# Influence of temperature on growth and siderophore production of Pseudomonas fluorescens-putida strains, and consequence for root colonization

J. Mattar, Bernard Digat

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J. Mattar, Bernard Digat. Influence of temperature on growth and siderophore production of Pseudomonas fluorescens-putida strains, and consequence for root colonization. 2. International Workshop on Plant Growth-Promoting Rhizobacteria - Progress and Prospects, Organisation Internationale de Lutte Biologique et Intégrée contre les Animaux et les Plantes Nuisibles (OILB). FRA., Oct 1990, Interlake, Switzerland. 418 p. hal-01607164

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# INFLUENCE OF TEMPERATURE ON GROWTH AND SIDEROPHORE PRODUCTION OF PSEUDOMONAS FLUORESCENS-PUTIDA STRAINS, AND CONSEQUENCE FOR ROOT COLONIZATION

J. MATTAR and B. DIGAT

Institut National de la Recherche Agronomique, Station de Pathologie Végétale et Phytobactériologie, Beaucouzé - 49000 Angers, France

## ABSTRACT

Comparing influence of temperature on tomato root colonization by a temperate and a tropical strain of *Pseudomonas fluorescens-putida*, it was observed that at both 30 °C and 20 °C, tropical strain So11 had a better root colonization capacity than temperate strain Ch4, when both strains were inoculated together. However, at both temperatures no clear difference in root colonization capacity was observed between the two strains when inoculated individually. Effects of this thermosensitivity on establishment, survival and development of PGPR populations in substrates and in soils are discussed.

## INTRODUCTION

In a previous work, a direct correlation was established between the geographical origin of *Pseudomonas fluorescens-putida* strains and their behaviour at different temperatures (1). In this study, more strains were used and results concerning growth and fluorescence at 35 °C as well as resistance to temperature stress were confirmed. Tropical strains grew normally and produced siderophores at 35 °C, temperate strains except for four did not. In addition, tropical strains resisted better than temperate ones at 41 °C for 48 hours (Table 1). On the other hand, in the rhizosphere, temperature may affect the dynamics of soilborne *Pseudomonas* (3). So, the dynamics of bacterial populations on tomato roots were examined at 20 °C and 30 °C as follows:

## MATERIALS AND METHODS

Tomato cultivar 'Tresor' seeds were disinfected by dipping them into calcium hypochlorite (5%) solution for 7 min and rinsing in sterile water. The seeds were germinated on sterile filter paper in the dark at 25 °C. When roots had just appeared, the seedlings were transplanted onto sterile rockwool cylinders (50 x 20 mm) in sterile glass tubes (150 x 22,5 mm), sealed with caps until the 28th day, and then transplanted into glass bottles (250 ml). The plants were then grown under the following conditions: ambient temperature 25 °C day, 20 °C night; substrate temperature stabilized by thermostated waterbaths at 20 °C or 30 °C;
light intensity of 10'000 lux with a 12 hr photoperiod; nutrient solution pH 6. with added FeEDTA (1 mg Fe per L). Because there is no direct evidence of specificity in the relationship between host-plant and *Pseudomonas fluorescens-putida*, a temperate strain Ch4, isolated from chicory rhizosphere (France), and a tropical strain Sol1, isolated from soya rhizosphere (Thailand), were used. Two days after transplanting, the rockwool cylinders were inoculated with a bacterial suspension so as to obtain 1.12 x 10⁷ and 1.77 x 10⁷ CFU per g rockwool for, respectively, the tropical strain Sol1 and the temperate strain Ch4. According to treatments, bacterial strains were inoculated singly or together. The experiment was continued for 43 days. Nine bacterial counts were obtained, with four samples per treatment at each count. The rockwool cylinders were opened and vigorously shaken in 150 ml of sterile water, and root pieces were measured and shaken in sterile water. Bacteria were counted after dilution and plating on King's medium B (2).

RESULTS

When inoculated individually, populations of both temperate Ch4 and tropical Sol1 strains reached and maintained two days after inoculation an average of 5 x 10⁵ CFU/cm root at 20 °C as well as at 30 °C (see Fig. 1). When inoculated together, the tropical strain Sol1 reached the same level of colonization (5 x 10⁵).

