The host-parasite physiology of the maize smut fungus, Ustilago maydis I. The effect of smut infection on maize growth

Physiological

Plant Pathology

(1978)

12, 93-102

The host-parasite physiology of the maize smut fungus, Ustilago maydis I. The effect of smut infection on maize growth E. ELLEN kmrrrt

and J. H. BURNETT

Department of Agrimltural Parks Road, Oxford OXI

Science, Univerti& 3PF, England

(Accepted

for publication

August

of Oxford,

1977)

The effect of U&ago maydis infection on the vegetative growth of young maize plants was examined. The dry weight of infected tissue increased gradually from 3 days after inoculation with the fungus until early sporulation, but this was paralleled by a decrease in weight of other developing plant parts. As infection progressed, unfolding of leaves from the me&tern was delayed and the maximal leaf area attained was reduced. The roots, however, suffered a proportionately larger reduction in their dry weight than the shoot. Infected areas of a leaf blade were chlorotic 3 to 5 days after inoculation, having lost 60% of their chlorophyll. Eventually, all chlorophyll disappeared from these infected areas, Tissue immediately adjacent to a leaf gall, however, resembled a “green island”, retaining more chlorophyll than the rest of the leaf when the leaf was senescing.

INTRODUCTION Ustilago maydis, an ecologically obligate biotroph which penetrates its host,
METHODS

material. Maize plants (
Plant

t Present address: Plant Sutton Bonington, Loughborough

Physiology LE12

and 5RD.

Environmental

Studies,

University

of Nottingham,

94

E. E. Billett and J. H. Burnett

Plants were grown in the glasshouse. The temperature was maintained at 25 + 4 “C and lighting was by natural daylight only. No significant temperature or light gradients were detected under these conditions so that an experimental layout to take into account such gradients was not necessary. Fungal material and inoculum. Ten days after sowing, when plants had three unfolded leaves plus one half-unfolded leaf, plants were inoculated with 1 ml of a suspension containing 1.5 x lo6 sporidia. The sporidial mixture was injected into the centre of the tightly rolled leaf whorl, just above the apical meristem [9]. Control plants were inoculated with heat-killed sporidia. The sporidial culture contained a mixture of wild type sporidial strains of opposing mating type prepared by germinating diploid spores on a complete nutrient medium as described by Holliday [1.5-j. The two main investigations undertaken are described below. In both investigations five replicates, each consisting of five healthy and five infected plants per pot, were arranged in a completely randomized block design along the bench. Since selection of plants with only one infected area facilitated physiological interpretation, only plants with a single stem gall and with no tillers were used. Changes in dry weight during growth. Plants were harvested and analysed on the day before inoculation (day - 1) and on days 3, 6, 10 and 19 after inoculation. The root system was carefully washed free of soil and seed remains were discarded prior to drying. The plants were then blotted and partitioned into: (i) Stem-tissue between the first (coleoptile) node and the tip of the first leaf sheath, including the basal parts of any other leaf sheaths and the apical meristem. The ligule of a leaf was taken to be the junction between the sheath and blade. (ii) Individual leaves-rest of sheath and blade, taken together. (iii) Roots-tissue at or below the first node, including prop roots. All plant parts were dried to constant weight at 90 “C. Leaf expansion and chlorophyll production during growth. The area and chlorophyll content of individual leaves (blade + sheath) were measured. Leaf area was measured photoelectrically [23]. Chlorophyll a and b were determined as described by Arnon [I]. Harvests were taken the day before inoculation (day - 1) and at 2, 4, 7, 11 and 20 days after inoculation. Chlorophyll content of leaves Owing to the onset of winter, plants were grown in a growth cabinet at a constant temperature of 25 + 1.5 “C, with a 16 h day and were illuminated by a battery of fluorescent tubes which gave an intensity of 15 600 to 17 800 lx at pot level. Plants were inoculated with a sporidial culture containing a mixture of two wild type sporidial strains, of opposing mating type, isolated from a single germinating spore. This source of inoculum resulted in a large number of leaf galls, unlike that derived directly from a mixture of germinating brandspores. Chlorophyll was extracted and measured as described earlier.

