The production of sclerotia by Sclerotium rolfsii

[ 3°3 ] Tr ans. Brit. mvcol, Soc. 48 ( 2 ) , :3° 3- :114 ( 1<)0:'» ) Printed ill Great Britain

THE PRODUCTION OF SCLEROTIA BY SCLEROTIUM ROLFSII II. THE RELATIONSHIP BETWEEN MYCELIAL GROWTH AND INITIATION OF SCLEROTIA

By B. E.J . W H EELE R

AN D J .

M. WA LLER*

Imperial College Field Station, Silwood Park, Sunninghill, Berks. (With II T ext -figur es) Th e relationship between mycelial growth and initiation of sclerotia in a single isolate of S. rolfsii Sacco was investigat ed ( I) in Petri dishes of different sizes, (2) in colonies (pa rt or whole) transferred to fresh m ed ia a nd/or Petri dish es, and (3) in colonies having di fferent rates of mycelial growth . In itiati on appears to be regul at ed by th e growing hyphae at th e periphery of th e mycelial mat. In dishes of 5, 7, 9 a nd [3 em. diam. initials did no t appear until the lat eral ext ensio n of th e myceliu m was restricted. In colonies on millipore filters transferred to fresh plates whe re furth er extension of th e mycelium could occur, initi at ion of sclerotia was suppressed. On central disks 5 em . diam. remo ved from colonies a t different stages ofgr ow th , initia tion bega n ea rlier than 011 un dis turbed coloni es of com parable age. Wh en th e growth ra te on a par ticular medium was altered , eithe r by in cubating at a low er temperature during the early stages or by usin g different inocul a , there was a corr esponding cha nge in th e nu m be r of initials formed. An a nalysis of data from seventee n exp eriments show ed a highly significant, positive correlation betw een growth rat e a nd number of initials produced . The significance of these resul ts is disc ussed. It is suggested that init ia tion of sclerotia de pends on th e acc umulat ion in th e hyphae of substances whic h a re metabolized in a d ifferent wa y from that involved in mycelial growt h.

'Wheeler & Sharan ( 1965) hav e suggested th at initiation of sclerotia within a colony of Sclerotium rolfsii on aga r is in som e way controlled by the actively gro wing hyphae at th e edge of the ext ending myceli al mat. In this paper are presented th e results of experim en ts in which various aspects of this rel ati onship were investiga ted . The term ' mycelial growth' as used here is restricted to th e formation of the fungus mat on the agar surface: it specifically excludes growth of hyphae which are visibly involved in th e formation of sclerotia. MATER IA LS AN D METHODS

The methods used were gen erally similar to those already described (W h eeler & Sharan , 1965) . The isola te of S. rolfsii (IMI 742 10) was maintained under oil on slopes of V 8 j uice ag ar and on ' X ' medium (Last & Hamley, 1956). Several media wer e used for in vestigation of sclerotial form ation: ( I) 2 % glucose nitrat e agar (g.n .a .) as previously defined.

* Present address : Scott

Agri cu ltural Laboratori es, Nairobi, K en ya .

304

Transactions British Mycological Society

(2 ) 0 ' 5 % g.n.a.: glucos e 0'5 % (w/v), NaN0 3 0 '05 %, and the oth er constituents at the sam e levels as in 2 % g.n.a. (3) V8 juice agar: 10 ml. V8 juice (Campbell' s Soups Ltd. ), 2 g. agar, go ml. distilled water. (4) Potato dextrose agar (p.d.a.} : 200 g. washed potatoes (not peeled) boiled until soft; the liquid decanted through muslin, 20 g. dextrose and r5 g. agar added to it, and the volume made up to I 1. with distilled water. (5) Plain agar: 1'5 g. agar, roo ml. distilled wat er. All media wer e autoclaved for 20 min. at 120° C. Inoculum was prepared by transferring a small portion of a stock culture to a pl at e of 0'5 % g.n .a. and using either 0·6 em. disks cu ' from the edge of this colon y or disks cut from daughter coloni es gro wn on medium of the same composition, on plain agar, or on V 8 juice agar. All cultures were incubated at 25 °. R ESULTS

