MORPHOLOGICAL, CYTOLOGICAL, AND CULTURAL ASPECTS OF CURVULARIA PALLESCENS

Abstracts

Curvularia pallescens Boedijn (Hyphomycetes) is redescribed with the aid of a scanning electron microscope, and the optimal cultural conditions for growing this fungus are discussed. Cytological analysis and nuclear condition, observed through the HCl-Giemsa technique, showed vegetative and reproductive structures (hypha and conidia) formed by uni, bi, tri, and multinucleated segments. Cultures of C. pallescens in Complete medium and in Potato dextrose agar varied on growth, on aspects of the border of the colonies and also on medium pigmentation. The Complete medium and the temperature between 25-28°C were the most indicated for growth of C. pallescens.

conidial development; fungi; electron microscopy; Hyphomycetes

MORPHOLOGICAL, CYTOLOGICAL, AND CULTURAL ASPECTS OF CURVULARIA PALLESCENS

Sônia Valéria Pereira Freire, Laura Mesquita Paiva, Elza Aúrea de Luna-Alves Lima, Leonor Costa Maia^*

Departamento de Micologia, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife, PE, Brasil

Approved: July 23, 1998

ABSTRACT

Curvularia pallescens Boedijn (Hyphomycetes) is redescribed with the aid of a scanning electron microscope, and the optimal cultural conditions for growing this fungus are discussed. Cytological analysis and nuclear condition, observed through the HCl-Giemsa technique, showed vegetative and reproductive structures (hypha and conidia) formed by uni, bi, tri, and multinucleated segments. Cultures of C. pallescens in Complete medium and in Potato dextrose agar varied on growth, on aspects of the border of the colonies and also on medium pigmentation. The Complete medium and the temperature between 25-28^oC were the most indicated for growth of C. pallescens.

Key words: conidial development, fungi, electron microscopy, Hyphomycetes.

INTRODUCTION

Species of Curvularia Boedijn are saprophytes or phytopathogenic, occur mostly in tropical and subtropical areas, and are isolated from soil, air, organic matter, plants and animals, including humans (22). Some Curvularia species have been more extensively studied since they are known as cellulase producers (2, 3, 7, 9, 14).

There are more than 30 species of Curvularia; many are anamorphic states of the Loculoascomycete genera Cochliobolus and Pseudocochliobolus (19). Farr et al. (6) reported that C. pallescens occurs on grasses and other substrata throughout tropical and subtropical regions. The species is isolated commonly from soil and decaying plant debris. However, it has been isolated from a disseminated infection in a 13 year-old boy (11).

Conidiogenesis has been studied in other species of Curvularia, including a study of spore development in C. inequalis (Shear) Boedijn using time lapse photography (10). Similar ontogenic studies have not been made of C. pallescens, however.

The aim of this work is to describe morphological and cytological features of vegetative and reproductive structures of C. pallescens observed with the light and the scanning electron microscope (SEM), and to indicate conditions for better growth of this species in culture.

MATERIALS AND METHODS

Fungal isolate, culture medium, and growth conditions.

A strain of C. pallescens, developed on filter paper during a soil potential experiment with an alluvial soil of a semi-arid area of northeast Brazil (4), was maintained in the culture collection (Micoteca URM - No. 3296) of the "Departamento de Micologia, Universidade Federal de Pernambuco, Brasil". Inocula from a monosporic culture were transferred to Petri dishes containing Potato dextrose agar (PDA) or Complete medium (CM) (17), modified by Azevedo and Costa (1) and incubated for 10 days at laboratory conditions (25^oC-28^oC; 12h day/night). For both media the pH was adjusted to 6.0 with 1N NaOH. Colony development was observed and measured daily. In order to determine the ideal temperature for growing this species, PDA cultures were maintained at 25°-28°C, 30°-32°C, and 42°-43°C, and colony diameters were measured during an eight day period.

