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Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes
Soil Biology & Biochemistry 36 (2004) 1339–1346 www.elsevier.com/locate/soilbio
Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes J.F. Slattery*, D.J. Pearce, W.J. Slattery Department of Primary Industries, Rutherglen Research Institute, Rutherglen, Vic. 3685, Australia
Abstract Communities of resident rhizobia capable of effective nodulation of pulse crops were found to vary considerably over a range of soil environments. These populations from soils at 50 sites in Southern Australia were evaluated for nitrogen fixing effectiveness in association with Pisum sativum, Vicia faba, Lens culinaris, Vicia sativa, Cicer arietinum and Lupinus angustifolius. The values for nitrogen fixing effectiveness could be related to soil pH as determined by soil type and location. It was found that 33% of paddocks had sufficient resident populations of Rhizobium leguminosarum bv viciae for effective nodulation of faba bean, 54% for lentils, 55% for field pea and 66% for the effective nodulation of the vetch host plant. Mesorhizobium cicer populations were very low with only 7% of paddocks surveyed having sufficient resident populations for effective nodulation. Low resident rhizobial populations (,10 rhizobia g21 soil) of R. leguminosarum bv viciae and M. cicer were found in acid soil conditions. In contrast, Bradyrhizobium populations increased as soil pH decreased. Inoculation increased faba bean yields from 0.34 to 4.4 t ha21 and from 0.47 to 2.37 t ha21 for chickpeas on acid soils. On alkaline soils, where resident populations were large there was no consistent response to inoculation. Observations at experimental field sites confirmed the findings from the survey data, stressing the importance of rhizobial inoculation, especially on the acid soils in south-eastern Australia. Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Rhizobial populations; pH; Rhizobial inoculation; Crop yield; Nodulation effectiveness
1. Introduction Pasture and crop legumes have been used extensively in Australian agriculture during the past century mainly to boost soil fertility and, as a consequence, crop and animal productivity. Many soils are often unable to sustain productive farming systems or limit crop production due to factors associated with low fertility, sodicity, salinity and extremes of acidity and alkalinity (Slattery et al., 2001). These same attributes can also have a negative effect on the legume-Rhizobium symbiotic relationship and thus reduce the ability of rhizobia to form nodules with optimal N2-fixing capacity (Brockwell et al., 1995). In temperate Australian soils, numbers of resident rhizobia can vary considerably in size, ranging from , 10 to . 106 rhizobia g21 soil (Gibson et al., 1975; Slattery et al., 1999). The size of the soil rhizobial community is dependent on many factors including field history, soil and environmental characteristics and the presence of host plants. Successful establishment of inoculant strains into the soil is also dependent on the numbers of resident populations, * Corresponding author. Fax: þ 61-2-60-304-600. E-mail address: [email protected] (J.F. Slattery).
host plant activity, as well as other soil characteristics (Brockwell et al., 1995). Where the resident populations of rhizobia are small (, 50 Rhizobium g21 soil) and specific to a target legume, the introduction of new strains by seed inoculation is usually successful. On the other hand, many authors have reported low recovery of the inoculant strain where resident rhizobial populations are large (. 103 rhizobia g21 soil) (Thies et al., 1991; Brockwell et al., 1995). The adoption of pulse (grain) legumes in Australia, and their production has increased rapidly as farmers appreciate the rotational and financial benefits that pulses provide to their production systems. In , 40 years, the area in Australia under pulse production has increased from zero to over 2 £ 106 ha in 1996 (Siddique and Sykes, 1997) with pulses now occupying more than 10% of the area cropped. The Australian continent contains a spectrum of geographic cropping environments with pulses produced over a wide range of climatic conditions and soil environments. Soils in the pulse growing regions of south-eastern Australia vary from strongly alkaline (NW Victoria and much of South Australia), neutral to slightly acid (Wimmera, central and southern Victoria) to highly acid (NE Victoria and southern NSW). This wide variation in soil pH together with salinity,
0038-0717/$ - see front matter Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.04.015
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sodicity and fertility has serious implications for rhizobial survival and effectiveness as well as the need for re-inoculation of following pulse crops. The capacity of inoculant strains to colonise soils in sufficient quantities to provide effective nodulation is highly dependent on the soil pH (Brockwell et al., 1995). Thus, a thorough understanding of the size and effectiveness of resident populations (defined as the naturalised or indigenous populations of rhizobia in the soil) will enable recommendations to be made regarding the need for re-inoculation of pulse legumes. We report on the effect of soil types on the persistence of resident rhizobial populations to form nodules and whether it is necessary for the re-inoculation of rhizobia when introducing pulse legumes into different soil environments. A comprehensive soil survey was conducted across the pulse legume growing regions of Victoria to provide a critical analysis of the soil chemical and resident rhizobial populations under different soil environments. This critical data provides a starting point for an assessment of inoculation and re-inoculation requirements of pulse crops.
2. Materials and methods There are two main experimental approaches described in this paper, one involved the survey of soil from 50 paddocks where grain legumes had previously been grown over the past 5 years (paddock history data not presented) and the other involved an investigation of a grain legume (pulse) yield response to inoculation at 6 field sites. 2.1. Field survey and soil sampling Soil rhizobial populations, soil chemical characteristics and classification were measured under a range of pulse crop and pasture legumes. Fifty paddocks were sampled during August 1997, over a wide geographical range for the pulse legume growing regions of Victoria. The number of paddocks sampled and regional location of each were as follows: Birchip, southern Mallee (11 sites); Walpeup, northern Mallee (10 sites); Horsham, Wimmera (7 sites); Elmore, central Victoria (7 sites); Charlton, north central (5 sites); Dookie, north east Victoria (7 sites); Rutherglen, north east Victoria (3 sites). Ten soil cores (10 cm depth £ 2.5 cm dia) were collected randomly using an aseptic technique (Slattery and Coventry, 1993) from each paddock, bulked, thoroughly mixed, sieved (, 2 mm) to remove stones and large pieces of undecomposed organic matter, air dried, then stored at room temperature before chemical and rhizobial analysis. Extreme care was taken to exclude the sampling of rhizosphere populations, by sampling away from any plants. An intact core to a depth of 110 cm was removed for soil characterisation using Isbell (1996) classification system.
2.2. Experimental sites In order to make recommendations for farmers on the need for the re-inoculation of subsequent legume crops additional treatments (^ inoculation) were imposed across farmer paddocks as demonstration strips in the year following a cereal crop or pasture. Inoculation response trials for six legumes (field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris), vetch (Vicia sativa), chickpea (Cicer arietinum) and narrow-leafed lupin (Lupinus angustifolius)) were each established at 6 sites in 2000. Locations, soil classification, soil pH and resident rhizobial population status for experimental field sites are described in Table 1. The rhizobium status was based upon a mean probable number determination (MPN) (Brockwell, 1963). At each site, and for each legume, the commercial inoculant was applied to the seed and sown as the inoculant treatment in the 36 m2 plots. For comparison untreated seed was sown as the nil treatment. When needed, annual grasses and broadleaf weeds were controlled with the appropriate chemicals according to registered recommendations. Ten individual plants were removed from each demonstration plot 10 –12 weeks after sowing for nodulation and plant dry matter (DM) measurements. Plant roots were scored for nodulation on a 0 – 5 scale, based on the nodule number, size, position, distribution and pigmentation of effective nodules on the crown and lateral roots (adaptation of Corbin et al. (1977)). Plant shoot material was dried in a fan-forced draught oven at 70 8C for 48 h, prior to DM measurement. At maturity grain seed yield was determined by the mechanical harvesting of the entire plot using a plot harvester and weigh bins. The MPN was used to estimate soil rhizobial populations collected from the soil survey and from the inoculation response trials (Brockwell, 1963). 2.3. Evaluation of nitrogen-fixing effectiveness—field survey Air-dried soil samples collected from each site were used to enumerate populations of rhizobia and to estimate Table 1 Location, soil pH and resident rhizobial populations in the surface 10 cm of soil of inoculation response trials conducted in the winter of 2000 Site location
Soil typea
Soil pHCa
Resident rhizobial populationsb
Birchip-F, NW Vic. Birchip-S, NW Vic. Walpeup-P, NW Vic. Walpeup-N, NW Vic. Walpeup-C, NW Vic. Rutherglen-B, NE Vic.
