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Cell wall composition of Fusarium oxysporum
.%ti &of. Btrrchrm. Vol. 21. No. 6, pp. (IV+-871. Prtnted in Gxtt B&tin. All nghtr reserved
I989
0038-0717,89 $3.00 + 0.00 Copyrtght c‘ 1989 Pcrgamon Press plc
SHORT COMMUNICATION CELL WALL COMPOSITION
OF FUSARIUM
ALEX SWAN+
OXYSPORUM
and ILAN CHET~
Department of Plant Pathology and Microbiology. The Faculty of Agriculture. The Hebrew University of Jerusalem, Rehovot 76100. Israel (Accepted IS Ju~ur_v 1989)
The morphology, physiology and ecology of Fusnrium os~spurum as a soil-borne plant pathogen have been described (Nelson et al., 1981). However, there are not suflicient data regarding the cell wall composition of Fusaria and particularly of F. o.r_vsporum. Fungal cell-wall structure contributes to the unique hyphal shape, the survival in soil and serves as the main barrier against microbial lysis in soil (Potgieter and Alexander, 1966: Chet cf al.. 1967). Cell walls of F. o.~.~spurunr were found to be more resistant to cell-wall degrading enzymes compared with those of Rhizocloniu sohi Kuhn and Sclerolium rorfii (Sivan and Chet, 1989). Therefore. our objective was to analyze the hyphal wall ~om~sition of F. u.~~.~~r~rn SchIecht f. sp. ffzelon~ Snyd. and Hans (E o. ~ie~o~i~)and F. os~s~orum f. sp. ~~~nfeef~ (Ark.) Snyd. and Hans (F. o. rasinfecrum) and to compare them with those of R. sofuni and S. rolfiii. The fungi were c&red on a yeast extract-glucose medium (YM) containing (g I-’ distilled water) yeast extract (Difco), 5; peplone (Difco). 5; glucose, IO; agar, 20 and were held at 27‘C. Hyphal walls were produced from 96 h-old mycelial mats grown in liquid YM (Chet and Huttcrmann. 1980). No residual glucose, protein and amino acids could bc detected in the supernatant of the washed cell walls as determined by glucose oxidase (Sigma). coomassie-brilliant blue (Sedmak and Grossberg, 1977) and Ninhydrin {Cocking and Yemm, 1954) reagents, respectively. Cell walls were deep-frozen and lyophilized. Cell wall preparations (20mg) of each fungus tested were se&vi in hydrolysis ampoules containing 2 ml of 2N H2S0, and heated al I05 C for 5 h. This resulted in the highest
*Present address: The Institutes of Applied Research, BenGurion University of The Ncgev, P.O. Box lO2S, BeerSheva S-11IO, Israel. tTo whom all correspondence should be addressed. T;lble
I
amount of released glucose as determined by the glucose-oxidase (Sigma) method according to the manufacturer instructions. The hydrolysates were neutralized with decreasing concentrations (2~. IN, 0.1~ and 0.05~) of NaOH. diluted to 25 ml and assayed for total carbohydrates (Dubois PI al., 1956). Hexosamines were determined (Elson and Morgan, 1933) in hydroysates neutralized with the same concentrations as above of hot Ba(OH). Amino acids were determined in 20mg cell-wall samples which were hydrolyzed in 6~ HCI for 24 h at 105°C. The hydrolysates were dried in a rotovapor and tested using a Biotronic amino acid analyzer. The protein content was determined from the amino acid profile by calculating the total amount of amino acids in cell wall hydrolyzates. Lipids were quantified according to Folch er al. (1957). The ash content weight was determined after heating the cell walls at 650’C. Infm-red spectra were obtained with a Nicolet IR-spectrophotometer using 2 mg of fungal cell wall powder which was mixed with 90 mg of KBr and pressed into a pellet. The results of the acid hydrolysis (Table I) showed a relatively high hexosamine content in cell walls of both Fusaria. The hexosamine content in cell walls of F. o. cvsinfecfum was 75.8% higher than in cell walls of F. o. meloink. On the other hand. cell walls of F. a melonis contained 94.4% more neutral sugars. These data correlate with other studies reporting the high content of N-acetyl-oglucosamine (45 and 39%) in cell walls of F. sold f. sp. p~f~~eo~i (Skujins e’tul.. 1965) and F. su~reum (Barran et al., 1975). respectively. Analysis of cell walls of R. solani and S. rogsii showed great differences between these and the Fusarial walls in almost any tested variable. Hyphal walls of R. solunni and S. ro@ii contained 57 and 58.5% neutral sugars and 6.1 and 5.4% of hexosamines, respectively. Similarly, previous reports demonstrated that most of the cell wall polysaccharides in S. ro&i (Chet CI u/.. 1967) and
