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Involvement of phenazine-1-carboxylic acid, siderophores and hydrogen cyanide in
suppression of Rhizoctonia solani and Pythium spp. damping-off by Pseudomonas
oryzihabitans and Xenorhabdus nematophila
Apostolos Kapsalis 1 *, Fotios Gravanis 1 and Simon Gowen 2
1 Technological Education Institution of Larissa, T.K. 411 10, Larissa, Greece. 2 School of Agriculture, Policy and Development,
University of Reading, Earley Gate, PO Box 236, Reading, RG6 6AR, Berkshire, UK.*Ermou 23, Pili Trikalon, T.K. 42032,
Thessaly, Greece. *e-mail: A.Kapsalis@teilar.gr, Kapsalis_tegos@yahoo.com, aap00ak@hotmail.com
Received 12 September 2007, accepted 5 December 2007.
Journal of Food, Agriculture & Environment Vol.6(1) : 168-171. 2008
Abstract
Entomopathogenic bacterial strains Pseudomonas (Flavimonas) oryzihabitans and Xenorhabdus nematophilus, both bacterial symbionts of the
entomopathogenic nematodes Steinernema abbasi and S. carpocapsae have been recently used for suppression of soil-borne pathogens. Bacterial
biocontrol agents (P. oryzihabitans and X. nematophila) have been tested for production of secondary metabolites in vitro and their fungistatic
effect on mycelium and spore development of soil-borne pathogens. Isolates of Pythium spp. and Rhizoctonia solani, the causal agent of cotton
damping-off, varied in sensitivity in vitro to the antibiotics phenazine-1-carboxylic acid (PCA), cyanide (HCN) and siderophores produced by
bacterial strains shown previously to have potential for biological control of those pathogens. These findings affirm the role of the antibiotics PCA,
HCN and siderophores in the biocontrol activity of these entomopathogenic strains and support earlier evidence that mechanisms of secondary
metabolites are responsible for suppression of damping-off diseases. In the present studies colonies of P. oryzihabitans showed production of
PCA with presence of crystalline deposits after six days development and positive production where found as well in the siderophore’s assay
when X. nematophila strain indicated HCN production in the in vitro assays. In vitro antifungal activity showed that bacteria densities of 10 5 to
10 6 cells/ml have antifungal activity in different media cultures. The results show further that isolates of Pythium spp. and R. solani insensitive to
PCA, HCN and siderophores are present in the pathogen population and provide additional justification for the use of mixtures of entomopathogenic
strains that employ different mechanisms of pathogen suppression to manage damping-off.
Key words: Pseudomonas oryzihabitans, Xenorhabdus nematophila, phenazine, cyanide, siderophores.
Introduction
In the first stages of development young seedlings (cotton) are
vulnerable to be attacked by a number of pathogens and especially
Pythium and Rhizoctonia species. P. oryzihabitans and X.
nematophila showed evidence of production of secondary, toxic
or other compounds that display anti-microbial effects to certain
fungal isolates from different families like Pythium and Fusarium
species 10,11, 31 .The potential of using strains of entomopathogenic
bacterial strains (Pseudomonas oryzihabitans and Xenorhabdus
nematophila) to suppress cotton soil-borne pathogens have been
tested in preliminary studies showed to have significant disease
control and antifungal activity 7 . Pseudomonas oryzihabitans
bacterium has been shown evidence to produce compounds that
display anti-microbial effects 1 . A number of bacterial antibiotics
produced by Pseudomonas spp., like pyoluteorin (Plt), pyrrolnitrin
(Prn), phenazine-1-carboxylic acid (PCA), 2,4-diacetyl
phloroglucinol (Phl), cyanide (HCN) and siderophores are
currently a key point for further research in biological control. A
great number of antibiotics and other antimycotic compounds
that are produced by Xenorhabdus and Photorhabdus species
have been isolated and characterised 16, 17, 19, 21 . X. nematophila
produces antibiotics during phase one that inhibit the growth of
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Botrytis cinerea, Ceratocystis ulmi, Ceratocystis dryocoetidis,
Mucor piriformis, Pythium coloratum, Pythium ultimum and
Trichoderma pseudokingii 4 . Xenocoumacin 1, isolated from X.
