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Induction of Sclerotium formation by acid staling compounds in Sclerotinia sclerotiorum and Sclerotium rolfsii
[ 413 ] Trans. Br. mycol. Soc. 68 (3) 413-420 (1977)'
Printed in Great Britain
INDUCTION OF SCLEROTIUM FORMATION BY ACID STALING COMPOUNDS IN SCLEROTINIA SCLEROTIORUM AND SCLEROTIUM ROLFSII By F. M. HUMPHERSON-JONES* AND R. C. COOKE Botany Department, The University, Sheffield Sterile medium staled by the growth of Sclerotinia sclerotiorum (Lib .) de Bary and Sclerotium rolfsii Sacco enhanced sclerotiwn production when applied to test colonies, the active components being acidic and heat stable. In S. sclerotiorum, sclerotium production appeared to be associated with secretion of organic acids, and two unidentified acids with morphogenetic properties were isolated from staled medium. In culture fungal hyphae usually grow in an environment which is modified by metabolites diffusing from them, and many of these substances can induce morphogenetic changes in the hyphae that are producing them. Stimulation of sclerotiwn formation by such compounds has been implicated in Sclerotinia sclerotiorum (Lib .) de Bary and Sclerotium rolfsii Sacco (Bedi, 1958 j Liu & Wu, 1971). However, it is possible that, particularly in the case of S. rolfsii, such stimulation could be due to the accumulation of morphogenetically active metabolites within the hyphae during growth rather than in the surrounding mediwn (Wheeler & Waller, 1965; Goujon, 1970). As well as there being confusion with respect to the location of accumulating morphogenetic compounds, their possible nature is generally unknown, although thermolabile morphogenetic factors have been detected in cell-free extracts of S. rolfsii (Gouion, 1970). There are also indications that organic acids might in some way be involved (Vega, Corsini & Le Tourneau, 1970). When S. sclerotiorum was grown in a liquid medium with glucose as sole carbon source sclerotia were formed, the pH of the medium fell rapidly, and this was accompanied by the appearance of oxalate, malate, succinate, fumarate and glycolate in culture filtrates. When mannitol was the sole carbon source no sclerotia were produced, the pH of the mediwn increased, and only oxalate, malate and glycolate were found in culture filtrates . These observations have been taken to mean that there is a relationship between pH, organic acid synthesis and sclerotiwn production. The work described here was carried out to ob-
* Present address: National Vegetable Research Station, Wellesbourne, Warwick.
tain more detailed information on the morphogenetic activity of staled media and to determine which staling compounds could induce sclerotium formation. MATERIALS AND METHODS
Fungi and media Five isolates of Sclerotinia sclerotiorum (Lib .) de Bary and one each of Sclerotium rolfsii Saccoand Sclerotium delphinii Welch were used. Four isolates of S. sclerotiorum were derived from single ascospores while the fifth (NS 3) was obtained from a single hyphal tip excised from a colony growing on agar . The morphological characteristics of these isolates are summarized in Table 1S. sclerotiorum was maintained on 2 % GAY agar, comprising glucose, 20 g; asparagine , 2 g; yeast extract, 1 g; KH 2POU 1 g; KCI, 0'5 g; MgS04 .7H20, 0'5 g; FeS0 4, 0'01 g; agar 20 g; distilled water 1 1. S . rolfsii and S. delphinii were grown on 2 % 'Oxoid' malt extract agar and homemade potato dextrose agar respectively (250 g potato tissue boiled in 500 ml tap water for 20 min, filtered, supplemented with 20 g glucose, made up to 1 I and solidified with 20 g agar). Where liquid media were used the agar was omitted. Effect of staled medium S. rolfsii, S. delphinii and isolates B1-7 and NS 3 of S. sclerotiorum were grown in 100 ml of their respective liquid media at 25°. Three replicate flasks were used for each fungus and inoculum for each flask comprised 25 pI of homogenized myceliwn obtained from a stock colony grown in liquid med ium. At the time of appearance of sclerotium initials (6 days growth for S . sclerotiorum and 10 days growth for the two Sclerotium
Sclerotium formation Table
1.
Origin and morphological chara cteristics of isolates of Sclerotinia sclerotiorum, Sclerotium rolfsii and S. delphinii
Origin Sclerotiniasclerotiorum Isolate Bt-7 Phaseolus vulgaris Isolates Bt-t5 Pt-tO Isolate Bt-46 Isolate NS 3
P. vulgaris Pisum satioum
P. vulgaris P. vulgaris
Sclerotium ro/fsii
Pennisetum purpureum
S . delphinii
Unknown
Characteristics Rapid growth, colony flat, abundant sclerotia scattered over surface Rapid growth, dense mycelium, somewhat floccose, few sclerotia formed Slow growth, sclerotia not formed Rapid growth, abundant mycelium, very floccose, sclerotia not formed Rapid growth, colony flat, abundant sclerotia scattered over surface Rapid growth, colony flat, numerous sclerotia formed towards margin of colony
species) stale media were collected and filtered through Whatman No. 1 filter paper. For isolate NS 3, the medium was collected when initials formed in isolate Bl-7. Media for each fungus were bulked and stored at - 10°. When required, a suitable amount was thawed at 5°, its pH was adjusted to 4'5 with either N-KOH or N-HCI, and it was then sterilized by passage through a Millipore disc of pore size 0'45 pm . The sclerotium-inducing capacity of staled media was then assessed. Medium from the sclerotial isolate Bl-7 of S. sclerotiorum was tested against colonies of Bl-7 itself, Bl-15, Pr-so and NS 3; medium from the entirely mycelial isolate NS 3 was tested against isolate Bl-7 and Bl-15. Medium from each Sclerotium species was tested against all of the Sclerotium isolates. Fungi to be used in bioassays were grown on 20 ml of the appropriate agar in 9 em diam Petri dishes and in 20 ml of liquid medium in 100 ml conical flasks. Inoculum for the flasks comprised homogenized mycelium. Petri dishes were each inoculated centraUy with a 1 cm diam disk cut from the margin of a 4-day-old stock colony growing on agar. For each fungus six replicate flasks and six replicate dishes were used and all were incubated at 25°. When a mycelial mat had formed in liquid cul.ture, 6 ml of prepared, staled medium was injected through three mats of each fungus using a sterile hypodermic syringe. The remaining three mats were used as controls, being similarly treated with 6 ml of one quarter strength unstaled medium. All flasks were then incubated at 25° for 23 days at which time sclerotia were counted, and, where possible, weighed after drying at 90° for 24 h . Assays using agar-grown colonies were carried out when these were 6-7 em diam. A peripheral ring of agar 1 em wide was removed from each of three dishes of each species and 6 ml of stale