Chemical composition of hyphal walls of F. o.r.~t~~urn f. sp. curiu~ectum;tnd E o.r.vs~orum f. sp. mehis as compttred with that of R. sohni and S. ro&ii Percentage of hyphal cell wall dry weight
Neutral sugars Henosamincs Proteins tipids Ash Non-hydrolyzed residue
19.5c 29.Oa 3.Sb 3.8b I .2b 32.8~1
37.9b 16Sb 5.&t 4.2b
Total
89.8
25. I b
.57.ou 6.k I .9d xc I.lb 8.4~
58.&t 5.4c t&c 7.5a l.la 12.2c
90.7
76.7
87.2
1.41
*In order to have a common basis for comparison with cell walls of F. o.r,~~~rurn cell wall analysis of S. d/ii was repeated in part after Chct er 01. (1967). Walues of each cell wall component followed by the same letter are not significantly different according to Duncan’s multiple range test (P = 0.05).
869
870
Short
Amino acid Aspartic acid Threonine Serine Glutamic acid Roline Glycine Alanine Cistine Valine Methioninc Leucine Tyrosinc Pheny~-alanine Histidine Lysine Arainine
‘g mg-’
wsinfecrum 2.41 3.15 I s2 3.52 2.74 1.14 &4
communications
melonis 0.65 0 0.8 0.75 0.96 0 0.69 2.92 1.87 I .08 0.66 0.48 0.73 23.2 0 0
3:17 0.77 1.32 1.18 0.8 29.6 0.5 I.1
dry wt of cell walls.
R. solani (Potgieter and Alexander, 1966) are glucans. Those studies, as well as others, also indicated the low percentages of chitin and protein in these fungi. Data provided by the amino acid analysis revealed that the protein content of cell walls of F. o~ysporum was up to 194 and 115%greater than those of R. solani and S. rolfsii, respectively, Other Fusaria aIso have high (‘i-28%) protein contents (Lamborda er ai.,
1974 Barran et at.. 1975; Schneider er al., 1977). Schneider et at. (1977) suggested that the high protein content in cell walls ofchlamydospores of F. sutfureum {21%) may increase resistance to microbial lysis in soil. Thus, F. o.~.vsporum may be less affected by indigenous or introduced mycolytic antagonists. The protein hydrolysate of cell walls of F. o. metonis and F. o. casinfeclum, comprised mainly of histidine flable 2), compared with 19 and 4.5% of this amino acid in cell walls of R. solani and S. rot/ii, respectively. Some of the chemical differences between cell walls of Fusarium and S. rolfii were confirmed by their IR spectra (Fig. I). The spectrum of chitin showed high absorbance at wavenumbers ranging from 1160to 1670 with peaks at: 1161.2,1317,l383. 1564 and 1666 which are characteristic to fungal chitin (Galat and Popowicz, 1978). However, this part of the IR spectrum showed a very low absorbance of laminarin. Cell walls of F. oxysporum had higher absorbance at wavenumbers 1317, 1383 and 1564 than these of S. rofiii. This indicates that Fusarial walls have a higher chitin-laminarin ratios and higher chitin contents compared to those of S. rotfii. In addition, cell walls of F. oxysporum were found to be highly resistant to sulphuric acid hydrolysis (Table 1). Presumably, there are different or additional bonds in or between polysaccharides in walls of F. oxysporum. Indeed, Chu and Alexander (1970) argued that cell walls of Fusarium consist of heterosaccharides which contribute to their resistance to lytic enzymes in soit. This, as well as the higher protein content, may explain the relatively high resistance of F. o.xyspmrn to Iytic enzymes (i.e. &I$-glucanase and chitinase) (Sivan and Chet. 1989). The high content ofchitin and protein in Fusarial walls should be taken into consideration when screening for antagonistic mycoparasites against this plant pathogen. Ac~nowle~gemenrs-This research was supported by a grant from the Binational Agricultural Research and Development Fund U.S.A.-Israel. We gratefully acknowledge the help in IR-spectrophotometry of L. Margulies, the critical suggestions of Y. Hadar and the technical assistance of J. Inbar.
