NOTES
Nematophagous fungi: three new species of Myzocytium Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/15/14 For personal use only.
G. L. BARRON Departmen! of EnvironmenfalBiology, University of Cuelph, Crtelph, Ontario NIG 2 W l Accepted January 16, 1976
BARRON, G. L. 1976. Nematophagous fungi: three new species of Myzocyrilrm. Can. J . Microbiol. 22: 752-762. Three species of Myzocyfilrtn parasitic on nematodes are described as new. In M . papillaflrm the zoospores encyst directly on the host cuticle before penetration. This species produces smooth, spherical oospores. In M. glufitlosporunl the biflagellate zoospores do not attack the host directly; after encystment they produce a spherical adhesive bud whichallows the spores to adhere to the cuticle ofpassing nematodes. This species produces echinulate, spherical oospores. In M . rtnomal~rmthe primary spores are aplanospores. After a dormant phase, and when suitably stimulated, these aplanospores change into biflagellate zoospores and the latter encyst on the host cuticle. No sexual state is known in this species. Persistence is by means of thick-walled, spherical chlamydospores. BARRON, G. L. 1976. Nematophagous fungi: three new species of Myzocyfi~rm.Can. J. Microbiol. 22: 752-762. Trois esptces de Myzocyfirrm parasites des nematodes sont dtcrites comme nouvelles. Chez M . papillatum, les zoospores s'enkystent directement sur la cuticule de I'hBte avant leur penetration. Cette espece produit des oospores sphkriques lisses. Chez M . glufinosponrm, les zoospores biflagellees n'attaquent pas I'hBte directement; apres leur enkystement, elles produisent un bourgeonnement spherique adhisif qui permet aux spores d'adherer a la cuticle lors du passage des nematodes. Cette espece produit des oospores spheriques Cchinulees. Chez M . anon~ul~rm les , spores primaires sont des aplanospores. Aprts une phase de dormance et une stimulation adequate, ces aplanospores se changent en zoospores biflagellies qui s'enkystent ultirieurement sur la cuticule de I'hBte. Le stade sexuel n'est par connu pour cette espece. La persistance est assuree grlce a des chlamydospores spheriques a parois tpaisses. [Traduit par le journal]
In studies of the nematophagous fungi of Ontario, three species of Myzocytium which have not been previously described were recorded. A point of particular interest is that all three species exhibit different patterns in their parasitic cycle. In the first species (M. papillatum) laterally biflagellate spores are produced and these encyst at any point on the nematode cuticle before penetration and infection. In the second species (M. glutinosporurn) biflagellate spores are again produced but these do not attack the host directly. After a swarming period, the zoospores encyst and produce an adhesive bud at the apical end. This adhesive bud allows the spore to adhere to the cuticle of passing nematodes and subsequent penetration initiates the infection cycle. In the third species (M. anomalum) the sporangia produce aplano'Received September 4, 1975.
spores which may be released as such or remain dormant in the sporangium until disintegration. After an interval of time and when suitably stimulated the aplanospores change into laterally biflagellate zoospores. These latter spores encyst on the host and penetrate directly through the cuticle in a manner similar to the first species. Myzocytium papillatum sp. nov. (Figs. 1-9) Thallus endoparasiticus, segmentis aut globosis aut ellipsoideis aut citriformibus, saepe cum papilla conspicua in singulis extremis 20-40 x 10-22 pm. Segmenta sporangialia proferentia zoosporas a latere biflagellatas, lentiformes. Zoosporae 8-10 pm in longitudine, evadentes ex brevi tub0 evacuationis, denique cystas fingentes in hospite et penetrantes recte per murum corporis. Segmenta antheridialia et oogonialia haud facile distincta segmentis ZOOsporangialibus. Antheridia minora quam oogo-
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FIGS.1-5. M y z o c y t i r ~ ~pnpillntutn. ?~ Figs. 1, 2. Mature thalli inside host. Some segments behave as zoosporangia and others as antheridia or oogonia. Mature oospores are spherical and thick-walled. Fig. 3. Laterally biflagellate zoospores. Fig. 4. Encysted zoospores on host cuticle. Fig. 5. Antheridia are adjacent to the oogonia with each oogonium containing a single oospore.
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FIGS. 6-9. Myzocytium papillatum. Figs. 6 , 7. Zoospores encysted on host cuticle appearing hatshaped. Fig. 8. Antheridia (a) and oogonia ( 0 )containing oospores (0s).Fig. 9. Papillate zoosporangia can be seen in the left half of the photo. Note that both oogonia and antheridia have originated from papillate segments.