medium was introduced into each of the trenches so formed. The remaining three dishes of each fungus were used as controls, and were similarly treated using 6 ml of sterile, quarter strength unstaled medium. All dishes were incubated at 25° for 23 days at the end of which period they were examined and the number of sclerotia counted. Fra ctionation of medium staled by S. sclerotiorum and S. rolfsii
Fractionation of 70 ml of medium staled by either isolate Bl-7 of S. sclerotiorum or S. rolfsii was carried out using 10 x o-B em columns of Amberlite anion- and cation-exchange resins IRA 400 and IR 120 prepared and used according to Bryant & Overell (1953). The volumes of the acidic, neutral, and basic fractions obtained were adjusted to 70 ml and their pH was then brought to 4'5 using either N-KOH or N-HCl. They were then sterilized by filtration and their morphogenetic activity was assayed on agar-grown colonies as described above. For fractions from Bl-7 medium, the weakly-sclerotial isolates of S. sclerotiorum Bl-15 and Pr-uo were used as test fungi, while fractions from S. rolfsii medium were tested against S. rclfsii. Organic acid production and sclerotium formation by S. sclerotiorum
The sclerotial isolate Bl-7 and the non-sclerotia! isolate NS 3 were grown on 20 ml of GAY liquid at 25° in 100 ml conical flasks. At intervals over a 24 day period three replicate flasks of each isolate were taken, the pH of the medium was measured, and the mycelial dry weight was determined after drying at 90° for 24 h. In a parallel experiment the same two isolates were grown in GAY liquid from which the yeast extract had been omitted. After 8 days incubation
F. M. Humpherson-Jones and R. C. Cooke Table 2, Effect of staled medium from a sclerotial isolate (B1-7) of S. sclerotiorum on weakly- or nonsclerotial isolates of the same species, Each figure is the average from three replicates Total sclerotia No. of sclerotia dry wt. per colony per Bioassay on (mg) colony agar-grown colonies Bl-7 Unstaled medium 60'7 4'7 126'2* Staled medium 17'3* o o NS 3 Unstaled medium o o Staled medium 12 '3 Bl-15 Unstaled medium 71'4 206'2* 21'7* Staled medium Pr -ao Unstaled medium 11'3 45'9 101'6* Staled medium 20'7* Bioassay on liquid-grown colonies Bl-7 Unstaled medium Staled medium NS 3 Unstaled medium Staled medium Bl-15 Unstaled medium Staled medium Pi -ro Unstaled medium Staled medium * Significant (P
415
treatment in an equilibrated steam cabinet for 3 h. Chromatograms were developed by spraying with either bromophenol blue, ammoniacal silver nitrate, ammonium vanadate, or acetic anhydride pyridine (Ranson, 1955). The morphogenetic activity of sterile, aqueous solutions (0'1 % w/v) of a range of known organic acids was assayed using agar grown colonies of the weakly sclerotial isolates B1-15 and P1-10. Acids occurring in staled media were similarly assayed after having been eluted in distilled water from line-loaded chromatograms which had been run using butanol: formic acid: water, and had then been cut into strips , The amount of these acids applied to each test colony (in 6 ml of distilled water) was approximately equivalent to that present in 6 ml of the original staled medium.
RESULTS
10'7 16'3* o 150 * (approx.) 22'3 20'7 17'7 12'7 =
226'3 206'1 o Not determined 113'8
121 '6 161'4 173'2
0'01).
(at the point when initials were forming) and after 14 days (when sclerotia were mature) medium from six flasks of each isolate was collected, and replicate filtrates were bulked. Non-volatile organic acids were purified from filtrates using a 10 x 0,8 em Amberlite IRA 400 anion-exchange column in the CO a form. Filtrates were passed through at a rate of 0'5 ml per min, the resin bed was then washed with 50 ml distilled water and acids were eluted from the column using 500 ml N(NH4)2COa, The eluate was evaporated to dryness under vacuum at 35° and the ammonium salts obtained were dissolved in 50 ml distilled water, Free acids were regenerated from this solution by passage through a column of Amberlite IR 120 cation-exchange resin, in the H + form (Bryant & Overell, 1953). Acids were identified by one-dimensional descending paper chromatography using Whatman No.1 paper and either butanol : formic acid: water (100: 30: 100 by vol.) or ethyl ether : acetic acid: water (150 :30: 10 by vol.), Solvent was removed from chromatograms by air-drying for 1-2 hand
Effect of staled medium Staled medium from the non-sclerotial isolate, NS 3, of S . sclerotiorum had no detectable effect on either sclerotium number or the dry weight of sclerotium tissue produced in the two isolates, B1-7 and Bl-15, used in the biossay. Results from the remaining bioassays are given in Tables 2 and 3. Agar-grown colonies of isolates Bl-7, Bl-5 and Pr-uo, when treated with staled medium from the sclerotial isolate B1-7, produced sclerotia 1-2 days sooner than control colonies. In addition, both the final number of sclerotia formed and their dry weight yield increased by exposure to staled medium. No morphogenetic effects were observed in isolate NS 3, In liquid-grown colonies, treatment with staled medium had a similar effect only on Bl-7, where sclerotium numbers were increased but not their dry weight yield, and on the non-sclerotial isolate NS 3 which, in contrast to its behaviour when growing on agar, produced many small, immersed sclerotia 1 mm diam within 5-7 days of exposure to staled medium (Figs. 1, 2). Staled medium of S. rolfsii increased sclerotium number and dry weight yield in agar- and liquidgrown colonies of both S, rolfsii and S. delphinii (Table 3). In S. delphinii sclerotia appeared within 48 h of exposure to staled med ium, 6-7 days earlier than in control colonies, while in S. rolfsii they were formed 2-3 days earlier than in controls. Medium from S. de/phinii had no stimulatory effect on either S. delphinii or S. rolfsii but reduced the dry weight yield of sclerotia in S. de/phinii. Autoclaving staled media did not reduce their sclerotium- inducing properties.