REFERENCES
Barran L. R., Schneider E. F., Wood P. J., Madhosingh C. and Miller W. R. (1975) Ceil wall of Fusarium su/phureum. I. Chemical com~sition of the hyphal wall. ~io~hirnt~a ef Biophysiea Acta 392, 146156. Cher I. and Huttermann A. (1980) Chemical composition of hyphal walls of Fames annosus. European Journal of Forest Pathology
IO, 65-70.
Chet I.. Henis Y. and Mitchell R. (1967) Chemical composition of hyphal and sclerotial walls of ScteroGum rotfii Sacc. Canadian Journal o/ Microbiology 13, 137-14 I, Chu S. B. and Alexander M. (1970) Resistance and susceptibility of fungal spores to lysis. Transaclions of rhe British .\lvcologicat
Society 58, 489-492.
Cocking E. C. and Yemm E. W. (1954) Estimation of amino acids by ninhydrin. Biochemical Journal 58, xii. Dubois M., Gillies K. A., Hamilton S. K., Rebers P. A. and Smith F. (1956) CoIorimetric method for determination of sugars and related substances. 3iochemicat Journal 28. 350-3.56.
uxo
XQO
2000 WAVENUMBER
,
I
WOO
Fig. I. IR spectra of KBr discs of chitin (Sigma) (A) and laminarin (Sigma) (B) as compared with hyphal walls of F. o.~ysporum f. sp. oasinfectum (C) and S. ro(jsii (D).
Elson L. A. and Morgan W. T. (1933) A cotorimetric method for the determination of glucosamine and condrosdamine. Biochemical Journal 27. 1824-1826. Folch J., Lees M. and Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemisrry 226, 497-509.
Galat A. and Popowicz J. (1978) Study of the infrared spectra of chitins. Butterin De L’Academie Polonaise des Sciences 28, 195-300.
871
Short cornlmunications Lamborda F.. Garcia-Acha I.. Uruburu F. and Villanueva J. R. (1974) Structure of the conidial wall of 1Cusurium culmorum. Transactions of rhe British M_vcologicai Society 62, 557-566. Nelson P. E., Tousson T. A. and Cook R. J. (1981) Fusarium: Diseases, Bioiogv and Taxonomy. Pennsylvania
State University Press, University Park. Potgieter H. J. and Alexander M. (1966) Susceptibility and resistance of several fungi to microbial lysis. lournal of Bucterio!ogy 91, 1526-l 532. Schneider E. F., Barran L. R.. Wood P. J. and Siddiqui I. R. (1977) Cell wall of Fusurium suiphureum II. Chemical
composition
of the conidial and chlamydospore
walls.
Canadian Journal of Microbiology 23. 763-769.
Sedmak J. J. and Grossberg S. E. (1977) A rapid, sensitive and versatile assay for protein using coomassie brilliant blue G250. Analyrical Biochemistry 79, 544-552. Sivan I. and Chet I. (1989) Degradation of fungal cell walls by lytic enzymes of Trichoderma hurziunum. Journal of General Microbiology In press. Skujins J. J.. Potgieter H. J. and Alexander M. (1965) Dissolution of fungal cell walls by a streptomycete chitinase and jl-(I-3)-glucanase. Arch&es of Biochemistry and Biophysics llf,
356-364.
