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Melilotus siculus (syn messanensis) is constrained by a lack of suitable rhizobia

Nigel Charman, Ross Ballard and Andrew Craig

South Australian Research and Development Institute, GPO Box 397, Adelaide, SA 5001.
Email charman.nigel@saugov.sa.gov.au

Abstract

Two experiments have shown that a lack of suitable rhizobia is contributing to poor plant vigour in regenerating Melilotus siculus field trials. Most probable number estimates of rhizobial populations in regenerating M. siculus plots have indicated that the rhizobial strains recently used have persisted poorly. The species has also been shown to form sub-optimal symbiosis when nodulated by the commercial annual medic inoculant (strain WSM1115). Twenty-two rhizobial strains sourced from salt affected soils have been tested for their compatibility with M. siculus in the greenhouse. Several strains had greater N2-fixation capacity (up to 2.7 fold), compared to WSM1115. Field-testing of these improved rhizobia is underway.

Key Words

Salinity, pasture legume, nodulation, symbiotic effectiveness.

Introduction

Melilotus siculus (syn Melilotus messanensis) is one of the most promising herbaceous annual pasture legumes being evaluated for use in salt affected pastures in southern Australia. This species has demonstrated the capacity to be productive and persist in these environments. However, observations of erratic plant growth, poor nodulation and N deficiency symptoms within field plots in South and Western Australia indicate that the rhizobial symbiosis is constraining the full potential of this species.

Methods

Rhizobial persistence

To determine if the erratic nodulation and growth of regenerating Melilotus siculus plots at a saline discharge site near Keith in the south east of South Australia was due to poor rhizobial persistence, the most probable number (MPN) of rhizobia was determined. The field plots were located on a sandy soil over-lying limestone, with mean pHw of 7.4 and peak (summer) EC level of 1.7 dS/m. The site is subject to periods of inundation in winter. M. siculus had been inoculated with a mixture of Sinorhizobium strains RRI128, WSM1115, SRDI118 and SU277, when sown in 2003. Soil samples were collected in August 2005 from three ‘good’ and three ‘poor’ areas within each of the 5 replicates. The ‘good’ areas contained vigorous green plants. Conversely, the ‘poor’ areas contained small yellow or brown plants. Each soil sample comprised 3 cores (1.5 cm diameter) of 10 cm depth. Ten-fold dilutions of the soil samples were used to inoculate 3 replicate plants of M. siculus accession SA36983. The frequency of nodulated plants 28 days after inoculation was used to estimate the number of rhizobia in the soil.

New strains of rhizobia

To obtain strains of rhizobia with improved soil persistence, soil samples were collected from salt affected pastures in South Australia (mean pHCa 7.1) and Western Australia (mean pHCa 5.6) (Table 2). M. siculus accession SA36983 was used to trap rhizobia from these soils. Following the authentication of the isolates, a detailed assessment was made of the N2-fixation capacity of 22 strains from 8 soils. Seed of M. siculus accession SA36983 was surface sterilised and germinated overnight. Four seedlings per pot were sown into a sterile, nitrogen deficient soil mix of coarse sand and vermiculite contained in 1.5 L ‘water-well’ pots. Rhizobial strains were suspended in water and 1 ml, containing approximately 106 cfu applied to each of the 4 plants in a pot. Sinorhizobium medicae strains WSM1115 and WSM688 (present and past AM medic inoculants) and an uninoculated treatment were included as controls. Pots were arranged in the greenhouse in a complete randomised block design with 3 replicates. Plant shoots were harvested 33 days after inoculation. Shoots were dried and weighed to give an indication of the N2-fixation capacity of each strain.

Results

The mean MPN of rhizobia in the ‘good areas’ was 500 per g soil (Table 1), with rhizobia detected in 10 of the 15 individual soil samples. In comparison, the mean MPN of rhizobia in the ‘poor areas’ was 6. No rhizobia were detected in 14 of the 15 samples.

Table 1. Most Probable Number (MPN) of rhizobia (per g soil) in regenerating plots of M. siculus growing near Keith in South Australia. Number of samples (out of 3) where rhizobia were detected is shown in parentheses.

Sample location

Plot replicate

 
 

1

2

3

4

5

Mean

Good area

1557 (3)

500 (1)

0 (0)

353 (3)

91 (3)

500

Poor area

0 (0)

0 (0)

0 (0)

31 (1)

0 (0)

6

Rhizobial strains sourced from salt affected soils resulted in shoot weights 6 to 12 times greater than the uninoculated treatment (Table 2). Strains isolated from soils of pHCa ≥ 6.9 resulted in a greater range of shoot weight (67 to 145 mg DM) than those from soils of pH ≤ 5.5 (78 to 102 mg DM). There was no relationship between soil salinity and shoot weight. Strain WSM1115 resulted in significantly less shoot weight than most of the soil derived strains. WSM688 and the uninoculated treatment produced a similar shoot weights.

Table 2. Effect of inoculation treatment on the shoot dry weight of Melilotus siculus.

Inoculation treatment

Characteristics of source soils

Shoot weight
(mg DM/plant)

 

Soil identity

Salinity, EC (dS/m)

pHCa

 

SRDI453

SA 269

3.9

7.3

145

SRDI532

WA 126

0.3

7.3

129

SRDI457

SA 269

3.9

7.3

110

SRDI468

SA 265

1.5

6.9

106

SRDI466

SA 265

1.5

6.9

104

SRDI524

WA 18

0.3

4.7

102

SRDI529

WA 138

2.3

5.4

102

SRDI530

WA 138

2.3

5.4

102

SRDI534

WA 141

4.5

5.5

99

SRDI535

WA 141

4.5

5.5

98

SRDI528

WA 141

4.5

5.5

95

SRDI465

SA 265

1.5

6.9

92

SRDI523

WA 18

0.3

4.7

90

SRDI526

WA 19

0.3

4.9

90

SRDI450

SA 270

0.9

7.2

88

SRDI522

WA 18

0.3

4.7

87

SRDI525

WA 18

0.3

4.7

87

SRDI446

SA 270

0.9

7.2

86

SRDI527

WA 141

4.5

5.5

78

SRDI531

WA 138

2.3

5.4

78

SRDI451

SA 270

0.9

7.2

67

SRDI533

WA 126

0.3

7.3

67

WSM1115

-

-

-

54

WSM688

-

-

-

24

Uninoc.

-

-

-

12

L.s.d. (P=0.05)

     

34

Discussion

The failure to detect any rhizobia in 14 of the 15 ‘poor’ areas of M. siculus growing on a salt affected soil indicates that poor rhizobial persistence is a factor contributing to variation in plant vigour, in the field. This finding is consistent with data from other salt affected field plots in both South and Western Australia (N Charman and RA Ballard, unpub. data). The greenhouse assessment of the new rhizobial strains has additionally shown that the symbiotic effectiveness of strain WSM1115 previously supplied for M. siculus was probably sub-optimal. This has been confirmed in the field where several of the new strains improved the nodulation and growth of M. siculus in the year of sowing. Persistence of these new strains is being assessed across a range of saline soils where M. siculus will be grown. Data will be available in 2007.

Acknowledgments

This work was funded by GRDC through the National Rhizobium Program. We thank Phil Nichols, Tony Albertson and Sean Miller for the supply of soil samples and Andrew Patterson for technical assistance.

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