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Chalara angustata sp. nov. from roots of Quercus petraea and Quercus robur
1112
Mycol. Res. 100 ( 9 ) :1112-1116 (1996) Printed in Great Britain
Chalara angustata sp. nov. from roots of Quercus petraea and Quevcus vobur
T. KOWALSKI' A N D E. HALMSCHLAGER2 Department of Forest Pathology, Faculty of Forestry, 29-Listopada 46, PL-31-425 Krakdw, Poland 'Institute of Forest Entomology, Forest Pathology and Forest Protection, UNI-BOKU Vienna, A-1190 Vienna, Austria
A new species of the hyphomycete genus Chalara is described. This fungus was isolated several times from roots of healthy and declining oaks (Quercus petraea and Q. robur). It differs from previously described species of Chalara by its septate conidia, the morphology of the phialophores and by colony characteristics.
A survey of fungi from roots of healthy and declining oak trees in Austria (E. Halmschlager & T. Kowalski, unpubl.) yielded several isolates of an apparently new species of Chalam (Corda) Rabenh., characterized by long chains of cylindric, 3-septated spores. Although the survey was carried out at two sites the fungus was found only at Patzmannsdorf (60 km north of Vienna) in an 80-yr-old oak stand (Q. pefraea [Matt.] Lieb., Q. robur L.) mixed with Corylus avellana L., Carpinus befulus L. and Ulrnus glabra Huds. Some species of Chalam are known as important plant pathogens (Upadhyay, 1981; Kile & Walker, 1987). The newly discovered Chalam sp. described here appears to be confined to fine roots. It was isolated from roots with a diam. not greater than 10 mm (Table 1). Mycelial growth of this fungus from root fragments in vifro did not occur earlier than three months after the start of incubation of the root fragments. The fungus could not be accommodated as a known species of Chalam in the monographs by Nag Raj & Kendrick (1975) and Upadhyay (1981) or in recently published papers by Kile & Walker (1987), Kirk (1985, 1986) and Holubovi-Jechovi (1984). It is therefore described here as a species new to science and compared to known species with similar characteristics.
Measurements were conducted on original cultures obtained by plating out root segments as well as on subcultures and single conidia isolates. Dimensions of conidiophores and conidia were measured for each strain in distilled water (30 replicates). Photomicrographs were taken using a Reichertm Polyvar microscope in bright field or interference contrast. Material for SEM was cut from agar with a cork borer (diam. 5 mm), fixed with 4% glutaraldehyde buffered by a 0.1 M phosphate buffer (pH = 7.1) and dehydrated in a graded ethanol series (15, 30, 50, 70, 80, 90, 96, 100%) followed by acetone (100%). Specimens were then critical point dried, mounted on stubs, coated with gold/palladium and viewed with a scanning electron microscope (JSM 5200). To examine the relationship to Cevafocysfis s. str., cycloheximide tolerance (Harrington, 1981) of the Chalara sp. was tested. The fungus (four strains) was inoculated onto MEA amended with 0.1 g cycloheximide I-'. Extension of colonies was measured after 30 d by taking the average of two colony diameters for each plate. Two Petri dishes per strain were used and the experiment was conducted at room temperature and diffuse daylight.
RESULTS Chalara angustata T. Kowalski & Halmschlager sp. nov.
MATERIALS A N D M E T H O D S
Etym.: derived from the Latin word meaning diminished
Investigations were carried out on six strains (Table 1) isolated from oak roots. After surface sterilization ( I min ethanol 96%, 5 min NaOCl4 %, 30 s ethanol 96 %) roots were plated out on the surface of 2% malt extract agar (MEA, 20 g 1-I malt extract Difco, 15 g 1-I agar Difco, supplemented with 100 mg I-' streptomycin sulphate) and incubated a t room temperature and diffuse daylight. Resulting isolates were transferred to new MEA plates and incubated at 20 OC in the dark.
Coloniae in agaro maltoso primum tenues, hyalinae, albidae aut albocinereae, paulo mycelio superficiali soluto usque aut floccoso, post sporulationem intensam aspectu pulverulento. In culturis sequentibus zonis concentricis. Facies aversa primum hyalina, deinde in centro grisea maculis cinereis vel brunneolis. Incrementurn coloniarurn variatione magna: post 21 dies 22-57 mm in diam. ad 20° in tenebris. Fungus potuit resistere dilutis cycloheximide, increment0 non derninuto in 0.1 g cycloheximide 1-l. Mycelium immersum vel superficiale, hyphis hyalinis aut subhyalinis, 1.2-3.0 pm latis, intumescentibus ad 4.8 pm, pariete tenui, laevibus et numerosis,
1113
T. Kowalski and E. Halmschlager Table 1. Origin of strains of Chalara angustata, isolated from roots of Quercm pefraea and Q . robur (Patzmannsdorf, Austria)
Strain no.
