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Drought tolerance ofRhizobium leguminosarum andR. meliloti

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Page 1: Drought tolerance ofRhizobium leguminosarum andR. meliloti

Folia Microbiol. 38 (4), 320-324 (1993)

Drought Tolerance of Rhizobium leguminosarum and R. meliloti J . N E CA SE K a, M . NI~MCOVA b, L. LISA. b, J . D U S B A B K O V A a, V. NA~INEC a a n d D . P O ~ ' ~ R K O V A a

alnstitute of Plant Molecular Biology, Academy of Sciettces of the Czech l~elmblic, 370 OS Cesk~" Bud~ovice blnstitute of Microbiology, Academy of Sciences of tile Czebh Rqmblic, 142 20 Prague 4

Received January 22, 1993 Rea,ised version April 28, 1993

ABSTRACT. Cell suspension of Rhizobium leguminosarunt by. viciae D-253, R. leguminosarum by. viciae D-560 and R. meliloti D-557 were incorporated into sterile diatomaceous earth (DE) and dried at room temperature. Initial numbers of colony- forming units (CFU), expressed as lOgl0, were 8.27, 8.36 and 8.51, respectively. After 5 months of storage the CFU numbers were 0.00, 5.99 and 7.43, respectively. R. meliloti D-557 showed only minor lowering of the CFU number even after 16 months of storage (logl0 = 7.07). After 7 months of storage in DE some single-colony isolates of D-253 produced 10-100 times higher CFU numbers than the original strain. The isolates of D-560 were much more drought-tolerant. The cells of the original strain died after 7 months of storage, lOgl0 of CFU was 6 - 7 in the isolates. In both strains some of their drought-tolerant isolates had the same specific acetylene-reducing activity of nodule tissue as the original strains. Diatomaceous earth seems to be a prospective carrier for the formulation of bacterization preparations.

The tolerance of rhizobia against stress factors is a characteristic important for the bacteriza- tion of seeds of leguminous plants (Bushby 1982; Mare~kov~i 1983). Predominantly the tolerance to low pH, often connected with a higher content of aluminum ions, was studied (Karanja and Wood 1988a,b; Vargas and Graham 1989; Taylor et al. 1990; Merbach et al. 1990). Although it was impossible to iso- late clones tolerant to low pH (Bromfield and Jones 1980), the existence of cells tolerant to stress function of hydrogen ions is supposed; these cells may be present in cell populations of individual strains (Howieson et aL 1988).

The carrier generally used in the formulation of commercial bacterization preparations is wet peat. Rhizobial cells have a low viability in dry carriers, e.g. phosphates, coal, talc, perlite, amberlite and diatomaceous earth (Vincent 1982; Roughley 1988). Kosanke et al. (1992) discovered that slow rehy- dration of cells improves their recovery in dry carriers, e.g. clay powder. The drying of soil samples before isolation of rhizobia (Vincent 1970, 1982) is a necessary but not suitable technique; their popu- lation structure may change during the loss of water.

The effect of moisture on cell viability was studied by Osa-Afiana and Alexander (1979) and Pefia-Cabriales and Alexander (1979). Lime silt loam was used as carrier. It was found that the curve expressing the number of cells in different time periods has a biphasic shape. After a rapid lowering of cell numbers in 4 -6 d another phase of very slow cell dying follows. If the water content is 40 % or less, the toxic effect of oxygen increases; the mechanism of the toxic effect of oxygen has not been elu- cidated.

The genetics of rhizobia is sufficiently but not completely investigated (Beringer et al. 1982). The symbiotic effectivity is controlled by 30-40 genes, which can change through mutagenesis, genome rearrangement and genetic exchange (Gibson et al. 1991). It is understandable that the same strain deposited in several culture collections may have a different colony morphology, growth rate, produc- tion of polysaccharides, competitiveness, etc. (Mullen and Wollum 1989). In a strain bred for high nitrogen-fixing activity the low competitive ability with other rhizobial soil populations often prohibits its application in agronomic practice (Paau 1991).

The studies of h~terstraht population genetics are based mainly on the determination of isozyme spectra (Young 1985; Harrison et al. 1989; Eardly et al. 1990). There is, however, no knowl- edge about hltrastrahz variability. A precise specification of individual substrain populations may be obtained through plasmid profile screening (Hartmann and Amarger 1991).