Calcarosol Calcarosol Calcarosol Calcarosol Calcarosol Kurosol
7.60 7.78 7.16 7.29 8.80 4.60
High High High High High Lowc
a
According to Isbell (1996). MPN (Brockwell, 1963) using the host plants listed in Section 2.2. c An average of resident rhizobial populations on six host plants were: Low (,10 rhizobium g21 soil), High (.103 rhizobium g21 soil). b
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symbiotic effectiveness. The method used to evaluate the effectiveness of N2-fixation of rhizobial populations in soil samples was a whole soil inoculation technique (WSIT) (Brockwell et al., 1988). The ability of the soils to support rhizobial populations that form nodules was tested using the plants listed in Section 2.2. Seeds were surface sterilised, germinated on 2% water agar plates at 22 8C, prior to planting. Single test plants were grown in seedling tubes (10 cm depth £ 3 cm2) in a semi-sterilised system (washed sand:vermiculite mixture, moistened with N-free nutrient salt solution (Slattery and Pearce, 2002)). In this system, the shoots are exposed to the atmosphere whilst the roots are grown under aseptic microbiological conditions. Each seedling was inoculated 5 days later with a 1 ml soil suspension (10 g soil in 90 ml N-free nutrient salt solution) of each isolate and four replicates. The plants were grown for a further 4 weeks in a glasshouse (range 12– 25 8C), with the moisture content of each plant maintained with either sterile distilled water or N-free nutrient solution. The same plants that were grown to determine nitrogen-fixing effectiveness were also used to assess nodulation. All plants were harvested, the roots examined for nodulation, and DM from the plant shoot material was measured after oven drying at 70 8C for 24 h. For each legume, there were three control treatments including an uninoculated control; uninoculated control with 1 ml of 0.05% KNO3 applied at weekly intervals after sowing and an inoculated control using the appropriate commercial strain (e.g. SU303, WSM1274, CC1192 or WU425).
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1999). Soils were identified according to the Australian soil classification system (Isbell, 1996). 2.5. Statistical analysis Soil analysis and rhizobial data were analysed for difference by analysis of variance, with the treatment means compared at the 95% probability ðP ¼ 0:05Þ (GENSTAT V).
3. Results 3.1. Field survey 3.1.1. Evaluation of nitrogen fixing effectiveness Soils vary in their ability to nodulate pulse crops as a result of soil pH and paddock history. Results from our survey showed a variation in the presence (or survival) of soil rhizobia at individual sites within a location or between rhizobial species and plants (Table 2). On some soil types, the soil rhizobia present were capable of infecting the host legume and forming nodules, but at other sites soil rhizobia were absent and plants in these soils failed to form nodules. In the experiments to numerate rhizobial populations, all nodulated test plants grew vigorously fixing N, indicator the effectiveness of the populations (rhizobial counts not presented). Averaging across the 50 paddocks surveyed; 33% of the paddocks had sufficient resident Rhizobium leguminosarum bv viciae to nodulate faba bean, 54% for lentils, 55% for field pea, and 66% for vetch (Table 2). At 17 sites (34%) no R. leguminosarum bv viciae were detected using WSIT (Brockwell et al., 1988). The presence of chickpea rhizobia, Mesorhizobium cicer, was very low with only 7% of paddocks surveyed having resident populations to chickpea by the WSIT. In the case of Bradyrhizobium spp., 38% of paddocks had sufficiently large resident populations (. 102 rhizobia g21 soil) to nodulate lupin.
2.4. Soil chemical analysis—field survey and experimental sites Soil pH Ca (10 mM CaCl 2) and soil pH W 1:5 soil:extractant was measured using an automated system, whilst Olsen P (0.5 M NaHCO3) 1:100 soil:extractant, extractable K (0.5 m NaHCO3) 1:100 soil:extractant, total N (Kjeldahl), mineral N (2 M KCl—Kjeldahl) and organic C were determined by standard procedures (Slattery et al., Table 2 Percentage of soil extracts ðn ¼ 50Þ forming nodules on six legume species Location
Mean of 50 paddocks Birchip Walpeup Horsham Charlton Elmore Dookie Rutherglen LSD ðP , 0:05Þ
Host plant Soil classification
Vetch
Field pea
Lentil
Faba bean
Lupin
Chickpea
Calcarosol Calcarosol Sodosol Dermosol Dermosol Ferrosol Kurosol
66 100 78 100 80 14 14 0 23
55 100 71 86 67 0 14 0 22
54 100 56 100 40 0 14 0 23
33 67 0 71 100 0 0 0 23
38 33 14 13 33 57 71 33 11
7 10 0 67 0 0 0 0 13
Soils were collected from seven locations in the pulse growing regions of Victoria.
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Table 3 Summary of soil chemical characteristics (range) in the surface 10 cm of soil collected from 50 paddocks at seven locations in the pulse growing regions of Victoria Location
Birchip Walpeup Horsham Charlton Elmore Dookie Rutherglen
Soil characteristics Number of locations
pHwater
pHCa
Org C (%)
Total N (%)
Olsen P (mg kg21)
Ex K (mg kg21)
11 10 7 5 7 7 3
6.8 –8.8 6.9 –8.8 7.6 –8.0 6.1 –7.5 5.4 –6.1 5.5 –5.3 NT
6.1 –8.0 5.9 –8.1 7.4 –7.7 5.8 –6.8 4.4 –4.7 4.4 –5.3 4.2 –4.9
0.4 –1.6 0.4 –1.0 1.5 1.3 –1.7 1.6 –2.8 1.3 –2.3 NT
0.04–0.09 0.05–0.05 0.19 0.06–0.13 0.08–0.38 0.08–0.19 NT
5–47 10–34 5 11–26 7–33 8–32 NT
168–618 162–600 789 248–640 192–386 274–510 NT
NT, soils not tested.
The major pulse growing regions in Victoria that require nodulation by R. leguminosarum bv viciae are in the central west and north west (Birchip and Horsham) of the State. Resident R. leguminosarum bv viciae populations in these regions have been considered present in sufficient numbers and in the past, so rhizobial inoculation has not been recommended. However, the survey results showed 23% of faba bean crops had inadequate R. leguminosarum bv viciae populations to nodulate faba bean, which would lead to a reduction in crop yield. R. leguminosarum bv viciae populations capable of infecting vetch, field pea, lentils and faba beans at the Elmore, Dookie and Rutherglen locations were extremely small (Table 2). The populations of chickpea rhizobia (M. cicer) were even lower in the Birchip regions, only 10% of paddocks compared to 67% of paddocks in the Horsham region (Table 2). Bradyrhizobium sp. populations, capable of infecting lupins were present in all soil types but varied in numbers with respect to the proportion of paddocks in which they were present (Table 2) (individual data not presented). 3.1.2. Soil chemical variables The range of values for the soil chemical measurements collected from 50 paddocks across seven locations in the pulse growing regions of Victoria showed some similarities and differences that could be related to soil type and region (Table 3). Surface soil pHCa at the Charlton site was in the pHCa range from 5.8 to 6.8 and higher than the surface soil pH at the Elmore site of 4.4– 4.7. The soils in the Charlton and Elmore region were classified as Dermosols, these soils tend to form surface crusting if not managed appropriately, the effects of which may influence rhizobial survival (Isbell, 1996). Surface soil pHCa in the Rutherglen region were the lowest with pHCa ranging from 4.2 to 4.9 and the soils were classified as Kurosol (Isbell, 1996). In the Birchip region surface soil pHCa for the 8 paddocks ranged from 6.1 to 8.0, with 63% of the sites being highly alkaline (pHCa . 7.5). Soil organic C ranged from 0.4 to 1.6%; the mean value of 1.1% considered low but typical for farming systems with a long history of continuous cropping, intense cereal rotations, together with low growing season