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nia. Oosporae laeves, crassitunicatae, globosae, 12-22 pm in diametro. TYPE: slides in Herbarium (OAC 10904) HABITAT: from barnyard soil, Aberfoyle, Ontario; parasitic on Rhabditis terricola Dujardin. Myzocytium papillatum was maintained for several months in nonaxenic culture in association with its nematode host (Rhabditis terricola Dujardin). The zoospores are lens-shaped, laterally biflagellate, and measure 8-10 pm long (Fig. 3). The stimulus that attracts the zoospores to the nematode host is not known but eventually the zoospores encyst on the host cuticle and then appear somewhat hat-shaped with the hemispherical cyst having a flattened ridge in contact with the host (Figs. 4, 6, 7). The encysted spore behaves as an appressorium from which a peg penetrates through the host cuticle. The contents of the cyst are then transferred into the body of the host to form the young infection thallus. The cyst now appears as an empty shell on the surface of the host and disappears with time. The infection thallus grows the entire length of the host and at maturity becomes septated into a number of segments. Each segment swells and becomes constricted at the septa to the point where adjacent segments often disarticulate. Segments may be globose to subglobose but are often lemon-shaped and distinctly papillate at one or both ends. Sporangia measure 2040 x 10-22 pm and are usually in irregular linear arrangements in the body of the host. In segments which behave as zoosporangia 2 single evacuation tube is produced. The zoospores may mature inside the zoosporangium and swim out but in some cases the entire content of a zoosporangium is expelled through the evacuation tube to gather at the mouth in a vesicle (Fig. 5). No vesicular membrane was observed. Some of the segments behave as antheridia and oogonia. Such segments are in no way different from those which are destined to be zoosporangia. Two adjacent segments behave as the sex organs. When the segments are of different sizes usually the smaller of the two becomes the antheridium. Sexual reproduction is by conjugation and the antheridial protoplast migrates into the oogonium to fuse with the oogonial protoplast. When the entire contents of the antheridium have been transferred into the oogonium a wall is secreted by the zygote. The resulting oospores are thick-walled, smooth,
755
spherical, and measure 12-22 pm in diameter. Whether the antheridial protoplast migrates through a pore in the contiguous wall or the entire wall breaks down at the point of contact is not known. In Fig. 8 the antheridium on the extreme left seems to have extended its normal shape to meet and fuse with the oogonium. It appears likely in this case that the entire point of contact between the two breaks down. No sign of a fertilization tube was found in any of the oogonial-antheridial associations. Myzocytium glutinosporum sp. nov. (Figs. 10-22) Thallus endoparasiticus, simplex aut exigue ramosus. Segmenta variabilia ab globosis aut ellipsoideis aliquando ad cylindrica, 18-38 x 14-30 pm. Segmenta sporangialia proferentia zoosporas pyriformes, a latere biflagellatas. Zoosporae 8-12 pm in longitudine cystas rapide fingentes. Omnis spora cystata proferens bulbum tenacem aut seriem bulborum tenacium in apice. Sporae ad corpus hospitale adhaerentes. Segmenta antheridialia et oogonialia similia segmentis zoosporangialibus, antheridiis plerumque minoribus quam oogoniis. Oosporae crassitunicatae, globosae, minute echinulatae, 15-22 pm in diametro. TYPE: slides in Herbarium (OAC 10905) HABITAT: recovered from barnyard soil, Aberfoyle, Ontario; parasitic on Rhabditis terricola Dujardin. In M. glutinosporum the zoospores are broader at the base and taper slightly towards the apex. They measure 8-12 pm long and are biflagellate with the flagellae attached in the upper half of the spore (Fig. 10a). The zoospores are relatively poor swimmers and encyst after a short swarming phase with encystment sometimes taking place within minutes of release. Each encysted zoospore produces a spherical adhesive bud (Fig. lob) at the apical end by means of which it adheres to the cuticle of passing nematodes. If the spore does not attach immediately, the apex continues to proliferate until a series of buds is produced (Fig. 10c). Once attached, a peg from the spore passes directly through the host cuticle (Figs. 11, 17, 18, 19). The contents of the spore now pass into the body of the host to initiate the infection thallus with the spore remaining as an empty shell on the outside. The spore shell is frequently sloughed off but leakage of nutrients through the infection site often encourages a bacterial colony
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FIGS. 10-16. Myzocyfil~rnglufinospot~~o~~. Fig. 10. Zoospores (a), encysted zoospores with adhesive buds (b), and older spores with series of buds (c). Fig. 11. Nematode with spores adhering to the body and young infection thalli developing inside. Figs. 12, 13. Mature sporangia. Fig. 14. Empty zoosporangia with evacuation tubes. Some segments remain in a resting phase. Figs. 15, 16. Antheridia are attached t o and immediately adjacent to the oogonia. Oospores are spherical and echinulate.
in the vicinity of the attached spore which trails like a streamer behind a moving nematode. This bacterial streamer is a useful indicator when looking for early infection stages in living nematodes.
The infection thallus grows the entire length of the host body and at maturity becomes septated with each segment swelling markedly and becoming constricted at the septa to the point where adjacent segments may completely
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NOTES
FIGS. 17-22. Myzocytium glutinosporum. Figs. 17-19. Adhesive spores attached to the cuticle of the host. The first stage of penetration can be clearly seen in Fig. 19. Figs. 20, 21. These show the remarkable uniformity in the development of the zoosporangial segments inside the body of the host. Fig. 22. Mature oospores are spherical and echinulate; the antheridia in this specimen have disintegrated.