nematophila is a noticeable antibiotic, which is active against
pathogenic fungi 16, 17 . A great number of bacteria strains have
been recorded already with antifungal activity capable of
producing strong antibiotics, like phenazines, which are capable
of reducing disease incidence 19, 24 . The production of
siderophores has been linked to the disease suppression ability
of certain fluorescent Pseudomonas spp. 14 . Questions that are
remaining unresolved are: a) which are the compounds that are
released by P. oryzihabitans and X. nematophila and b) what is
the main effect of those compounds on soil-borne pathogens
and in particular on damping-off pathogens. The principal
objectives of the present study were to determine the production
of secondary metabolites and especially phenazine-1-carboxylic
acid (PCA), cyanide (HCN) and siderophores from P.
oryzihabitans and X. nematophila strains and the potential of
using these strains to suppress Pythium and Rhizoctonia species
causing cotton damping-off.
168 Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008

Materials and Methods
P. oryzihabitans and X. nematophila were cultured as described
by Kapsalis 8 . In the first assay, the production of phenazine-1-
carboxylic acid (PCA) by X. nematophila and P. oryzihabitans
was done in Petri dish cultures following the Thomashow and
Weller technique 28 . In vitro evaluations for cyanide (HCN)
production were done by selecting single colonies which were
streaked on Petri dish (9 cm in diameter) cultures of nutrient agar
(NA) with glycine (4.4 g 1 -1) . A Whatman No. 1 filter paper was
soaked in copper ethylo-acetate (5 mg ml -1 ) and 4,4-methylene-
bis-N,N-dimethylalanine (5 mg ml -1 ) in chloroform and air dried 32 .
For siderophore production two methods of assay protocol were
followed. Firstly colonies of X. nematophila and P. oryzihabitans
were placed in CAS medium 25 and siderophore production was
displayed by the colour change of the indicators from blue to
orange. In the second assay, bacterial cells were suspended in 10
µg MgSO 4 on NA plates . The study was based at the possible
influence of iron-regulated metabolites, such as siderophores on
the antifungal activity of the bacterial strains. NA agar
supplemented with 100 µM FeCl 3 was spot-inoculated with clear
cell bacterial suspensions by pipetting two droplets of 5 µl of the
suspension on the plate. Detection of siderophores was made
after incubation at 27°C for 48 h.
In vitro inhibition of R. solani, Pythium ultimum and P.
aphanidermatum by the two tested bacteria was measured in
dual cultures on three different media [water agar (WA), potato
dextrose agar (PDA) and nutrient agar (NA)] 2 . Bacterial strains
were obtained from 2-day-old nutrient broth (NB) No 2. A clear
cell suspension was extracted and used throughout the study
with bacteria CFU estimated at 1 x 10 6 cells per ml. Four bacterial
cell densities (10 6 to 10 3 cells ml -1 ) with 10 replications per treatment
were spot inoculated in NA cultures for 3 days. Assessments
were made after 4 days after adding the fungal plugs onto the
culture dishes. In the second in vitro assay, bacterial strains were
tested for their ability to inhibit the growth of soil-borne pathogens
using a double-layer agar method 27 . Bacterial strains obtained
with the same technique as above and four bacterial cell densities
(10 6 to 10 3 cells ml -1 ) were spot inoculated in NA cultures and
incubated for 48 h. Bacteria were killed by inverting the plates
over 3 ml of chloroform for 1 h. After drying, the plates were
covered with 15 ml molten 1% water agar containing a mycelia
plug (4 mm) of each pathogen tested with ten replications per
bacterial density. Estimations were made after 3 days at 28°C. The
experiments were performed twice and estimations were made by
analysis of variance (ANOVA) to compare treatment means (SPSS
12.0). Tukey and Duncan multiple range tests were used to separate
the means at the 5% confidence level 7 .
Results
Entomopathogenic bacteria and especially P. oryzihabitans have
been reported to control plant diseases caused by Pythium spp.
and Rhizoctonia solani 8, 31 , evidence that this bacterium has a
broad spectrum of activity against microorganisms. In the present
assay for investigating possible PCA production, colonies of P.
oryzihabitans under an UV light showed a slightly positive effect
on PCA production (Plate 1a) with presence of crystalline deposits
after 6 days when the other entomopathogenic bacterial strain
(X. nematophila) showed no presence of discoloration as a result
of negative production of PCA. In the assay for HCN, X.
nematophila gave a slight indication of HCN presence and P.
oryzihabitans in the same assay didn’t produce any quantity of
HCN. At the last assay’s testing the ability of entomopathogenic
bacterial strains to produce siderophores, for P. oryzihabitans
colonies were noticed characteristic orange halos after 30 h of
incubation as a result of siderophore production but for the other
bacterial strain (X. nematophila) there were no evidence of
producing siderophores.