rainfall (GSR) and low plant productivity as experienced in the Walpeup region. The mean total N values were above those considered limiting for pulse crops, with many of the paddocks also having large resident rhizobial populations for effective nodulation of pulse crops, it is therefore likely that available N was not limiting crop yield. There was a 10fold difference between the maximum and minimum values of extractable P, with 75% of sites having a low Olsen P value of , 17 mg kg21. With the exception of one property, soils in the Birchip region were classified as Calcarosols derived from limestone and containing dissolved and particulate calcium carbonate present throughout the profile (Isbell, 1996). The survey data showed a significant relationship between the effective nodulation of soil rhizobia for a particular plant host and soil pH, and to a lesser extent the relationship between effective nodulation of soil rhizobia with the other soil measurements (Fig. 1). From Fig. 1, it can be seen that there is an interaction between soil rhizobial populations capable of effective nodulation of pulse crops and pH (Fig. 1a), where soil pHCa and R. leguminosarum bv viciae populations at the Elmore, Dookie and Rutherglen sites were low. In contrast, background populations capable of effective nodulation of Bradyrhizobium spp. were higher on the acidic soils and with the exception of the sites in the Horsham region, the resident populations of M. cicer were low (Fig. 1b). 3.2. Experimental sites The site data demonstrated that in the major pulse growing regions of Victoria, resident populations of rhizobia capable of effective nodulation of pulse crops can be high (Table 1) and any response to inoculation might not be evident. For this reason, field data has been presented for the Kurosol and only one of the Calcarosol sites (Tables 4 and 5). On the acid Kurosol at Rutherglen, the nodulation derived from the resident rhizobial population varied between plant species (Table 4). For example, inoculation increased faba bean yields from 0.34 t ha21 for the nil treatment to 4.4 t ha21 and root DM from 1.39 to
J.F. Slattery et al. / Soil Biology & Biochemistry 36 (2004) 1339–1346
Fig. 1. (a) Relationship between soil pHCa at seven regional locations in Victoria and nodulation for Pisum sativum, Vicia faba, Lens culinaris and Vicia sativa. Vertical bars represent the least significant differences at P ¼ 0:05: (b) Relationship between soil pHCa at seven regional locations in Victoria and nodulation for Cicer arietinum and Lupinus angustifolius. Vertical bars represent the least significant differences at P ¼ 0:05:
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4.21 g pl21 with inoculation. Nodulation (nodule number and nodule score) and herbage DM increased at P ¼ 0:1: A visual response to inoculation was observed for the chickpea crop with an increase in grain yield from 0.47 to 2.37 t ha21 and increases for nodulation and root DM. Adequate nodulation occurred in the nil treatment for field pea, lentil and vetch. Although visible growth differences were observed late in the growing season for lentils, these differences could not be related to increased grain yields as the farmers’ sheep unfortunately grazed the lentil crop prior to harvest. On the alkaline Calcarosols responses to inoculation were not visually evident, although at the Birchip-F site (Table 5) where resident populations under lupins were low, inoculation increased nodulation. However, as a consequence of the high resident populations of R. leguminosarum bv viciae, inoculation did not improve the nodulation of faba bean, field pea, lentil or vetch. Inoculation with SU303 for these cultivars improved the yields in each case compared to the nil treatment. In the case of lentils, grains yields for WSM1455 and WSM1483 were higher (statistically not significant) compared to WSM1274, the commercial inoculant at the time. For chickpeas, resident populations were low and inoculation significantly increased nodulation (and improved spring DM production). Overall crop grain yields were lower at the Birchip site where the annual GSR of 238 mm was 43 mm less than the 50-years average compared a GSR of 423 mm at Rutherglen.
Table 4 Nodule number, nodule score, shoot dry weight (g pl21), root dry weight (g pl21) and grain yield (t ha21) for the farmer inoculation response trial at the Rutherglen site (2000) Rutherglen: legume
Rhizobial strain
Nodule number
Nodule score
Root DM (g pl21)
Top DM (g pl21)
Crop yield (t ha21)
Lupin
Nil WU425
16 20 ns
2.6 3.2 ns
0.63 0.50 ns
1.80 1.91 ns
0.71 0.98 ns
Nil WSM1274
10 35 ns
2.6 4.2 ns
1.39 4.21 1.95
4.33 18.50 ns
0.34 4.41 1.6
Nil SU303
43 54 ns
4.1 4.3 ns
0.55 0.46 ns
6.28 7.43 ns
2.15 2.87 ns
Nil WSM1274
10 22 ns
2.2 3.2 ns
0.11 0.11 ns
0.89 0.86 ns
0.15a 0.12a ns
Nil SU303
34 64 ns
3.5 4.8 1.4
0.66 0.51 ns
5.44 7.09 ns
7.87b 8.07b ns
Nil CC1192
2 24 7.5
0.6 4.0 2.5
0.88 2.20 1.0
3.13 3.78 ns
0.47 2.37 2.0
LSD ðP , 0:05Þ Faba bean LSD ðP , 0:05Þ Field pea LSD ðP , 0:05Þ Lentil LSD ðP , 0:05Þ Vetch LSD ðP , 0:05Þ Chickpea LSD ðP , 0:05Þ a b
Crop grazed by sheep, reduced grain yield. Vetch dry matter t ha21, vetch cut prior to grain harvest.
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Table 5 Nodule number, nodule score, shoot dry weight (g pl21), root dry weight (g pl21) and grain yield (t ha21) for the farmer inoculation response trial at the Birchip-F site (2000) Birchip-F: legume
Rhizobia strain
Nodule number
Nodule score
Root DM (g pl21)
Top DM (g pl21)
Crop yield (t ha21)
Lupin
Nil WU425
0.1 6 ns
0.1 2.4 1.07
0.33 0.40 ns
1.80 1.62 ns
0.74 0.81 ns
Nil WSM1274 SU303 RRI294 RRI339
24 38 26 20 20 ns
4.3 4.3 4.0 3.7 4.0 ns
0.99 0.78 0.92 0.65 0.89 ns
6.01 5.15 5.61 4.09 5.48 ns
0.26 0.59 0.73 0.27 0.46 ns
Nil SU303 RRI294 RRI339
31 24 22 22 ns
4.0 4.2 4.2 4.0 ns
0.40 0.28 0.28 0.23 ns
6.57 6.19 6.57 5.92 ns
0.78 0.92 0.81 0.81 ns
Nil WSM1274 WSM1455 WSM1483
24 19 25 21 ns
4.2 3.6 4.0 3.5 ns
0.09 0.10 0.09 0.09 ns
1.03 1.09 1.03 1.00 ns
0.83 1.01 1.17 1.35 ns
Nil SU303
21 14 ns
4.2 3.4 ns
0.17 0.12 ns
2.40 2.14 ns
1.76 1.81 ns
Nil CC1192
0.2 4 ns
0.2 2.0 0.9
0.23 0.26 ns
1.30 1.61 ns
NH NH
LSD ðP , 0:05Þ Faba bean
LSD ðP , 0:05Þ Field pea
LSD ðP , 0:05Þ Lentil
LSD ðP , 0:05Þ Vetch LSD ðP , 0:05Þ Chickpea LSD ðP , 0:05Þ
NH, not harvested, cut and removed due to Ascochyta damage.
