Maataloustieteellinen A ikakauskirja
Vol. 59: 199—208, 1987

The biological control of seed-borne Alternaria brassicicola of
cruciferous plants with a powdery preparation of Streptomyces sp.

TAHVONEN, R. and AVIKAINEN, H.
Department of Plant Pathology, Agricultural Research Centre,
SF-31600 JOKIOINEN, Finland

Abstract. The effectiveness of a powdery preparation of a Streplomyces sp. isolate as a
seed dressing agent against seed-borne Alternaria brassicicola on different Brassica species was
investigated in the study. The preparation was made by freeze-drying and milling the biomass
produced in a fermentor into a form suitable for use as a dusting agent.

Seed dressing was 80—90 % successful in controlling damping-off from seeds artificially
infected with A. brassicicola. The effectiveness of dressing remained unchanged on seeds stored
under dry conditions for 5—6 weeks, but subsequently decreased slowly and was ca. 50 %

sixmonths after dusting. Streplomycesdressing controlled, in a manner comparable to chemical
dressing with thiram, damping-off caused by Alternaria fungi on seedlings which were grown
from commercial seed lots of different origin. The results of biological control of damping-
off did not vary in the peat lots of different origin whose natural disease suppressivity varied
considerably. The control result was the same or better than chemical dressing with thiram.
The acidity of the substrate (pH 4.8—8.6) had no effect on the effectiveness of biological control.
The results obtained against Alternaria damping-off were the same in other substrates clay,
fine sand and mull as in peat.

Index words: Streplomyces sp. Alternaria brassicicola, biological control, Brassica sp.

Introduction

An isolate of Streplomyces sp., obtained
from light-coloured, Finnish Sphagnum horti-
culturalpeat, has proved to be a potential bio-
logical control agent against plant pathogens
(Tahvonen 1982 a, 1982 b, Tahvonen and
Uon 1983, Tahvonen 1985), and especially
effective against seed-borne Alternaria on
cruciferous plants. A. brassicicola is the most

common seed-borne pathogen of Brassica sp.
throughout the world (Neergaard 1945). It is
also common and the most serious seed-borne
damping-off fungus on cruciferous plants in
Finland (Tahvonen 1979), and is routinely
controlled by chemical dusting with thiram
(Tahvonen 1985).

A research programme into the practical
application of Streplomyces sp. isolated from
peat was started at the beginning of 1985.

199

JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND

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A dry, powdery preparation in which the num-
ber of living particles was of the same order
of magnitude as in the spore suspensions used
in earlier experiments (Tahvonen 1982 b),
was developed by Kemira Co. The dry, pow-
dery preparation was given the commercial
name »Mycostop».

The aim of this study was to determine the
effectiveness of the powdery Streptomyces
preparation as a dressing agent in the control
ofAlternaria on cruciferous plants. The study
was carried out at the Department of Plant
Pathology, Agricultural Research Centre,
during 1985—86.

Material and methods

A powdery preparation suitable for use as
a seed dressing agent was prepared from a
Streptomyces griseoviridis Anderson et al.
isolate. Following culture in a fermentor, the
cell mass was separated from the culture me-
dium by centrifugation and then freeze-dried.
Compounds designed to improve adhesion to
the seeds and to maintain the vitality of the
dry preparation, were added in connection
with drying and milling. The final product
contained 10B—l8 —109 living particles/g. Details
of the manufacture of the preparation are not
given here.

Preliminary and methodological tests were
carried out on »Erfurter 291» or »Suuri Tans-
kalainen» cauliflower seeds, which were arti-
ficially infected with A. brassicicola in order
to ensure an even degree of infection (Tahvo-
nen 1982 b). Commercial lots of cruciferous
seed with a wide range of natural Alternaha
infection (Fig. 4) were obtained from the State
Seed Testing Institute and from a number of
commercial seed suppliers. Ten lots of cabbage
seed, 8 lots of cauliflower, and 9 lots of other
Brassica species and their varieties were tested.
A number of Chinese cabbage, swede and
turnip seed lots were also included, but their
results are not presented here because the
degree of infection of the seeds was too low
from the point of view of the experiment.

