# Influence of nature and physico-chemical properties of substrates on the bacterization with Pseudomonas fluorescens

Bernard Digat, Francis Lemaire

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Bernard Digat, Francis Lemaire. Influence of nature and physico-chemical properties of substrates on the bacterization with Pseudomonas fluorescens. Second Symposium on Horticultural Substrates and their Analysis, International Society for Horticultural Science (ISHS). INT.; International Society for Horticultural Science (ISHS). INT., Sep 1990, Guernesey, United Kingdom. hal-01604514

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<figure><img src="image_1.png" /></figure>
Second
Symposium on Horticultural
Substrates and their Analysis

Editor
Denis Smith

10 - 14 September 1990
Guernsey, United Kingdom

XXIIIrd International
Horticultural Congress

Editor
Dr Ir O. Verdonck

August 1990
Florence, Italy
INFLUENCE OF NATURE AND PHYSICO-CHEMICAL PROPERTIES OF SUBSTRATES ON THE BACTERIZATION WITH *PSEUDOMONAS FLUORESCENS*

DIGAT B. and LEMAIRE F.

Institut National de la Recherche Agronomique (INRA)
Rue Georges Morel
49070 BEAUCOUZE
FRANCE

## Abstract

In soilless cultures, most materials used as substrates are microorganisms-poor because of their making process. To avoid future contamination by pathogenic or deleterious microorganisms, it seems interesting to supply substrates with beneficial ones for example bacteria such as PGPR before and during the growing. Dynamics of a bacterial population of *Pseudomonas fluorescens-putida*, chosen because of its antagonistic effects on the pathogen microflora and its promoting effect on the plant growth, have been studied in different kinds of substrates : sphagnum peat, french brown peat, Hortifibre, pine barks, sand, vermiculite, perlite, rockwool in sterilized or non-sterilized conditions. Bacteria inoculum has been introduced in form of granules. The effects of pH, salinity, form and amount of nitrogen in the substrate have been tested on to the establishment of the bacteria without plant. Moreover, an experiment of bacterization of two substrates (Hortifibre and rockwool) has been made in conditions of cucumber soilless growing. In sterilized organic substrates, *Pseudomonas* population was higher than that of non-sterilized materials and the level of bacteria was keeping the same value from the day 60th except in the pine barks where it gradually decreased with the time. In the mineral substrates, higher bacterial population was in vermiculite. In the non sterilized mineral substrates, the kind of substrate acted on the dynamic of bacteria released from the granules : quick in vermiculite and perlite, slower in sand, none in rockwool during the first four days after inoculation. Then, the bacterial population increased in rockwool, became stable in vermiculite and perlite and decreased in sand. pH, salinity, nitrogen form and amount had little influence in the experiment conditions : ph 5 and 7 ; osmotic pressure, 1 and 10 bars ; nitric and urea nitrogen amounts (22 and 90 mg/l). During the Cucumber growing, the study of dynamics of *Pseudomonas* population pointed out that it was necessary to make several boosters to maintain a satisfactory level of bacterial inoculum.

## 1. Introduction

Microflora is generally poorer in substrates than in soils "in situ". Substrates manufactured at high temperature (rockwool, perlite, Hortifibre) contain very few organisms. The bacterization of substrates with beneficial strains of *Pseudomonas* could be a way to establish a bacterial population which can stimulate the plant growth and give an antagonistic buffer effect against soil pathogens (DIGAT, 1988). The physico-chemical properties of substrates have been well studied (LEMAIRE *et al*, 1990) but few works are existing on the microbial properties. Particularly, the conditions of bacterial population establishment are not yet well determined in relation with the nature and the physico-chemical properties of substrates. The purpose of this study is to elucidate the incidence of some abiotic factors on to the dynamics of an introduced population of *Pseudomonas fluorescens-putida* in controlled conditions without and with a plant growing.
## 2. Material and methods

### 2.1. Experiments in vitro without plant

* **Substrate type** : Four organic substrates were used : Sphagnum peat, french brown peat (Mazerolles), Hortifibre (wood fibers of *Pinus sylvestris*) described by LEMAIRE *et al* (1989) ; and non-composted ground pine barks of *Pinus pinaster* (LEMAIRE *et al*, 1980). Four mineral substrates were chosen : gravelly coarse sand (from the Loire valley), perlite, vermiculite and rockwool cultilene. Physical and chemical characteristics of these substrates are given in the table 1. The acid material was neutralized at pH 6,5 by adding calcium carbonate.

