Wood–Inhabiting Marine Fungi from the Coast of Shandong Ⅱ

Wood–inhabiting marine fungi from the coast of Shandong, China Ⅲ

SUN Su-Li JIN Jing* LI Bao-Du LU Bing-Sheng College of Plant Protection, Qingdao Agricultural University, Qingdao 266109, China

Abstract: Five species of higher marine fungi were observed on the incubated drift and intertidal woods collected from the coasts of Yellow Sea and Bohai Sea. Among them, Halosphaeriopsis was a genus newly recorded for China. Taxonomy and morphology of these species were discussed in this paper. The specimens were deposited in Mycology Herbarium at Qingdao Agricultural University (MHQAU). Key words: new record, driftwood, intertidal wood, gelatinous sheath, ascospore appendage

1 INTRODUCTION The marine fungi have been perfectly known in Europe (England, France and Denmark), North America (USA and Canada), South America (Brazil and Chile), Oceania (Australia and New Zealand) and some countries of Asia (Thailand, India, Malaysia, Japan and Brunei). Whereas the marine fungi in China were still imperfectly documented. The early reports on species, distribution, seasonality pattern and the impact of hydrological factors about lignicolous marine fungi were made by Vrijmoed et al. (1982a, 1982b, 1986a, 1986b). In recent years, a number of marine fungi from mangrove along the coast of Hong Kong were recorded (Hyde et al. 1992; Vrijmoed et al. 1994; Hyde & Pointing 2000; Lu et al. 2000; Jones & Vrijmoed 2003), and some compounds which were unique and novel in structure were found (Vrijmoed et al. 1990; Lin et al. 2001). There were no other reports on marine fungi from Bohai Sea, Yellow Sea and East China Sea except the papers published by Huang (1994),

Supported by the National Natural Science Foundation of China (No. 30500004) *Corresponding author. E-mail: [email protected] Received: 26-11-2007, accepted: 20-12-2007

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Jin (2004) and Liu (2006).

2 MATERIALS AND METHODS Driftwoods and intertidal woods were collected from the four sandy beaches of Longkou (Bohai Sea), Yantai, Weihai and Qingdao (Yellow Sea) and placed in clean ‘snap lock’ polythene bags. The samples were taken to the laboratory and examined under stero microscope as soon as possible. Some samples in which fungi were not observed were put into the sterile moist chambers containing sands and seawater in order to induce the production of ascocarps or conidiomata. The chambers were kept at the room temperature. Sterile seawater was sprayed regularly on the samples to prevent from dry.

3 RESULTS AND DISCUSSIONS Five fungal species are obtained and identified from the incubated samples, which belong to Ascomycota and mitosporic fungi. The morphological characteristics are described as follows. Halosphaeriopsis T.W. Johnson, J. Elisha Mitchell Sci. Soc. 74: 44, 1958 Perithecia innate, dark brown or black, necked, subglobose or ellipsoid, non-paraphysate. Asci 2-8-spored, irregularly clavate, unitunicate, deliquescing. Ascospores ellipsoid, hyaline, 1-3-septate, guttulate; each spore with three stiff, straight or curved, hyaline, setose, equatorial appendages. Type species: Halosphaeriopsis mediosetigera (A.B. Cribb et J.W. Cribb) T. W. Johnson, J. Elisha Mitchell Sci. Soc. 74: 44, 1958. Halosphaeriopsis mediosetigera (A.B. Cribb et J.W. Cribb) T.W. Johnson, J. Elisha Mitchell Sci. Soc. 74: 44, 1958. Fig. 1: A-L ≡Halosphaeria mediosetigera A.B. Cribb et J.W. Cribb, University Queensland Paper of the Department of Botany, 3: 100, 1956. ＝Halosphaeria mediosetigera var. grandispora Kohlm., Nova Hedwigia 2: 10, 1956. Anamorph: Trichocladium achrasporum (Meyers et R.T. Moore) M. Dixon ex Shearer et J.L. Crane, Mycologia, 63: 244, 1971. Ascomata solitary or gregarious, embedded or superficial, black, subglobose or globose, subcarbonaceous, 280-615 × 207-510μm (mean 426 × 415µm), necked, the ascocarp wall consists of 4-7 layers of irregular, polygonal, thick-walled cells, forming a textura angularis, non-paraphysate. Neck papillate, conoid or cylindric, 145-300 × 29-56μm, centric or eccentric, brown, occasionally more than one. Asci initial irregularly clavate, irregularly fusiform near maturity, unitunicate, thin-walled, early deliquescing, 50-65 × 23-36μm, eight-spored. Ascospores long-ellipsoid or subfusiform, hyaline, 24-37 × 9-11µm, 2-celled, with a large guttule in each cell, slightly constricted at the septa. Aquatorial appendages three, setose, curved, nonflexous, hyaline, terminal appendages cap-like. Chlamydospore chains fuscous, 5-26-cell, mostly apical. Johnson (1958) considered the species in Halosphaeria had gelatinous, flexuous and deliquescing terminal and meridional appendages, whereas Halosphaeria mediosetigera had bristle-like, curved but non-flexous, persistent meridional appendages formed from exospore and the

