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Science and Technology<br />
Meri-Rastilantie 3 B, FI-00980<br />
Helsinki, Finland<br />
e-mail: info@world-<strong>food</strong>.<strong>net</strong><br />
<strong>Involvement</strong> <strong>of</strong> <strong>phenazine</strong>-1-<strong>carboxylic</strong> <strong>acid</strong>, siderophores and hydrogen cyanide in<br />
suppression <strong>of</strong> Rhizoctonia solani and Pythium spp. damping-<strong>of</strong>f by Pseudomonas<br />
oryzihabitans and Xenorhabdus nematophila<br />
Apostolos Kapsalis 1 *, Fotios Gravanis 1 and Simon Gowen 2<br />
1 Technological Education Institution <strong>of</strong> Larissa, T.K. 411 10, Larissa, Greece. 2 School <strong>of</strong> Agriculture, Policy and Development,<br />
University <strong>of</strong> Reading, Earley Gate, PO Box 236, Reading, RG6 6AR, Berkshire, UK.*Ermou 23, Pili Trikalon, T.K. 42032,<br />
Thessaly, Greece. *e-mail: A.Kapsalis@teilar.gr, Kapsalis_tegos@yahoo.com, aap00ak@hotmail.com<br />
Received 12 September 2007, accepted 5 December 2007.<br />
Journal <strong>of</strong> Food, Agriculture & Environment Vol.6(1) : 168-171. 2008<br />
Abstract<br />
Entomopathogenic bacterial strains Pseudomonas (Flavimonas) oryzihabitans and Xenorhabdus nematophilus, both bacterial symbionts <strong>of</strong> the<br />
entomopathogenic nematodes Steinernema abbasi and S. carpocapsae have been recently used for suppression <strong>of</strong> soil-borne pathogens. Bacterial<br />
biocontrol agents (P. oryzihabitans and X. nematophila) have been tested for production <strong>of</strong> secondary metabolites in vitro and their fungistatic<br />
effect on mycelium and spore development <strong>of</strong> soil-borne pathogens. Isolates <strong>of</strong> Pythium spp. and Rhizoctonia solani, the causal agent <strong>of</strong> cotton<br />
damping-<strong>of</strong>f, varied in sensitivity in vitro to the antibiotics <strong>phenazine</strong>-1-<strong>carboxylic</strong> <strong>acid</strong> (PCA), cyanide (HCN) and siderophores produced by<br />
bacterial strains shown previously to have potential for biological control <strong>of</strong> those pathogens. These findings affirm the role <strong>of</strong> the antibiotics PCA,<br />
HCN and siderophores in the biocontrol activity <strong>of</strong> these entomopathogenic strains and support earlier evidence that mechanisms <strong>of</strong> secondary<br />
metabolites are responsible for suppression <strong>of</strong> damping-<strong>of</strong>f diseases. In the present studies colonies <strong>of</strong> P. oryzihabitans showed production <strong>of</strong><br />
PCA with presence <strong>of</strong> crystalline deposits after six days development and positive production where found as well in the siderophore’s assay<br />
when X. nematophila strain indicated HCN production in the in vitro assays. In vitro antifungal activity showed that bacteria densities <strong>of</strong> 10 5 to<br />
10 6 cells/ml have antifungal activity in different media cultures. The results show further that isolates <strong>of</strong> Pythium spp. and R. solani insensitive to<br />
PCA, HCN and siderophores are present in the pathogen population and provide additional justification for the use <strong>of</strong> mixtures <strong>of</strong> entomopathogenic<br />
strains that employ different mechanisms <strong>of</strong> pathogen suppression to manage damping-<strong>of</strong>f.<br />
Key words: Pseudomonas oryzihabitans, Xenorhabdus nematophila, <strong>phenazine</strong>, cyanide, siderophores.<br />
Introduction<br />
In the first stages <strong>of</strong> development young seedlings (cotton) are<br />
vulnerable to be attacked by a number <strong>of</strong> pathogens and especially<br />
Pythium and Rhizoctonia species. P. oryzihabitans and X.<br />
nematophila showed evidence <strong>of</strong> production <strong>of</strong> secondary, toxic<br />
or other compounds that display anti-microbial effects to certain<br />
fungal isolates from different families like Pythium and Fusarium<br />
species 10,11, 31 .The potential <strong>of</strong> using strains <strong>of</strong> entomopathogenic<br />
bacterial strains (Pseudomonas oryzihabitans and Xenorhabdus<br />
nematophila) to suppress cotton soil-borne pathogens have been<br />
tested in preliminary studies showed to have significant disease<br />
control and antifungal activity 7 . Pseudomonas oryzihabitans<br />