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FIGS.23-28. Myzocytirlrn arzornal~~rn. Fig. 23. Primary aplanospores just after release ( a ) , in a transitional stage ( b ) ,and as fully developed zoospores (c). Fig. 24. Mature stages in the development of the sporangia with aplanospores now differentiated inside the sporangia. Developing chlamydospores are seen at each end of the host. Fig. 25. Young (a) and mature (b) chlamydospores. Fig. 26. Two thalli developing inside the host and beginning to form segments. Fig. 27. Mature sporangia following development of evacuation tubes. Aplanospores have been released from one sporangium. Fig. 28. Nematode with zoospores encysted on the cuticle.
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NOTES
FIGS.29-32. Myzocyiiurn anomalum. Fig. 29. Nematode with zoospores encysted on the cuticle. A young infection thallus can be seen inside the host. Fig. 30. Several thalli inside host becoming septated into spindle-shaped segments. Figs. 31, 32. Stages in maturation of the sporangia.
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disarticulate. Segments may be globose, subglobose, ellipsoid, ovoid, or irregular in shape (Figs. 12, 13) and may measure 18-38 x 1430 pm. Shape of mature segments depends on the number of infection thalli per host. When only a single infection occurs then a linear series of spherical segments of remarkable symmetry is produced (Figs. 12, 20, 21). When several simultaneous infections occur competition between developing thalli results in a great variety in size and shape of segments. Each zoosporangial segment produces a single exit tube which emerges at more or less right angles to the long axis of the nematode (Fig. 14). The zoospores differentiate inside the zoosporangium and an external vesicle was never observed. Development of sexual spores in this species is by conjugation and is similar in all essential details to that previously described for M. papillatum and M , humicola (Barron and Percy 1975). The oospores resulting from conjugation are thick-walled, spherical, and echinulate and measure 15-22 pm in diameter. In this species oospores are infrequent in occurrence even in older cultures. In those antheridialoogonial associations studied no fertilization tube was observed. Myzocytium glutinosporum is closest to M. vermicola Zopf (Dangeard 1903) and M. humicola Barron and Percy. It differs from both of these species in having echinulate oospores. Both zoospores and resulting adhesive spores tend to be shorter and less tapered than those of M. humicola. Myzocytium vermicola is distinguished from the other two adhesive-spored species in having reticulate oospores and a fertilization tube from the antheridium to the oogonium.
Myzocythm anomalum sp. nov. (Figs. 23-36) Thallus endoparasiticus, simplex, in maturitate septatus ut producat seriem linearem segmentorum a fusiformibus ad triangularia variabilia. Segmenta 25-55 x 10-15 pm, cum brevi truncato tub0 evacuationis. Sporangia proferentia aplanosporas 8-10 x 4-6 pm. Sporae aut ellipsoideae aut ovoideae aut irregulares. Aplanosporae transeuntes in zoosporas a latere biflagellatas, lentiformes, quae cystas recte in hospite fingunt et per murum penetrant. Status sexualis ignotus. Sphaericae chlamydosporae 12-18pm in diametro in culturis vetustis formatae.
TYPE: slides in Herbarium (OAC 10906) HABITAT: from barnyard soil, Aberfoyle,
Ontario; parasitic on an unidentified nematode. Inside the host the infection thallus of M. anomalum is unbranched and becomes septate at maturity to form a series of elongate segments (Figs. 26, 30). These are mostly spindle-shaped to triangulate but may be ellipsoid to ovoid or occasionally irregular. Segments may become swollen and constricted at the septa although adjacent segments tend to remain attached to form a linear series (Figs. 31, 32). In small nematodes often a number of infection thalli develop simultaneously and these may completely fill the body of the host (Fig. 32). At maturity each segment becomes a sporangium. Sporangia measure 25-55 x 10-15 pm. At maturity the protoplasm inside each sporangium cleaves to form numerous non-motile spores which are ellipsoid to ovoid or sometimes irregular in shape because of mutual pressure (Figs. 33, 34). The spores measure 8-10 x 4-6 pm (Figs. 23, 35). Each sporangium produces a short broad evacuation tube about 8-10 pm broad and up to 25 p long (Figs. 24, 27, 33). Aplanospores may be expelled to the exterior through the evacuation tube but often remain inside the sporangia which produce them and are eventually released following disintegration of the host and sporangial walls (Fig. 34). Aplanospores may remain in a dormant state for prolonged periods. Under suitable conditions, however, such as the presence of abundant fresh nematode hosts, they change slowly into lens-shaped laterally biflagellate zoospores very similar in appearance to those of M. papillatum. This change may take place outside or inside the sporangium such that in some conditions zoospores may be seen inside the sporangia but they are apparently never the primary spore formed. Zoospores measure 8-10 pm long and encyst at any point on the host cuticle to form hemispherical cysts (Figs. 28, 29). Penetration is similar to that of M. papillatum. In older cultures instead of sporangial segments, the infection thallus may produce a series of spherical segments which become thick-walled chlamydospores (Figs. 24, 25, 36). The chlamydospores are 12-18 pm in diameter and are apparently the persistent phase of the fungus. No sexual state was found.