Sclerotium formation
416
Table 3. Effect of staled medium from S. rolfsii or S. delphinii on sclerotium production Each figure is the average from three replicates Medium from S. delphinii
Medium from S. rolfsii
J-
Bioassay on agar-grown colonies S . rolfsii Unstaled medium Staled medium S. delphinii Unstaled medium Staled medium Bioassay on liquid-grown colonies S. rolfsii Unstaled medium Staled medium S. delphinii Unstaled medium Staled medium
Number of sclerotia per colony
Total sclerotium dry wt. per colony (mg)
Number of sclerotia per colony
Total sclerotium dry wt, per colony (mg)
117'0 164'7*
96'4 132'6*
129'3 119'7
84'S 89'3
7'0 18'3*
64'2 120'8*
9'3 8'7
59'8 69'9
66'7 84'7
59'2 78,6*
53'3 49'7
47'2 45'8
61'1 5'0 11'3* 92 '5* * Significant (P = 0'01).
9'3 8'3
78'1 5 1'6*
Table 4. Effect of staled medium and medium fractions on sclerotium production Each figure is the mean from five replicates Control
Stale medium
Acid fraction
Medium staled by S, sclerotiorum (B1- 7) Test isolate P1-10 Number of sclerotia 8'2 21'6* 14'2* per colony Av. dry wt. sclerotia 38'9 103'2* 61'8* per colony (mg) Bl-15 Number of sclerotia 9,6 18'2* 12,8 per colony 72'1 173'6* 103'6* Av. dry wt. sclerotia per colony (mg) Medium staled by S. rolfsii Test isolate S. rolfsii Number of sclerotia 86'2 129'9* 65'2* per colony 61'8 109'6* 60'1 Av. dry wt. sclerotia per colony (mg) * Significantly different from corresponding control (P Effect of fractions from staled medium
The effects of acidic, neutral and basic fractions from media staled by S, sclerotiorum (B1-7) and S. rolfsii are given in Table 4. Although complete
Neutral fraction
Basic
fraction
9'8 42'6
7'4
12'4
93'8
69'4 58'6 =
0'01) .
staled medium from S . rolfsii had a marked effect on sclerotium formation, none of the three fractions were stimulatory, and the acid fraction reduced sclerotium numbers significantly in test colonies, In contrast, the acid fraction from
F. M. Humpherson-Jones and R. C. Cooke
417
2 Fig. Fig.
15
1. 2.
Mycelial mat of non-sclerotial isolate NS 3 grown on untreated liquid medium . Mycelial mats of isolate NS 3 grown on liquid medium containing filtrate from a 6-day-old liquid culture of the sclerotial isolate B1-7.
MYC
68
Sclerotium formation
4 18
300
81-7 6
200
5
4
l1J
100
3
bO
'oj
5
1: Ci3
.Eeo
<-
.;:;
0
~
:I:
...»
0.
7.
"0
~ -;:; (J »
NS3
~
6
200
5
4
100
3
2
4
6
8
10 12 14 16 Growth period (days)
18
20
22
Fig. 3. Changes in mycelial dry weight (-0-) and pH of medium (--e-) during growth of a sclerotial isolate,Bl-7, and a non-sclerotial isolate,NS 3, of S. sclerotiorum. The broken line indicates the point at which initials appeared. medium staled by S. sclerotiorum stimulated sclerotium production, although to a lesser extent than did whole, staled medium. It also increased dry weight yield of sclerotia in isolate Bt-t5, but the slight rise in the number of sclerotia produced was non-significant.
