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The influence of cotyledons, roots and leaves on the susceptibility of hypocotyls of bean (Phaseolus vulgaris) to compatible races of Colletotrichum lindemuthianum
Physiological
Plant Pathology
(1963)
23,245-256
The influence of cotyledons, roots and leaves on the susceptibility of hypocotyls of bean (Phaseolus vulgaris) to compatible races of Colletotrichum lindemuthianum P. M.
ROWELL
and J. A.
Long Ashton Research Station, (Acceptedfor
publication
March
BAILEY
University
of Bristol,
Long Ashton,
Bristol,
BS18 9AF
1983)
Hypocotyl segments which were excised from bean seedlings supported extensive biotrophic colonization by compatible races of Colletotrichum lindemuthianum. Subsequent death of infected tissues was followed by production of small amounts of phytoalexins, water-soaked spreading lesions and eventual complete rotting of the hypocotyls. When cotyledons or, to a lesser extent, leaves, were retained the hypocotyls did not rot. Superficial flecking occurred on segments which retained only their cotyledons, whilst large restricted lesions occurred when roots and cotyledons or only leaves were retained. Greatest concentrations of the phytoalexins, phaseollin, phaseollidin and phaseollinisoflavan, occurred in hypocotyls bearing cotyledons. Light also enhanced the production of these compounds. However, in contrast to previous results, phaseollinisoflavan was obtained from both Colletotrichum and tobacco necrosis virus-infected hypocotyls maintained in total darkness. It is suggested that symptom development is controlled by complex interactions between the extent of biotrophic colonization, which appears to be enhanced by roots but reduced by cotyledons, by the ability of necrotrophic hyphae to kill tissues quickly and hence reduce the production of phytoalexins, and by the ability of the healthy tissues which surround lesions to produce large quantities of phytoalexins, a process enhanced by both cotyledons and leaves.
INTRODUCTION Colletottichum lindemuthianum
(Sacc. et Magn.) Bri. et Cav. is a facultative biotrophic (hemibiotroph) [4]. On hypocotyls of susceptible cultivars, compatible races are capable ofestablishing biotrophic infection hyphae within epidermal cells and later within underlying cortical cells. The period of biotrophy can extend for several days, after which, the infected tissues die, and dark brown lesions appear. These may become water-soaked and the entire hypocotyl soon rots. This extreme symptom of susceptibility occurs on young hypocotyls following excision above the roots and below the cotyledons, and incubation in diffuse light or darkness at temperatures between 15 and 18 “C [ 2, 51. Under other conditions the symptoms are more variable and often much less extensive. For example, in race surveys using intact plants [IO], or with hypocotyls incubated at temperatures above 22 “C [5, 131, symptoms of resistance, i.e.
fungus
localized
flecking
or limited
lesions,
have
been
reported.
Similarly,
a great
range
of
symptoms can occur on different aged hypocotyls and at different positions on the same hypocotyl ([13, 14; see also research with hypocotyls of Glycine max inoculated with Phytophthora megasperma var. so@ [II, 281). 0048-4059/83/050245
+ 12 so3.00/0
0
1983 Academic
Press Inc.
(London)
Limited
246
P. M. Rowe11 and J. A. Bailey
The mechanisms controlling the intercellular growth of C. lindemzithianum in susceptible cultivars and the factors influencing symptom expression are not well understood, although the accumulation of phytoalexins to fungitoxic concentrations and the increased resistance to penetration of cell walls of older tissues probably contribute to cessation of fungal growth in limited lesions [I]. The present paper examines the influence of cotyledons, leaves, roots and light on infection of bean hypocotyls by a compatible race of C. lindemuthianum, including effects on biotrophy, expression of symptoms and accumulation of phytoalexins. A previous paper showed that virusinfected hypocotyls which retained cotyledons and were incubated in light produced most phytoalexin [26].
MATERIALS AND METHODS Growth of plants andfungi Seedlings of Phaseolus vulgaris
L. cvs Kievitsboon Koekoek, Tendergreen and Tendercrop were grown in sterilized Vermiculite either in an illuminated cabinet (Fisons 9600), at 25 “C, rh 80% and periods of light ( 16 h, 40 w m-2 s-r) and darkness (8 h) for 6 to 7 days so that the primary leaves had emerged from between the cotyledons but had not expanded, or in darkness in a dark room protected by a light trap [I61 at 22°C for 6 or 7 days. Seeds of cv. Kievitsboon Koekoek were harvested from plants grown at Long Ashton Research Station, those of Tendergreen and Tendercrop were a gift from Charles Sharpe and Co. Ltd., Sleaford, Lines. Only seedlings with two undamaged cotyledons were used. Seedlings were removed from the Vermiculite and used intact. Hypocotyl segments were obtained by excising the seedlings 3 cm above the roots and 1 cm above the cotyledonary node. All cut surfaces were sealed with wax. Seedlings or hypocotyl segments were placed on their sides in transparent plastic boxes and the central areas of the hypocotyl were inoculated with five drops (5 fl) of a spore suspension (5 x lo5 spore ml-‘), each placed 10 mm apart. When roots were present they were enclosed in moist tissue paper. The p and Y races of C. lindemuthianum were used; their growth and sporulation have been described [Z]. Tissues were macerated in ethanol and after filtration and solvent evaporation the ethanol-soluble material was subjected to thin-layer chromatography (tic) using SiGel 60 (Merck 5715) developed in ethanol :chloroform (100 :3 v/v) [Z]. This achieved separation of phaseollin, phaseollidin and phaseollinisoflavan, so that their concentrations could be measured spectrophotometrically [Z].
RESULTS E$ect of cotyledons on symptoms expressed on bean hypocotyl segments
Hypocotyl segments were obtained as described above. Both cotyledons were removed from half the segments, and the symptoms produced on entire segments and on segments without cotyledons were compared. Symptoms on hypocotyls of cv. Kievitsboon Koekoek inoculated with race Y, with or without cotyledons are shown in Fig. 1. Large ( > 10 mm diameter) dark brown water-soaked lesions, which spread to cause complete rotting after 14 days, occurred
Susceptibility
of hypocotyls
247
of bean
FIG. 1. Symptoms on hypocotyls of cv. Kievitsboon Koekoek, with C. lindemuthianum, race Y. (a) Severe symptoms on excised lacked cotyledons, roots and leaves; (b) limited flecking on segments All hypocotyls were trimmed immediately before being photographed.
10 days after inoculation hypocotyl segments which which retained cotyledons.
on hypocotyls without cotyledons. The symptoms on segments with their cotyledons varied from small (1 to 3 mm) limited lesions to a scattering of isolated necrotic cells, and had the appearance of an incompatible (hypersensitive) reaction [17]. Cotyledons also decreased symptom development on other cultivars. After inoculation with race f-?,lesions on hypocotyls of cv. Tendercrop lacking cotyledons were paler brown than on cv. Kievitsboon Koekoek and coalesced rapidly causing the entire hypocotyl to rot within 12 days. When cotyledons were present, symptoms were restricted to small (5 to 10 mm) dark brown lesions. Cultivar Tendergreen was more resistant to rotting than the other cultivars tested. In the absence of cotyledons, lesions due to race b were dark brown, turning black and some (30%) became restricted in size (10 to 15 mm), so that healthy tissue remained between the lesions: despite this many hypocotyls rotted. In the presence of cotyledons the lesions were smaller (1 to 5 mm) and no hypocotyls rotted. Thus in these three cultivars, the presence of cotyledons decreased disease development. The mechanisms responsible for enhanced resistance were examined in the following experiments.
