High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils

RESEARCH ARTICLE High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils Steven Wakelin1¤a*, Guyléne Tillard2, Robert van Ham3, Ross Ballard4, Elizabeth Farquharson4, Emily Gerard1, Rene Geurts3, Matthew Brown1, Hayley Ridgway5¤b, Maureen O’Callaghan1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 AgResearch Ltd, Lincoln Science Centre, Christchurch, New Zealand, 2 Institut National Superieur des Sciences Agronomiques, Dijon, France, 3 Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands, 4 South Australian Research and Development Institute, Urrbrae, South Australia, Australia, 5 Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand ¤a Current address: Scion Research Ltd, Christchurch, New Zealand ¤b Current address: Plant & Food Research, Christchurch, New Zealand * OPEN ACCESS Citation: Wakelin S, Tillard G, van Ham R, Ballard R, Farquharson E, Gerard E, et al. (2018) High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils. PLoS ONE 13(2): e0192607. https://doi.org/ 10.1371/journal.pone.0192607 Editor: Tzen-Yuh Chiang, National Cheng Kung University, TAIWAN Received: November 29, 2017 Accepted: January 28, 2018 Published: February 28, 2018 Copyright: © 2018 Wakelin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The NZ wide soil data set is pre-published. This work builds on the soil properties, and then adds new information regarding the Rhizobium population size and its symbiotic performance. The entire data set, including all new soil data, and rhizobia population size and performance (SP), are available in supplementary S1–S5 Tables. Abstract Biological nitrogen fixation through the legume-rhizobia symbiosis is important for sustainable pastoral production. In New Zealand, the most widespread and valuable symbiosis occurs between white clover (Trifolium repens L.) and Rhizobium leguminosarum bv. trifolii (Rlt). As variation in the population size (determined by most probable number assays; MPN) and effectiveness of N-fixation (symbiotic potential; SP) of Rlt in soils may affect white clover performance, the extent in variation in these properties was examined at three different spatial scales: (1) From 26 sites across New Zealand, (2) at farm-wide scale, and (3) within single fields. Overall, Rlt populations ranged from 95 to >1 x 108 per g soil, with variation similar at the three spatial scales assessed. For almost all samples, there was no relationship between rhizobia population size and ability of the population to fix N during legume symbiosis (SP). When compared with the commercial inoculant strain, the SP of soils ranged between 14 to 143% efficacy. The N-fixing ability of rhizobia populations varied more between samples collected from within a single hill country field (0.8 ha) than between 26 samples collected from diverse locations across New Zealand. Correlations between SP and calcium and aluminium content were found in all sites, except within a dairy farm field. Given the general lack of association between SP and MPN, and high spatial variability of SP at single field scale, provision of advice for treating legume seed with rhizobia based on field-average MPN counts needs to be carefully considered. Funding: Work at Whangara was supported by the P21 programme ‘Legumes in challenging PLOS ONE | https://doi.org/10.1371/journal.pone.0192607 February 28, 2018 1 / 16 Spatial variation in Rhizobium leguminosarum population size and symbiotic performance environments’ (SW), and access to the farm was provided by the Whangara B5 group. The wider aspects of this programme are funded by the New Zealand Ministry of Business Innovation and Employment (MBIE) (SW) and DairyNZ within the “Improving forage legume-rhizobia performance” programme (C10X1308) (SW). None of the authors work for commercial enterprises; we are all either university or government funded staff or students. Competing interests: We declare no competing interests. The authors hold no commercial appointments nor received commercial funding for this work. The work, and authors, are solely funded by government, university, and sector-based research grants. Introduction Legumes, in symbiosis with rhizobia bacteria, can fix atmospheric nitrogen into plant available forms [1]. Over time, biological nitrogen fixation (BNF) increases the fertility and productive capacity within natural and managed ecosystems [2]. In New Zealand, where pastoral agriculture is extensively used to support livestock production, BNF supplied from the legume-rhizobia symbiosis drives growth of non-leguminous forages species such as grasses and herbs [3]. Furthermore, BNF inputs of N into farming systems from can have a lower environmental cost compared with chemical N fertilisers [3]. Thus, effective legume-rhizobia symbiosis can support the productivity and profitably of pastoral farming, whilst reducing the environmental footprint [4]. Many factors can affect legume establishment and growth in agricultural soils. Constraints can include hostile soil conditions (e.g. pH, high Al3+ content, P-infertility), adverse climatic conditions (soil moisture deficit, extremes of temperature), through to pests, diseases, and weeds (see review by Tozer et al. 2013 [5] and references therein). In many cases, these can be addressed through careful selection of legume species that match the soil and environmental conditions, and effective management of the farming system such as timing of over-sowing and controlling of grazing pressure [5]. However, the success of legumes in agricultural systems is also affected by the availability of sufficient population sizes of nodule-forming (nod+) and nitrogen-fixing (fix+) rhizobia in soils [6,7]. These not only determine if legumes can enter nodule-forming symbioses, but also how effectively the symbiosis can fix nitrogen. White clover (Trifolium repens L.) is the most widely grown pasture legume in New Zealand [4]. The species is phenotypically diverse and cultivars suited to many grazing and management systems are available [8]. An agronomic strength of white clover is its phenotypic plasticity, underpinned by its high genetic diversity arising from being an obligate out-crosser [9]. This gives rise to locally-selected populations that can be adapted to a range of different pastoral agroecosystems [10]. The rhizobia capable of forming root nodule symbiosis with white clover (Rhizobium leguminosarum bv. trifolii; Rlt) were likely introduced into New Zealand on soil and plant material du (...truncated)