'Journal of General Microbiology (1981), 122, 55-60.
Printed in Great Britain
55
Effects of Fungi on Barley Seed Germination
By S . H . T . H A R P E R A N D J . M. L Y N C H *
Agricultural Research Council Letcombe Laboratory, Wantage, Oxfordshire OX12 9JT
(Received 20 June 1980)
Barley seeds placed in contact with straw buried in soil imbibed water more slowly and had a
greater fungal biomass under the husk at germination than did seeds which had been in
contact with soil only. The growth of fungi under the husk increased during imbibition and
continued when the seed was fully imbibed; such growth resulted in poor seed germination. A
common soil saprophyte, Gliocladium roseurn, inoculated on to the outer surfaces of the seed
husk, suppressed germination by competing with the embryo for available oxygen. The
prevention of seed germination by some micro-organisms seems to be related to the microbial
affinity for oxygen.
INTRODUCTION
The examination of seeds for colonization by micro-organisms has been limited largely to
samples of grain stored before feeding to animals or sowing. The seed microflora are
predominantly fungi (Christensen, 1972) and relatively few bacteria are found (Mundt &
Hinkle, 1976). The fungi can be identified and their location ascertained by incubating seed or
portions of seed on agar (Mulinge & Chesters, 1970; Tempe & Limonard, 1973; Flannigan,
1974) but this does not give a quantitative measure of colonization. Methods of direct
observation have been described; the outer layers of seed containing the fungi are removed or
seed sections are prepared and the fungi are then stained (Hyde, 1950; Warnock & Preece,
1971; Mulinge & Chesters, 1970). The colonization of barley and oat seed can be studied by
removing and then examining the husk. This allows examination of the colonization of the
entire husk rather than that of small cross-sections; also a relatively large number of seeds
can be examined. The method is based on the assumption that colonization is predominantly
on the husk with relatively little on the husk-covered pericarp.
In addition to the colonization of seed on the ear or during storage, micro-organisms can
develop during seed germination, and when the supply of oxygen is limited they can compete
for oxygen and inhibit germination (Gaber & Roberts, 1969; Harper & Lynch, 1979; Lynch
& Pryn, 1977; Matthews & Collins, 1975). The husk may limit the supply of oxygen to barley
seed (Roberts, 1969) and fungi beneath the husk are better placed to compete with the embryo
for oxygen than are the soil microflora. These fungi also have the first chance to utilize the
carbonaceous energy-rich exudates released from the seed during germination (Simon, 1974).
We have now studied the course and effect of colonization beneath the husk by seed-borne
fungi and the influence of the husk on oxygen uptake by germinating barley seed.
Colonization on the outer surface of the husk by saprophytic fungi, such as Gliocladium
roseum, which compete with the seed for oxygen can inhibit germination (Lynch & Pryn,
1977). Since seed is particularly prone to invasion by such primary colonizers when drilled
into decomposing mats of crop residues, the effect of seed contact with straw on germination
in a simulated drill slit has also been examined.
METHODS
Measurements of colonization. Seeds were dehusked manually, dry seed being first soaked for 1 h in 0.01%
aqueous eosin. The husk was stained for 1 min in phenol/acetic acid/aniline blue (Jones et al., 1948), washed for
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56
S. H. T. H A R P E R A N D J . M. L Y N C H
1 min in water, mounted between two microscope slides and then examined immediately. Fungal biomass was
calculated from the length of hyphae by the method of Warnock (1971) which assumes a mean hyphal
cross-section of 1-96 x lo-' cm2, a density of 1.34 g fresh weight
and a dry weight of 10% fresh weight. The
degree of colonization was assessed by comparison with a slide of five husks which provided standards for the
following scale of mycelial dry wt b g seed)-': 0, no growth (< 1); 1, five or less short hyphal units (1); 2, localized
colonization at the embryo end of the seed (10); 3, moderate localized or slight overall colonization (100);4, heavy
localized or moderate overall colonization (300); 5, heavy overall colonization (1OOO). At least 30 seeds from each
treatment were examined. Preliminary examination showed that the colonization of lemma and palea were closely
correlated ( r = 0.709 for 50 seeds). Therefore, subsequently, only the lemma was examined and seed colonization
was calculated on the basis that the lemma comprised 60% of the surface area of the husk.
Colonization of seed by seed-borne fungi. Seed was incubated in an atmosphere of high relative humidity to
increase moisture content and promote fungal colonization without providing adequate water for germination.