Organic acid production and sclerotium formation by S. sclerotiorum Changes in the pH of the medium and mycelial dry weight yield during growth of isolates Bl-7
and NS 3 are shown in Figure 3. In the sclerotial isolate Bl-7, medium pH decreased initially but rose as growth proceeded and finally attained a value close to the initial pH. Initials appeared at day 6 when the pH was near minimum. In NS 3 the pH rose initially and then gradually declined, again finally reaching a value close to the original pH of the medium. An assessment of the acids detected by paper chromatography in medium taken after 8 days growth indicated that isolate Bt-7 secreted large
F. M. Humpherson-Jones and R. C. Cooke Table 5. Effect of organic acids on sclerotium production in S, sclerotiorum Each figure is the mean of five replicates Isolate Pr-ro
Isolate Bl-1S
.-~
Number of sclerotia per colony
Av, sclerotium dry wt per colony (mg)
a-ketoglutaric 10'6 7°'4 Citric 42'6 10'4 6,8 Fumaric 40'1* 7,6 Glycolic 48'3 Glyoxylic 70'1 9'2 Lactic 11'2 64'2 Malic 34'6* 8'4 Oxalic 10'0 69'4 Succinic 10'2 7°'2 Chromatogram eluates 100'8* 18'2* Unknown 1 Unknown 2 22'8 99'8* Citric 10'2 7°'0 Malic+oxalic 7'6 44'3 6,8 Succinic 48'1 Fumaric 60'3 9'2 Control 61'3 9'8 * Significant (P = 0'01), amounts of fumaric, malic, and succinic acid into the medium together with some citric and oxalic acid. The non-sclerotial isolate NS 3 secreted only oxalic and malic acids and these were in trace amounts. In addition to these identified organic acids, two large acid spots with low R, values (x 100) of 8-10 (unknown 1) and 11-18 (unknown 2) respectively in ethyl ether: acetic acid: water, were found in medium staled by Bl-7, These two unknowns were also present in medium from NS 3 but only in trace amounts. Chromatography of media taken after 14 days growth showed a marked decrease in all organic acids secreted by isolate Bl-7, citric acid disappearing entirely, The effect of organic acids, including the two unknown acids, on sclerotium production in the two weakly sclerotial isolates, Bl-15 and Pr-ao are shown in Table 5. Nine known organic acids were tested, together with the four identified and the two unknown acids from chromatogram eluates. The latter two compounds had a marked stimulatory effect on sclerotium production. Unknown 1 increased sclerotium number and dry weight yield in both isolates, while unknown 2 increased numbers only in isolate Pr-ao but increased yield in both isolates, In addition, both unknowns caused sclerotia of both isolates to
Av. sclerotium
Number of sclerotia per colony
per colony (mg)
16'0 14'6 10'4 12'6 12,8 15'0 9,6 9'8 12'0
90'1 86'3 80'4 72'1 80'6 69'6 61'4 78'1 96'8
22'4* 13'6 12'8 15'0 16'0 13'4 12'2
141'6* 146'2* 94'3 104'6* S9'2 61'4 78'2
drywt
appear 24 h sooner than those on control colonies. Fumaric and malic acids at a concentration of 0'1 % significantly reduced dry weight yield of sclerotia without significantly altering sclerotium number, yet the mixture of malic and oxalic acids eluted from chromatograms increased dry weight yield in isolate Bl-15 but did not alter the number of sclerotia formed. DISCUSSION
It is now clear that media staled by S. sclerotiorum and S. rolfsii contain heat-stable factors which accelerate the appearance of initials, increase their numbers, and enhance the final dry weight yield of sclerotia, However, in S. rolfsii and S. delphinii their role in sclerotium morphogenesis does not appear to be a simple one. For instance, S, delphinii does not produce such factors, although it is closely-related to (if not synonymous with) S, rolfsii. This indicates that they are not necessarily essential for sclerotium formation, yet S, delphinii responds markedly to staling compounds produced by S, rolfsii, In addition, the morphogenetic activity of staled medium is lost when the medium is fractionated. This could be due to the retention of active compounds within the ion-exchange columns or, equally likely, it indicates the 15-2
42 0
Sclerotium formation
synergisnc action of a number of compounds in the stimulation of sclerotium formation. With S. sclerotiorum the situation appears to be much more straightforward, and there seems to be a direct link between the ability to secrete organic acids to the medium and sclerotium formation. The existence of such a link is strongly suggested by the observation that there is acid staling during sclerotium formation by the sclerotial isolate B1-7, but alkaline staling during growth of the nonsclerotial isolate NS 3 over an equivalent period. Further evidence is provided by the fact that the morphogenetic activity of medium staled by isolate B1-7 resides in the acid fraction of culture filtrates, and is due in particular to two unidentified organic acids of low chromatographic mobility. Since sclerotium production by S. sclerotiorum remains unaltered over a range of pH 3'1-6'9, it is highly likely that these two acids are acting directly on morphogenetic processes rather than modifying these through a pH effect (Humpherson-jones, 1976). The identity of the two acids remains to be determined, but they are not acids of the TeA cycle, and the suggestion that TCA acids are directly involved in sclerotium morphogenesis is not substantiated by the assays carried out here (Chet & Henis, 1975). The possible importance of these acid compounds in sclerotium formation in S. sclerotiorum is further emphasized by the apparent absence from its mycelium of extractable morphogenetic compounds even during the phase of formation of initials (Humpherson-Jones, 1976). The existence of morphogenetic factors in media staled by some sclerotium-forming fungi is now adequately confirmed. In such species, explanations of sclerotium initiation which ignore the effects of staled media must be re-examined. In add ition, future experiments on the effects of putative morphogenetic 'triggers' should be carried out either under conditions that preclude the
accumulation of active staling factors, or using species or isolates which do not respond to their own staling products. We wish to thank the Science Research Council for the award of a postgraduate studentship (to F.M.H.- J.) during the tenure of which this work was carried out . REFERENCES
BEDI, K. S. (1958). The role of stale products in the formation of sclerotia of Sclerot inia sclerotiorum (Lib.) de Bary, Indian Phytopathology 2, 29-36. BRYANT, F. & OVERELL, B. T. (1953). Quantitative chromatographic analysis of organic acids in plant extracts. Biochimica et Biophysica Acta 10, 471-476. CHET, I. & HENIS, Y. (1975). Sclerotial morphogenesis in fungi. Annual Review of Phytopathology 13, 169192 • GoUJON, M. (1970). Mecanismes physiologiques de la formation des sclerotes chez le Corticium rolfsii (Sacc.) Curzi. Physiologie Vegetale 8, 349-360. HUMPHERSON-]ONES, F. M. (1976). The physiology of morphogenesis of fungal sclerotia. Ph.D. Thesis, University of Sheffield. LIU, T. M . & Wu, L. C. (1971). The effect of am ino acids on the growth and morphogenesis of Sclerotium rolfsii Sacco Plant Protection Bulletin 13, 87-96. RANSON, S. L. (1955). Non volatile mono-, di- and tricarboxylic acids (Chromatographic and ion exchange methods). Modern Merhods of Plant Analysis, vol. II (ed. K. Peach and M. V. Tracey), pp, 539582. Springer-Verlag. VEGA, R. R., CORSINI, D. & LE TOURNEAU, D . (1970). Non-volatile organic acids produced by Sclerotinia sclerotiorum in synthetic liquid media. Mycologia 62, 332-338. WHEELER, B. E.]. & WALLER,]. M. (1965). The production of sclerotia by Sclerotium rolfsii, II. The relationship between mycelial growth and initiation of sclerotia. Tran sactions of the British Mycolog ical Society 48, 303-314.