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NOTES
FIGS. 33-36. Myzocyfilrm atrotnalum. Fig. 33. Mature sporangia with broad, truncate evacuation tubes. Spores have been released from one of the sporangia. Fig. 34. Mature aplanospores inside the body of the host with some sporangial walls appearing to have disintegrated. Fig. 35. Aplanospores. Fig. 36. Young chlamydospores.
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Acknowledgments -
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The writer is indebted to the National Research for to carry out these studies and to Dr. V. J. Matthews for translating the diagnoses into Latin. It is a
pleasure to thank Jim Holley and Yvonne Dierkes for technical assistance. BARRON,G. L . , and J. G. PERCY.1975. Nematophagous fungi: a new Myzocyti~rm.Can. J. Bot. 53: 1306-1309. DANGEAR,, P. A. 1903. Recherches sur le developpernent dupCrithtcechezlesAscomycetes.Botaniste,9: 1-303.
Regulation of acetyl-CoA synthetase of Saccharomyces cerevisiael Deparrtnet~tof Microbiology, Miami University, Oxford, Ohio45056 Accepted December 3 I, 1975
C O L E M A NJ., S . , and J. K. BHATTACHARIEE. 1976. Regulation of acetyl-CoA synthetase of Saccharomyces cercvisiae. Can. J. Microbiol. 22: 762-764. Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure. T h e activity in vitro was inhibited significantly by NADPH, NADH, or AMP and to a lesser extent by NADP, NAD, o r ADP. Glutarnic acid and a-ketoglutaric acid were not inhibitory. T h e enzyme level was repressed when the cells were grown in a complex nutrient medium a s opposed t o the minimal medium. However, a glutamic acid auxotroph glrrl, when grown in excess glutamic acid, demonstrated a fivefold increase of acetyl-CoA synthetase. COLEMAN,J. S., e t J. K. BHATTACHARIEE. 1976. Regulation of acetyl-CoA synthetase of Saccharomyces cerevisine. Can. J. Microbiol. 22: 762-764. L'activitC d e I'acCtyl-coenzyme A synthetase ( E C 6.2.1. I) d e Sacchorotnyces cerevisioe a i t 6 dCterminCe par une methode d e dosage radioactive. L'activitC in vitro est inhibie d e f a ~ o n significative par le NADPH, le NADH ou I'AMP et a un moindre d e g r i par le NADP, le N A D o u I'ADP. L'acide glutamique et I'acide a-~Ctoglutariquen'inhibent pas I'activitC de I'enzyme. L e taux d'enzyme est rCprimi lorsque les cellules sont cultivies dans un milieu nutritif complexe mais non dans un milieu minimal. Cependant, lorsqu'un auxotrophe a 1'Cgard de I'acide glutamique glrtl est cultivi en prksence d'un e x c t s d'acide glutamique, le taux de I'acetyl-CoA synthetase augmente de cinq fois. [Traduit par le journal]
Several enzymes involved in lipogenesis and ketogenesis including acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) are inhibited by longchain fatty acid CoA esters (6, 8). Little is known about the nucleotide and amino acid regulation of ACS. Regulation of citrate synthase (EC 4.1.3.7) by nucleotides, glutamic acid, or aketoglutaric acid is well known (2, 9). We report here the regulation of ACS of Saccharomyces cerevisiae in vitro and in vivo by various nucleotides and glutamic acid, respectively. A prototrophic strain, X2180, and a glutamic acid auxoJroph, MO-1-9B (glul), of S. cerevisiae were used in this study (1, 3). Cell extract was prepared from late log phase culture by procedures described elsewhere (7). Regulation of 'Received September 23, 1975.
ACS in vitro was determined in extracts prepared from X2 180 grown in the minimal medium, composed of Difco yeast nitrogen base without amino acids, 0.67%, and glucose, 1%. For the regulation of the biosynthesis of ACS, extracts were prepared from X2180 grown in the minimal medium with different amounts of glucose as well as in the complex nutrient medium, composed of peptone, 0.6%; yeast extract, 0.4%; MgS04.7H20, 0.1%; KH2P04, 0.2%; and glucose, 4%. Since the mutant glul lacks aconitase activity and requires glutamic acid supplementation for growth (3), the specific effect of glutamic acid on the biosynthesis of ACS was determined in extracts prepared from MO-1-9B grown in the minimal medium with known concentrations of glutamic acid. The ACS activity was determined by a radio-
Nematophagous fungi: three new species of Myzocytium Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/15/14 For personal use only.
