White Clover (*Trifolium repens* L.) Benefits in Grazed Pastures and Potential Improvements

*John R. Caradus, Marissa Roldan, Christine Voisey and Derek R. Woodfield*

#### **Abstract**

White clover has been, and continues to be, a valuable component of grazed pastures through improving feed quality and nutritive value, improving seasonal dry matter distribution, and providing biologically fixed nitrogen that benefits not only white clover itself but also the surrounding plants. The contribution of white clover to sustainability and environmental goals is a growing focus of breeding programs. The use of genome mapping and genotyping by sequencing to determine genetic variation and population structure in clover improvement programs needs to be expanded to improve breeding efficiencies. Seed yields also need to be improved while maintaining the selected agronomic performance traits to ensure that commercial cultivars remain cost-effective with other crops and land uses. Beneficial traits not available within the white clover genome may be provided through genetic modification and gene editing, particularly traits that contribute towards addressing challenges associated with animal nutrition and health, water quality and climate change. The inherent benefits of white clover as well as the potential for including additional beneficial traits will be described.

**Keywords:** biologically fixed nitrogen, breeding, environment, genetic modification, nutritive value

#### **1. Introduction**

White clover (*Trifolium repens* L.) is a critical component of grazed pastures in most temperate areas of the world [1]. The value of white clover in mixed species grazed pastures is due to its contribution towards improving feed quality and nutritive value, complementary seasonal dry matter distribution, and the provision of biologically fixed nitrogen (**Figure 1**). A meta-analysis demonstrated that including white clover in perennial ryegrass swards maintained or increased milk solids yield at lower stocking rates and lower N application rates [2]. This can lead to economic benefit and improved profitability of pastoral farming systems. White clover plant breeding programs have sought to increase dry matter yield, improve feed quality, improve persistence, or a combination of these. However, with changing management

**Figure 1.** *White clover in a mixed sward, a highly nutritious component of multi-species grassland pastures.*

systems, resource constraints, environmental imperatives, and climate variability continued improvement will be required. New genomic and breeding techniques provide an opportunity to achieve new and better outcomes and will ensure that white clover continues to be a species of value and importance in grazed temperate pastures. The aim here is to describe the origin of white clover, its domestication, overview breeding objectives, describe the known advantages of white clover, and then discuss future improvements that will be required, and how they can be achieved, to ensure white clover continues to deliver benefit.

#### **2. Origin**

White clover is an allotetraploid (2n = 4x = 32), originating from hybridisation of two diploid *Trifolium* species [3]. As an amphidiploid, white clover has the full diploid set of chromosomes from each parent species [4]. The identity of those two ancestral species was purported to be *T. nigrescens* and *T. uniflorum* [5], or *T. nigrescens* and *T. occidentale* [6]. However, the most likely ancestral species based on DNA sequence analyses, molecular cytogenetics, interspecific hybridization are a diploid alpine species (*T. pallescens*) and a diploid coastal species (*T. occidentale*), which probably occurred during the last major glaciations 13,000–130,000 years ago [7–9]. F1 hybrids between *T. pallescens* and *T. occidentale* have been created using embryo rescue which produced a significant frequency of unreduced gametes, indicating this as the likely mode of polyploidisation, and these hybrids were themselves inter-fertile with white clover [8]. The increased genetic diversity created by allopolyploidisation can confer enhanced fitness, phenotypic plasticity, and adaptability, and has been correlated with survival in stressful environments [9].

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

White clover originated from multiple hybridisation events in Mediterranean glacial refugia characterised by fertile soils, good soil moisture, and the presence of grazing animals which conceivably led to its spread through Europe and into western Asia and North Africa [9–11].

#### **3. Domestication**

White clover is a perennial stoloniferous plant which can also seed prolifically. It is the most important pasture legume in many temperate parts of the world, particularly where defoliation is through grazing. Domestication of white clover occurred at least 400 years ago in the Netherlands and spread globally with European colonisation [12]. The wide habitat tolerance of white clover has ensured its success in temperate [13], Mediterranean [14] and some subtropical regions [15].

Trait inheritance for white clover is disomic where chromosome pairing during meiosis is similar to that of nonhomologous pairs of chromosomes in diploids [16]. White clover has a strong gametophytic self-incompatibility mechanism such that only a small proportion of plants in a population are self-compatible [17]. The outcrossing and disomic inheritance of white clover results in populations that are a heterogeneous mixture of heterozygous individuals [11].

#### **3.1 Types of white clover**

White clover cultivars and ecotypes have been categorised largely on leaf size as Small, Intermediate, Large and Ladino types [1, 18]. Cyanogenesis, the production of hydrogen cyanide in damaged leaves [19], has been another trait used to classify white clover. Plants can quantitatively vary from having high levels to no expression (acyanogenic types) of hydrogen cyanide. While both Ladino and Large categories of white clover are large-leaved, Ladino types are characterised by being completely acyanogenic [18, 20]). Large leaved types generally have moderate to high cyanogenesis, and Small and Intermediate types may display low to high cyanogenesis [18].