Host
R255/1 R255/2 R255/3 R255/4 R2.5515 R255/6
Q. petraea Q. petraea Q. petraea Q. pefraea Q. robur Q. pefraea
Symptoms of oak decline1
Diam. class of SoiI horizon (cm) root (mm)
-
+ + +
-
Rooi tissue
Year of isolation
Bark Wood Bark Bark Bark Bark
1993 1993 1993 1993 1994 1994
-No symptoms, +symptoms of oak decline
parvis, lateralibus gibberibus, 3-11 x 1.2-2.0 wn omatis. Chlamydosporis nullis. Phialophora solitaria vel gregaria, cylindrica elongata, recta vel leniter flexuosa, mononematosa, non rarnosa, laevia, ferruginea ad brunnea, 1-4 (15) septis in parte basali, lI(r160 ( - 212) pm longa. Cellular basalis 6.2-11.2 pm lata vel valde contracta et 1.2-1.8 pm lata. Phialidae subcylindricae, interdum arnpulliformes, 96-125 Dm longae; venter subcylindricus vel ellipsoideus, 38-60 x 10-12 pm; collum cylindricum, 58-87 x 5.k7.0 pm, obscurius quam venter; transitio ex ventre ad collum gradatim, raro abrupta; ratio longitudinis colli et ventris = 1.4: 1. Phialoconidia in catenis longis dissolventibus formata, cylindrica, ad apicem rotundata, ad basim abscissa collo pusillo, 3- interdum 2-, 4- vel 6-septata, hyalina aut subhyalina, laevia 22-42 x 4.8-6 pm; ratio conidii longitudinis/latitudinis = 5.4 : 1. Ex radicibus Quercus petraeae (Matt.) Lieb., Patzmannsdorf/Austria, legunt Halmschlager & Kowalski, 24 June 1993. Holotypus (colonia exsiccata) in herbario ZT, cultura viva in CBS Baam.
Colonies on MEA initially thin, hyaline, white to whitish-grey, with some patches becoming grey to dark grey, with sparse, fluffy hairy-villose to lanuginose aerial mycelium, looking powdery in the case of abundant sporulation, with distinct concentric zones of different densities of aerial mycelium (Figs 1, 2). Single spore cultures forming soft, moist, radial folded colonies without aerial mycelium but with tufts of phialophores. Reverse at first hyaline, later becoming grey in the centre with grey or brownish dots. Extension rate of colonies showed high variability: Original cultures attaining 22-57 mm diam. in 21 d on MEA at 20' in the dark (Figs 1, 2) whereas growth of subcultures was always slower. No reduction in growth was observed at the tested concentration of cycloheximide (Table 2). Vegetative hyphae hyaline or subhyaline, 1.2-3-0 ym wide with swellings up to 4.8 ym; thin-walled, smooth with several small lateral burls 3-11 x 1.2-2.0 ym; septate with septa 4-18 ym apart. Chlamydospores absent. Phialophores arising directly on the superficial or slightly immersed vegetative hyphae or in older cultures from cells of a pseudoparenchymatous stroma; solitary and scattered or in tufts, erect, straight or slightly bent, rust-brown to brown with 1-4 (15) septa in the basal part, wall smooth, without constriction at the septa, 110-160 (-212) ym long, terminating in a phialide (Figs 3, 7, 9). Basal cell of the phialophore 6.2-11.2 pm wide or strongly constricted only 1.2-1.8 pm wide (Figs 3, 73. Percurrent proliferation of phialophores rare (Fig. 8). Phialides subcylindrical, occasionally lageniform, 96-125 (109) urn long. Venter subcylindrical or ellipsoid 38-60 (46) x 10-12 (11)ym. Cellarette cylindrical 58-87 (65) x 5.67.0 ym, darker than venter; ratio of mean length of collarette
to venter = 1.4: 1.Transition from venter to collarette gradual, occasionally abrupt. Phialoconidia catenate, formed in long, readily fragmenting chains (Figs 10, 11); cylindrical with obtuse apex and a truncate base bearing a marginal basal frill (Figs 4, 12, 13); 3septate, occasionally 2-, 4- or 6-septate, hyaline to subhyaline (in old cultures with a yellow-greenish tinge), filled with fine granular cytoplasm or with oil droplets, smooth walled; 22-42 (29.8) x 4.8-6 ( - 7) (5.5) bm (Fig. 4); mean conidium lengthlwidth ratio = 5.4: I. Sterile structures, like deformed or underdeveloped phialophores, are formed particularly in cultures that have been stored under cool conditions (6') for several months, sometimes also in young cultures. Two types are found most frequently: Type 1is clavate, 53-125 ym long with 2-6 septa, 7.5-8.7 ym wide at the base enlarging towards the apex to 10-12.5 pm, apical wall closed or breaking open (Fig. 6). Type 2 is cylindrical, up to 380 ym long with up to 20 septa, unicolour, light brown to brown (Fig. 5). From roots of Quercus pefraea (Matt.) Lieb., Patzmannsdorf/ Austria, legunt Halmschlager & Kowalski, 24 June 1993. Holotypus (colonia exsiccata) in herbarium ZT, cultura viva CBS Baam.