248
P. M. Rowell
and J. A. Bailey
EJect of removitlg cotyledons on the production
of phytoalexins by bean hypocotyls infected with C. lindemuthianum Hypocotyl segments of cv. Kievitsboon Koekoek were inoculated with race 7. Cotyledons were removed as required and the development of symptoms and the concentrations of phaseollin, phaseollidin and phaseollinisoflavan which had accumulated in the infected tissue were assessed. Results are presented in Table 1. Symptoms developed as in previous experiments: on hypocotyls without cotyledons, symptoms were visible as dark brown spreading lesions; but when cotyledons were not removed only flecking occurred (see Fig. 1) .
Effect
of removing
cotyltdons
TABLE 1 on accumulation of phytoaltxinr in bean hypocotyl compatible TUCEof C. lindemuthianum
segments infected with n
Phytoalexin concentrations (pg g-’ ‘infected tissue) Period of incubation (days) 9
12 14
Treatment + + + -
Cotyledons Cotyledons Cotyledons Cotyledons Cotyledons Cotyledons
Mean fresh weight of infected tissue (mg) 9.4 30.0 7.1 35.7 7.2 27.1
Symptomsa 1 3 1 3 l,L 3s
Phaseollin 382 80 742 111 439 125
Phaseollidin 166 28 195 14 290 8
Phaseollinisoflavan 117 3 445 7 644 9
Hypocotyl segments ofcv. Kiev&boon Koekoek, obtained by cutting above the roots and above the cotyledonary node, were inoculated with race y. The cotyledons were immediately removed from halfthe segments and at intervals symptoms were recorded, infected tissue was excised and the concentrations of phytoalexins were determined. ‘Symptom categories are: 1, single or groups of scattered brown cells; 3, lesions larger than site of inoculation. L and S indicate, respectively, that symptoms were limited in extent or continued to spread.
Greatest concentrations of all phytoalexins occurred in hypocotyls with cotyledons, i.e. in the more resistant tissues. When the total amounts of the three phytoalexins are considered, their concentrations after 9, 12 and 14 days were 111, 132 and 142 Ng-1 tissue in hypocotyls without cotyledons and 665, 1382 and 1373 pgg-’ tissue in hypocotyls with cotyledons. In most hypocotyls, the concentrations of each phytoalexin increaased during the incubation period, but occasionally, e.g. phaseollin in hypocotyls bearing cotyledons, their concentrations seemed lower after 14 days than after 12 days. Effect of removing cotyledons, at intervals after inoculation, segments to C. lindemuthianum
Hypocotyl incubated. Symptoms terized as
on the susceptibility
of hypocotyl
segments of cv. Kievitsboon Koekoek were inoculated with race Y and Both cotyledons were removed from some of them at different intervals. were assessed6, 7.5, 8.5 and 10 days after inoculation and were characfollows: 1, single or groups of scattered brown cells (small flecks) ; 2,
Susceptibility Effect
of hypocotyls
of removing cotyledons,
Period of incubation (days)
of bean TABLE 2 at intmrals after inoculation, on the susceptibility to a compatible race ofC. lindemuthianum
Time after inoculation when cotyledons removed (h) 0 12 36 60
6.0 Cotyledons
retained 0 12 36 60
7.5 Cotyledons
retained 0 12 36 60
8.5 Cotyledons
retained 0 12 36 60
IO.0 Cotyledons
249
retained
No. of inoculation sites showing symptom? 5/40 14 19 10 25 15 31 34 35 38 29 36 38 36 38 36 39 38 36 39
of bean hypocotyl
segments
No. of sites showin i as: symptoms categorized 1 2 3 5 14 19 10 25 0 8 26 25 14 5 7 0 18 11 0 0 0 5,L 11,L
0 0 0 0 0 3 6 0 10 24 2 4 12 18 27 0 0 12,L 31,L 28,L
0 0 0 0 0 12 17 8 0 0 22 25 26 0 0 36,s 39,s 26,L 0 0
Hypocotyl segments of cv. Kievitsboon Koekoek, obtained by excision above the roots and above the cotyledonary node were inoculated with race 7. At intervals both cotyledons were removed and symptoms subsequently recorded. aFive sites on eight hypocotyls. ‘Symptom categories are: 1, single or groups of scattered brown cells; 2, confluent areas of necrosis within the site of inoculation; 3, lesions larger than site of inoculation. L and S, respectively, indicate that symptoms were limited in extent or continued to spread.
confluent brown areas within the initial sites of inoculation, which were approximately 3 mm long (large flecks) ; 3, lesions larger than the site of inoculation. The eventual limited or spreading nature of the infections was also recorded. Results are shown in Table 2. They confirm that the development of lesions in hypocotyls with cotyledons was restricted, whilst hypocotyls whose cotyledons had been removed at the time of inoculation eventually rotted due to the presence of water-soaked spreading lesions. Spreading lesions and subsequent rotting also occurred on hypocotyls from which cotyledons were removed 12 h after inoculation, but removal thereafter, did not prevent the restriction in lesion development and the hypocotyls did not rot. These results also indicate that symptoms became visible on hypocotyls with cotyledons before those on hypocotyls without cotyledons. For example, after 6 days, 62.5% of sites on hypocotyls with cotyledons showed symptoms, whereas symptoms were present on only 12.5% of sites on hypocotyls without cotyledons.
P. M. Rowell
250 TABLE Effect
of removing cotyledons, at inkwals
Time after inoculation when cotyledons removed (h) 0 12 30 60 72 Cotyledons
retained
Mean fresh weight of infected tissue (4 30.0 35.0 12.4 8.9 9.5 9.4
3
af&r inoculation,
segments infected with a compatible
and J. A. Bailey
on accumulation of phaseollin race of C. lindemuthianum
Symptoms
in bean hypocotyl
Phaseollin concentration ( pgg-1 infected tissue) 79 119 177 198 406 382
3,s 3s I-2,L l,L 1,L l,L
Hypocotyl segments of cv. Kievitsboon Koekoek were obtained by cutting above the roots and above the cotyledonary node and inoculated with race Y. Cotyledons were removed at the times indicated and after 9 days incubation symptoms were recorded (see Table l), infected tissue was excised, weighed and concentrations of phaseollin determined.