Ladino types of white clover all originate from the Po Valley, particularly in the area of southern Lombardy close to the town of Lodi [21] in northern Italy [20]. This large leaved acyanogenic type of clover is believed to have developed during the fifteenth and sixteenth centuries with white clover grown under a management system involving mowing to feed an intensive dairy system supporting Parmesan cheese production [22]. Ladino types were widely adopted in North America where the strong, erect growth proved popular [10].

Significant relationships between yield of white clover populations in perennial ryegrass (*Lolium perenne*) swards and shoot morphology and cyanogenesis traits have been observed [18, 23]. Of note were significant (P < 0.05) negative correlations between leaf size and stolon growing point number of −0.66 and −0.46, in years one and two respectively after planting; positive correlations between leaf size and clover percentage content of +0.73 and +0.25, in years one and two respectively after planting; and interestingly in year one, a negative correlation between stolon growing point density and clover percentage content of −0.55, but in year two a positive correlation of +0.41 [23].

#### **3.2 Breeding objectives**

Plant breeding of white clover has occurred in most countries with temperate environments. Traditionally, plant breeding has sought to improve on-farm productivity,

primarily through increased DM yield, improved feed quality, improved persistence, or a combination of these [24]. This has led to successful outcomes for both yield and clover percentage content in a grass sward [25]. Breeding programs have also been undertaken seeking improvements in resistance to pests and diseases, tolerance of drought, aluminium toxic soils, and improved yield at low levels of soil phosphorus.

#### *3.2.1 Yield and combining ability with grass*

The negative association between yield potential and persistence of white clover [26] has led to breeding programs seeking to break this relationship resulting in high yielding and persistent cultivars [27].

A study comparing 20 cloned genotypes from each of 15 white clover cultivars in three different grass swards (*L. perenne*, *Festuca arundinacea* and *Agrostis capillaris*) that were intermittently grazed by sheep over 18 months from planting showed that, despite not detecting differences in the effect of grass species on particular white clover cultivars, there was a strong difference in the spread of genotypes within each cultivar and that there may be a differential response to grasses at an individual genotype level [28]. This provides scope for improving competitive ability and reinforces the importance of selecting white clover under grass competition rather than as spaced white clover plants growing in monoculture. Competition effects of grasses maybe stronger under grazing than cutting, and competition effects become severe when grasses shade stolons [29].

#### *3.2.2 Persistence*

Persistence can be adversely affected by grazing management, diseases and pests, competition from other species, deficiencies and toxicities of nutrients and climatic factors [26]. As a stoloniferous species white clover depends on its taproot only in its seedling stage, with the taproot lasting less than 18-months from establishment [30]. The nodal root systems then maintain the remaining plant parts. Rooting frequency of nodes is positively correlated with stolon branching frequency [31]. However, it is the combination of the timing of death of the seminal tap root and the development of stolons that determines the persistence of white clover rather than the absolute survival of the seminal taproot [32].

Selection for increased stolon development while maintaining leaf size is seen as the key to improving both yield and persistence of white clover selections [30]. In general, leaf size along with plant height, both positively related to yield at least initially [33], are negatively correlated with stolon growing point density [18] which is intuitively associated with vegetative persistence [34].

In pasture systems using nitrogen from both white clover biological nitrogen fixation and from nitrogen fertiliser, pasture management can maintain clover content and as a result, pasture nutritive quality. For example, when up to 200 kg N ha−1 yr−1 of nitrogen fertiliser is used on dairy pastures, clover content can be maintained by ensuring additional pasture is fully utilised, particularly in spring [35] so that the grass component does not shade the clover and put it at a competitive disadvantage.

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

#### *3.2.3 Seed production*

Monitoring seed production potential of white clover cultivars is crucial in commercial delivery of any agronomically superior cultivar. White clover seed production yield is a product of inflorescence density and yield per inflorescence [36]. However, each node is only capable of producing either a flower or a stolon branch [37] so increased yield per inflorescence is a preferred strategy for large leaved cultivars with fewer nodes per unit area [36]. Vigorous and continued flowering results in plants being less persistent vegetatively [37]. While inflorescence density and seed yield per inflorescence are under independent genetic control and can be utilised to increase seed yield of new white clover cultivars [36]. Increases in seed yield are mostly associated with increases in inflorescence density (inflorescences/m2 ), and to a lesser extent with increased seed yield/inflorescence [38]. However, importantly seed yield can be achieved while maintaining desirable morphological features and improving the uniformity of the cultivars.

#### *3.2.4 Plant morphology*

The success of white clover as a perennial legume in grazed swards is largely reliant on its ability to spread vegetatively through profusely branching stolons (**Figure 2**). Selections have been made within both a large and a small leaved cultivar of white clover for high and low proportions of nodal branches, long and short internodes, and large and small leaf size [23]. Heritability estimates were higher for leaflet size and internode length than for proportion of nodes branching, indicating that increasing shoot density and therefore persistence should focus on selection for reduced internode length rather

#### **Figure 2.**

*White clover with a part of the canopy removed to expose the stolon network which allows the plant to persist, spread and vegetatively replicate.*

than increased proportion of nodes branching. Indeed, developing white clover cultivars with higher stolon growing point densities at a particular leaf size should improve persistence while maintaining the greater yield potential [27, 39].

