This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Carisse, O. Articles by Bernier, J. Search for Related Content PubMed Articles by Carisse, O. Articles by Bernier, J. Agricola Articles by Carisse, O. Articles by Bernier, J.

Microsphaeropsis ochracea sp. nov. associated with dead apple leaves

     Agriculture and Agri-Food Canada, Horticultural Research and Development Center, 430 Gouin Blvd., Saint-Jean-sur-Richelieu, Québec, Canada, J3B 3E6. Tel: (450) 346-4494. Fax: (450) 346-7740

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 

A new saprophyte species of Coelomycetes, Microsphaeropsis ochracea, is described based on an isolate recovered from apple leaf litter from Quebec, Canada. Although this isolate possesses conidia similar to those of Microsphaeropsis arundinis (Ahmad), there are several differences between the two species. M. ochracea forms pycnidia of 70 µm up to 120 µm into senescent apple leaf tissue and unlike M. arundinis they are not ostiolate when grown either on a apple leaf or on culture media. Conidiogenous cells are 4.5–10 x 2.5–4.5 µm, which is much larger than those described for M. arundinis. Reverse side of the colony presents a pale luteous to ochreous pigment that diffuses through the media. The description of this new isolate was compared with the published description of M. arundinis as well as with the dry specimen of the paratype.

Key words: biocontrol, Coelomycetes, saprophyte, taxonomy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
A fungal isolate (strain P130A) recovered from apple litter (Bernier et al 1996 ) has been shown to be a potential biological control agent against Venturia inaequalis, the causal agent of apple scab (Benyagoub et al 1997 , Philion et al 1997 , Carisse et al 2000 ). This isolate was sent to the IMI (International Mycological Institute, England) for identification. Identification of isolate P130A using the identification key developed by Sutton (1980a) led to the species Microsphaeropsis arundinis (Ahmad) because of its very small, smooth-walled, cylindrical conidia. Based on published descriptions of Microsphaeropsis species, arundinis is the only species producing this type of conidia (Crous and van der Linde 1993 , Hammounda 1992 , Heiny et al 1992 , Laudon 1984 , Morgan-Jones 1974 , Morgan-Jones and White 1987 , Someya et al 1997 , Sutton 1971, 1974, 1980b , Watson et al 1974).

When comparing the two published descriptions of this species (Ahmad 1971 , Sutton 1980a ) it was clear that there is a lack of concordance between the two descriptions. Moreover, there is no example of holotype culture of M. arundinis available for detailed taxonomic studies. Finally, a M. arundinis paratype IMI 337189 is available only as a dry specimen and unfortunately it was impossible to examine the specimen on a microscopic mount since it is dried up. Consequently, because no work was possible using the holotype/paratype specimens, other strains previously identified as M. arundinis or a closely related species were ordered from different culture collections in order to compare our strain to other living cultures of M. arundinis.

In a recent study, morphological, physiological and biochemical differences among isolates corresponding to the description of M. arundinis revealed the need for the description of a new species. These fungal isolates differed in several characters, including size of pycnidia, presence of ostiole, pigment production, growth rate, secondary metabolites production, and RFLPs (Bernier, Lévesque, Carisse unpubl).