Initiation if sclerotia in plates if different sizes Experiment r In previous experiments (Wheeler & Sharan, 1965) 9 cm. diam . Petri dishes were used and it could be argued that this determined the pattern of mycelial growth and initiation of sclerotia. A similar sequ ence of events was observed, however, when S. rolfsii was grown on 0'5 % g.n .a. in 5, 7, 9 and r3 em. diam. dishes (Fig. I). Sclerotial initials did not appea r within the colonies until the mycelium approached the edge of the dish.

I ~

....------...---.---,/,.....i .f ' 13

4

1} ;i::~. '.·-·.-.~ ::~-·.: :~::~71 ~ 7 ' I ~l r /: ; ' ~ '"' ,' 00

: ' r r

'5 5· ·, · ··· · · · · ·· ·· · ·

2

I

/: ::

3 /~ ~

!

'

&v .

.

r~-.j

I

:

I ~2

5~7~

Days

Fig .

I

-

~~

":

:J ~ ~

0

j:t 0 /

--: 3 .§

0

•

2

~ "•

•

0'--+2- 4!--"';'6---:S!--"" ' 0=--=,"'2---}-H,..--J

Area of colony at transfer (in un its of TT em.") Fig.

2

Fig . I. Mycelial growth ( + ) and initiation of sclerotia (0) on 0'5% glu cose-ni trate agar in Petri dish es of different sizes. There were four replicates of each size of Petri dish . The va lues for initials are the means ofeach set of four replicates; tho se for mycelial growth the means of all plates available for assessment. Fig. 2 . Sclerotial production on cellophane disks in rela tion to colon y size a t time of transfer to plain agar (means of six repli cates).

Experiment 2 Initials appeared after 5 days on plates 0[2 % g.n.a. 9 em, and 13 em. in diameter (Table r). By day 6, the number of initials on the 9 em. plates had risen rapidly and this coincided with th e lateral restriction of the

Sclerotium rolfsii. II. B. E.]. Wheeler and]. M. Waller

305

mycelium; but, in contrast, there was a relatively small increase in the number of initials on the 13 em. plates. Large numbers did not appear on these plates until day 7 when the agar had been covered by mycelium.

Experiment :) In dishes of p.d.a. 5, 7, 9 and 13 em. diam.the appearance of the first initials again coincided with the lateral restriction of the mycelium in all but the 13 em. plates in which some initials appeared when the mean colony diameter was 8'9 em" but as in the previous experiment there was no substantial increase in number of initials until the agar surface was completely covered by mycelium, Initiation of sclerotia thus appears to be associated with the lateral restriction in colony growth, but the degree of this association can be modified if different media are used. Table

I.

j~ycelial growth

and initiation of sclerotia on 2 nitrate agar in 9 and 13 em, dishes

%

glucose .

Days .,

:)

.J

Ii

7

<) em,

plate Colony diarn. CIllo Illitial~ I:, ern, plate Colon v diarn. ern. [nitial~

2'()

1':1

1'+

'.!'H

,t"~

,t'!>

6'3

7' j

g"S

.,

'r:, ,

'J'o (i](;