bacterium has been shown evidence to produce compounds that<br />
display anti-microbial effects 1 . A number <strong>of</strong> bacterial antibiotics<br />
produced by Pseudomonas spp., like pyoluteorin (Plt), pyrrolnitrin<br />
(Prn), <strong>phenazine</strong>-1-<strong>carboxylic</strong> <strong>acid</strong> (PCA), 2,4-diacetyl<br />
phloroglucinol (Phl), cyanide (HCN) and siderophores are<br />
currently a key point for further research in biological control. A<br />
great number <strong>of</strong> antibiotics and other antimycotic compounds<br />
that are produced by Xenorhabdus and Photorhabdus species<br />
have been isolated and characterised 16, 17, 19, 21 . X. nematophila<br />
produces antibiotics during phase one that inhibit the growth <strong>of</strong><br />
www.world-<strong>food</strong>.<strong>net</strong><br />
Botrytis cinerea, Ceratocystis ulmi, Ceratocystis dryocoetidis,<br />
Mucor piriformis, Pythium coloratum, Pythium ultimum and<br />
Trichoderma pseudokingii 4 . Xenocoumacin 1, isolated from X.<br />
nematophila is a noticeable antibiotic, which is active against<br />
pathogenic fungi 16, 17 . A great number <strong>of</strong> bacteria strains have<br />
been recorded already with antifungal activity capable <strong>of</strong><br />
producing strong antibiotics, like <strong>phenazine</strong>s, which are capable<br />
<strong>of</strong> reducing disease incidence 19, 24 . The production <strong>of</strong><br />
siderophores has been linked to the disease suppression ability<br />
<strong>of</strong> certain fluorescent Pseudomonas spp. 14 . Questions that are<br />
remaining unresolved are: a) which are the compounds that are<br />
released by P. oryzihabitans and X. nematophila and b) what is<br />
the main effect <strong>of</strong> those compounds on soil-borne pathogens<br />
and in particular on damping-<strong>of</strong>f pathogens. The principal<br />
objectives <strong>of</strong> the present study were to determine the production<br />
<strong>of</strong> secondary metabolites and especially <strong>phenazine</strong>-1-<strong>carboxylic</strong><br />
<strong>acid</strong> (PCA), cyanide (HCN) and siderophores from P.<br />
oryzihabitans and X. nematophila strains and the potential <strong>of</strong><br />
using these strains to suppress Pythium and Rhizoctonia species<br />
causing cotton damping-<strong>of</strong>f.<br />
168 Journal <strong>of</strong> Food, Agriculture & Environment, Vol.6 (1), January 2008
Materials and Methods<br />
P. oryzihabitans and X. nematophila were cultured as described<br />
by Kapsalis 8 . In the first assay, the production <strong>of</strong> <strong>phenazine</strong>-1-<br />
<strong>carboxylic</strong> <strong>acid</strong> (PCA) by X. nematophila and P. oryzihabitans<br />
was done in Petri dish cultures following the Thomashow and<br />
Weller technique 28 . In vitro evaluations for cyanide (HCN)<br />
production were done by selecting single colonies which were<br />
streaked on Petri dish (9 cm in diameter) cultures <strong>of</strong> nutrient agar<br />
(NA) with glycine (4.4 g 1 -1) . A Whatman No. 1 filter paper was<br />
soaked in copper ethylo-acetate (5 mg ml -1 ) and 4,4-methylene-<br />
bis-N,N-dimethylalanine (5 mg ml -1 ) in chlor<strong>of</strong>orm and air dried 32 .<br />
For siderophore production two methods <strong>of</strong> assay protocol were<br />
followed. Firstly colonies <strong>of</strong> X. nematophila and P. oryzihabitans<br />
were placed in CAS medium 25 and siderophore production was<br />
displayed by the colour change <strong>of</strong> the indicators from blue to<br />
orange. In the second assay, bacterial cells were suspended in 10<br />
µg MgSO 4 on NA plates . The study was based at the possible<br />
influence <strong>of</strong> iron-regulated metabolites, such as siderophores on<br />
the antifungal activity <strong>of</strong> the bacterial strains. NA agar<br />
supplemented with 100 µM FeCl 3 was spot-inoculated with clear<br />
cell bacterial suspensions by pipetting two droplets <strong>of</strong> 5 µl <strong>of</strong> the<br />
suspension on the plate. Detection <strong>of</strong> siderophores was made<br />
after incubation at 27°C for 48 h.<br />
In vitro inhibition <strong>of</strong> R. solani, Pythium ultimum and P.<br />
aphanidermatum by the two tested bacteria was measured in<br />
dual cultures on three different media [water agar (WA), potato<br />
