UPPI. Bact. 1976, 40, 129-134

Production of Plant Growth Regulators by Rhizosphere Phosphate-solubilizing Bacteria

J. M. BAREA, E. NAVARRO AND E. MONTOYA Microbiology Department, Experimental Station of Zaidin and Faculty of Sciences, Granada, Spain Received 14 M a r c h 1975 and accepted 28 October 1975

Culture supernatant fluids of 50 phosphate-dissolving bacteria isolated from rhizospheres of crop plants were examined for IAA, gibberellins and cytokinins. These bacteria possessed phytase activity and 27 could dissolve rock phosphate. Twenty bacteria synthesized all 3 types of plant hormones, 43 produced IAA, 29 formed gibberellinsand 45 cultures produced cytokinin-like substances. Of the 50 bacteria tested 28 decomposed IAA. Plant growth inhibitors were detected in cultures of some isolates. The ecological significance of these rhizosphere bacteria and their mode of action when used as inoculants is considered.

BAREA,A Z C ~ & N HAYMAN (1975) found that in low phosphate soils plant growth and phosphate uptake were greater after inoculating with both Endogone and phosphate dissolving bacteria than after inoculating with either organisms separately. Further work (Axon, Barea & Hayman, 1976) using inoculants of phosphate-solubilizing bacteria together with sparingly soluble rock phosphate added to the soil showed that the bacteria enhanced the root/shoot ratio of lavender plants and the dry weight of plants increased steadily with the amount of rock phosphate, although the %P in the plants was the same irrespective of the amount of rock phosphate. The authors suggested two possible explanations : (i) the bacteria produced plant growth regulating substances which promoted root growth, the active root system then explored more soil and zones where phosphate ions were liberated from the rock phosphate particles; (ii) the bacteria solubilized phosphate. Because there is doubt about the ability of bacteria to solubilize complex inorganic P compounds or use organic phosphates to increase the overall pool of soluble P in soil (Greaves & Webley, 1969; Martin, 1973) it was suggested (Brown, 1974) that the increases in plant growth sometimes observed after inoculation with phosphate solubilizing bacteria were primarily the result of synthesis of plant growth regulators. As yet there has been no systematic survey of plant hormone production by the rhizosphere population of phosphate-dissolving bacteria (Sperber, 1958 ; Louw & Weblcy, 1959; Greaves & Webley, 1965; Barea, Ramos & Callao, 1 9 7 0 ~ Louw, ; 1970) and this paper presents evidence of the widespread ability of this population to produce auxins, gibberellin and cytokinin-like substances. Selected phosphate-dissolving bacteria were grown in liquid culture and the supernatant fluids examined for growth regulators and for substances that could inhibit plant growth. Ability of bacteria to break down IAA was also studied, because it was recently suggested that bacteria with this property may be ecologically signi[ 1291



ficant in rhizospheres lacking organic substrates (Strzelczyk, Kainpert & Dahm, 1973).

Materials and Methods Isolation of phosphate-dissolving bacteria from the root region

Rhizosphere soils from several crop plants were sampled for bacteria able to solubilize calcium phytate. Representative parts of root systems with adhering soil were shaken in distilled water and 10-fold dilutions of the soil suspensions plated on RC medium (Ramos & Callao, 1967) containing calcium phytate (Greaves & Webley, 1965). After 14 days' incubation at 25 " colonies surrounded by clear zones were isolated, checked for purity and frequently subcultured. Hydrolysis of sodium phytate and solubilization of rock phosphate by the isolated organisms

Hydrolysis of sodium phytate was determined using the technique described by Greaves, Anderson & Webley (1963). Solubilization of rock phosphate by 6 bacteria able to dissolve calcium phytate was tested on RC agar medium containing 0.2 % (w/v) of this sparingly soluble phosphate; plates were incubated for 7 days at 25 ".Organisms were also tested for rock phosphate solubilization in liquid RC medium. The bacteria were grown for 7 days on a rotary shaker at 25" in 250 ml Erlenmeyer flasks containing 50 ml medium with 0.5 g rock phosphate (1 "/, w/v). Soluble P in the supernatant fluid was then determined photocolorimetrically (Barea, Ramos & Callao 1970b). Selected bacteria were identified to genus level using the manuals of Gibbs & Skinner (1966), Gibbs & Shapton (1968) and Harrigan & McCance (1969). Determination of growth regulators production

The selected strains were grown for 12 days on a rotary shaker at 25" in 250ml flasks containing 70 ml of medium described by Brown (1972). Cultures were centrifuged at 3000 g for 40 min, each centrifugate was acidified to pH 3.0 and divided into two equal fractions, A and B. Fraction A was extracted for gibberellins (Brown & Burlingham, 1968) and fraction B extracted with ethyl acetate (Brown, 1972). The organic phase of fraction B was examined for auxins and both organic and aqueous phases for cytokinins (Barea & Brown, 1974). The different extracts were analysed by descending paper chromatography and chromatograms cut into a sequence of 10 RF values which were used in bioassays (Brown, 1972). The method of Barea, Navarro, Palomares & Montoya (1974) was used for a preliminary screening for growth substances and the following specific bioassays were then used: length increase of wheat coleoptile segments for auxins (Nitsch & Nitsch, 1956), extension of lettuce hypocotyls for gibberellin-like substances (Frankland & Wareing, 1960) and the retention of chlorophyll in the excised first leaves of oat for cytokinins (Wheeler, 1972). In the cytokinin bioassay interference by auxins and gibberellins was minimized by heating the chromatogram strips representing each RF value to 115" for 20 min (Azc6n & Barea, 1975).



