Characterization of the nutrients required by Botrytis cinerea to infect broad bean leaves

Physiological. Plant Pathology (1981) 19,153-167

Characterization of the nutrients required by Botrytis cinerea to infect broad bean leaves A. M.

HARPER

U R. N. STRANGE

Department of Botany and Microbiology; University Callege Landon, Gotoer Street, London WC1E 6BT, U.K.

and P.

LANGCAKE

ShellResearch Limited, ShellBiosciences Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent ME9 BAG, U.K. (Accepted faT publication April 1981)

Spores of Botryiis cinerea, suspended in sucrose solution, generally did not cause lesions on detached leaves of Vicia faba and gave rise to little mycelial growth in vitro. In contrast, the addition of Vogel's mineral salts medium to spores suspended in sucrose solution allowed the development of aggressive lesions in vivo and considerable growth in vitro. Glucose, fructose, raffinose and maltose in the presence ofVogel's medium were effective carbon sources for both virulence enhancement and growth in vitro but lactose, ribose, galactose, inositol, mannitol and sorbitol were not. Extracts of wheat germ, pollen, grapes and oranges, which allowed B. cinerea to cause aggressive lesions on leaves of V.faba, contained sugars which were active in this respect. The fungus was also versatile in its requirement for a nitrogen source, nitrate or ammonium supporting good growth in vitro and a mixture of amino acids allowing the development of aggressive lesions in vivo. Neither wheat germ extract, sucrose plus Vogel's medium nor sucrose alone desensitized germ tube extension to the inhibitory effects of wyerone acid, wyerone 01' medicarpin,

INTRODUCTION

"Chocolate spot" of broad bean is usually caused by Botrytis fabae hut under certain conditions the necrotic spreading lesions, associated with the disease caused by this fungus, may also be produced by B. cinerea. In particular, the presence of exogenous nutrients appears to be a major factor in the promotion of infection. Since the work of Chou & Preece [5], several workers have shown that pollen contains factors that enhance virulence both in this host-parasite system and others [4, 14, 18, 39], but the active components have never been defined. Recently, wheat germ, which is more readily available for experimental work than pollen, has also been found effective [34]. The aim of the investigation, therefore, was to define those components present in wheat germ and other plant extracts which enabled B. cinerea to cause spreading lesions on detached leaves of Vicia faba and to attempt to elucidate the underlying mechanism of this phenomenon.

t Present address: Haematology Department, School of Medicine, University College London, 98 Ohenies Mews, London, WCIE 6HX, U.K. :j: nee Deramo. 1981 Academic Press Inc. (London) Limited 0048-4059/81/050153+ 15 $02.00/0

e

154

A. M. Harper, R. N. Strange and P. Langcake

MATERIALS AND METHODS

Growth. of plants and culture ofB. cinerea Leaves of broad beans (Vicia faba cv. Sutton's Colossal or Exhibition Long Pod) were harvested from plants grown as previously described [34] , except that in some instances they were obtained during May and June from field-grown plants which were 6 to 8 weeks old. The latter were used exclusively in the studies ofphytoalexin accumulation. Leaves of lettuce (Lactuca sativa cv. Webbs Wonderful) and French bean (Phaseolus vulgaris cv. Canadian Wonder) were also obtained from field-grown plants which were 4 weeks old. B. cinerea was grown on agar medium and spore s from the cultures washed and harvested as previously described for Fusarium graminearum [36].