The seeds were dressed by carefully shaking
them together with the dusting agent in a
glass jar. When small lots of seeds were to be
treated, a small batch was first dressed in
order to ensure that there was a constant
amount of the preparation on the wall of the
jar. The seeds used in the experiment depicted
in Fig. 2 were coated by a special method
developed at Kemira Co’s Vaasa factory in
order to ensure that all the added amount
of dressing agent had adhered to the seeds.
According to tests carried out by Kemira Co,
adhesion of the preparation in the other ex-
periments varied from 50—70 °7o. In addition
to undressed controls, the experiments also
included undressed uninfected controls and a
chemical control with a thiram dressing of
4 g/kg on naturally infected seeds.

Retention of the dressing effectiveness on
seeds stored under dry conditions was studied
over a period of o—lB0—18 weeks after dressing.
The dressed seeds were stored in the dark at
+ 5 °C and at -I- 20 °C. Since the storage tem-
perature did not effect the effectiveness of
control, the results presented in Figs. 1 and
2 are the means of two test factors.

In order to test the effect of substratepH on
the effectiveness of the Streptomyces isolate,
the peat was limed with varying amounts of
dolomite limestone and calcium hydroxide.
The pH of the substrate following this treat-
ment, as determined from a 0.02 N CaCl2
extract, can be seen in Fig. 5. The effect
of soil type on the effectiveness of the dress-
ing agent was tested using the 6 different
substrates listed in Table 2. In addition, the
effectiveness of Streptomyces dressing was
tested in 12 non-disinfected peat lots of dif-
ferent origin (Fig. 6).

Unless otherwise stated in connection with
the results or in the tables, the seeds were sown
in steam-disinfected light-coloured Sphagnum
peat, which was given a basic fertilization of
10 g dolomite limestone/1, and 1.5 g of Peat
Super Y fertilizer/l following steam disinfec-
tion. The seedlings were raised in 1 1 plastic
pots or in 30 X 40 cm plastic boxes. There were
three or four replications, each replication

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consisting of 50 seeds. The seedlings were
grown from seed in thermostatically con-
trolled greenhouses at 18—20 °C under ar-
tificial illumination of 8000 lux. This meant
that the lots raised at different times were
grown under as exactly the same conditions
as possible. In addition to the substrate tests,
4 x 25 naturally infected seeds were sown on
water agar in 9 cm petri dishes. The degree
of fungal infection of the seedlings was deter-
mined under the microscope, as well as the
mortality rate, two weeks after the seeds had
been sown.

The degree of damping-off infection was
determined on the seedlings at the end of the
substrate experiments using the scale o—2,0 —2,
where 0 = healthy, 1 = slightly infected, 2 =

dead or non-viable. In addition, the fresh
weight of those seedlings which had developed
from naturally infected seed was determined.
These results are not presented here because
there were no differences in the weights of the
healthy seedlings between different dusting
treatments, and the dryweight results for the
infected seedlings were correlated with the
degree of infection.

The infection index, viable seedling %

(which includes healthy + slightly infected
seedlings as a °Io of the number of seeds
sown), and the control effectiveness % (cal-
culated from the formula (A—B):(C—B)x
100, where A is the infection index or vitality
of the dressed seedlings, B that of the infected
control and C that of the healthy control), are
presented in the tables. Where necessary, the
statistical significance of the results has been
tested using variance analysis.

Results

Control of Alternaria damping-off
with the Streptomyces preparation

Dressing the seeds with the Streptomyces
preparation controlled artificial infection by
seed-borne A. brassicicola. Dressing levels
greater than 5 g/kg seed had no effect on the
effectiveness of dressing when sowing was
done on the same day or within one week after
dressing (Table 1, Fig. 2). A dressing level of
1 g/kg seed resulted in significantly more
healthy seedlings than the control, but was

Fig. I. Retention of Streptomycesseed dressing (15 g/kg)
effectiveness on seed artificially infected with
Alternaria brassicicola in seedling experiments.