Effect of removing cotyledons, at intervals after inoculation, bean hypocotyl segments infected with C. lindemuthianum
on accumulation
of phaseollin
in
As in earlier experiments, water-soaked spreading lesions were produced on hypocotyl segments of cv. Kievitsboon Koekoek, whose cotyledons were removed immediately after inoculation with race 7 or 12 h later (Table 3). When cotyledons were retained for at least 30 h, the hypocotyls were more resistant, symptoms varying from small to large flecks. Retention of cotyledons for 30 h or longer also led to the accumulation of greater quantities of phaseollin. A close association occurred between the amounts of phytoalexin produced and the time when cotyledons were removed: the greatest amounts of phaseollin were produced by hypocotyls which retained their cotyledons for 72 h or throughout the experiment. Although not subjected to quantitative analysis, the amounts of phaseollidin and phaseollinisoflavan, asjudged by spraying tic plates with diazotized-p-nitroaniline, appeared greater when cotyledons were retained for longer than 72 h. Effect of removing
cotyledons, roots and/or leaves on the susceptibility of bean seedlings to C. lindemuthianum Seedlings of cv. Kievitsboon Koekoek were removed from the Vermiculite. Roots, cotyledons and/or leaves were excised and the hypocotyls inoculated with race Y and incubated. The type of symptom which developed after 5, 6, 7, 8 and 10 days are shown in Table 4. The numbers ofinoculation sites showing symptoms after a particular period of incubation were analysed using a generalized linear model with binomial error structure. After 5 days the presence of each organ had a highly significant effect independently (P
E&IA of removing
Period of incubation (days)
Cotyledons
Leaves
Roots
+ .+ + -t + + +
+ + + +
+ + + + + + + + + + + + + + + + + + + +
+ + + f
+ + + +
+ + +
KoekoekC + f + + + + + +
6
+ + + +
7
8
+ t + + -
IO
(b) Tendergreend
10
a b c d
TABLE 4 leaves and/or roots from bean seedlings on the susceptibility C. lindemuthianum
Tissue remaining or removeda
(a) cv. Kievitsboon
5
cotyledons,
+ + + + + + + + t + + + +
+
No. of inoculation sites showing symptoms
o/50 0 16 5 30 7 32 14
of hypocotyls
to
No. of sites showin symptoms categorized % as 1 2 3
16 5 30 J 32 14
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
44 18 42 30 41 18 40 17
30 10 42 23 41 18 40 17
14 8 0 7 0 0 0 0
0 0 0 0 0 0 0 0
50 28 50 43 50 50 50 50
23 20 42 30 50 29 50 37
10 0 0 0 0 0 0 0
17 8 8 13 0 21 0 13
50 48 50 50 50 50 50 50
6 4 28 23 50 16 48 34
3 5 0 17 0 0 2 0
41 39 22 10 0 34 0 16
50 50 50 50 50 50 50 50
0 0 %L 0 43,L 5,L 46,L 15,L
0 0 0 0 0 0 0 0
50,s 50,s 41,L 40,L : 10,s 7,L 45,L 4,L 35,L
29/30 27 27 30 29 29 29 30
4,L 0 0 0 4,L 4,L ll,L
0 0 2J,L 16,L 24,L 0 18,L 0
Organs were removed from seedlings and the hypocotyls inoculated. Symptoms were recorded and categorized as in Table 2. + Indicates that an organ was retamed, -indicates that an organ Symptoms categorized as in Table 2. Five sites on ten hypocotyls. Five sites on six hvoocotvls.
0 0
6,L
was removed.
25,s 27,s 0 14,L 0 25,L 0 24,L
252
P. M. Rowell
and J. A. Bailey
FIG. 2. Symptoms on hypocotyls of cv. Kievitshoon Koekoek, 10 days after inoculation with C. lindemudianum, race Y. (a) Severe symptoms on excised hypocotyls which retained only roots; (h) large limited lesions on segments which retained roots and cotyledons; (c) flecking on segments which retained only cotyledons. All hypocotyls were trimmed immediately before being photographed.
The data in Table 4 also show that after 10 days extensive water-soaked spreading lesions were present at all sites on seedlings without cotyledons, roots and leaves or without cotyledons and leaves. The retention of cotyledons conferred resistance, symptoms being restricted to small flecks or limited lesions. The presence of leaves produced a similar pattern of increased resistance, although when cotyledons were present and roots absent the effect of leaves was not large enough to be statistically different. In contrast to cotyledons and leaves, the presence of roots led to increased tissue damage except on those hypocotyls without cotyledons or leaves, when all sites produced spreading lesions. For example, lesion development was always restricted on hypocotyls with only leaves, but if roots were also present, 20% of sites developed spreading lesions. Similarly, on seedlings with cotyledons, although no spreading lesions were formed, those with roots produced 70 and 90% limited lesions and 30 and 10% small flecks in the presence and absence of leaves respectively, whilst those lacking roots produced 8 and 14% 1imited lesions, but 92 and 86% small flecks. Typical symptoms are illustrated on Fig. 2. Similar trends were observed with cv. Tendergreen. Efect of darkness on production to C. lindemuthianum
of phytoalexins
in bean hypocotyl segments and their susceptibility
Hypocotyl segments of cv. Kievitsboon Koekoek were obtained from seedlings, grown in light or total darkness, by cutting above the roots and above the cotyledons. The cotyledons were removed from half of these segments, which were then inoculated with race Y and returned to light or darkness. The symptoms which developed and the
Susceptibility
of hypocotyls
253
of bean TABLE
Effect
phytolexinr
and susceptibility
to a compatible Phytoalexin
Treatment (i)
(ii)
5
of removing cotyledons from bean hypocotyl segments, grow
Inoculated with C. lindemuthianum Light + cotyledons Light - cotyledons Lightb + cotyledons Lightb - cotyledons Dark + cotyledons Dark - cotyledons Inoculated with TNV Lightb + Lightb - cotyledons Dark + cotyledons Dark - cotyledons
Symptomsa
l-2
in light or darkness, on accumulation race of C. lindemuthianum or TNV concentrations
Phaseollin
Ocgg-l
Phaseollidin
infected
of
tissue)
Phaseollinisoflavan
Phaseollinisoflavan (%)
(L) 3 (S) 2-3 (L) 3 (9 2-3 (L) 3 (3
142 67 279 22 108 42
55 17 64 11 90 36
64 5 127 4 64 9
25 6 27 I1 24 10
-
325 193 72 84
191 86 56 47
30 7 5 4
6 3 4 3
Hypocotyl segments ofcv. Kievitsboon Koekoek grown in light or darkness, obtained by cutting above the roots and above the cotyledonary node were inoculated. The cotyledons were immediately removed from half the segments and after 10 days symptoms were recorded and the concentration of phytoalexins were determined. %ymptom categories for C. lindcmuthianum as described in Table 2; symptoms for TNV were extensive pigmentation throughout all hypocotyls. bHypocotyls grown in darkness and transferred to light after Inoculation.
of phaseollin, phaseollidin and phaseollinisoflavan produced in these tissues and in tobacco necrosis virus (TNV)-infected hypocotyls incubated under identical conditions are presented in Table 5. TNV infections were made for a comparison with previous work [ 261. In both light and darkness, the presence of cotyledons led to increased resistance; symptoms being expressed as flecks on light-grown hypocotyls and as limited lesions on dark-grown tissues. All hypocotyls without cotyledons, whether incubated in light or darkness, produced water-soaked spreading Iesions. Largest quantities of all three phytoalexins were produced in resistant tissues. The proportion of phaseollinisoflavan (24 to 27%) was greatest in Colletotrichum-infected hypocotyls bearing cotyledons. Virus infected hypocbtyls consistently contained large quantities of phaseollin and phaseollidin but the proportion of phaseollinisoflavan was always less than 6%.