Potato dextrose agar plates received 0.1ml of a conidial suspension of C. pallescens. Five squares (2 x 2 cm) of previously autoclaved dialyses membranes, were placed over the suspension in the Petri dishes. Using cotton blue dye, fungal structures developing on the membranes were examined 72 hr later. The HCl-Giemsa technique (8) modified by Luna and Azevedo (13) was employed to stain vegetative and reproductive structures of the fungus. The process included fixation in an ethanol, lactic acid, and acetic acid solution (6:1:1), hydration through an alcoholic series (90%, 70%, 50%), hydrolysis in a 1N HCl solution for 8 min at 27°C, and for 6 min in water bath (60°C - 63°C). The material was washed three times with distilled water and transferred to phosphate buffer (pH 7.0) for 60 min. For staining, the material was immersed for 20 min in a HCl-Giemsa solution (1:10) prepared with phosphate buffer. After washing in the same buffer, the material was mounted on a glass slide and observed with a light microscope. In order to stain hyphal nuclei, dialysis membranes (previously autoclaved) were placed over 0.1 ml of a conidial suspension in Petri dishes with PDA. After 72 hours the mycelium grown on the membranes was stained with HCl-Giemsa. To stain conidial nuclei a thin layer of an albumin water solution (5%) was placed over glass slides and slightly dried. The side of the glass slide with albumin was pressed over the colonies previously developed and stained with HCl-Giemsa.

Scanning electron microscope (SEM) studies

Cultures of C. pallescens grown on pieces of dialysis membranes were placed over PDA as described above. Seventy two hours later the membranes with developed cultures were processed for SEM. Live material was fixed for 2 h at 25^oC in 2.5% glutaraldehyde, washed three times with phosphate buffer, postfixed in aqueous 0.1% OsO₄, washed with the same buffer, dehydrated in an acetone series (50-70-90-100%), critical point dried, sputter-coated with gold, and then observed using a JEOL SEM at 80 Kv.

RESULTS

Cultures of C. pallescens developed in PDA and MC showed different growth rates, colony morphologies and pigmentations of the media. When maintained in MC the fungus developed colonies of compact texture, brown on both surfaces, irregular borders, and without exudates. Formation of sectors and concentric zones were observed during all stages of growth (Figs. 1, 2). When the strain was cultivated in PDA the colonies were grey in color, and black on the backside, presented irregular borders, cottony growth with concentric zones, and production of exudates (Figs. 1, 2). From the first to the 4th day the cultures in PDA and CM had different growth rates: those in PDA developed larger colonies; between the 5th and the 7th day the size of colonies was similar in both media. However at the 10th day, colonies developed on CM were greater in diameter (Fig. 3).

Figures 1 - 2: Colonies of C. pallescens after 10 days of growth in MC (a) and PDA (b). 1 - Front view. See sectors and zonations (arrowed). 2 - Back view.

As expected, growth of the strain of C. pallescens varied according to cultural conditions. On PDA, temperature between 25-28°C was more suitable for growth: colonies 9.0 cm wide formed after 8 days of incubation. When the cultures were maintained at 30-32°C, the colonies did not develop more than 1.6 cm, and when incubated at 42-43°C the fungus did not grow.

After 72h of growth on dialyses membranes over PDA, colonies of C. pallescens produced well developed mycelium with some anastomosis (Fig. 4) and formation of conidia on sympodially proliferating conidiogenous cells (Figs. 5, 6). Colonies were effuse, gray, becoming black when older. Mycelium was mostly immersed, forming straight or flexuous and smooth conidiophores, which appeared roughed when observed with the SEM (Figs. 6-9) as a result of condensation of exudates. The conidiogenous cell was broader at the apex and seemed to be inflated at first, with sympodial formation of conidia (Fig. 7). Conidia were slightly curved, three septate, with the medium septum somewhat off-center. In general, central cells were broader and darker than the end cells (Fig. 8). Some mature conidia were slightly collapsed after being liberated from the conidiophore (Fig. 9). The conidia observed were in different stages of development, measuring 12-28 X 6-12 mm.

Figures 4-11: Morphological and cytological aspects of C. pallescens grown in culture and on dialysis membranes, observed with the SEM (Figs. 4, 6, 7, 8, 9) and the LM (Figs. 5, 10, 11). 4. Hyphal anastomosis (X 1200). 5. Conidiophore with sympodially formed conidia (X 380). 6. Slightly curved and roughened conidiophore with many conidia (X 1900). 7. A primordium (P) and an older conidiophore as seen with the SEM (X 1900). 8. A sympodially branched conidiophore with different ages of conidia (X 3300). 9. Conidiogenous cell slightly inflated with sympodially formed conidia (X 3300). 10. Mycelium showing multinucleate condition of hyphal segments and conidium with three germinating cells, all developing multinucleate germ tubes (X 600). 11. Hyphal differentiation showing bi, tri, and multinucleate segments (X 1000). Figs. 4, 6, 7, 8, 9, (scale bar = 10 µm).

