A New Endophytic Species of Neostagonospora (Pleosporales) from the Coastal Grass Spinifex Littoreus in Taiwan

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A new endophytic species of Neostagonospora (Pleosporales) from the coastal grass Spinifex littoreus in Taiwan

Journal: Botany

Manuscript ID cjb-2015-0246.R2

Manuscript Type: Article

Date Submitted by the Author: 19-May-2016

Complete List of Authors: Yang, Jun-Wei; National Central University Yeh, Yu-Hung;Draft National Central University Kirschner, Roland ; Department of Life Sciences, , National Central University, Taiwan

coastal dunes, coelomycetes, culture-dependent, culture-independent, Keyword: Dothideomycetes

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A new endophytic species of Neostagonospora (Pleosporales)

from the coastal grass Spinifex littoreus in Taiwan

JunWei Yang, YuHung Yeh, and Roland Kirschner

Jun-Wei Yang, Yu-Hung Yeh, and Roland Kirschner. Department of Life Sciences,

National Central University, Jhongli District, Taoyuan City 32001, Taiwan (R.O.C.).

Corresponding author: Roland Kirschner (email: [email protected]). Draft

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Abstract: A new species of Neostagonospora associated with Spinifex littoreus was found. Endophytic isolates remained sterile, but a specimen collected on living leaves could be used for morphological characterization and cultivation. The ITS sequences from the the cultures as well as from direct total DNA extraction from the plant indicated conspecificity and a broader geographic distribution than the single field collection, but yielded low support on the genus level in phylogenetic analyses.

Analysis of the RPB2 sequences, however, sufficiently supported a placement of the species in Neostagonospora . The shape of conidiogenous cells as well as numbers of septa and sizes of conidia are usefulDraft characters for distinguishing Neostagonospora species.

Key words : coastal dunes, coelomycetes, culturedependent, cultureindependent,

Dothideomycetes.

Introduction

Endophytes are microorganisms that live asymptomatically within plant tissues

(Narayan et al. 2006). Some endophytes may transform into pathogens causing plant diseases, whereas others may become saprobic decomposers after senescence and death of the plant. In other cases, however, endophytes benefit their host plants by producing a plethora of substances that provide protection and improve the survival of

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the plants (Waller et al. 2005). These features indicate that endophytes play an

important role in ecological communities.

Most of studies on endophytes have been conducted using a culturedependent

approach. It can, however, be expected that the actual diversity of endophytes

colonizing plants is as high as or higher than that suggested by cultivationbased

approaches (Zimmerman and Vitousek 2012). The cultureindependent approach

relies on DNA sequences obtained from the plant sample. The culturedependent

approach usually yields a high proportion of sterile mycelia without characteristics

suitable for morphological identification.Draft Because of the lack of DNA sequence data

for ca. 80% of the described fungal species (Hawksworth 2004), attempts often fail to

identify species with DNA sequences. In this study, we combined the

culturedependent, cultureindependent and field collection approaches to investigate

the diversity of endophytes and to identify species which could not be identified with

a single approach alone.

On the coast of Taiwan, Spinifex littoreus (Poaceae) grows on sandy beaches and

dunes. To propagate and overcome sand burial, the species produces vertical and

horizontal rhizomes and stolons that contribute to the formation of dunes (Maun

2009). Besides its importance for stabilizing and protecting coastal areas, S. littoreus

has also been shown to have antiinflammatory properties (Yogamoorthi and Priya

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2006). Little, however, is known about the organisms associated with this plant.

Through culturedependent and cultureindependent approaches and morphological study of a single specimen gathered in the field, a species was identified as a member of Neostagonospora , but was not identical to any of the previously described species of that genus.

The genus Neostagonospora was introduced by Quaedvlieg et al. (2013) and is characterized by immersed, pycnidial conidiomata, phialidic conidiogenous cells with a periclinal apical wallthickening, and hyaline, septate conidia. The genus currently includes two species and in thisDraft study, an additional species, Neostagonospora spinificis is proposed based on molecular analyses and morphological characteristics.