amounts
DISCUSSION
In all these experiments, hypocotyls ofsusceptible cultivars were inoculated with a compatible (virulent) race of C. lindemuthiunum. However, it is clear from the results that typical symptoms of susceptibility, i.e. extensive tissue damage, were not a necessary consequence of infection, and that symptoms associated with resistance were simple to produce. Thus the presence of cotyledons, and, to a lesser extent, leaves, reduced the extent of tissue damage. Water-soaked spreading lesions were produced on hypocotyls whose cotyledons and leaves had been removed. When either of these organs
254
P. M. Rowell
and J. A. Bailey
were retained, symptoms were restricted, appearing as brown flecks, which were similar to the hypersensitive reaction which occurred on resistant cultivars [16], or as dark brown limited lesions. A clear distinction between these symptoms was not always possible and their appearance was influenced by which organs were removed, by the cultivar used and by the length of time the cotyledons were retained. In contrast, hypocotyls which retained roots ultimately suffered more damage, although it was slower to develop, than those lacking roots; an effect readily observed if cotyledons were also present (Table 4). The differing extents of symptom development were associated with two factors : the length of the period of biotrophy, as indicated by the time which elapsed before browning occurred, and the amounts of phytoalexins which accumulated during the subsequent necrotrophic phase. For example, on cv. Kievitsboon Koekoek, restricted symptom development, due to the presence of cotyledons, followed a shorter period of biotrophy, whilst enhanced tissue damage, due to the presence of roots, was associated with an extended biotrophic period. In addition, in the presence of cotyledons, when symptoms were restricted, infected tissues contained greater concentrations of phytoalexins than the infected tissue ofhypocotyls without cotyledons. Phaseollin was usually the predominant inhibitor and its concentration was enhanced four to seven times by the presence ofcotyledons. However, the enhancement ofphaseollidin (five to 36 times) and phaseollinisoflavan accumulation (40 to 70 times) were even greater. Experiments comparing symptom development in light and darkness, showed that light also reduced the amounts of damage caused. In contrast to our previous report [16], when we failed to detect the isoflavan in virus-infected hypocotyls grown and incubated in total darkness, we have now found small (4 to 30 pgg-l) but significant quantities of this compound under these conditions. The reasons for the different results are not known but our present data negate our previous conclusion that production of the isoflavan requires light. It is of interest, however, that the isoflavan was not produced by roots [7]. Detailed studies on the amounts of phytoalexin produced and their localization in infected tissueswere not attempted during this work, but the data are consistent with the conclusion that accumulated phytoalexins prevent pathogen activity, which in turn restricts symptom development [4]. If phytoalexins are responsible for inhibition of pathogen growth, it becomes important to assessthe mechanisms by which phytoalexins accumulate to high concentrations in some tissues but to low concentrations in others. Equally, the differences in type of tissue damage reported in this and previous papers [I, 6, IO, 12, 13, 14, 15, 19, 201 also require consideration. A probable explanation relies on proposals that synthesis of bean phytoalexins occurs in healthy cells, which are adjacent to dead or dying infected tissues [3] and that greater quantities of phytoalexin are produced by metabolically active tissues [26]. Thus it can be envisaged that in hypocotyls bearing newly formed necrotic lesions, the amounts of phytoalexin produced will depend on the outcome of interactions between the ability of the surrounding living bean cells to produce these secondary metabolites and the ability of advancing necrotrophic hyphae to disrupt such cellular metabolism before significant amounts can be formed. Colletotrichum lindemuthianum is a facultative biotroph, capable of biotrophic, as well as necrotrophic growth. In the absence of cotyledons there is extensive biotrophic colonization of the hypocotyl tissue which has a poor capacity for producing
Susceptibility
of hypocotyls
of bean
255
phytoalexins. As a result, when necrosis occurs phytoalexin production by the hypocotyl is slow and the large amount of established hyphae ensures rapid necrotrophic fungal growth, continuing death of surrounding healthy cells, little accumulation of phytoalexin and eventual rotting of the entire hypocotyl. In the presence of cotyledons, premature necrosis of infected cells ensures that biotrophic establishment is reduced. Since cotyledons also enhance the capacity of tissue to produce phytoalexins, necrosis is followed by rapid accumulation of phytoalexins which quickly prevent further pathogen development and symptoms become severely restricted. In the absence of cotyledons, roots have little effect on the eventual development of spreading lesions, although the initial period of biotrophic growth may be greater. However, if both roots and cotyledons are retained, the presence of roots leads to an extended period of biotrophy, but when cellular collapse occurs subsequent necrotrophic fungal growth is soon restricted by the enhanced production of phytoalexins, resulting from the presence of cotyledons. As a result, symptoms were evident as relatively large but limited lesions. Differences in the extents of biotrophy and phytoalexin accumulation may also contribute to the different symptoms which have been reported on many other occasions, including suggestions that some bean cultivars give a response “intermediate” between resistance and susceptibility. The symptoms on such cultivars were restricted and usually appeared as small flecks or limited lesions [8, 20, 191. This study suggests that “intermediate” symptoms probably represent cultivars which support initial biotrophic growth, but which prevent extensive necrotrophic development. It has also been observed that symptoms vary along the length of a hypocotyl, being most extensive in the upper younger tissues [unpublished data]. Similar variations occurred onsoybean hypocotyls inoculated with Phytophthora megasperma, another facultative biotroph [II]. Again these different symptoms could be controlled by different amounts of biotrophic and necrotrophic fungal growth. In conclusion, symptoms on potentially susceptible bean hypocotyls varied considerably, being expressed as flecks, as limited lesions or as spreading lesions. By altering the physiological activity of the infected tissue (see [5] for effects of higher temperatures), symptoms were produced which could classify the same cultivar as either resistant or susceptible to the same race of C. lindemuthianum. This suggests that observations of visible symptoms alone may be inappropriate for designating genetic relationships between cultivars of bean and races of C. lindemuthianum. These specific relationships seem reliant on the establishment of biotrophy or not (hypersensitivity), phenomena which can only be reliably assessedby observations of the cellular interactions between fungal hyphae and infected host cells. On this basis, biotrophy can be regarded as an integral part of host-pathogen recognition controlled by complementary genes ofhost and pathogen. Subsequent symptom expression is controlled by a complex interaction between the extent of biotrophy and factors influenced by independent genotypic and phenotypic properties of the host and/or pathogen, e.g. the ability of the pathogen to kill cells quickly or of the host to produce more or less phytoalexin. The present results do not explain how roots, cotyledons and temperature influence either the extent of biotrophy or the amounts of phytoalexins produced, although the role of plant growth regulators deserves investigation [9]. However, the
256
P. M. Rowell
and J. A. Bailey
effects described in this and previous research [5] p resent great potential as probes into the biochemical nature of biotrophy itself, a process essential to the establishment of most important fungal pathogens. We thank G. M. Arnold for statistical analyses and S. C. Teague for photographic work. REFERENCES 1. ALLARD, C. (1974). Etude vulgaris) a l’anthracnose
histologique en microscopic optique de la resistance du Haricot (Collelotrichum lindemuthiuaum) gouvernie par le gene Cornell.
(Phaseolus Annales
de Phytopathologie 6, 35%383. 2. BAILEY, J. A. (1974). The relationship between symptom expression and phytoalexin concentration in hypocotyls of Phaseolus vulgaris. Physiological Plant Pathology 4, 477488. 3. BAILEY, J. A. (1982a). Mechanisms of phytoalexin accumulation. In Phytoalewins, Ed. by J. A. Bailey
and J. W. Mansfield, pp. 289-318. Blackie & Son, Glasgow and London. 4. BAILEY, J. A. (1982b). Physiological and biochemical events associated with the expression of resistance to disease. In Active Defence Mechanisms in Plants, Ed. by R. K. S. Wood, pp. 3965 Plenum Press, London and New York. 5. BAILEY, J. A., ROWELL, P. M. & ARNOLD, G. M. (1980). The temporal relationship between host cell death, phytoalexin accumulation and fungal inhibition during hypersensitive reactions of Phaseolus uulgaris to Colletotrichum lindemuthianum. Physiological Plant Pathology 17, 329-339. 6. CHARRIER, A. & BANNEROT, H. (1970). Contribution a l’etude des races physiologiques de l’anthracnose du haricot. Annals de Phytopathologie 2,489-506. 7. DEWICK, P. M. & STEELE, M. J. (1982). Biosynthesis of the phytoalexin phaseollin in Phnreolus uulgaris. Phytochemistry 21, 1599-1603. 8. DIXON, G. R. & DOODSON, J. K. (1973). A technique used for testing dwarf bean cultivars for resistance to anthracnose (Colletotrichum lindemuthianum). Journal of the National Institute of Agricultural Botany 1187-89. 9. GOOSENS, J. F. V.
J. C. (1982). Effects of abscisic acid, cytokinins and light on isoaccumulation in Phaseolus vulgaris L. Planta lH, 441446. 10. KRUGER, J., HOFFMANN, G. M. & HUBBELING, N. (1977). The Kappa race of Colletotrichum lindemuthianum and sources of resistance to anthracnose in PhaFeolus bean. Euphytica 26,23-25. 11. LAZAROVITS, G., STOSSEL, P. & WARD, E. W. B. (1981). Age-related changes in specificity and glyceollin production in the hypocotyl reaction of soybeans to Phytophthora megasperma var sojae, flavonoid
Phytopathology
12. MERGER, 13.
14.