In the in vitro assay, significant interactions (P = 0.05) were
recorded between the bacteria and fungi tested. X. nematophila
was found to inhibit growth of the fungi. Bacteria densities of 10 5
to 10 6 cells/ml showed antifungal activity in all the media tested
but especially in PDA and NA. However, the other two
concentrations (10 4 and 10 3 ) seemed to have as good antifungal
activity and inhibition zones were visible with moderate and in
some cases poor inhibition. P. oryzihabitans also had a significant
effect on the mycelium growth of the fungi. In the NA and PDA
assay the lower bacterial concentrations failed to produce strong
inhibition zones. The higher bacteria concentrations, especially
10 5 to 10 6 cells/ml, proved very effective against mycelium growth
and inhibition zones overlapped the fungi growth (Fig. 1). In the
second assay exploring the residues of dead cells of P.
oryzihabitans and X. nematophila (applied at 10 5 and 10 6 cells/
ml) the same antifungal activity was shown against the three
pathogens. However, with R. solani, due to the relatively slower
rate of mycelium growth or due to the diffusible compounds
produced, inhibition zones were smaller. Lower cell densities (10 4
and 10 3 cells/ml) did not have such strong antifungal activity and
inhibition zones were relatively small compared with the higher
cell concentrations. Similarly the higher cell densities (10 5 and
10 6 ) of X. nematophila caused increased inhibition of Pythium
ultimum, P. aphanidermatum and Rhizoctonia solani. X.
nematophila cells seemed to be very aggressive, particularly
against the Pythium spp.
Discussion
Pythium spp. are the causal agents of pre-and postemergence
damping-off of a number of crops. Seeds rot before or shortly
after germination (preemergence damping-off) and newly emerged
seedlings collapse (postemergence damping-off) 27 . Antibiotics
are produced by different bacterial strains and have been shown
to have an important role in the biological control of fungal plant
pathogens and especially in our work against seedling damping-
off.
Phenazine-1-carboxylic acid (PCA) is major determinant of
biological control of soil-borne plant pathogens by strains of
fluorescent Pseudomonas spp. 30 . Many bacterial strains with
antifungal activity produce phenazines, for instance, phenazine-
1-carboxylate was implicated in the control of take-all disease
caused by Gaeumanomyces graminis var. tritici 29 . Our findings
confirm previous studies 23, 34 , that the production of PCA by
Pseudomonas spp. are the primary contributors to the biological
control of different pathogens and especially damping-off
pathogens. Pseudomonads, as well capable of producing
antibiotics or hydrogen cyanide (HCN), compete with a pathogen
for niches and nutrients or induce systemic acquired resistance
in the plant after successful colonization of the roots 3, 20 . Large
concentrations of hydrogen cyanide are found in agricultural
soils, which can sometimes be really toxic 27 . Microbial HCN
Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008 169

a b
P. oryzihabitans P. oryzihabitans
X. nematophila
Plate 1. Identification for production of siderophores and phenazine by Pseudomonas
oryzihabitans and Xenorhabdus nematophila. a. Orange pigmentation and crystalline deposits
in the centre of P. oryzihabitans colony after the period of 2 weeks. b. Strains spot inoculated
with bacteria cell to identify for presence of orange pigment which indicates positive
siderophore production.
Inhibition zone (mm)
20
15
10
5
0
P. ultimum P. aphanidermatum R.solani
a b a b a b
Figure 1. Effect of P. oryzihabitans (%) and X. nematophila (%) dead
cells assay on mycelium growth of P. ultimum and P. aphanidermatum
and R.solani. *Source is significantly different from the overall
distribution at the 0.05 level. 1 Bacterial strains a = 10 6 and b = 10 5 cells/
ml.
biosynthesis has been demonstrated in fungi and in bacteria
species of the genus Pseudomonas. Pseudomonads need
sufficient iron to produce HCN 12 . Rhizobacteria with inhibition
effect like pseudomonads play an important role in the biological
control of soil-borne fungal plant pathogens. In different cases,
this effect has been attributed due to the siderophore production 6,
13, 15 of the biocontrol strains. For example, there are strains like P.