Preparation and analysis of extracts Wheat germ extract (WGE) was prepared by stirring wheat germ (Viga-Vyte, Prewett's, Stone Flour Mills, Horsham, Sussex; 40 g) with methanol (3 x200 ml ), The combined extracts were filtered through muslin and Whatman No. I filter paper on a Buchner funnel, evaporated to dryness on a rotary film evaporator (40 to 50 00) and the gum dissolved in a I : I mixture ofether and water (3 X 100 ml) , Adjustment of the pH to 4 ·5 with H 2S0 4, allowed the 2 phases to separate. The a queous fraction was washed with ether (3 x 50 ml) and the washings combined with the ether phase. Before discarding the ether fraction it was washed with water (3 X 50 ml) and the washings added to the aqueous phase. The concentration of the aqueous fraction was then adjusted to 20 mg solute ml -1 and the pH of the extract increased to 6·5 wi th NaOH. Pollen (2,5 g) from Dactylis glomerate (supplied by St Mary's Hospital Medical School, Paddington, London W.2) was extracted by a procedure similar to that used for wheat germ. Orange and grape extracts were made by squeezing fresh fruit and filtering the expressed juices. Sugars were determined by gas chromatographic analysis of trimethylsilyl derivatives [2]. Metallic cations were measured on a Unicam SP-90 atomic absorption spectrophotometer and carbon, hydrogen, nitrogen and sulphur were determined on a Perkin Elmer 240 elemental analyser. Phosphorus was estimated by the method of Saliman [32J and amino acids analyzed by a Locarte auto-analyser based on a design by Spackman et al. [33]. Virulence enhancement assay Virulence enhancement activity of extracts and solutions was determined on detached leaves under humid conditions, essentially as previously described [34]. Briefly, a dilution series of the sample to be tested was made and each dilution added to a spore suspension in McIlvaine buffer (0'01 M citric acid mixed with 0·02 M disodium hydrogen orthophosphate) or sodium hydrogen maleate-NaOH buffer (0'05 M) [6] at pH 6·5. Droplets (1,5 J1l) containing 1000 spores, buffer and test solutions or their dilutions were placed on one half of detached leaves while the other halves, which served as controls, received similar droplets containing water in place of test solutions. Five half leaves, each with 10 inoculation sites were used for each dilution of test solution, giving a total of 50 inoculation sites in all. Sites were scored for the

Characterization of nutrients

155

development of both total and spreading lesions 5 days after inoculation and the scores converted to probits [13] after correction for any control sites which developed lesions [34]. When the probit percentage lesion formation was plotted against the logarithm of the concentration oftest solution a straight line was obtained from which the ED 50 value could be determined [34]. This value, expressed as the logarithm of the dilution factor, was termed the titre of the test solution.

Measurement offungal growth in vivo and in vitro The mycelial content ofleaflesions was determined by chitin estimation and expressed as glucosamine equivalents [28]. The glucosamine values were converted to fungal mass by using a value of32 Jlg glucosamine mg r! dry wt offungus which was derived from cultures of the organism grown on Vogel's medium [38J with 2% sucrose as carbon source. Growth in shake culture was measured by nephelometry since absorbance of homogenized cultures was linearly related to mycelium weight, 1 absorbance unit being the equivalent of 0·6 mg dry wt of fungus ml -1 suspension. Erlenmeyer flasks (loa ml) containing spore suspension (5 ml ; 5 X 105 spores ml r-), buffer (5 ml; McIlvaine or sodium hydrogen maleate, pH 6'5) and nutrients (10 ml) were incubated on a reciprocal shaker (80 strokes min -1; 3 em throw) for varying periods of time at 20°C. Mycelial mats were homogenized for 60 s in a Sorvall Omnimixer (DuPont UK Ltd., Hitchin, Herts.) at three-quarters maximum speed, made up to 20 ml in distilled water and the absorbance measured on an EEL colorimeter used with white light. Assessment

ofnecrosis

Necrosis was scored on a 1 to 10 scale based on that of Mansfield and Deverall [25J (Table 1). An average figure for replicates was obtained by multiplying the number TABLE

1

Necrosis scale for scoring lesions caused hy Botrytis cinerea on broadbean leaves (qfter Mansfield and Deoerall [25]) Class

o 1 2 3 4 5 6 7 8 9 10

Lesion type No necrosis 1-12% necrosis" 13-25% necrosis" 26-50% necrosis" 51-75% necrosis" 76-99% necrosis" 100% necrosis" Spreading initiated but <1 mrn" Spreading 1 mm-<2 mrn" Spreading 2 mm-<3 mm" Spreading >3 mm"

Figures refer to area of necrosis as a percentage of the leaf area under the Inoculum droplet. b Figures refer to the distance between. the periphery of the inoculum droplet and the periphery of the lesion.

a

156

A. M. Harper, R. N. Strange and P. Langcake

of lesions in each class by the class number and dividing the sum of these by the number of inoculation sites.