II'') IOC,

(j,)H

1:,'0 II'l4

:vkalls ol lour replicates

Transfer of part or whole oj the colon)' tofresh media and/or plates Experiment

I

Fifteen 5 em. diam. Petri plates, each containing 6 ml. ofz % g.n.a. were prepared. Fifteen millipore disks (grade AAO, diam. 47 mm.) were washed in boiling distilled water, placed singly between filter papers (\\Thatman no, I, 7 em. diam.) in a dish, and autoclaved for 15 min. at 120 c. The disks were then placed, one in each of the prepared plates, on the surface of the agar and inoculated centrally. When the first initials appeared, the disks from 12 plates were peeled off and the colonies transkrred to lresh media as follows (:3 replicates of each) : g.n.a. in 5 em. diam. plates; A, on fresh 2 B, on plain agar in :") em. diam. plates; C, on fresh 2 % g.n.a. in 9 em. plates, centrally placed; D, on plain agar in 9 em. plates, centrally placed; E, not removed frorn r; em. plates (control). The effects of these treatments on the formation of sclerotia are shown in Table 2. Each figure is the mean of the three replicates. The number of initials already formed on the millipore disks at the time of transfer was subtracted from the number recorded each day. There are three main points of interest:

':u

306

Transactions British Mycological Society

(I) Fewer sclerotial initials were formed during the next 24 hr. on disks transferred to fresh media than on disks not transferred. Though care was taken in removing disks it is likely there was some damage to the colony, and this might account for the lower rate of initiation. (2) Where, on transfer, there was fresh medium for further mycelial extension, the rate of initiation during the first 2 days was further depressed. The number of initials then rose rapidly when the mycelium had covered the available agar surface. Table

2.

Days after transfer

Effeet on number of initials and sclerotia of transferring 5 em. disks tofresh media Transferred to 5 em. diam. agar plates A

Plain

Nutrient

Not transferred (control)

Transferred to 9 em. diam. agar plates

,

Plain

Nutrient

(a) Initials I

2 3 4 5 (b) Sclerotia 5 6 7 8 9 10

88 336 5 22 67° 7°3

73 253 588 64 8 773

153 226 420 59 1 648

47 107 49 2 74 1 81 3

31 79 564 1683 1898

° 5 18 33 4° 40

0 ° 4 36 136 206

° 7 44 114 128 151

4 39 89 124 136 152

0 ° ° 12 23 3 4 17

(3) Transferring disks to 5 cm. diam. plates had little effect on the total number of initials but mature sclerotia were fewer on disks transferred to plain agar. This suggests that there is further absorption of nutrients from the medium after the mycelial mat is formed. On the other hand, there is an apparent discrepancy here because on disks transferred to 9 ern. diam. plates of plain agar the number of mature sclerotia formed was similar to that of the control plates. This is being investigated.

Experiment 2 Sheets of cellophane (British Cellophane Ltd., grade PT 300), 25 x 20 cm., were washed in two changes of boiling, distilled water. From these sheets, 30 disks 9 em, diam. were cut and soaked overnight in distilled water, then laid singly between filter papers (Whatman no. I, 9 cm. diam.) in a sterile dish and sterilized by steaming for It-2 hr. When cool, one cellophane disk was placed on the agar surface in each ofthirty 9 em, plates of 2 % g.n.a. (20 ml.) and inoculated centrally. At five different stages of growth the disks bearing the growing colonywere taken from six plates chosen at random and transferred to fresh gcm. plates ofplain agar. The number of initials and mature sclerotia which subsequently formed were assessed at intervals, and finally, 20 days after the inoculation of the g.n.a. plates. The interest in this experiment centres on the relationship between these assessments of sclerotial formation and the amount of mycelial growth at

Sclerotium rolfsii. II. B. E. J. Wheeler and J. M. Waller

307

the tim e of tr an sfer (Fig. 2). The cor rela tion between ma ture sclero tia eventually formed and colony size a t tra nsfer is highly significant (r = 0'98 9, P < 0'01 ), but not that betwee n tota l initials and colony size.

200

/ 9 f

or.

]

.~ 100

-

.If I

-

.1:)

Jf .

,,' .1 ..:0 ._·6 ..·· 0' .

I .

':

,<'

I

-:

r :

o

r

'

/ 10 '"

I o

j

/

/.:' I . I

:

':

I '

i ,'.. . i p.. . i /.6

•

1 0'

f ,,..''