4. Discussion 4.1. Soil rhizobial populations and effectiveness It is clear from this survey that soils vary in their ability to nodulate pulse crops as a result of soil pH and previous frequency of pulse crop. R. leguminosarum bv viciae populations were resident in most (83%) of the 50 soils collected. It can be assumed that for the remaining soils, rhizobial numbers were insufficient to allow effective nodulation of the host plant. Whilst there was a large variation in the presence of soil rhizobia, shown by the percentage of paddocks exhibiting effective nodulation of the host plant, the highest frequency of resident populations were located in the more alkaline soils of the major pulse growing regions of Victoria. With the exception of the central Mallee or Horsham regions, the extremely low M. cicer resident population highlights the fact that chickpea has not yet been extensively grown in these paddocks. Consequently, there is a need to inoculate when chickpea crops are first introduced to paddocks in this region. The chickpea-M. cicer symbiosis is highly specific and extensive studies have demonstrated the uniqueness of the chickpea rhizobia with 99% of the chickpea isolates nodulating only the original host plant
and not species belonging to the Fabaceae and Mimosaceae families (Gaur and Sen, 1979). This chickpea host specificity has been confirmed by Nour et al. (1994) using molecular techniques. The successful nodulation of lupin requires the presence of a sizeable resident Bradyrhizobium population capable of infecting lupins. Bradyrhizobium strains capable of infecting and nodulating lupins may be indigenous to many parts of Western Australia, but do not occur naturally in Eastern Australia (Gault et al., 1986; Slattery and Coventry, 1989). Except for the Dookie location where 71% of sites had sufficient populations, the survey data suggests that inoculation of lupin seed is essential when growing lupins for the first time in these regions. 4.2. Soil chemical variables When survey data were compared from each location the presence of resident root-nodule bacteria could be related to soil pH, with acid soils being typically devoid of resident populations. Poor rhizobial persistence of R. leguminosarum bv viciae and M. cicer indicates a need for inoculation when sowing into acid soil environments. Soil acidity can effect survival, growth, persistence and nitrogen fixation potential of rhizobia (Rai, 1991), or nodule
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initiation and N2-fixation effectiveness (Coventry and Evans, 1989) whilst nutritional disorders affect the legume-Rhizobium symbiosis. The survey has shown that not only is there a need to inoculate pulse crops in acid soils, but research continues for more adapted rhizobia and the matching of the rhizobia with the plant host. The selection of root nodule bacteria for Biserrula (Biserrula pelecinus) is a good example of the need to match a rhizobial species with a pasture species in the acid duplex soils of Western Australia (Howieson et al., 1995). The good growth in vitro at low pH of the rhizobia is indicative of an ability to maintain intracellular pH when exposed to acid stress, a characteristic that offers an advantage for bacterial survival in acid soils. In the field, soil acidity limits rhizobial survival and persistence in soils, and subsequent root colonisation, infection and nodule activity (Brockwell et al., 1991). The correct range of soil pH is crucial for the survival of rhizobial spp., and in adverse soil pH environments, strains of rhizobia differ in their ability to infect the host plant (Brockwell et al., 1995). In contrast to these acid soil conditions, in alkaline soils there are usually large resident rhizobial populations capable of infecting pulse crops and the inoculation of pulses is not always considered necessary. This soil survey identified considerable variation in both soil type and the presence of rhizobia in the soil. However, there is still a need to understand the effects and interaction that these factors have for farmers regarding the need for re-inoculation when sowing into a paddock with a previous history of legumes. If there are sufficient effective rhizobia or if the soil is sodic and contains a high EC, is there a need to re-inoculate? The complex interactions between soil condition and rhizobial survival and persistence are not fully understood and will require further detailed research. These issues are especially important in Australian farming systems where intensified cropping is often accompanied by an increased reliance on chemicals for weed, pest and disease control, fertiliser inputs or the control of chemical toxicities. 4.3. Experimental sites In an attempt to answer the question of re-inoculation for subsequent legume crops, the additional treatments of ^ inoculation were imposed on these same farmer paddocks. The experimental sites provided us with field evidence to support the survey data. Soil pH, paddock history, disease control and rainfall all contribute to successful crops and grain yields. The presence of resident rhizobia is not always sufficient in itself, to ensure optimal N2-fixing capacity in the host legume. It is usually accepted that it is difficult to introduce superior strains by inoculation when there is a large resident population and well-adapted rhizobia, and nodules occupied by the inoculant strain may decline in the years following inoculation (Unkovich and Pate, 1998; Slattery and Coventry, 1999).
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Soil surveys in high pH soil environments in some areas of southeast Australia show that the lack of effective rhizobia may limit the performance of annual medics (Ballard and Charman, 1996; Slattery et al., 1999). In these areas, the naturalised medic rhizobial populations are generally high (. 104 rhizobia g21 soil), but the annual medic species frequently do not achieve an effective symbiosis with the naturalised rhizobia (Ballard and Charman, 1996; Slattery et al., 1999). The reasons for this poor effectiveness of naturalised medic populations are uncertain at this stage, but may be related to soil chemical characteristics at high pH. It is clear that to obtain optimum N2-fixation for a range of soil environments the soil chemical status and resident rhizobial populations must first be investigated. Only then can rational conclusions be made regarding the effectiveness of new strains for specific soil conditions. On the acid Kurosol inoculation increased chickpea grain yields. In general, chickpea crops are not usually grown in acid soils, especially where the soil pHCa is 4.6 or lower. However, when the growing seasonal rainfall is below average and crop disease is suitably controlled, chickpea yields of 2.37 t ha21 are an economic option for farmers in northeast Victoria. M. cicer survival in the soil and growth in the rhizosphere is most important. Rai (1991) investigating chickpea symbiosis in India demonstrated that only 5% of the strains examined were found to be suitable for nodulation and growth in such strongly acid soil environments. In contrast to these findings, the presence of nodules on plants does not necessarily mean that sufficient N2 is being fixed for maximum benefit to the host plant. In India, groundnut and pigeonpea are nodulated naturally at most locations due to the cross-species promiscuity of the cowpea rhizobia (Wani et al., 1995), as well as the promiscuity and ease of nodulation of the host plants. However, it may still be necessary to introduce superior strains of rhizobia to ensure adequate N2-fixation for maximum growth and yield of the host plant. When pulse crops are sown, responses can be variable and the decisions driving the need to inoculate must be clearly understood. In soils where effective indigenous strains of pulse rhizobia are insufficient or absent, inoculation is essential to ensure adequate nodulation and N2-fixation especially when crops are sown into acid soils. Significant responses to inoculation have also been reported where indigenous rhizobial populations are small (Thies et al., 1991). Cropping systems in Australia are intensifying with longer periods of the crop phase used in a rotation. Associated with this intensification is the higher input of chemicals, increased reliance on N-fertiliser, less tillage and more flexibility in sowing management. Large-scale increases in pulse legume yields are unlikely unless the plant breeding programs incorporate resistance to fungal diseases, for example, Ascochyta and Fusarium genotypes. Nonetheless, continued research on the effectiveness of
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rhizobia is needed, as often the symbiosis is not optimal. Selection and evaluation of more effective rhizobial inoculants can only be obtained under a wider range of environmental conditions when all of the soil factors that have the potential to restrict rhizobial growth together with nutrient needs are considered in the selection programs for new inoculum strains.
Acknowledgements We acknowledge the financial support of the Grains Research and Development Corporation (GRDC) to undertake this work.
References Ballard, R., Charman, N., 1996. Are the Rhizobium in Australian soils limiting the performance of annual medics? In: Farming Systems Developments. Proceedings of the Workshop on Farming Development Systems of Southern Australia, 26–28 March 1996, pp. 107 –108. Brockwell, J., 1963. Accuracy of a plant-infection technique for counting populations of Rhizobium trifolii. Applied Microbiology 11, 377–383. Brockwell, J., Holliday, R.A., Pilka, A., 1988. Evaluation of the symbiotic nitrogen-fixing potential of soils by direct microscopic means. Plant and Soil 108, 163– 170. Brockwell, J., Pilka, A., Holliday, R.A., 1991. Soil pH is a major determinant of the numbers of naturally occurring Rhizobium meliloti in non-cultivated soils in central New South Wales. Australian Journal of Experimental Agriculture 31, 211 –219. Brockwell, J., Bottomley, P.J., Thies, J.E., 1995. Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant and Soil 174, 143 –180. Corbin, E.J., Brockwell, J., Gault, R.R., 1977. Nodulation studies on chickpea (Cicer arietinum). Australian Journal Experimental Agriculture and Animal Husbandry 17, 126–134. Coventry, D.R., Evans, J., 1989. Symbiotic nitrogen fixation and soil acidity. In: Robson, A.D., (Ed.), Soil Acidity and Plant Growth, Academic Press, Sydney, pp. 103–137. Gault, R.R., Corbin, E.J., Boundy, K.A., Brockwell, J., 1986. Nodulation studies on legumes exotic to Australia: Lupinus and Ornithopus spp. Australian Journal of Experimental Agriculture 26, 37–48. Gaur, Y.D., Sen, A.N., 1979. Cross-inoculation group specificity in Cicer rhizobium symbiosis. New Phytologist 83, 745–754. Gibson, A.H., Curnow, B.C., Bergersen, F.J., Brockwell, J., Robinson, A.C., 1975. Studies of field populations of Rhizobium: effectiveness of strains of Rhizobium trifolii associated with Trifolium subterraneum L. pastures in south-eastern Australia. Soil Biology and Biochemistry 7, 95–102.