#### *3.2.5 Phenotypic plasticity*

Phenotypic plasticity describes the ability, or not, of plants to respond to their environment by making changes to their morphology and physiology [40, 41]. 'Differential phenotypic plasticity' is the extent to which phenotypic plasticity occurs with a species [42]. White clover grows in a wide range of environments and has a high level of phenotypic plasticity, a trait common to many clonal species [43, 44]. Using soil phosphorus level as the environmental variable, all plant characters of white clover measured exhibited plasticity with yield-related characters in general being more plastic than characters associated with plant morphology [42]. Of the morphological characteristics measured internode length and leaf size were the most plastic. The large variation observed for phenotypic plasticity indicated that breeding for an increase or decrease in plasticity of plant traits in white cover is achievable. Plasticity of plant traits has been identified as important in the yield of white clover during adaptation to environmental and seasonal fluctuations [45].

#### *3.2.6 Pest and disease resistance*

In many environments high cyanogenesis is associated with greater persistence than acyanogenic clovers [26]. Cyanogenic white clover plants are avoided by slugs and/or snails [46, 47], voles (*Arvicola terrestris*) [48], and larvae of alfalfa weevils (*Hypera postica*) [49]. However, cyanogenic expression while slowing symptom expression of pepper spot caused by *Stemphylium sarciniforme* did not provide long term resistance [50].

Several invertebrate pests can seriously affect white clover production and concomitantly nitrogen fixation. In New Zealand pastures this includes slugs (*Deroceras recticulatum*), clover flea (*Sminthurus viridus*), grass grub (*Costelytra giveni*), porina (*Wiseana* spp.), clover weevil (*Sitona obsoletus*), black field cricket (*Teleogryllus commodus*) and root nematodes such as clover cyst (*Heterodera trifolii*) and root-knot (*Meloidogyne hapla*) [51–53]. Simply removing nematode effects using nematicides has shown increases of 40% for annual white clover yield and 57% for nitrogen fixed [54, 55]. Selection for resistance to root feeding insects and nematodes has been challenging. While variation has been observed under controlled conditions for resistance/tolerance to nematodes [56, 57], and insect pests such as grass grub (*Costelytra zealandica*) [58], none have resulted in commercial releases due to the recessive genetic control of clover cyst and root-knot nematode resistance.

#### *3.2.7 Abiotic tolerances*

White clover plants adapted to cold environments have little to no cyanogenic expression [59–61]. Heritability for tolerance to frost in white clover is high, ranging from 0.75 to 0.93 [62], as it is for many other species such as wheat [63] and rice [64]. During hardening, prior to exposure to frost, increases occur for dry matter content, soluble carbohydrates, sucrose and proline levels in stolons [65].

Drought can have significant effects on clover persistence, with the quantum of impact associated with grazing management [66, 67]. Under set stocking with sheep,

#### *White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

loss of stolon dry weight was much lower than for plants managed under rotational grazing, where stolon dry weight decreased by 75–90%, and white clover content in the sward reduced from 15 to 2%. Selections for improved tolerance to drought in white clover has had marginal success. White clover is a shallow rooted creeping legume where seedling taproots and nodal root size is positively correlated with leaf size [30]. Selection for specific root characteristics has improved yield and persistence in drought prone environments. For example, white clover populations developed by divergent selection for taproot diameter and for root weight ratio (proportion root weight to total plant weight), when assessed under grazing in a drought-prone environment and in a controlled-environment study, respectively, demonstrated that selection for medium leaf size and large taproot diameter gave yields 70% better in moist conditions and 35% better under dry conditions than that of the standard cultivar, Grasslands Huia [68]. Selection for increased root weight ratio was also effective in improving growth and survival in drought prone environments. Including ecotypes collected from drought-prone sites has been part of the development of cultivars in Australia, New Zealand and USA for heat and drought affected environments [69–71].

White clover is a species that requires high levels of soil phosphorus for optimal yields, particularly when grown in competition with grasses [72, 73]. Differences in response to added phosphorus among white clover genotypes has been shown in controlled environments [74–78], but this has not been effectively transferred to benefits in grazed pastures [79]. Selection for increased root hair length in white clover has been achieved [80] but when used in a field environment any benefit related to phosphorus uptake is negated by mycorrhizal infection in low phosphorus soils [81]. While mycorrhizal infection is important for white clover growth and survival in low phosphorus soils, selection for clovers able to develop more effective relationships with mycorrhiza has not yet been achievable [82].

Similarly for aluminium tolerance in acid soils, differences between white clover cultivars in controlled environments can be shown but these do not necessarily result in differences in field trials [83].