G. L. BARRON Departmen! of EnvironmenfalBiology, University of Cuelph, Crtelph, Ontario NIG 2 W l Accepted January 16, 1976
BARRON, G. L. 1976. Nematophagous fungi: three new species of Myzocyrilrm. Can. J . Microbiol. 22: 752-762. Three species of Myzocyfilrtn parasitic on nematodes are described as new. In M . papillaflrm the zoospores encyst directly on the host cuticle before penetration. This species produces smooth, spherical oospores. In M. glufitlosporunl the biflagellate zoospores do not attack the host directly; after encystment they produce a spherical adhesive bud whichallows the spores to adhere to the cuticle ofpassing nematodes. This species produces echinulate, spherical oospores. In M . rtnomal~rmthe primary spores are aplanospores. After a dormant phase, and when suitably stimulated, these aplanospores change into biflagellate zoospores and the latter encyst on the host cuticle. No sexual state is known in this species. Persistence is by means of thick-walled, spherical chlamydospores. BARRON, G. L. 1976. Nematophagous fungi: three new species of Myzocyfi~rm.Can. J. Microbiol. 22: 752-762. Trois esptces de Myzocyfirrm parasites des nematodes sont dtcrites comme nouvelles. Chez M . papillatum, les zoospores s'enkystent directement sur la cuticule de I'hBte avant leur penetration. Cette espece produit des oospores sphkriques lisses. Chez M . glufinosponrm, les zoospores biflagellees n'attaquent pas I'hBte directement; apres leur enkystement, elles produisent un bourgeonnement spherique adhisif qui permet aux spores d'adherer a la cuticle lors du passage des nematodes. Cette espece produit des oospores spheriques Cchinulees. Chez M . anon~ul~rm les , spores primaires sont des aplanospores. Aprts une phase de dormance et une stimulation adequate, ces aplanospores se changent en zoospores biflagellies qui s'enkystent ultirieurement sur la cuticule de I'hBte. Le stade sexuel n'est par connu pour cette espece. La persistance est assuree grlce a des chlamydospores spheriques a parois tpaisses. [Traduit par le journal]
In studies of the nematophagous fungi of Ontario, three species of Myzocytium which have not been previously described were recorded. A point of particular interest is that all three species exhibit different patterns in their parasitic cycle. In the first species (M. papillatum) laterally biflagellate spores are produced and these encyst at any point on the nematode cuticle before penetration and infection. In the second species (M. glutinosporurn) biflagellate spores are again produced but these do not attack the host directly. After a swarming period, the zoospores encyst and produce an adhesive bud at the apical end. This adhesive bud allows the spore to adhere to the cuticle of passing nematodes and subsequent penetration initiates the infection cycle. In the third species (M. anomalum) the sporangia produce aplano'Received September 4, 1975.
spores which may be released as such or remain dormant in the sporangium until disintegration. After an interval of time and when suitably stimulated the aplanospores change into laterally biflagellate zoospores. These latter spores encyst on the host and penetrate directly through the cuticle in a manner similar to the first species. Myzocytium papillatum sp. nov. (Figs. 1-9) Thallus endoparasiticus, segmentis aut globosis aut ellipsoideis aut citriformibus, saepe cum papilla conspicua in singulis extremis 20-40 x 10-22 pm. Segmenta sporangialia proferentia zoosporas a latere biflagellatas, lentiformes. Zoosporae 8-10 pm in longitudine, evadentes ex brevi tub0 evacuationis, denique cystas fingentes in hospite et penetrantes recte per murum corporis. Segmenta antheridialia et oogonialia haud facile distincta segmentis ZOOsporangialibus. Antheridia minora quam oogo-
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NOTES
FIGS.1-5. M y z o c y t i r ~ ~pnpillntutn. ?~ Figs. 1, 2. Mature thalli inside host. Some segments behave as zoosporangia and others as antheridia or oogonia. Mature oospores are spherical and thick-walled. Fig. 3. Laterally biflagellate zoospores. Fig. 4. Encysted zoospores on host cuticle. Fig. 5. Antheridia are adjacent to the oogonia with each oogonium containing a single oospore.
CAN. J. MICROBIOL. VOL. 22. 1976
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FIGS. 6-9. Myzocytium papillatum. Figs. 6 , 7. Zoospores encysted on host cuticle appearing hatshaped. Fig. 8. Antheridia (a) and oogonia ( 0 )containing oospores (0s).Fig. 9. Papillate zoosporangia can be seen in the left half of the photo. Note that both oogonia and antheridia have originated from papillate segments.