15.
16.
17.
18.
19. 20.
& VENDRIC,
phytoalexin
71. 94-97.
P. C., UJOD, R. K. S., & GREENWOOD, A. D. (1975). Ultrastructure of the parasitism of Phaseolus uulgaris by Colletotrichum lindemuthianum. Physiological Plant Pathology 5, 203-2 14. RAHE, J. E. (1973). Occurrence and levels of the phytoalexin phaseollin in relation to delimitation at sites of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Canadian Journal of Botany 51, 2423-2430. RAHE, J. E. & Kuc J. (1970). Metabolic nature of the infection-limiting effect of heat on bean anthracnose. Phytopathology 66, 100~1009. RAHE, J. E. & Ku&, J., CHUANG, C. M. & WILLIAMS, E. B. (1969). Correlation of phenolic metabolism with histological changes in Phaseolus vulgaris inoculated with fungi. Netherlands Journal of Plant Pathology 75, 58-7 1. ROWELL, P. M. & BAILEY, J. A. (1982). Influence of light and excision of organs on accumulation of phytoalexins in virus-infected hypocotyls of bean (Phareolus vulgaris). Physiological Plant Pathology 21, 75-84. SKIPP, R. A. & DEVERALL, B. J. (1972). Relationships between fungal growth and host changes visible by light microscopy during infection of bean hypocotyls (Phaseolus vulgaris) susceptible and resistant to physiological races of Colletotrichum lindemuthianum. Physiological Plant Pathology 2, 357-374. STOSSEL, P., LAZAROVITS, G. & WARD, E. W. B. (1980). Penetration and growth of compatible and incompatible races of Phytophthora megasperma var sojee in soybean tissue differing in age. Canadian Journal of Botany 56,2594-260 1. THEODOROU, M. K. & SMITH, I. M. (1979). The response of French bean varieties to components isolated from races of Colletotrichum lindemuthianum. Physiological Plant Pathology 15, 297-309. Tu, J. C. & AYLESWORTH, J. W. (1980). An effective method of screening white (pea) bean seedlings (Phaseolus vulgaris L.) for resistance to Colletotrichum lindemuthianum. Phytopathologische Zeitschrift 99, 13 l137.
Plant Pathology
(1963)
23,245-256
The influence of cotyledons, roots and leaves on the susceptibility of hypocotyls of bean (Phaseolus vulgaris) to compatible races of Colletotrichum lindemuthianum P. M.
ROWELL
and J. A.
Long Ashton Research Station, (Acceptedfor
publication
March
BAILEY
University
of Bristol,
Long Ashton,
Bristol,
BS18 9AF
1983)
Hypocotyl segments which were excised from bean seedlings supported extensive biotrophic colonization by compatible races of Colletotrichum lindemuthianum. Subsequent death of infected tissues was followed by production of small amounts of phytoalexins, water-soaked spreading lesions and eventual complete rotting of the hypocotyls. When cotyledons or, to a lesser extent, leaves, were retained the hypocotyls did not rot. Superficial flecking occurred on segments which retained only their cotyledons, whilst large restricted lesions occurred when roots and cotyledons or only leaves were retained. Greatest concentrations of the phytoalexins, phaseollin, phaseollidin and phaseollinisoflavan, occurred in hypocotyls bearing cotyledons. Light also enhanced the production of these compounds. However, in contrast to previous results, phaseollinisoflavan was obtained from both Colletotrichum and tobacco necrosis virus-infected hypocotyls maintained in total darkness. It is suggested that symptom development is controlled by complex interactions between the extent of biotrophic colonization, which appears to be enhanced by roots but reduced by cotyledons, by the ability of necrotrophic hyphae to kill tissues quickly and hence reduce the production of phytoalexins, and by the ability of the healthy tissues which surround lesions to produce large quantities of phytoalexins, a process enhanced by both cotyledons and leaves.
INTRODUCTION Colletottichum lindemuthianum
(Sacc. et Magn.) Bri. et Cav. is a facultative biotrophic (hemibiotroph) [4]. On hypocotyls of susceptible cultivars, compatible races are capable ofestablishing biotrophic infection hyphae within epidermal cells and later within underlying cortical cells. The period of biotrophy can extend for several days, after which, the infected tissues die, and dark brown lesions appear. These may become water-soaked and the entire hypocotyl soon rots. This extreme symptom of susceptibility occurs on young hypocotyls following excision above the roots and below the cotyledons, and incubation in diffuse light or darkness at temperatures between 15 and 18 “C [ 2, 51. Under other conditions the symptoms are more variable and often much less extensive. For example, in race surveys using intact plants [IO], or with hypocotyls incubated at temperatures above 22 “C [5, 131, symptoms of resistance, i.e.
fungus
localized
flecking
or limited
lesions,
have
been
reported.
Similarly,
a great
range
of
symptoms can occur on different aged hypocotyls and at different positions on the same hypocotyl ([13, 14; see also research with hypocotyls of Glycine max inoculated with Phytophthora megasperma var. so@ [II, 281). 0048-4059/83/050245
+ 12 so3.00/0
0
1983 Academic
Press Inc.
(London)
Limited
246
P. M. Rowe11 and J. A. Bailey
The mechanisms controlling the intercellular growth of C. lindemzithianum in susceptible cultivars and the factors influencing symptom expression are not well understood, although the accumulation of phytoalexins to fungitoxic concentrations and the increased resistance to penetration of cell walls of older tissues probably contribute to cessation of fungal growth in limited lesions [I]. The present paper examines the influence of cotyledons, leaves, roots and light on infection of bean hypocotyls by a compatible race of C. lindemuthianum, including effects on biotrophy, expression of symptoms and accumulation of phytoalexins. A previous paper showed that virusinfected hypocotyls which retained cotyledons and were incubated in light produced most phytoalexin [26].
MATERIALS AND METHODS Growth of plants andfungi Seedlings of Phaseolus vulgaris
L. cvs Kievitsboon Koekoek, Tendergreen and Tendercrop were grown in sterilized Vermiculite either in an illuminated cabinet (Fisons 9600), at 25 “C, rh 80% and periods of light ( 16 h, 40 w m-2 s-r) and darkness (8 h) for 6 to 7 days so that the primary leaves had emerged from between the cotyledons but had not expanded, or in darkness in a dark room protected by a light trap [I61 at 22°C for 6 or 7 days. Seeds of cv. Kievitsboon Koekoek were harvested from plants grown at Long Ashton Research Station, those of Tendergreen and Tendercrop were a gift from Charles Sharpe and Co. Ltd., Sleaford, Lines. Only seedlings with two undamaged cotyledons were used. Seedlings were removed from the Vermiculite and used intact. Hypocotyl segments were obtained by excising the seedlings 3 cm above the roots and 1 cm above the cotyledonary node. All cut surfaces were sealed with wax. Seedlings or hypocotyl segments were placed on their sides in transparent plastic boxes and the central areas of the hypocotyl were inoculated with five drops (5 fl) of a spore suspension (5 x lo5 spore ml-‘), each placed 10 mm apart. When roots were present they were enclosed in moist tissue paper. The p and Y races of C. lindemuthianum were used; their growth and sporulation have been described [Z]. Tissues were macerated in ethanol and after filtration and solvent evaporation the ethanol-soluble material was subjected to thin-layer chromatography (tic) using SiGel 60 (Merck 5715) developed in ethanol :chloroform (100 :3 v/v) [Z]. This achieved separation of phaseollin, phaseollidin and phaseollinisoflavan, so that their concentrations could be measured spectrophotometrically [Z].