#### *3.2.8 Introgression using interspecific hybridisation*

To increase genetic variation in white clover concerted attempts have been made to create interspecific hybrids with 11 related *Trifolium* species, notably *T. nigrescens*, *T. uniflorum*, *T. occidentale*, *T. pallescens*, and *T. ambiguum* [84, 85]. These species range from annuals to long-lived, hardy perennials, some with adaptations to stressful environments, providing new traits for breeding more resilient cultivars of white clover for seasonally dry, infertile grassland environments. However, to date only one cultivar derived from interspecific hybridisation has been commercially released. Named Aberlasting, this cultivar was derived from crosses between *T. repens* and *T. ambiguum.* Initial results suggested enhanced persistence under grazing, possibly due to the presence of rhizomes in the hybrids [86], although other agronomic trials have not shown expected benefits [87]. Adequate seed production remains a major hurdle for successful commercialisation of *Trifolium* hybrids [88, 89].

#### **4. Benefits of white clover**

White clover has been the go-to legume in grazed pastures because of its ability to withstand defoliation and compete with companion species, but also due to being able to effectively fix nitrogen, have a high nutritive value, provide complementary seasonal yield with companion grasses, and improve on-farm profitability. Combining white clover with not just grasses but also forage herbs such as chicory (*Cichorum intybus*) and plantain (*Plantago lanceolata*) can contribute additional micro- and macro-minerals to livestock diets [90]. It has been proposed, with some evidence, that for white clover to make any significant contribution to the nitrogen economy and feed quality of a pasture it should make up at least 30% total dry matter [91]. White clover is a component of most diverse pasture mixes and one that tends to dominate over time due to the loss of herbs and other shorter-lived species [90]. The inclusion of herbs such as plantain with biological nitrification inhibition may help further reduce N emissions in diverse pastures [92].

#### **4.1 Nitrogen fixation**

White clover has an affinity for *Rhizobium leguminosarum* bv. *trifolii*, and this nitrogen-fixing symbiosis can produce on average 80–100 kg N/ha/year (range 10–270 kg N/ha/year) in grazed permanent clover/grass pastures in temperate regions of the world [93, 94]. Compared with white clover monocultures, grass competition has been shown to markedly increase the proportion of clover nitrogen derived from symbiotic nitrogen fixation [95]. This phenomenon is the result of strong competitiveness by ryegrass for soil nitrogen such that white clover in the cloverryegrass mixtures becomes more dependent on symbiotic nitrogen fixation than when grown in monoculture [96].

Severe defoliation can cause rapid degradation of leghaemoglobin in nodules resulting in decreased nitrogen fixation capacity [97]. However, less severe defoliation may preferentially influence symbiotic nitrogen fixation, as opposed to the uptake of mineral nitrogen from the soil [96]. This observation led to the conclusion that symbiotic nitrogen fixation does not limit the supply of nitrogen to clover and hence its growth. Therefore, symbiotic nitrogen fixation in white clover is regulated more by the demand for nitrogen rather than by the availability of carbohydrate reserves in the plant.

The application of nitrogen fertiliser also reduces the level of biologically fixed nitrogen from clover. In a mixed grass-clover sward grazed by sheep the application of up to 390 kg N ha−1 yr−1 (applied at 30 kg N ha−1 after each grazing) has been shown to decrease annual nitrogen fixation by nearly 60% [98]. Similarly, in a mixed grassclover sward grazed by dairy cows the application of 400 kg N ha−1 yr−1 (applied at approximately 40 kg N ha−1 after each grazing) decreased annual nitrogen fixation from 154 kg N ha−1 yr−1 when no nitrogen was added to 39 to 53 kg N ha−1 yr−1 [99]. In a UK study, it was determined that under intensive grazing, the maximum applied N rate that optimised herbage yield while having minimal effects on white clover content and nitrogen fixation rates was 60–120 kg N ha−1 [100].

Low temperatures also have a detrimental effect on biological nitrogen fixation. In a controlled environment study higher shoot temperatures (23°C vs. 13°C day temperatures) resulted in increased nitrogen fixed irrespective of whether or not root temperature was increased in parallel [101]. Low root temperature (5°C) however did result in a lower proportion of nitrogen derived from biological nitrogen fixation.

Grass-legume swards containing white clover produce higher grass and total sward yield than mixtures containing red clover, alfalfa or birdsfoot trefoil [102]. This is potentially due to the higher N fixation and a faster release of N from roots of white clover than alfalfa. Louarn et al. [103] reported 60% less transfer of N fixed by alfalfa to the associated grasses than white clover despite the alfalfa having twice the biomass of the white clover.