Materials and Methods

Altogether, 21 individuals of Spinifex littoreus were collected in four locations on sandy sites of the northern and eastern coast of Taiwan, i.e. Taipei City, Taoyuan,

Miaoli and Yilan Counties from 2011 to 2014. Plants were removed with a trowel, individually placed in bags, returned to the laboratory and kept at 4 °C until further processing within 48 hours after sampling. Seventeen of these plants were used exclusively to study endophytes in pure culture. These plants were washed and divided into root, stem, leaf sheath, and leaf lamina before being surface sterilized as

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in Yeh and Kirschner (2014). Three parts of additional plants collected in 2014 were

cut transversally into six fragments. Three of the fragments were used for the

culturedependent approach and the remaining three for the cultureindependent one.

An additional living plant showing leaf spots was collected in 2013.

For the isolation and cultivation of endophytes, 1.3% maltextract agar was used

for culturing from surfacesterilized plant parts. Extraction of DNA and PCR

amplification of internal transcribed spacer (ITS) and large subunit (LSU) ribosomal

DNA regions, and gel electrophoresis were done as in Yeh and Kirschner (2014). For

the RNA polymerase II secondDraft largest subunit (RPB2) gene, PCR amplification

conditions were as in Quaedvlieg et al. (2013). The ITS region was amplified with

primers ITS1F and ITS4, the partial LSU ribosomal DNA with primers NL4 and NL1,

and the partial RPB2 gene with primers fRPB25F and fRPB2414R (White et al.

1990, O'Donnell 1993, Quaedvlieg et al. 2013). Sequencing of PCR amplicons was

done by Mission Biotech (Nankang, Taipei) with the same primers for the PCR. To

culture from the leaf spots, leaves with inconspicuous lesions were cut transversally in

order to open the conidiomata. Conidia from the opened conidiomata were aseptically

transferred with a flamed acupuncture needle to corn meal agar with 0.2%

chloramphenicol. Then the specimen was dried and deposited in the Natural Museum

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of Natural Science, Taichung, Taiwan (TNM). The culture was deposited in the

Bioresource Research and Collection Centre, Hsinchu, Taiwan (BCRC).

Total genomic DNA was extracted from surfacesterilized plant fragments with

Genomic DNA Spin Kit (Plant), according to the modified manufacturer’s protocol

(Bioman Scientific Co., Ltd., Taiwan). The primer pair ITS1F/ITS4 was used for amplification of the ITS rDNA (Gardes and Bruns 1993, White et al. 1990). The PCR products were separated on a 2.0% agarose gel and the band of interest was excised from the gel and purified with the Clean/Gel Extraction Kit (BioKit Biotechnology

Co., Ltd., Taiwan). The purified productsDraft were ligated into the pJET1.2/blunt Cloning

Vector and then recombinants were transformed into Escherichia coli DH5 alpha competent cells with CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Inc.,

USA). The positive transformants of ITS rDNA libraries were screened by PCR amplification of inserts using the pJET1.2 forward/ pJET1.2 reverse sequencing primer pair. PCR amplification products containing the correct size of insert were digested with 10 U of restriction enzyme Msp I l −1 for 1 h at 37 °C. The digested

DNA fragments were separated on 2.0% agarose gels and the analyses of restriction fragment length polymorphism (RFLP) patterns were performed manually. The different PCR amplification products were sequenced by Mission Biotech (Nankang,

Taipei).

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All DNA sequences were edited using CodonCode Aligner version 4.0.1

(CodonCode Corporation, USA). DNA sequences generated in this study were

deposited in the DNA Data Bank of Japan and in GenBank (Table 1). The following

ITS rDNA sequences of the species treated in this paper were deposited in GenBank:

KP676041 and KP676042 obtained by the culturedependent approach, KP676043

and KP676044 by the cultureindependent approach, and KP676045 from a culture

derived from leaf lesions. A partial nuLSU rRNA gene sequence was also obtained

from this culture and deposited in GenBank (KP676046). The ITS sequences were

useful for comparison between species,Draft but not for phylogenetic resolution (FIGURE

2). Following Quaedvlieg et al. (2013), RPB2 gene sequences were also generated

and deposited in DDBJ for the culture derived from leaf lesions (LC055104) and

cultures Z0809 (LC055105) and Z1104 (LC055106). Taxon sampling was based on a

recent phylogenetic treatment of the Phaeosphaeriaceae (Quaedvlieg et al. 2013).