Effect of maize smut on maize growth

95

Chlorophyll content of healthy and infected leaves. Five replicate samples of healthy and infected fourth-leaf blades were taken in the morning 4 and 6 days after inoculation. The leaf area was determined by tracing the outline on graph paper and weighing. Chlorojhyll content of smutted and non-smutted areas of infected leaves. The fourth-leaf blades from seven healthy and seven infected plants were harvested 6 days after inoculation, following a 24 h period in the light or in the dark. Tissue up to 5 mm wide adjacent to the galled areas (A.G.) and tissue, of equivalent area, at least 2 cm away from the gall (D.G.) were dissected away. Comparable samples were taken from completely healthy leaves for further comparison, and the area and chlorophyll content of each sample measured as above. Statistical treatment of results Standard errors were calculated for the relevant means and the significance of differences between population means was assessed by a pooled variance test and t-test after ensuring that the individual variances were the same within the limits P< 0.05 [2]. RESULTS

Growth ana&+ All infected plants had visible stem galls 7 days after inoculation. In a few cases the leaf sheath bases of the younger leaves, surrounding the galled stem, were also galled. However, there were no galled leaf blades. Sporulation in the galled stem occurred mainly between day 10 and day 20. Dry weight. Total dry weight of infected plants was slightly greater than that of healthy plants at day 3, was significantly less than that of healthy plants at day 6 and was less than half that of healthy plants 13 days later (Table 1). Both root weight and total leaf weight were reduced but the shoot (stem+leaf)/root ratio demonstrates that the roots eventually suffered a proportionately greater reduction than the shoot. Total leaf weight, however, was significantly reduced as early as 6 days after inoculation, whereas the roots were not reduced until day 10. Dry

weight of healthy

and infected

TABLE 1 maize plants and plant parts at dz&rent

times after inoculationa

Days after inoculation

Plant

Total

Root

Leaf

Stem

-1

H

411

193

137

81

3

H

1000+ 17* 1109+44

336+ 17 363+26

6

H I

1741+37*** 1634+67

457k 18 417*40

1070f28** 943*27

10

H I

2871+172** 2421+56

742f64*** 604+ 13

1844+ 14282

19

H I

9477*343-a 4106+ 363

o Dry weight expressed errors are given where * P
534k 16 575+_29

2396+ 143*** 699 + 70

as mg per five relevant. +** PcO.01.

maize

plants.

120* 142

6134f261*** 2238k 174 H,

healthy;

Mean shoot/root 1.0

130+ 1.0 17126.7

1.98 2.06

214*7-O*** 2742 11.0

2.81 2.92

285k 389+

2.87 3.01

14** 13

947 + 58 1169-+215 I, infected.

2.96 4.87 Standard

E. E. Billett

96

and

J. H. Burnett

Only leaves which unfolded after inoculation were reduced in dry weight (Table 2) and the younger the leaf, the greater was the reduction. By day 19, leaf 4, leaf 5 and leaves 6, 7 and 8 of infected plants weighed respectively 37%, 50% and 78% less than the corresponding leaves on healthy plants. Leaves 1 and 3 were equal in healthy and infected plants at all harvests. Leaf 2, on the other hand, although it was not galled, was slightly heavier in the infected plants, but this difference was only significant at day 6. TABLE

Dry

after

weight

Days

inoculation

of individual

Plant

leaves of healthy

Leaf

1

Leaf

2

or infected plants

2

Leaf

3

at dz$rent

Leaf

4

times after

Leaf

5

irweulation” Leaves and

6, 7 8

H

62

57

19

9

3

H I

83 89

179+4 19Ok6

181 193

86 95

6

H I

80 88

199+5* 218+7

326+ 325+9

10

H I

78 84

192+7 227+21

350&21 352 + 16

527*40*** 350* 13

454f20**+ 206+39

234 + 15*** 105+ 16

19

H I

297 323

478 f 62 418+41

1052+164** 632 + 36

592+87*** 655 f 68

2394+263*** 670; 29

,-I

D Dry weight expressed as mg per five maize errors are given where relevant. * P
10

plants.

324k 238+

5 8 16*** 11

H, healthy;

-

122+8*** 64+5

I, infected.

19 10

Standard

An infected stem was significantly heavier than a healthy stem as early as 3 days after inoculation (Table 1). By day 19, however, when the stem gall(s) had sporulated and was beginning to dry up, the stem’s weight was not significantly different from that of a healthy stem. Nevertheless, even at this stage, the galled stem contributed about 24% of the total dry weight of the infected plant, whereas the stem of the healthy plant represented only 10% of its dry weight. In other experiments [3], not reported here, it was found that if a leaf was infected, the infected areas were heavier per unit area than the corresponding areas on a healthy plant. and chlorophyll content. Infection resulted in a significant reduction in total leaf expansion 7 days after inoculation (Table 3) and the total leaf area of infected plants was only 27% that of healthy plants at the day 20 harvest. Once infection had become established, the rate of expansion of leaves 3 and 4 was less in infected than in healthy plants and the onset of contraction earlier. Infection caused a great reduction in the rate of expansion of the later leaves and these leaves stopped expanding earlier in infected than in healthy plants. Infection also reduced the rate at which leaves unfolded from the meristem (Table 4). Nevertheless, chlorophyll content per unit area of leaves was similar both in infected plants with stem galls and in healthy plants throughout the period examined [Table 5(a)]. No physiological significance is attached to the two anomalous values for leaves 2 and 4 at day 20.