DISCUSSION Chalara is characterized by sessile or stalked phialides with basal venters and long collarettes, deep-seated conidiogenous loci and usually cylindrical, hyaline, subhyaline or pale brown, unicellular or septate phialoconidia (Nag Raj & Hughes, 1974; Holubovi-Jechovi, 1984). There are two related genera bearing phialophores mixed with sterile elements. In Chaetochalara setae-like structures are found. Sporoschisma is characterized by capitate hyphae with apical, hyaline mucilaginous-like caps (Holubovi-Jechovi, 1973; Nag Raj & Hughes, 1974). Sterile elements found in cultures of the fungus described here have neither the character of capitate hyphae as in Sporoschisma nor the seta-like hyphae found in Chaetochalara. Structures in this case are very variable in shape and can be interpreted as degenerate or underdeveloped phialophores as a result of growth conditions. This point of view is also supported by the increase of sterile structures in subcultures and older cultures. The fungus described here is therefore accommodated in Chalara. This also fits well for the hyaline to subhyaline phialospores which are coloured in Sporoschisma (Nag Raj & Kendrick, 1975).
Chalara angustata sp. nov.
Figs 1-9. Colony characteristics, phialophores, phialoconidia and sterile structures of Chalara angustata. Figs 1, 2. slow and fast growing colony of Ch. angusfafa with distinct concentric zones grown on MEA at 20' in the dark after 21 d. Fig. 3. Light microg~ raph of Ch. angustata showing a tuft of phialophores (bar, 20 pm). Fig. 4. Light micrograph of 3-septate phialoconidia with oil droplets; base of conidia bearing a marginal basal frill [arrows] (bar, 10 pm). Fig. 5. Cylindrical sterile structure (bar, 20 pm). Fig. 6. Clavate sterile structure (bar, 10 um). Fig. 7. Single phialophore with constricted basal cell [arrow] (bar, 20 urn). Fig. 8. Percurrent proliferation of phialophores (bar, 20 pm). Fig. 9. Phialophore with 15 septa in the basal part (bar, 20 pm).
T. Kowalski and E. Halmschlager Table 2. Colony diam. (mm) of subcultures of Chalara angwfata after 30 d on MEA and the same medium amended with 0.1 g cycloheximide I-' Strain no.
MEA
Cycloheximide agar
Chalara angusfafa bears some resemblance to Ch. unicolor s. Hughes & Nag Raj (Nag Raj & Hughes, 1974). Ch. unicolor is, however, easily distinguished by its brown colony colour and its phialophores composed of a single stalk cell without constriction at the base. Furthermore venter and collarette are concolorous and wider in Ch. unicolor (Nag Raj & Kendrick, 1975) compared with Ch. angwfafa. Chalara species are known as anarnorphs in a few diverse taxonomic groups of Ascomycetes, e.g. in Cerafocysfis (Upadhyay, 1981; Nag Raj & Kendrick, 1993). Sensitivity of species in Ceratocysfis s. slr. to cycloheximide has been an important characteristic in the taxonomy of this group (Harrington, 1981). The tolerance of Ch. angusfafa to cycloheximide suggests that the species is unrelated to Ceratocysfis s. str.