Cytological analysis of C. pallescens showed that the mycelium formed by hypha had uninucleate to multinucleate segments, and globose to elliptical nuclei (Fig. 10). Multinucleate conidia and germ tubes were observed during hyphal differentiation (Figs.10, 11). The germination of conidia did not show polarity, with germ tubes developing usually from the central cells (Fig. 10) and giving rise to multinucleated hyphal segments.

DISCUSSION

The behavior of a strain of C. pallescens may differ according to the nutrient growth medium used in the laboratory. There was observed formation of sectors, which may indicate a high genetic variability in the species. Differences in growth of the strain indicate that a richer media promote better development of colonies, as expected. The natural medium PDA, is the most used for growing fungi in laboratory and was employed for growth of Curvularia pallescens; however it promoted low sporulation of the strain. Because of that a semi-synthetic and richer medium (CM) was employed and better sporulation was obtained. Then, the Complete medium is more suitable than PDA for growing and sporulation of Curvularia pallescens. Cytological analysis showed higher conidial production when cultures of C. pallescens were grown on a rich nutrient medium (15). The same results were observed with other species of Hyphomycetes (12,18). In the same way, the ideal temperature for growth of fungi varies according to cultural conditions. Based on the observed results C. pallescens can be classified as a mesophilic species - it reached optimum growth when temperature was between 25-28°C. Olufolaji (15) observed increase in spore numbers of C. pallescens when cultures were maintained at 24°C. However significant decrease in sporulation was observed when cultures were maintained at a minimum temperature of 9°C and at a maximum of 30°C. In some fungal species, elevated temperature stimulates enzymatic activity and development of the germ tubes (20).

The size of conidia produced by C. pallescens during this study (12-28 X 6-12 mm), was a little different from that cited by Ellis (5), who mentioned that the size of conidia in this species varies between 17-32 mm long and 1-12 mm wide, but all others characteristics confirmed the species name. The size of conidia varies according to pH of medium, ratio of C:N, nutrients, and relative air humidity (16) and because of that it represents a complementary taxonomic character.

The occurrence of uni to multinucleate segments with globose and elliptical nuclei seems to indicate that there were interphase nuclei in some segments of the mycelium of the stain of C. pallescens. This multinucleate condition suggests a phenomenon related with successive mitotic divisions, without division of the conidium (21). Such a condition makes it difficult to do genetic studies on this species. In the same way, the high number of nuclei in a single conidium makes it also difficult to obtain mutants. Luna (12) mentioned that in Metarhizium anisopliae (Metsch.) Sorokin strain M5 it was hard to obtain mutants, as a result of this same multinuclear condition. Germination of conidia without polarity, showing germ tubes developing from the central cells and giving rise to multinucleated hyphal segments constitute an important cytological characteristic of this species. There are no data regarding nuclear behavior of other Curvularia species; studies on this area are needed for better cytological characterization of this genus.

ACKNOWLEDGEMENTS

Special thanks to Dr. James W. Kimbrough for reading the manuscript and making valuable suggestions. The facilities provided by the Laboratório de Imunopatologia Keizo Asami (LIKA/UFPE) for the SEM studies are appreciated. The authors are gratefull to CNPq and CAPES for grant support.

RESUMO

Aspectos morfológicos, citológicos e culturais de Curvularia pallescens

Curvularia pallescens Boedijn (Hyphomycetes) é redescrito com auxílio de Microscopia de Luz e Eletrônica de Varredura e as condições ótimas de cultivo para crescimento deste fungo são discutidas. Análise citológica e condição nuclear, utilizando a técnica de HCl-Giemsa, revelou estruturas vegetativas e reprodutivas (hifas e conídios) formadas por segmentos uni, bi, tri, tetra e multinucleados. Culturas de C. pallescens, desenvolvidas em Meio completo e em Meio de batata dextrose agar, apresentaram variação no crescimento, no aspecto dos bordos das colônias e na pigmentação do meio. O Meio completo e a temperatura entre 25-28° C foram os mais indicadas para cultivo de C. pallescens.

Palavras-chave: Conidiogênese, condições de cultivo, microscopia eletrônica, Hyphomycetes.

REFERENCES

Exercícios práticos de genética

2. Banerjee, U. C. Production of beta-glucosidade (cellobiase) by Curvularia sp. Lett. Appl. Microbiol. 10: 197-199, 1990.

3. Banerjee, U. C. and Chakrabarti, S. Production and properties of carboxymethylcellulase 3 (endo-1,4-b-glucanase) from Curvularia lunata. World J. Microbiol. Biotech. 8: 423-424, 1992.

4. Cavalcanti, M. A., and Maia, L. C. Cellulolytic fungi isolated from an alluvial soil in a semi-arid area of the northeast of Brazil. Rev. Microbiol. 25: 251-251, 1994.