Howieson, J.G., Loi, A.C., Carr, S.J., 1995. Biserrula pelecinus L.—a legume pasture species with potential for acid, duplex soils which is nodulated by unique root-nodule bacteria. Australian Journal of Agricultural Research 46, 997 –1009. Isbell, R.F., 1996. The Australian Soil Classification, CSIRO Publishing, Collingwood. Nour, S.M., Fernandez, M.P., Normand, P., Cleyet-Marel, J.-C., 1994. Rhizobium ciceri sp. nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.). International Journal of Bacteriology 44, 511 –522. Rai, R., 1991. Effects of soil acidity factors on interaction of chickpea (Cicer arietinum L.) genotypes and Rhizobium strains: symbiotic Nfixation, grain quality and grain yield in acid soils. In: Wright, R.J., Baliger, V.C., Murrmann, R.P. (Eds.), Plant–Soil Interactions at Low pH, Proceedings of the Second International Symposium on Plant –Soil Interactions at Low pH, Kluwer Academic Publishers, Dordrecht, pp. 597 –601. Siddique, K.H.M., Sykes, J., 1997. Pulse production in Australia past, present and future. Australian Journal of Experimental Agriculture 37, 103 –111. Slattery, J.F., Coventry, D.R., 1989. Populations of Rhizobium lupini in soils used for cereal-lupin rotations in north-east Victoria. Soil Biology and Biochemistry 21, 1009–1010. Slattery, J.F., Coventry, D.R., 1993. Variations of soil populations of Rhizobium leguminosarum bv trifolii and the occurrence of inoculant rhizobia in nodules of subterranean clover after pasture renovation in north-eastern Victoria. Soil Biology and Biochemistry 25, 1725– 1730. Slattery, J.F., Coventry, D.R., 1999. Persistence of introduced strains of Rhizobium leguminosarum bv trifolii in acidic soils of north-eastern Victoria. Australian Journal of Experimental Agriculture 39, 829 –837. Slattery, J.F., Pearce, D.J., 2002. Development of elite inoculant strains of Rhizobium in southeastern. In: Herridge, D., (Ed.), Inoculants and Nitrogen Fixation of Legumes in Vietnam, Proceedings of a Workshop Proceedings, ACIAR Proceedings No. 109e, pp. 86 –94. Slattery, J.F., Slattery, W.J., Carmody, B.C., 1999. Influence of soil chemical characteristics on medic rhizobia in the alkaline soils of south eastern Australia. In: Martinez, E., Hernandez, G. (Eds.), Highlights of Nitrogen Fixation Research, Kluwer Academic/Plenum Publishers, New York, pp. 243– 249. Slattery, J.F., Coventry, D.R., Slattery, W.J., 2001. Rhizobial ecology as affected by the soil environment. Australian Journal of Experimental Agriculture 41, 289 –298. Thies, J.E., Singleton, P.W., Bohlool, B., 1991. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field grown legumes. Applied and Environmental Microbiology 57, 19 –28. Unkovich, M.J., Pate, J.S., 1998. Symbiotic effectiveness and tolerance to early season nitrate in indigenous populations of subterranean clover rhizobia from SW Australian pastures. Soil Biology and Biochemistry 30, 1435–1443. Wani, S.P., Rupela, O.P., Lee, K., 1995. Sustainable agriculture in the semiarid tropics through biological nitrogen fixation in grain legumes. Plant and Soil 174, 29 –49.
Effects of resident rhizobial communities and soil type on the effective nodulation of pulse legumes J.F. Slattery*, D.J. Pearce, W.J. Slattery Department of Primary Industries, Rutherglen Research Institute, Rutherglen, Vic. 3685, Australia
Abstract Communities of resident rhizobia capable of effective nodulation of pulse crops were found to vary considerably over a range of soil environments. These populations from soils at 50 sites in Southern Australia were evaluated for nitrogen fixing effectiveness in association with Pisum sativum, Vicia faba, Lens culinaris, Vicia sativa, Cicer arietinum and Lupinus angustifolius. The values for nitrogen fixing effectiveness could be related to soil pH as determined by soil type and location. It was found that 33% of paddocks had sufficient resident populations of Rhizobium leguminosarum bv viciae for effective nodulation of faba bean, 54% for lentils, 55% for field pea and 66% for the effective nodulation of the vetch host plant. Mesorhizobium cicer populations were very low with only 7% of paddocks surveyed having sufficient resident populations for effective nodulation. Low resident rhizobial populations (,10 rhizobia g21 soil) of R. leguminosarum bv viciae and M. cicer were found in acid soil conditions. In contrast, Bradyrhizobium populations increased as soil pH decreased. Inoculation increased faba bean yields from 0.34 to 4.4 t ha21 and from 0.47 to 2.37 t ha21 for chickpeas on acid soils. On alkaline soils, where resident populations were large there was no consistent response to inoculation. Observations at experimental field sites confirmed the findings from the survey data, stressing the importance of rhizobial inoculation, especially on the acid soils in south-eastern Australia. Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Rhizobial populations; pH; Rhizobial inoculation; Crop yield; Nodulation effectiveness
1. Introduction Pasture and crop legumes have been used extensively in Australian agriculture during the past century mainly to boost soil fertility and, as a consequence, crop and animal productivity. Many soils are often unable to sustain productive farming systems or limit crop production due to factors associated with low fertility, sodicity, salinity and extremes of acidity and alkalinity (Slattery et al., 2001). These same attributes can also have a negative effect on the legume-Rhizobium symbiotic relationship and thus reduce the ability of rhizobia to form nodules with optimal N2-fixing capacity (Brockwell et al., 1995). In temperate Australian soils, numbers of resident rhizobia can vary considerably in size, ranging from , 10 to . 106 rhizobia g21 soil (Gibson et al., 1975; Slattery et al., 1999). The size of the soil rhizobial community is dependent on many factors including field history, soil and environmental characteristics and the presence of host plants. Successful establishment of inoculant strains into the soil is also dependent on the numbers of resident populations, * Corresponding author. Fax: þ 61-2-60-304-600. E-mail address: [email protected] (J.F. Slattery).
host plant activity, as well as other soil characteristics (Brockwell et al., 1995). Where the resident populations of rhizobia are small (, 50 Rhizobium g21 soil) and specific to a target legume, the introduction of new strains by seed inoculation is usually successful. On the other hand, many authors have reported low recovery of the inoculant strain where resident rhizobial populations are large (. 103 rhizobia g21 soil) (Thies et al., 1991; Brockwell et al., 1995). The adoption of pulse (grain) legumes in Australia, and their production has increased rapidly as farmers appreciate the rotational and financial benefits that pulses provide to their production systems. In , 40 years, the area in Australia under pulse production has increased from zero to over 2 £ 106 ha in 1996 (Siddique and Sykes, 1997) with pulses now occupying more than 10% of the area cropped. The Australian continent contains a spectrum of geographic cropping environments with pulses produced over a wide range of climatic conditions and soil environments. Soils in the pulse growing regions of south-eastern Australia vary from strongly alkaline (NW Victoria and much of South Australia), neutral to slightly acid (Wimmera, central and southern Victoria) to highly acid (NE Victoria and southern NSW). This wide variation in soil pH together with salinity,
0038-0717/$ - see front matter Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.04.015
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sodicity and fertility has serious implications for rhizobial survival and effectiveness as well as the need for re-inoculation of following pulse crops. The capacity of inoculant strains to colonise soils in sufficient quantities to provide effective nodulation is highly dependent on the soil pH (Brockwell et al., 1995). Thus, a thorough understanding of the size and effectiveness of resident populations (defined as the naturalised or indigenous populations of rhizobia in the soil) will enable recommendations to be made regarding the need for re-inoculation of pulse legumes. We report on the effect of soil types on the persistence of resident rhizobial populations to form nodules and whether it is necessary for the re-inoculation of rhizobia when introducing pulse legumes into different soil environments. A comprehensive soil survey was conducted across the pulse legume growing regions of Victoria to provide a critical analysis of the soil chemical and resident rhizobial populations under different soil environments. This critical data provides a starting point for an assessment of inoculation and re-inoculation requirements of pulse crops.
2. Materials and methods There are two main experimental approaches described in this paper, one involved the survey of soil from 50 paddocks where grain legumes had previously been grown over the past 5 years (paddock history data not presented) and the other involved an investigation of a grain legume (pulse) yield response to inoculation at 6 field sites. 2.1. Field survey and soil sampling Soil rhizobial populations, soil chemical characteristics and classification were measured under a range of pulse crop and pasture legumes. Fifty paddocks were sampled during August 1997, over a wide geographical range for the pulse legume growing regions of Victoria. The number of paddocks sampled and regional location of each were as follows: Birchip, southern Mallee (11 sites); Walpeup, northern Mallee (10 sites); Horsham, Wimmera (7 sites); Elmore, central Victoria (7 sites); Charlton, north central (5 sites); Dookie, north east Victoria (7 sites); Rutherglen, north east Victoria (3 sites). Ten soil cores (10 cm depth £ 2.5 cm dia) were collected randomly using an aseptic technique (Slattery and Coventry, 1993) from each paddock, bulked, thoroughly mixed, sieved (, 2 mm) to remove stones and large pieces of undecomposed organic matter, air dried, then stored at room temperature before chemical and rhizobial analysis. Extreme care was taken to exclude the sampling of rhizosphere populations, by sampling away from any plants. An intact core to a depth of 110 cm was removed for soil characterisation using Isbell (1996) classification system.