At the moment Chalara includes species bearing conidia with basal frills as well as conidia without frills. As can be deduced from the marginal basal frill of the conidia in Ch. angusfafa, this species has a mode of conidiurn development similar to the Chalara-state of Ceratocysfis autographa (Wingfield ef al., 1995). In that case basal frills lead to the adherence of conidia to each other forming what Subramanian (1972) and Minter, Kirk & Sutton (1982, 1983) called 'false chains '. Ch. angustafa appears to be confined to h e roots, where the fungus remains causing no symptoms. This symptomless endophytic infection in the roots may be typical of the fungus at certain stages in its life cycle (Fisher, Petrini & Webster, 1991). It is of interest that the fungus has so far not been reported as a root endophyte. This may be because it is species specific to oak roots which have not been intensively examined yet. A more likely explanation is the long incubation required in vifro before the fungus can be detected, leading to it being overlooked in earlier assays of the roots of other plants. Slow growth of Ch. angusfata in vitro and the fact that the fungus grew only after a long incubation period of the root fragments suggests that a selective medium might help to detect the incidence of this species in the host plant. Kile & Walker (1987) listed 11 Chalara species from oak. Two further species from oak, Ch. ovoidea Nag Raj & W. B. Kendr. and Ch. breviclavafa Nag Raj & W. B. Kendr. have
Figs 10-13. Conidia and conidiogenous cells of Ch. angustafa. Fig. 10. SEM of cylindrical conidium at the apex of a tubular collarette. Fig. 11. SEM showing a false chain of phialoconidia. Fig. 12. SEM of obtuse apex of a phialoconidium. Fig. 13. SEM of truncate base
of a phialoconidium.
Chalara angusfata sp. nov. been described by Holubovi-Jechovi (1984) and Traue & Arnold (1991). Ch. angusfata described here is the first species from oak with multiseptate phialospores. We thank Professor Dr H. Butin for the Latin diagnosis and the Austrian Ministry of Science, Research and Arts for financial support.
REFERENCES Fisher, P. I., Petrini, 0 . & Webster, J. (1991). Aquatic hyphomycetes and other fungi in living aquatic and terrestrial roots of Alnus glutinosa. Mycological Research 95,543-547. Harrington, T.C. (1981). Cycloheximide sensitivity as a taxonomical character in Ceratocystis. Mycologia 73, 1123-1129. (1973). Lignicolous hyphomycetes from Holubovii-Jechova, V. Czechoslovakia. 3. Sporoschisma, Sporoschismopsis and Cutenularia. Folia Geobotanica et Phytotaxonomica 8, 209-218. Holubovii-Jechovii, V. (1984). Lignicolous hyphomycetes from Czechoslovakia. 7. Chalara, Exochlara, Fwichalara and Dictyochaeta. Folia Geobotanica et Phytotaxonomica 19, 387-438. Kile, G. A. & Walker, J. (1987). Chalara australis sp. nov. (Hyphomycetes), a vascular pathogen of Nothofagw cunninghamii (Fagaceae) in Australia and its relationship to other Chalara species. Awtralian Journal of Botany 35, 1-32.
(Accepted 15 February 1996)
1116 Kirk, P.M. (1985). New or interesting microfungi. XIV. Dematiaceous hyphomycetes from Mt Kenya. Mycotaxon 23,305-352. Kirk, P. M. (1986). New or interesting microfungi. XV. Miscellaneous hyphomycetes from the British Isles. Transactions of the British Mycological Society 86, 409-428. Minter, D. W., Kirk, P. M. & Sutton, B. C. (1982). Holoblastic phialides. Transactions of the British Mycological Society 79, 75-93. Minter, D. W., Kirk, P. M. & Sutton, B. C. (1983). Thallic phialides. Transactions of the British Mycological Society 80, 39-66. Nag Raj, T. R. &Hughes, S. J. (1974). New Zealand Fungi. 21. Chalara (Corda) Rabenhorst. New Zealand]ournal of Botany 12, 115-129. Nag Raj, T. R. & Kendrick, B. (1975). A Monograph of Chalara and Allied Genera. Wilfnd Laurier University Press: Waterloo, Ontario, Canada. Nag Raj, T. R. & Kendrick, B. (1993). The anamorph as generic determinant in the holomorph: the Chalara connection in the ascomycetes, with special reference to the ophiostomatoid fungi. In Ceratocystis and Ophiostoma (ed. M . J.Wingfield, K. A. Seifert & J. F. Webber), pp. 61-70. American Phytopathological Society: St Paul, MN. Subramanian, C. V. (1972). Conidial chains, their nature and significance in the taxonomy of hyphomycetes. Current Science 41,43-49. Traue, H. & Arnold, G. R. W. (1991). Chalara ovoidea Nag Raj & Kendrick eine weitere Pilzart im Krankheitsgeschehen der Trauben- und Stieleichen. Feddes Repertorium 102, 133-136. Upadhyay, H. P. (1981). A Monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press: Athens, GA. Wingfield, M. J., Benade, E., Van Wyk, P. S. & Visser, C. (1995). Conidium development in Ceratocystis autographa. Mycological Research 99, 1289-1294.
