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Short communication

The laboratory medium used to grow biocontrol

Pseudomonas

sp.

Pf153 in¯uences its subsequent ability to protect cucumber from

black root rot

J.-G. Fuchs

a, 1

, Y. MoeÈnne-Loccoz

a, b

, G. DeÂfago

a,

*

a

Phytopathology group, Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), UniversitaÈtstr. 2, CH-8092 ZuÈrich, Switzerland

b

UMR CNRS Ecologie Microbienne du Sol, UniversiteÂ Claude Bernard (Lyon 1), BaÃt. 741, 43 Bd du 11 Novembre, 69622 Villeurbanne cedex, France

Received 19 March 1999; received in revised form 23 July 1999; accepted 16 August 1999

Keywords:Growth medium; Fermentation; Formulation; Biocontrol; Inoculant; Cucumber; Rhizosphere;Pseudomonas;Phomopsis

Certain root-colonising bacteria can protect plants from soil-borne pathogens when used as inoculants (Keel et al., 1989; Slininger et al., 1996). However, the properties of the formulation used to deliver these bio-control agents can in¯uence the success of the inocu-lation (Shah-Smith and Burns, 1997; MoeÈnne-Loccoz et al., 1999). Formulations of biocontrol agents have been designed to promote their survival in soil (Tre-vors et al., 1992), colonisation of the rhizosphere (Russo et al., 1996), production of antimicrobial com-pounds (Russo et al., 1996) and eective disease sup-pression (Slininger et al., 1996). The incorporation of nutrients into formulations improved survival of Bra-dyrhizobium japonicum (Fouilleux et al., 1996) and Pseudomonas ¯uorescens(MoeÈnne-Loccoz et al., 1999), but unexpectedly did not enhance (Slininger et al., 1996) or even decreased the ecacy of biocontrol pseu-domonads (MoeÈnne-Loccoz et al., 1999). Whether this observation could be extended to the conditions in which cells are being produced (i.e. prior to formulat-ing the cells) was investigated in the current work.

This was achieved by comparing the ability of cells of Pseudomonas sp. Pf153 grown in dierent laboratory media to protect cucumber from Phomopsis sclero-tioides-mediated black root rot.

The fungus P. sclerotioides v. van Kest. strain L3 (kindly provided by H.-P. Lauber, FAW WaÈdenswil, Switzerland) was grown at 248C on potato dextrose agar (PDA; Difco). Fungal plugs were used to inocu-late autoclaved millet seeds (Maurhofer et al., 1992), which were incubated in the dark for 2 d at 27₈C. The colonized millet seeds were ground aseptically with a spatula and used as inoculum in the experiments.

The ¯uorescent Pseudomonassp. Pf153 was isolated from the root of tobacco grown in Morens soil (Fri-bourg canton, Switzerland) suppressive toThielaviopsis basicola-mediated black root rot of tobacco. Strain Pf153 inhibits P. sclerotioides L3 on PDA and King's B agar (KBA; King et al., 1954). Antifungal com-pounds synthesized by Pf153 include an extracellular protease (identi®ed on skim milk agar) and hydrogen cyanide (method of Castric and Castric, 1983). Unlike other pseudomonads from Morens (e.g. strain CHA0), Pf153 does not produce 2,4-diacetylphloroglucinol or pyoluteorin (determined as described by Keel et al., 1996). Pf153Nal is a spontaneous mutant of Pf153 re-sistant to nalidixic acid (1 mg mlÿ1) and that grows like Pf153 under in vitro conditions. Bacteria were rou-tinely grown in liquid King's B medium (KBB).

Soil Biology & Biochemistry 32 (2000) 421±424

www.elsevier.com/locate/soilbio

1

Current address: Biophyt SA, Schulstr. 13, CH-5465 Mellikon, Switzerland.

* Corresponding author. Tel.: 3869; fax: +41-1-632-1108.

E-mail addresses: [email protected] (Y.