I989
0038-0717,89 $3.00 + 0.00 Copyrtght c‘ 1989 Pcrgamon Press plc
SHORT COMMUNICATION CELL WALL COMPOSITION
OF FUSARIUM
ALEX SWAN+
OXYSPORUM
and ILAN CHET~
Department of Plant Pathology and Microbiology. The Faculty of Agriculture. The Hebrew University of Jerusalem, Rehovot 76100. Israel (Accepted IS Ju~ur_v 1989)
The morphology, physiology and ecology of Fusnrium os~spurum as a soil-borne plant pathogen have been described (Nelson et al., 1981). However, there are not suflicient data regarding the cell wall composition of Fusaria and particularly of F. o.r_vsporum. Fungal cell-wall structure contributes to the unique hyphal shape, the survival in soil and serves as the main barrier against microbial lysis in soil (Potgieter and Alexander, 1966: Chet cf al.. 1967). Cell walls of F. o.~.~spurunr were found to be more resistant to cell-wall degrading enzymes compared with those of Rhizocloniu sohi Kuhn and Sclerolium rorfii (Sivan and Chet, 1989). Therefore. our objective was to analyze the hyphal wall ~om~sition of F. u.~~.~~r~rn SchIecht f. sp. ffzelon~ Snyd. and Hans (E o. ~ie~o~i~)and F. os~s~orum f. sp. ~~~nfeef~ (Ark.) Snyd. and Hans (F. o. rasinfecrum) and to compare them with those of R. sofuni and S. rolfiii. The fungi were c&red on a yeast extract-glucose medium (YM) containing (g I-’ distilled water) yeast extract (Difco), 5; peplone (Difco). 5; glucose, IO; agar, 20 and were held at 27‘C. Hyphal walls were produced from 96 h-old mycelial mats grown in liquid YM (Chet and Huttcrmann. 1980). No residual glucose, protein and amino acids could bc detected in the supernatant of the washed cell walls as determined by glucose oxidase (Sigma). coomassie-brilliant blue (Sedmak and Grossberg, 1977) and Ninhydrin {Cocking and Yemm, 1954) reagents, respectively. Cell walls were deep-frozen and lyophilized. Cell wall preparations (20mg) of each fungus tested were se&vi in hydrolysis ampoules containing 2 ml of 2N H2S0, and heated al I05 C for 5 h. This resulted in the highest
*Present address: The Institutes of Applied Research, BenGurion University of The Ncgev, P.O. Box lO2S, BeerSheva S-11IO, Israel. tTo whom all correspondence should be addressed. T;lble
I
amount of released glucose as determined by the glucose-oxidase (Sigma) method according to the manufacturer instructions. The hydrolysates were neutralized with decreasing concentrations (2~. IN, 0.1~ and 0.05~) of NaOH. diluted to 25 ml and assayed for total carbohydrates (Dubois PI al., 1956). Hexosamines were determined (Elson and Morgan, 1933) in hydroysates neutralized with the same concentrations as above of hot Ba(OH). Amino acids were determined in 20mg cell-wall samples which were hydrolyzed in 6~ HCI for 24 h at 105°C. The hydrolysates were dried in a rotovapor and tested using a Biotronic amino acid analyzer. The protein content was determined from the amino acid profile by calculating the total amount of amino acids in cell wall hydrolyzates. Lipids were quantified according to Folch er al. (1957). The ash content weight was determined after heating the cell walls at 650’C. Infm-red spectra were obtained with a Nicolet IR-spectrophotometer using 2 mg of fungal cell wall powder which was mixed with 90 mg of KBr and pressed into a pellet. The results of the acid hydrolysis (Table I) showed a relatively high hexosamine content in cell walls of both Fusaria. The hexosamine content in cell walls of F. o. cvsinfecfum was 75.8% higher than in cell walls of F. o. meloink. On the other hand. cell walls of F. a melonis contained 94.4% more neutral sugars. These data correlate with other studies reporting the high content of N-acetyl-oglucosamine (45 and 39%) in cell walls of F. sold f. sp. p~f~~eo~i (Skujins e’tul.. 1965) and F. su~reum (Barran et al., 1975). respectively. Analysis of cell walls of R. solani and S. rogsii showed great differences between these and the Fusarial walls in almost any tested variable. Hyphal walls of R. solunni and S. ro@ii contained 57 and 58.5% neutral sugars and 6.1 and 5.4% of hexosamines, respectively. Similarly, previous reports demonstrated that most of the cell wall polysaccharides in S. ro&i (Chet CI u/.. 1967) and