Mycol. Res. 100 ( 9 ) :1112-1116 (1996) Printed in Great Britain
Chalara angustata sp. nov. from roots of Quercus petraea and Quevcus vobur
T. KOWALSKI' A N D E. HALMSCHLAGER2 Department of Forest Pathology, Faculty of Forestry, 29-Listopada 46, PL-31-425 Krakdw, Poland 'Institute of Forest Entomology, Forest Pathology and Forest Protection, UNI-BOKU Vienna, A-1190 Vienna, Austria
A new species of the hyphomycete genus Chalara is described. This fungus was isolated several times from roots of healthy and declining oaks (Quercus petraea and Q. robur). It differs from previously described species of Chalara by its septate conidia, the morphology of the phialophores and by colony characteristics.
A survey of fungi from roots of healthy and declining oak trees in Austria (E. Halmschlager & T. Kowalski, unpubl.) yielded several isolates of an apparently new species of Chalam (Corda) Rabenh., characterized by long chains of cylindric, 3-septated spores. Although the survey was carried out at two sites the fungus was found only at Patzmannsdorf (60 km north of Vienna) in an 80-yr-old oak stand (Q. pefraea [Matt.] Lieb., Q. robur L.) mixed with Corylus avellana L., Carpinus befulus L. and Ulrnus glabra Huds. Some species of Chalam are known as important plant pathogens (Upadhyay, 1981; Kile & Walker, 1987). The newly discovered Chalam sp. described here appears to be confined to fine roots. It was isolated from roots with a diam. not greater than 10 mm (Table 1). Mycelial growth of this fungus from root fragments in vifro did not occur earlier than three months after the start of incubation of the root fragments. The fungus could not be accommodated as a known species of Chalam in the monographs by Nag Raj & Kendrick (1975) and Upadhyay (1981) or in recently published papers by Kile & Walker (1987), Kirk (1985, 1986) and Holubovi-Jechovi (1984). It is therefore described here as a species new to science and compared to known species with similar characteristics.
Measurements were conducted on original cultures obtained by plating out root segments as well as on subcultures and single conidia isolates. Dimensions of conidiophores and conidia were measured for each strain in distilled water (30 replicates). Photomicrographs were taken using a Reichertm Polyvar microscope in bright field or interference contrast. Material for SEM was cut from agar with a cork borer (diam. 5 mm), fixed with 4% glutaraldehyde buffered by a 0.1 M phosphate buffer (pH = 7.1) and dehydrated in a graded ethanol series (15, 30, 50, 70, 80, 90, 96, 100%) followed by acetone (100%). Specimens were then critical point dried, mounted on stubs, coated with gold/palladium and viewed with a scanning electron microscope (JSM 5200). To examine the relationship to Cevafocysfis s. str., cycloheximide tolerance (Harrington, 1981) of the Chalara sp. was tested. The fungus (four strains) was inoculated onto MEA amended with 0.1 g cycloheximide I-'. Extension of colonies was measured after 30 d by taking the average of two colony diameters for each plate. Two Petri dishes per strain were used and the experiment was conducted at room temperature and diffuse daylight.
RESULTS Chalara angustata T. Kowalski & Halmschlager sp. nov.
MATERIALS A N D M E T H O D S
Etym.: derived from the Latin word meaning diminished
Investigations were carried out on six strains (Table 1) isolated from oak roots. After surface sterilization ( I min ethanol 96%, 5 min NaOCl4 %, 30 s ethanol 96 %) roots were plated out on the surface of 2% malt extract agar (MEA, 20 g 1-I malt extract Difco, 15 g 1-I agar Difco, supplemented with 100 mg I-' streptomycin sulphate) and incubated a t room temperature and diffuse daylight. Resulting isolates were transferred to new MEA plates and incubated at 20 OC in the dark.
Coloniae in agaro maltoso primum tenues, hyalinae, albidae aut albocinereae, paulo mycelio superficiali soluto usque aut floccoso, post sporulationem intensam aspectu pulverulento. In culturis sequentibus zonis concentricis. Facies aversa primum hyalina, deinde in centro grisea maculis cinereis vel brunneolis. Incrementurn coloniarurn variatione magna: post 21 dies 22-57 mm in diam. ad 20° in tenebris. Fungus potuit resistere dilutis cycloheximide, increment0 non derninuto in 0.1 g cycloheximide 1-l. Mycelium immersum vel superficiale, hyphis hyalinis aut subhyalinis, 1.2-3.0 pm latis, intumescentibus ad 4.8 pm, pariete tenui, laevibus et numerosis,
1113
T. Kowalski and E. Halmschlager Table 1. Origin of strains of Chalara angustata, isolated from roots of Quercm pefraea and Q . robur (Patzmannsdorf, Austria)
Strain no.