5. Ellis, M. B. 1971. Dematiaceous Hyphomycetes. CAB. Commonwealth Mycological Institute, Kew, Surrey, 1971, 608p.

6. Farr, D. F.; Bills, G. F.; Chamuris, G. P.; Rossman, A. Y. Fungi on plants and plant products in the United States. APS Press, St. Paul, MN., 1989, 1252p.

7. Freire, S. V. P. Atividade celulolítica e aspectos citológicos de Curvularia pallescens. Recife, 1995, 95p. (Dissertação Mestrado. Departamento de Biologia, Universidade Federal Rural de Pernambuco).

8. Furtado, J. S. Basidial cytology of Exidia nudeata. Mycologia 60: 9-15, 1968.

9. Jyoti, S.; Sing, R. P. Temperature relation and cellulolytic activity of weed fungi occurring in the beds of Pleurotus sajor caju. Indian J. Plant Pathol. 3: 7-10, 1985.

10. Kendrick, W. B.; Cole, G. T. Conidium ontogeny in hyphomycetes. The sympodulae of Beauveria and Curvularia. Can. J. Bot. 46: 1297-1301, 1968.

11. Kwon-Chung, K. J.; Bennett, J. E.. Medical Mycology. Lea and Febiger, Philadelphia, PA., 1992, 866p.

12. Luna, E. A. 1985. Caracteristicas citológicas e genéticas de linhagens selvagens, mutantes e diplóides de Metarhizium anisopliae (Metsch.) Sorokin. Rio de Janeiro, 1985. 260p. (Tese Doutorado. Departamento de Genética, UFRJ).

13. Luna, E. A.; Azevedo, J. L. Técnicas para coloração nuclear em estruturas vegetativas de Metarhizium anisopliae (Metsch.) Sorokin. Bol. Grupo Pesq. Control Biol. 4: 20-21, 1983.

14. Nitharwal, P. D.; Gour, H. N.; Agarwal, S. Effects of different factors on the production of cellulase by Curvularia lunata. Folia Microbiol. 36: 357-361, 1991.

15. Olufolaji, D. B. Sporulation and growth of Curvularia pallescens as affected by media, temperature, and nitrogen sources. Phytopathology 74: 260-263, 1983.

16. Phillips, D. J.; D. A. Margosan, D. A.; Mackey, B. E. Size, nuclear number, and agressiveness of Botrytis cinerea spores produced on media of varied glucose concentrations. Phytopathology 77: 1606-1608, 1987.

17. Pontecorvo, G.; Roper, J. A.; Hemons, L. M.; MacDonald, W. D.; Button, A. N. J. The genetics of Aspergillus nidulans. Adv. Gen. 5: 141-238, 1953.

18. Ribeiro, S. M. A.; Luna-Alves Lima, E. A.; Assunção, W. T. G.; Lima, D. M. M. Behavior and characteristics of a wild strain of Metarhizium anisopliae. Rev. Microbiol. 23: 97-100, 1992.

19. Rossman, A. Y.; M. E. Palm, M. E.; Spielman, L. J. A literature guide for the identification of plant pathogenic fungi. APS Press, St. Paul, MN, 1987, 252p.

20. Sing, K. V.; Bhatt, S. K. Studies on amylolytic ability of some plant pathogens. Indian J. Mycol. Pl. Pathology 14: 143-145, 1984.

21. Tebeest, D.O.; Shilling, C. W.; Riley, L. H.; Weidemann, G. J. The number of nuclei in spores of three species of Colletotrichum. Mycologia 81: 147-149, 1989.

22. Travis, W.D.; Kwon-Ghung, K. J.; Kleiner, D. E.; Geber, A.; Lawson, W.; Pass, H. I.; Henderson, H. D. Unusual aspects of allergic bronchopulmonary fungal disease. Report of two cases due to Curvularia organisms associated with allergic fungal sinusitis. Human Pathol. 22: 1240-1248, 1991.