Leaf expansion

Days

H I

H I

H I

7

11

20

as cm* per ** P
H I

4

H

Plant

H I

inoculation

2

-1

0 Area expressed *** P
after

Total

five maize * P
plants.

2854+ 186*** 769+215

1108+39*+* 632 i 70

580*26*+* 4085 19

34Qk 13 309+20

192 200

Total

H, healthy;

-

36 37

35 36

35 35

31 34

30

Leaf

baf area and areas of individual 1

Leaf

2

I, infected.

Standard

66+3.1 47 + 10.00

84 84

79_+4 93+11

85 86

63 69

37

Leaf

3

errors

Leaf

97*4** 66+9

27 25

-

4

times aftr

where

335+32’** lllk28 relevant.

273f 19*** 145+22

189_+ 14*** 102+9

at di$erent

arc given

136f8** 86+ 17

176+8*** 129+9

151+5*** 121+11

115 110

68 73

16

TABLE 3 leaves of healthy and infecfed plants Leaf

705+49*** 231+44

366+25*** 165233

109+46*** 50+5

8 12

-

-

inocukztion” 5

Leaves

6,7

and

848+76*** 131 f 79

153+48 72+ 18

17 8

-

-

-

8

E. E. Billett TABLE

Number

of leaves in healthy

Days

after

and infectcd plants

(a) Chlorofihyll

at dzyerent Number H

inoculation

2-3 5-6 10-11 19-20 B H, healthy;

3-4 6-7 7-8 8-9

of leaves I 3-4 5-6 5-6or6-7 6-7 or 7-8

I, infected.

TABLE 5 Chlorophyll content #er unit area of 1eaveP per unit area of leaves of healthy and infected giants without inoculation

leaf galls

Plant

-1

H

28

16

9

H I

39 45

35 36

20 20

15 17

7

H I

42 37

27 26

19k2.0 20+2*0

11

H I

39 42 40 37

37 34

28 26

20

H I

-

11&l** 21*2

18 15

2

Leaf

(b) Chlorophyll

after

inoculation

1

Leaf

2

Leaf

3

Leaf

Chlorophyll

at di&ent

Leaf

5

times after

Leaves 6, 7, 8 and 9

-

1

1

-

12 13

22+2 18+2

13*3 13+2 17+3 14-t 1

20*2* 13+2

15+1 18k2

9 11

content per unit area of whole ungalbd

Plant

4

-

per unit

7 6

or galled leaves

area

Chlorophyll Chlorophyll

4

H I

22.6kO.8 20.8kO.7

3.7f.O.2* 4.2 f 0.2

6

H I

23.0 f 0.6*** 18.4* 1.9

3.7+0*1 3.7 * 0.2

Fourth leaves sampled at 4 and 6 days after inoculation. * P
Chlorophyll

J. H. Burnett

times after inoculationa

Days after inoculation

Days

and

4

I, infected.

Standard

a b

errors

content of infected leaves

Chlorophyll content of smutted leaves. Chlorophyll content per unit area of whole infected fourth-leaf blades was slightly less than in healthy blades 4 days after inoculation, the difference becoming significant 2 days later [Table 5(b)]. The chlorophyll a/ chlorophyll b ratio was similar in infected and healthy blades. Chlorophyll content of smutted and non-smutted areaS of infected leaves. Chlorophyll content per unit area of fourth-leaf blades which had been exposed to a 24 h continuous light period (Table 6) was noticeably higher than the values obtained earlier [Table

Effect of maize smut on maize growth

99 TABLE

Chlorophyll

6

content jer unit area of smutted and mm-smutted &ark treatmenP

Tissue

Pre-treatment

Chlorophyll

areas of infected leaves fobwing

per unit

area

Chlorophyll Chlorophyll

H A.G.I. D.G.I.

Light Light Light

41.6 42.2 37.9

1.1 0.9 1.0

H A.G.I. D.G.I.

Dark Dark Dark

21.7 28.4 21.2

3-o 2.5 2.5

light or

a 6

a Results expressed as ug chlorophyll per cm a. Healthy (H) and infected (I) fourth leaves sampled after 24 h in the light or in the dark. Harvesting undertaken 6 days after inoculation. A.G., areas adjacent to leaf galls; D.G., areas 2 cm away from leaf galls.