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Biocontrol experiments were done in microcosms containing 400 g of arti®cial sandy-loam soil (Keel et al., 1989) or a natural loam from Eschikon (Natsch et al., 1994). Seven dierent laboratory media were used to grow Pf153 i.e. liquid M1 (i.e. (lÿ1) 0.98 g K2HPO43H2O, 0.4 g MgSO47H2O, 0.4 g CaCO3, 3 g

yeast extract and 10 g sucrose; Roughley, 1970), yeast extract-mannitol broth (YEM; i.e. (lÿ1) 0.65 g K2HPO43H2O, 0.1 g MgSO47H2O, 0.2 g NaCl, 0.2 g

CaCl26H2O, 0.017 g FeCl36H2O, 1 g yeast extract

and 10 g mannitol, pH adjusted to 6.9; Vincent, 1970), nutrient-yeast extract broth (NYB; i.e. (lÿ1) 25 g Difco nutrient broth and 5 g yeast extract; Stanisich and Holloway, 1972), KBB (i.e. (lÿ1) 20 g Difco proteose peptone No. 2, 1.5 g K2HPO43H2O, 1.5 g

MgSO47H2O and 8.4 ml of a 87% glycerol solution),

20% KBB (i.e. ®fth-strength KBB), KBA (i.e. KB con-taining 17 g agar lÿ1) and 20% KBA (i.e. ®fth-strength KBA but with 17 g agar lÿ1). All liquid cultures were incubated with shaking (140 rpm). Cells from over-night cultures (278C) were washed three times in sterile distilled water and used to inoculate soil at 107 colony-forming units (CFU) cmÿ3

soil. On the same day, fun-gal inoculum (prepared as ground inoculated millet seed) was added to soil at the rate of 1.5 mg cmÿ3soil. The soil was mixed thoroughly and used to prepare the microcosms. Cucumber was planted 3 d later.

Seeds of cucumber (Cucumis sativusL.) cv. Pandorex (Samen Mauser, Winterthur, Switzerland) were sur-face-disinfected in 1% sodium hypochlorite for 30 min and rinsed several times in sterile distilled water. They were germinated on water agar at 248C for 2 d and used directly in the experiment performed with arti®-cial soil. When microcosms consisted of natural soil, the two-day-old seedlings were transferred to auto-claved Potgrond BF4 4C potting mix (M. de Baat B.V., Coevorden, The Netherlands) and grown for another 5 d prior to planting into soil. A greenhouse chamber set at 70% relative humidity with 16 h of light (24₈C) and 8 h of dark (20₈C) was used. In ad-dition to the seven inoculated treatments described above, each experiment comprised an uninoculated control (no inoculation at all) and an inoculated con-trol (only the fungus added).

At approximately two months after sowing, the severity of black root rot was assessed for each plant by scoring the percentage of the root covered by lesions, using the following rating scale (Maurhofer et al., 1995): 0no lesion_; ₂:5 0<xR5%_,

12:5 5<xR20%_, ₃₀₂₀<xR40%_,

50 40<xR60%_, ₇₀₆₀<xR80%_,

87:5 80<xR95%_{, 97}:5 95<x<100%, 100 100%_{lesion (plant dead).}

Two microcosms (containing three plants each) were studied per treatment in the experiment in arti®cial soil and six microcosms (containing two plants each) were

used per treatment in the experiment in natural soil. Within each biocontrol experiment (i.e. in arti®cial soil or in natural soil), the distribution of the microcosms in the growth chamber followed a randomized block design. Each biocontrol experiment was repeated twice.

The results obtained in arti®cial soil microcosms and in natural soil microcosms were studied separ-ately. For each treatment, ¯uctuation of data between microcosms within a repeated experiment was smaller than that between repeated experiments. Therefore, for statistical analysis the three rep-etitions of each experiment were considered as three replications (Maurhofer et al., 1992). Means from each repeated experiment were expressed as the

per-Fig. 1. In¯uence of the laboratory medium used to grow Pseudomo-nas sp. Pf153 on the subsequent ability of the strain to protect cucumber against black root rot in arti®cial soil under gnotobiotic conditions. Hatching is used to emphasise bacterial treatments in which cells were grown in diluted broth or on solid medium derived from KBB. Vertical bars represent standard errors (calculated from the means obtained in each repeated experiment). The statistical re-lationship between treatments is indicated with letters a to c.

Fig. 2. In¯uence of the laboratory medium used to grow Pseudomo-nas sp. Pf153 on the subsequent ability of the strain to protect cucumber against black root rot in natural soil. Hatching is used to emphasise bacterial treatments in which cells were grown in diluted broth or on solid medium derived from KBB. Vertical bars represent standard errors (calculated from the means obtained in each repeated experiment). The statistical relationship between treatments is indi-cated with letters a to d.

