Developmental Morphology of Conidiomata in <em>Phyllosticta</em> Caryotae

Research Article

J Bacteriol Mycol. 2016; 3(4): 1038.

Developmental Morphology of Conidiomata in Phyllosticta Caryotae

Murugan M¹*, Arumugam P² and Arunkumar K³

¹Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, India

²Department of Zoology, School of Life Science, Bharathiar University, Coimbatore, Tamil Nadu, India

3Department Of Plant Science, School of Biological science, Central University of Kerala, Kerala, India

*Corresponding author: Murugan M, Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, India

Received: October 31, 2016; Accepted: December 09, 2016; Published: December 12, 2016


Coelomycetes are anamorphic Ascomycota that produce conidia within a flask- shaped ostiolate pycnidial conidiomata, acervular conidiomata or on a mass of vegetative hyphae (stroma). Coelomycetes fungi play an important role in terrestrial ecosystems. The objective of this work was to clarify the development of conidiomata in Phyllosticta caryota using light microscopical techniques. Light microscopic observations shows symphogenous type of pycnidial formation. The conidia within young pycnidia were found with the sheath surrounding them. The mature conidium is multinucleate. The conidiogenous cells which are produced initially inside the cavity are referred to as “temporary conidiogenous cells”. Subsequently, the cells lining the cavity elongate to form a palisade of permanent conidiogenous cells, which then start producing conidia. During dehiscence of the conidioma, these inner hyaline layers in the papilla dissolve to form the ostiole and the outer thick walled cells from the beak like structure of the conidioma. The papilla may also exert pressure to rupture the epidermis of the host for releasing the conidia outside.

Keywords: Appendages; Coelomycetes; Conidium ontogeny


Traditional classification of Coelomycetes was based on the morphology and was thus subjective, often resulting in artificial generic and species boundaries [1]. In view of the variability and diversity of the coelomycete conidiomata it is no easy task to accommodate them in a system of classification based on conidiomata satisfactorily. The fungi coelomycetes have conidia formed within a cavity lined by fungal or fungal/host tissue. The conidia-bearing structure (conidioma) is classified into five types according to exterior morphology: pycnidial, pycnothyrial, acervular, cuplate, and eustromatic [2] (Watanabe et al. 1998). Many researchers have described pycnidial-type conidiomatal development [3-8] (Mercer 1913). These facts suggest that the morphogenesis of conidioma has taxonomic value. In view of the variability and diversity in the form of the Coelomycete conidiomata it is no easy task to accommodate them in a system of classification based on conidiomata satisfactorily. Development is divided into three stages: primordia, cavity formation, and conidiogenesis, with each pycnidial fungus having a determinate mode in each stage.

The genus Phyllosticta (Teleomorph: Guignardia Viala & Ravaz was established by Persoon in 1818). Many species of Phyllosticta have been known as the causal fungi of leaf spot diseases of various plants [9]. The genus - Phyllosticta is also known as the endophytic fungus in a wide range of host plants, including Ericaceae [10-14]. Plant diseases caused by species of Phyllosticta are reported worldwide. Saccardo [15,16] revised the generic concept of Phyllosticta. The revision of Saccardo was based on perithecia which are subepidermal, lenticular, thin membranous, ostiolate, punctiform, areolate on leaf or rarely on stem, with small, ovoid to oblong, aseptate, hyaline to greenish, conidia. However, most subsequent investigators described the species based on the concept as “All leaf inhabiting pycnidial fungi with hyaline, one celled conidia should be classified in Phyllosticta, whereas morphologically comparable fungi on stem in Phoma” [9]. The genus concept however was reconstructed by van der Aa and collaborators [17].