It is well documented in the literature that the present state of taxonomy of the Microsphaeropsis and Coniothyrium genera is unsettled (Sutton 1971, 1980a,b , Morgan-Jones 1974, 1985 , Verkley and Van der Aa 1997 ). Currently, Coniothyrium is distinguished from Microsphaeropsis by the presence of annelides (Morgan-Jones 1974 , Jones 1976 ). Although it is well documented that there is a need to transfer some Coniothyrium species into other genera such as Microsphaeropsis (Morgan-Jones 1987 , Phillips 1985 , Sutton 1971 ), no attempt has been made to do so because of the hundreds of Coniothyrium described, and also because there is some question about the priority of Microsphaeropsis (Sutton 1980a ). Therefore, isolate P130A was compared only with the described species of Microsphaeropsis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Optical microscopy – Microsphaeropsis isolate P130A was grown on potato dextrose agar (PDA, Difco Laboratories, Detroit, USA) under light for two weeks to allow pycnidial production. Mycelium, pycnidia, phialides and spores were examined and measured using light microscopy. At least 10 hyphae and phialides and 20 to 30 pycnidia and conidia originating from three different plates were observed for measurements, and the observation was done twice. The localization and size of pycnidia within the tissue was also achieved by inoculation of a spore suspension on five apple leaves discs which had been sterilized by irradiation (4Mrad, for 10 to 12 h). The leaves were placed on water agar (WA, Difco Laboratories, Detroit, USA) at 25 C for ten days. To determine the level of colonization in the leaf, fifteen pycnidia present on the adaxial surface of the leaves were observed using a binocular loupe and then transferred to a microscope slide for measurements. Pycnidia diameter was calculated using the average of width and length.

Transmission electron microscopy – Samples from agar blocs and apple leaf discs were fixed with 3% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 2 h at room temperature and overnight at 4 C and then rinsed in cacodylate buffer. Post-fixation was performed in osmium tetroxide 1% (w/v) in the same buffer for 1 h at 4 C. Samples were then dehydrated in a graded ethanol series. Infiltration was done in increasing concentrations of propylene oxide and Epon 812 resin. For light microscopy, thin sections of about 5–10 µm were collected on microscopic slides and stained with 1% toluidine blue, or cultures were mounted directly in 1% toluidine blue. Ultra-thin sections of about 0.1 µm were used. Observations were done using a JEOL 1200 EX electron microscope (Tokyo, Japan) operated at 80 KV (Benyagoub et al 1998 ).

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Microsphaeropsis ochracea Carisse & Bernier sp.nov.

(Figs. 1 – 3 )

View larger version (76K): [in this window] [in a new window]    FIG. 1. Pycnidia of Microsphaeropsis isolate P130A. a) On inoculated dead apple leaves, 5.76x; b) Trans-section within apple leaf disc, 22.4x

View larger version (74K): [in this window] [in a new window]    FIG. 2. Trans-section of a pycnidium of isolate P130A using transmission electron microscopy. a) Global view and number of cell layers, 1.227x; b) Longitudinal-section of a conidium emerging from a phialide, 13678x

View larger version (65K): [in this window] [in a new window]    FIG. 3. Isolate P130A through optical microscopy from a culture. a) Pycnidium with a brown mass of spores and the blue cell wall, 20x; b) Phialides and parts of a cell wall, 1250x; c) Conidia, 1250x

Conidiomata occuring on fallen leaves. Pycnidia globose to subglobose, 70–120 µm diameter, numerous, separate, immersed to semi-immersed (Fig. 1 ), black-brown, glabrous; wall of textura angularis, 11–17 µm thick, two-layered, outer layer 1–2 cells thick and dark brown, inner layer 1–3 cells thick and hyaline (Fig. 2a ). Ostiole not seen. Conidiophores absent. Conidiogenous cells ampulliform to doliiform, somewhat variable, 4.5–10 x 2.5–4.5 µm, phialidic, determinate, discrete, hyaline, smooth-walled, collarette present (Figs. 2b, 3b ). Conidia cylindrical to bacilliform, 3.5–5.5 x 1–2 µm, hyaline with a faint smoke-olivaceaous color, thin-walled, smooth, bi-guttulate, aseptate (Fig. 3c ). Colonies on potato dextrose agar pale greyish rosy buff (Rayner 1970 ), white margin, with sparse aerial mycelium. Slow-growing, producing a pale luteous to ochreous pigment in the agar which turns purple when 1N NaOH is added. Mycelial hyphae branched, septate, smooth, hyaline, 1.5–2.5 µm wide. Pycnidia superficial to semi-immersed, brown to dark brown, 150–600 µm, numerous, often aggregated, conidial exudate black (Fig. 3a ).