Phytoalexin accumulation in lesions Leaf discs bearing lesions (0,5 to 1·0 g) were excised with a No.3 cork borer (6 rom diameter) and the tissue ground with a pestle and mortar in 70% methanol (10 ml). After centrifuging, the supernatant was evaporated to dryness on a rotary evaporator at 30 °0 and taken up in an equal mixture of water and ether (5 ml). The pH was adjusted to 4·5 with H SP04 and the aqueous phase partitioned twice more against ether (2 x5 ml), The combined ether phases were washed with 5 rnl H 20 and the ether fraction dried in a current ofNz' The residue was dissolved in methanol (1 g fresh tissue ml -1) and assayed by high performance liquid chromatography for wyerone acid and wyerone on a column (25 X 0-45 em Ld.) of Hypersil ODS (5 11m particle size, Anachem Ltd., Luton, Bedfordshire) using methylcyanide: 1 % formic acid (1: 1) as mobile phase with a flow rate of2 ml min -1. The eluate was monitored with a Pye Unlearn LC-UV monitor (Pye Unicam Ltd., Cambridge) fitted with a microflow cell (8 111), operating at 350 nm, the absorbance being relayed to a Tekman recorder (Tekman Electronics Ltd., Bicester, Oxfordshire), 10mV full-scale deflection. Wyerone and wyerone acid concentrations were calculated from their peak areas relative to those of a quantitative internal standard (butyrophenone, which was included with the samples) after correction for the relative detector response to the compounds.

Isolation if phytoalexins Crude phytoalexin preparations were obtained from pod endocarp inoculated with

B. cinerea by the method of Letcher et al. [23]. The crude extracts were fractionated by chromatography on a column (100)< 3 em) of Sephadex LH20 with 95 % ethanol as eluant. Fractions were chromatographed on silica gel thin-layer chromatography using hexane : acetone (2: 1) followed by CHels : petroleum spirit (2 : 1) as solvents [17] and zones of antifungal activity located on test strips by spraying the plates with a thick spore suspension of Cladosporium cucumerinum in double strength Czapek-Dox broth before incubating at 25°C in moist conditions for 48 h [1]. Further purification of the Sephadex fractions was achieved on a column of silica gel (100-200 mesh) (25)< 1·5 ern) eluted sequentially with hexane, hexane: acetone (99 : 1, 98 : 2, 95 : 5, 90 : la, 75 : 25, 50 : 50) and acetone. Stock solutions of the phytoalexins were prepared in methanol and stored at -20°C. Concentrations were determined using published extinction coefficients [12, 15,23J.

Spore germination in thepresence oj phytoalexins Inoculum mixture (10 ul) containing nutrient (5 mg solute ml r-), McIlvaine buffer (pH 4·5 or 6,5) and spore suspension (5 X 104 spores ml -1) in the ratio 2 : 1 : 1, was placed in the wells of microtest trays [W. Sarstedt (UK) Ltd., 165 Scudarnore Road, LeicesterJ and the lids lined with damp filter paper to prevent evaporation of the droplets. Phytoalexins in 2% methanol (10 111) were added either immediately or after an incubation period of 15 h at 20 00. Spores were scored for germination after

Characterization of nutrients

157

incubation for 15 h and were considered germinated if their germ tubes exceeded one spore length. Lengths of hyphae were determined immediately before and 8 h after the addition of phytoalexin using a X 10 objective and a graticule eyepiece ( X 10) and the percentage inhibition of germ tube elongation calculated by the following equation:

C-x

- - X 100 = percentage inhibition

C-Cl

where C=the final length of germ tubes incubated in nutrients, buffer and distilled water, X=the final length of germ tubes incubated in nutrients, buffer and phytoalexin and C1 =the length of germ tubes at the time of phytoalexin addition. RESULTS

The effect of plant extracts andJungal culture media on the virulence and growth in vitro of B. cinerea Extracts of wheat germ, pollen, grapes and oranges, when added to spore suspensions of B. cinerea in maleate buffer, caused the development oflesions, many of which. were of the spreading type, on detached leaves of V.Jaba (Table 2). Fungal culture media were also effective, the highest titre for spreading lesions being given by Vogel's medium (Vg) with 2% sucrose as carbon source (SVg) (Table 2). This medium also TAllLE

2

The development of lesions on detached leaves tifVicia faba in response to inoculation with aqueous spore suspensions ofBotrytis cinerea supplemented with plant extracts orfungal culture media Extract" or Culture medium"

pH

Wheat germ Pollen Grape Orange Gzapek-Dox Medium XJ SVg Sucrose Vg

6·5 6·5 6·5 6·5 6·6 6·2 6·3 6·5 6·0

Total lesions Maximum response" Titre" (%)