(Accepted/or publication
12
November 1976)
Printed in Great Britain
INDUCTION OF SCLEROTIUM FORMATION BY ACID STALING COMPOUNDS IN SCLEROTINIA SCLEROTIORUM AND SCLEROTIUM ROLFSII By F. M. HUMPHERSON-JONES* AND R. C. COOKE Botany Department, The University, Sheffield Sterile medium staled by the growth of Sclerotinia sclerotiorum (Lib .) de Bary and Sclerotium rolfsii Sacco enhanced sclerotiwn production when applied to test colonies, the active components being acidic and heat stable. In S. sclerotiorum, sclerotium production appeared to be associated with secretion of organic acids, and two unidentified acids with morphogenetic properties were isolated from staled medium. In culture fungal hyphae usually grow in an environment which is modified by metabolites diffusing from them, and many of these substances can induce morphogenetic changes in the hyphae that are producing them. Stimulation of sclerotiwn formation by such compounds has been implicated in Sclerotinia sclerotiorum (Lib .) de Bary and Sclerotium rolfsii Sacco (Bedi, 1958 j Liu & Wu, 1971). However, it is possible that, particularly in the case of S. rolfsii, such stimulation could be due to the accumulation of morphogenetically active metabolites within the hyphae during growth rather than in the surrounding mediwn (Wheeler & Waller, 1965; Goujon, 1970). As well as there being confusion with respect to the location of accumulating morphogenetic compounds, their possible nature is generally unknown, although thermolabile morphogenetic factors have been detected in cell-free extracts of S. rolfsii (Gouion, 1970). There are also indications that organic acids might in some way be involved (Vega, Corsini & Le Tourneau, 1970). When S. sclerotiorum was grown in a liquid medium with glucose as sole carbon source sclerotia were formed, the pH of the medium fell rapidly, and this was accompanied by the appearance of oxalate, malate, succinate, fumarate and glycolate in culture filtrates. When mannitol was the sole carbon source no sclerotia were produced, the pH of the mediwn increased, and only oxalate, malate and glycolate were found in culture filtrates . These observations have been taken to mean that there is a relationship between pH, organic acid synthesis and sclerotiwn production. The work described here was carried out to ob-
* Present address: National Vegetable Research Station, Wellesbourne, Warwick.
tain more detailed information on the morphogenetic activity of staled media and to determine which staling compounds could induce sclerotium formation. MATERIALS AND METHODS
Fungi and media Five isolates of Sclerotinia sclerotiorum (Lib .) de Bary and one each of Sclerotium rolfsii Saccoand Sclerotium delphinii Welch were used. Four isolates of S. sclerotiorum were derived from single ascospores while the fifth (NS 3) was obtained from a single hyphal tip excised from a colony growing on agar . The morphological characteristics of these isolates are summarized in Table 1S. sclerotiorum was maintained on 2 % GAY agar, comprising glucose, 20 g; asparagine , 2 g; yeast extract, 1 g; KH 2POU 1 g; KCI, 0'5 g; MgS04 .7H20, 0'5 g; FeS0 4, 0'01 g; agar 20 g; distilled water 1 1. S . rolfsii and S. delphinii were grown on 2 % 'Oxoid' malt extract agar and homemade potato dextrose agar respectively (250 g potato tissue boiled in 500 ml tap water for 20 min, filtered, supplemented with 20 g glucose, made up to 1 I and solidified with 20 g agar). Where liquid media were used the agar was omitted. Effect of staled medium S. rolfsii, S. delphinii and isolates B1-7 and NS 3 of S. sclerotiorum were grown in 100 ml of their respective liquid media at 25°. Three replicate flasks were used for each fungus and inoculum for each flask comprised 25 pI of homogenized myceliwn obtained from a stock colony grown in liquid med ium. At the time of appearance of sclerotium initials (6 days growth for S . sclerotiorum and 10 days growth for the two Sclerotium
Sclerotium formation Table
1.
Origin and morphological chara cteristics of isolates of Sclerotinia sclerotiorum, Sclerotium rolfsii and S. delphinii
Origin Sclerotiniasclerotiorum Isolate Bt-7 Phaseolus vulgaris Isolates Bt-t5 Pt-tO Isolate Bt-46 Isolate NS 3
P. vulgaris Pisum satioum
P. vulgaris P. vulgaris
Sclerotium ro/fsii
Pennisetum purpureum
S . delphinii
Unknown
Characteristics Rapid growth, colony flat, abundant sclerotia scattered over surface Rapid growth, dense mycelium, somewhat floccose, few sclerotia formed Slow growth, sclerotia not formed Rapid growth, abundant mycelium, very floccose, sclerotia not formed Rapid growth, colony flat, abundant sclerotia scattered over surface Rapid growth, colony flat, numerous sclerotia formed towards margin of colony
species) stale media were collected and filtered through Whatman No. 1 filter paper. For isolate NS 3, the medium was collected when initials formed in isolate Bl-7. Media for each fungus were bulked and stored at - 10°. When required, a suitable amount was thawed at 5°, its pH was adjusted to 4'5 with either N-KOH or N-HCI, and it was then sterilized by passage through a Millipore disc of pore size 0'45 pm . The sclerotium-inducing capacity of staled media was then assessed. Medium from the sclerotial isolate Bl-7 of S. sclerotiorum was tested against colonies of Bl-7 itself, Bl-15, Pr-so and NS 3; medium from the entirely mycelial isolate NS 3 was tested against isolate Bl-7 and Bl-15. Medium from each Sclerotium species was tested against all of the Sclerotium isolates. Fungi to be used in bioassays were grown on 20 ml of the appropriate agar in 9 em diam Petri dishes and in 20 ml of liquid medium in 100 ml conical flasks. Inoculum for the flasks comprised homogenized mycelium. Petri dishes were each inoculated centraUy with a 1 cm diam disk cut from the margin of a 4-day-old stock colony growing on agar. For each fungus six replicate flasks and six replicate dishes were used and all were incubated at 25°. When a mycelial mat had formed in liquid cul.ture, 6 ml of prepared, staled medium was injected through three mats of each fungus using a sterile hypodermic syringe. The remaining three mats were used as controls, being similarly treated with 6 ml of one quarter strength unstaled medium. All flasks were then incubated at 25° for 23 days at which time sclerotia were counted, and, where possible, weighed after drying at 90° for 24 h . Assays using agar-grown colonies were carried out when these were 6-7 em diam. A peripheral ring of agar 1 em wide was removed from each of three dishes of each species and 6 ml of stale