dextrose agar (PDA) and nutrient agar (NA)] 2 . Bacterial strains<br />
were obtained from 2-day-old nutrient broth (NB) No 2. A clear<br />
cell suspension was extracted and used throughout the study<br />
with bacteria CFU estimated at 1 x 10 6 cells per ml. Four bacterial<br />
cell densities (10 6 to 10 3 cells ml -1 ) with 10 replications per treatment<br />
were spot inoculated in NA cultures for 3 days. Assessments<br />
were made after 4 days after adding the fungal plugs onto the<br />
culture dishes. In the second in vitro assay, bacterial strains were<br />
tested for their ability to inhibit the growth <strong>of</strong> soil-borne pathogens<br />
using a double-layer agar method 27 . Bacterial strains obtained<br />
with the same technique as above and four bacterial cell densities<br />
(10 6 to 10 3 cells ml -1 ) were spot inoculated in NA cultures and<br />
incubated for 48 h. Bacteria were killed by inverting the plates<br />
over 3 ml <strong>of</strong> chlor<strong>of</strong>orm for 1 h. After drying, the plates were<br />
covered with 15 ml molten 1% water agar containing a mycelia<br />
plug (4 mm) <strong>of</strong> each pathogen tested with ten replications per<br />
bacterial density. Estimations were made after 3 days at 28°C. The<br />
experiments were performed twice and estimations were made by<br />
analysis <strong>of</strong> variance (ANOVA) to compare treatment means (SPSS<br />
12.0). Tukey and Duncan multiple range tests were used to separate<br />
the means at the 5% confidence level 7 .<br />
Results<br />
Entomopathogenic bacteria and especially P. oryzihabitans have<br />
been reported to control plant diseases caused by Pythium spp.<br />
and Rhizoctonia solani 8, 31 , evidence that this bacterium has a<br />
broad spectrum <strong>of</strong> activity against microorganisms. In the present<br />
assay for investigating possible PCA production, colonies <strong>of</strong> P.<br />
oryzihabitans under an UV light showed a slightly positive effect<br />
on PCA production (Plate 1a) with presence <strong>of</strong> crystalline deposits<br />
after 6 days when the other entomopathogenic bacterial strain<br />
(X. nematophila) showed no presence <strong>of</strong> discoloration as a result<br />
<strong>of</strong> negative production <strong>of</strong> PCA. In the assay for HCN, X.<br />
nematophila gave a slight indication <strong>of</strong> HCN presence and P.<br />
oryzihabitans in the same assay didn’t produce any quantity <strong>of</strong><br />
HCN. At the last assay’s testing the ability <strong>of</strong> entomopathogenic<br />
bacterial strains to produce siderophores, for P. oryzihabitans<br />
colonies were noticed characteristic orange halos after 30 h <strong>of</strong><br />
incubation as a result <strong>of</strong> siderophore production but for the other<br />
bacterial strain (X. nematophila) there were no evidence <strong>of</strong><br />
producing siderophores.<br />
In the in vitro assay, significant interactions (P = 0.05) were<br />
recorded between the bacteria and fungi tested. X. nematophila<br />
was found to inhibit growth <strong>of</strong> the fungi. Bacteria densities <strong>of</strong> 10 5<br />
to 10 6 cells/ml showed antifungal activity in all the media tested<br />
but especially in PDA and NA. However, the other two<br />
concentrations (10 4 and 10 3 ) seemed to have as good antifungal<br />
activity and inhibition zones were visible with moderate and in<br />
some cases poor inhibition. P. oryzihabitans also had a significant<br />
effect on the mycelium growth <strong>of</strong> the fungi. In the NA and PDA<br />
assay the lower bacterial concentrations failed to produce strong<br />
inhibition zones. The higher bacteria concentrations, especially<br />
10 5 to 10 6 cells/ml, proved very effective against mycelium growth<br />
and inhibition zones overlapped the fungi growth (Fig. 1). In the<br />
second assay exploring the residues <strong>of</strong> dead cells <strong>of</strong> P.<br />
oryzihabitans and X. nematophila (applied at 10 5 and 10 6 cells/<br />
ml) the same antifungal activity was shown against the three<br />
pathogens. However, with R. solani, due to the relatively slower<br />
rate <strong>of</strong> mycelium growth or due to the diffusible compounds<br />
produced, inhibition zones were smaller. Lower cell densities (10 4<br />
and 10 3 cells/ml) did not have such strong antifungal activity and<br />
inhibition zones were relatively small compared with the higher<br />