1·11 1·07 1·29 1·35 1040 1'72 1'27 <0·27· <0·00

95 96 96 98 89 90 98 55 5

Spreading lesions Maximum response" Titre" (%)

0·59 0·51 0·4{) 0'67 <0·37" <0·38" 0·84 <0·00 <0·00

72 69 63 80 59 56 71 34 1

"Plant extracts at pH 6'5 contained 20 mg solute ml- 1 and were buffered with 0·05 M sodium hydrogen maleate-NaOH at pH 6·5. b All culture media apart from Vg contained 20 mg sugar ml (Ozapek-Dox contains sucrose, medium contains glucose), and were buffered between pH 6·0 and 6·6 with 0'05 M sodium hydrogen maleate-NaOH. " Titres are expressed as the logarithm of the dilution factor of an extract which gave rise to lesions at 50% of the inoculation sites after correcting for any lesions that occurred on control half leaves. Each figure is the mean of 2 replicates. d Maximum response is the highest number of lesions developed for any dilution of an extract expressed as a percentage of the number of inoculation sites after correction for controls as in note c. Each figure is the mean of 2 replicates. " Value of one replicate <0·00 but taken as 0·00 for calculation of mean. r Last and Hamley's medium X [22]. r

',

A. M. Harper, R. N. Strange and P. Lanqcake

158

stimulated limited lesion formation by B. cinerea on lettuce leaves and spreading lesions on French bean leaves. The activity of sucrose in virulence enhancement was found to be variable and periodically, high lesion titres resulted from inoculum droplets containing sucrose and McIlvaine buffer. Titres were reduced, however, when maleate buffer was used (Table 3). It appeared, therefore, that in the presence of sucrose the constituents of McIlvaine buffer, disodium hydrogen orthophosphate and citric acid promoted lesion development. Titres for WGE were also lower in maleate compared with McIlvaine buffer but this was not so for SVg which contained sodium, phosphate and citrate ions (Table 3). The importance of KHaP04 and NH4NO a in both virulence enhancement and growth in vitro of B. cinerea was established by separately omitting each of the constituent salts from complete SVg (Table 4). In vivo, the requirement for NH 4NO s TABLE

The

effe~t

3

if Mclluaine and maleate brdJer on the development if lesions caused by aqueous spore suspensions ofBotrytis cinerea supplemented with WGE, sucrose orSVg

Inoculum supplement

Buffer (pH 6'5)

WGE"

McIlvaine Maleate McIlvaine Maleate McIlvaine Maleate

Sucrose" SVg

Total lesions Maximum response" Titre" ('Yo) 1·44 0·98 0·76 <0·00 1·09 1·28

90 88 76 31 87 97

Spreading lesions Maximum response" Titre" ('Yo) 0·70 0·35 <0.34 8 <0·00 0'67 0·63

68 72 44 11

71 66

" 20 mg solute ml- 1 • b See note a afTable 2. "See note d of Table 2. 8 See note e of Table 2. TABLE 4 The effect of the omission of single components if SVg on the development if lesions on detached leavesqfVicia faba in response toinoculation with Botrytis cinerea compared with theyield if the fungusgrown in vitro onthe same medium

Inoculum supplement"

b

WGE Sucrose SVg SVg less NH.NO a SVg less KHnP0 4 SVg lesssodium citrate SVg less biotin SVg less MgSO. SVg less c,or, .~.

I

Total lesions Maximum response/ Titre" (%) 0·98 <0·00 1·28 0'30 0'65 1·00 0·86 0·92 1'l6

88 31 97 59 76 94 84 89 92

Spreading lesions Maximum Yield in vitro' response" (mgper20 ml culture medium) Titre" (%) 0·35 <0·00 0·63 <0·00 <0·00 0·82 <0·00 <0'09" <0·47"

71 11

48·0

66

77-0

15

2-4 2·4 44-0 60·0 2·4 77·0

32 84 48 53 76

2-4

See notes a to e respectively of Table 2. Fungus was grown for 3 days in shake culture at 20°C. Each figure is the mean of 2 replicates.