J.-G. Fuchs et al. / Soil Biology & Biochemistry 32 (2000) 421±424

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centage of the value (obtained in the corresponding repeated experiment) for the treatment where only the pathogen was added, to account for dierences in disease pressure (shown by standard errors of the fungal control treatment in Figs. 1 and 2) between repetitions of the experiments. These percentages were arcsine-transformed and analyses of variance were performed, followed by Fisher's LSD tests to compare treatments (P< 0.05).

The biocontrol experiment in natural soil described above was also carried out with cells of Pf153Nal grown in KBB, 20% KBB or KBA. Per-sistence of Pf153Nal in the microcosms was investi-gated at the end of the experiment (i.e. about two months after planting). Since roots were physically weakened in certain treatments due to black root rot, determinations were carried out on whole microcosm samples (about 10 g each) without separ-ating roots from soil. Sterile distilled water was added to the samples (soil:water ratio of 1:10) to extract bacteria (30 min shaking at 140 rpm) and to dilute the extracts prior to spread plating onto KBA containing nalidixic acid (1 mg mlÿ1). Three samples (each from a dierent microcosm) were stu-died per treatment (i.e. three replicates in total) in each of the three runs of the experiment. No colony was found on plates supplemented with nalidixic acid in the treatments without inoculation of Pf153Nal. Col-ony counts were log-transformed prior to analysis of variance (P< 0.05).

In arti®cial soil,Pseudomonas sp. Pf153 reduced the severity of black root rot from 81% (inoculated con-trol) to less than 18%, regardless of the laboratory medium used to grow the strain (Fig. 1). In natural soil however, cells of Pf153 grown in M1 protected better than cells from KBB (Fig. 2). In addition, KBB-grown cells of Pf153 protected less than cells KBB-grown in less rich media (e.g. 20% KBB) or in KBA, in which the agar physically reduces diusion of nutrients com-pared with KBB. No further improvement was achieved when cells from 20% KBA were used. These observations are in accordance with the results of MoeÈnne-Loccoz et al. (1999), who showed that a nutri-ent-amended formulation of P. ¯uorescens F113 did not protect sugarbeet from Pythium-mediated damp-ing-o, whereas the unamended formulation did.

Survival of P. ¯uorescens R1 in soil can be in¯u-enced by the type of laboratory medium used to grow the strain (Wessendorf and Lingens, 1989), and thus the biocontrol results obtained here might be explained by dierences in survival of Pf153 in soil and/or the rhizosphere. The nalidixic acid-resistant mutant Pf153Nal and the media KBB, 20% KBB and KBA were used to evaluate this hypothesis. This was appro-priate because the eect of Pf153Nal on disease sever-ity was statistically identical to that of Pf153,

regardless of the medium used (i.e. KBB, 20% KBB or KBA; data not shown). In natural soil, the dierence in disease suppressiveness between cells of Pf153Nal grown in KBB, 20% KBB or KBA was not linked to dierences in the population size of the strain in the microcosms at the end of the experiment, since Pf153Nal was recovered at 8105 CFU cmÿ3 soil in all three treatments within each repetition of the exper-iment.

These results indicate that the in¯uence of the lab-oratory medium on the biocontrol ecacy of Pf153 involved the interactions of the pseudomonad with the resident soil microbiota. Growth of Pf153 was essen-tially similar in all media studied, and there was no correlation (Pearson's coecient) between the eect of laboratory medium on disease suppression by Pf153 in natural soil and growth characteristics of the pseudo-monad in these media (i.e. log-phase doubling time, time from inoculation to completion of log phase, or population size de Pf153 at 12 h after inoculation; data not shown). We hypothesize that the laboratory medium had an eect on the physiological state of the cells, which in¯uenced the subsequent biocontrol abil-ity of Pf153 in the rhizosphere. Further work will be needed to understand the mechanisms responsible for this eect.

In conclusion, the laboratory conditions used to pre-pare the inoculum need to be considered carefully when optimizing production of a biocontrol pseudo-monad. An improvement in biocontrol ecacy may be achieved when richer laboratory media are replaced with less rich media, which in addition may lower manufacturing costs of biocontrol products.

Acknowledgements

We are grateful to B. PaÈrli and D. Kospe for techni-cal help and discussion of data. This work was sup-ported by the Bundesamt fuÈr Landwirtschaft of Switzerland.

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