2.2. Experimental sites In order to make recommendations for farmers on the need for the re-inoculation of subsequent legume crops additional treatments (^ inoculation) were imposed across farmer paddocks as demonstration strips in the year following a cereal crop or pasture. Inoculation response trials for six legumes (field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris), vetch (Vicia sativa), chickpea (Cicer arietinum) and narrow-leafed lupin (Lupinus angustifolius)) were each established at 6 sites in 2000. Locations, soil classification, soil pH and resident rhizobial population status for experimental field sites are described in Table 1. The rhizobium status was based upon a mean probable number determination (MPN) (Brockwell, 1963). At each site, and for each legume, the commercial inoculant was applied to the seed and sown as the inoculant treatment in the 36 m2 plots. For comparison untreated seed was sown as the nil treatment. When needed, annual grasses and broadleaf weeds were controlled with the appropriate chemicals according to registered recommendations. Ten individual plants were removed from each demonstration plot 10 –12 weeks after sowing for nodulation and plant dry matter (DM) measurements. Plant roots were scored for nodulation on a 0 – 5 scale, based on the nodule number, size, position, distribution and pigmentation of effective nodules on the crown and lateral roots (adaptation of Corbin et al. (1977)). Plant shoot material was dried in a fan-forced draught oven at 70 8C for 48 h, prior to DM measurement. At maturity grain seed yield was determined by the mechanical harvesting of the entire plot using a plot harvester and weigh bins. The MPN was used to estimate soil rhizobial populations collected from the soil survey and from the inoculation response trials (Brockwell, 1963). 2.3. Evaluation of nitrogen-fixing effectiveness—field survey Air-dried soil samples collected from each site were used to enumerate populations of rhizobia and to estimate Table 1 Location, soil pH and resident rhizobial populations in the surface 10 cm of soil of inoculation response trials conducted in the winter of 2000 Site location
Soil typea
Soil pHCa
Resident rhizobial populationsb
Birchip-F, NW Vic. Birchip-S, NW Vic. Walpeup-P, NW Vic. Walpeup-N, NW Vic. Walpeup-C, NW Vic. Rutherglen-B, NE Vic.
Calcarosol Calcarosol Calcarosol Calcarosol Calcarosol Kurosol
7.60 7.78 7.16 7.29 8.80 4.60
High High High High High Lowc
a
According to Isbell (1996). MPN (Brockwell, 1963) using the host plants listed in Section 2.2. c An average of resident rhizobial populations on six host plants were: Low (,10 rhizobium g21 soil), High (.103 rhizobium g21 soil). b
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symbiotic effectiveness. The method used to evaluate the effectiveness of N2-fixation of rhizobial populations in soil samples was a whole soil inoculation technique (WSIT) (Brockwell et al., 1988). The ability of the soils to support rhizobial populations that form nodules was tested using the plants listed in Section 2.2. Seeds were surface sterilised, germinated on 2% water agar plates at 22 8C, prior to planting. Single test plants were grown in seedling tubes (10 cm depth £ 3 cm2) in a semi-sterilised system (washed sand:vermiculite mixture, moistened with N-free nutrient salt solution (Slattery and Pearce, 2002)). In this system, the shoots are exposed to the atmosphere whilst the roots are grown under aseptic microbiological conditions. Each seedling was inoculated 5 days later with a 1 ml soil suspension (10 g soil in 90 ml N-free nutrient salt solution) of each isolate and four replicates. The plants were grown for a further 4 weeks in a glasshouse (range 12– 25 8C), with the moisture content of each plant maintained with either sterile distilled water or N-free nutrient solution. The same plants that were grown to determine nitrogen-fixing effectiveness were also used to assess nodulation. All plants were harvested, the roots examined for nodulation, and DM from the plant shoot material was measured after oven drying at 70 8C for 24 h. For each legume, there were three control treatments including an uninoculated control; uninoculated control with 1 ml of 0.05% KNO3 applied at weekly intervals after sowing and an inoculated control using the appropriate commercial strain (e.g. SU303, WSM1274, CC1192 or WU425).
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1999). Soils were identified according to the Australian soil classification system (Isbell, 1996). 2.5. Statistical analysis Soil analysis and rhizobial data were analysed for difference by analysis of variance, with the treatment means compared at the 95% probability ðP ¼ 0:05Þ (GENSTAT V).
3. Results 3.1. Field survey 3.1.1. Evaluation of nitrogen fixing effectiveness Soils vary in their ability to nodulate pulse crops as a result of soil pH and paddock history. Results from our survey showed a variation in the presence (or survival) of soil rhizobia at individual sites within a location or between rhizobial species and plants (Table 2). On some soil types, the soil rhizobia present were capable of infecting the host legume and forming nodules, but at other sites soil rhizobia were absent and plants in these soils failed to form nodules. In the experiments to numerate rhizobial populations, all nodulated test plants grew vigorously fixing N, indicator the effectiveness of the populations (rhizobial counts not presented). Averaging across the 50 paddocks surveyed; 33% of the paddocks had sufficient resident Rhizobium leguminosarum bv viciae to nodulate faba bean, 54% for lentils, 55% for field pea, and 66% for vetch (Table 2). At 17 sites (34%) no R. leguminosarum bv viciae were detected using WSIT (Brockwell et al., 1988). The presence of chickpea rhizobia, Mesorhizobium cicer, was very low with only 7% of paddocks surveyed having resident populations to chickpea by the WSIT. In the case of Bradyrhizobium spp., 38% of paddocks had sufficiently large resident populations (. 102 rhizobia g21 soil) to nodulate lupin.
2.4. Soil chemical analysis—field survey and experimental sites Soil pH Ca (10 mM CaCl 2) and soil pH W 1:5 soil:extractant was measured using an automated system, whilst Olsen P (0.5 M NaHCO3) 1:100 soil:extractant, extractable K (0.5 m NaHCO3) 1:100 soil:extractant, total N (Kjeldahl), mineral N (2 M KCl—Kjeldahl) and organic C were determined by standard procedures (Slattery et al., Table 2 Percentage of soil extracts ðn ¼ 50Þ forming nodules on six legume species Location
Mean of 50 paddocks Birchip Walpeup Horsham Charlton Elmore Dookie Rutherglen LSD ðP , 0:05Þ
Host plant Soil classification
Vetch
Field pea
Lentil
Faba bean
Lupin
Chickpea
Calcarosol Calcarosol Sodosol Dermosol Dermosol Ferrosol Kurosol
66 100 78 100 80 14 14 0 23
55 100 71 86 67 0 14 0 22
54 100 56 100 40 0 14 0 23
33 67 0 71 100 0 0 0 23
38 33 14 13 33 57 71 33 11
7 10 0 67 0 0 0 0 13
Soils were collected from seven locations in the pulse growing regions of Victoria.
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Table 3 Summary of soil chemical characteristics (range) in the surface 10 cm of soil collected from 50 paddocks at seven locations in the pulse growing regions of Victoria Location
Birchip Walpeup Horsham Charlton Elmore Dookie Rutherglen
Soil characteristics Number of locations
pHwater
pHCa
Org C (%)
Total N (%)
Olsen P (mg kg21)
Ex K (mg kg21)
11 10 7 5 7 7 3
6.8 –8.8 6.9 –8.8 7.6 –8.0 6.1 –7.5 5.4 –6.1 5.5 –5.3 NT
6.1 –8.0 5.9 –8.1 7.4 –7.7 5.8 –6.8 4.4 –4.7 4.4 –5.3 4.2 –4.9
0.4 –1.6 0.4 –1.0 1.5 1.3 –1.7 1.6 –2.8 1.3 –2.3 NT
0.04–0.09 0.05–0.05 0.19 0.06–0.13 0.08–0.38 0.08–0.19 NT
5–47 10–34 5 11–26 7–33 8–32 NT
168–618 162–600 789 248–640 192–386 274–510 NT
NT, soils not tested.
