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Long-chain fatty acids in spores of Penicillium
312
Transactions British Mycological Society
rings and depends entirely on knobs for capture of nematodes. Results were similar to D. candida except that the knobs were not as readily detached and as a result proportionately more nematodes were held fast to the knobs without breaking them off. Even in this species however a large number of nematodes had one or more detached knobs stuck to the cuticle as evidence of previous' escapes'. In some cases in D. haptotyla detached knobs did not penetrate the host cuticle but germinated in situ to produce a secondary knob on a short stalk (PI. 42, fig. 8). These knobs were also functional and occasionally nematodes could be seen swimming in tandem, held together by the two knobs with the stalk as a connective. It is probable that the knobs are detachable in other species of predaceous fungi. The occurrence of detachable knobs is a distinct advantage to the biological success of the parasite. A nematode can travel a significant distance on the micro scale before penetration and growth of the fungus incapacitates it. Thus the parasite is not only transported to a new site for further predation but arrives with an immediately available food source and can quickly produce additional trapping devices. Detachable knobs are obviously especially important for species which depend entirely on knobs for predation. The writer thanks the National Research Council of Canada for financial support. REFERENCES
DRECHSLER, C. (1937). Some hyphomycetes that prey on free-living terricolous nematodes. Mycologia 29, 447-552. DUDDINGTON, C. L. (1962). Predaceous fungi and the control of eelworms. Viewpoints in Biology I, 151-200. EXPLANATION OF PLATE
42
Fig. I. Dactylaria haptotyla. Captured nematode filled with hyphae of parasite. Fig. 2. D. haptotyla. Freshly caught nematode held at five locations. Fig. 3. D. haptotyla. Freshly caught nematode stilI struggling but held by knob in tail region. Note number of detached knobs attached to forward part of body indicating previous' escapes'. Fig. 4. Dactylaria candida. Nematode with more than twenty detached knobs adhering to cuticle. Fig. 5. D. candida. Detached knobs have penetrated cuticle and produced subcuticular swellings. Note deterioration of oesophageal musculature in vicinity of infection bulbs. Fig. 6. D. candida. Non-constricting ring. Fig. 7. D. candida. Free-swimming nematode with detached knobs adhering to cuticle. Fig. 8. D. haptotyla. Free-swimming nematode with detached knobs germinating to produce secondary knobs.
LONG-CHAIN FATTY ACIDS IN SPORES OF PENICILLIUM R. K. DART
Department ofChemistry, University of Technology, Loughborough, Leicestershire, U.K. The Penicillia can be divided into a number of subgroups (Raper & Thorn, 1949; Pitt, 1973) all of which have small spores usually lacking any Trans. Br. mycol, Soc. 65 (2), (1975). Printed in Great Britain
Tran s. Br. mycol. Soc.
Vol. 65.