Alignments of the RPB2 gene sequences were produced with the default options of

MUSCLE implemented in MEGA6 (Tamura et al. 2013) without manual editing and

after trimming the alignment block to a size of 310 characters. A maximum likelihood

analysis was done on the alignment block using the Kimura 2parameter model,

gamma distributed with invariant sites, complete deletion of gaps, and 1000 bootstrap

replicates. The tree shown in FIGURE 1 was rooted with Dothistroma pini Hulbary

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(Mycosphaerellaceae, Capnodiales). The alignment file has been deposited in

TreeBASE (number 17770). In addition, the ITS sequences were also compared with other species using the NCBI Nucleotide BLAST tool.

Description of micromorphology is based on dry leaf material. The samples were sectioned by hand and mounted in 5–10% (w/v) aqueous KOH solution and 1% phloxine. The sections were examined at 1000x magnification with an Olympus

CX41 light microscope. The statistical treatment of measurements was done with 30 measurements of the conidia which are given as mean value ± standard deviation with extreme values in brackets. DrawingsDraft were made using scaled paper and processed with Adobe Photoshop.

Results

All three approaches used in this work resulted in the identification of a species of Neostagonospora . Three strains of this species had identical RPB2 gene sequences, but these strains did not sporulate in culture and could not be compared morphologically with the known species. In the phylogenetic analysis, sequences derived from the new species clustered within the Phaeosphaeriaceae of the

Pleosporales (FIGURE 1). On the other hand, the five ITS sequences of the new species formed a well supported clade, but the support for the genus was low

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(FIGURE 2). The infraspecific ITS sequence variation for the new species was 0–1

positions. When comparing ITS sequences exceeding 400 positions with their most

similar matches from BLAST searches, sequences of the new species differed for

24–25 positions (4%) from those of N. caricis and for 32–33 positions (6%) from

those of N. elegiae . According to the phylogenetic tree based on the RPB2 gene,

“Septoria ” arundinacea CBS 133.68 probably belongs to Neostagonospora . Its ITS

sequence differed for 35–36 positions (6%) from those of the undescribed taxon.

Species of the next most closely related genus belonged to Parastagonospora . The

ITS sequences of these ParastagonosporaDraft species differed for at least 39 positions

(7%) from those of the Neostagonospora species. In addition to molecular data, the

morphological characteristics of the new species were different from those of the

other Neostagonospora species. In contrast to the high morphological similarity

between the new fungus from S. littoreus and Ascochyta species, their ITS sequence

similarity was low (less than 90%). Based on the congruent results of the three

approaches used in this work, a new species of Neostagonospora is proposed.

Taxonomy

Neostagonospora spinificis R. Kirschner, JunW. Yang and Yu Hung Yeh, sp. nov.

FIGURE 3

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Index Fungorum IF550947

Type:—Taiwan, Miaoli County, Zhunan Township, sand beach north of Dragon and Phoenix Port (24.7017°N, 120.8589°E), on living leaves of Spinifex littoreus ,

30.03.2013, R. Kirschner 3867 (TNM, holotype!). Extype strain: Bioresource

Collection and Research Centre Taiwan (BCRC) FU30120. ITS rDNA sequence:

GenBank KP676045. LSU rDNA sequence: GenBank KP676046. RPB2 gene sequence: DNA Data Bank of Japan (DDBJ) LC055104.

Etymology: —Derived from the host genus, Spinifex .