HOLOTYPE: TRTC 52744 (Cryptogamic Herbarium of the Royal Ontario Museum), ATCC 74412 (Microsphaeropsis sp. strain P130A) isolated from leaves of Malus domestica, from an abandoned orchard, St-Joseph-du-Lac, Quebec, Canada, 20 April 1993.

Specimens examined. INDIA. MADHYA: Pradesh, Jabalpur. Stem of Arundo donax, 1990, N.D. Sharma, Microsphaeropsis arundinis, strain number IMI 337189. CANADA. QUEBEC: Ottawa. Core rot of Red Delicious apple, 19 Sept 1988, J. H. Ginns/R.A Shoemaker, Microsphaeropsis sp. DAOM 198536. USA. WISCONSIN: Madison. Apple leaf, 1985, J.H. Andrews, Microsphaeropsis arundinis IMI 294735. CANADA.QUEBEC: St-Joseph-du-Lac, dead apple leaf, 20 April 1993, O. Carisse, Microsphaeropsis sp. P176A.

Etymology – The name ochracea was chosen because of the distinctive ochreous pigment produced when grown on culture media.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
The genus name Microsphaeropsis Höhn. was used for pycnidial fungi with small aseptate conidia formed from phialides (Sutton 1971, 1974, 1980a , Morgan-Jones 1974) as a distinction from Coniothyrium characterized by small, often septate conidia produced from annellides. Of the known species of Microsphaeropsis (Sutton 1971 , Morgan-Jones 1974 , Someya et al 1974, 1980b, 1997, Watson et al 1974, Laudon 1984 , Morgan-Jones and White 1987 , Hammounda 1992 , Heiny et al 1992 , Crous and van der Linde 1993 ) and from the taxonomic key provided by Sutton (1980a) , only M. arundinis species have similar cylindrical thin-walled conidia when compared to our isolate. On the other hand, the description of the holotype of M. arundinis differs in several features from the description of M. arundinis in Sutton's key (1980a) , among which shape and size of conidia are described as oblong and 6.5–7.8 x 1.8–2.5 µm by Ahmad (1971) , but are described as cylindrical, 4–4.5 x 1.5 µm in Sutton's key. In the original description (Ahmad 1971 ), pycnidia were described as 130–165 x 165–230 µm, however, in Sutton's key pycnidia are less than 175 µm in diameter. The isolate described here produces pycnidia smaller than 120 µm on the host.

There are no genera or species from the same host (Malus sylvestris) that are similar to our isolate found in the literature, although the possibility exists that this species or a similar species have been formally described from another host (Arundo donax) (Ahmad 1971 ). Although size of conidia matches the description provided by Sutton, this isolate cannot be M. arundinis for several reasons. First, description of M. arundinis (Sutton 1980a ) differs from strain P130A by the absence of an ostiole and by the size of the phialides, which are 2 to 3 times larger. Second, the description differs from Ahmad's (1971) by the size of pycnidia, absence of ostiole(s), and the shape and size of conidia (described as oblong, which is different from a cylindrical bacilliform shape) (Hawksworth et al 1983 ). Finally, there are no taxonomic criteria based on the same host, or on culture media, which could have enabled comparisons of more suitable morphological characters of this strain. It is presently impossible to compare properly any strains with M. arundinis using its present state of description and the availability and condition of the specimens. This will be true for identification of other strains producing pycnidia and phialidic, cylindrical-shaped conidia. Considering that the holotype is available only as a dry specimen and that the referring microscopic mounts do not allow precise observations, it is not possible to confirm that the described species is M. arundinis.

We propose that this isolate be considered as a new species of Microsphaeropsis and we suggest the name Microsphaeropsis ochracea Carisse & Bernier.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. G. Kinsey from the CABI Bioscience, England, for discussion on the taxonomy, for proof reading of the manuscript and for the Latin diagnosis. We are also grateful to Dr. N. Benhamou and Alain Goulet from Laval University, Quebec, Canada, for assistance with the electron microscopy.