Plate 42
(Facing p. 312)
Notes and Brief Articles
313
distinctive feature. A considerable amount of work has been carried out on th e commercial importance of Penicillium species but most authors studying the chemistry of this genus do not distinguish between mycelial products and spore products (K om an & Betina, 1972). The following species of Penicillium were used: Penicillium charlesii 4°232 , P. chrysogenum 2621 I, P. patulum 28808, P.lavendulum 4°57°, P. thomii 40027 and P. lanosooiride 398 I 9iL All the cultures were obtained from the Commonwealth Mycological Institute, Ferry Lane, Kew, Surrey. The organisms were grown on a semi-defined m edium which had been extensively defatted before use . The medium con tained in I I distilled water: glucose, 10 g; KN0 3 , 2'0 g; K aP0 4 , 2'0 g; NH 4NO a, 2'0 g; CaCI 2 , 0'25 g; yeast extract,s g; cas amino acids,s g; and trace metals solution, 0'1 ml. pH was adjusted to 5'0. Both yeast and cas amino acids were defatted with chloroform: diethyl ether 50/50 before use. The organisms were grown in static culture for 4 weeks at a temperature of 24°. At the end of this period a thick mat of mycelium covered with spores had grown over the surface of the medium in all cases. The entir e medium was tipped into a blender which was operated at full speed for 2 x lOS. This was found to be sufficient to release the spores from the mycelial mass. The mixture was then filtered twice through loose cotton-wool plugs. Microscopic examination of the filtrate showed a pure susp ension of spores. These wer e harvested by centrifugation, washed twice with distilled water and dried. The methyl esters of the fatty acids were prepared b y treating the spores with anhydrous methanol containing 2 % cone. H 2S0 4 for 24 h at 55 °. The mixture was then treated with an equal volume of saturated NaCl solution and extracted with an equal volume of n-hexane. The hexane fraction was reduced to a small volume which was used for gas-liquid chromatography. Gas-liquid chromatography was carried out on a Pye 104 using either a 10 % diethylene gl ycol succinate column at qoo or an Apiazon L column at 180°. Peaks were identified from their retention times and co-ch ro m a tography with authentic samples. Th e results are shown in Table I. The area of each peak on the gasliquid chromatography trace was determined by triangulation and the amount of each methyl ester was determined as a percentage of the total for each species under consideration. The mean value for each fatty acid was calculated and the standard deviation of each from the mean was determined. The major saturated fatty acids found were palmitic and stearic and the major unsaturated fatty acids were C I 8 : 1 ' C 18 : 2 , C I 8 : a. These three unsaturated fatty acids co-chromatographed with oleic acid, linoleic and a-linolenic acid, but the positions of the double bonds were not determined. In addition to the fatty acids shown in Table I, all species showed traces of n-C 14 , n-C 15 and C I 7 : 1 , with the exception of Penicillium chrysogenum which contained 0'4 % of C I 7 : 1 • Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain
Transactions British Mycological Society
314 Table
I.
Percentages iffatly acids in spores if Penicillium species with the standard deviation if each from the mean n-C 16
P. thomii
%
C16 : 1
n-C l1
n-C 18
C 18 : 1
C18: 2
C 16 : 3
Trace
7'3 -0'10
21'8 0'27
38'1 0'79
4',,) 0'47
2·8 -C'82
3 0'S 1'4 1
53'::l 0,67
0·8 -0·81
Trace
19'5 1·85
14'9 -0·64
33'0 -1'28
3'2 0'02
25"4 0'5 0
3'0
10'5 -1'53
0'7
%
29'4 1'05
Trace
%
28'S 0'93
1'0
1'0
9'4 0'23
II'S -1'08
4°'7 -0'54
8'0 1-69
%
18-6 -0'42
Trace
Trace
6'3 0'26
13-6 -0·81
59'2 1'23
2'3 -0'29
%
17,8 -0'53 21'7 7'34
Trace
Trace
2'4 -0·89
26'2 0·85 19'7 7,63
53.6 0'70 4 6'3 10'43
-1'09 3'13 2·88
Nov of s.n.s. P. chrysogenum
%
Nov of s.n.s
1'0
P. lavendulum
No.ofs.D.s P. lanosoviride No,ofs.D.S P. patulum No.ofs.D.S P. charlesii No.ofs,D.S
Av. % S.D.
