TECHNIQUES TO STUDY MARINE FUNGI

Techniques to study marine fungi
There are 2 broad techniques for the study of marine fungi, namely the direct detection and indirect culture techniques. For studying higher marine fungi direct examination under a stereozoom microscope (sensu Kohlmeyer, 1979) should be followed. Fungi isolated through culture techniques are generally the ubiquitous terrestrial fungi. Hence, while using any particular technique in marine mycology, the specific purpose of application will have to be borne in mind, since different methods are used for achieving different goals.
Collection Collection is best carried out during low-tide periods after consulting the Tide time. In this way material from the intertidal zone can be sampled. Appropriate footwear (tennis shoes) is recommended for collection in the slushy intertidal regions of mangroves. Gum boots and sandals with straps have not been found useful in our experience as they will remain stuck to sticky mud. A ruck sack should be used in order to allow free hands. Small pieces of wood of manageable size, leaf detritus and algae can be collected. Small pieces may be chipped off from wood structures using a hammer and chisel. Samples are collected in sterile, 30 x 40 cm polythene bags. Wood and leaf samples covered with mud in the intertidal mangrove sites, should be thoroughly rinsed with in situ water in the field to remove the mud. Barnacles and wood-borers that may be present in the wood are removed with forceps or a blunt knife later in the laboratory.
In addition to the collection of naturally occurring wood, marine fungi may also be studied by submerging wooden panels placed in the sea attached to a rope or by employing litter bags in order to submerge substrates such as mangrove and seagrsss leaves and other plant parts. In the latter case, nylon bags of approximately 16 mm² mesh (Newell and Fallon, 1989) or 1 mm² (Sathe-Pathak et al., 1993) may be employed. These methods are extremely useful in ecological studies and yield information on fungal colonization of different substrata and their relation to depth, salinity and other environmental parameters.
Arenicolous fungi are studied by collecting sand in polythene bags, taking care not to break the sample into pieces (Tokura, 1984). Spores of lignicolous marine fungi may be collected from beach foam. Beach foam for this purpose is collected using a broad metallic spatula and placed in broad-mouthed, 200 ml capacity glass jars. The foam is then allowed to settle and "dissolve" in the refrigerator for about 12 h. Enough foam is collected to yield 5-10 ml of water. A method to quantify arenicolous fungi by analysing foam samples has been described by Kirk (1983).
Identification
Monographs and illustrated keys for the identification of lignicolous marine fungi, viz., Kohlmeyer and Kohlmeyer (1979), Kohlmeyer and Volkmann-Kohlmyer (1991), Hyde and Sarma (1999), Sarma and Hyde (1999) and the numerous papers published by marine mycologists should be consulted. In addition, the following texts will be useful and should be consulted for further information (Hyde, 1996; Hyde and Lee, 1995; Hyde et al., 1998; Hyde et al., 1999; Jones, 1976, 1993, 1995; Jones and Alias, 1997; Moss, 1986). Original papers should be consulted whenever there is a doubt. Regular literature survey should be carried out to keep abreast of the taxonomy of these fungi. Computerised literature search from databases in floppy diskettes, such as those provided by ‘Current Contents’ and "Aquatic Sciences and Fisheries Abstracts" (ASFA) is of enormous help in this respect. Major characters that should be observed for identification of marine ascomycetes are listed in the Table.

Table: Important characters for identification of obligately marine lignicolous Ascomycetes
ASCOMATA	ASCI	ASCOSPORES
Ascomata: Cliestothecial, perithecial or apothecial.	Persistent or deliquescent ascus.	Number of cells
Size, colour and presence of papillae	Unitunicate or bitunicate	Size, shape and colour.
Texture: eg., carbonaceous or coriaceous	Size and shape.	Wall nature: thin or thick, smooth or striate
Location on substratum: superficial, immersed	Number of ascospores present. Presence or absence of an apical ring or apical thickening Reaction to Melzer's reagent positive or negative	Presence, nature and location of appendages: eg., mucilaginous, sheath-like, cap-like, as chambered apices, tube-like, bristle- or setae-like, flexuous or cilia-like; position on ascospores; persistent or deciduous.
Presence or absence of periphyses, catenophyses
Peridial structure of ascocarp.
Development
Host: wood, algae etc.