medium was introduced into each of the trenches so formed. The remaining three dishes of each fungus were used as controls, and were similarly treated using 6 ml of sterile, quarter strength unstaled medium. All dishes were incubated at 25° for 23 days at the end of which period they were examined and the number of sclerotia counted. Fra ctionation of medium staled by S. sclerotiorum and S. rolfsii
Fractionation of 70 ml of medium staled by either isolate Bl-7 of S. sclerotiorum or S. rolfsii was carried out using 10 x o-B em columns of Amberlite anion- and cation-exchange resins IRA 400 and IR 120 prepared and used according to Bryant & Overell (1953). The volumes of the acidic, neutral, and basic fractions obtained were adjusted to 70 ml and their pH was then brought to 4'5 using either N-KOH or N-HCl. They were then sterilized by filtration and their morphogenetic activity was assayed on agar-grown colonies as described above. For fractions from Bl-7 medium, the weakly-sclerotial isolates of S. sclerotiorum Bl-15 and Pr-uo were used as test fungi, while fractions from S. rolfsii medium were tested against S. rclfsii. Organic acid production and sclerotium formation by S. sclerotiorum
The sclerotial isolate Bl-7 and the non-sclerotia! isolate NS 3 were grown on 20 ml of GAY liquid at 25° in 100 ml conical flasks. At intervals over a 24 day period three replicate flasks of each isolate were taken, the pH of the medium was measured, and the mycelial dry weight was determined after drying at 90° for 24 h. In a parallel experiment the same two isolates were grown in GAY liquid from which the yeast extract had been omitted. After 8 days incubation
F. M. Humpherson-Jones and R. C. Cooke Table 2, Effect of staled medium from a sclerotial isolate (B1-7) of S. sclerotiorum on weakly- or nonsclerotial isolates of the same species, Each figure is the average from three replicates Total sclerotia No. of sclerotia dry wt. per colony per Bioassay on (mg) colony agar-grown colonies Bl-7 Unstaled medium 60'7 4'7 126'2* Staled medium 17'3* o o NS 3 Unstaled medium o o Staled medium 12 '3 Bl-15 Unstaled medium 71'4 206'2* 21'7* Staled medium Pr -ao Unstaled medium 11'3 45'9 101'6* Staled medium 20'7* Bioassay on liquid-grown colonies Bl-7 Unstaled medium Staled medium NS 3 Unstaled medium Staled medium Bl-15 Unstaled medium Staled medium Pi -ro Unstaled medium Staled medium * Significant (P
415
treatment in an equilibrated steam cabinet for 3 h. Chromatograms were developed by spraying with either bromophenol blue, ammoniacal silver nitrate, ammonium vanadate, or acetic anhydride pyridine (Ranson, 1955). The morphogenetic activity of sterile, aqueous solutions (0'1 % w/v) of a range of known organic acids was assayed using agar grown colonies of the weakly sclerotial isolates B1-15 and P1-10. Acids occurring in staled media were similarly assayed after having been eluted in distilled water from line-loaded chromatograms which had been run using butanol: formic acid: water, and had then been cut into strips , The amount of these acids applied to each test colony (in 6 ml of distilled water) was approximately equivalent to that present in 6 ml of the original staled medium.
RESULTS
10'7 16'3* o 150 * (approx.) 22'3 20'7 17'7 12'7 =
226'3 206'1 o Not determined 113'8
121 '6 161'4 173'2
0'01).
(at the point when initials were forming) and after 14 days (when sclerotia were mature) medium from six flasks of each isolate was collected, and replicate filtrates were bulked. Non-volatile organic acids were purified from filtrates using a 10 x 0,8 em Amberlite IRA 400 anion-exchange column in the CO a form. Filtrates were passed through at a rate of 0'5 ml per min, the resin bed was then washed with 50 ml distilled water and acids were eluted from the column using 500 ml N(NH4)2COa, The eluate was evaporated to dryness under vacuum at 35° and the ammonium salts obtained were dissolved in 50 ml distilled water, Free acids were regenerated from this solution by passage through a column of Amberlite IR 120 cation-exchange resin, in the H + form (Bryant & Overell, 1953). Acids were identified by one-dimensional descending paper chromatography using Whatman No.1 paper and either butanol : formic acid: water (100: 30: 100 by vol.) or ethyl ether : acetic acid: water (150 :30: 10 by vol.), Solvent was removed from chromatograms by air-drying for 1-2 hand
Effect of staled medium Staled medium from the non-sclerotial isolate, NS 3, of S . sclerotiorum had no detectable effect on either sclerotium number or the dry weight of sclerotium tissue produced in the two isolates, B1-7 and Bl-15, used in the biossay. Results from the remaining bioassays are given in Tables 2 and 3. Agar-grown colonies of isolates Bl-7, Bl-5 and Pr-uo, when treated with staled medium from the sclerotial isolate B1-7, produced sclerotia 1-2 days sooner than control colonies. In addition, both the final number of sclerotia formed and their dry weight yield increased by exposure to staled medium. No morphogenetic effects were observed in isolate NS 3, In liquid-grown colonies, treatment with staled medium had a similar effect only on Bl-7, where sclerotium numbers were increased but not their dry weight yield, and on the non-sclerotial isolate NS 3 which, in contrast to its behaviour when growing on agar, produced many small, immersed sclerotia 1 mm diam within 5-7 days of exposure to staled medium (Figs. 1, 2). Staled medium of S. rolfsii increased sclerotium number and dry weight yield in agar- and liquidgrown colonies of both S, rolfsii and S. delphinii (Table 3). In S. delphinii sclerotia appeared within 48 h of exposure to staled med ium, 6-7 days earlier than in control colonies, while in S. rolfsii they were formed 2-3 days earlier than in controls. Medium from S. de/phinii had no stimulatory effect on either S. delphinii or S. rolfsii but reduced the dry weight yield of sclerotia in S. de/phinii. Autoclaving staled media did not reduce their sclerotium- inducing properties.