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NOTES
nia. Oosporae laeves, crassitunicatae, globosae, 12-22 pm in diametro. TYPE: slides in Herbarium (OAC 10904) HABITAT: from barnyard soil, Aberfoyle, Ontario; parasitic on Rhabditis terricola Dujardin. Myzocytium papillatum was maintained for several months in nonaxenic culture in association with its nematode host (Rhabditis terricola Dujardin). The zoospores are lens-shaped, laterally biflagellate, and measure 8-10 pm long (Fig. 3). The stimulus that attracts the zoospores to the nematode host is not known but eventually the zoospores encyst on the host cuticle and then appear somewhat hat-shaped with the hemispherical cyst having a flattened ridge in contact with the host (Figs. 4, 6, 7). The encysted spore behaves as an appressorium from which a peg penetrates through the host cuticle. The contents of the cyst are then transferred into the body of the host to form the young infection thallus. The cyst now appears as an empty shell on the surface of the host and disappears with time. The infection thallus grows the entire length of the host and at maturity becomes septated into a number of segments. Each segment swells and becomes constricted at the septa to the point where adjacent segments often disarticulate. Segments may be globose to subglobose but are often lemon-shaped and distinctly papillate at one or both ends. Sporangia measure 2040 x 10-22 pm and are usually in irregular linear arrangements in the body of the host. In segments which behave as zoosporangia 2 single evacuation tube is produced. The zoospores may mature inside the zoosporangium and swim out but in some cases the entire content of a zoosporangium is expelled through the evacuation tube to gather at the mouth in a vesicle (Fig. 5). No vesicular membrane was observed. Some of the segments behave as antheridia and oogonia. Such segments are in no way different from those which are destined to be zoosporangia. Two adjacent segments behave as the sex organs. When the segments are of different sizes usually the smaller of the two becomes the antheridium. Sexual reproduction is by conjugation and the antheridial protoplast migrates into the oogonium to fuse with the oogonial protoplast. When the entire contents of the antheridium have been transferred into the oogonium a wall is secreted by the zygote. The resulting oospores are thick-walled, smooth,
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spherical, and measure 12-22 pm in diameter. Whether the antheridial protoplast migrates through a pore in the contiguous wall or the entire wall breaks down at the point of contact is not known. In Fig. 8 the antheridium on the extreme left seems to have extended its normal shape to meet and fuse with the oogonium. It appears likely in this case that the entire point of contact between the two breaks down. No sign of a fertilization tube was found in any of the oogonial-antheridial associations. Myzocytium glutinosporum sp. nov. (Figs. 10-22) Thallus endoparasiticus, simplex aut exigue ramosus. Segmenta variabilia ab globosis aut ellipsoideis aliquando ad cylindrica, 18-38 x 14-30 pm. Segmenta sporangialia proferentia zoosporas pyriformes, a latere biflagellatas. Zoosporae 8-12 pm in longitudine cystas rapide fingentes. Omnis spora cystata proferens bulbum tenacem aut seriem bulborum tenacium in apice. Sporae ad corpus hospitale adhaerentes. Segmenta antheridialia et oogonialia similia segmentis zoosporangialibus, antheridiis plerumque minoribus quam oogoniis. Oosporae crassitunicatae, globosae, minute echinulatae, 15-22 pm in diametro. TYPE: slides in Herbarium (OAC 10905) HABITAT: recovered from barnyard soil, Aberfoyle, Ontario; parasitic on Rhabditis terricola Dujardin. In M. glutinosporum the zoospores are broader at the base and taper slightly towards the apex. They measure 8-12 pm long and are biflagellate with the flagellae attached in the upper half of the spore (Fig. 10a). The zoospores are relatively poor swimmers and encyst after a short swarming phase with encystment sometimes taking place within minutes of release. Each encysted zoospore produces a spherical adhesive bud (Fig. lob) at the apical end by means of which it adheres to the cuticle of passing nematodes. If the spore does not attach immediately, the apex continues to proliferate until a series of buds is produced (Fig. 10c). Once attached, a peg from the spore passes directly through the host cuticle (Figs. 11, 17, 18, 19). The contents of the spore now pass into the body of the host to initiate the infection thallus with the spore remaining as an empty shell on the outside. The spore shell is frequently sloughed off but leakage of nutrients through the infection site often encourages a bacterial colony
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FIGS. 10-16. Myzocyfil~rnglufinospot~~o~~. Fig. 10. Zoospores (a), encysted zoospores with adhesive buds (b), and older spores with series of buds (c). Fig. 11. Nematode with spores adhering to the body and young infection thalli developing inside. Figs. 12, 13. Mature sporangia. Fig. 14. Empty zoosporangia with evacuation tubes. Some segments remain in a resting phase. Figs. 15, 16. Antheridia are attached t o and immediately adjacent to the oogonia. Oospores are spherical and echinulate.
in the vicinity of the attached spore which trails like a streamer behind a moving nematode. This bacterial streamer is a useful indicator when looking for early infection stages in living nematodes.