The major pulse growing regions in Victoria that require nodulation by R. leguminosarum bv viciae are in the central west and north west (Birchip and Horsham) of the State. Resident R. leguminosarum bv viciae populations in these regions have been considered present in sufficient numbers and in the past, so rhizobial inoculation has not been recommended. However, the survey results showed 23% of faba bean crops had inadequate R. leguminosarum bv viciae populations to nodulate faba bean, which would lead to a reduction in crop yield. R. leguminosarum bv viciae populations capable of infecting vetch, field pea, lentils and faba beans at the Elmore, Dookie and Rutherglen locations were extremely small (Table 2). The populations of chickpea rhizobia (M. cicer) were even lower in the Birchip regions, only 10% of paddocks compared to 67% of paddocks in the Horsham region (Table 2). Bradyrhizobium sp. populations, capable of infecting lupins were present in all soil types but varied in numbers with respect to the proportion of paddocks in which they were present (Table 2) (individual data not presented). 3.1.2. Soil chemical variables The range of values for the soil chemical measurements collected from 50 paddocks across seven locations in the pulse growing regions of Victoria showed some similarities and differences that could be related to soil type and region (Table 3). Surface soil pHCa at the Charlton site was in the pHCa range from 5.8 to 6.8 and higher than the surface soil pH at the Elmore site of 4.4– 4.7. The soils in the Charlton and Elmore region were classified as Dermosols, these soils tend to form surface crusting if not managed appropriately, the effects of which may influence rhizobial survival (Isbell, 1996). Surface soil pHCa in the Rutherglen region were the lowest with pHCa ranging from 4.2 to 4.9 and the soils were classified as Kurosol (Isbell, 1996). In the Birchip region surface soil pHCa for the 8 paddocks ranged from 6.1 to 8.0, with 63% of the sites being highly alkaline (pHCa . 7.5). Soil organic C ranged from 0.4 to 1.6%; the mean value of 1.1% considered low but typical for farming systems with a long history of continuous cropping, intense cereal rotations, together with low growing season
rainfall (GSR) and low plant productivity as experienced in the Walpeup region. The mean total N values were above those considered limiting for pulse crops, with many of the paddocks also having large resident rhizobial populations for effective nodulation of pulse crops, it is therefore likely that available N was not limiting crop yield. There was a 10fold difference between the maximum and minimum values of extractable P, with 75% of sites having a low Olsen P value of , 17 mg kg21. With the exception of one property, soils in the Birchip region were classified as Calcarosols derived from limestone and containing dissolved and particulate calcium carbonate present throughout the profile (Isbell, 1996). The survey data showed a significant relationship between the effective nodulation of soil rhizobia for a particular plant host and soil pH, and to a lesser extent the relationship between effective nodulation of soil rhizobia with the other soil measurements (Fig. 1). From Fig. 1, it can be seen that there is an interaction between soil rhizobial populations capable of effective nodulation of pulse crops and pH (Fig. 1a), where soil pHCa and R. leguminosarum bv viciae populations at the Elmore, Dookie and Rutherglen sites were low. In contrast, background populations capable of effective nodulation of Bradyrhizobium spp. were higher on the acidic soils and with the exception of the sites in the Horsham region, the resident populations of M. cicer were low (Fig. 1b). 3.2. Experimental sites The site data demonstrated that in the major pulse growing regions of Victoria, resident populations of rhizobia capable of effective nodulation of pulse crops can be high (Table 1) and any response to inoculation might not be evident. For this reason, field data has been presented for the Kurosol and only one of the Calcarosol sites (Tables 4 and 5). On the acid Kurosol at Rutherglen, the nodulation derived from the resident rhizobial population varied between plant species (Table 4). For example, inoculation increased faba bean yields from 0.34 t ha21 for the nil treatment to 4.4 t ha21 and root DM from 1.39 to
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Fig. 1. (a) Relationship between soil pHCa at seven regional locations in Victoria and nodulation for Pisum sativum, Vicia faba, Lens culinaris and Vicia sativa. Vertical bars represent the least significant differences at P ¼ 0:05: (b) Relationship between soil pHCa at seven regional locations in Victoria and nodulation for Cicer arietinum and Lupinus angustifolius. Vertical bars represent the least significant differences at P ¼ 0:05:
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4.21 g pl21 with inoculation. Nodulation (nodule number and nodule score) and herbage DM increased at P ¼ 0:1: A visual response to inoculation was observed for the chickpea crop with an increase in grain yield from 0.47 to 2.37 t ha21 and increases for nodulation and root DM. Adequate nodulation occurred in the nil treatment for field pea, lentil and vetch. Although visible growth differences were observed late in the growing season for lentils, these differences could not be related to increased grain yields as the farmers’ sheep unfortunately grazed the lentil crop prior to harvest. On the alkaline Calcarosols responses to inoculation were not visually evident, although at the Birchip-F site (Table 5) where resident populations under lupins were low, inoculation increased nodulation. However, as a consequence of the high resident populations of R. leguminosarum bv viciae, inoculation did not improve the nodulation of faba bean, field pea, lentil or vetch. Inoculation with SU303 for these cultivars improved the yields in each case compared to the nil treatment. In the case of lentils, grains yields for WSM1455 and WSM1483 were higher (statistically not significant) compared to WSM1274, the commercial inoculant at the time. For chickpeas, resident populations were low and inoculation significantly increased nodulation (and improved spring DM production). Overall crop grain yields were lower at the Birchip site where the annual GSR of 238 mm was 43 mm less than the 50-years average compared a GSR of 423 mm at Rutherglen.
Table 4 Nodule number, nodule score, shoot dry weight (g pl21), root dry weight (g pl21) and grain yield (t ha21) for the farmer inoculation response trial at the Rutherglen site (2000) Rutherglen: legume
Rhizobial strain
Nodule number
Nodule score
Root DM (g pl21)
Top DM (g pl21)
Crop yield (t ha21)
Lupin
Nil WU425
16 20 ns
2.6 3.2 ns
0.63 0.50 ns
1.80 1.91 ns
0.71 0.98 ns
Nil WSM1274
10 35 ns
2.6 4.2 ns
1.39 4.21 1.95
4.33 18.50 ns
0.34 4.41 1.6
Nil SU303
43 54 ns
4.1 4.3 ns
0.55 0.46 ns
6.28 7.43 ns
2.15 2.87 ns
Nil WSM1274
10 22 ns
2.2 3.2 ns
0.11 0.11 ns
0.89 0.86 ns
0.15a 0.12a ns
Nil SU303
34 64 ns
3.5 4.8 1.4
0.66 0.51 ns
5.44 7.09 ns
7.87b 8.07b ns
Nil CC1192
2 24 7.5
0.6 4.0 2.5
0.88 2.20 1.0
3.13 3.78 ns
0.47 2.37 2.0
LSD ðP , 0:05Þ Faba bean LSD ðP , 0:05Þ Field pea LSD ðP , 0:05Þ Lentil LSD ðP , 0:05Þ Vetch LSD ðP , 0:05Þ Chickpea LSD ðP , 0:05Þ a b
Crop grazed by sheep, reduced grain yield. Vetch dry matter t ha21, vetch cut prior to grain harvest.
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Table 5 Nodule number, nodule score, shoot dry weight (g pl21), root dry weight (g pl21) and grain yield (t ha21) for the farmer inoculation response trial at the Birchip-F site (2000) Birchip-F: legume
Rhizobia strain
Nodule number
Nodule score
Root DM (g pl21)
Top DM (g pl21)
Crop yield (t ha21)
Lupin
Nil WU425
0.1 6 ns
0.1 2.4 1.07
0.33 0.40 ns
1.80 1.62 ns
0.74 0.81 ns
Nil WSM1274 SU303 RRI294 RRI339
24 38 26 20 20 ns
4.3 4.3 4.0 3.7 4.0 ns
0.99 0.78 0.92 0.65 0.89 ns
6.01 5.15 5.61 4.09 5.48 ns
0.26 0.59 0.73 0.27 0.46 ns
Nil SU303 RRI294 RRI339
31 24 22 22 ns
4.0 4.2 4.2 4.0 ns
0.40 0.28 0.28 0.23 ns
6.57 6.19 6.57 5.92 ns
0.78 0.92 0.81 0.81 ns
Nil WSM1274 WSM1455 WSM1483
24 19 25 21 ns
4.2 3.6 4.0 3.5 ns
0.09 0.10 0.09 0.09 ns
1.03 1.09 1.03 1.00 ns
0.83 1.01 1.17 1.35 ns
Nil SU303
21 14 ns
4.2 3.4 ns
0.17 0.12 ns
2.40 2.14 ns
1.76 1.81 ns
Nil CC1192
0.2 4 ns
0.2 2.0 0.9
0.23 0.26 ns
1.30 1.61 ns
NH NH
LSD ðP , 0:05Þ Faba bean
LSD ðP , 0:05Þ Field pea
LSD ðP , 0:05Þ Lentil
LSD ðP , 0:05Þ Vetch LSD ðP , 0:05Þ Chickpea LSD ðP , 0:05Þ
NH, not harvested, cut and removed due to Ascochyta damage.