http://journals.im.ac.cn/jwxtcn 68 Mycosystema terminal appendages were cap-like. Therefore he established the genus Halosphaeriopsis based on Halosphaeria mediosetigera. At present, the genus Halosphaeriopsis is monotypic, cosmopolitan. In Johnson’s description the ascospore has four, rarely five appendages, whereas in our observation under SEM the ascospore has only three appendages distributed proportionally in the equator of ascospore. The appendages are setose or bristle-like under light microscope, but under SEM the base of the appendage is wide and tapered towards to the top (Fig. 1-E). Therefore, the characteristics of the genus need to be emended. The isolates on the GPY (Glucose 10g, Peptone 5g, Yeast 1g; 1000mL seawater), YGA (Yeast 4g, Glucose 20g, Agar 20g; 1000mL seawater) and PDA (Potato 200g, Dextrose 20g, Agar 20g; 1000mL seawater) are obtained by dilution method and cultivation at 20℃-22℃. Ascocarps are easily produced in one month after subinoculation. Ascocarps in culture media have one or more necks, generally 1-3 (Fig. 1-C, D), occasionally 4-5, whereas the ascocarps on natural substrates generally have one neck. (Fig. 1-A). This is a new record for China. Specimen examined: Yantai; on unidentified woods; incubated for three and a half years; Jin Jing; January 22, 2003; YT2001. Ceriosporopsis halima Linder, in Barghoorn & Linder, Farlowia, 1: 409, 1944. Fig. 2: A-F =Ceriosporopsis barbata Höhnk, Veroëff. Inst. Meereforsch. Bremerhaven, 3: 210, 1955. Anamorph：Zalerion maritimum (Linder) Anastasiou, Can. J. Bot. 41:1136, 1963. ≡Helicoma salinum Linder, Farlowia 1: 405, 1944. Perithecia solitary or gregarious, immersed, partly immersed or superficial, subglobose to elongate-ellipsoid, youth hyaline, memebranous, maturity black to the naked eye or dark brown to dark under the microscope, coriaceous, 120-190 × 120-180µm (mean 159 × 153µm), necked, papillate, long-conoid or cylindric, centric or somewhat eccentric, variable in size, 20-160 × 19-39µm. Ascospores enveloped a sheath, ellipsoid, hyaline, 16.9-27.5 × 8.5-16.3µm, 2-celled, constricted at the septa, each cell usually with a single large vacuole, with a stout, hyaline, tapering, straight, curved or flexous appendages at each end, the polar appendages appears to be an outgrowth of the spore wall, sometimes gelatinized and either deliquescent or dehiscent, enveloped by the sheath which is strap-like, collar-like or wristband-like at the bases of the polar appendages. It is one of the most common marine fungi and exists in various habitats. Barghoorn & Linder (1944) and Johnson & Sparrow (1961) found that Helicoma salinum in artificial medium was the imperfect stage of C. halima. Since then, the anamorph was not found once again although the species was observed many times (Anastasiou 1963). Jones (1962) observed that the perithecia of C. halima have multi-ostiole, even more than 16 ostioles, and most of ostioles have branches. We observed that some perithecia were buried in the substrata, and some necks were exposed through the substrata and the upper part was lighter in color. Most perithecia had only one ostiole with or without two branches, and some had two ostioles. On SWA (seawater and agar) medium,