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

#### **4.2 Nutritive value**

Forage legumes are generally considered to be of higher nutritive value than grasses due to a higher intake, a higher ratio of protein/energy absorbed [104–108] and higher digestibility [109]. Dry matter intake has been shown to be at its greatest when white clover is about 60% of the feed mixture consumed [110]. Increased intakes of clover with higher nutritive value are the main contributing factors leading to increased milk yields [111] and lamb growth rates [112] associated with high clover diets. Mixtures of clover and forage herbs such as chicory (*Cichorium intybus*) and plantain (*Plantago lanceolata*) resulted in higher growth rates of sheep and cattle particularly during summer and autumn compared with ryegrass/white clover pastures due to the higher nutritive value [113]. Herb and clover mixes, while having a similar crude protein content compared with ryegrass/white clover pastures, have lower fibre content and higher organic matter digestibility and metabolisable energy levels.

Compared with a concentrate diet offered *ad libitum* lambs fed a cocksfoot (*Dactylis glomerata*) and white clover pasture mix resulted in carcasses with less fat and more protein [114]. However, comparison of lambs fed either white clover or perennial ryegrass found that clover-fed lambs had 40% greater slaughter weights but also had higher amounts of fat resulting from the greater production of rumen-reticulum volatile fatty acids [115]. A lower stocking rate associated with lambs grazing grass/clover compared with grass fertilised with 190 kg N ha−1 was compensated for by higher live-weight gain and carcass weight without changes in fatty acid composition of carcass tissues [116]. However, polyunsaturated fatty acid concentrations are often higher in white clover than alfalfa (*Medicago sativa*) and grasses [117]. Higher liveweight gain and earlier slaughter of lambs grazing clover-dominant swards tend to outweigh any fatness disadvantages relative to ryegrass-dominant pastures due to high feed conversion efficiency [118].

Methods to select for improved nutritive value of white clover have been developed [119]. This could allow the identification of germplasm with proteins that are relatively insoluble and resistant to rumen degradation leading to increased levels of amino acids that are available for absorption from the intestine.

#### **4.3 Seasonal yield**

Asynchronous seasonal biomass production of components in mixed species pastures has been related to increased yield [120] and yield stability [121] of sown grasslands. However, a study comparing perennial ryegrass pure stands and eight populations of white clover either in pure stands or in mixture with perennial ryegrass over three years at three sites concluded that it was doubtful if genetic variability of seasonal growth patterns within white clover can be used to increase the performance of clover-ryegrass mixtures [122].

#### **4.4 Economic benefits**

The inclusion of *Trifolium* species in grazed grass swards has been demonstrated to improve both productivity and profitability compared with grass-only swards for both sheep and dairy production systems [2, 123, 124]. Introduction of more persistent white clovers into south-eastern USA pastures added US\$86/ha through increased cattle liveweight gain and reduced N fertiliser requirements [125]. In New Zealand the annual financial contribution of white clover through fixed nitrogen,

forage yield, seed production and honey production was estimated to be NZ\$3.095 billion [126]. The contribution of white cover to New Zealand's direct and dependent industry Gross Domestic Product has been estimated to be NZ\$2.35 billion in 2015/16 [127] when milk solid payout was about NZ\$4.40 per kg milk solids whereas now that is closer to NZ\$9 per kg milk solids [128]. The inclusion of white clover in low-fertility hill country pasture in New Zealand has been modelled to result in a 17% increase in spring and summer forage consumption generating a 32% greater cattle carcass weight production per ha and leading to a 49% improvement in farm system profit [129]. This represents a positive net present value of over NZ\$360,000 for the original investment in white clover establishment into existing pastures.

#### **5. Future improvements**

Future breeding aims need to align with increasing environmental challenges and the regulations imposed on farming operations such as limits of nitrogen fertiliser use, protection of waterways, identification of plants that will mitigate against or be adapted to predicted climate change, and the effect this will have on plant performance, species requirements, and the resulting changes that will happen in farm systems [24].

#### **5.1 Breeding techniques**

#### *5.1.1 Genomics and trait mapping*

Genome mapping and genotyping by sequencing to determine genetic variation and population structure in clover improvement programs provide the opportunity to improve breeding efficiencies [130–135]. However, there are limited examples of successes despite gene markers being identified for seed yield [136, 137], or drought tolerance using quercetin glycoside accumulation gene markers [138].

Genomic selection utilises DNA markers and trait data to estimate the breeding value and kinship of genotypes without having to phenotype them. In white clover Ehoche et al. [133] demonstrated the potential of genomic selection to be at least double the rate of genetic gain for DM yield in white clover compared with a conventional half-sib breeding scheme. Once validated in the field, this can shorten the breeding cycle and improve efficiency of breeding for many multigenic traits by enabling access to within family variation. The lack of access to within-family genetic variation has been identified as a major reason for the poor genetic gain in forages [139].

A potential setback to the implementation of genomic selection is the increase in net inbreeding per year as the reduction in generation interval decreases genetic variance faster [140]. Phenotypic selection typically takes 3 years or more to perform one cycle of selection, while two cycles per year can be completed with genomic selection. Breeding schemes for white clover aim to increase the frequency of desirable alleles in a population while maintaining heterozygosity. Consequently, breeders are faced with a dilemma of increasing genetic gain by selection whilst preserving or even increasing genetic diversity. Practically, this problem can be ameliorated by initiating selections in populations with high genetic diversity, or simultaneously running pre-breeding activities so that new genetic variability can be introduced as a plateau in the response to selection is reached [140, 141].