* **Substrate pH** : samples of french brown peat were put at two pH values 5 and 7 by adding calcium carbonate.

* **Substrate salinity** : samples of Hortifibre were put at two salinity levels expressed as osmotic pressure : 1 and 10 bars by adding potassium chloride or sodium chloride solutions.

* **Nitrogen supply** : two kinds of nitrogen (nitrate and urea) were supplied with two concentrations (22 and 90 mg/l N) in sterilized or non-sterilized Hortifibre.

### 2.2. Experiment with plant in soilless culture

Fifty Hortifibre (100 x 19 x 7,5 cm) and rockwool cultilene (100 x 20 x 7,5 cm) slabs were used. Cucumber cv "Corona" was grown in soilless culture (gullies) conditions : drip irrigation with complete nutrient solution (pH 5.5 ; EC 3ms) and three plants per slab. This experiment was conducted in the greenhouses of the experimental station of CTIFL (Carquefou, France) during 209 days from January up to the end of July.

### 2.3. Bacterial inoculum

The L 26-1 bacterial strain was isolated from a lettuce rhizosphere grown in a sandy loam (pH 5.9) at Brain sur l'Authion (France). The strain was characterized as belonging to the *Pseudomonas fluorescens-putida* group, according to the classic determinative scheme (STANIER *et al*, 1966). It was selected because its capacity of *in vitro* antagonism against a broad host range of pathogens (DIGAT and GARDAN, 1987). The bacterial strain was grown in a shaken (160 rpm) medium (MISAGHI *et al*, 1983). The stationary phase of 10⁹ c.f.u./ml was reached after 48 hours at 26°C and bacterial concentration was adjusted by turbidity (Turbidimeter HACH 43900). The bacterial suspension was encapsulated according to a new method such as bacterial granules containing about 10⁷ bacteria each were obtained (DIGAT, 1988).

### 2.4. Substrate bacterization

* **Sterilization effect**

For each substrate, forty pyrex sterilized tubes were filled with forty ml of substrate. Twenty tubes filled with substrate were heat sterilized at 120°C during 1 hour. Inoculation of substrates was made by ploughing four bacterial granules per tube at a same level in the tube. Sterile water was supplied to each tube to obtain 70 % water capacity of the substrate. Tubes were hermetically closed and laid down in a dark room where temperature and relative humidity were maintained respectively at 20°C ±5°C and 80 % ± 10 %. Five tubes of sterilized substrate and five tubes of non-sterilized substrate were kept as non-bacterized controls.
<table><caption>Table 1 - Physical and chemical characteristics of used substrates</caption><thead><tr><th></th><th>Sphagnum<br>peat</th><th>French<br>brown<br>peat</th><th>Pine<br>barks</th><th>Hortifibre*</th><th>Perlite</th><th>Vermiculite</th><th>Sand</th><th>French<br>rockwool</th></tr></thead><tbody><tr><td>• Bulk density kg/l</td><td>0,074</td><td>0.158</td><td>0.173</td><td>0.047</td><td>0.09</td><td>0.13</td><td>1.63</td><td>0.09</td></tr><tr><td>• Porosity</td><td>94.4</td><td>92.6</td><td>89</td><td>96.6</td><td>96.4</td><td>95.1</td><td>38.3</td><td>96.7</td></tr><tr><td>• Water volume %</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- 10 cm tension</td><td>78.0</td><td>80.5</td><td>34.1</td><td>32.5</td><td>34.6</td><td>74.9</td><td>31.7</td><td>81.8</td></tr><tr><td>- 100 cm tension</td><td>35.9</td><td>44.2</td><td>24.3</td><td>9.8</td><td>22.6</td><td>64.9</td><td>4.0</td><td>4.0</td></tr><tr><td>• Air volume %</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>- 10 cm tension</td><td>16.4</td><td>12.1</td><td>54.9</td><td>64.1</td><td>61.8</td><td>10.2</td><td>6.6</td><td>14.9</td></tr><tr><td>• Available water</td><td>42.1</td><td>36.3</td><td>9.8</td><td>22.7</td><td>12</td><td>10</td><td>27.1</td><td>77.8</td></tr><tr><td>• pH H<sub>2</sub>O</td><td>4.6</td><td>4.6</td><td>5.6</td><td>5.1</td><td>9.0</td><td>7.2</td><td>7.1</td><td>8.5</td></tr><tr><td>• C.E.C. meq/l</td><td>136</td><td>218</td><td>89</td><td>21</td><td>12</td><td>60</td><td>0</td><td>5</td></tr></tbody></table>