Draft

Conidiomata completely immersed, filling the substomatal chamber, forming globose pycnidia, up to 100 µm diam.; wall formed of one layer of pale brown textura angularis. Conidiophores reduced to conidiogenous cells that are broadly compressed ampulliform, brown, smooth, 5–8 µm wide and 2–4 µm high; slightly tapering at apex, with apical wallthickening. Conidia hyaline, smooth, thinwalled, narrowly ellipsoidal or almost cylindrical, rarely slightly narrowing to the apex, apex subobtuse, base truncate, 1septate at the median, measuring (17–)19–21(–24) × (3–)3.5(–4) µm

(n = 30, av. ± SD: 19.7 ± 2.04 × 3.8 ± 0.4 µm). Habitat, living leaf blades of Spinifex littoreus , associated with green tissues or inconspicuous yellow discoloration around a pale black spot. Hitherto known only from Taiwan.

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Discussion

When studying the fungal biodiversity of sand dune soil, Prenafeta Boldú et al.

(2014) found that the culturedependent approach favors easily cultivable saprobic

fungi, whereas the fungi revealed by the DNAbased approach depended on the

selected primers, and the field collections revealed macrofungi that were not detected

by the other two approaches. Both the culturedependent and cultureindependent

approaches yield quick and relatively seasonindependent insights into the diversity of fungi associated with a plant speciesDraft by counting different operational taxonomic units (OTUs) based on different colony morphologies in culture and/or different DNA

sequences. The species found with these two approaches differ considerably with only

few species being recovered by both approaches (Gao et al. 2005). For a large

proportion of these OTUs, however, further progress towards characterization and

identification of species is not possible with these methods, because many cultures

remain sterile or form in vitroartifacts. In addition, ca. 80% of the described species

lack DNA data (Hawksworth 2004). In order to overcome these obstacles, the

traditional approach of field collection with morphological characterization remains

indispensable. Field collection, however, has the disadvantage that morphologically

recognizable specimens might be present only at a certain place and time. As an

example, the fungus described in this paper was detected several times using the

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culturedependent as well as cultureindependent approaches, but only once as traditional field specimen. In all approaches, the fungus was detected only in the leaves, not from stems or roots, which indicates a high specificity towards leaf tissue.

Since a culture was successfully isolated from the field specimen, the ITS and RPB2 gene sequences that were obtained from the sterile cultures can be linked to the new species and a morphological description can be presented.

Based on its morphology, particularly on the oneseptate conidia, the fungus was first tentatively identified as a species of Ascochyta . Among the accepted species of

Ascochyta occurring on Poaceae,Draft however, shapes and sizes do not match with those of the species from S. littoreus (Mel′nik 2000; Punithalingam 1979). The Ascochyta species most similar with respect to conidium size have low genetic similarity to the species with available DNA data (Didymella exitialis (Morini) E. Müll.). The species lacking DNA data differ by having a proven Didymella teleomorph ( D. graminicola

Punith.), or by having apically narrowing conidia (A. arundinariae Tassi, A. hordeicola Punith.), and minor differences of sizes of conidia or pycnidia

(Punithalingam 1979). In addition, when the sequences from N. spinificis are compared with those available from Ascochyta species that exceed 400 positions, the similarity was less than 90%. Ascochyta is a polyphyletic genus within the

Pleosporales, but the type species and close relatives are connected to Didymella in

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the Didymellaceae (De Gruyter et al. 2009), whereas Neostagonospora

phylogenetically belongs to the Phaeosphaeriaceae (Quaedvlieg et al. 2013).

According to Sutton (1980), conidiogenous cells of Stagonospora species in the

former, broad sense, are holoblastic (i.e. without apical wallthickenings),

occasionally annellidic, and have conidia with at least two septa. In comparison, the

species on S. littoreus forms conidiogenous cells with apical wallthickenings and

oneseptate conidia. For morphological reasons, the fungus isolated from S. littoreus

cannot be confused with the species formerly described in Stagonospora .