159

Characterization of nutrients

could be satisfied by a mixture of amino acids (Table 5), whereas, although MgS0 4 was essential for growth in vitro and for ,the development of spreading lesions, the formation of limited lesions was only partially reduced in its absence (Table 4). Of the other constituents of SVg, biotin appeared to be necessary for lesion formation but not for growth. In contrast, omission of sodium citrate had little effect on lesion titres but did reduce the yield of the fungus in vitro. Removal of calcium chloride from the medium had no effect on growth or total lesion titres and a variable effect on spreading lesion titres. No individual salt was consistently found to replace the complete salt mixture (results not shown). In further experiments

TABLE

5

The ejfect if replacement of NH.NO s with amine acids in SVg 011 the development of lesions on detachedleaves ofVieia faba in responseto inoculation with aqueous sporesuspensions qfBotrytis

cinerea Total lesions Maximum Inoculum supplement"

Titre"

(%)

Spreading lesions Maximum response" TitreC (%)

1·11 <0·27' 1·27 0·93 1·35 0·44

95 55 98 94 99 62

0·59 <0·00 0·84 0·42 0·83 <0·00

response'[ b

Wheat germ Sucrose SVg SVg less NH,NO s SVg less NH,NOs+amino acids' Vg less NH4NO a+ amino acids'

72 34 71 60 88 18

.-, See notes a to e respectively of Table 2. r Amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine) Nitrogen content ssnitrogen content of NH,NO s in SVg (i.e, 0·7 mg ml- 1 ) . TABLE

6

The effect of omission of single ionsfrom Vg on the growth ofBotrytis cinerea in shake culture

Inoculum supplement"

Buffer

SVg SVg less NH,NO s SVg less NH, + (+0·058% Na +) SVg less NOs- (+0'089% 01-) SVg less KH~PO, SVg less K+ (+0·085% Na +) SVg less PO.a- (+0·13% 01-) SVg less MgSO, SVg less MgH (+0-0042% Na+) SVg less SO,3- (+0'0056% 01-) SVg + 0·085% Na + +0,36% PO,oSVg + 0'13% 01-+0·14% K+ SVg SVg less POl- (+0·13% 01-)

McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine McIlvaine Maleate Maleate

• All culture media contained 20 mg sucrose ml- 1 • b See noteJofTable 4.

Yield in vitrob (mgper20ml culture medium)

77-0 2·458·0

88·0 2·42·458·0 2·4 2·4 +8 59·0 76·0 52·0 2·4

A. M. Harper, R. N. Strange and P. Langcake

160

in shake culture, individual ions were omitted from the complete mixture and replaced with either sodium or chloride ions. Little growth occurred when either potassium, magnesium, sulphate or phosphate ions were omitted, but the fungus grew well when NH4NO a was substituted by either NH4Cl or NaNO a (Table 6).

The effect of sugars on the virulence and growth in vitro qfB . cinerea In the presence of McIlvaine buffer, glucose, fructose, raffinose and maltose, as well as sucrose, promoted the virulence of B. cinerea. A further increase in lesion titres was obtained when these sugars were supplemented with Vg and these combinations allowed good growth of the fungus in vitro (Table 7). Lactose, sorbitol, mannitol, galactose, inositol and ribose were inactive in vivo and in vitro whether or not they were combined with Vg (Table 7).

TABLE

7

The development oflesions on detached leaves qfVicia faba in rtsponSt to inoculation with aqueous sport suspensions ofBotrytis cinerea supplemented with sugars together with Vg compared with the yield of thefungus grown in vitro on the samemedia Total lesions Maximum response" T itre" (%)

Inoculum supplemen t"

1·58 1·31 1·38 1·25 1·10

Sucrose-l-Vg Glucose-l-Vg Fructose-]- Vg

Raffinose+ Vg Maltose-l-Vg Lactose, sorbitol, mannitol, galactose, inositol, ribose + Vg

<0·00

Spreading lesions Maximum Yield in uitro" response" (mg per 20 ml Titre b culture medium ) (%)

100 94: 79 87 100

1·14 0·91 0·52 0'82 <0·00

91 81 53 79 46

34-0 23·0 23·0 23·0 35·0

16-47

<0 ·00

0-40

<2 '0

" All culture media contained 20 mg sugar ml- 1 and inocul a were buffered in McIlvaine buffer, 0·01 M, pH 6'5 . be See notes c and d respectively of Table 2. d See notef of Table 4:. TABLE

8

Tile sugar constituents of WGE, pollen, grape and orange extracts

Extract

Sucrose

WGE Pollen Grape Orange

440

0

0

380

78 460 260

140

±c

360

Glucose

Sugar (mg g -l dry wt)" Mannitol Fructose Inositol or sorbitol" Raffinose

520 240

±c 100 0 0

52 0 0 0

• Each value is the mean of 2 determinations. t ThC3e compounds were not separated on the gas chromatograph. C ±, Compound barely detected.