• ¹
  Azevedo, J. L., and Costa, S. O. P. 1973. Exercícios práticos de genética Editora da Universidade de São Paulo, São Paulo. 1973. 288p.
• ²
  Banerjee, U. C. Production of beta-glucosidade (cellobiase) by Curvularia sp. Lett. Appl. Microbiol 10: 197-199, 1990.
• ³
  Banerjee, U. C. and Chakrabarti, S. Production and properties of carboxymethylcellulase 3 (endo-1,4-b-glucanase) from Curvularia lunata. World J. Microbiol. Biotech 8: 423-424, 1992.
• ⁴
  Cavalcanti, M. A., and Maia, L. C. Cellulolytic fungi isolated from an alluvial soil in a semi-arid area of the northeast of Brazil. Rev. Microbiol 25: 251-251, 1994.
• ⁵
  Ellis, M. B. 1971. Dematiaceous Hyphomycetes CAB. Commonwealth Mycological Institute, Kew, Surrey, 1971, 608p.
• ⁶
  Farr, D. F.; Bills, G. F.; Chamuris, G. P.; Rossman, A. Y. Fungi on plants and plant products in the United States APS Press, St. Paul, MN., 1989, 1252p.
• ⁷
  Freire, S. V. P. Atividade celulolítica e aspectos citológicos de Curvularia pallescens Recife, 1995, 95p. (Dissertação Mestrado. Departamento de Biologia, Universidade Federal Rural de Pernambuco).
• ⁸
  Furtado, J. S. Basidial cytology of Exidia nudeata. Mycologia 60: 9-15, 1968.
• ⁹
  Jyoti, S.; Sing, R. P. Temperature relation and cellulolytic activity of weed fungi occurring in the beds of Pleurotus sajor caju. Indian J. Plant Pathol 3: 7-10, 1985.
• ¹⁰
  Kendrick, W. B.; Cole, G. T. Conidium ontogeny in hyphomycetes. The sympodulae of Beauveria and Curvularia. Can. J. Bot 46: 1297-1301, 1968.
• ¹¹
  Kwon-Chung, K. J.; Bennett, J. E.. Medical Mycology Lea and Febiger, Philadelphia, PA., 1992, 866p.
• ¹²
  Luna, E. A. 1985. Caracteristicas citológicas e genéticas de linhagens selvagens, mutantes e diplóides de Metarhizium anisopliae (Metsch.) Sorokin. Rio de Janeiro, 1985. 260p. (Tese Doutorado. Departamento de Genética, UFRJ).
• ¹³
  Luna, E. A.; Azevedo, J. L. Técnicas para coloração nuclear em estruturas vegetativas de Metarhizium anisopliae (Metsch.) Sorokin. Bol. Grupo Pesq Control Biol. 4: 20-21, 1983.
• ¹⁴
  Nitharwal, P. D.; Gour, H. N.; Agarwal, S. Effects of different factors on the production of cellulase by Curvularia lunata. Folia Microbiol 36: 357-361, 1991.
• ¹⁵
  Olufolaji, D. B. Sporulation and growth of Curvularia pallescens as affected by media, temperature, and nitrogen sources. Phytopathology 74: 260-263, 1983.
• ¹⁶
  Phillips, D. J.; D. A. Margosan, D. A.; Mackey, B. E. Size, nuclear number, and agressiveness of Botrytis cinerea spores produced on media of varied glucose concentrations. Phytopathology 77: 1606-1608, 1987.
• ¹⁷
  Pontecorvo, G.; Roper, J. A.; Hemons, L. M.; MacDonald, W. D.; Button, A. N. J. The genetics of Aspergillus nidulans. Adv. Gen. 5: 141-238, 1953.
• ¹⁸
  Ribeiro, S. M. A.; Luna-Alves Lima, E. A.; Assunção, W. T. G.; Lima, D. M. M. Behavior and characteristics of a wild strain of Metarhizium anisopliae. Rev. Microbiol 23: 97-100, 1992.
• ¹⁹
  Rossman, A. Y.; M. E. Palm, M. E.; Spielman, L. J. A literature guide for the identification of plant pathogenic fungi APS Press, St. Paul, MN, 1987, 252p.
• ²⁰
  Sing, K. V.; Bhatt, S. K. Studies on amylolytic ability of some plant pathogens. Indian J. Mycol. Pl. Pathology 14: 143-145, 1984.
• ²¹
  Tebeest, D.O.; Shilling, C. W.; Riley, L. H.; Weidemann, G. J. The number of nuclei in spores of three species of Colletotrichum. Mycologia 81: 147-149, 1989.
• ²²
  Travis, W.D.; Kwon-Ghung, K. J.; Kleiner, D. E.; Geber, A.; Lawson, W.; Pass, H. I.; Henderson, H. D. Unusual aspects of allergic bronchopulmonary fungal disease. Report of two cases due to Curvularia organisms associated with allergic fungal sinusitis. Human Pathol 22: 1240-1248, 1991.

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Received
  11 Aug 1997
• Reviewed
  02 July 1998