cell concentrations. Similarly the higher cell densities (10 5 and<br />
10 6 ) <strong>of</strong> X. nematophila caused increased inhibition <strong>of</strong> Pythium<br />
ultimum, P. aphanidermatum and Rhizoctonia solani. X.<br />
nematophila cells seemed to be very aggressive, particularly<br />
against the Pythium spp.<br />
Discussion<br />
Pythium spp. are the causal agents <strong>of</strong> pre-and postemergence<br />
damping-<strong>of</strong>f <strong>of</strong> a number <strong>of</strong> crops. Seeds rot before or shortly<br />
after germination (preemergence damping-<strong>of</strong>f) and newly emerged<br />
seedlings collapse (postemergence damping-<strong>of</strong>f) 27 . Antibiotics<br />
are produced by different bacterial strains and have been shown<br />
to have an important role in the biological control <strong>of</strong> fungal plant<br />
pathogens and especially in our work against seedling damping-<br />
<strong>of</strong>f.<br />
Phenazine-1-<strong>carboxylic</strong> <strong>acid</strong> (PCA) is major determinant <strong>of</strong><br />
biological control <strong>of</strong> soil-borne plant pathogens by strains <strong>of</strong><br />
fluorescent Pseudomonas spp. 30 . Many bacterial strains with<br />
antifungal activity produce <strong>phenazine</strong>s, for instance, <strong>phenazine</strong>-<br />
1-carboxylate was implicated in the control <strong>of</strong> take-all disease<br />
caused by Gaeumanomyces graminis var. tritici 29 . Our findings<br />
confirm previous studies 23, 34 , that the production <strong>of</strong> PCA by<br />
Pseudomonas spp. are the primary contributors to the biological<br />
control <strong>of</strong> different pathogens and especially damping-<strong>of</strong>f<br />
pathogens. Pseudomonads, as well capable <strong>of</strong> producing<br />
antibiotics or hydrogen cyanide (HCN), compete with a pathogen<br />
for niches and nutrients or induce systemic acquired resistance<br />
in the plant after successful colonization <strong>of</strong> the roots 3, 20 . Large<br />
concentrations <strong>of</strong> hydrogen cyanide are found in agricultural<br />
soils, which can sometimes be really toxic 27 . Microbial HCN<br />
Journal <strong>of</strong> Food, Agriculture & Environment, Vol.6 (1), January 2008 169
a b<br />
P. oryzihabitans P. oryzihabitans<br />
X. nematophila<br />
Plate 1. Identification for production <strong>of</strong> siderophores and <strong>phenazine</strong> by Pseudomonas<br />
oryzihabitans and Xenorhabdus nematophila. a. Orange pigmentation and crystalline deposits<br />
in the centre <strong>of</strong> P. oryzihabitans colony after the period <strong>of</strong> 2 weeks. b. Strains spot inoculated<br />
with bacteria cell to identify for presence <strong>of</strong> orange pigment which indicates positive<br />
siderophore production.<br />
Inhibition zone (mm)<br />
20<br />
15<br />
10<br />
5<br />
0<br />
P. ultimum P. aphanidermatum R.solani<br />
a b a b a b<br />
Figure 1. Effect <strong>of</strong> P. oryzihabitans (%) and X. nematophila (%) dead<br />
cells assay on mycelium growth <strong>of</strong> P. ultimum and P. aphanidermatum<br />
and R.solani. *Source is significantly different from the overall<br />
distribution at the 0.05 level. 1 Bacterial strains a = 10 6 and b = 10 5 cells/<br />
ml.<br />
biosynthesis has been demonstrated in fungi and in bacteria<br />
species <strong>of</strong> the genus Pseudomonas. Pseudomonads need<br />
sufficient iron to produce HCN 12 . Rhizobacteria with inhibition<br />
effect like pseudomonads play an important role in the biological<br />
control <strong>of</strong> soil-borne fungal plant pathogens. In different cases,<br />
this effect has been attributed due to the siderophore production 6,<br />
13, 15 <strong>of</strong> the biocontrol strains. For example, there are strains like P.<br />
aeruginosa 7NSK2 with good biocontrol effect against Pythium<br />
induced damping-<strong>of</strong>f <strong>of</strong> tomato which produces three<br />
siderophores, pyoverdin, pyochelin and salicylic <strong>acid</strong>, under<br />
iron limitation 8 . Siderophores produced by bacterial strains and<br />
especially Pseudomonas spp. have been implicated in the<br />
biological control <strong>of</strong> soil-borne pathogens as reported in wheat<br />
grown in Pythium-infested soils 3 and tomato (Lycopersicon<br />