Fig. 2. Effectiveness of Streptomyces seed dressing on
seed artificially infected to different extents with
Allernaria brassicicola.

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Table I. Effect of the Streptomyces seed dressing dose on damping-offcaused by Alternaria brassicicola on cauliflower
raised in peat.

Seed Streptomyces dose, g/kg seed
0 I 5 10 15 20

Viable seedlings, %

A Iternuna- in feet ed
Non-infected

30 62 90 94 90 86 F = 139.6***
86 F = 1.088 86 78

however poorer than dressing levels of 5 g/kg
or more. Dressing had no effect on the num-
ber of healthy seedlings nor the dry weight in
the case of healthy, uninfected seed in any of
the experiments.

The effectiveness of the Streptomyces pow-
der remained unchanged on dressed seed stored
for a period of 4—6 weeks. The treatment was
still effective on dressed seed stored for as long
as 18 weeks, although less effective than after
a short storage period (Figs. 1 and 2). Higher
dressing levels of 10 and 15 g/kg seed had a
better effectiveness after 6 weeks storage than
the level of 5 g/kg seed (Fig. 2).

Dressing naturally infected Brassica sp.
seeds with the Streptomyces powder com-
pletely controlled damping-off on the peat
substrate. The result was fully comparable
with that obtained with chemical dressing with
thiram. A slight damage was found at the
base of seedlings grown from some dressed
seed lots. This was insignificant from the point
of view of seedling growth, and in fact oc-
curred with both biological and chemical
dressing. The effect of dressing on A. bras-
sicicola on seeds grown on the water agar
medium also proved to be effective (Fig. 4).
Both 5 g and 10g of the Streptomyces prep-
aration/kg seed were sufficent to prevent the
growth of the pathogen. Almost the same
result was obtained with thiram dressing, but
the effectiveness of chemical dressing was lost
in the case of some seed lots and there was
more Alternaria in these petri dishes than
in seeds dressed with Streptomyces. The mor-
tality of the seedlings on the agar medium was
completely dependent on the Alternaria con-
tent.

The effect of pH and the substrate
on the effectiveness of
the Streptomyces preparation

The acidity of the substrate had no effect
on the ability of Streptomyces to control
Alternaria damping-off over the pH range
4.8 —8.6 (Fig. 5). There was more damping-
off on undressed seeds grown on acidic or
slightly alkaline substrates than on a neutral
substrate. However, the result was not statis-
tically significant.

Streptomyces dressing controlled damping-
off on cauliflower on both the organic and
inorganic substrates in a similar fashion to
thiram dressing. There was no difference as
regards the control of Alternaria between the
steam-sterilized and unsterilized substrate
(Table 2).

A. brassicicola caused damping-off to a
varying degree on the peat substrate of dif-
ferent origins. Some of the peat lots inhibited
Alternaria by 60—70 % compared to the
steam-sterilized peat or peat lots susceptible
to this pathogen. Streptomyces dressing con-
trolled damping-off in the different peat lots
in the same way as chemical dressing with
thiram despite the natural antipathogenic
properties of the substrate. Dressing resulted
in similar numbers of viable seedlings as were
obtained when healthy seeds were used (Fig. 6).

Discussion

The powdery preparation made from Strep-
tomyces griseoviridis bacteria proved to be
highly effective in controlling seed-borne
Alternaria brassicicola growing on cruciferous

202



plants. This is in good agreement with the re-
sults of earlier experiments in which a spore
suspension was used in treating the seeds

(Tahvonen 1982 b). The effectiveness against
damping-off was the same on both artificially
infected and naturally infected seed. On the

Fig. 3. Effect of seed dressing with thiram or different
amounts of the Streptomyces preparation on the
control of seed-borne Alternaria brassicicola in
different lots of Brassica sp. seed. Naturally in-
fected seed.