/./ .-

,r

2

Da ys after tra nsfer Fig. 3. Formation of ini tia ls on 5 em . diam. disks removed fro m colonies of S. rolfsii at differen t stages of growth . D isks removed when colony c . 6 em . diam . (- -) , 7'S em. d iam . (- - - - -}, <) em . di am . ( , '- a nd when initials began to a ppear ( _. ;.

Experiment 3 Fift een 9 em. dia m . plat es of '2 (:/~ g.n.a . ( 2 0 ml.) wer e inoculat ed cen tr ally. At four differen t stages of growth a 5 em . diam. disk was cut from the cen tr e of eac h of three selected pla tes, and transferred to a sterile dish of the sa me diam eter. T he four stages of growth wer t : A, colony di am eter, c. 6 em . (pla te 1, 5' 9 cm. , plate 2, 6'4 cm ., plate 3, 6'3 em .); 20-2

Transactions British Mycological Society B, colony diameter, c. 7'5 cm. (plate I, 7.8 cm., plate 2, 7'2 cm., plate 3, 7"5 em.); C, colony diameter just 9 cm.; D, sclerotial initials beginning to appear. Disks were not cut from three plates and these provided a check of sclerotiaI formation on this medium. There are four main points of interest in the results of this experiment: (I) In A, Band C initiation of sclerotia began earlier on the cut disks than on the control plates. (2) There was a marked' lag phase' in the production of initials on disks of A, not observed in the other treatments (Fig. 3). Table 3. Sclerotial production on 5 em. diam. disks cutfrom colonies at different stages of growth Growth stage A

A Total initials Mature sclerotia

224 160

Means of three replicates

(3) Generally the number of initials and mature sclerotia finally formed decreased from A to D, i.e. delay in cutting the disks from the colony reduced the ability of the excised portion of the colony to form sclerotia (Table 3). (4) The distribution of sclerotia on the cut disks varied from a fairly broad band at the periphery (A) to a narrow band at the very edge (C and D). In D, in particular, some of the mature sclerotia were malformed; this suggests that, though no initials were visible in this particular area when the disks were cut, the sites of sclerotial formation were already determined. The general significance of these results will be discussed later.

Initiation of sclerotia in relation to different growth rates A number of incidental observations made during this investigation suggested that there was a connexion between the rate at which the mycelium grew over a particular medium and its ability to form sclerotia. This was therefore examined. Experiment

I

Different growth rates were obtained by incubating one set of 2 % g.n.a. cultures at 15° C. for the first 5 days then transferring to 25°, and incubating another set continuously at 25° (each of6 replicates). The rate ofinitiation and the final number of initials formed (Fig. 4) was lower on colonies with the slower growth rate but there was no significant difference in the number of sclerotia which matured.

Sclerotium rolfsii. II. B. E. J. Wheeler and J. M . Waller Experiment

309

'2

Different growth rates were induced by usin g inocula prepared as foll ows. A subc ulture on 0'5 % g.n.a., started from the stock culture of S. rolfsii, was incubated for 4 d ays at 25°; then two 0·6 em . disks cut from the edge of the colony were used to ino culate centrall y pl ates of plain agar a nd 0'5 % g .n.a. The two plat es were th en in cubat ed at 25° for 3 d ays. For

.0--.-. 0

6

- . - - - - - ---

~ -0

51

0'

IF d _--.--- -- ..•-

2

". -,

o

2

4

6

8

10

• I

12

14

16

18

20

22

24

Days Fig. 4. Growth and prod uct ion or sclero tia on :.1(~·o glucose-uiuarc agar. Colonies inc uouted at 25 ' co nt inuo usly ( - - " colo nies incu ba ted a t IS· lor lirst.) days, then at 25 ° (- - - - -) .: m ycelial growth ( + .). in it ia ls (O) , mature scle r o tia (e! . Si g n ifica n t differences lo r fina l number of ini tia ls : J:;7 at P = 0 ' 0 5 : ]( I:.! a t P = o-u r .

