217 218 223
C. lumnitzericola MFLUCC 17-0508 A A G C T C C G T C T C G A A A C A
C. thailandica MFLUCC 17-0262 A G - - T T T T T T T C A A A - C A
C. thailandica MFLUCC 17-0263 A G - - T T T T T T T C A A A - C A
C. xylocarpi MFLUCC 17-0251 G G - - A A C T C C A T A T G - A -
Taxon Strain ACT RPB2
224 225 231 234 242 245 246 4 18 33 42 57 84 85 96 102 108 120
C. lumnitzericola MFLUCC 17-0508 C G C - - A A T T C T C C T T C G A
C. thailandica MFLUCC 17-0262 T T C T G T G T C A T C T C T C A G
C. thailandica MFLUCC 17-0263 T T C T G T G T C A T C T C T C A G
C. xylocarpi MFLUCC 17-0251 T T A C G T A C T C C T T C C A A G
Taxon Strain RPB2
123 126 129 144 153 171 174 177 204 210 213 216 222 231 237 243 246 279
C. lumnitzericola MFLUCC 17-0508 C G C G T G C C G C T C T T C T C T
C. thailandica MFLUCC 17-0262 T A T A C G T C G T C C C T T T T C
C. thailandica MFLUCC 17-0263 T A T A C G T C G T C C C T T T T C
C. xylocarpi MFLUCC 17-0251 C A C A T A T T C C C T C G C C C T
Taxon Strain RPB2
282 294 306 309 336 339 342 351 352 357 378 390 393 396 402 405 435 441
C. lumnitzericola MFLUCC 17-0508 C A T C T C G T C G A C C G T T C T
C. thailandica MFLUCC 17-0262 T G C T C A A C T C G C T A T C C T
C. thailandica MFLUCC 17-0263 T G C T C A A C T C G C T A T C C T
C. xylocarpi MFLUCC 17-0251 T A C C T C G T C C A T T A C T T G
Taxon Strain RPB2
456 465 468 492 498 510 516 517 543 561 570 576 603 612 613 615 627 633
C. lumnitzericola MFLUCC 17-0508 C T C G T T A T T A A G T T C C C G
C. thailandica MFLUCC 17-0262 C C G C C C A T C A G A C C T G C G
C. thailandica MFLUCC 17-0263 C C G C C C A T C A G A C C T G C G
C. xylocarpi MFLUCC 17-0251 T T T G T C G C C G G G T C T G G A
Taxon Strain RPB2
651 663 675 678 690 693 699 702 711 732
C. lumnitzericola MFLUCC 17-0508 T A C T T G T C C T
C. thailandica MFLUCC 17-0262 C G T C G A C T C C
C. thailandica MFLUCC 17-0263 C G T C G A C T C C
C. xylocarpi MFLUCC 17-0251 C A T C T A C C T T

Acknowledgements

Chada Norphanphoun would like to thank the National Natural Science Foundation of China (No. 31760014) and the Science and Technology Foundation of Guizhou Province (No. [2017]5788); the Mushroom Research Foundation (MRF), Chiang Rai, Thailand, the Thailand Research Fund grant no RSA5980068 entitled “Biodiversity, Phylogeny and role of fungal endophytes on above parts of Rhizophora apiculata and Nypa fruticans” and Mae Fah Luang University for a grant “Diseases of mangrove trees and maintenance of good forestry practice” (Grant number: 60201000201) for support. We would like to thank Dr. Wijarn Meepol from the Ranong Mangrove Forest Research Center, Ranong and the Sirindhorn International Environmental Park, Cha-am, Cha-am District, Phetchaburi, Thailand. R Jeewon thanks Mae Fah Luang University and University of Mauritius for research support.

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