Host
R255/1 R255/2 R255/3 R255/4 R2.5515 R255/6
Q. petraea Q. petraea Q. petraea Q. pefraea Q. robur Q. pefraea
Symptoms of oak decline1
Diam. class of SoiI horizon (cm) root (mm)
-
+ + +
-
Rooi tissue
Year of isolation
Bark Wood Bark Bark Bark Bark
1993 1993 1993 1993 1994 1994
-No symptoms, +symptoms of oak decline
parvis, lateralibus gibberibus, 3-11 x 1.2-2.0 wn omatis. Chlamydosporis nullis. Phialophora solitaria vel gregaria, cylindrica elongata, recta vel leniter flexuosa, mononematosa, non rarnosa, laevia, ferruginea ad brunnea, 1-4 (15) septis in parte basali, lI(r160 ( - 212) pm longa. Cellular basalis 6.2-11.2 pm lata vel valde contracta et 1.2-1.8 pm lata. Phialidae subcylindricae, interdum arnpulliformes, 96-125 Dm longae; venter subcylindricus vel ellipsoideus, 38-60 x 10-12 pm; collum cylindricum, 58-87 x 5.k7.0 pm, obscurius quam venter; transitio ex ventre ad collum gradatim, raro abrupta; ratio longitudinis colli et ventris = 1.4: 1. Phialoconidia in catenis longis dissolventibus formata, cylindrica, ad apicem rotundata, ad basim abscissa collo pusillo, 3- interdum 2-, 4- vel 6-septata, hyalina aut subhyalina, laevia 22-42 x 4.8-6 pm; ratio conidii longitudinis/latitudinis = 5.4 : 1. Ex radicibus Quercus petraeae (Matt.) Lieb., Patzmannsdorf/Austria, legunt Halmschlager & Kowalski, 24 June 1993. Holotypus (colonia exsiccata) in herbario ZT, cultura viva in CBS Baam.
Colonies on MEA initially thin, hyaline, white to whitish-grey, with some patches becoming grey to dark grey, with sparse, fluffy hairy-villose to lanuginose aerial mycelium, looking powdery in the case of abundant sporulation, with distinct concentric zones of different densities of aerial mycelium (Figs 1, 2). Single spore cultures forming soft, moist, radial folded colonies without aerial mycelium but with tufts of phialophores. Reverse at first hyaline, later becoming grey in the centre with grey or brownish dots. Extension rate of colonies showed high variability: Original cultures attaining 22-57 mm diam. in 21 d on MEA at 20' in the dark (Figs 1, 2) whereas growth of subcultures was always slower. No reduction in growth was observed at the tested concentration of cycloheximide (Table 2). Vegetative hyphae hyaline or subhyaline, 1.2-3-0 ym wide with swellings up to 4.8 ym; thin-walled, smooth with several small lateral burls 3-11 x 1.2-2.0 ym; septate with septa 4-18 ym apart. Chlamydospores absent. Phialophores arising directly on the superficial or slightly immersed vegetative hyphae or in older cultures from cells of a pseudoparenchymatous stroma; solitary and scattered or in tufts, erect, straight or slightly bent, rust-brown to brown with 1-4 (15) septa in the basal part, wall smooth, without constriction at the septa, 110-160 (-212) ym long, terminating in a phialide (Figs 3, 7, 9). Basal cell of the phialophore 6.2-11.2 pm wide or strongly constricted only 1.2-1.8 pm wide (Figs 3, 73. Percurrent proliferation of phialophores rare (Fig. 8). Phialides subcylindrical, occasionally lageniform, 96-125 (109) urn long. Venter subcylindrical or ellipsoid 38-60 (46) x 10-12 (11)ym. Cellarette cylindrical 58-87 (65) x 5.67.0 ym, darker than venter; ratio of mean length of collarette
to venter = 1.4: 1.Transition from venter to collarette gradual, occasionally abrupt. Phialoconidia catenate, formed in long, readily fragmenting chains (Figs 10, 11); cylindrical with obtuse apex and a truncate base bearing a marginal basal frill (Figs 4, 12, 13); 3septate, occasionally 2-, 4- or 6-septate, hyaline to subhyaline (in old cultures with a yellow-greenish tinge), filled with fine granular cytoplasm or with oil droplets, smooth walled; 22-42 (29.8) x 4.8-6 ( - 7) (5.5) bm (Fig. 4); mean conidium lengthlwidth ratio = 5.4: I. Sterile structures, like deformed or underdeveloped phialophores, are formed particularly in cultures that have been stored under cool conditions (6') for several months, sometimes also in young cultures. Two types are found most frequently: Type 1is clavate, 53-125 ym long with 2-6 septa, 7.5-8.7 ym wide at the base enlarging towards the apex to 10-12.5 pm, apical wall closed or breaking open (Fig. 6). Type 2 is cylindrical, up to 380 ym long with up to 20 septa, unicolour, light brown to brown (Fig. 5). From roots of Quercus pefraea (Matt.) Lieb., Patzmannsdorf/ Austria, legunt Halmschlager & Kowalski, 24 June 1993. Holotypus (colonia exsiccata) in herbarium ZT, cultura viva CBS Baam.