5(b)] for both healthy and infected leaves. Since chlorophyll synthesis has a lightdependent step, this is not surprising. In the light, chlorophyll per unit area of tissue adjacent to the galls (A.G.) was similar to that of healthy leaves, whilst that of galled (D.G.) tissue was reduced. In the dark, on the other hand, A.G. tissue had retained more chlorophyll than either D.G. or healthy tissue. The chlorophyll u/chlorophyll b ratio was similar for healthy and infected tissue in the light, whilst in the dark it was smaller in the infected tissue. Since chlorophyll b is preferentially degraded in most tissues [a, its breakdown in the dark is apparently less in infected than in healthy tissue. DISCUSSION A definitive interpretation of the changes, just described, in the dry weights of various plant parts of healthy and smutted maize plants is not possible without a knowledge of the patterns of transport of organic and other nutrients. Since the translocation of f*C-labelled assimilates is described in a subsequent paper [4], the discussion here is confined to a limited number of topics only. Plants with galled stem Such infection resulted in an increase in the stem’s dry weight 3 days after inoculation and it continued to increase more rapidly after infection until at least 10 days after inoculation, when the galls had started to sporulate. The smut fungus occupies a very small volume in the plant during early stages of infection and, even during early sporulation, it occupies less than 10% of the host tissue [3]. This suggests that the fimgal contribution to the increased dry weight cannot be very large as appears to be the case in some other tissues infected with obligate biotrophs [II, 25, 301. Increased stem weight of smutted maize was accompanied by a decrease both in dry weight and area of young leaves. Leaf expansion was eventually delayed, and final leaf size reduced. Increased dry weight of tissues infected with obligate biotrophs

100

E. E. Billett

and

J. H. Burnett

[18] and a reduction in total leaf area [12, 20, 211 is common. The rate of leaf development is also reduced in some cases [II, 121 but not in others [IO]. Reduction in leaf area is likely to result in a reduction in the assimilate available for future plant growth. Such an effect was shown by the restricted root growth subsequent to the reduction in leaf area in smutted plants. A reduction in the root system is a normal reaction to treatments which cause a reduction in the carbohydrate level of the shoot, for example, shading and defoliation [ZZ]. It is also a common response to foliar infections by obligate biotrophs such as powdery mildew on barley and on wheat [17, ZU], rust and loose smut on wheat [IO, 121 and rust on beans [31]. The younger the maize leaf at the time of inoculation of the plant, the greater was the reduction in the growth of this leaf. One hypothesis to account for this can be suggested. Biotrophs are thought to compete with their hosts for essential metabolites, particularly phosphorus compounds [13] and carbohydrates [IB]. Since the smut fungus grows initially in the apical meristem, it is probable that it would compete first with the developing leaf primordia, and hence the primordium least advanced at the time of inoculation would be most affected. Other hypotheses can be suggested, e.g. reduced translocation of carbohydrates into leaves throughout the growing period, or toxic effects caused by the fungus, but there is no evidence to suggest that such effects occur. For example, leaf 3 of smutted plants did not reach its normal size and started to shrink earlier than the third leaf of healthy plants. However, its chlorophyll concentration and its total dry weight were the same as that of the third leaf of healthy plants. In addition, the second leaf of smutted plants was sometimes heavier than the second leaf of healthy plants. It is possible that these leaves were photosynthesizing at an increased rate per unit chlorophyll to compensate for the decrease in the photosynthetic capacities of younger leaves, in a similar manner to that recorded in rust-infected bean plants [19]. An increase of stem weight, if it is principally in host tissue, could result from a reduced rate of respiration in the tissue, from an increased rate of transport of organic substances from the leaves and of inorganic substances via the roots into the stem or to decreased export from the stem. The present data do not enable a discrimination to be made between these possibilities but it may be noted that respiration is usually increased, rather than reduced, in infected tissue [24]. Plants with galled leaves

These were obtained in growth room experiments and are not strictly comparable with the results already discussed which were obtained from plants raised in similar glasshouse conditions but exclusively with stem galls. The reduction in the chlorophyll content of infected leaves is as expected since chloroplasts disintegrate in infected areas in maize [3, 81. Somewhat less expected was the demonstration of areas in infected leaves comparable to the “green islands” produced by rusts and mildews [5, 71 which are detectable after leaf senescence has been accelerated by darkness [ 71. Smutted maize leaves kept in the dark for an extended period retained more chlorophyll in leaf tissue adjacent to the gall(s) than did either healthy leaves, or infected leaves at some distance from the gall(s). Even though photosynthesis is unlikely to be stimulated in infected leaves, because of their reduced chlorophyll content, it is

Effect

of maize

smut

on maize

growth

101

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