Barley seed (Hordeum vulgare var. Proctor) was suspended on stainless-steel wire mesh (5 mm) 2 cm above water
at 20 "C. Samples of 30 seeds were taken at intervals and germinated on coarse sand (1.0 to 1.5 mm, 1 kg)
moistened with water (225 ml) in conical flasks (2 1 capacity) at 20 OC. Oxygen concentrations in the flasks were
maintained by continuously passing air or 2-5% oxygen in nitrogen at 100 ml min-'. Germination percentage was
recorded after 48 h; seed was said to have germinated when the coleorhiza penetrated the husk.
Colonization of seed in soil under laboratory conditions. Plastic columns (40 cm x 10 cm diam.) were sealed at
the base where a hole (5 mm) was drilled to allow drainage. The columns were filled within 3 cm of the top with
clay loam soil (Denchworth series) sieved to < 3 - 3 5 mm. The drainage hole was sealed and water was added until
the soil was waterlogged; the hole was then opened and the columns were allowed to drain standing in water such
that the water level was 30 cm below the soil surface. The columns were incubated for 7 d before planting seed.
Ten seeds were placed in each column at a depth of 3 cm. In some columns, straw (10 g) chopped into 5 cm
lengths was spread over the surface; a steel plate was then forced into the soil to form a 3 cm deep straw-lined slit
into which the seed was planted. The columns were incubated at 20 or 10 OC. The bulk density of soil in columns
Seed
. was sampled at intervals up to germination, and moisture content was estimated by
was 0.8 to 0.95 g ~ m - ~
drying at 105 OC for 1 h. All seeds were examined for colonization.
Germination of seed inoculated with a saprophytic fungus. Gliocladium roseum Bain. (IMI 196507) was
cultured in malt extract broth at 25 "C and barley seeds were soaked ( 5 min) in washed suspensions (5 g 1-') of the
fungus. Seed was germinated on sand at 20 "C as described above.
Oxygen uptake by saprophytic fungi. Gliocladium roseum, Mucor hiemalis (ATCC 26035) and a Penicillium sp.
isolated from barley seed were grown in batch culture (1 1 culture vessel) on a glucose and mineral salts medium
(Lynch & Harper, 1974). The specific rate of oxygen udzation (40,)was found from culture dry weight and the
difference between oxygen in effluent and influent gas streams.
Respirometric studies. Oxygen uptake was measured in a Gilson respirometer as described earlier (Harper &
Lynch, 1979). Husks were removed from some seeds manually or a small part of the husk (the plug) at the embryo
end of the seed was removed leaving the rest of the husk intact. When Gliocladium roseum was added to seed the
inoculum density was 7 g dry wt 1-'.
Histology of seeds. Generally, seeds were embedded in wax and longitudinal sections (15 ,um thick) were stained
with safranin and fast green. Alternatively, the seeds were embedded in plastic resin ('Spurr's'; Taab Laboratories,
Reading) and longitudinal sections (2 ,um thick) were stained with toluidine blue.
RESULTS
Colonization and germination of seed incubated at high relative humidity. When seed was
incubated over water the weight of fungus colonizing each seed increased with seed moisture
content. Colonization increased linearly with time after the moisture content had reached a
maximum (Fig. 1). The experiment was terminated after 1 0 d since some seed had
germinated. The germination percentage of seed removed at intervals and planted on
moistened sand declined progressively as colonization proceeded, particularly if the oxygen
supply to seed was limited (Fig. 2). The maximum depression of germination percentage in
2.5 % (v/v) oxygen was reached after 4 d incubation; more than 80% of these seeds carried
100,ug fungus under the husk.
This experiment also showed the importance of the husk together with associated fungal
colonization in impairing germination of seed in low oxygen concentrations. Germination
after incubation over water for 8 d was <30%in 2.5 % (v/v) oxygen, compared with 75 % in
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Fungi and seeds
57
100
..-c
Y
80
60
M
W
5
40
Y
0
2
a"
20
I-
I
50
0
100
150
200
250
Preincubation time (h)
Fig. 1
Fig. 2
Fig. 1. Imbibition (0)
and colonization (0)of barley seed incubated at high relative humidity.
Fig. 2. Germination of barley seed prehcubated at high relative humidity in 21 96 (0)
and 2.5% (0)
oxygen.
Table 1. Percentage of seed carrying more than 100 pg fungal biomass at
germination in contact with soil or separated from soil by straw
Percentage of seeds with more
than 100 pg fungi
Incubation
temperature
("C)
10
20
A
Seed planted
in contact
with soil only
Seed planted
in contact
with straw
25
10
75
70
air, but when the husks were removed germination always exceeded 80% even in 2-5 % (v/v)
oxygen.
Colonization of seed separated from soil by straw. When seed was separated from soil by a
layer of straw the rate of imbibition was slowed and germination was delayed at both 10 and
20 OC (Fig. 3). In addition, fungal colonization at germination was greatly promoted by the
presence of straw (Table 1).