Table 1. Production <strong>of</strong> antifungal compounds by P. oryzihabitans and X. nematophila.<br />
PCA HCN Siderophores<br />
P. oryzihabitans + a - +++<br />
Symptoms Orange colonies, No discoloration <strong>of</strong> Orange halos after 48 h<br />
crystalline deposits the filter paper growth in NA<br />
X. nematophila - + -<br />
Symptoms No discoloration in<br />
the colonies<br />
Slightly blue color in<br />
the indicator<br />
esculentum) in Pythium-induced damping-<strong>of</strong>f.<br />
Also production <strong>of</strong> pyoverdin has proven to be<br />
important in the biological control <strong>of</strong> Pythium<br />
damping-<strong>of</strong>f <strong>of</strong> cotton by Pseudomonas<br />
fluorescens 3551 14 . A great need for<br />
investigating further production <strong>of</strong><br />
siderophores like pyochelin and pyoverdin by<br />
the entomopathogenic bacteria are necessary<br />
as a way to achieve high levels <strong>of</strong> protection<br />
against Pythium postemergence damping-<strong>of</strong>f.<br />
Previous studies showed that mycelial growth 18<br />
but not sporangial germination 22 <strong>of</strong> Pythium<br />
spp. is inhibited by iron starvation. In our study,<br />
isolates <strong>of</strong> P. oryzihabitans were classified as<br />
capable <strong>of</strong> producing an amount <strong>of</strong> PCA<br />
antibiotic and siderophores (Plate 1b). On the other hand, through<br />
in vitro assays for antifungal antibiotics/metabolites X.<br />
nematophila gave only a small indication <strong>of</strong> possible HCN<br />
production (Table 1).<br />
In previous studies only single antifungal metabolites produced<br />
by the biocontrol agent proved to be the main mechanism <strong>of</strong><br />
pathogen inhibition 9, 12, 26, 29, 32 . The results <strong>of</strong> antifungal activity<br />
<strong>of</strong> this study suggest that in some cases variation in sensitivity<br />
<strong>of</strong> the target pathogen to antifungal metabolites, produced by<br />
entomopathogenic bacterial strains, may contribute to the<br />
inconsistent performance <strong>of</strong> these biocontrol agents observed<br />
in vitro. This also provides evidence that in vitro assays for the<br />
activity <strong>of</strong> a microbial metabolite against a target pathogen may<br />
be useful in a program to predict biocontrol potential <strong>of</strong> the<br />
producing microbial agent, but this assay alone may not always<br />
provide an accurate prediction <strong>of</strong> disease suppression. In<br />
conclusion, further investigation need to take place with thin-<br />
layer chromatographic [TLC] analysis, for specific detection and<br />
isolation <strong>of</strong> PCA, HCN and siderophores. Knowledge <strong>of</strong> the<br />
ecology <strong>of</strong> entomopathogenic bacterial strains that harbor specific<br />
biocontrol traits will contribute to improving the efficacy <strong>of</strong><br />
existing biocontrol agents and find new ways through the mode<br />
<strong>of</strong> action <strong>of</strong> the organisms to specific soils and/or host-pathogen<br />
systems. Additional experiments will be required to determine in<br />
more detail the extent <strong>of</strong> conservation <strong>of</strong> <strong>phenazine</strong>-cyanide-<br />
siderophore biosynthetic genes in strains that produce<br />
structurally different antibiotics.<br />
No orange halos noticed<br />
after 48 h<br />
aCompounds production rating (+++ Highly production symptoms, ++ Medium production symptoms,+ Minor production symptoms ,<br />
- Negative production symptoms)<br />
Acknowledgements<br />
This study was conducted with grants<br />
awarded by the IKY (Greek State<br />
Scholarships Foundation) for the first<br />
year <strong>of</strong> MSc course and the following<br />
two year’s period <strong>of</strong> the PhD. We are<br />
grateful to Dr. Jama and Ms Barbara<br />
Pembroke for their support and for<br />
valuable comments on the current<br />
research process. As well special<br />
acknowledgements to all the<br />
personnel <strong>of</strong> University <strong>of</strong> Reading<br />
(Department <strong>of</strong> Agriculture and Plant<br />
Sciences) and especial to those who<br />
provided isolates used in this study.<br />
170 Journal <strong>of</strong> Food, Agriculture & Environment, Vol.6 (1), January 2008
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