	FOOTNOTES

 
¹ Corresponding author, Email: carisseo{at}em.atr.ca

Accepted for publication July 24, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY DISCUSSION LITERATURE CITED

 
Ahmad S., 1971 Contribution to the fungi of west Pakistan Biologia 17:1-26

Bernier J, Carisse O, Paulitz TC., 1996 Fungal communities isolated from dead apple leaves from orchards in Quebec Phytoprotection 77:129-134

Benyagoub M, Benhamou N, Carisse O., 1998 Cytochemical investigation of the antagonistic interaction between Microsphaeropsis sp. (isolate P130A) and Venturia inaequalis Phytopathology 88:605-613

Carisse O, Philion V, Rolland D, Bernier J., 2000 Effect of fall application of fungal antagonists on spring ascospore production of the apple scab pathogen, Venturia inaequalis Phytopathology: 90:31-37

Crous PW, van der Linde EJ., 1993 New and interesting records of South African Fungi. XI. Eucalyptus leaf fungi S Afr J Bot 59:300-304

Hammounda AM., 1992 Microsphaeropsis sp. causing leaf spot of banana in Oman FAO Plant Prot Bull 40:1-2

Hawsworth DL, Sutton BC, Ainsworth GC., 1983 Ainsworth & Bisby's Dictionary of the fungi. 7th ed Commonwealth Mycological Institute Kew. 445 p

Heiny DK, Mintz AS, Weidemann GJ., 1992 Redisposition of Aposphaeria amaranthi in Microsphaeropsis Mycotaxon 44:137-154

Jones JP., 1976 Ultrastructure of conidium ontogeny in Phoma pomorum, Microsphaeropsis olivaceum, and Coniothyrium fuckelii Can J Bot 54:831-851

Laudon G., 1984 Diplodia pittosporum and Diplodia pittospori Trans Br Mycol Soc 82:164-166

Morgan-Jones G., 1974 Concerning some species of Microsphaeropsis Can J Bot 52:2575-2579

———, White JF., 1987 Notes on Coelomycetes. III. Concerning Microsphaeropsis concentrica: morphology and ultrastructure Mycotaxon 30:177-187

Philion V, Carisse O, Paulitz TC., 1997 In vitro evaluation of fungal isolates for their ability to influence leaf rheology, production of pseudothecia, and ascospores of Venturia inaequalis European Journal of Plant Pathology 103:441-452

Phillips AJL., 1985 Coniothyrium minitans on sclerotia of Sclerotinia sclerotiorum in South Africa Phytophylactica 17:217-219

Rayner RW., 1970 A mycological colour chart Commonwealth Mycological Institute, Kew

Someya A, Yaguchi T, Udagawa S., 1997 Microsphaeropsis rugospora, a new species from Japanese soil Mycoscience 38:429-431

Sutton BC., 1971 Coelomycetes. IV. The genus Harknessia and similar fungi on Eucalyptus Mycol Pap 123:1-46

———. 1974 Miscellaneous Coelomycetes on Eucalyptus Nova Hedwigia 25:161-172

———. 1980a The Coelomycetes. Fungi Imperfecti with pycnidia, acervuli and stromata New York: CABI Publishing. 696 p

———. 1980b Microsphaeropsis clidemiae sp.nov., associated with leaf lesions on Clidemia hirta Trans Br Mycol Soc 74:645-647

Verkley GJM, Van der Aa HA., 1997 Striosphaeopsis mirabilis anam. gen. et sp. nov., a new Coelomycete isolated from soil in Papua New Guinea Mycotaxon 65:113-119

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Carisse, O. Articles by Bernier, J. Search for Related Content PubMed Articles by Carisse, O. Articles by Bernier, J. Agricola Articles by Carisse, O. Articles by Bernier, J.