Table 4. Sclero tuil production in relation to growth rate Inoculum so u rce O' ;:-l

Plain agar

t~~

glucose-

nitra te ag ar

.I'im c (h r. : ta ke n to grow to (J em , d iarn . *

To tal in itials formed M ature sclerotia Significan t differ ences for to ta l initials : 5 0 at P = 0' 0 5 . 71 at P * Ca lcula ted fro m regression or ~ (col on v dia ru. ',' time (F ig. .'» .

= 0 ' 0 1.

the experiment, five pla tes of 0'5 % g.n.a, were inocul ated with 0·6 ern , disks from th e edge of th e plain aga r colony and five pl at es fro m th e o-5 % g.n .a. colony . Colonies d erived from plain agar ino cul a grew more slowly and produced fewer initi als tha n those from inocul a grown exclu -

310

Transactions British Mycological Society sively on O's % g.n.a. (Table 4). There was, however, no significant difference in the number of mature sclerotia produced. Throughout the investigation many experiments with o'S % g.n.a. and inocula prepared in different ways were made; the design varied, but in each five plates were inoculated in the standard way and incubated at 2So, mycelial growth was measured, and the number of initials and mature sclerotia recorded until sclerotial formation ceased. The results of seventeen such experiments were analysed with respect to growth and number of initials. The inocula were derived from either plain agar or o'S % g.n.a. colonies. They varied in their past history or the time at which, and the position in the colony from which, they were taken. .'

2 /,t

~:,..

// ..-

/

,/

s "0 2

/

~ 1

j .'

./

f'

./

i

/~:

1

+/. ,,#

I

,

,

1

2

, , 3 4 Days (a)

//

I

~/

.//

1

:'

, .'

0

,

1I

, 5

J

"

/

" ,1,: 0

" 3 4 Da ys (b)

2

5

Fig. S. Growth of S. rolfsii on O's % glucose-nitrate agar. (a), Inoculum from 3-day-old culture on o- 5 % glucose-nitrate agar; (b), inoculum from 3-day-old culture on V 8 juice agar; (c), inoculum from 14-day-old culture on 0'5 % glucose-nitrate agar. Colony diam. (- - - - -), ~(colony diam.) (--).

The main problem was to find, for each experiment, some measure of the overall growth rate across the medium. The method finally adopted was to take the cube roots of the mean colony diameters and to plot these transformed readings against time. By doing this the growth curve obtained from each experiment was converted to a straight line (Fig. sa), which could be accurately fitted by the method of least squares, and the slope of which gave a measure of the growth rate. In all seventeen experiments, a straight line was obtained including the size ofinoculum as origin. Further investigation showed two instances where inclusion of inoculum size gave misleading results, one was in an experiment using inoculum from V 8 juice agar, the other using 14-day-old inoculum on o·S % g.n.a. In these situations inoculum size at day 0 is omitted in the relevant calculations (Fig. Sb, c). In Fig. 6 the mean number of initials obtained in each of the seventeen experiments is plotted against the appropriate estimate of growth. There is clearly a significant correlation between the two. The change in number of initials corresponding to a slight change in growth rate is relatively large. In terms of the time taken to colonize an agar surface of q em. diam., a difference of 48 hr. can be expected to lead to a mean difference of 300 initials per plate.