The infection thallus grows the entire length of the host body and at maturity becomes septated with each segment swelling markedly and becoming constricted at the septa to the point where adjacent segments may completely
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NOTES
FIGS. 17-22. Myzocytium glutinosporum. Figs. 17-19. Adhesive spores attached to the cuticle of the host. The first stage of penetration can be clearly seen in Fig. 19. Figs. 20, 21. These show the remarkable uniformity in the development of the zoosporangial segments inside the body of the host. Fig. 22. Mature oospores are spherical and echinulate; the antheridia in this specimen have disintegrated.
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FIGS.23-28. Myzocytirlrn arzornal~~rn. Fig. 23. Primary aplanospores just after release ( a ) , in a transitional stage ( b ) ,and as fully developed zoospores (c). Fig. 24. Mature stages in the development of the sporangia with aplanospores now differentiated inside the sporangia. Developing chlamydospores are seen at each end of the host. Fig. 25. Young (a) and mature (b) chlamydospores. Fig. 26. Two thalli developing inside the host and beginning to form segments. Fig. 27. Mature sporangia following development of evacuation tubes. Aplanospores have been released from one sporangium. Fig. 28. Nematode with zoospores encysted on the cuticle.
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NOTES
FIGS.29-32. Myzocyiiurn anomalum. Fig. 29. Nematode with zoospores encysted on the cuticle. A young infection thallus can be seen inside the host. Fig. 30. Several thalli inside host becoming septated into spindle-shaped segments. Figs. 31, 32. Stages in maturation of the sporangia.
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disarticulate. Segments may be globose, subglobose, ellipsoid, ovoid, or irregular in shape (Figs. 12, 13) and may measure 18-38 x 1430 pm. Shape of mature segments depends on the number of infection thalli per host. When only a single infection occurs then a linear series of spherical segments of remarkable symmetry is produced (Figs. 12, 20, 21). When several simultaneous infections occur competition between developing thalli results in a great variety in size and shape of segments. Each zoosporangial segment produces a single exit tube which emerges at more or less right angles to the long axis of the nematode (Fig. 14). The zoospores differentiate inside the zoosporangium and an external vesicle was never observed. Development of sexual spores in this species is by conjugation and is similar in all essential details to that previously described for M. papillatum and M , humicola (Barron and Percy 1975). The oospores resulting from conjugation are thick-walled, spherical, and echinulate and measure 15-22 pm in diameter. In this species oospores are infrequent in occurrence even in older cultures. In those antheridialoogonial associations studied no fertilization tube was observed. Myzocytium glutinosporum is closest to M. vermicola Zopf (Dangeard 1903) and M. humicola Barron and Percy. It differs from both of these species in having echinulate oospores. Both zoospores and resulting adhesive spores tend to be shorter and less tapered than those of M. humicola. Myzocytium vermicola is distinguished from the other two adhesive-spored species in having reticulate oospores and a fertilization tube from the antheridium to the oogonium.
Myzocythm anomalum sp. nov. (Figs. 23-36) Thallus endoparasiticus, simplex, in maturitate septatus ut producat seriem linearem segmentorum a fusiformibus ad triangularia variabilia. Segmenta 25-55 x 10-15 pm, cum brevi truncato tub0 evacuationis. Sporangia proferentia aplanosporas 8-10 x 4-6 pm. Sporae aut ellipsoideae aut ovoideae aut irregulares. Aplanosporae transeuntes in zoosporas a latere biflagellatas, lentiformes, quae cystas recte in hospite fingunt et per murum penetrant. Status sexualis ignotus. Sphaericae chlamydosporae 12-18pm in diametro in culturis vetustis formatae.
TYPE: slides in Herbarium (OAC 10906) HABITAT: from barnyard soil, Aberfoyle,
Ontario; parasitic on an unidentified nematode. Inside the host the infection thallus of M. anomalum is unbranched and becomes septate at maturity to form a series of elongate segments (Figs. 26, 30). These are mostly spindle-shaped to triangulate but may be ellipsoid to ovoid or occasionally irregular. Segments may become swollen and constricted at the septa although adjacent segments tend to remain attached to form a linear series (Figs. 31, 32). In small nematodes often a number of infection thalli develop simultaneously and these may completely fill the body of the host (Fig. 32). At maturity each segment becomes a sporangium. Sporangia measure 25-55 x 10-15 pm. At maturity the protoplasm inside each sporangium cleaves to form numerous non-motile spores which are ellipsoid to ovoid or sometimes irregular in shape because of mutual pressure (Figs. 33, 34). The spores measure 8-10 x 4-6 pm (Figs. 23, 35). Each sporangium produces a short broad evacuation tube about 8-10 pm broad and up to 25 p long (Figs. 24, 27, 33). Aplanospores may be expelled to the exterior through the evacuation tube but often remain inside the sporangia which produce them and are eventually released following disintegration of the host and sporangial walls (Fig. 34). Aplanospores may remain in a dormant state for prolonged periods. Under suitable conditions, however, such as the presence of abundant fresh nematode hosts, they change slowly into lens-shaped laterally biflagellate zoospores very similar in appearance to those of M. papillatum. This change may take place outside or inside the sporangium such that in some conditions zoospores may be seen inside the sporangia but they are apparently never the primary spore formed. Zoospores measure 8-10 pm long and encyst at any point on the host cuticle to form hemispherical cysts (Figs. 28, 29). Penetration is similar to that of M. papillatum. In older cultures instead of sporangial segments, the infection thallus may produce a series of spherical segments which become thick-walled chlamydospores (Figs. 24, 25, 36). The chlamydospores are 12-18 pm in diameter and are apparently the persistent phase of the fungus. No sexual state was found.