<footnote>* Hortifibre is a ligno-cellulosic material</footnote>
## • Physico-chemical conditions

Two types of substrates were used: Hortifibre and french brown peat. Twenty four plastic containers were filled with 1 liter of each substrate. One hundred bacterized granules were added per container. Each treatment was repeated once. Six non-bacterized containers were used as control. Sterile water was added to each substrate to obtain 70 % water capacity. Containers were covered by a plastic sheet to prevent evaporation and external contamination, and were put in a greenhouse at 20°C ± 5°C with 70 % ± 10 relative humidity.

## • Soilless culture conditions

Half number of slabs of each substrate were bacterized with bacterial granules; the other half was used as non-bacterized control. The granules were dispatched on slabs surface after fifty-one days of growing so as to obtain a theoretical content of $10^6$ bacteria per gramme of substrate. During the growing, the bacterial population was maintained on the cucumber roots at a level of $10^3$ c.f.u. per root cm. To realize this condition, several boosters were perfected by supplying one hundred granules to the plant stalk at periodical times: 92, 112, 140 and 162th growing days.

## 2.5. Sampling and bacterial numeration

In tube and container conditions, a 5 ml substrate sample was taken at day 4, 8, 16, 32, 64 and 130 after inoculation. Two tubes per substrate were sampled. In cucumber growing conditions, a sample of root system (30 cm long) of four plants per treatment was taken before each booster (nine sampling during the culture). The substrate and root samples were put in a sterile phosphate buffer solution at pH 6.5 and were shaken strongly during 5 minutes. The supernatant was discarded. By serial dilution and streaking on KING'B medium (KING *et al*, 1954), bacterial colonies were obtained after 48 hours at 26°C, identified by the sero-agglutination technique using a specific antiserum made from a bacterial glycoprotein extract as antigen (DIGAT and CAMBRA, 1976) and finally numerated.

# 3. Results

## 3.1. Experiments in vitro without plant

### a) Effect of substrate type and sterilization

#### • Organic substrates (fig. 1)

In sterilized conditions, the bacteria release from the granules was fast and the *Pseudomonas* population reached a maximum in substrate 8 days after inoculation. Then, for 140 days, the population kept a value of $10^6$ c.f.u./g sphagnum, french brown peat and hortifibre while it decreased in the pine barks down to $1.2 \times 10^3$ c.f.u./g after 140 days (significant level at 1 %). In non sterilized substrates, the release of bacteria from the granules was as fast as in the sterilized ones but the maximum of the population after 10 days was lower ($10^4$ to $10^5$ c.f.u./g). In the four substrates, the number of bacteria decreased to the day 64. Then it kept the same value ($10^3$ c.f.u./g) till the days 140. The population in hortifibre was slightly higher than in the other substrates but non significantly.