Chemical composition of hyphal walls of F. o.r.~t~~urn f. sp. curiu~ectum;tnd E o.r.vs~orum f. sp. mehis as compttred with that of R. sohni and S. ro&ii Percentage of hyphal cell wall dry weight
Neutral sugars Henosamincs Proteins tipids Ash Non-hydrolyzed residue
19.5c 29.Oa 3.Sb 3.8b I .2b 32.8~1
37.9b 16Sb 5.&t 4.2b
Total
89.8
25. I b
.57.ou 6.k I .9d xc I.lb 8.4~
58.&t 5.4c t&c 7.5a l.la 12.2c
90.7
76.7
87.2
1.41
*In order to have a common basis for comparison with cell walls of F. o.r,~~~rurn cell wall analysis of S. d/ii was repeated in part after Chct er 01. (1967). Walues of each cell wall component followed by the same letter are not significantly different according to Duncan’s multiple range test (P = 0.05).
869
870
Short
Amino acid Aspartic acid Threonine Serine Glutamic acid Roline Glycine Alanine Cistine Valine Methioninc Leucine Tyrosinc Pheny~-alanine Histidine Lysine Arainine
‘g mg-’
wsinfecrum 2.41 3.15 I s2 3.52 2.74 1.14 &4
communications
melonis 0.65 0 0.8 0.75 0.96 0 0.69 2.92 1.87 I .08 0.66 0.48 0.73 23.2 0 0
3:17 0.77 1.32 1.18 0.8 29.6 0.5 I.1
dry wt of cell walls.
R. solani (Potgieter and Alexander, 1966) are glucans. Those studies, as well as others, also indicated the low percentages of chitin and protein in these fungi. Data provided by the amino acid analysis revealed that the protein content of cell walls of F. o~ysporum was up to 194 and 115%greater than those of R. solani and S. rolfsii, respectively, Other Fusaria aIso have high (‘i-28%) protein contents (Lamborda er ai.,
1974 Barran et at.. 1975; Schneider er al., 1977). Schneider et at. (1977) suggested that the high protein content in cell walls ofchlamydospores of F. sutfureum {21%) may increase resistance to microbial lysis in soil. Thus, F. o.~.vsporum may be less affected by indigenous or introduced mycolytic antagonists. The protein hydrolysate of cell walls of F. o. metonis and F. o. casinfeclum, comprised mainly of histidine flable 2), compared with 19 and 4.5% of this amino acid in cell walls of R. solani and S. rot/ii, respectively. Some of the chemical differences between cell walls of Fusarium and S. rolfii were confirmed by their IR spectra (Fig. I). The spectrum of chitin showed high absorbance at wavenumbers ranging from 1160to 1670 with peaks at: 1161.2,1317,l383. 1564 and 1666 which are characteristic to fungal chitin (Galat and Popowicz, 1978). However, this part of the IR spectrum showed a very low absorbance of laminarin. Cell walls of F. oxysporum had higher absorbance at wavenumbers 1317, 1383 and 1564 than these of S. rofiii. This indicates that Fusarial walls have a higher chitin-laminarin ratios and higher chitin contents compared to those of S. rotfii. In addition, cell walls of F. oxysporum were found to be highly resistant to sulphuric acid hydrolysis (Table 1). Presumably, there are different or additional bonds in or between polysaccharides in walls of F. oxysporum. Indeed, Chu and Alexander (1970) argued that cell walls of Fusarium consist of heterosaccharides which contribute to their resistance to lytic enzymes in soit. This, as well as the higher protein content, may explain the relatively high resistance of F. o.xyspmrn to Iytic enzymes (i.e. &I$-glucanase and chitinase) (Sivan and Chet. 1989). The high content ofchitin and protein in Fusarial walls should be taken into consideration when screening for antagonistic mycoparasites against this plant pathogen. Ac~nowle~gemenrs-This research was supported by a grant from the Binational Agricultural Research and Development Fund U.S.A.-Israel. We gratefully acknowledge the help in IR-spectrophotometry of L. Margulies, the critical suggestions of Y. Hadar and the technical assistance of J. Inbar.