Fig. 4. Effect of seed dressing with thiram or different
amounts of the Streptomyces preparation on the
Alternaria brassicicola content of seedlings grown
from seed on agar for two weeks. Different seed
lots.

203



basis of this, the results obtained with artifi-
cially infected seed can be generalized to cover
normal commercial seed.

Allernaria fungi (A. brassicae, A. brassi-
cicola, A. raphani) are the most common seed-
borne pathogens on cruciferous plants (Neer-
gaard 1945), and are controlled chemically

by dressing the seed with dithiocarbamate
(Heinze 1974, Dixon 1981) or iprodione and
fempropimorph fungicides (Maude et al.
1984). In the present experiments biological
control of naturally infected seed was equally
successful with the Streptomyces preparation,
or in some cases even better, than chemical
dressing with thiram. The result was the same
in both in vitro and in vivo experiments. Since
the antagonistic effect of S. griseoviridis
against different pathogens is very wide in
vitro (Tahvonen 1982 a), there is no reason
to assume that it would behave differently
against A. brassicae and A. raphani as against
A. brassicicola. The fungi in question are
biologically and systematically very close to
each other (Neergaard 1949). On the basis of
this, Streptomyces dressing would appear to
be effective against all seed-borne Allernaria
fungi on cruciferous plants, as is the case with
chemical dressing. However, Phoma lingam
can act as a seed-bornepathogen of cruciferous
plants (Neergaard 1977), and its susceptibility
to this biological control method could not
be estimated here. Nowadays, however, the
fungus does not have to be controlled since it
is of no significance as a seed-borne pathogen
in seedling production in Finland (Tahvonen
1979) for which most of the seed material is
imported from Sweden, Denmark and Hol-
land. In the experiments carried out here, for
instance, none of the 33 seed lots examined
was infected by P. lingam.

Table 2. Effectiveness of Streptomyces seed dressing in controlling Allernaria brassicicola on cauliflower raised
in different types of soil.

Seed dressing Type of soil

Peat Steam-sterilized Fine sand Fine sand + Clay Mull
peat 30 % peat

Healthy, viable seedlings

Healthy seeds 86a 90a 86a 91a 75a 85a
Untreated 26b 35b 58b 25b 38b 72a
Slreptomyces
5 g/kg 84a 83a 74a 85a 70a 78a
Slreptomyces
10 g/kg 79a 80a 80a 86a 71a 88a

Thiram
4 g/kg 85a 83a 90a 80a 77a 85a

204

Fig. 5. Effect of Streptomyces seed dressing on the
control of damping-off caused by Allernaria
brassicicola on cauliflower seedlings grown in
peat of different pH. F values; dressing =

151.7***; pH = 1.06.



The Streptomyces isolate used in these
experiments was strongly antagonistic on
agar against all the pathogens tested earlier
(Tahvonen 1982 b). Turhan and Grossmann
(1986) reported that their Streptomyces isolates
were always antagonistic to a number of patho-
gens if they were strongly antagonistic towards
Rhizoctonia and Alternaria. This would in-
dicate that the dressing method employed here
could also be effective against other fungi,
such as Fusahum spp., Phoma spp. and
Stemphylim spp, which are all significant
seed-borne fungi on a number of plants
(Neergaard 1977).

Bacteria proper and Actinomycetes usual-
ly thrive best in neutral or slightly alkaline
substrates (Baker and Cook 1974). For this
reason, it would be expected that Actino-
mycetes would be an ineffective biological
control agent in acidic substrates such as peat,
where the pH at which cultivation normally
takes place is in the range 5.5—6.5 (Puusx-

järvi 1974). In the experiments described in
this paper, the Streptomyces sp. isolated from
naturally acidic peat was effective against Al-
ternaria over the wide pH range of 4.8—8.6.
This is a very satisfactory result as regards
the practical application of the control method
in substrates of varying acidity. However,
the question of whether it is a case of Actino-
mycetes thriving in the favourable conditions
of the plants’ rhizosphere, or an example of
adaptation to different acidity levels in the
soil, can only be answered through more
detailed investigations.