Establishment of IAA destruction

IAA degradation by selected phosphate-dissolving bacteria was studied in the same medium as used for growth regulator production with the addition of a filter-sterilized solution of IAA to give 100 pg/ml. After 5 days at 25" (Strzelczyk et al., 1973) IAA breakdown was assessed using Salkowski's reagent (Libbert & Risch, 1969).

Results Phosphate-dissolving ability of the isolates

One hundred and eight strains of bacteria isolated from rhizosphere soil produced clear zones of solubilization on RC agar medium containing calcium phytate. Of these, 50 bacteria hydroiysed sodium phytate, that is, possessed phytase activity; 27 of these 50 were able to solubilize rock phosphate. Of the 58 bacteria unable to produce phytase only 18 could dissolve rock phosphate. Cultures of the 50 bacteria with phytase activity were examined for growth regulator production. IdentiJcation of selected bacteria

Bacteria belonging to many genera hydrolysed sodium phytate (Table 1). The 27 rock phosphate solubilizers were identified as strains of Bacillus (1 I), Pseudomonas (9, Chromobacterium (3), Flavobacterium (2), Acinetobacter (l), Aerobacter (l), Corynebacterium (I), Aeromonas (I), Micrococcus (1) and Agrobacterium (1). Production of growth regulators

Table 1 shows the number of bacteria producing plant growth regulating substances. TABLE1 Production of growth regulators and IAA-decomposition by selected phosphobacteria Number of bacteria: A

Producing Bacteria* Bacillus (23) Pseudornonas (8) Chrornobacterium (5) Acinetobacter (2) Aerobucter (2) Fluvobacterium (2) Arthrobacter (2) Micrococcus (2) Corynebucterium (I) Xanthornonus (1) Aeromonas (1) Agrobucterium (1) Total (50) Percentage






17 8 4 2 2 2 2 2 1 1 1 1 43 86

15 3 1 1 1 2 2 2 0 0 1 1 29 58

21 6 5 2 2 2 2 1 1 1 1 1 45 90

7 0 1 0 1 0 1 0 0 0 0 0 10 20

17 2 3 2 0 0 0 1 1 1 0 1 28 56

* Genera of the 50 bacteria hydrolysing sodium phytate, the parentheses contain thenumber of isolates



Calculated from the response curve of the bioassay plant material to standard amounts of IAA, GA3 and kinetin, the amounts of growth substances in the different supernatant fluids ranged, in pg/ml, from 0.01-0.09 of IAA, 0.008-0-2 of GA3 and 0-01-0.1 of kinetin. Ten bacteria, 20% of the total tested, formed substances which significantly inhibited lettuce hypocotyl extension. Filtrates of 7 of these bacteria contained a mixture of gibberellin-like substances and substances inhibiting plant growth. Table 2 shows that phosphate-dissolving bacteria produced plant growth regulating substances irrespective of their ability to dissolve rock phosphate. TABLE2 Solubilization of rock phosphate by producers of growth substances and IAA-deconposers Number of bacteria Type of bacteria*



23 12 24 4 11

20 17 21 6 17

Auxin-producers (43) Gibberellin-producers(29) Cytokinin-producers (45) Inhibitor-producers (10) IAA-decomposers (28)

* In parentheses total number of theselected bacteriawith this property. TABLE3 Phosphobacteria producing the 3 types of plant hormones (auxins, gibberellins and cytokinins) Hormones produced

Auxins-gibberellinsxytokinins Auxins-gibberellins Auxinsxytokinins Gibberellins-cytokinins Only auxins Only gibberellins Only cytokinins None of the three substances

Number of producers 20 3 19 5 1 1 1 0

Phosphate dissolving bacteria would probably be more effective as inoculants if they produced several growth regulators rather than only one or two. The cultures were therefore examined for this ability and results are given in Table 3. Brown (1972) showed that bacteria can produce more than one substance with gibberellin activity and cultures of phosphate dissolving bacteria were examined similarly. Table 4 shows the number of these organisms able to produce 4, 3, 2 or 1 substances with properties of a gibberellin. Table 4 also shows results of a similar study of cytokinin-like substances. All gibberellin-producing bacteria synthesized one substance with an RF value on the chromatograins similar to GA3 (Brown & Burlingham, 1968). Gibberellin substances were also found in 5 other zones on chromatograms with peaks of activity at



TABLE 4 Bacteria producing 4, 3, 2 or I diflerent gibberellins or cytokinins Number of bacteria producing different: A


Number of substances produced



_.d_____7 r-A ,

Total ._

4 3 2 1






6 12 7 4

12 24 14 8

7bT -


21 41 23 14

7 14 17 7

14 28 34 14


Production of plant growth regulators by rhizosphere phosphate-solubilizing bacteria.

J. UPPI. Bact. 1976, 40, 129-134 Production of Plant Growth Regulators by Rhizosphere Phosphate-solubilizing Bacteria J. M. BAREA, E. NAVARRO AND E...
377KB Sizes 0 Downloads 0 Views