#### *5.1.2 Genetic modification and gene editing*

Beneficial traits not available within the white clover genome may be provided through genetic modification and gene editing. Initial genetic modification of white clover sought to improve insect resistance [142, 143] and virus resistance [144, 145]. Breeding strategies for developing genetically modified white clover cultivars have been considered [146]. However, traits that contribute towards addressing challenges associated with animal nutrition and health, water quality, drought tolerance and climate change have become increasingly important [147–149].

#### **5.2 Environmental benefits**

Pastoral agriculture has been criticised for exacerbating both air and water issues through methane production from ruminants contributing to increasing greenhouse gas levels, and through nitrogen movement to waterways [150–153]. White clover as a high protein component of grazed pasture contributes to this concern but it also has the opportunity to provide solutions. One study has laid to rest the concern that biological nitrogen fixation might exacerbate N2O emissions, this appears to be more influenced by soil carbon content and surplus nitrogen levels [154].

Reducing emissions of the methane from ruminants grazing pastures is a serious research target in some countries. An example of this is the utilisation of plant secondary compounds such as condensed tannins. Condensed tannins are found in the leaves of several forage legumes, but not to any significant extent in white clover. They are known to bind proteins, protecting them from degradation in the rumen where methane producing-microbes are active [155, 156]. White clover synthesises condensed tannins, which occurs naturally in the flowers, and in trichomes on the under-surface of leaves [157]. A recent advance, using a molecular biology approach, has identified a transcription factor or master switch that can 'turn on' the condensed tannin pathway present in white clover leaves, and with the appropriate promoters allows biologically significant levels of condensed tannin expression in leaf tissue [148, 158, 159]. *In vitro* tests have demonstrated that the condensed tannins produced in white clover leaves can bind to protein at pH 6.5, as found in the rumen, and then release them at pH 2.0, the pH in the abomasum This suggests that protein protection in the rumen is possible, and that when released in the acidic abomasum, these proteins will be digested into essential amino acids for absorption in the small intestine of the animal [160, 161]. These studies also demonstrated that these condensed tannins could reduce methane production by up to 15% in the first 6 hours of incubation in rumen fluid under laboratory conditions (**Figure 3**). While the use of genetically modified organisms in many jurisdictions is regulated, this development has the potential to improve environmental, animal health and animal productivity outcomes from grazed pasture systems.

#### **5.3 Persistence, yield and competitive ability**

Although past breeding programs have been successful in selecting for persistence and yield [25, 69], future programs will continue to focus on these traits, particularly in mixed species swards and under grazing [24]. The use of grass competition and grazing in selection trials has been important in identifying persistent and high yielding cultivars particularly when grown in stressed environments [26, 39, 162]. This has resulted in the production of cultivars such as Durana [69], Trophy [70], and

#### **Figure 3.**

*Effect of condensed tannins in white clover leaves (WC-1 and WC-2) on methane production after 6 hours of incubation in rumen fluid in vitro, compared with untransformed control (white clover cv Mainstay). Means that do not share a letter are significantly different at p < 0.01 using Tukey's multiple comparison test. Graph redrawn as part of data published in Roldan et al. [160].*

Tribute [163]. The challenge for breeders is to identify effective screening and selection processes, sourcing new genetic variation and integrating genotyping by sequencing to link important traits to gene markers to improve selection efficiencies.

#### **5.4 Microbial associations**

#### *5.4.1 Rhizobium symbiosis*

The provision of effective rhizobium strains, along with novel seed-coating technology that extends shelf life, can led to increased symbiotic capacity in white clover [164]. Selecting rhizobium strains that are competitive with naturalised rhizobia strains to ensure both their persistence in the soil and superior nodule occupancy is key [165]. A better understanding of the genes associated with ensuring a competitive and effective symbiosis would be beneficial [166]. Spill-over benefits of rhizobium have been demonstrated, with the white clover *Rhizobium* strain TA1 conferring tolerance against Cd toxicity, an impurity in phosphate fertilisers which may have toxic effects on both plant growth and rhizobia activity [167]. Whether variation in nitrogen fixation capacity by genotypes within populations of white clover can be exploited for more effective symbiotic outcomes is yet to be determined [168].

#### *5.4.2 Mycorrhiza*

Synergistic effects between arbuscular mycorrhiza and rhizobium strains has shown increased nitrogen acquisition by white clover, as well as increased shoot and root growth, and increased amino acids levels in roots [169]. Mycorrhiza, such as *Glomus mosseae,* have been shown to extend the soil phosphorus depletion zone to

#### *White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

nearly 12 cm compared with non-mycorrhizal white clover roots which may extend to 1 cm [170]. However, mycorrhizae are only effective at low soil phosphorus levels [171–173]. There is some evidence that mycorrhiza may not only aid uptake of phosphorus, but also may enhance growth and drought tolerance of white clover [174]. It is unlikely however that mycorrhiza are a route for transfer of nitrogen between white clover and grasses [175].

