Biocontrol of zoosporic plant pathogens with the

biosurfactant-producing Pseudomonas koreensis 2.74

Malin Hultberg1*, Therese Bengtsson2 and Beatrix Alsanius1
1 Dept. of Horticulture, Microbial Horticultural Laboratory, Swedish

University of Agricultural Sciences, Alnarp, Sweden

2 Dept. of Plant Protection, Swedish University of Agricultural Sciences,

Alnarp, Sweden *Malin.Hultberg@ltj.slu.se

Zoospore-producing oomycetes are major plant pathogens in a wide variety of crops. They are of particular concern in
hydroponic systems due to the re-use of excess nutrient solution and a low microbial biodiversity in the cultivation system. This
group also includes the notorious plant pathogen Phytophthora infestans causing potato late blight, one of the most destructive
plant diseases world-wide [1]. In recent years biosurfactants, surfactants produced by various groups of microorganisms, have
attracted increased attention for biological control of zoospore-producing pathogens [2]. The zoospore is particularly sensitive to
surfactant interactions since it lacks the protection of a cell wall [3]. The strain Pseudomonas koreensis 2.74 has previously been
isolated from a closed hydroponic system [4] and produces high amounts of a surface-active compound [5]. The biocontrol effect
of Pseudomonas koreensis 2.74 and its biosurfactant was evaluated towards Phytophthora infestans using the Euca-blight
detached leaf-protocol (cv. Bintje and cv. Ovatio) and in a tomato hydroponic system infected with a zoospore-suspension of
Pythium ultimum.
Result
100
Conclusion
In the detached leaf assay a 90
P e rc e n t n e c ro s i s o f th e l e a f

80 a

significant reduction of the diameter 70 The use of biosurfactant-producing strains

60
of the lesion was observed when cv. 50 b cv. Bintje could be a part in the development of a
a cv. Ovatio
b
sustainable management of zoosporic plant
.
40 b a
Bintje were treated with 100 µg/ml 30
a
a

of biosurfactant. Increasing the 20 pathogens. Pseudomonas koreensis 2.74 is not

10

concentration of biosurfactant 0 capable of growth at 37ºC and therefore a

Control 0.1 mg/ml 0.5 mg/ml 1.0 mg/ml
further reduced the lesion diameter, suitable alternative for formulation and large-
though not significantly. Less effect Fig. 1 The biocontrol effect of a biosurfactant produced by
Pseudomonas koreensis 2.74 on potato leaves infected with a
scale experiments.
zoospore suspension of Phytophthora infestans
was observed on cv. Ovatio (fig 1).
The treatment with a bacterial
100
suspension reduced the diameter of 90 a Method
P erc en t n e cro sis o f th e leaf

80
lesion significantly (fig 2). In the 70
60 b
treatment where the densest bacterial 50
b b cv. Bintje The biosurfactant was recovered from the bacterial
a
a cv. Ovatio
suspension (OD620 2.0) was used 40 growth medium and the critical micelle concentration
30 b
b (CMC) was determined by measurements with a
brownish lesion was occasionally 20
10 SITA DynoTester (SITA, Messtechnik, GmbH) in
observed on the leaves. 0
Control 0.5 OD620 1.0 OD620 2.0 OD620
distilled water at 22 °C. The surface tension was
plotted as a function of surfactant concentration and
In the hydroponic system supply of Fig. 2 The biocontrol effect of the strain Pseudomonas koreensis 2.74 on CMC was considered the point at which the slope of
potato leaves infected with a zoospore suspension of Phytophthora
the biosurfactant as a crude extract in infestans the curve abruptly changed. The biosurfactant used in
the concentration of 100 µg/ml the hydroponic cultivation system was recovered
a a through acid precipitation and the CMC was 700
protected the plant towards disease. 100
mg/l, the concentration used in the plant cultivation
W e i g h t% c o m p a r e d to C o n tr o l +

90
This effect was more evident 80
a a
system was 100 mg/l. The biosurfactant used for the
ab ab
comparing root weight than plant 70 detached leaf assay was obtained through adsorption
60
weight. Also addition of the 50
b b Root and the CMC was 210 mg/l. In the detached leaf
Plant assay the biosurfactant concentrations 0.1, 0.5 and 1
biosurfactant-producing bacteria to 40
30 mg/ml were evaluated.
the nutrient solution, log 7 cfu/ml, 20
For preparation of the bacterial suspension the strain
10
protected the plants against disease. 0
Pseudomonas koreensis 2.74 was incubated for 48
The protection against disease was Control+ Biosurfactant Bacteria Control - hours in 250 ml of medium (20 mM NH4Cl, 20 mM
KCl, 1.6 mM MgSO4, 14.7 mM KH2PO4, 11.5 mM
equal to the effect obtained when the Fig. 3 The effect of the strain Pseudomonas koreensis 2.74 or its
K2HPO4, 0.5 % glucose,1% peptone, pH 7.2) in a 1
biosurfactant on hydroponically cultured tomato infected with a zoospore
crude extract was used (fig 3). suspension of Pythium ultimum. L Erlenmeyer flask at room temperature on a rotary
shaker (160 rpm). The bacterial cells were removed
1. van West P., A.A. Appiah and N.A.R. Gow. Advances in research on oomycete root pathogens. Physiol Mol Plant Pathol, 2003, 62: p. 99-113. by centrifugation at 10 000 g, 4°C for 15 minutes
2. Tran T.T.H. Interactions between biosurfactant-producing Pseudomonas and Phytophthora species. PhD Thesis, 2007, Wageningen (Avanti J-20, Beckman Coulter, CA, USA) and
University. The Netherlands washed with 0.8 % NaCl. In the hydroponic
3. Stanghellini M.E. and R.M. Miller. Biosurfactants: Their identity and potential efficacy in the biological control of zoosporic plant pathogens.
Plant Dis, 1997, 81: p. 4-12. cultivation system the bacteria was added to a density
4. Hultberg M., K.J. Bergstrand, S. Khalil and B. Alsanius. Characterization of biosurfactant-producing strains of fluorescent pseudomonads in of log 7 cfu/ml of nutrient solution. In detached leaf
a soilless cultivation system. Antonie van Leeuwenhoek, 2008, 94: p. 329-334. assay the concentrations 0.5, 1.0 and 2.0 OD620 was
5. Hultberg M., T. Alsberg, S. Khalil and B. Alsanius. Suppression of disease in tomato infected by Pythium ultimum with a biosurfactant
produced by Pseudomonas koreensis. Under revision for BioControl. evaluated.

Acknowledgement This research has been funded by FORMAS, Sweden