aeruginosa 7NSK2 with good biocontrol effect against Pythium
induced damping-off of tomato which produces three
siderophores, pyoverdin, pyochelin and salicylic acid, under
iron limitation 8 . Siderophores produced by bacterial strains and
especially Pseudomonas spp. have been implicated in the
biological control of soil-borne pathogens as reported in wheat
grown in Pythium-infested soils 3 and tomato (Lycopersicon
Table 1. Production of antifungal compounds by P. oryzihabitans and X. nematophila.
PCA HCN Siderophores
P. oryzihabitans + a - +++
Symptoms Orange colonies, No discoloration of Orange halos after 48 h
crystalline deposits the filter paper growth in NA
X. nematophila - + -
Symptoms No discoloration in
the colonies
Slightly blue color in
the indicator
esculentum) in Pythium-induced damping-off.
Also production of pyoverdin has proven to be
important in the biological control of Pythium
damping-off of cotton by Pseudomonas
fluorescens 3551 14 . A great need for
investigating further production of
siderophores like pyochelin and pyoverdin by
the entomopathogenic bacteria are necessary
as a way to achieve high levels of protection
against Pythium postemergence damping-off.
Previous studies showed that mycelial growth 18
but not sporangial germination 22 of Pythium
spp. is inhibited by iron starvation. In our study,
isolates of P. oryzihabitans were classified as
capable of producing an amount of PCA
antibiotic and siderophores (Plate 1b). On the other hand, through
in vitro assays for antifungal antibiotics/metabolites X.
nematophila gave only a small indication of possible HCN
production (Table 1).
In previous studies only single antifungal metabolites produced
by the biocontrol agent proved to be the main mechanism of
pathogen inhibition 9, 12, 26, 29, 32 . The results of antifungal activity
of this study suggest that in some cases variation in sensitivity
of the target pathogen to antifungal metabolites, produced by
entomopathogenic bacterial strains, may contribute to the
inconsistent performance of these biocontrol agents observed
in vitro. This also provides evidence that in vitro assays for the
activity of a microbial metabolite against a target pathogen may
be useful in a program to predict biocontrol potential of the
producing microbial agent, but this assay alone may not always
provide an accurate prediction of disease suppression. In
conclusion, further investigation need to take place with thin-
layer chromatographic [TLC] analysis, for specific detection and
isolation of PCA, HCN and siderophores. Knowledge of the
ecology of entomopathogenic bacterial strains that harbor specific
biocontrol traits will contribute to improving the efficacy of
existing biocontrol agents and find new ways through the mode
of action of the organisms to specific soils and/or host-pathogen
systems. Additional experiments will be required to determine in
more detail the extent of conservation of phenazine-cyanide-
siderophore biosynthetic genes in strains that produce
structurally different antibiotics.
No orange halos noticed
after 48 h
aCompounds production rating (+++ Highly production symptoms, ++ Medium production symptoms,+ Minor production symptoms ,
- Negative production symptoms)
Acknowledgements
This study was conducted with grants
awarded by the IKY (Greek State
Scholarships Foundation) for the first
year of MSc course and the following
two year’s period of the PhD. We are
grateful to Dr. Jama and Ms Barbara
Pembroke for their support and for
valuable comments on the current
research process. As well special
acknowledgements to all the
personnel of University of Reading
(Department of Agriculture and Plant
Sciences) and especial to those who
provided isolates used in this study.
170 Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008

References
1 Andreoglou, F.I., Samaliev, H.Y. and Gowen, S.R. 2000. Temperature
response of the bacterium Pseudomonas oryzihabitans from the
entomopathogenic nematodes Steinernema abbasi, on the potato cyst
nematode Globodera rostochiensis. Aspects of Applied Biology 59:67-
73.
2 Anith, K.N. and Manomohandas, T.P. 2001. Combined application of
Trichoderma harzianum and Alcaligenes sp. Strain AMB 8 for
controlling nursery rot disease of black pepper. Indian Phytopathology
54:335–339.