7'9 6'26
The overall pattern found in the fatty-acid content of spores of Penicillium species is similar, even though there is considerable individual variation and the species come from several different subgroups (Pitt, 1973). In all species studied the major fatty acid is a C I 8 diene. Similar results have been found by other workers. The spores of Penicillium atrovenetum contained 65'7 % linoleic acid (Van Etten & Gottlieb, 1965). A similar pattern has been noticed in other genera, 63 % linoleic acid in the fatty acids of spores of Tilletia oetens (Tulloch & Ledingham, 1960) and 60 % linoleic acid in the fatty acids of the sclerotia of Sclerotium roifsii (Howell & Fergus, 1964). The yeast extract and cas amino acids were extensively defatted before use in the medium, suggesting that all fatty acids were synthesized de novo. In many groups offungi the spores have been shown to contain unusual fatty acids. Tulloch & Ledingham (1960, 1962) showed that spores of Uredinales contained up to 41 % of cis 9,10-epoxyoctadecanoic acid whilst spores of members of the Erysiphales contained nearly 42 % behenic (docosanoic) acid. No unusual fatty acids could be found in the spores of the Penicillium species tested, apart from trace amounts of odd numbered saturated and unsaturated fatty acids. REFERENCES
D. M. & FERGUS, C. L. (1964). The component fatty acids found in sclerotia of Sclerotium rolfsii. Canadian Journal of Microbiology IO, 616-618. KOMAN, V. & BETINA, V. (1972). Diphasic production of secondary metabolites by Penicillium notatum. Westling 5-52. Folia Microbiologic I8, 133-141. HOWELL,
Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain
Notes and Brief Articles PITT, ]. I. (1973). An appraisal of identification methods for Penicillium species. Novel taxonomic criteria based on temperature and water relations, Mycologia 65, I 1351157· RAPER, K. B. & THOM, C. (1949). Manual ofthe Penicillia. Baltimore: Williams & Wilkins. TULLOCH, A. P. & LEDINGHAM, G. A. (1960). The component fatty acids of oils found in spores of plant rusts and other fungi. Canadian Journal of Microbiology 6, 425-434. TULLOCH, A. P. & LEDINGHAM, G. A. (1962). The component fatty acids of oils found in spores of plant rusts and other fungi. Part II. Canadian Journal of Microbiology 8, 379-3 87. VAN ETTEN,]. L. & GOTTLIEB, D. (1965). Biochemical changes during the growth of fungi. II. Ergosterol and fatty acids in Penicillium atrovenetum. Journal of Bacteriology 89, 4°9-4 14.
GROWTH OF FIVE PHYTOPATHOGENIC FUNGI IN LIQUID MEDIA CONTAINING A URONIC ACID AS THE SOLE CARBOHYDRATE ANNE RATTIGAN AND P. G. AYRES
Department
if Biological Sciences, University of Lancaster
In the primary cell wall of plants the two major non-cellulosic fractions are the pectic and the hemicellulosic polysaccharides (Talmadge, Keegstra, Bauer & Albersheim, 1973). On hydrolysis the former yield galacturonic acid and the latter, glucuronic acid. The two groups have been recognized because different methods are used for their extraction, but this separation may not reflect a clear distinction in vivo. Studies on the production of non-cellulosic polysaccharide degrading enzymes by phytopathogenic fungi have usually examined only the ability of such enzymes to degrade model pectic substrates (Bateman & Millar, 1966). The emphasis of these studies has been upon the effect that the enzymes have on the structure of the cell wall of the host and the potential pathway for the growth of the fungus that this creates. The possibility that the fungus could utilize as a nutrient source the uronic acids released by the degradation of host cell walls has rarely been examined. We report here an experiment that examined the ability of five phytopathogenic fungi, known to produce