In this study it was found that cells tolerant to drought are present in rhizobial populations. Although most drought-tolerant isolates showed lowered symbiotic activity it was possible to identify among them some that were comparable with the original strain.

Page 2: Drought tolerance ofRhizobium leguminosarum andR. meliloti

1993 DRO U G t tT TOLERANCE OF R. leguminosarum AND R. meliloti 321

M A T E R I A L S A N D METHODS

Strab~s. Strains R. legumb~osamm by. viciae D-253, R. legumhzosamm bv. viciae D-560 and R. meliloti D-557 (Mare~kov~i and Slepi~kov~i 1983) used for commercial production of bactcrization preparations, were a kind gift from the Research hlstitute of Crop Production, Prague-Ruzyn6.

Media and cultivation. For the cultivation and maintenance of individual strains and their sin- gle-colony isolates a modified Fred-Wachsman medium (medium C) was used. Medium C contained (g/L): glucose 10, K2HPO4 0.5, MgSO4"7H20 0.2, NaCI 0.1, yeast hydrolyzate (Imuna) 0.2, casein hydrolyzate (Imuna) 1.2. For solidification 2 % agar was used.

Bacterial suspensions were cultivated in Erlenmeyer flasks (volume 250 mL) with 100 mL of medium C on a rotary shaker (1.7 Hz, excenter 25 mm) at 28 ~ Inoculated cultures (original concen- tration of CFU 105/mL) were incubated for 3 d (final concentration of CFU was 108/mL); other flasks were inoculated with 1 mL of the cell suspension. After 4 d of incubation the concentration of colony- forming units (CFU) determined on solidified medium C was 108-109/mL.

hzcorporation of cells hzto the carder. From a set of possible carriers diatomaceous earth (DE) was chosen. This carrier was kindly supplied by the hzstitute of Antibiotics and Biotransfonnations, Roztoky near Prague. DE (10 g sterilized in 100 mL of water) was filtered in a vacuum funnel with 120 mm diameter. A DE layer approximately 3 mm thick was used for subsequent vacuum filtration of the bacterial suspension. Samples of DE with bacterial cells were transferred to Petri dishes and allowed to dry at room temperature. After 4 - 6 weeks the dry mass of DE containing bacterial cells was more than 95 %. The temperature was 18-22 ~ and the relative humidity in the room was 40-60 % during the storage; day-and-night light regime was not regulated. CFU were counted using the plate-count method. For this purpose 500-800 mg of material was weighed, suspended in distilled water and inoculated in several dilutions on solidified medium C in 2 - 3 Petri dishes. The concentra- tion of CFU is presented in base-ten logarithms (Paczkowski and Berryhill 1979; Bushby 1982; Hoben et al. 1991). The dry mass of the carrier with bacterial cells was determined after 4 h of drying at 95 ~

Nitrogen fixation. Test plants (Pisum sativum cv. Bohat~r) were grown in plastic tubes con- taining 216 mL perlite saturated with a nutrient solution (Skrdleta et al. 1980) containing 76 mg/L Ca(NO3)2"4H20 - a nodulation and dinitrogen fixation supporting nitrate level (Skrdleta et al. 1984). The tube bottoms were closed with a cotton plug allowing roots to grow through. The nutrient solution was exchanged twice a week. Plants were grown under environmentally controlled conditions (,~krdleta et al. 1991) in the SIOH Conviron chamber. Two-d-old seedlings were inoculated with 3 mL of suspen- sion of the respective Rhizobhtm strains or isolates at a rate of 109 bacteria per seedling.

Five h after the start of the light period, six 31-d-old plants were sampled from each inoculum treatment. To assay their total acetylene-reducing activity (TAR) nodulated roots were separated and immediately incubated in 100 mL serum bottles at 23~ for 30 min under 10 kPa acetylene as described elsewhere (~krdleta et al. 1991). After incubation, the counts and fresh mass of root nodules were determined.