Sclerotium formation
416
Table 3. Effect of staled medium from S. rolfsii or S. delphinii on sclerotium production Each figure is the average from three replicates Medium from S. delphinii
Medium from S. rolfsii
J-
Bioassay on agar-grown colonies S . rolfsii Unstaled medium Staled medium S. delphinii Unstaled medium Staled medium Bioassay on liquid-grown colonies S. rolfsii Unstaled medium Staled medium S. delphinii Unstaled medium Staled medium
Number of sclerotia per colony
Total sclerotium dry wt. per colony (mg)
Number of sclerotia per colony
Total sclerotium dry wt, per colony (mg)
117'0 164'7*
96'4 132'6*
129'3 119'7
84'S 89'3
7'0 18'3*
64'2 120'8*
9'3 8'7
59'8 69'9
66'7 84'7
59'2 78,6*
53'3 49'7
47'2 45'8
61'1 5'0 11'3* 92 '5* * Significant (P = 0'01).
9'3 8'3
78'1 5 1'6*
Table 4. Effect of staled medium and medium fractions on sclerotium production Each figure is the mean from five replicates Control
Stale medium
Acid fraction
Medium staled by S, sclerotiorum (B1- 7) Test isolate P1-10 Number of sclerotia 8'2 21'6* 14'2* per colony Av. dry wt. sclerotia 38'9 103'2* 61'8* per colony (mg) Bl-15 Number of sclerotia 9,6 18'2* 12,8 per colony 72'1 173'6* 103'6* Av. dry wt. sclerotia per colony (mg) Medium staled by S. rolfsii Test isolate S. rolfsii Number of sclerotia 86'2 129'9* 65'2* per colony 61'8 109'6* 60'1 Av. dry wt. sclerotia per colony (mg) * Significantly different from corresponding control (P Effect of fractions from staled medium
The effects of acidic, neutral and basic fractions from media staled by S, sclerotiorum (B1-7) and S. rolfsii are given in Table 4. Although complete
Neutral fraction
Basic
fraction
9'8 42'6
7'4
12'4
93'8
69'4 58'6 =
0'01) .
staled medium from S . rolfsii had a marked effect on sclerotium formation, none of the three fractions were stimulatory, and the acid fraction reduced sclerotium numbers significantly in test colonies, In contrast, the acid fraction from
F. M. Humpherson-Jones and R. C. Cooke
417
2 Fig. Fig.
15
1. 2.
Mycelial mat of non-sclerotial isolate NS 3 grown on untreated liquid medium . Mycelial mats of isolate NS 3 grown on liquid medium containing filtrate from a 6-day-old liquid culture of the sclerotial isolate B1-7.
MYC
68
Sclerotium formation
4 18
300
81-7 6
200
5
4
l1J
100
3
bO
'oj
5
1: Ci3
.Eeo
<-
.;:;
0
~
:I:
...»
0.
7.
"0
~ -;:; (J »
NS3
~
6
200
5
4
100
3
2
4
6
8
10 12 14 16 Growth period (days)
18
20
22
Fig. 3. Changes in mycelial dry weight (-0-) and pH of medium (--e-) during growth of a sclerotial isolate,Bl-7, and a non-sclerotial isolate,NS 3, of S. sclerotiorum. The broken line indicates the point at which initials appeared. medium staled by S. sclerotiorum stimulated sclerotium production, although to a lesser extent than did whole, staled medium. It also increased dry weight yield of sclerotia in isolate Bt-t5, but the slight rise in the number of sclerotia produced was non-significant.
Organic acid production and sclerotium formation by S. sclerotiorum Changes in the pH of the medium and mycelial dry weight yield during growth of isolates Bl-7
and NS 3 are shown in Figure 3. In the sclerotial isolate Bl-7, medium pH decreased initially but rose as growth proceeded and finally attained a value close to the initial pH. Initials appeared at day 6 when the pH was near minimum. In NS 3 the pH rose initially and then gradually declined, again finally reaching a value close to the original pH of the medium. An assessment of the acids detected by paper chromatography in medium taken after 8 days growth indicated that isolate Bt-7 secreted large
F. M. Humpherson-Jones and R. C. Cooke Table 5. Effect of organic acids on sclerotium production in S, sclerotiorum Each figure is the mean of five replicates Isolate Pr-ro
Isolate Bl-1S
.-~
Number of sclerotia per colony
Av, sclerotium dry wt per colony (mg)
a-ketoglutaric 10'6 7°'4 Citric 42'6 10'4 6,8 Fumaric 40'1* 7,6 Glycolic 48'3 Glyoxylic 70'1 9'2 Lactic 11'2 64'2 Malic 34'6* 8'4 Oxalic 10'0 69'4 Succinic 10'2 7°'2 Chromatogram eluates 100'8* 18'2* Unknown 1 Unknown 2 22'8 99'8* Citric 10'2 7°'0 Malic+oxalic 7'6 44'3 6,8 Succinic 48'1 Fumaric 60'3 9'2 Control 61'3 9'8 * Significant (P = 0'01), amounts of fumaric, malic, and succinic acid into the medium together with some citric and oxalic acid. The non-sclerotial isolate NS 3 secreted only oxalic and malic acids and these were in trace amounts. In addition to these identified organic acids, two large acid spots with low R, values (x 100) of 8-10 (unknown 1) and 11-18 (unknown 2) respectively in ethyl ether: acetic acid: water, were found in medium staled by Bl-7, These two unknowns were also present in medium from NS 3 but only in trace amounts. Chromatography of media taken after 14 days growth showed a marked decrease in all organic acids secreted by isolate Bl-7, citric acid disappearing entirely, The effect of organic acids, including the two unknown acids, on sclerotium production in the two weakly sclerotial isolates, Bl-15 and Pr-ao are shown in Table 5. Nine known organic acids were tested, together with the four identified and the two unknown acids from chromatogram eluates. The latter two compounds had a marked stimulatory effect on sclerotium production. Unknown 1 increased sclerotium number and dry weight yield in both isolates, while unknown 2 increased numbers only in isolate Pr-ao but increased yield in both isolates, In addition, both unknowns caused sclerotia of both isolates to