REFERENCES
Barran L. R., Schneider E. F., Wood P. J., Madhosingh C. and Miller W. R. (1975) Ceil wall of Fusarium su/phureum. I. Chemical com~sition of the hyphal wall. ~io~hirnt~a ef Biophysiea Acta 392, 146156. Cher I. and Huttermann A. (1980) Chemical composition of hyphal walls of Fames annosus. European Journal of Forest Pathology
IO, 65-70.
Chet I.. Henis Y. and Mitchell R. (1967) Chemical composition of hyphal and sclerotial walls of ScteroGum rotfii Sacc. Canadian Journal o/ Microbiology 13, 137-14 I, Chu S. B. and Alexander M. (1970) Resistance and susceptibility of fungal spores to lysis. Transaclions of rhe British .\lvcologicat
Society 58, 489-492.
Cocking E. C. and Yemm E. W. (1954) Estimation of amino acids by ninhydrin. Biochemical Journal 58, xii. Dubois M., Gillies K. A., Hamilton S. K., Rebers P. A. and Smith F. (1956) CoIorimetric method for determination of sugars and related substances. 3iochemicat Journal 28. 350-3.56.
uxo
XQO
2000 WAVENUMBER
,
I
WOO
Fig. I. IR spectra of KBr discs of chitin (Sigma) (A) and laminarin (Sigma) (B) as compared with hyphal walls of F. o.~ysporum f. sp. oasinfectum (C) and S. ro(jsii (D).
Elson L. A. and Morgan W. T. (1933) A cotorimetric method for the determination of glucosamine and condrosdamine. Biochemical Journal 27. 1824-1826. Folch J., Lees M. and Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemisrry 226, 497-509.
Galat A. and Popowicz J. (1978) Study of the infrared spectra of chitins. Butterin De L’Academie Polonaise des Sciences 28, 195-300.
871
Short cornlmunications Lamborda F.. Garcia-Acha I.. Uruburu F. and Villanueva J. R. (1974) Structure of the conidial wall of 1Cusurium culmorum. Transactions of rhe British M_vcologicai Society 62, 557-566. Nelson P. E., Tousson T. A. and Cook R. J. (1981) Fusarium: Diseases, Bioiogv and Taxonomy. Pennsylvania
State University Press, University Park. Potgieter H. J. and Alexander M. (1966) Susceptibility and resistance of several fungi to microbial lysis. lournal of Bucterio!ogy 91, 1526-l 532. Schneider E. F., Barran L. R.. Wood P. J. and Siddiqui I. R. (1977) Cell wall of Fusurium suiphureum II. Chemical
composition
of the conidial and chlamydospore
walls.
Canadian Journal of Microbiology 23. 763-769.
Sedmak J. J. and Grossberg S. E. (1977) A rapid, sensitive and versatile assay for protein using coomassie brilliant blue G250. Analyrical Biochemistry 79, 544-552. Sivan I. and Chet I. (1989) Degradation of fungal cell walls by lytic enzymes of Trichoderma hurziunum. Journal of General Microbiology In press. Skujins J. J.. Potgieter H. J. and Alexander M. (1965) Dissolution of fungal cell walls by a streptomycete chitinase and jl-(I-3)-glucanase. Arch&es of Biochemistry and Biophysics llf,
356-364.