pectic enzymes in vivo (see Table I for references), to grow when the only carbohydrate available to them was either glucuronic acid or galacturonic acid. Each fungus was grown in a liquid medium that previously (see Table I for references) had been found to support rapid growth. Cultures were grown in 100 ml conical flasks containing 40 ml of liquid medium. After growth for 2 I days at 23°C the mycelial mat was removed and aseptically washed in distilled water before being cut into 14 mm disks. Each disk was used to inoculate a flask of fresh medium the same composition as had been used before for that fungus except that the carbohydrates were replaced by 1'0,0'1 or 0'01 % (w/v) glucuronic acid or galacturonic acid (Sigma Chemical Corp.). The dry weight of the mycelial discs was Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain 21
MYC
65
Transactions British Mycological Society
rings and depends entirely on knobs for capture of nematodes. Results were similar to D. candida except that the knobs were not as readily detached and as a result proportionately more nematodes were held fast to the knobs without breaking them off. Even in this species however a large number of nematodes had one or more detached knobs stuck to the cuticle as evidence of previous' escapes'. In some cases in D. haptotyla detached knobs did not penetrate the host cuticle but germinated in situ to produce a secondary knob on a short stalk (PI. 42, fig. 8). These knobs were also functional and occasionally nematodes could be seen swimming in tandem, held together by the two knobs with the stalk as a connective. It is probable that the knobs are detachable in other species of predaceous fungi. The occurrence of detachable knobs is a distinct advantage to the biological success of the parasite. A nematode can travel a significant distance on the micro scale before penetration and growth of the fungus incapacitates it. Thus the parasite is not only transported to a new site for further predation but arrives with an immediately available food source and can quickly produce additional trapping devices. Detachable knobs are obviously especially important for species which depend entirely on knobs for predation. The writer thanks the National Research Council of Canada for financial support. REFERENCES
DRECHSLER, C. (1937). Some hyphomycetes that prey on free-living terricolous nematodes. Mycologia 29, 447-552. DUDDINGTON, C. L. (1962). Predaceous fungi and the control of eelworms. Viewpoints in Biology I, 151-200. EXPLANATION OF PLATE
42
Fig. I. Dactylaria haptotyla. Captured nematode filled with hyphae of parasite. Fig. 2. D. haptotyla. Freshly caught nematode held at five locations. Fig. 3. D. haptotyla. Freshly caught nematode stilI struggling but held by knob in tail region. Note number of detached knobs attached to forward part of body indicating previous' escapes'. Fig. 4. Dactylaria candida. Nematode with more than twenty detached knobs adhering to cuticle. Fig. 5. D. candida. Detached knobs have penetrated cuticle and produced subcuticular swellings. Note deterioration of oesophageal musculature in vicinity of infection bulbs. Fig. 6. D. candida. Non-constricting ring. Fig. 7. D. candida. Free-swimming nematode with detached knobs adhering to cuticle. Fig. 8. D. haptotyla. Free-swimming nematode with detached knobs germinating to produce secondary knobs.
LONG-CHAIN FATTY ACIDS IN SPORES OF PENICILLIUM R. K. DART
Department ofChemistry, University of Technology, Loughborough, Leicestershire, U.K. The Penicillia can be divided into a number of subgroups (Raper & Thorn, 1949; Pitt, 1973) all of which have small spores usually lacking any Trans. Br. mycol, Soc. 65 (2), (1975). Printed in Great Britain
Tran s. Br. mycol. Soc.
Vol. 65.