The placement of a strain designatedDraft as “Septoria ” arundinacea by Quaedvlieg

et al. (2013) in the Neostagonospora clade is confirmed in our study, but further data

about this strain are lacking. If new strains with the same DNA sequences are isolated

in the future and can be characterized morphologically, they could be placed within

Neostagonospora .

The two known species of Neostagonospora were described only from culture,

which might cause some artifacts, whereas the description of N. spinificis is based on

material collected in nature. Unfortunately, the original specimens from field

collections used by Quaedvlieg et al. (2013) were not preserved and details about the

‘symptomatic leaves’ indicating whether the fungi were rather parasitic or saprobic

were not presented. Neostagonospora spinificis differs from the known species by at

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least 24 different positions (4%) of the ITS region. Morphologically, N. spinificis and

N. caricis differ from N. elegiae by their oneseptate conidia, whereas conidia of N. elegiae are (0–)3septate. Neostagonospora spinificis and N. caricis can be distinguished by the length of their conidia, i.e. no more than 19 µm in N. caricis and mostly no less than 19 µm for N. spinificis . Furthermore, the compressed shape of the conidiogenous cells of N. spinificis also differs from the ampulliform conidiogenous cells of the other two species. The deviating shape of conidiogenous cells as well as number of cell layers of the pycnidium (one in N. spinificis , more than one in the other species) are characters thatDraft vary inter as well as infraspecifically within the same genus (Punithalingam 1979, Sutton 1980).

Acknowledgements

This work was financed by the Ministry of Science and Technology (formerly

National Science Council) of Taiwan (NSC102–2621–B–008–001–MY3). The

Yongkang Hiking Group of Zhongli is thanked for the opportunity to collect the specimen at Miaoli.

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TABLE 1. Origins and accession numbers of DNA sequences obtained for Neostagonospora spinificis from leaves of Spinifex littoreus in Taiwan.

Collection DNA sequence accession number Approach * Locality/date number ITS LSU rDNA RPB2 gene Zhuangwei Township, Yilan DDBJ ** Z0809 CD County, LC055105 29.10.2011 Gongliao GenBank Z0907 CD District, Taipei KP676041 City, 29.10.2011 Zhuangwei Township, Yilan Z1104 CD DDBJ LC055106 County, Draft29.10.2011 Houlong Township, GenBank Z1305 CD Miaoli County, KP676042 09.04.2012 Guanyin Township, GenBank 501LS1 CI Taoyuan County, KP676043 01.05.2014 Guanyin Township, GenBank 501L1 CI Taoyuan County, KP676044 01.05.2014 Zhunan RK3867 = Township, GenBank GenBank BCRC FC DDBJ LC055104 Miaoli County, KP676045 KP676046 FU30120 30.03.2013

* CD=culturedependent, CI=cultureindependent, FC=field collection

**DDBJ=DNA Data Bank of Japan 19

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Legends

FIGURE 1. Maximum Likelihood tree showing estimated relationships of

Neostagonospora spinificis among Pleosporales based on RPB2 gene sequences, the names given with quotes follow Quaedvlieg et al. (2013). Bootstrap values above

50% (1,000 replicates) are indicated at the nodes. The tree was rooted with

Dothistroma pini .

FIGURE 2. Maximum Likelihood tree showing estimated relationships of

Neostagonospora spinificis among Pleosporales based on ITS sequences, the names given with quotes follow Quaedvlieg et al. (2013). Bootstrap values above 50% (1,000 replicates) are indicated Draft at the nodes. The tree was rooted with Vrystaatia aloeicola .

FIGURE 3. Neostagonospora spinificis . A–B. Photographs of pycnidia. C.

Pycnidium under stoma. D. Pycnidium under epidermis. E. Conidiogenous cells. F–G.

Conidia. Scale bars: A–B = 10 m, C = 20 m, D = 10 m, E = 2.5 m, F–G = 10

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Draft

FIGURE 1. 250x250mm (300 x 300 DPI)

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Draft

Figure 2 274x218mm (300 x 300 DPI)

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Draft

Figure 3 150x216mm (300 x 300 DPI)

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