Sclerotium rolfsii. II. B. E. J. Wheeler and J. M. Waller

3II

The relationship between the number of initials formed and the number of these which matured, in the same group of experiments, is also of interest (Fig. 7). As might be expected there is a high degree of correlation between the two, the more initials formed the greater the number which mature. A very large increase in the number of initials, however, gave only a small increase in the number of mature sclerotia, e.g. an increase of 500 initials gave an increase of only seven mature sclerotia. It seems likely that the nutrient status of the medium itselflargely governs the number of sclerotia which mature, whereas the total number of initials formed is more directly related to the way in which the fungus grows on the particular medium. 10 0

9

8 7

.!6
0

.... 6

-o

§ 6

j

0

4-

o

0

]

0

.:S "I

o

0

o

000

o

o

~

§

0

2

0

] ....
'ill ..,.. 7

8

9

10

11

Growth rate ( x

12

~

1(3)

!

!

!

!

!

!

!

!

!

2

3

4

5

6

7

8

9

10

Initials (hundreds) Fig. 7

Fig. 6

Fig. 6. Relationship between growth (as rate of mycelial extension) and number of initials formed on 0'5 % glucose-nitrate agar. r = 14'28X- 871, r = 0'7229, P < 0'01. Fig. 7. Relationship between total number ofinitials formed and number which matured on 0'5 % glucose-nitrate agar. r"" 128·8+0·014X, r = 0'9299, P < 0'001.

Distribution

of sclerotia within the colony

Though the exact distribution of sclerotia on the mycelial mat appears to vary with the medium used (Wheeler & Sharan, 1965), the general basic pattern is a band of sclerotia towards the periphery of the plate. A few, preliminary investigations on this aspect of sclerotial formation were carried out with 0'5 %g.n.a. The distribution was recorded as follows. The position of each sclerotium was first marked with indian ink on the base of the dish. A perspex disk, accurately etched with concentric circles at a spacing of 2 mm. was then placed over the marked surface, so that its centre coincided with the position of the inoculum, and the number of sclerotia in each concentric zone counted. The results were expressed as number/unit area of the zone counted. An example is given in Fig. 8. In

312

Transactions British Mycological Society

this part icular experiment growth rate was high , a large number of in iti als was formed , and th e first a ppeared befor e th e mycelium reached the edge of th e ag ar. There were two zones of sclerotia, a small zon e round th e in oculum and a large, broad zone towards th e periphery. The position of the mature sclerotia indica ted that th e first-formed initials were those th at ma tu red . 3 2

102 hr.

j

0

3 ..s

CJ

~

;; 2. ~

E CJ

<:i

sr:

0

3

2

34 2 Co lony rad ius (cm .)

Fig . 8. Distribution of sclerotia on 0'5 % glucose-nitrate agar. Total in itials (0 ), mature sclerotia (. ). R ad ius of colony indicated by vert ical arr ow .

DI SCUSSION

The results pres ented here strongly suggest th at th e initia tion ofsclero tia is regul ated by th e actively growing hyphae of the mycelial mat. Fo r example : (I) Initiation does not occur in Petri dishes of 5, 7, 9 or 13 em . diam. un til hyphae approach or reach th e edge of the agar. (2) Transfer of mycelium which has begun to form scleroti a to condi-