Vol.27 No.1 69 perithecia were produced in fifty days, with 1-3 necks which were 1mm long and variable in position on the perithecia. The ascospore appendages wavedly extended to 100µm in length in seawater (Fig.2-D, E). The fungus was reported from blocks of bamboo and masson pine immersed in seawater from Hong Kong in 1982 (Vrijmoed et al. 1982a, 1982b) and it was the second record from China. Specimen examined: Weihai; on unidentified woods; incubated for two months; Sun Su-Li; January 27, 2007; WH2106. Zalerion R.T. Moore & Meyers, Can. J. Microbiol. 8: 408, 1962 emend. Anastasiou, Can. J. Bot. 41: 1136, 1963. Conidiophores of up to several cells; conidia sessile, non-hygroscopic, pleuricellular, monacrogenous, filaments variable in length and septation, cell wall constricted at the septa, sometimes branched, coiled irregularly in three dimensions, often producing a knot or ball of cells, subhyaline to fuscous. Although the majority of the Helicosporeae are terrestrial, some are marine, such as Cirrenalia, Cumulospora, Helicorhoidion and Zalerion. It is clearly distinguished by morphological characteristics among the four genera. Z. maritimum and Z. varium have been reported in Hong Kong (Vrijmoed et al. 1982a, 1982b, 1986a, 1986b). Zalerion maritimum (Linder) Anastasiou, Can. J. Bot., 41: 1136, 1963. Fig. 2: G-K ≡Helicoma maritimum Linder, Farlowia, 1: 405, 1944. ＝Helicoma salinum Linder, Farlowia, 1: 406, 1944. Telemorph: Ceriosporopsis halima Linder, in Barghoorn & Linder, Farlowia, 1: 409, 1944. Lulworthia uniseptata Nakagiri, Trans. Mycol. Soc. Japan, 25: 382, 1984. Colonies effuse, sparse, circular, mycelium branching, dilutely to deep fuscous. Conidiophores variable in size, up to 50µm long, usually hyaline or pallid-fuscous, with erect or irregularly bent branches from the sterile mycelium, distantly or not septate. Conidiogenous cells monoblastic, usually uniform with basal cell in color, occasionally connect with basal cell of conidia when it dropped from the conidiophores. Conidia acrogenous, solitary, dilutely to deep fuscous, cell wall smooth, 7-28-septate, the septa deep fuscous or black, deeper than all cells of conidia, thick-walled and strongly constricted at the septa, each cell 7.3-12.5 × 7.1-9.0µm (mean 10.7 × 7.9µm)，spore filament 1-3 times coiled, coil 18.3-36.7µm in diameter, conidia occasionally fragment into separate cells. The fungus was identified as Helicoma maritimum by Linder (1944). Moore & Meyers (1962) erected Zalerion including four species: Z. nepura, Z. eistla, Z. xylestrix and Z. raptor. Later, Anastasiou (1963) emended Zalerion after examining the hundreds of intergrading speciments, and he considered Helicoma salinum, which had been reported to be the anamorph of Ceriosporopsis halima Linder (1944), was actually synonymous with H. maritimum. He also considered Z. nepura, Z. eistla, Z. xylestrix and Z. raptor as physiological races of H. maritimum, and combined H. maritimum

http://journals.im.ac.cn/jwxtcn 70 Mycosystema as Z. maritima, and regarded Z. maritima as the type of Zalerion. His opinions were admitted by many mycologists (Ellis 1976; Kohlmeyer & Kohlmeyer 1979; Molina & Hughes 1982; Goos 1985; Hyde & Pointing 2000). Conidium ontogeny of Z. maritimum was observed by Cole (1976). Molina & Hughes (1982) studied growth responses of Z. maritimum to salinity and temperature, he concluded that optimal growth condition was in 29.7% and 33.3% at 20℃ and 25℃ respectively. Nakagiri (1984) reported Z. maritimum was also the anamorph of Lulworthia uniseptata. The cultural colonies of the fungus on PDA agar were darker than those on GPY agar and grew quickly to 4.5cm in diameter within 20 days. The authors found the immature hyaline perithecia of Ceriosporopsis halima in the culture. Specimen examined: Longkou; on unidentified woods; incubated for three and a half years; Jin Jing; November 28, 2003; LK2020, Zalerion varium Anastasiou, Can. J. Bot., 41: 1136, 1963. Fig. 2: L-P Colonies sparse, black on substrate. Mycelium hyaline, septate, branched. Conidiophores hyaline, simple, septate, occasionally absent. Conidia solitary, embedded in substrata or on effuse mycelium, brown to dark brown, black in mass, 20.2-100.5 × 10.6-30.5µm, constricted at the septa, septa dark-colored, conidial filament irregularly coiled in three dimensions, and forming a tangled knot or ball of cells, visible to the naked eyes, sometimes branched and occasionally subtending an additional conidium; conidial cells 4.0-9.2 × 3.4-7.5µm (mean 5.7 × 4.5µm). Z. varium differs from Z. maritimum in two important characteristics. The filament of the conidia usually turns to produce a lateral spiral in Z. maritimum and a terminal spiral in Z. varium. In addition, Z. varium always produces more gyres and complex spores embedded in its natural woody substrata. Some conidia may be composed of several hundred cells and are very large. Aerial spiral conidia of the Z. varium are looser than those embedded in substrata, and often fail to coil and are long, many-celled strands, or break into short fragments (Anastasiou 1963). Z. varium occurred frequently on incubated woods from Weihai, and the colonies were large and can be seen by the naked eyes on substrata. It is the second report of the fungus in China. Specimen examined: Weihai; on unidentified woods; incubated for 2 months; Li Bao-Du; January 27, 2007; WH2111. Torula herbarum (Pers.) Link, Magazin Ges. naturf. Freunde, Berlin 3(1-2): 19,1809 Fig. 1: M-Q ≡Monilia herbarum Pers., Syn. meth. fung.. (Göttingen) 2: 693, 1801. Colonies black, velvety. Conidiophores distantly or not septate, when septa absent the conidiophores become globose and swelling. Conidia catenate, acropetally matured, straight or slightly curved, more or less cylindrical, rounded at the ends, brown to dark, verruculose or finely echinulate, 14.5-35.4 × 6.0-8.7µm, 3-6- (mostly 3-4-) septate, deep constricted at the septa, septa black, the basal cell of conidia cupulate when departed from conidiogenous cells. T. herbarum is very common on dead herbaceous stems, occasionally on leaves and woods, and