However, there is still controversy for the species concept. The species of teleomorph Guignardia and its anamorph are host species, with a host range confined to species of a single family, from the results of inoculation tests [18-21], whereas some are endophytic Phyllosticta cultures from 67 plant species in 54 genera of 38 families. All these cultures were identified as a single species, Phyllosticta capitalensis Henn, based on morphology and sequence analysis of ribosomal DNA internal transcribed spacer regions. Baayen et al. [10], revealed that Guignardia citricarpa Kiely (anamorph: P.citricarpa (McAlpine) Aa was different from nonpathogenic species Guignardia mangiferae. Roy isolated from citrus, based on cultural characteristics, growth rate, and thickness of mucoid sheath surrounding the conidial wall. Also, they are different in nucleotide sequence data, although the size of their conidia was same. On the other hand, delimitation of the species of Phyllosticta relationship is not yet available. Hence, in this study, we applied Van der Aa and Vanev species concept [17,9] wherein the species epithet was given on the basis of fungus morphology on diseased leaf of host plant, cultural morphology on diseased leaf of host plant, cultural characterististics, and connection with teleomorph species.

Cryptosporiopsis radicicola produces only excipular covering conidioma-like tissue with adhesive amorphous material and setae. Synnematous conidiomata with abundant macroconidia dominate the colony of Cryptosporiopsis ericae. Cryptosporiopsis rhizophila is different in its globose to subglobose conidiomata, consisting of loosely aggregated vegetative hyphae developing macroconidial conidiophores. Cryptosporiopsis grisea, being the only teleomorph connected species, differs from the others in its distinct columnar surface [22].

Therefore, as more and more data on this effect becomes available, the distinction between Hyphomycetes and Coelomycetes may be abandoned because of the presence of intermediary stages between hyphomycetes, acervular, stromatic and cupulate conidiomata. In this investigation developmental stages of the conidioma in the Phyllosticta caryotae state of the fungus were investigated. This forms part of a programme of work on developmental morphology and taxonomy of Coelomycetes which is in progress in this department.

Materials and Methods

Culture characters and identification

A culture of Phyllosticta caryotae Chen was isolated from leaf of Citrus sinensis from kodaikanal, India. It was grown on Oat Meal Agar (OMA) and Potato Dextrose Agar (PDA) in Petri dishes at room temperature (28°C). Cultures were incubated at 24°C in continuous light, and cultural morphology was examined after 7 days. Spore size was determined by measuring the length and width of 30 to 40 arbitrarily selected conidia from a conidial suspension. The isolates were identified initially by comparing morphological and cultural characteristics (i.e., size of conidia, thickness and length of apical appendages).

Germination study

The initial stages of the development of conidiomatal primordia were studied by slide cultures [23]. For germination studies, conidia were collected aseptically from the teased out conidiomata in 1% glucose solution and allowed to germinate in cavity slides were kept at room temperature (28°C) and were examined every 5hrs for 36hrs to study germination.

Light microscope

To study the development of conidiomata, selected conidiomata with agar were trimmed into 2 mm square blocks and fixed in 2% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) for 2hrs at room temperature (27°C) and 1h at 4°C and post fixed for 12hrs in 1% osmium tetroxide. Specimen were dehydrated through an ascending series of acetone (30-100%) at room temperature, each changes at 30min intervals, followed by 2-3 changes in fresh Spurr in the ratio of 3:1 (Acetone : Spurr) for 6hrs, followed by two changes with absolute Spurr mixture for 24hrs each lasting for 8hrs and polymerised in fresh spurr at 70°C for 8hrs. The samples infiltrated with the resin were transferred to a vacuum chamber for 1h for complete removal of air bubbles. Thin sections (0.5μm) were cut from these blocks and stained with 1% aqueous toluidine blue to study the development of conidiomata and conidiogenesis under the light microscope.


Description of the fungus

Conidiomata pycnidial, pycnidia black, spherical to subspherical, 75-120 μm in diam. sometimes 200 μm diam. with a small ostiole. In vertical section, the pycnidial wall 3-4 layered, the outer layer brown in colour, becoming pale brown to hyaline towards inside. Conidiogenous cells lining the pycnidial cavity, 1-2 celled, short, erect, 5-12 x 2.5 μm, aseptate. Conidia formed singly at the apex of the conidiogenous cell, hyaline, aseptate, 7-13 x 5-10 μm. The appendage arises from the apex of the conidium while the conidium is undergoing development and still attached to the conidiogenous cell (Figure 1).