#### • Mineral substrates (fig. 2)

In sterilized substrates, the *Pseudomonas* population kept a higher value in vermiculite and perlite than in sand and in french rockwool. In non sterilized substrates, the speed of bacteria release and the dynamic of survival were depending on the kind of substrate: fast bacteria release and stable high value ($10^5$ c.f.u./g) in vermiculite; lower value ($10^4$ c.f.u./g) in perlite; strong decrease after five days and stable low value ($10^2$
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /><figcaption>Figure 1 - Dynamics of the *Pseudomonas* population according to the type of sterilized (A) or non sterilized (B) organic substrate (each point is the mean of two replications)</figcaption></figure>
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /><figcaption>Figure 2 - Dynamics of the *Pseudomonas* population according to the type of sterilized (A) or non sterilized (B) mineral substrate (each point is the mean of two replications)</figcaption></figure>
c.f.u./g) in sand; no release of bacteria during the first five days after the inoculation and then increasing number of bacteria to reach $10^3$ c.f.u./g value after 60 days in the french rockwool.

b) Effect of substrate pH (fig. 3)

In non sterilized french brown peat, pH 5 had a greater incidence than pH 7 on the drop of *Pseudomonas* population survival specially from 40 days after inoculation.

c) Effect of salinity (table 2)

Salinity had no incidence on the release of bacteria from granules during the first days, but influenced the survival of bacteria after 68 days. The increasing osmotic pressure value decreased the survival of bacteria, more with NaCl than with Kcl. It could be due probably to the toxicity of Na ions.

d) Effect of nitrogen supply (table 3)

In sterilized hortifibre, no difference was occurring between the treatments. In non sterilized hortifibre, after 38 days, the increasing N supply decreased slightly the survival of *Pseudomonas* in the substrate. The N form had no effect.

## 3.2. Experiments with plant in soilless culture (fig. 4)

Between the days 51 and 92 after plantation, a high decrease of the population survival was observed in Hortifibre and rockwool Cultilène as well from $10^6$ to $10^2$ c.f.u./cm root, but it decreased dramatically at day 133 in Hortifibre. After, from day 150 and till the end of the experiment, the *Pseudomonas* population kept the same level in the two kinds of substrate : $10^3$ c.f.u./cm root.

## Discussion

The results obtained in controlled conditions allowed to check that bacterial granules released fastly but progressively the inoculum in substrates and that the release speed was influenced by the kind of substrate. In soilless culture, where the loss of inoculum can be important because of high drainage by nutrient solution in the case of supplying with liquid form, bacteria granules allowed to keep the inoculum near the roots. From another hand, the effect of sterilization on the dynamic of *Pseudomonas* population in substrate without plant is depending on the kind of used substrate. In organic substrates, level and survival of *Pseudomonas* population was higher with sterilized conditions probably because of the competitive effect of resident microflora which was suppressed and also because of nutrient elements which were solubilized from organic compounds; in the case of pine barks, compounds (tannins, polyphenols) were appearing and could explain the inoculum drop. In mineral substrates, the effect of sterilization was weak because mineral compounds were relatively heat stable. Among the mineral substrates, it has been observed that the dynamics of colonization by *Pseudomonas* were different in vermiculite and in french rockwool. Mineral ions such as Mg$^{++}$ in vermiculite and Ca$^{++}$ in rockwool can have different effects on the mechanism of bacteria release from the granules, the membrane of which is composed of Calcium Alginate and Kaolin. To release bacteria in the substrate, the Alginate membrane must be solubilized by chemical reactions which are increased by Mg$^{++}$ ions and inhibited by Ca$^{++}$ ions; those last ions
<figure><img src="image_6.png" /><figcaption>Figure 3 - Dynamics of the *Pseudomonas* population in french brown peat according to the substrate pH</figcaption></figure>