Fig. 6. Effect of seed dressing with thiram or different
amounts of the Streptomyces preparation on the
control of damping-off caused by Alternaria
brassicicola on cauliflower grown in different
peat lots. Peat lots: 1 = steamed peat, 2—13 peat
lots of different origin.

Fig. 7. Growth of Streptomycessp. on cauliflower seeds
and agar.

Fig. 8. Control of Alternaria damping-off with Strep-
tomyces seed treatment. At the bottom untreated
seeds and at the top seeds treated with Strep-
tomyces-preparate.

205



The properties of the substrate, especially
pH, moisture content, airspace and the organic
matter, have a fundamental effect on the
activities of antagonists in the soil (Baker
and Cook 1974). For this reason it would be
very important to demonstrate that Strepto-
myces sp. also functions in different types of
substrate, such as fine sand and clay soils, and
in mull mixtures. Similarly, the effectiveness
of biological control of the pathogen was good
in peat lots of different origin, irrespective of
the peat’s own disease suppressive properties
which have been found in earlier studies to
vary considerably from lot to lot (Tahvonen
1982 a). It is obvious that the natural antago-
nists in the peat do not inhibit the activities
of the Streptomyces antagonist carried by the
seed.

It has been possible in these experiments
to demonstrate, using a wide range of strate-
gies, that the Streptomyces sp. isolated from
peat is effective in controlling seed-borne
A. brassicicola on Brassica sp. plants. Other
antagonists, such as Gliocladium, Trichoderma
and Penicillium spp., have also given good
results in similar applications (Wu and Lu
1984).

Other species of Streptomyces have been
successfully used on cabbage (Kundu and
Nandi 1984) and pea (Rothrock and Gottlieb
1984) in the control of Rhizoctonia damping-
off. In preliminary trials the Streptomyces
sp. used here also controlled Rhizoctonia
damping-off (Tahvonen 1982 b). The Strep-
tomyces antagonist clearly has a good poten-
tial in commercial applications as an alterna-
tive to the chemical dressing of cruciferous
plants. The present results suggest that more
extensive applications to other plants and
pathogens are also possible (Tahvonen and
Uoti 1983, Tahvonen 1985).

Fig. 9. Effect of Streptomyces and thiram seed treatment
on seed-borne Atternaria brassicicota. At the top
Streptomyces, in the middle thiram treated seeds
and at the bottom naturally infected cauliflower
seeds.

206



References

Baker, F.B. & Cook, R.J. 1974. Biological control of
plant pathogens. 433 p. San Francisco.

Dixon, G.R. 1981. Vegetable Crop Diseases. 404 p.
Salisbury, Wiltshire.

Heinze, K. 1974. Leitfaden der Schädlingsbekämpfung.
Band I. Schädlinge und Krankheiten im Bemiisebau.
360 p. Stuttgart.

Kundu, P.K. & Nandi, B. 1984. Control of cauliflower
damping-offby using antagonist coated seeds. Pedobiol.
27: 43—48.

Rothrock, C.S. & Gottlieb, D. 1984. Rise of antibiosis
in antagonism of Slreotimyces hygroscopicus var.
geldanus to Rhizoctonia solani in soil. Can. J. Microbiol.
30: 1440—1447.

Maude, R. 8., Humpherson, J.F.M. & Shurino, C.G.
1984. Treatments to control Phoma and Alternaria in-
fections of brassica seeds. PI. Path. 33: 525—535.

Neergaard, P. 1945. Danish species of Alternaria and
Slemphylium. 560 p. Cobenhagen.

1977. Seed Patholgy. 1187 p. Surrey.
Puustjärvi, V. 1974. Kasvuturve ja sen käyttö. 172 p.

Helsinki.
Tahvonen, R. 1979. Seed-borne fungi on cruciferous cul-

tivated plants in Finland and their importance in seedling
raising. J. Scient. Agric. Soc. Finl. 51: 327—379.

1982 a. The suppressiveness of Finnish light coloured
Sphagnum peat. J. Scient. Agric. Soc. Finl. 54:
345—356.