3 Bakker, P. A. H. M., Van Peer, R. and Schippers, B. 1991. Suppression
of soil-borne plant pathogens by fluorescent pseudomonads:
Mechanisms and prospects. In Beemster, A. B. R., Bollen, G. J.,
Gerlach, M., Ruissen, M. A., Schippers, B. and Tempel, A. (eds).
Biotic Interactions and Soil-Borne Diseases. Proceedings of the First
Conference of the European Foundation for Plant Pathology. Elsevier,
Amsterdam, pp. 217–230.
4 Becker, J.O. and Cook, R.J. 1984. Pythium control by siderophore-
producing bacteria on roots of wheat. (Abstract) Phytopathology
74:806.
5 Chen, G., Dunphy, G.B. and Webster, J.M. 1994. Antifungal activity of
two Xenorhabdus species and Photorhabdus luminescens, bacteria
associated with the nematodes Steinernema species and Heterorhebditis
megidis. Biological Control 4:157-162.
6 Elad, Y. and Baker, R. 1985. The role of competition for iron and carbon
in suppression of chlamydospore germination of Fusarium sp. by
Pseudomonas spp. Ecol. Epidemiol. 75:1053–1059.
7 Green, B.S. and Salkind, N.J. 2003. Using SPSS for Windows and
Macintosh: Analyzing and Understanding Data. 3 rd edn. Prentice Hall,
New Jersey.
8 Hofte, M., Buysens, S., Koedam, N. and Cornelis, P. 1993. Zinc affects
siderophore-mediated high affinity iron uptake systems in the
rhizosphere Pseudomonas aeruginosa 7NSK2. Biometals 6:85–91.
9 Howie, W.J. and Suslow, T.V. 1991. Role of antibiotic biosynthesis in
the inhibition of Pythium ultimum in the cotton spermosphere and
rhizosphere by Pseudomonas fluorescens. Mol. Plant-Microbe
Interact. 4:393–399.
10 Kapsalis, A.V., Gowen, S.R. and Gravanis, F.T. 2002. Nematode
entomopathogenic symbiotic bacteria acting as biological agents against
tomato seedlings fungal pathogens. Proceedings of the BCPC
Conference - Pests & Diseases 2002 2:749-752.
11 Kapsalis, A.V., Gravanis, F.T. and Gowen, S.R. 2003. Seed treatment
with a bacterial antagonist for reducing cotton damping-off caused by
Pythium spp. Proceedings of the BCPC International Congress – Crop
Science and Technology (Glasgow, UK) 1:655-658.
12 Keel, C., Voisard, C., Berling, C., Kahr, H. G. and De´fago, G. 1989.
Iron sufficiency, a prerequisite for the suppression of tobacco black
root rot by Pseudomonas fluorescens strain CHA0 under gnotobiotic
conditions. Phytopathology 79:584–589.
13 Kloepper, J.W., Leong, J., Teintze, M. and Schroth, M.N. 1980.
Pseudomonas siderophores: A mechanism explaining disease-
suppressive soils. Curr. Microbiol. 4:317–320.
14 Loper, J.E. and Buyer, J.S. 1991. Siderophores in microbial interactions
on plant surfaces. Mol. Plant-Microbe Interact. 4:5–13.
15 Leong, J. 1986. Siderophores: Their biochemistry and possible role in
the biocontrol of plant pathogens. Annu. Rev. Phytopathol. 24:187–
209.
16 McInerney, B.V., Gregson, R.P., Lacey, M.J., Akhurst, R.J., Lyons,
G.R., Rhodes, S.H. and Smith, D.R.J. 1991. Biologically active
metabolites from Xenorhabdus spp. Part 1. Dithiolopyrrolone
derivatives with antibiotic activity. Journal of Natural Products
(Lloydia) 54:774-784.
17 McInerney, B.V., Taylor, W.C., Lacey, M.J., Akhurst, R.J. and Smith,
D.R.J. 1991. Biologically active metabolites from Xenorhabdus spp.
Part 2. Benzopyran-1-one derivatives with gastroprotective activity.
Journal of Natural Products (Lloydia) 54:785-795.
18 Meyer, J.M., Halle´, F., Hohnadel, D., Lemanceau, P. and Ratefiarivelo,
H. 1987. Siderophores of Pseudomonas—biological properties. In
Winkelmann, G., van der Helm, D. and Neilands, J. B. (eds). Iron
Transport in Microbes, Plants and Animals. Verlagsgesellschaft mbH,
Weinheim, Germany, pp. 189–205.