RESULTS E$ect of cotyledons on symptoms expressed on bean hypocotyl segments
Hypocotyl segments were obtained as described above. Both cotyledons were removed from half the segments, and the symptoms produced on entire segments and on segments without cotyledons were compared. Symptoms on hypocotyls of cv. Kievitsboon Koekoek inoculated with race Y, with or without cotyledons are shown in Fig. 1. Large ( > 10 mm diameter) dark brown water-soaked lesions, which spread to cause complete rotting after 14 days, occurred
Susceptibility
of hypocotyls
247
of bean
FIG. 1. Symptoms on hypocotyls of cv. Kievitsboon Koekoek, with C. lindemuthianum, race Y. (a) Severe symptoms on excised lacked cotyledons, roots and leaves; (b) limited flecking on segments All hypocotyls were trimmed immediately before being photographed.
10 days after inoculation hypocotyl segments which which retained cotyledons.
on hypocotyls without cotyledons. The symptoms on segments with their cotyledons varied from small (1 to 3 mm) limited lesions to a scattering of isolated necrotic cells, and had the appearance of an incompatible (hypersensitive) reaction [17]. Cotyledons also decreased symptom development on other cultivars. After inoculation with race f-?,lesions on hypocotyls of cv. Tendercrop lacking cotyledons were paler brown than on cv. Kievitsboon Koekoek and coalesced rapidly causing the entire hypocotyl to rot within 12 days. When cotyledons were present, symptoms were restricted to small (5 to 10 mm) dark brown lesions. Cultivar Tendergreen was more resistant to rotting than the other cultivars tested. In the absence of cotyledons, lesions due to race b were dark brown, turning black and some (30%) became restricted in size (10 to 15 mm), so that healthy tissue remained between the lesions: despite this many hypocotyls rotted. In the presence of cotyledons the lesions were smaller (1 to 5 mm) and no hypocotyls rotted. Thus in these three cultivars, the presence of cotyledons decreased disease development. The mechanisms responsible for enhanced resistance were examined in the following experiments.
248
P. M. Rowell
and J. A. Bailey
EJect of removitlg cotyledons on the production
of phytoalexins by bean hypocotyls infected with C. lindemuthianum Hypocotyl segments of cv. Kievitsboon Koekoek were inoculated with race 7. Cotyledons were removed as required and the development of symptoms and the concentrations of phaseollin, phaseollidin and phaseollinisoflavan which had accumulated in the infected tissue were assessed. Results are presented in Table 1. Symptoms developed as in previous experiments: on hypocotyls without cotyledons, symptoms were visible as dark brown spreading lesions; but when cotyledons were not removed only flecking occurred (see Fig. 1) .
Effect
of removing
cotyltdons
TABLE 1 on accumulation of phytoaltxinr in bean hypocotyl compatible TUCEof C. lindemuthianum
segments infected with n
Phytoalexin concentrations (pg g-’ ‘infected tissue) Period of incubation (days) 9
12 14
Treatment + + + -
Cotyledons Cotyledons Cotyledons Cotyledons Cotyledons Cotyledons
Mean fresh weight of infected tissue (mg) 9.4 30.0 7.1 35.7 7.2 27.1
Symptomsa 1 3 1 3 l,L 3s
Phaseollin 382 80 742 111 439 125
Phaseollidin 166 28 195 14 290 8
Phaseollinisoflavan 117 3 445 7 644 9
Hypocotyl segments ofcv. Kiev&boon Koekoek, obtained by cutting above the roots and above the cotyledonary node, were inoculated with race y. The cotyledons were immediately removed from halfthe segments and at intervals symptoms were recorded, infected tissue was excised and the concentrations of phytoalexins were determined. ‘Symptom categories are: 1, single or groups of scattered brown cells; 3, lesions larger than site of inoculation. L and S indicate, respectively, that symptoms were limited in extent or continued to spread.
Greatest concentrations of all phytoalexins occurred in hypocotyls with cotyledons, i.e. in the more resistant tissues. When the total amounts of the three phytoalexins are considered, their concentrations after 9, 12 and 14 days were 111, 132 and 142 Ng-1 tissue in hypocotyls without cotyledons and 665, 1382 and 1373 pgg-’ tissue in hypocotyls with cotyledons. In most hypocotyls, the concentrations of each phytoalexin increaased during the incubation period, but occasionally, e.g. phaseollin in hypocotyls bearing cotyledons, their concentrations seemed lower after 14 days than after 12 days. Effect of removing cotyledons, at intervals after inoculation, segments to C. lindemuthianum
Hypocotyl incubated. Symptoms terized as
on the susceptibility
of hypocotyl
segments of cv. Kievitsboon Koekoek were inoculated with race Y and Both cotyledons were removed from some of them at different intervals. were assessed6, 7.5, 8.5 and 10 days after inoculation and were characfollows: 1, single or groups of scattered brown cells (small flecks) ; 2,
Susceptibility Effect
of hypocotyls
of removing cotyledons,
Period of incubation (days)
of bean TABLE 2 at intmrals after inoculation, on the susceptibility to a compatible race ofC. lindemuthianum
Time after inoculation when cotyledons removed (h) 0 12 36 60
6.0 Cotyledons
retained 0 12 36 60
7.5 Cotyledons
retained 0 12 36 60
8.5 Cotyledons
retained 0 12 36 60
IO.0 Cotyledons
249
retained
No. of inoculation sites showing symptom? 5/40 14 19 10 25 15 31 34 35 38 29 36 38 36 38 36 39 38 36 39
of bean hypocotyl
segments
No. of sites showin i as: symptoms categorized 1 2 3 5 14 19 10 25 0 8 26 25 14 5 7 0 18 11 0 0 0 5,L 11,L
0 0 0 0 0 3 6 0 10 24 2 4 12 18 27 0 0 12,L 31,L 28,L
0 0 0 0 0 12 17 8 0 0 22 25 26 0 0 36,s 39,s 26,L 0 0
Hypocotyl segments of cv. Kievitsboon Koekoek, obtained by excision above the roots and above the cotyledonary node were inoculated with race 7. At intervals both cotyledons were removed and symptoms subsequently recorded. aFive sites on eight hypocotyls. ‘Symptom categories are: 1, single or groups of scattered brown cells; 2, confluent areas of necrosis within the site of inoculation; 3, lesions larger than site of inoculation. L and S, respectively, indicate that symptoms were limited in extent or continued to spread.
confluent brown areas within the initial sites of inoculation, which were approximately 3 mm long (large flecks) ; 3, lesions larger than the site of inoculation. The eventual limited or spreading nature of the infections was also recorded. Results are shown in Table 2. They confirm that the development of lesions in hypocotyls with cotyledons was restricted, whilst hypocotyls whose cotyledons had been removed at the time of inoculation eventually rotted due to the presence of water-soaked spreading lesions. Spreading lesions and subsequent rotting also occurred on hypocotyls from which cotyledons were removed 12 h after inoculation, but removal thereafter, did not prevent the restriction in lesion development and the hypocotyls did not rot. These results also indicate that symptoms became visible on hypocotyls with cotyledons before those on hypocotyls without cotyledons. For example, after 6 days, 62.5% of sites on hypocotyls with cotyledons showed symptoms, whereas symptoms were present on only 12.5% of sites on hypocotyls without cotyledons.