DISCUSSION Chalara is characterized by sessile or stalked phialides with basal venters and long collarettes, deep-seated conidiogenous loci and usually cylindrical, hyaline, subhyaline or pale brown, unicellular or septate phialoconidia (Nag Raj & Hughes, 1974; Holubovi-Jechovi, 1984). There are two related genera bearing phialophores mixed with sterile elements. In Chaetochalara setae-like structures are found. Sporoschisma is characterized by capitate hyphae with apical, hyaline mucilaginous-like caps (Holubovi-Jechovi, 1973; Nag Raj & Hughes, 1974). Sterile elements found in cultures of the fungus described here have neither the character of capitate hyphae as in Sporoschisma nor the seta-like hyphae found in Chaetochalara. Structures in this case are very variable in shape and can be interpreted as degenerate or underdeveloped phialophores as a result of growth conditions. This point of view is also supported by the increase of sterile structures in subcultures and older cultures. The fungus described here is therefore accommodated in Chalara. This also fits well for the hyaline to subhyaline phialospores which are coloured in Sporoschisma (Nag Raj & Kendrick, 1975).
Chalara angustata sp. nov.
Figs 1-9. Colony characteristics, phialophores, phialoconidia and sterile structures of Chalara angustata. Figs 1, 2. slow and fast growing colony of Ch. angusfafa with distinct concentric zones grown on MEA at 20' in the dark after 21 d. Fig. 3. Light microg~ raph of Ch. angustata showing a tuft of phialophores (bar, 20 pm). Fig. 4. Light micrograph of 3-septate phialoconidia with oil droplets; base of conidia bearing a marginal basal frill [arrows] (bar, 10 pm). Fig. 5. Cylindrical sterile structure (bar, 20 pm). Fig. 6. Clavate sterile structure (bar, 10 um). Fig. 7. Single phialophore with constricted basal cell [arrow] (bar, 20 urn). Fig. 8. Percurrent proliferation of phialophores (bar, 20 pm). Fig. 9. Phialophore with 15 septa in the basal part (bar, 20 pm).
T. Kowalski and E. Halmschlager Table 2. Colony diam. (mm) of subcultures of Chalara angwfata after 30 d on MEA and the same medium amended with 0.1 g cycloheximide I-' Strain no.
MEA
Cycloheximide agar
Chalara angusfafa bears some resemblance to Ch. unicolor s. Hughes & Nag Raj (Nag Raj & Hughes, 1974). Ch. unicolor is, however, easily distinguished by its brown colony colour and its phialophores composed of a single stalk cell without constriction at the base. Furthermore venter and collarette are concolorous and wider in Ch. unicolor (Nag Raj & Kendrick, 1975) compared with Ch. angwfafa. Chalara species are known as anarnorphs in a few diverse taxonomic groups of Ascomycetes, e.g. in Cerafocysfis (Upadhyay, 1981; Nag Raj & Kendrick, 1993). Sensitivity of species in Ceratocysfis s. slr. to cycloheximide has been an important characteristic in the taxonomy of this group (Harrington, 1981). The tolerance of Ch. angusfafa to cycloheximide suggests that the species is unrelated to Ceratocysfis s. str.