Plate 42
(Facing p. 312)
Notes and Brief Articles
313
distinctive feature. A considerable amount of work has been carried out on th e commercial importance of Penicillium species but most authors studying the chemistry of this genus do not distinguish between mycelial products and spore products (K om an & Betina, 1972). The following species of Penicillium were used: Penicillium charlesii 4°232 , P. chrysogenum 2621 I, P. patulum 28808, P.lavendulum 4°57°, P. thomii 40027 and P. lanosooiride 398 I 9iL All the cultures were obtained from the Commonwealth Mycological Institute, Ferry Lane, Kew, Surrey. The organisms were grown on a semi-defined m edium which had been extensively defatted before use . The medium con tained in I I distilled water: glucose, 10 g; KN0 3 , 2'0 g; K aP0 4 , 2'0 g; NH 4NO a, 2'0 g; CaCI 2 , 0'25 g; yeast extract,s g; cas amino acids,s g; and trace metals solution, 0'1 ml. pH was adjusted to 5'0. Both yeast and cas amino acids were defatted with chloroform: diethyl ether 50/50 before use. The organisms were grown in static culture for 4 weeks at a temperature of 24°. At the end of this period a thick mat of mycelium covered with spores had grown over the surface of the medium in all cases. The entir e medium was tipped into a blender which was operated at full speed for 2 x lOS. This was found to be sufficient to release the spores from the mycelial mass. The mixture was then filtered twice through loose cotton-wool plugs. Microscopic examination of the filtrate showed a pure susp ension of spores. These wer e harvested by centrifugation, washed twice with distilled water and dried. The methyl esters of the fatty acids were prepared b y treating the spores with anhydrous methanol containing 2 % cone. H 2S0 4 for 24 h at 55 °. The mixture was then treated with an equal volume of saturated NaCl solution and extracted with an equal volume of n-hexane. The hexane fraction was reduced to a small volume which was used for gas-liquid chromatography. Gas-liquid chromatography was carried out on a Pye 104 using either a 10 % diethylene gl ycol succinate column at qoo or an Apiazon L column at 180°. Peaks were identified from their retention times and co-ch ro m a tography with authentic samples. Th e results are shown in Table I. The area of each peak on the gasliquid chromatography trace was determined by triangulation and the amount of each methyl ester was determined as a percentage of the total for each species under consideration. The mean value for each fatty acid was calculated and the standard deviation of each from the mean was determined. The major saturated fatty acids found were palmitic and stearic and the major unsaturated fatty acids were C I 8 : 1 ' C 18 : 2 , C I 8 : a. These three unsaturated fatty acids co-chromatographed with oleic acid, linoleic and a-linolenic acid, but the positions of the double bonds were not determined. In addition to the fatty acids shown in Table I, all species showed traces of n-C 14 , n-C 15 and C I 7 : 1 , with the exception of Penicillium chrysogenum which contained 0'4 % of C I 7 : 1 • Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain
Transactions British Mycological Society
314 Table
I.
Percentages iffatly acids in spores if Penicillium species with the standard deviation if each from the mean n-C 16
P. thomii
%
C16 : 1
n-C l1
n-C 18
C 18 : 1
C18: 2
C 16 : 3
Trace
7'3 -0'10
21'8 0'27
38'1 0'79
4',,) 0'47
2·8 -C'82
3 0'S 1'4 1
53'::l 0,67
0·8 -0·81
Trace
19'5 1·85
14'9 -0·64
33'0 -1'28
3'2 0'02
25"4 0'5 0
3'0
10'5 -1'53
0'7
%
29'4 1'05
Trace
%
28'S 0'93
1'0
1'0
9'4 0'23
II'S -1'08
4°'7 -0'54
8'0 1-69
%
18-6 -0'42
Trace
Trace
6'3 0'26
13-6 -0·81
59'2 1'23
2'3 -0'29
%
17,8 -0'53 21'7 7'34
Trace
Trace
2'4 -0·89
26'2 0·85 19'7 7,63
53.6 0'70 4 6'3 10'43
-1'09 3'13 2·88
Nov of s.n.s. P. chrysogenum
%
Nov of s.n.s
1'0
P. lavendulum
No.ofs.D.s P. lanosoviride No,ofs.D.S P. patulum No.ofs.D.S P. charlesii No.ofs,D.S
Av. % S.D.