Sclerotium rolfsii. II. B. E. J. Wheeler and J. M. Waller

313

tion s wher e further mycelial extension is possible markedl y reduces th e d evelopment of initials. This occurs whether th e tr ansfer is made to a nutrient agar from which materials could be absorbed for further mycelial growth, or to plain agar where an y further growth depends on nutrients in th e existing mycelium. (3) Wh en a central zone of 5 em. diam. is removed Irorn a colony and transferred to a Petri dish of the sam e size, initiation begin s earlier than in undisturbed colonies of compara ble age. The main problem is th e nature of this regulating mechanism . Two possible explanations are th at (a) the hypha I tips produce some substance which inhibits sclerotial formation, and (b) the hyph al tips are competing tor som e metabolite (s) essential for sclerotial production . The evidence available hardly supports the first hypothesis. Mycelial growth and initiation of sclerotia can occur simultan eously, e.g. on potato d extrose agar in plates of 13 em. diam., and in fast-growing colonies on 0'5 % g.n.a. in 9 em. plates. The rapid production of initials which is seen after the mycelium reaches the edge of the agar suggests that if such a substa nce is involved it is not very stable. On the other hand, it must operate over an extensive ar ea of th e colony especially in a plate of 13 em. diam. Ther efore, either it diffus es back through th e mycelium ra pidly or it is a gas. In either event one would expect a diffusion gra d ien t de cr easing from th e tip to the inoculum, and initials, therefore, to develop from the centre to th e periphery of th e colony . This does not occur. Though some initials a ppear near the inoculum, th e majority appear and develop first in a zon e a t th e edge of the agar. This distribution of sclerotia is more readily explained by th e second hypothesis. If there is such a metabolite then one might expect the highest conce n tration to be near the site where it was previously used , i.e. the hy phal tip s at the periphery of th e colony. There is circumstan tial evidence Jar the mobilization of such a substance (or substances) at the growing tips Irom the experiment in which central portions were removed from colonies a t -d ifferent stages of growth. Not onl y was th ere a d ecr ease in the number of sclerotia produced on th e disk by delaying its removal but the zone of sclcrotial production becam e more narrowly restricted towards the edge. It follows from this hypothesis that there must be a certa in amount of this substance present in the mycelium before initiation can take place. This will depend on its rate of production and its rat e of utilization in mycelial growth. The lag ph ase in sclerotial production on disks cut from colonies ofapprox. 6 em. diam. suggests that at this stage th e a moun t of the substa nce available is limited and meets only the requ irem ents of mycelial growth . If th e substance is utiliz ed in mycelial growth its rate of production is likely to influence the rate at which the colon y grows across a particular agar medium. It is significant in this respect that high growth rates are correla ted with the production of large numbers of initials, and that in rapidly growing colonies initials often appear befor e the mycelium completely covers the medium . It is difficult to accept this second hypothesis unl ess one also accepts that different metabolic pat hwavs a rc involved in the utiliz ati on of this sub-

314

Transactions British Mycological Society

stance in mycelial growth and in sclerotial formation. Otherwise, once the lateral extension of the mycelial mat is restricted, there seems to be no reason why an accumulation of the substance should not lead to continued growth of the mycelial type elsewhere in the colony. As a basis for future exp eriments the following scheme is thus proposed: metabolic pathway A

. edilate Hr----~· mycelium rnterrn Nutrients;---->-. [ metabolitetsi j L--->~ -initials metabolic pathway B

One small advance towards substantiating this hypothesis would be made if materials were found which would inhibit initiation but allow mycelial growth to continue. One such material has been found and its effects will be described in a further paper. It is pertinent to emphasize here that this hypothesis involves the accumulation of some metabolite within the hyphae. The idea that initiation of sclerotia results from the accumulation in the medium of some 'staling ' product is rejected for the following reasons: (I) Growth of S. rolfsii on the media used is not of the ' staling ' type. (2) The behaviour in Petri plates of different sizes, and on millipore disks transferred to fresh agar in plates of the same size as the disks, is not consistent with the accumulation of a sclerotia-inducing material in the medium. (3) All attempts by the authors to demonstrate such a material have yielded negative results. The work carried out by]. M. Waller was undertaken as part of the requirements for the Diploma of Imperial College in mycology and plant pathology. B. E.]. Wheeler is indebted to Mr E. Green and Miss B. Curtis for technical assistance, and to the Central Research Fund of the University of London for a grant to purchase equipment. REFERENCES

LAST, F. T. & HAMLEY, R. E. (1956). A local lesion technique for measuring the infectivity of Botrytis fabae Sardina. Ann. appl. Biol. 44, 410-418. WHEELER, B. E. J. & SHARAN, N. (1965). The production of sclerotia by Sclerotium rolfsii I. Effects of varying the supply of nutrients in an agar medium. Trans. Brit. mycol. Soc. 48, 291-3°1.

(Acceptedfor publication

2

October 1964)