280 0

0 0

Total 772 698 980 860

o

::T

~

D>

....~

j;jIII

~.

a

:J

o

:J C

9 AnalYsis of extracts compared withthecompositirm rifdefined media TABLE

S.

(l)

:J

ih Component (mg

ml- 1 )

Total Amino Sugar Extract (20 mg ml-~) WGE Pollen Grape Orange Media (20 mg ml- 1 sugar) SVg Medium Xb Czapek-Dox 4

b

N

N

P

Mg

16 14 19 16

0-28 0-048 0-05 0-002 0-36 0·098 0-09 0·002 0-07 0·032 0-06 0-002 0-18 0-114 0-12 0·008

20 20 20

0-70 0 2-00 0 0·22 0

HO 0-02 0·68 0·10 0·05 0-04

S

Ca

K

0-12 0·002 0·23 0'15 0'005 0'51 0 0·010 0-21 0 0·029 0'39

Na

0·10 0-44 0·340'45

Cl Citrate Ash Total

.:»

-

_4

-

.:» .:»

a a

-•

_4

0·03 0-030 1·40 0·70 0-05 1·93 0·14- 0 1-40 3'20 0·50 0 0·04- 0 0·28 0-36 0·16 0

1·9 0 1·0 0

18-7 15·6 20·7 17-2 26·0 28·0 21·2

Not determined. Last and Hamley's medium X [22].

en .....

A. M. Harper, R. N. Strange and P. Langcake

162

Analysis if the sugar and elemental composition of plant extracts which cause virulence enhancement Sugars were the major constituents of WGE, pollen, grape and orange extracts and the predominant ones, sucrose, glucose, fructose and raffinose, were those which caused virulence enhancement as well as good growth in vitro (compare Tables 7 and 8). The elements necessary for virulence enhancement and growth in vitro implicated by the data in Table 6 were nitrogen, phosphorus (as phosphate), magnesium, sulphur (as sulphate) and potassium. In general plant extracts had lower concentrations of these elements than the 3 defined media, SVg, Czapek-Dox and Last and Hamley's medium X (Table 9). Grape extract was particularly deficient in nitrogen. Sulphuric acid was added to WGE and pollen in the extraction procedure and notably these extracts, but not those from grapes and oranges, contained sulphur. The growth if B. cinerea in vivo in relation to inoculum nutrition, necrosis and phytoalexin accumulation Necrosis developed rapidly from inocula supplemented with SVg or WGE compared with those suspended in sucrose or distilled water and high necrosis scores were associated with high values for mycelial weight (Fig. I). Wyerone acid was the major phytoalexin in lesions and concentrations increased as necrosis developed while lesions remained limited. In the absence of nutrients no lesions were formed and no phytoalexins accumulated [Fig. I (a)]. When sucrose was included with the inoculum, wyerone acid accumulated until the 3rd day post-inoculation and dropped thereafter [Fig. I (b) J. A similar but much earlier rise and fall was noted with SVg and a much to

20

·10 ( b)

(0 )

8

8

-;- 6 ::l ~

~

T

'" '"

5.

4

.

0 10

!9 g' 8 ,a 0

i ~ C>

4

2.

6 4

2.

0

/ - ".--..i>• y'"

."......-

2. III

';;;

..e 0

z

•

(c)

8 6

.

/~. / .

6

III

s: III

16

•

./

-

2

.-. 3

4

5

8

s:

:fl

4 r

(d)

•6

::l III

.!!!

./::

0 20

.~/' --.-....

Q;

12

16

12 8

'"

'":l. c: ';< .,

I"5'0 "C

0;

>= 4

3

4

5

6

0

Days after inoculation

FIG. 1. Changes in necrosis (.4), fungus content (e) and wyerone acid concentration (.) in lesions. Inocula consisted of spore suspension in McIlvaine buffer pH 6·5 supplemented with distilled water (a); sucrose (20 mg ml- 1 ) (b); SVg (c) and WOE (20 mg ml- 1) (d).