Vol.27 No.1 71 isolated from air and soil. It is cosmopolitan, especially exists in temperate regions (Ellis 1971). T. herbarum is similar to T. herbarum f. quaternella Sacc., the distinct characters are listed in Table 1.

Table 1 Comparison between Torula herbarum and Torula herbarum f. quaternella

Species Torula herbarum f. quaternella Torula herbarum

Size of conidia 12.2-19.8 × 3.8-5.5µm 14.5-35.4 × 6.0-8.7µm

Surface of conidia smooth verruculose or finely echinulate

Number of septa 1-3 3-6

Colour of conidia yellowish to brown brown to dark

T. herbarum was isolated from the wood collected from the coast of Qingdao. Colonies are more or less circular, and very different in color on PDA and GPY media. Colonies on PDA have plenty and flourish hyphae, initially subhyaline or pallid, deep brown later, with a conspicuous protuberance in the middle which is darker in colour (Fig. 1-N, right). Abundant conidia were produced under the mycelial mass, and the colonies had mushroom-like odor. The colonies on GPY are subhyaline, pallid or pallid-flavous, with sparse mycelia but plentiful conidia (Fig. 1-N, left). Specimen examined: Qingdao, on unidentified woods; incubated for two months; Sun Su-Li, April 22, 2007; YK2034.

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Fig. 1 A-L: Halosphaeriopsis mediosetigera; A. Perithecium on wood; B. Culture on PDA (seawater); C-D. Cultural perithecia with two and three necks; E. Ascospore with three aquatorial and two terminal cap-like appendages under SEM; F. Section of ascocarp through ostiole canal with brown layer; G. Section of ascocarp peridial wall; H. Chains of chalamydospores; I. Young clavate asci; J. Sub-mature fusiform ascus; K. Mature ascus with spores and ascus wall prior to deliquescing; L. Ascospore with aquatorial appendages. M-Q: Torula herbarum; M. Colonies on wood; N. Configuration of conidium and conidiogenous cell; O. Culture on GPY (left) and PDA (right, seawater); P. Four- and six-celled conidia with two teminal cupulate cells; Q.

Seven-celled conidium with the cupulate basal cell. (F, G, J, K, L, P, Q under Nikon interference microscope, the others under

Olympus bright field. Scale bars: B, M, O=2cm; A, C, D=200μm; F, H=100μm; G, I-K, P, Q =20μm; E, L, N=10μm).

http://journals.im.ac.cn/jwxtcn 74 Mycosystema

Fig. 2 A-F: Ceriosporopsis halima; A. Immersed perithecia with exposed necks on wood; B. Superficial perithecium on substrate; C. Sub-mature asci; D, E. Wave-likely extended polar appendages; F. Ascospore with enveloping exosporial sheath which is collar-like at the base of polar appendages. G-K: Zalerion maritimum; G. Colonies on wood; H. Culture on PDA (seawater); I. Configuration of conidium and conidiophore; J. 1-coiled conidium; K. 2.5-coiled conidium. L-P: Z. varia; L. Colonies on wood; M, N. Conidia; O, P. A tangled knot conidium and a terminal spiral conidium under SEM. (E under Nikon contrast microscope; C, D, F, M, N under Nikon interference microscope; the others under Olympus bright field. Scale bars: H=2cm; A,B, G, L=100μm; C-F, M, N=20μm; I-K, O, P=10μm).