Germination and respiration of seed inoculated with Gliocladium roseum. The uptake of
oxygen by seeds inoculated with the fungus was 8 pl h-' seed-' (Fig. 4). Oxygen uptake by
control seed increased slowly to reach 3.5 p1 h-I seed-' after 16 h when seed germinated.
Oxygen uptake by inoculated seed did not increase and germination was completely
suppressed.
Afinity of fungi for oxygen. The qo, values for Gliocladium roseum and Mucor hiemalis
were 3-0 and 6.6 mmol (g dry wt)-' h-l, respectively, and that for a Penicillium sp. isolated
from seed was 1.1 mmol (g dry wt)-' h-l.
Histological examination. We found no evidence of fungal penetration through the
pericarp-testa (the layer beneath the husk). When seed was inoculated with Gliocladium
roseum, the fungus grew predominantly at the tip of the seed containing the embryo. Here, a
continuation of the inner layer of the husk forms a 'plug' that seems to be the point of oxygen
entry into the seed (Lynch & Pryn, 1977) (Fig. 5). The plug is fragile and we have never been
able to prepare a section where the plug remained attached to both sides of the outer layer of
the husk. No evidence has been found for fungal mycelium penetrating the plug.
Respiration of seed after removal of the husk or plug. All seed germinated when oxygen
uptake had reached 3 to 4pl h-' seed-'. Since oxygen uptake by the seed increased after
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58
S . H. T. HARPER AND J. M. LYNCH
60
80
100
0
5
10
15
20
25
Time (h)
Time (h)
Fig. 3
Fig. 4
Fig. 3. Imbibition of barley seed in contact with soil or separated from soil by straw: 0 , 10 OC in
contact with soil; 0 , 20 OC in contact with soil; 0 , 10 O C in contact with straw: M, 20 OC in contact
with straw. Arrows indicate time of germination.
Fig. 4. Oxygen uptake by barley seed inoculated with Gliocladium roseum: 0,control; 0,inoculated.
0
20
40
Fig. 5. Longitudinal section of barley seed in the region of the embryo. Bar marker represents 0.1 mm.
10
15
20
25
Time (h)
husk removed; A, plug removed.
Fig. 6. Oxygen uptake by barley seed: 0,control (husk present); 0,
Arrows indicate time of germination.
0
5
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Fungi and seeds
59
removal of the husk or of the plug at the embryo end of the seed, these treatments resulted in
earlier germination (Fig. 6).
DISCUSSION
Our observations show that when fungi colonize barley seed beneath the husk, germination
can be impaired. Similar effects observed with wheat were attributed to damage by seed-borne
species of Aspergillus and Penicillium (Grimn, 1966), but long periods of incubation were
necessary before germination was affected. Competition for oxygen between the embryo and
fungal population seems to provide the most likely explanation for impaired germination
during the early stages of colonization by seed-borne fungi examined here. When seeds are
separated from soil by straw, fungal colonization is promoted and hence the susceptibility to
low concentrations of oxygen is increased. We have found that seeds recovered from the field
and which have not germinated are most heavily colonized by fungi (Lynch et al., 1980).
The present respirometric results add further support to the hypothesis that competition
between the seed and fungus for oxygen is probably the mechanism by which germination is
suppressed. In our experience aspergilli and penicillia, which have small specific rates of
oxygen uptake, produce relatively little effect on germination when colonizing the outer husk,
but when these fungi colonize the seed beneath the husk they might compete with the seed for
the limited supply of oxygen that penetrates the 'plug'. The maximum reduction in
germination percentage in low concentrations of oxygen was recorded when more than 80 %
of the seed carried 100 pg fungus or more beneath the husk. If this fungus was Penicillium, it
would account for a maximum oxygen uptake of 2.5 pl h-' seed-', closely comparable to the
3.5 pl h-' seed-' uptake by uncolonized seed at germination. However, if Mucor were the
colonist, the oxygen uptake by the fungus would be 15p1 h-' seed-', greatly in excess of the
seed uptake.
These observations are consistent with the effect of the bacterium Azotobacter
chroococcum on barley seed (Harper & Lynch, 1979). Among the bacteria the qo, values of
Azotobacteraceae are large, up to 223 mmol (g dry wt)-' h-l (Williams & Wilson, 1954),
more than an order of magnitude greater, for example, than that of Escherichia coli and
KlebsieZZa aerogenes (Harrison, 1972). Again A . chroococcum is notable amongst the
bacteria we have tested in preventing seed germination.
We thank Mr S. F. Young for help with the histology.
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