Av. sclerotium
Number of sclerotia per colony
per colony (mg)
16'0 14'6 10'4 12'6 12,8 15'0 9,6 9'8 12'0
90'1 86'3 80'4 72'1 80'6 69'6 61'4 78'1 96'8
22'4* 13'6 12'8 15'0 16'0 13'4 12'2
141'6* 146'2* 94'3 104'6* S9'2 61'4 78'2
drywt
appear 24 h sooner than those on control colonies. Fumaric and malic acids at a concentration of 0'1 % significantly reduced dry weight yield of sclerotia without significantly altering sclerotium number, yet the mixture of malic and oxalic acids eluted from chromatograms increased dry weight yield in isolate Bl-15 but did not alter the number of sclerotia formed. DISCUSSION
It is now clear that media staled by S. sclerotiorum and S. rolfsii contain heat-stable factors which accelerate the appearance of initials, increase their numbers, and enhance the final dry weight yield of sclerotia, However, in S. rolfsii and S. delphinii their role in sclerotium morphogenesis does not appear to be a simple one. For instance, S, delphinii does not produce such factors, although it is closely-related to (if not synonymous with) S, rolfsii. This indicates that they are not necessarily essential for sclerotium formation, yet S, delphinii responds markedly to staling compounds produced by S, rolfsii, In addition, the morphogenetic activity of staled medium is lost when the medium is fractionated. This could be due to the retention of active compounds within the ion-exchange columns or, equally likely, it indicates the 15-2
42 0
Sclerotium formation
synergisnc action of a number of compounds in the stimulation of sclerotium formation. With S. sclerotiorum the situation appears to be much more straightforward, and there seems to be a direct link between the ability to secrete organic acids to the medium and sclerotium formation. The existence of such a link is strongly suggested by the observation that there is acid staling during sclerotium formation by the sclerotial isolate B1-7, but alkaline staling during growth of the nonsclerotial isolate NS 3 over an equivalent period. Further evidence is provided by the fact that the morphogenetic activity of medium staled by isolate B1-7 resides in the acid fraction of culture filtrates, and is due in particular to two unidentified organic acids of low chromatographic mobility. Since sclerotium production by S. sclerotiorum remains unaltered over a range of pH 3'1-6'9, it is highly likely that these two acids are acting directly on morphogenetic processes rather than modifying these through a pH effect (Humpherson-jones, 1976). The identity of the two acids remains to be determined, but they are not acids of the TeA cycle, and the suggestion that TCA acids are directly involved in sclerotium morphogenesis is not substantiated by the assays carried out here (Chet & Henis, 1975). The possible importance of these acid compounds in sclerotium formation in S. sclerotiorum is further emphasized by the apparent absence from its mycelium of extractable morphogenetic compounds even during the phase of formation of initials (Humpherson-Jones, 1976). The existence of morphogenetic factors in media staled by some sclerotium-forming fungi is now adequately confirmed. In such species, explanations of sclerotium initiation which ignore the effects of staled media must be re-examined. In add ition, future experiments on the effects of putative morphogenetic 'triggers' should be carried out either under conditions that preclude the
accumulation of active staling factors, or using species or isolates which do not respond to their own staling products. We wish to thank the Science Research Council for the award of a postgraduate studentship (to F.M.H.- J.) during the tenure of which this work was carried out . REFERENCES
BEDI, K. S. (1958). The role of stale products in the formation of sclerotia of Sclerot inia sclerotiorum (Lib.) de Bary, Indian Phytopathology 2, 29-36. BRYANT, F. & OVERELL, B. T. (1953). Quantitative chromatographic analysis of organic acids in plant extracts. Biochimica et Biophysica Acta 10, 471-476. CHET, I. & HENIS, Y. (1975). Sclerotial morphogenesis in fungi. Annual Review of Phytopathology 13, 169192 • GoUJON, M. (1970). Mecanismes physiologiques de la formation des sclerotes chez le Corticium rolfsii (Sacc.) Curzi. Physiologie Vegetale 8, 349-360. HUMPHERSON-]ONES, F. M. (1976). The physiology of morphogenesis of fungal sclerotia. Ph.D. Thesis, University of Sheffield. LIU, T. M . & Wu, L. C. (1971). The effect of am ino acids on the growth and morphogenesis of Sclerotium rolfsii Sacco Plant Protection Bulletin 13, 87-96. RANSON, S. L. (1955). Non volatile mono-, di- and tricarboxylic acids (Chromatographic and ion exchange methods). Modern Merhods of Plant Analysis, vol. II (ed. K. Peach and M. V. Tracey), pp, 539582. Springer-Verlag. VEGA, R. R., CORSINI, D. & LE TOURNEAU, D . (1970). Non-volatile organic acids produced by Sclerotinia sclerotiorum in synthetic liquid media. Mycologia 62, 332-338. WHEELER, B. E.]. & WALLER,]. M. (1965). The production of sclerotia by Sclerotium rolfsii, II. The relationship between mycelial growth and initiation of sclerotia. Tran sactions of the British Mycolog ical Society 48, 303-314.
(Accepted/or publication
12
November 1976)