Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

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Abstract

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×103 rhizobia g−1 of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g−1 soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N2 fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5–9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N2 fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N2 fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.

Introduction

Field pea (Pisum sativum L.) is the major pulse crop grown in South Australia (SA). There are 18 cultivars available, which are segregated into Dun, White and Blue seeded types. In 2000, 114,000 ha of field pea were grown in SA and produced 190,000 t of grain (O'Connell, 2001), an average yield of 1.7 t ha−1. Field pea is also valued because subsequent wheat crops are higher yielding and have higher N content than wheat on wheat rotations (Evans et al., 1991, Rowland et al., 1994). These benefits are partly attributable to changes in soil N balance after field pea, which has been estimated to add on average 44 kg N ha−1 (Evans et al., 2001b), with variation in N contributions strongly linked to biomass production of the legume crop (McCallum et al., 2000, Peoples et al., 2001).

Field pea is nodulated by strains of Rhizobium leguminosarum bv. viciae. The same rhizobia also form nodules with other legumes grown in SA, including faba bean (Vicia faba L.), vetch (Vicia sativa L.), lentil (Lens culinaris Medik.) and the infrequently grown grass pea (Lathyrus sativus L.). Numerous strains (SU302, SU331, SU364, SU390, TA101 and SU391) of R. l. bv. viciae have been used in commercial inoculants for field pea since the 1950s (Date, 1969). Strain SU303 (syn. NA533) replaced strain SU391 in 1990, when the poor symbiotic effectiveness of strain SU391 with faba bean was recognised (Silsbury, 1991). Subsequently, strain WSM1274 was introduced as a separate and more effective inoculant strain for faba bean from 1997 to 2000. While there is little doubt that commercial inoculants were used extensively and played a critical role in the early development of the pea industry, only about 30% of the SA pea crop is now inoculated with rhizobia (Gary Bullard, Bio-Care Technology). This is in part due to farmers' recognition that many of SA's alkaline soils now contain naturalised populations of pea rhizobia, and that the seed dressings which are required to control fungal disease can be detrimental to rhizobial survival (Stovold and Evans, 1980, Evans et al., 1989).

Yield trends point to a significant decline in grain yield of field pea (Peck and McDonald, 1998), especially in SA. While investigations have implicated disease build-up (Davidson and Ramsey, 2000) and sensitivity to herbicide residues (Gonzalez et al., 1996), there has been no consideration of the occurrence and efficacy of the soil rhizobia, which are relied onto nodulate many pea crops.

We describe the size and effectiveness of R. l. bv. viciae populations which have become naturalised in SA soils. We also examined the diversity of naturalised populations of rhizobia isolated from five soils, and determined their genetic relatedness to both former and current inoculant strains and to each other.

Section snippets

Collection and characterisation of soils

Thirty-three soils (32 from SA and one from Victoria) were used in the experiment (Fig. 1). Soils 1–26 were sub-sampled from bulk soil samples (about 2 kg) collected from April to May in 2000, from paddocks with a history of pea cropping during a study on soil borne-diseases of field pea. The remaining soils were collected directly from the paddock for use in rhizobial studies. Soil 30 was collected in 2001; soils 29, 31 and 32 were collected in 2000; soil 33 was collected in 1996; and soils 27

Most probable number of Rhizobium leguminosarum bv. viciae

The MPN of R. l. bv. viciae ranged from not detectable (six soils) to 32×103 g−1 soil (Table 1) with 24 of the 33 soils containing more than 1×102 rhizobia g−1 dry soil.

For soils which had grown a host legume and for which a reliable paddock history was available, there was no significant association (P=0.45, d.f.=25) between the time since the legume host was grown (either field pea, faba bean, vetch or lentil) and the MPN of pea rhizobia g−1 soil. For example, no pea rhizobia were detected in

Size of the rhizobial populations

In SA soils with a cropping history of field pea, faba bean, vetch or lentil, significant (detectable by MPN) residual populations of rhizobia were nearly always present. In some cases, substantial populations of pea rhizobia (>1×103 g−1 dry soil for soils 6 and 25) were measured five or more years after cropping with a host legume. Clearly, factors other than presence of the legume host influenced the size of these rhizobial populations. For example, a number of sites cropped to a legume host

Conclusions

Based on the generally good effectiveness of the pea rhizobia populations with the cultivar Parafield, we conclude that the N2 fixation capacity of the naturalised soil rhizobia is unlikely to be a major factor in declining field pea yields. However, not all soils contained populations of sufficient size needed for prompt nodulation. Hence, inoculation should still be recommended at least for pea crops sown on western Eyre Peninsula or where there is no history of a host legume having been

Acknowledgements

We would like to thank Mr Neil Schubert, Mrs Christine Schutz and Mrs Marzena Kaczmarek for excellent technical assistance. Financial assistance for this work was provided by the Grains Research and Development Corporation and the South Australian Grains Industry Trust. We also thank the staff of the Australian Legume Inoculants Research Unit for supplying cultures of strains that have been used in commercial pea inoculants.

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