#### *5.4.3 Bioprotectants*

Microbial bioprotectants can enhance plant growth, improve nutrient uptake, and suppress disease and pests [176–178]. Inoculation with plant growth-promoting rhizobacter (e.g. *Bacillus aryabhattai* and *Azotobacter vinelandii*) along with effective *Rhizobium* strains has been shown to significantly increase nitrogenase activity plus potassium, calcium, and magnesium contents in shoots when grown in phosphorus deficient soils [179]. Use of bioprotectants, other than *Rhizobium*, to improve white clover growth and persistence is a research area requiring attention [179].

#### **5.5 Use as a cover crop**

Perennial legumes such as white clover have been used as cover crops for improving soil properties, increasing future crop production, and positively impacting environmental aspects of any farming operation [180]. For example, mixtures of white clover and perennial ryegrass have been successfully used as a living mulch to achieve high yields, with sufficient irrigation and additional fertilisation, while increasing the inputs of nitrogen through biological nitrogen fixation into the entire cropping system [181]. Consistent yields in maize on unfertilised soil where white clover had previously been used as a living mulch, was shown to be the result of effective mycorrhizal fungus colonisation leading to improved phosphorus uptake by maize [182, 183].

#### **6. Conclusion and future perspectives**

White clover will continue to be a crucial component of grazed pastures in the temperate world, particularly as consumer demands for improved environmental and animal welfare outcomes in agricultural production systems become more strident. Use of new molecular and genomic methodologies for more effective and efficient selection of beneficial traits will remain a priority. Reliance on legume-based swards resulting in reduced inorganic nitrogen use will become increasingly important in reducing nutrient runoff into waterways and nitrous oxide emissions. Including traits into white clover that reduce both methane emissions and nitrogen losses can be achieved through genetic modification. Traits such as leaf expression of condensed tannins in forage can simultaneously deliver to these environmental goals, plus enhance animal health by reducing bloat, and increase production of meat, milk and fibre. The challenge then will be balancing the perceived risk of using genetic modification against the benefits of improving the environmental footprint of livestock farming and animal health and productivity. In addition, enhancements in white clover performance using microbial technologies may create more sustainable farming outcomes through reduced synthetic fertiliser and pesticidal inputs.

#### **Conflict of interest**

John Caradus and Derek Woodfield are employed by organisations that breed and own the intellectual property associated with some white clover cultivars mentioned in this review. Christine Voisey and Marissa Roldan are involved in the condensed tannin expression research outlined above.

### **Author details**

John R. Caradus1 \*, Marissa Roldan<sup>2</sup> , Christine Voisey2 and Derek R. Woodfield3

1 Grasslanz Technology Ltd., Palmerston North, New Zealand

2 AgResearch, Palmerston North, New Zealand

3 PGG Wrightson Seeds Ltd., Palmerston North, New Zealand

\*Address all correspondence to: john.caradus@grasslanz.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

#### **References**

[1] Caradus JR. Genetic diversity within white clover (*Trifolium repens* L.). Proceedings of Agronomy Society of New Zealand. 1994;**24**:1-6

[2] Dineen M, Delaby L, Gilliland T, McCarthy B. Meta-analysis of the effect of white clover inclusion in perennial ryegrass swards on milk production. Journal of Dairy Science. 2018;**101**(2):1804-1816. Epub 2017/11/28. DOI: 10.3168/jds.2017-12586

[3] Ansari HA, Ellison NW, Reader SM, Badaeva ED, Friebe B, Miller TE, et al. Molecular cytogenetic organization of 5S and 18S-26S rDNA loci in White clover (*Trifolium repens* L.) and related species. Annals of Botany. 1999;**83**(3):199-206. DOI: 10.1006/anbo.1998.0806

[4] Attwood SS, Hill HD. The regularity of meiosis in microsporocytes of *Trifolium repens*. American Journal of Botany. 1940;**27**(9):730-735. DOI: 10.1002/j.1537-2197.1940.tb10943.x

[5] Badr A, Sayed-Ahmed H, El-Shanshouri A, Watson IE. Ancestors of white clover (*Trifolium repens* L.), as revealed by isozyme polymorphisms. Theoretical and Applied Genetics. 2002;**106**(1):143-148. Epub 2003/02/13. DOI: 10.1007/s00122-002-1010-5

[6] Williams WM, Ansari HA, Hussain SW, Ellison NW, Williamson ML, Verry IM. Hybridization and introgression between two diploid wild relatives of White clover, *Trifolium nigrescens* Viv. and *T. occidentale* coombe. Crop Science. 2008;**48**(1):139-148. DOI: 10.2135/cropsci2007.05.0295

[7] Ellison NW, Liston A, Steiner JJ, Williams WM, Taylor NL. Molecular phylogenetics of the clover genus

(Trifolium--Leguminosae). Molecular Phylogenetics and Evolution. 2006;**39**(3):688-705. Epub 2006/02/18. DOI: 10.1016/j.ympev.2006.01.004