163

Characterization of nutrients

smaller but also early response with WGE [Fig. I (c) and (d)]. These data suggest the more rapid the growth of the fungus the quicker the accumulation and dissipation of wyerone acid. The level of wyerone acid detected, however, was very low (a maximum of20 J..lg g-l fresh tissue) compared to the values (100 to 200 Jlg g-1 fresh tissue) obtained by Hargreaves et al. [17]. When the same experiment was repeated with field-grown leaves, the rate of lesion development was slower, sucrose did not stimulate spreading lesion formation and higher maximum concentrations of wyerone acid were recorded (100 to 150 Jlg g -1 fresh tissue). AJ; in the previous experiments, the wyerone acid content increased with necrosis while lesions remained limited but only low concentrations were found in spreading lesions. Again, very little necrosis resulted when nutrients were absent.

The effect of nutrients on the inhibition of B. cinerea in vitro by broad bean phytoalexins Wyerone and medicarpin at concentrations up to 20 and 50 ug ml -1 respectively (bioassayed at pH 6,5) were ineffective inhibitors of spore germination in WGE (5 mg ml r-), In contrast concentrations of >4 Jlg ml r! wyeroneand >10 ug 00-1 medicarpin strongly inhibited spore germination in sucrose or SVg (Fig. 2). As

100 (0)

80 60 40 20 °0

...

_-"--

II

-~

,,~.,. -.--. 4

8

60

E

40

~ s: C. .c

.a

.Q

g

0

£

20

°0

==\•~10 20 30 40 50

__

.66 .,.?--I ...llrf . ./'" ~._/

'i 20

e

'" Z 400

.~

"" '" "" ~ e

-66

..t::.

Ql

(b)

IDa 80

12 16 20 ~

c

III

400

............

00

4

8

12 16 20

.a

E 100 ~ 80 'l5 .~

-59 -58

:13?7~1

:B :E

.S

'" '" e ~

(c)

'"

I

10 20 30 40 50 Concentration of phyloole~in (1"9

mrl)

FIG. 2. Inhibition of spore germination (I) or germ tube growth of pregerminated spores (II and III) in the presence of Sucrose (5 mg ml- 1 ) ( . ) ; SVgj4:, i.e. quarter strength (e). and WGE (5 mg ml r") (A). Wyerone (a) and medicarpin (b) assays were buffered at pH 6·5 and wyerone acid (c) assays at pH 4·5 with McIlvaine. Figures on Graphs III indicate average length of germ tubes (J.UIl) at the time of phytoalexin addition.

164

A. M. Harper, R. N. Strange and P. Langcake

wyerone acid was inactive at pH 6·5 (results not shown) in agreement with the findings of Deverall & Rogers [8], its activity was additionally determined at pH 4·5. A concentration of >30 J1.g ml-1 wyerone acid strongly inhibited germination in all 3 nutrients. Only small differences existed between the percentage inhibition of germ tube growth of pregerrninated spores by wyerone and medicarpin at pH 6·5, or wyerone acid at pH 6'5 (results not shown), or pH 4·5 in WGE, sucrose or SVG. In terms of absolute growth, however, WGE stimulated a much greater amount of growth than sucrose or SVg after 24 h (Fig. 2). DISCUSSION