At the moment Chalara includes species bearing conidia with basal frills as well as conidia without frills. As can be deduced from the marginal basal frill of the conidia in Ch. angusfafa, this species has a mode of conidiurn development similar to the Chalara-state of Ceratocysfis autographa (Wingfield ef al., 1995). In that case basal frills lead to the adherence of conidia to each other forming what Subramanian (1972) and Minter, Kirk & Sutton (1982, 1983) called 'false chains '. Ch. angustafa appears to be confined to h e roots, where the fungus remains causing no symptoms. This symptomless endophytic infection in the roots may be typical of the fungus at certain stages in its life cycle (Fisher, Petrini & Webster, 1991). It is of interest that the fungus has so far not been reported as a root endophyte. This may be because it is species specific to oak roots which have not been intensively examined yet. A more likely explanation is the long incubation required in vifro before the fungus can be detected, leading to it being overlooked in earlier assays of the roots of other plants. Slow growth of Ch. angusfata in vitro and the fact that the fungus grew only after a long incubation period of the root fragments suggests that a selective medium might help to detect the incidence of this species in the host plant. Kile & Walker (1987) listed 11 Chalara species from oak. Two further species from oak, Ch. ovoidea Nag Raj & W. B. Kendr. and Ch. breviclavafa Nag Raj & W. B. Kendr. have
Figs 10-13. Conidia and conidiogenous cells of Ch. angustafa. Fig. 10. SEM of cylindrical conidium at the apex of a tubular collarette. Fig. 11. SEM showing a false chain of phialoconidia. Fig. 12. SEM of obtuse apex of a phialoconidium. Fig. 13. SEM of truncate base
of a phialoconidium.
Chalara angusfata sp. nov. been described by Holubovi-Jechovi (1984) and Traue & Arnold (1991). Ch. angusfata described here is the first species from oak with multiseptate phialospores. We thank Professor Dr H. Butin for the Latin diagnosis and the Austrian Ministry of Science, Research and Arts for financial support.
REFERENCES Fisher, P. I., Petrini, 0 . & Webster, J. (1991). Aquatic hyphomycetes and other fungi in living aquatic and terrestrial roots of Alnus glutinosa. Mycological Research 95,543-547. Harrington, T.C. (1981). Cycloheximide sensitivity as a taxonomical character in Ceratocystis. Mycologia 73, 1123-1129. (1973). Lignicolous hyphomycetes from Holubovii-Jechova, V. Czechoslovakia. 3. Sporoschisma, Sporoschismopsis and Cutenularia. Folia Geobotanica et Phytotaxonomica 8, 209-218. Holubovii-Jechovii, V. (1984). Lignicolous hyphomycetes from Czechoslovakia. 7. Chalara, Exochlara, Fwichalara and Dictyochaeta. Folia Geobotanica et Phytotaxonomica 19, 387-438. Kile, G. A. & Walker, J. (1987). Chalara australis sp. nov. (Hyphomycetes), a vascular pathogen of Nothofagw cunninghamii (Fagaceae) in Australia and its relationship to other Chalara species. Awtralian Journal of Botany 35, 1-32.
(Accepted 15 February 1996)
1116 Kirk, P.M. (1985). New or interesting microfungi. XIV. Dematiaceous hyphomycetes from Mt Kenya. Mycotaxon 23,305-352. Kirk, P. M. (1986). New or interesting microfungi. XV. Miscellaneous hyphomycetes from the British Isles. Transactions of the British Mycological Society 86, 409-428. Minter, D. W., Kirk, P. M. & Sutton, B. C. (1982). Holoblastic phialides. Transactions of the British Mycological Society 79, 75-93. Minter, D. W., Kirk, P. M. & Sutton, B. C. (1983). Thallic phialides. Transactions of the British Mycological Society 80, 39-66. Nag Raj, T. R. &Hughes, S. J. (1974). New Zealand Fungi. 21. Chalara (Corda) Rabenhorst. New Zealand]ournal of Botany 12, 115-129. Nag Raj, T. R. & Kendrick, B. (1975). A Monograph of Chalara and Allied Genera. Wilfnd Laurier University Press: Waterloo, Ontario, Canada. Nag Raj, T. R. & Kendrick, B. (1993). The anamorph as generic determinant in the holomorph: the Chalara connection in the ascomycetes, with special reference to the ophiostomatoid fungi. In Ceratocystis and Ophiostoma (ed. M . J.Wingfield, K. A. Seifert & J. F. Webber), pp. 61-70. American Phytopathological Society: St Paul, MN. Subramanian, C. V. (1972). Conidial chains, their nature and significance in the taxonomy of hyphomycetes. Current Science 41,43-49. Traue, H. & Arnold, G. R. W. (1991). Chalara ovoidea Nag Raj & Kendrick eine weitere Pilzart im Krankheitsgeschehen der Trauben- und Stieleichen. Feddes Repertorium 102, 133-136. Upadhyay, H. P. (1981). A Monograph of Ceratocystis and Ceratocystiopsis. University of Georgia Press: Athens, GA. Wingfield, M. J., Benade, E., Van Wyk, P. S. & Visser, C. (1995). Conidium development in Ceratocystis autographa. Mycological Research 99, 1289-1294.