RESULTS AND DISCUSSION

Survival of bacterial cells expressed as log10 of CFU is shown in Table I. Strain R. legumh;osantm bv. viciae D-253 had a low survival ability. After 5 months of storage in DE there were no CFU even in plates inoculated with undiluted carrier suspension (0.5 g in 10 m'L of water). Colonies no. 1 - 9 were isolated after 3 months of storage; at this time the CFU numbers decreased approximately 1000 times in comparison with the original number of CFU. The strain R. legumb~osarum bv. viciae D-560 had a higher drought tolerance. After 3 months of storage the num- ber of CFU decreased to 1%. The colonies were isolated after 3 months (no. 11-15) and 7 months (no. 16-20) of storage. In contrast, the survival ability ofR. meliloti D-557 was high. After 3 months of storage the CFU number decreased to 10 % and after 16 months of storage it decreased to 3 %.

Isolates of the strain R. leguminosamm bv. viciae D-253 and D-560 were incorporated into DE using the same technique as the original cultures. In several intervals their CFU concentration was determined (Table II and III). Isolates of R. legumbzosamm bv. viciae D-253 after 3 months of storage showed a drought tolerance a little higher than the original culture; after 7 months of storage two iso- lates (no. 1 and 2) died and the others produced 10-100 times more CFU than the original strain D-253. Isolates ofR. leguminosarum bv. viciae D-560 display 106-107 CFU/g after 7 months of storage; no CFU were found in the original strain D-560 after this time of storage. There is a difference

Page 3: Drought tolerance ofRhizobium leguminosarum andR. meliloti

322 J. NEt~,/ISEK a al. Vol, 38

between the survival of D-253 and D-560 in experiments shown in Table I and in Tables II and III; the technique of drying was the same and the reason of this difference is unknown.

Table I. CFU concentrations a of R. leguminosarum by. viciae D-253, R. legumblosarum by. viciae D-560 and R. meliloti D-557

in 1 g of dry matter of diatomaceous earth

Time of storage, months b

Strain 0 1 3 5 7 9 12 16

D-253 8.27 7.98 5.07 0 - - -

D-560 8.36 8.04 6.30 5.99 5.00 4.39 3.94 3.07

D-557 8.51 7.69 7.50 7.43 7.32 7.20 7.11 7.07

aExpressed as lOgl0. trI'ime 0 indicates the CFU concentration immediately after filtration (dry matter 30-40 %).

Table II. Drought tolerance in single-colony isolates of R. legumhzosarum by. viciae D-253 a

Time of storage, months

Isolate

0 1 3 5 7

1 7.74 7.73 5.61 2.50 0

2 7.86 7.50 5.34 1.73 0 3 7.74 7.44 6.13 5.94 4.84

4 7.27 7.64 6.23 6.07 5.47

5 7.89 7.83 5.90 5.27 4.72

6 7.68 6.63 5.64 5.11 4.89 7 7.61 7.38 5.00 5.73 5.17

8 7.68 7.34 5.70 5.74 4.71

9 7.25 7.79 5.79 5.77 4.77

Mean b 7.63c 7.47c 5.70b 4.87ab 3.84a

D-253 c 8.04 7.76 4.94 4.53 3.36

aThe concentrations of CFU are expressed as IOgl0 in 1 g dry matter of

diatomaceous earth.

b rhe means not indexed with the same letter are significantly different at

95 % intervals (Student- Newman- Keuls test). CControl.

The dinitrogen fixation ability of isolates D-253 and D-560 is shown in Tables IV and V. Obviously, there is no correlation between the drought tolerance and the dinitrogen f'Lxation ability.

The different ability of strains of R. meliloti to tolerate absence of water was described by Mary et al. (1985). It is clear that this result has a broader validity.

The strains R. legumbzosarum bv. viciae D-253 and D-560 differ in their tolerance to desiccation considerably. The first was more sensitive to stor- age in dry DE than the latter (Table I). In both of them, however, the drought tolerance of their single- colony isolates, surviving the storage, is higher than the drought tolerance of the original strains. In this respect the cell populations of the two strains are not uniform. The single-colony isolates may be not genetically uni- form clones but rather mixtures of several clones; one CFU may contain

several bacterial cells adhering to a single microscopic particle of DE. The level of its genetic diversity may show a next round of selection. Strain R. meliloti is surprisingly drought-tolerant without any manipulation.

