ISSN 0253-6749
MISCELLANEOUS REPORTS 41
BIOLOGICAL CONTROL OF CROWN GALL
IN STONEFRUIT NURSERIES
N. Vouzounis
"
,
AGRICULTURAL RESEARCH INSTITUTE
MINISTRY OF AGRICULTURE AND NATURAL RESOURCES
CYPRUS
NICOSIA
SEPTEMBER 1990
BIOLOGICAL CONTROL OF CROWN GALL
IN STONEFRUIT NURSERIES
N. Vouz ounis
SUMMARY
Biological control of crown gall, caused by Agrobacterium tumefaciens, on cherry root­
stocks namely mazart was accomplished with strain K84 of the Agrobacterium rhizogenes. In
the nursery seed inoculation, before sowing, in a suspension of the antagonistic strain K84
resulted in 81 % control of the disease while root or combined seed and root inoculation
achieved 100% control. The effectiveness of the method was assessed by the difference be­
tween the dry weight of gall tissue on inoculated and non-inoculated plants expressed as a
percentage of the weight of gall tissue on non-inoculated plants.
nEPIAII'PH
BLOAOYLxi] xcna:n:oAEf!llOll rou crown gall, rtou :n:QoxaAELTaL ana TO Agrobacterium tu­
mefaciens, OE unoxetuev« xEQaOLu~ mazart Em~E~aLWSllXE XQllOLf!o:n:OLWVta~ TO strain K84
tou Agrobacterium rhizogenes. LTO qJ'llTWQLO AXEAELa~ Ef!~U:n:TLOll TlOV o:n:aQlOv OE ClLUA'llf!f!a
tou aVtaYlOVLOTLXOU ~axTllQtO'll ECllOOE 81% ~LOAOYLXi] xaTa:n:oAEf!llOll Tll~ aOSEVELa~ EVW
E~a:n:TLOll uovo QL<;'WV i] af!qJoTEQlOV o:n:aQlOv XaL QL<;'WV EtXE ocv a:n:oTEAWf!a 100%
xaTa:n:oAEf!llOll. H cnotsseoucnxotnrc Tll~ f!ESaClO'll EXTLf!i]SllxE ~UO£L Tll~ ClLaqJ0QU~ f!ETa~U
rou ~t]QOU ~UQ0'll~ TlOV xaQXLVlOf!UTlOV TlOV qJ'llTWV rtou Ef!~a:n:TtOTllxav OTO ClLllA'llf!f!a xm
EXELVlOV rroi: ClEV Ef!~a:n:TtoTllxav (f!UQT'llQE~), Ex:n:EqJQaOf!EVll~ (%) rou ~uQ0'll~ TlOV
xaQxLVlOf!UTlOV TlOV f!aQTuQlOv.
- 2 ­
INTRODUCTION
the optimum time to inoculate the plant material.
In cyprus, deciduous fruit tree seedlings are
raised mainly in government nurseries. About sixty
thousand stonefruit seedlings are issued to growers
annually, but demand always exceeds production.
MATERIALS AND METHODS
The work was carried out at the Akhelia gov­
ernment nursery where the soil was naturally infest­
ed with A. tumefaciens. The nonpathogenic strain
K84 of A. rhizogenes was grown in the laboratory
on a, special solid medium (nutrient broth 0.8 g and
agar 1.6 g, made up to 100 mI with distilled water
and autoclaved) using a pure culture provided in
ampoules as dry powder by the National Collection
of Plant Pathogenic Bacteria, U.K.. Inoculum was
subsequently prepared by washing a 3-day growth
with distilled water to give a concentration of ap­
proximately lOS colony forming units/rnl. One mI of
Tween - 20 per liter of inoculum was added to im­
prove its wettability (Dhanvantari, 1976). Young
seedlings or seeds were immersed in the solution for
five seconds and immediately planted in the nursery
plots.
One of the limiting factors in satisfying de­
mand for stonefruit (Prunus spp.) seedlings is infec­
tion by the A. tumefaciens which causes the crown
gall disease. The bacterium lives in the soil, enters
plants through wounds and induces unregulated cell
division, leading to massive gall formation just be­
low the soil surface at the crown of the plant (Kerr,
1980). The young seedlings are thus unable to grow
well and die. The disease occurs whenever these
crops are grown, but infection generally starts in
the nurseries (Schroth et el., 1971). Soil fumigation
or moving to new ground have not provided consis­
tent control of the disease (Schroth et el., 1976).
However, it has recently been shown that crown
gall can be controlled biologically, by treating young
plants or seeds before planting with a closely relat­
ed non-pathogenic bacterium, A. thizogenes, strain
K84 (Kerr, 1972; New and Kerr, 1972; Kerr and
Htay, 1974).
The four treatments were: no inoculation, seed
inoculation alone, root inoculation alone and both
seed and root inoculation. Non-inoculated plants
served as controls. The Randomized Complete
Block Design was used with three replications for
each treatment while the number of cherry seeds in­
itially sown was 300. Sowing of cherry seeds in pots .