1982 b. Preliminary experiments into the use of
Slreplomyces spp. isolated from peat in the biological
control of soil and seed-borne diseases in peat culture.
J. Scient. Agric. Soc. Finl. 54: 357—369.
1985. Mycostop ett biologiskt bekämpningsmedel

mot svampsjukdomar. Växtskyddsnotiser 49: 86—90,
93.
& Uoti, J. 1983. The use of Streptomyces as a bio-

logical control agent. 10th Intern. Congr. PI. Protect.
1983: 2, 795.

Turhan, G. & Grossmann, F. 1986. Investigation of
great number of actinomycete isolates on their antag-
onistic effect against soil-borne fungal plant pathogens
by an improved method. J. Phytopath. 116; 238—243.

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Ms received August 19, 1987

SELOSTUS

Allernaria Brassicicola -sienen biologinen
torjunta kaalikasvien siemeniltä jauhemaisella
.SVre/t/owjcey-hakteerivalmisteella

Risto Tahvonen ja Hanna Avikainen
Kasvilautiosaslo
Maatalouden tutkimuskeskus,
31600 Jokioinen

Turpeesta eristetystä Slreplomyces-bakteerista valmis-
tettua jauhemaista bio-peittausainetta tutkittiin kaali-
kasveilla siemenlevintäisen Allernaria brassicicola -sienen
torjumiseksi. Valmiste oli tehty fermentaattorissa kas-
vatetusta biomassasta pakastekuivaamalla ja jauhamal-
la peittausaineeksi soveltuvaksi pulveriksi, jonka kaupal-
linen nimi on Mycostop. Ainetta käytettiin peittauksissa

1—l5 g/siemenkilo. Taimipoltteen torjuntatehoa verrat-
tiin kemialliseen tiraami-peittaukseen sekä keinosaastu-
tetuilla että luontaisesti saastuneilla siemenillä ravinto-
alusta ja taimikasvatustestein. Valmisteen biologisen te-
hokkuuden säilyminen varastoiduilla, peitatuilla sieme-
nillä tutkittiin o—60—6 kk peittauksen jälkeen tehdyillä kyl-
vöillä höyrytettyyn turpeeseen. Eri alkuperää olevien tur-

peiden, eri kivennäisalustojen sekä turpeenpH:n vaiku-
tusta torjuntatehoon selvitettiin.

Siemenen peittaus S/repro/nycer-valmisteella torjui
80—90 %:sti taimipoltteen voimakkaasti Alternaria-sie-
nellä saastutetuilta siemeniltä turvealustalla kasvatettu-
na. Peittauksen teho säilyi kuivana varastoiduilla sieme-
nillä 5—6 viikkoa muuttumattomana, minkä jälkeen te-
ho laski hitaasti ollen noin 50 % 6 kk;n kuluttua peli-
tauksesta. Slreptomyces-pe'Maus torjui taimipoltteen sa-
malla tehokkuudella kuin kemiallinen tiraami-peittaus
luontaisesti saastuneilta kauppasiemeneriltä. Taimipolt-
teen biologinen torjuntatulos ei vaihdellut eri alkuperää
olevissa turve-erissä suoritetuissa taimikasvatuksissa, vaik-
ka turpeiden luontainen taudinestokyky vaihteli voimak-

207



kaasti. Torjuntatulos oli sama tai parempi kuin kemial-
lisella tiraami-peittauksella. Kasvualustan happamuudella
pH-a1uee11a44.8.6 ei ollut vaikutusta biologisen peit-
tauksen tehoon. Muissa kasvualustoissa, savi, hieta ja
multaseos, oli Alternaria-taimipoltteen torjuntatulos sa-

ma kuin turpeessa.
Tehdyt tutkimukset osoittivat, että kaalikasveilla voi-

daan kemiallinen peittaus korvata biologisella peittaus-
menetelmällä, joka ei käytännön toteutukselta kuitenkaan
poikkea perinteisistä työtavoista.

208