19 Nealson, K.H., Schmidt, T.M. and Bleakley, B. 1990. Physiology and
Biochemistry of Xenorhabdus. In Gaugler, R. and Kaya, H.K. (eds).
Entomopathogenic nematodes in biological control. CRC Press, Inc.,
Boca Raton, Fla, pp. 271-284.
20 O’Sullivan, D. J. and O’Gara, F. 1992. Traits of fluorescent Pseudomonas
spp. involved in suppression of plant root pathogens. Microbiol. Rev.
56:662–676.
21 Paul, V.J., Frautschy, S., Fenical, W. and Nealson, K.H. 1981. Antibiotics
in microbial ecology: Isolation and structure assignment of several
antibacterial compounds from the insect symbiotic bacteria
Xenorhabdus spp. Journal of Chemical Ecology 7:589-597.
22 Paulitz, T.C. and Loper J.E. 1991. Lack of a role for fluorescent
siderophore production in the biological control of Pythium damping-
off of cucumber by a strain of Pseudomonas putida. Phytopathology
81:930–935.
23 Pierson, L.S. and Thomashow, L.S. 1992. Cloning and heterologous
expression of the phenazine biosynthetic locus from Pseudomonas
aureofaciens 30-84. Mol. Plant-Microbe Interact. 5:330–339.
24 Powell, J.F., Vargas, J.M., Nair, M.G., Detweiler, A.R. and Chandra, A.
2000. Management of dollar spot on creeping bentgrass with
metabolites of Pseudomonas aureofaciens (TX-1). Plant Disease 84:19-
24.
25 Schwyn, B. and Neilands, J.B. 1987. Universal chemical assay for the
detection and determination of siderophores. Analytical Biochemistry
160:47-56.
26 Shanahan, P., O’Sullivan, D.J., Simpson, P., Glennon, J. and O’Gara, F.
1992. Isolation of 2,4-diacetylphloroglucinol from a fluorescent
pseudomonad and investigation of physiological parameters influencing
its production. Appl. Environ. Microbiol. 58:353–358.
27 Smith, I.M., Dunez, J., Lellioth, R.A., Phillips, P.H. and Archer S.A.
(eds). 1988. European Handbook of Plant Disease. Blackwell Scientific
Publications, Oxford.
28 Tambong, J.T. and Hofte, M. 2001. Phenazines are involved in biocontrol
of Pythium myriotylum on cocoyam by Pseudomonas aeruginosa
PNA1. European Journal of Plant Pathology 107:3499-3508.
29 Thomashow, L.S. and Weller, D.M. 1988. Role a phenazine antibiotic
from Pseudomonas fluorescens in biological control of
Gaeumannomyces graminis var. tritici. Journal of Bacteriology
170:3499-3508.
30 Thomashow, L.S. and Weller, D.M. 1996. Current concepts in the use
of introduced bacteria for biological disease control: Mechanisms and
antifungal metabolites. In Stacey, G. and Keen, N. T. (ed.). Plant-
Microbe Interactions. vol. 1. Chapman & Hall, Ltd., London, United
Kingdom, pp. 187–236.
31 Vagelas, I.K., Kapsalis, A.V., Gravanis, F.T. and Gowen, S.R. 2004.
Biological control of Rhizoctonia solani damping-off with a bacterium
symbiotically associated with Steinernema abbasi. International
Organization for Biological Control (IOBC/wprs) Bulletin 27(1):285-
290.
32 Voisard, C., Keel, C., Haas, D. and Defago, G. 1989. Cyanide production
by Pseudomonas fluorescens helps suppress black root rot of tobacco
under gnotobiotic conditions. EMBO J. 8:351–358.
33 Vidaver, A.K., Mathys, M.L., Thomas, M.E. and Schuster, M.L. 1972.
Bacteriocins of the phytopathogens Pseudomonas syringae, P. glycinea,
and P. phaseolina. Canadian Journal of Microbiology 18:705-713.
34 Vincent, M.N., Harrison, L.A., Brackin, J.M., Kovacevich, P.A.,
Mukerji, P., Weller, D.M. and Pierson, E.A. 1991. Genetic analysis of
the antifungal activity of a soil-borne Pseudomonas aureofaciens strain.
Appl. Environ. Microbiol. 57:2928–2934.
Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008 171