<table><caption>**Table 1.** Growth, siderophore production and resistance to temperature stress of *Pseudomonas fluorescens-putida* strains.</caption><thead><tr><th>Zone and country of origine</th><th>Number of tested strains</th><th>growth and fluorescence at (35 °C) growth* with Fluo</th><th>growth without Fluo</th><th>very weak or no growth</th><th>Temperature stress resistance**</th></tr></thead><tbody><tr><td>Temperate France</td><td>31</td><td>4</td><td>11</td><td>16</td><td>0</td></tr><tr><td>Mediterranean</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sicily</td><td>5</td><td>4</td><td>1</td><td>0</td><td>2</td></tr><tr><td>Liban</td><td>8</td><td>6</td><td>2</td><td>0</td><td>5</td></tr><tr><td>Tropical</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Thailand</td><td>10</td><td>10</td><td>0</td><td>0</td><td>8</td></tr><tr><td>Vietnam</td><td>2</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>La Reunion</td><td>8</td><td>8</td><td>0</td><td>0</td><td>8</td></tr><tr><td>Guyana</td><td>9</td><td>9</td><td>0</td><td>0</td><td>9</td></tr></tbody></table>

<footnote>*Visible growth and fluorescence on King's medium B (2).</footnote>

<footnote>**After 48 hr stay at 41 °C, visible growth and fluorescence at 26 °C in Misaghi's medium (4).</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1. Dynamics of tropical strain So11 and temperate strain Ch4 at 20 °C and 30 °C on to-mato roots. When inoculated together, tropical strain So11 maintained the higher level of col-onization at 20 °C and 30 °C as well.</figcaption></figure>

CFU/cm root) two days after inoculation as it did when inoculated individually. On the contrary, the temperate strain Ch4 only reached an average of 5 x 10⁴ CFU/cm root.

DISCUSSION

Comparing the dynamics of tropical strain So11 (Thailand) and of temperate Ch4 (France) on tomato roots, no clear difference was observed between 20 °C and 30 °C, nor between the two strains when inoculated individually. However, when inoculated together the colonization ability of the tropical strain was higher at both 30 °C and 20 °C. Therefore, concerning the influence of temperature, we speculate that the rhizosphere competence of tropical populations of *P. fluores-cens-putida* could be favoured under field conditions not only at high tempera-tures but also under temperate conditions.
REFERENCES

1. Digat, B., and Mattar, J. 1990. Effects of temperature on growth and siderophore production of *Pseudomonas fluorescens-putida* strains. Symbiosis 9:207-213.

2. King, E. O., Ward, M. K., and Raney, D. E. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44:301-307.

3. Loper, J. E., Haack, C., and Schroth, M. N. 1985. Population dynamics of soil *Pseudomonas* in the rhizosphere of potato (*Solanum tuberosum* L.). Appl. Environ. Microbiol. 49:416-422.

4. Misaghi, I. J., Stowel, L. J., Grogan, R. G., and Spearman, L. C. 1982. Fungistatic activity of water soluble fluorescent pigments of fluorescent *Pseudomonas*. Phytopathology 72:33-36.
UNION INTERNATIONALE DES SCIENCES BIOLORIQUES

ORGANISATION INTERNATIONALE DE LUTTE
BIOLOGIQUE ET INTEGREE CONTRE LES ANIMAUX
ET LES PLANTES NUISIBLES

ELECTION REGIONALE OUEST PALEARCTIQUE

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Plant Growth-Promoting Rhizobacteria -
Progress and Prospects

ISBN 92-9067-042-8

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The Second International Workshop
on Plant Growth-Promoting Rhizobacteria

Interlaken, Switzerland
October 14-19, 1990

WPRS Bulletin
Bulletin SROP

1991/XVII/8

INTERNATIONAL UNION OF BIOLOGICAL SCIENCES

INTERNATIONAL ORGANISATION FOR BIOLOGICAL
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