(each point is the mean of two replications)
<table><caption>Table 2 - Effect of osmotic pressure and type of added salt on the *Pseudomonas* population* in non sterilized Hortifibre</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="2">Osmotic pressure (bars)</th></tr><tr><th>1</th><th>10</th></tr></thead><tbody><tr><td>NaCl</td><td>4</td><td>2</td></tr><tr><td>KCl</td><td>4.7</td><td>4</td></tr></tbody></table>

* Bacteria are numerated (log cfu/f fresh substrate) on the day 68 after inoculation (Each value is the mean of two replications)

<table><caption>Table 3 - Effect of nitrogen supply and form on the *Pseudomonas* population* in non sterilized Hortifibre</caption><thead><tr><th rowspan="2">Treatment</th><th colspan="3">N (mg/l)</th></tr><tr><th>0</th><th>22</th><th>90</th></tr></thead><tbody><tr><td>Calcium nitrate</td><td>4</td><td>4.5</td><td>3</td></tr><tr><td>Urea</td><td>4</td><td>3.8</td><td>3.2</td></tr></tbody></table>

* Bacteria are numerated (log cfu/f fresh substrate) on the day 38 after inoculation (Each value is the mean of two replications)
<figure><img src="image_7.png" /><figcaption>Figure 4 - Dynamic of the *Pseudomonas* population on cucumber roots in hydroponic culture according to the type of substrate (hortifibre and rockwool)</figcaption></figure>
form Ca⁺⁺ bridges between the alkylates molecules. So the delay for releasing bacterial cells in the french rockwool would be explained by its high Ca⁺⁺ content, the favourable effect in vermiculite would be due to the high Mg⁺⁺ content and to the CEC, which can fix Ca⁺⁺ ions.

Finally, the results of this study show that the *Pseudomonas* strain L26-1 has good adaptation capacity to the kind of substrate and to some physico-chemical characteristics.

Several other parameters such as temperature (DIGÂT and MATTAR, 1990) and moisture stress resistances, must be studied before controlling the *Pseudomonas* population and obtaining the beneficial effects on the plant growth as well as a possible biocontrol of pathogens.

## References

Digat, B., 1988. The bacterization of horticultural substrates and its effects on plant growth. Acta Horticulturae, 221 : 279-288.

Digat, B., and Gardan, L., 1987. Caractérisation, variabilité et sélection des souches bénéfiques de *Pseudomonas fluorescens* et *P. putida*. Bulletin OEPP/EPPO.
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Misaghi, J., Stowell, L.J., Grogan, R.G., and Speaman, L.C., 1982. Fungistatic activity of water soluble fluorescent pigments of fluorescent Pseudomonads. Phytopathology 78 : 33-36.

Stanier, R.Y., Palleroni, N.J., and Doudoroff, M., 1966. The aerobic Pseudomonads, a taxonomic study. J. Gen. Microbiol., 43 : 159-271.

Digat, B., 1988. Structure for the inclusion of active substances. International patent. PCT n° WO 88/08876.

Digat, B., and Mattar, J., 1900. Effects of temperature on growth and siderophore production of *Pseudomonas fluorescens-putida* strains. Symbiosis vol 9 (in press).

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King, E.O., Ward, M.K., and Raney, D.E. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. Journal of Laboratory and Clinical Medicine 44 : 301-307.

Lemaire, F., Dartigues, A., and Rivière, L.M., 1989. Properties of substrates with ground pine barks. Acta Horticulturae, 99 : 67-80.

Lemaire, F., Dartigues, A., and Rivière, L.M., 1989. Physical and chemical characteristics of a ligno-cellulosic material. Acta Horticulturae, 238 : 9-22.

Lemaire, F., Dartigues, A., Rivière, L.M., et Charpentier, S., 1990. Cultures en pots et conteneurs : principes agronomiques et applications. INRA et PHM-Revue Horticole Ed., 181 p.

Lemarchand, F., 1989. Dynamique des populations de *Pseudomonas fluorescens* introduites dans les substrats horticoles. Mémoire Maîtrise. Université d'Angers. 29 p.