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NOTES
FIGS. 33-36. Myzocyfilrm atrotnalum. Fig. 33. Mature sporangia with broad, truncate evacuation tubes. Spores have been released from one of the sporangia. Fig. 34. Mature aplanospores inside the body of the host with some sporangial walls appearing to have disintegrated. Fig. 35. Aplanospores. Fig. 36. Young chlamydospores.
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Acknowledgments -
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The writer is indebted to the National Research for to carry out these studies and to Dr. V. J. Matthews for translating the diagnoses into Latin. It is a
pleasure to thank Jim Holley and Yvonne Dierkes for technical assistance. BARRON,G. L . , and J. G. PERCY.1975. Nematophagous fungi: a new Myzocyti~rm.Can. J. Bot. 53: 1306-1309. DANGEAR,, P. A. 1903. Recherches sur le developpernent dupCrithtcechezlesAscomycetes.Botaniste,9: 1-303.
Regulation of acetyl-CoA synthetase of Saccharomyces cerevisiael Deparrtnet~tof Microbiology, Miami University, Oxford, Ohio45056 Accepted December 3 I, 1975
C O L E M A NJ., S . , and J. K. BHATTACHARIEE. 1976. Regulation of acetyl-CoA synthetase of Saccharomyces cercvisiae. Can. J. Microbiol. 22: 762-764. Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure. T h e activity in vitro was inhibited significantly by NADPH, NADH, or AMP and to a lesser extent by NADP, NAD, o r ADP. Glutarnic acid and a-ketoglutaric acid were not inhibitory. T h e enzyme level was repressed when the cells were grown in a complex nutrient medium a s opposed t o the minimal medium. However, a glutamic acid auxotroph glrrl, when grown in excess glutamic acid, demonstrated a fivefold increase of acetyl-CoA synthetase. COLEMAN,J. S., e t J. K. BHATTACHARIEE. 1976. Regulation of acetyl-CoA synthetase of Saccharomyces cerevisine. Can. J. Microbiol. 22: 762-764. L'activitC d e I'acCtyl-coenzyme A synthetase ( E C 6.2.1. I) d e Sacchorotnyces cerevisioe a i t 6 dCterminCe par une methode d e dosage radioactive. L'activitC in vitro est inhibie d e f a ~ o n significative par le NADPH, le NADH ou I'AMP et a un moindre d e g r i par le NADP, le N A D o u I'ADP. L'acide glutamique et I'acide a-~Ctoglutariquen'inhibent pas I'activitC de I'enzyme. L e taux d'enzyme est rCprimi lorsque les cellules sont cultivies dans un milieu nutritif complexe mais non dans un milieu minimal. Cependant, lorsqu'un auxotrophe a 1'Cgard de I'acide glutamique glrtl est cultivi en prksence d'un e x c t s d'acide glutamique, le taux de I'acetyl-CoA synthetase augmente de cinq fois. [Traduit par le journal]
Several enzymes involved in lipogenesis and ketogenesis including acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) are inhibited by longchain fatty acid CoA esters (6, 8). Little is known about the nucleotide and amino acid regulation of ACS. Regulation of citrate synthase (EC 4.1.3.7) by nucleotides, glutamic acid, or aketoglutaric acid is well known (2, 9). We report here the regulation of ACS of Saccharomyces cerevisiae in vitro and in vivo by various nucleotides and glutamic acid, respectively. A prototrophic strain, X2180, and a glutamic acid auxoJroph, MO-1-9B (glul), of S. cerevisiae were used in this study (1, 3). Cell extract was prepared from late log phase culture by procedures described elsewhere (7). Regulation of 'Received September 23, 1975.
ACS in vitro was determined in extracts prepared from X2 180 grown in the minimal medium, composed of Difco yeast nitrogen base without amino acids, 0.67%, and glucose, 1%. For the regulation of the biosynthesis of ACS, extracts were prepared from X2180 grown in the minimal medium with different amounts of glucose as well as in the complex nutrient medium, composed of peptone, 0.6%; yeast extract, 0.4%; MgS04.7H20, 0.1%; KH2P04, 0.2%; and glucose, 4%. Since the mutant glul lacks aconitase activity and requires glutamic acid supplementation for growth (3), the specific effect of glutamic acid on the biosynthesis of ACS was determined in extracts prepared from MO-1-9B grown in the minimal medium with known concentrations of glutamic acid. The ACS activity was determined by a radio-