Isolates of both strains R. leg~unhlosarum bv. viciae show a different ability of nodulation and dinitrogen fixation. Three out of nine isolates of R. leguntinosantm bv. viciae D-253 lost the nodulation ability, the other six show great differences in the total acetylene-reducing activity (TAR) (1.04-26.56, Table IV) and in specific acetylene-reducing activity (SAR) (1.71-23.36, Table IV). In contrast, the isolates of R. legumbzosamm bv. viciae D-560 have a lower variability in TAR (4.54-11.67, Table V) and in SAR (7.27-18.60, Table V). Some isolates (no. 6, 9, 16) are drought-tolerant and their SAR is comparable with the values of the original strains. In some isolates (3, 12, 17) the nodule number (NN) is very high without any pronounced effect on SAR. The isolates of R. legunzbtosantm bv. viciae D-253 show great differences in nodule fresh mass (NFM) and nodule number (NN). The loss of nodulation ability in some isolates may be an extreme situation of low NFM and NN. The question of competition ability remains, however, uncertain (Paau 1991).

Page 4: Drought tolerance ofRhizobium leguminosarum andR. meliloti

1993 D R O U G I I T T O L E R A N C E OF R, legltntinosanan AND R. meliloti 3 2 3

Moreover, the results show that it is possible to use a dry carrier for the formulation of a bacterization prepa- ration containing rhizobial cells. The use of diatomaceous earth is pro- mising and may eliminate problems connected with the use of peat as a "wct" carrier (Brockwell 1982).

Table l l l . Drought tolerance in single-colony isolates of R. leguminos- arttm by. viciae D-560 a

Time of storage, months Isolate

0 1 3 5 7

11 8.20 8.20 7.10 6.18 6.15

12 8.30 8.26 7.57 7.00 6.53 13 8.25 7.65 6.65 6.94 6.87 14 8.23 7.41 6.91 6.68 6.49

I5 8.60 8.13 6.47 7.00 6.81 16 8.38 8,75 Z90 7.87 6.17 12 8.77 8,73 7.56 7.57 7.76

18 8.56 8.61 8.11 7.96 7.78 19 8.56 7.76 7.57 7,78 6.30 20 8.56 8.56 7.82 7.55 7.74

Mean b 8.41b 8.16b 7.36a 7.26a 6.85a

D-560 c 7.88 7.51 4.20 3.89 0

a,bFor explanation see Table II. CControl.

Table IV. Dinitrogen fixation in isolates of R. leguminos- arum by. viciae D-253 a,b

Table V. Dinitrogen fixation in isolates of R. leguminosamm by. viciae D-560 a,b

Isolate NFM NN T A R SAR

1 0 0 0 0 2 0.16a 99ab 1.04a 6.50a 3 0.50a 291c 3.39.2,2 6.64a

4 0 0 0 0 5 0 0 0 0 6 1.1 lb 226bc 26.56b 23.92,b

7 0.35a 221a - c 0.87a 2.48a

8 0.18a 73a 0.67a 3.72a 9 1.20b 173a - c 24.13b 20.10b

D-253 c 0.59a 103ab 10.67a 18.08b

aNFM = nodule fresh mass (g per root), NN = nodule

number (per root), T A R = total acetylene-reducing activ- ity (,,umol C2H4 per Ikper root), SAR = specific acetylene- reducing activity of nodule tissue (/,tmol C2H4 per h per g

nodule fresh mass). h i 'he means not indexed with the same letter are signifi- cantly different at 95 '7o intervals ( S t u d e n t - N e w m a n -

Keuls test). CControl.

Isolate NFM NN T A R SAR

11 0.68a 196a - c 7.33a 10,77ab

12 0.83a 283c 9.75a 11.74a - c 13 0.52a 197a - c 7.05a 13,55a - c

14 0.53a 175ab 4.54a 8.56a 15 0.53a 205a - c 6.58a 12,41a-c 16 0.58a 148ab 11.67a 20,12c

17 0.69a 258bc 7.53a 10.91a - c 18 0.62,a 234bc 7.60a 12.25a - c 19 0.63a 233bc 7.03a l l .15a - c 20 0.68a 155ab 5.89a 8.66a

D-560 c 0.84b 117a 21.52b 25,61bc

a'bFor explanation see Table IV. CControl.

We are grateful to Dr. V. Skrdleta (Institute of Microbiology, Academy of Sciences of the Czech Republic) for generous

help with Ihe assays of nitrogen fixation and for critical reading of the manuscript.

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324 J. NE~ASEK et al. Vol. 38

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