7'9 6'26
The overall pattern found in the fatty-acid content of spores of Penicillium species is similar, even though there is considerable individual variation and the species come from several different subgroups (Pitt, 1973). In all species studied the major fatty acid is a C I 8 diene. Similar results have been found by other workers. The spores of Penicillium atrovenetum contained 65'7 % linoleic acid (Van Etten & Gottlieb, 1965). A similar pattern has been noticed in other genera, 63 % linoleic acid in the fatty acids of spores of Tilletia oetens (Tulloch & Ledingham, 1960) and 60 % linoleic acid in the fatty acids of the sclerotia of Sclerotium roifsii (Howell & Fergus, 1964). The yeast extract and cas amino acids were extensively defatted before use in the medium, suggesting that all fatty acids were synthesized de novo. In many groups offungi the spores have been shown to contain unusual fatty acids. Tulloch & Ledingham (1960, 1962) showed that spores of Uredinales contained up to 41 % of cis 9,10-epoxyoctadecanoic acid whilst spores of members of the Erysiphales contained nearly 42 % behenic (docosanoic) acid. No unusual fatty acids could be found in the spores of the Penicillium species tested, apart from trace amounts of odd numbered saturated and unsaturated fatty acids. REFERENCES
D. M. & FERGUS, C. L. (1964). The component fatty acids found in sclerotia of Sclerotium rolfsii. Canadian Journal of Microbiology IO, 616-618. KOMAN, V. & BETINA, V. (1972). Diphasic production of secondary metabolites by Penicillium notatum. Westling 5-52. Folia Microbiologic I8, 133-141. HOWELL,
Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain
Notes and Brief Articles PITT, ]. I. (1973). An appraisal of identification methods for Penicillium species. Novel taxonomic criteria based on temperature and water relations, Mycologia 65, I 1351157· RAPER, K. B. & THOM, C. (1949). Manual ofthe Penicillia. Baltimore: Williams & Wilkins. TULLOCH, A. P. & LEDINGHAM, G. A. (1960). The component fatty acids of oils found in spores of plant rusts and other fungi. Canadian Journal of Microbiology 6, 425-434. TULLOCH, A. P. & LEDINGHAM, G. A. (1962). The component fatty acids of oils found in spores of plant rusts and other fungi. Part II. Canadian Journal of Microbiology 8, 379-3 87. VAN ETTEN,]. L. & GOTTLIEB, D. (1965). Biochemical changes during the growth of fungi. II. Ergosterol and fatty acids in Penicillium atrovenetum. Journal of Bacteriology 89, 4°9-4 14.
GROWTH OF FIVE PHYTOPATHOGENIC FUNGI IN LIQUID MEDIA CONTAINING A URONIC ACID AS THE SOLE CARBOHYDRATE ANNE RATTIGAN AND P. G. AYRES
Department
if Biological Sciences, University of Lancaster
In the primary cell wall of plants the two major non-cellulosic fractions are the pectic and the hemicellulosic polysaccharides (Talmadge, Keegstra, Bauer & Albersheim, 1973). On hydrolysis the former yield galacturonic acid and the latter, glucuronic acid. The two groups have been recognized because different methods are used for their extraction, but this separation may not reflect a clear distinction in vivo. Studies on the production of non-cellulosic polysaccharide degrading enzymes by phytopathogenic fungi have usually examined only the ability of such enzymes to degrade model pectic substrates (Bateman & Millar, 1966). The emphasis of these studies has been upon the effect that the enzymes have on the structure of the cell wall of the host and the potential pathway for the growth of the fungus that this creates. The possibility that the fungus could utilize as a nutrient source the uronic acids released by the degradation of host cell walls has rarely been examined. We report here an experiment that examined the ability of five phytopathogenic fungi, known to produce pectic enzymes in vivo (see Table I for references), to grow when the only carbohydrate available to them was either glucuronic acid or galacturonic acid. Each fungus was grown in a liquid medium that previously (see Table I for references) had been found to support rapid growth. Cultures were grown in 100 ml conical flasks containing 40 ml of liquid medium. After growth for 2 I days at 23°C the mycelial mat was removed and aseptically washed in distilled water before being cut into 14 mm disks. Each disk was used to inoculate a flask of fresh medium the same composition as had been used before for that fungus except that the carbohydrates were replaced by 1'0,0'1 or 0'01 % (w/v) glucuronic acid or galacturonic acid (Sigma Chemical Corp.). The dry weight of the mycelial discs was Trans. Br. mycol. Soc. 65 (2), (1975). Printed in Great Britain 21
MYC
65