In nature, B. cinerea is capable of growing on a variety of moribund tissues from a wide range of plant species, the colonized tissue serving as a base from which contiguous live tissue may be attacked [21]. Examples include wounded cabbage leaves [40], anthers on strawberry fruits [5] and flower remains on tobacco [18], onion [11], apple [37], snap bean [3] and Dendrobium [20]. Similarly, previous experiments showed that the presence of partially extruded anthers were crucial for the infection of wheat by Fusarium graminearum [35]. In this host-parasite system choline and betaine were identified as the compounds in anthers which promoted not only growth of the parasite in vitro but also in vivo. By contrast, in the present study no specific factors were found to be essential for growth and virulence of B. cinerea. Instead, infection and limited lesion production occurred when inocula were supplemented with a variety of simple sugars (sucrose, glucose, fructose, raffinose or maltose) recalling the recent work of Rossall & Mansfield [30]. They found that germination of spores and hyphal extension of B. cinerea were inhibited on the surface of V.faba leaves and demonstrated that the main cause was the epiphytic microflora while a minor cause was an unidentified inhibitor which accumulated in inoculum droplets. The inhibition was reversed by the addition of glucose to the inoculum droplets. In our experiments, when Vogel's saIts were added to inocula in addition to an appropriate sugar the number of successful infections was increased, and the fungus also grew well in vitro. Higher concentrations of nutrients but no additional compounds were required for the production of spreading as opposed to limited lesions. Four plant extracts which promoted virulence contained sugars which caused virulence enhancement when supplemented with Vogel's salts. Thus it seems that B. cinerea is nutritionally opportunistic, a range of sugars commonly encountered in plants serving as suitable carbon sources for growth in both saprophytic and parasitic modes. The avirulence of B. cinerea for leaves of V. faba has often been contrasted with the virulence ofBifabae for the same tissue [17]. Reasons for this difference have been variously attributed to the larger spore size of B.fabae [27], its relative insensitivity to wyerone acid [7, 9, 10, 15, 16, 17, 29, 31] and its greater ability to degrade this phytoalexin [10, 26], an effect which was subsequently shown to be more apparent than real [29]. Very recently the virulence of the 2 species has been closely correlated with the number of epidermal cells killed between 7·5 and 8'5 h after inoculation [19]. The findings of Chou & Preece [5] that B. cinerea becomes as virulent as B.fabae when inocula were supplemented with pollen diffusates not only prompted questions concerning the nature of the active principle (discussed above) but also the

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mechanism by which it acted. Mansfield & Deverall [24] confirmed that the addition of pollen diffusate to inocula of B. cinerea caused the fungus to become extremely aggressive but they found higher concentrations ofwyerone acid in aggressive lesions compared with the limited lesions resulting from inoculation with spores suspended in distilled water. They suggested that B. cinerea was rendered insensitive to wyerone acid by the pollen diffusate and corroborated this interpretation by experiments ill vitro in which they showed that far higher concentrations of wyerone acid were required to inhibit spore germination and genu tube extension in the presence of pollen diffusate than in water. In our experiments the addition of nutrients to inocula profoundly affected the concentration ofwyerone acid produced. Spores suspended in water did not cause the accumulation of appreciable concentrations of the phytoalexin, possibly because they were inhibited on the leaf surface [30] but spores suspended in the 3 nutrients all caused increases. The increase was small for spores suspended in WOE and had fallen by the 2nd day after inoculation. Similar results occurred with spores suspended in SVg but the level of wyerone acid reached 1 day after inoculation was higher. A more sustained increase in wyerone acid accumulation was caused by inocula suspended in sucrose but levels fell again by the 4th day after inoculation. In all 3 instan~es when nutrients were added to inocula wyerone acid concentrations fell just before the time at which lesions became aggressive. The reasons for this fall are unknown but could be the result of metabolism by the fungus or host and possibly suppression of elicitation. The latter might be caused by the killing of host cells in line with the observations of Hutson and Mansfield on B.fabae [19]. Studies in uitro showed that although WOE overcame the inhibition of spore germination caused by wyerone and medicarpin it did not do so when the inhibition was caused by wyerone acid buffered at pH 4·5. None of the nutrients reversed the inhibition of germ tube extension caused by the 3 phytoalexins, although germ tubes in WOE grew more extensively than those in the other 2 nutrients. These results, therefore, do not support the concept that nutrients may desensitise spores and germIings to inhibition by wyerone acid. Our experiments suggest that the conclusion of Yoder & Whalen [40] that "nutrients appeared to be the key ... for infection" by B. cinerea is correct and have shown that these nutrients may be simple and various. Possibly the reason for their effect on virulence is to allow the fungus to synthesize a sufficient amount of toxin to kill surrounding cells. These dead cells would provide further nutrients and allow the synthesis of more toxin thus explaining the non limited lesions formed in the presence of nutrients. A similar reasoning may apply to infections of B. fabae but here the spore which is larger than that of B. cinerea may have sufficient endogenous reserves necessary for the synthesis and export of the proposed toxin in amounts capable of killing host cells. One unexplained result is the much greater resistance of field-grown plants compared with greenhouse-grown ones. This appears to be related to the greater accumulation of wyerone acid in the former but the reason for this is unknown. We wish to thank the SRC for a grant which enabled us to purchase high performance liquid chromatography apparatus and for a studentship held by one of us (AMH).

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