P. M. Rowell
250 TABLE Effect
of removing cotyledons, at inkwals
Time after inoculation when cotyledons removed (h) 0 12 30 60 72 Cotyledons
retained
Mean fresh weight of infected tissue (4 30.0 35.0 12.4 8.9 9.5 9.4
3
af&r inoculation,
segments infected with a compatible
and J. A. Bailey
on accumulation of phaseollin race of C. lindemuthianum
Symptoms
in bean hypocotyl
Phaseollin concentration ( pgg-1 infected tissue) 79 119 177 198 406 382
3,s 3s I-2,L l,L 1,L l,L
Hypocotyl segments of cv. Kievitsboon Koekoek were obtained by cutting above the roots and above the cotyledonary node and inoculated with race Y. Cotyledons were removed at the times indicated and after 9 days incubation symptoms were recorded (see Table l), infected tissue was excised, weighed and concentrations of phaseollin determined.
Effect of removing cotyledons, at intervals after inoculation, bean hypocotyl segments infected with C. lindemuthianum
on accumulation
of phaseollin
in
As in earlier experiments, water-soaked spreading lesions were produced on hypocotyl segments of cv. Kievitsboon Koekoek, whose cotyledons were removed immediately after inoculation with race 7 or 12 h later (Table 3). When cotyledons were retained for at least 30 h, the hypocotyls were more resistant, symptoms varying from small to large flecks. Retention of cotyledons for 30 h or longer also led to the accumulation of greater quantities of phaseollin. A close association occurred between the amounts of phytoalexin produced and the time when cotyledons were removed: the greatest amounts of phaseollin were produced by hypocotyls which retained their cotyledons for 72 h or throughout the experiment. Although not subjected to quantitative analysis, the amounts of phaseollidin and phaseollinisoflavan, asjudged by spraying tic plates with diazotized-p-nitroaniline, appeared greater when cotyledons were retained for longer than 72 h. Effect of removing
cotyledons, roots and/or leaves on the susceptibility of bean seedlings to C. lindemuthianum Seedlings of cv. Kievitsboon Koekoek were removed from the Vermiculite. Roots, cotyledons and/or leaves were excised and the hypocotyls inoculated with race Y and incubated. The type of symptom which developed after 5, 6, 7, 8 and 10 days are shown in Table 4. The numbers ofinoculation sites showing symptoms after a particular period of incubation were analysed using a generalized linear model with binomial error structure. After 5 days the presence of each organ had a highly significant effect independently (P
E&IA of removing
Period of incubation (days)
Cotyledons
Leaves
Roots
+ .+ + -t + + +
+ + + +
+ + + + + + + + + + + + + + + + + + + +
+ + + f
+ + + +
+ + +
KoekoekC + f + + + + + +
6
+ + + +
7
8
+ t + + -
IO
(b) Tendergreend
10
a b c d
TABLE 4 leaves and/or roots from bean seedlings on the susceptibility C. lindemuthianum
Tissue remaining or removeda
(a) cv. Kievitsboon
5
cotyledons,
+ + + + + + + + t + + + +
+
No. of inoculation sites showing symptoms
o/50 0 16 5 30 7 32 14
of hypocotyls
to
No. of sites showin symptoms categorized % as 1 2 3
16 5 30 J 32 14
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
44 18 42 30 41 18 40 17
30 10 42 23 41 18 40 17
14 8 0 7 0 0 0 0
0 0 0 0 0 0 0 0
50 28 50 43 50 50 50 50
23 20 42 30 50 29 50 37
10 0 0 0 0 0 0 0
17 8 8 13 0 21 0 13
50 48 50 50 50 50 50 50
6 4 28 23 50 16 48 34
3 5 0 17 0 0 2 0
41 39 22 10 0 34 0 16
50 50 50 50 50 50 50 50
0 0 %L 0 43,L 5,L 46,L 15,L
0 0 0 0 0 0 0 0
50,s 50,s 41,L 40,L : 10,s 7,L 45,L 4,L 35,L
29/30 27 27 30 29 29 29 30
4,L 0 0 0 4,L 4,L ll,L
0 0 2J,L 16,L 24,L 0 18,L 0
Organs were removed from seedlings and the hypocotyls inoculated. Symptoms were recorded and categorized as in Table 2. + Indicates that an organ was retamed, -indicates that an organ Symptoms categorized as in Table 2. Five sites on ten hypocotyls. Five sites on six hvoocotvls.
0 0
6,L
was removed.
25,s 27,s 0 14,L 0 25,L 0 24,L
252
P. M. Rowell
and J. A. Bailey
FIG. 2. Symptoms on hypocotyls of cv. Kievitshoon Koekoek, 10 days after inoculation with C. lindemudianum, race Y. (a) Severe symptoms on excised hypocotyls which retained only roots; (h) large limited lesions on segments which retained roots and cotyledons; (c) flecking on segments which retained only cotyledons. All hypocotyls were trimmed immediately before being photographed.
The data in Table 4 also show that after 10 days extensive water-soaked spreading lesions were present at all sites on seedlings without cotyledons, roots and leaves or without cotyledons and leaves. The retention of cotyledons conferred resistance, symptoms being restricted to small flecks or limited lesions. The presence of leaves produced a similar pattern of increased resistance, although when cotyledons were present and roots absent the effect of leaves was not large enough to be statistically different. In contrast to cotyledons and leaves, the presence of roots led to increased tissue damage except on those hypocotyls without cotyledons or leaves, when all sites produced spreading lesions. For example, lesion development was always restricted on hypocotyls with only leaves, but if roots were also present, 20% of sites developed spreading lesions. Similarly, on seedlings with cotyledons, although no spreading lesions were formed, those with roots produced 70 and 90% limited lesions and 30 and 10% small flecks in the presence and absence of leaves respectively, whilst those lacking roots produced 8 and 14% 1imited lesions, but 92 and 86% small flecks. Typical symptoms are illustrated on Fig. 2. Similar trends were observed with cv. Tendergreen. Efect of darkness on production to C. lindemuthianum
of phytoalexins
in bean hypocotyl segments and their susceptibility
Hypocotyl segments of cv. Kievitsboon Koekoek were obtained from seedlings, grown in light or total darkness, by cutting above the roots and above the cotyledons. The cotyledons were removed from half of these segments, which were then inoculated with race Y and returned to light or darkness. The symptoms which developed and the
Susceptibility
of hypocotyls
253
of bean TABLE
Effect
phytolexinr
and susceptibility
to a compatible Phytoalexin
Treatment (i)
(ii)
5
of removing cotyledons from bean hypocotyl segments, grow
Inoculated with C. lindemuthianum Light + cotyledons Light - cotyledons Lightb + cotyledons Lightb - cotyledons Dark + cotyledons Dark - cotyledons Inoculated with TNV Lightb + Lightb - cotyledons Dark + cotyledons Dark - cotyledons
Symptomsa
l-2
in light or darkness, on accumulation race of C. lindemuthianum or TNV concentrations
Phaseollin
Ocgg-l
Phaseollidin
infected
of
tissue)
Phaseollinisoflavan
Phaseollinisoflavan (%)
(L) 3 (S) 2-3 (L) 3 (9 2-3 (L) 3 (3
142 67 279 22 108 42
55 17 64 11 90 36
64 5 127 4 64 9
25 6 27 I1 24 10
-
325 193 72 84
191 86 56 47
30 7 5 4
6 3 4 3
Hypocotyl segments ofcv. Kievitsboon Koekoek grown in light or darkness, obtained by cutting above the roots and above the cotyledonary node were inoculated. The cotyledons were immediately removed from half the segments and after 10 days symptoms were recorded and the concentration of phytoalexins were determined. %ymptom categories for C. lindcmuthianum as described in Table 2; symptoms for TNV were extensive pigmentation throughout all hypocotyls. bHypocotyls grown in darkness and transferred to light after Inoculation.