4. Discussion 4.1. Soil rhizobial populations and effectiveness It is clear from this survey that soils vary in their ability to nodulate pulse crops as a result of soil pH and previous frequency of pulse crop. R. leguminosarum bv viciae populations were resident in most (83%) of the 50 soils collected. It can be assumed that for the remaining soils, rhizobial numbers were insufficient to allow effective nodulation of the host plant. Whilst there was a large variation in the presence of soil rhizobia, shown by the percentage of paddocks exhibiting effective nodulation of the host plant, the highest frequency of resident populations were located in the more alkaline soils of the major pulse growing regions of Victoria. With the exception of the central Mallee or Horsham regions, the extremely low M. cicer resident population highlights the fact that chickpea has not yet been extensively grown in these paddocks. Consequently, there is a need to inoculate when chickpea crops are first introduced to paddocks in this region. The chickpea-M. cicer symbiosis is highly specific and extensive studies have demonstrated the uniqueness of the chickpea rhizobia with 99% of the chickpea isolates nodulating only the original host plant
and not species belonging to the Fabaceae and Mimosaceae families (Gaur and Sen, 1979). This chickpea host specificity has been confirmed by Nour et al. (1994) using molecular techniques. The successful nodulation of lupin requires the presence of a sizeable resident Bradyrhizobium population capable of infecting lupins. Bradyrhizobium strains capable of infecting and nodulating lupins may be indigenous to many parts of Western Australia, but do not occur naturally in Eastern Australia (Gault et al., 1986; Slattery and Coventry, 1989). Except for the Dookie location where 71% of sites had sufficient populations, the survey data suggests that inoculation of lupin seed is essential when growing lupins for the first time in these regions. 4.2. Soil chemical variables When survey data were compared from each location the presence of resident root-nodule bacteria could be related to soil pH, with acid soils being typically devoid of resident populations. Poor rhizobial persistence of R. leguminosarum bv viciae and M. cicer indicates a need for inoculation when sowing into acid soil environments. Soil acidity can effect survival, growth, persistence and nitrogen fixation potential of rhizobia (Rai, 1991), or nodule
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initiation and N2-fixation effectiveness (Coventry and Evans, 1989) whilst nutritional disorders affect the legume-Rhizobium symbiosis. The survey has shown that not only is there a need to inoculate pulse crops in acid soils, but research continues for more adapted rhizobia and the matching of the rhizobia with the plant host. The selection of root nodule bacteria for Biserrula (Biserrula pelecinus) is a good example of the need to match a rhizobial species with a pasture species in the acid duplex soils of Western Australia (Howieson et al., 1995). The good growth in vitro at low pH of the rhizobia is indicative of an ability to maintain intracellular pH when exposed to acid stress, a characteristic that offers an advantage for bacterial survival in acid soils. In the field, soil acidity limits rhizobial survival and persistence in soils, and subsequent root colonisation, infection and nodule activity (Brockwell et al., 1991). The correct range of soil pH is crucial for the survival of rhizobial spp., and in adverse soil pH environments, strains of rhizobia differ in their ability to infect the host plant (Brockwell et al., 1995). In contrast to these acid soil conditions, in alkaline soils there are usually large resident rhizobial populations capable of infecting pulse crops and the inoculation of pulses is not always considered necessary. This soil survey identified considerable variation in both soil type and the presence of rhizobia in the soil. However, there is still a need to understand the effects and interaction that these factors have for farmers regarding the need for re-inoculation when sowing into a paddock with a previous history of legumes. If there are sufficient effective rhizobia or if the soil is sodic and contains a high EC, is there a need to re-inoculate? The complex interactions between soil condition and rhizobial survival and persistence are not fully understood and will require further detailed research. These issues are especially important in Australian farming systems where intensified cropping is often accompanied by an increased reliance on chemicals for weed, pest and disease control, fertiliser inputs or the control of chemical toxicities. 4.3. Experimental sites In an attempt to answer the question of re-inoculation for subsequent legume crops, the additional treatments of ^ inoculation were imposed on these same farmer paddocks. The experimental sites provided us with field evidence to support the survey data. Soil pH, paddock history, disease control and rainfall all contribute to successful crops and grain yields. The presence of resident rhizobia is not always sufficient in itself, to ensure optimal N2-fixing capacity in the host legume. It is usually accepted that it is difficult to introduce superior strains by inoculation when there is a large resident population and well-adapted rhizobia, and nodules occupied by the inoculant strain may decline in the years following inoculation (Unkovich and Pate, 1998; Slattery and Coventry, 1999).
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Soil surveys in high pH soil environments in some areas of southeast Australia show that the lack of effective rhizobia may limit the performance of annual medics (Ballard and Charman, 1996; Slattery et al., 1999). In these areas, the naturalised medic rhizobial populations are generally high (. 104 rhizobia g21 soil), but the annual medic species frequently do not achieve an effective symbiosis with the naturalised rhizobia (Ballard and Charman, 1996; Slattery et al., 1999). The reasons for this poor effectiveness of naturalised medic populations are uncertain at this stage, but may be related to soil chemical characteristics at high pH. It is clear that to obtain optimum N2-fixation for a range of soil environments the soil chemical status and resident rhizobial populations must first be investigated. Only then can rational conclusions be made regarding the effectiveness of new strains for specific soil conditions. On the acid Kurosol inoculation increased chickpea grain yields. In general, chickpea crops are not usually grown in acid soils, especially where the soil pHCa is 4.6 or lower. However, when the growing seasonal rainfall is below average and crop disease is suitably controlled, chickpea yields of 2.37 t ha21 are an economic option for farmers in northeast Victoria. M. cicer survival in the soil and growth in the rhizosphere is most important. Rai (1991) investigating chickpea symbiosis in India demonstrated that only 5% of the strains examined were found to be suitable for nodulation and growth in such strongly acid soil environments. In contrast to these findings, the presence of nodules on plants does not necessarily mean that sufficient N2 is being fixed for maximum benefit to the host plant. In India, groundnut and pigeonpea are nodulated naturally at most locations due to the cross-species promiscuity of the cowpea rhizobia (Wani et al., 1995), as well as the promiscuity and ease of nodulation of the host plants. However, it may still be necessary to introduce superior strains of rhizobia to ensure adequate N2-fixation for maximum growth and yield of the host plant. When pulse crops are sown, responses can be variable and the decisions driving the need to inoculate must be clearly understood. In soils where effective indigenous strains of pulse rhizobia are insufficient or absent, inoculation is essential to ensure adequate nodulation and N2-fixation especially when crops are sown into acid soils. Significant responses to inoculation have also been reported where indigenous rhizobial populations are small (Thies et al., 1991). Cropping systems in Australia are intensifying with longer periods of the crop phase used in a rotation. Associated with this intensification is the higher input of chemicals, increased reliance on N-fertiliser, less tillage and more flexibility in sowing management. Large-scale increases in pulse legume yields are unlikely unless the plant breeding programs incorporate resistance to fungal diseases, for example, Ascochyta and Fusarium genotypes. Nonetheless, continued research on the effectiveness of
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rhizobia is needed, as often the symbiosis is not optimal. Selection and evaluation of more effective rhizobial inoculants can only be obtained under a wider range of environmental conditions when all of the soil factors that have the potential to restrict rhizobial growth together with nutrient needs are considered in the selection programs for new inoculum strains.
Acknowledgements We acknowledge the financial support of the Grains Research and Development Corporation (GRDC) to undertake this work.
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