The objective of this study was to test the ef­
fectiveness of the antagonistic strain K84 in control­
ling crown gallon cherry seedlings and to establish
Table 1. The effect of inoculation by the
Treatment
1. No inoculation (control)
2. Root inoculation
3. Seed and root inoculation
4. Seed inoculation
Seedlings
examined
192
132
132
225
si&in
K84 of A. rhizogenes on crown gall of cherry seedlings
Mean dry weight
of gall tissue
per plant
(g)
1.73b
oc
oc
0.3 a
Control
Infected
plants
(%)
(%)
100
100
81
11.2
0
0
0.3
a Means having the same letter are not significantly different, (P<O.05) using Duncan's Multiple Range Test.
-3 ­
and in the nursery plots was made on the 16th Feb­
ruary, 1987, while transplanting of young seedlings
from the pots to the nursery plots took place two
months later. Transplanted seedlings were covered
with a net to avoid plant shock from high tempera­
tures and prolonged sunshine. All seedlings were up­
rooted and examined for gall formation ten months
.after the last inoculation. The number of seedlings
examined for gall formation at the end of the trial
ranged from 132-225 (Table 1). Treatment effect
was assessed by the difference between the dry
weight of gall tissue on inoculated and non­
inoculated plants expressed as a percentage of the
weight of gall tissue on non-inoculated plants.
RESULTS AND DISCUSSION
Root inoculation with the bacterium K84,
alone or in combination with seed inoculation, pre­
vented completely the formation of gall tissue on
the root system of young cherry seedlings while the
percentage of non-inoculated (control) seedlings in­
fected with the pathogenic bacterium reached 11.2
(Table 1). Also, seedlings raised from seed inoculat­
ed with the antagonistic bacterium were less infected
(0.3%) compared with the controls. Inoculation of
seed alone reduced infection by 81%.
The present trials suggest that under local con­
ditions inoculation of seed with the antagonistic bac­
terium K84 can reduce the infection of seedlings
from crown gall disease considerably. However,
treatments of young seedlings in the nursery involv­
ing root inoculation alone or combined with seed
inoculation have disadvantages inspite of their in­
creased effectiveness in controlling the disease. This
procedure is time-consuming, costly and limits the
degree of success on transplanting the young seed­
lings because of exposure to high air temperatures
which have a drying effect on the root system. De­
spite its great effectiveness in controlling the disease
root inoculation during active growth does not offer
a practical solution because of this disadvantage. It
can, however, be made in the dormant season when
seedlings are transplanted from the nursery to the
orchard. This was confirmed by Moore, (1977) and
Schroth et el., (1976) who showed that root inocula­
tion of dormant rootstocks in a suspension of strain
K84 before planting ill their permanent place in the
field prevents infection through wounds caused prior
to planting. Since disease inception in stonefruit can
occur at any time during the life of the tree as a
result of wound caused by growth or mechanical in­
juries it is difficult to state with certainty whether
infection of a tree has occurred in the nursery or in
the orchard.
In conclusion, seed inoculation with the antag­
onistic bacterium K84 just before sowing the seeds
in the nurseries is recommended. However, addi­
tional inoculation of the rootstocks just prior to
their planting in the orchard would certainly prevent
infection of stonefruit seedlings. It must be empha­
sized that care should be taken to avoid damaging
the seedlings during cultivation since entry of the
pathogenic bacterium into the root is enhanced by
root injuries.
ACKNOWLEDGEMENTS
Thanks are due to Chr. Chrysostomou for
technical assistance and to the staff of the Depart­
ment of Agriculture at Akhelia, for their coopera­
tion.
REFERENCES
Dhanvantari, B.N. 1976. Biological control of crown
gall of peach in Southwestern Ontario. Plant
Disease Reporter 60:549-551.
Kerr, A. 1972. Biological control of crown gall:
Seed inoculation. Journal of Applied Bacteri­
ology 35:493-497.
Kerr, A. 1980. Biological control of crown gall:
Production of Agrocin 84. Plant Disease 64:25­
40.
Kerr, A. and K. Htay. 1974. Biological control of
crown gall through bacteriocin production.
Physiological Plant Pathology 4:37-44.
Moore, L.W. 1977. Prevention of crown gallon
Prunus roots by bacterial antagonists. Phytopa­
thology 67:139-144.
New, P.B. and A. Kerr. 1972. Biological control of
crown gall: Field measurements and glasshouse
experiments. Journal of Applied Bacteriology
35:279-287.
Schroth N.M. and l.W. Moller. 1976. Crown gall
controlled in the field with a nonpathogenic
bacterium. Plant Disease Reporter 60:275-278.
Schroth, N.M., A.R. Weinhold, A.H. McCain, D.C.
Hildebrand and W.Ross. 1971. Biology and
Control of Agrobacterium tumefaciens. Hilger­
dia 40 (15):537-552.
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