of phaseollin, phaseollidin and phaseollinisoflavan produced in these tissues and in tobacco necrosis virus (TNV)-infected hypocotyls incubated under identical conditions are presented in Table 5. TNV infections were made for a comparison with previous work [ 261. In both light and darkness, the presence of cotyledons led to increased resistance; symptoms being expressed as flecks on light-grown hypocotyls and as limited lesions on dark-grown tissues. All hypocotyls without cotyledons, whether incubated in light or darkness, produced water-soaked spreading Iesions. Largest quantities of all three phytoalexins were produced in resistant tissues. The proportion of phaseollinisoflavan (24 to 27%) was greatest in Colletotrichum-infected hypocotyls bearing cotyledons. Virus infected hypocbtyls consistently contained large quantities of phaseollin and phaseollidin but the proportion of phaseollinisoflavan was always less than 6%.
amounts
DISCUSSION
In all these experiments, hypocotyls ofsusceptible cultivars were inoculated with a compatible (virulent) race of C. lindemuthiunum. However, it is clear from the results that typical symptoms of susceptibility, i.e. extensive tissue damage, were not a necessary consequence of infection, and that symptoms associated with resistance were simple to produce. Thus the presence of cotyledons, and, to a lesser extent, leaves, reduced the extent of tissue damage. Water-soaked spreading lesions were produced on hypocotyls whose cotyledons and leaves had been removed. When either of these organs
254
P. M. Rowell
and J. A. Bailey
were retained, symptoms were restricted, appearing as brown flecks, which were similar to the hypersensitive reaction which occurred on resistant cultivars [16], or as dark brown limited lesions. A clear distinction between these symptoms was not always possible and their appearance was influenced by which organs were removed, by the cultivar used and by the length of time the cotyledons were retained. In contrast, hypocotyls which retained roots ultimately suffered more damage, although it was slower to develop, than those lacking roots; an effect readily observed if cotyledons were also present (Table 4). The differing extents of symptom development were associated with two factors : the length of the period of biotrophy, as indicated by the time which elapsed before browning occurred, and the amounts of phytoalexins which accumulated during the subsequent necrotrophic phase. For example, on cv. Kievitsboon Koekoek, restricted symptom development, due to the presence of cotyledons, followed a shorter period of biotrophy, whilst enhanced tissue damage, due to the presence of roots, was associated with an extended biotrophic period. In addition, in the presence of cotyledons, when symptoms were restricted, infected tissues contained greater concentrations of phytoalexins than the infected tissue ofhypocotyls without cotyledons. Phaseollin was usually the predominant inhibitor and its concentration was enhanced four to seven times by the presence ofcotyledons. However, the enhancement ofphaseollidin (five to 36 times) and phaseollinisoflavan accumulation (40 to 70 times) were even greater. Experiments comparing symptom development in light and darkness, showed that light also reduced the amounts of damage caused. In contrast to our previous report [16], when we failed to detect the isoflavan in virus-infected hypocotyls grown and incubated in total darkness, we have now found small (4 to 30 pgg-l) but significant quantities of this compound under these conditions. The reasons for the different results are not known but our present data negate our previous conclusion that production of the isoflavan requires light. It is of interest, however, that the isoflavan was not produced by roots [7]. Detailed studies on the amounts of phytoalexin produced and their localization in infected tissueswere not attempted during this work, but the data are consistent with the conclusion that accumulated phytoalexins prevent pathogen activity, which in turn restricts symptom development [4]. If phytoalexins are responsible for inhibition of pathogen growth, it becomes important to assessthe mechanisms by which phytoalexins accumulate to high concentrations in some tissues but to low concentrations in others. Equally, the differences in type of tissue damage reported in this and previous papers [I, 6, IO, 12, 13, 14, 15, 19, 201 also require consideration. A probable explanation relies on proposals that synthesis of bean phytoalexins occurs in healthy cells, which are adjacent to dead or dying infected tissues [3] and that greater quantities of phytoalexin are produced by metabolically active tissues [26]. Thus it can be envisaged that in hypocotyls bearing newly formed necrotic lesions, the amounts of phytoalexin produced will depend on the outcome of interactions between the ability of the surrounding living bean cells to produce these secondary metabolites and the ability of advancing necrotrophic hyphae to disrupt such cellular metabolism before significant amounts can be formed. Colletotrichum lindemuthianum is a facultative biotroph, capable of biotrophic, as well as necrotrophic growth. In the absence of cotyledons there is extensive biotrophic colonization of the hypocotyl tissue which has a poor capacity for producing
Susceptibility
of hypocotyls
of bean
255
phytoalexins. As a result, when necrosis occurs phytoalexin production by the hypocotyl is slow and the large amount of established hyphae ensures rapid necrotrophic fungal growth, continuing death of surrounding healthy cells, little accumulation of phytoalexin and eventual rotting of the entire hypocotyl. In the presence of cotyledons, premature necrosis of infected cells ensures that biotrophic establishment is reduced. Since cotyledons also enhance the capacity of tissue to produce phytoalexins, necrosis is followed by rapid accumulation of phytoalexins which quickly prevent further pathogen development and symptoms become severely restricted. In the absence of cotyledons, roots have little effect on the eventual development of spreading lesions, although the initial period of biotrophic growth may be greater. However, if both roots and cotyledons are retained, the presence of roots leads to an extended period of biotrophy, but when cellular collapse occurs subsequent necrotrophic fungal growth is soon restricted by the enhanced production of phytoalexins, resulting from the presence of cotyledons. As a result, symptoms were evident as relatively large but limited lesions. Differences in the extents of biotrophy and phytoalexin accumulation may also contribute to the different symptoms which have been reported on many other occasions, including suggestions that some bean cultivars give a response “intermediate” between resistance and susceptibility. The symptoms on such cultivars were restricted and usually appeared as small flecks or limited lesions [8, 20, 191. This study suggests that “intermediate” symptoms probably represent cultivars which support initial biotrophic growth, but which prevent extensive necrotrophic development. It has also been observed that symptoms vary along the length of a hypocotyl, being most extensive in the upper younger tissues [unpublished data]. Similar variations occurred onsoybean hypocotyls inoculated with Phytophthora megasperma, another facultative biotroph [II]. Again these different symptoms could be controlled by different amounts of biotrophic and necrotrophic fungal growth. In conclusion, symptoms on potentially susceptible bean hypocotyls varied considerably, being expressed as flecks, as limited lesions or as spreading lesions. By altering the physiological activity of the infected tissue (see [5] for effects of higher temperatures), symptoms were produced which could classify the same cultivar as either resistant or susceptible to the same race of C. lindemuthianum. This suggests that observations of visible symptoms alone may be inappropriate for designating genetic relationships between cultivars of bean and races of C. lindemuthianum. These specific relationships seem reliant on the establishment of biotrophy or not (hypersensitivity), phenomena which can only be reliably assessedby observations of the cellular interactions between fungal hyphae and infected host cells. On this basis, biotrophy can be regarded as an integral part of host-pathogen recognition controlled by complementary genes ofhost and pathogen. Subsequent symptom expression is controlled by a complex interaction between the extent of biotrophy and factors influenced by independent genotypic and phenotypic properties of the host and/or pathogen, e.g. the ability of the pathogen to kill cells quickly or of the host to produce more or less phytoalexin. The present results do not explain how roots, cotyledons and temperature influence either the extent of biotrophy or the amounts of phytoalexins produced, although the role of plant growth regulators deserves investigation [9]. However, the
256
P. M. Rowell
and J. A. Bailey
effects described in this and previous research [5] p resent great potential as probes into the biochemical nature of biotrophy itself, a process essential to the establishment of most important fungal pathogens. We thank G. M. Arnold for statistical analyses and S. C. Teague for photographic work. REFERENCES 1. ALLARD, C. (1974). Etude vulgaris) a l’anthracnose
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