[8] Williams WM, Ellison NW, Ansari HA, Verry IM, Hussain SW. Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding. BMC Plant Biology. 2012;**12**:55. DOI: 10.1186/1471-2229-12-55

[9] Griffiths AG, Moraga R, Tausen M, Gupta V, Bilton TP, Campbell MA, et al. Breaking free: The genomics of allopolyploidy-facilitated niche expansion in white clover. The Plant Cell. 2019;**31**(7):1466-1487. Epub 2019/04/27. DOI: 10.1105/tpc.18.00606

[10] Gibson PB, Cope WA. White Clover. Clover Science and Technology; 1985. pp. 471-490

[11] Williams WM. Adaptive variation. In: Baker MJ, Williams WM, editors. White Clover. Wallingford, UK: CAB Int; 1987. pp. 299-321

[12] Zeven AC. Four hundred years of cultivation of Dutch white clover landraces. Euphytica. 1991;**54**(1):93-99. DOI: 10.1007/BF00145635

[13] Brougham RW, Ball PR, Williams WM. The ecology and management of white clover based pastures. In: Wilson R, editor. Plant Relations in Pastures. Australia: CSIRO; 1978. pp. 309-324

[14] Davies EW, Young NR. The characteristics of European, Mediterranean and other populations of white clover (*Trifolium repens* L.). Euphytica. 1967;**16**(3):330-340. DOI: 10.1007/BF00028939

[15] O'Brien AD, editor. White clover (*Trifolium repens* L.) in a subtropical environment on the east coast of Australia. In: Proceedings 11th International Grasslands Congress. Australia: Surfers Paradise; 1970

[16] Williams WM, Mason KM, Williamson ML. Genetic analysis of shikimate dehydrogenase allozymes in *Trifolium repens* L. Theoretical and Applied Genetics. 1998;**96**(6):859-868. DOI: 10.1007/s001220050813

[17] Thomas RG. Reproductive development. In: Lancahsire JA, editor. White Clover. Wallingford: CAB International; 1987. pp. 63-123

[18] Caradus JR, MacKay AC, Woodfield DR, van den Bosch J, Wewala S. Classification of a world collection of white clover cultivars. Euphytica. 1989;**42**(1-2):183-196. DOI: 10.1007/BF00042631

[19] Till I. Variability of expression of cyanogenesis in white clover (*Trifolium repens* L.). Heredity. 1987;**59**(2):265-271. DOI: 10.1038/hdy.1987.122

[20] Annicchiarico P, Ruisi P, Di Miceli G, Pecetti L. Morpho-physiological and adaptive variation of Italian germplasm of sulla (*Hedysarum coronarium* L.). crop and pasture. Science. 2014;**65**(2):206- 213. DOI: 10.1071/CP13342

[21] Sansone A. Il ladino. Biblioteca Agraria Ottavi. Italy: Casale Monferrato; 1905

[22] Haussmann G. Il suolo d'Italia nella storia. Storia d'Italia I I caratteri originali. Torino, Italy: Einaudi; 1972

[23] Caradus JR, MacKay AC, editors. Selection for stolon branching and internode length in white clover. In: Proceedings 9th Australian Plant

Breeding Conference; 27 June-1 July 1988. Wagga Wagga: Agricultural Research Institute; 1988

[24] Caradus J, Bouton J, Brummer C, Faville M, George R, Hume D, et al., editors. Plant breeding for resilient pastures. In: Resilient Pasture Symposium, Grassland Research and Practice Series. Vol. 17. Karapiro, New Zealand: New Zealand Grassland Association; 2021. pp. 247-268. DOI: 10.33584/rps.17.2021.3441

[25] Woodfield DR, Caradus JR. Genetic improvement in white clover representing six decades of plant breeding. Crop Science. 1994;**34**(5):1205- 1213. DOI: 10.2135/cropsci1994.0011183X 003400050011x

[26] Caradus JR, Williams WM, editors. Breeding for legume persistence in New Zealand. 'Persistence of forage legumes' Proceedings of a Trilateral Workshop, Honolulu, Hawaii; 17-22 July 1988; Madison, Wisconsin. USA: American Society of Agronomy; 1989

[27] Caradus JR, Clifford PTP, Chapman DF, Cousins GR, Williams WM, Miller JE. Breeding and description of 'Grasslands Sustain', a medium-large-leaved white clover (*Trifolium repens* L.) cultivar. New Zealand Journal of Agricultural Research. 1997;**40**(1):1-7. DOI: 10.1080/00288233.1997.9513224

[28] Campbell BD, Caradus JR, Given MDJ, Karsten HD, Berthelsen HB, Williamson DY. Variation in the spread of white clover plants growing in competition with different grasses. Proceedings of the Agronomy Society of New Zealand. 1994;**24**:43-46

[29] Jackman RH, Mouat MCH. Competition between grass and clover for phosphate. I. Effect of browntop (*Agrostis tenuis* Sibth) on white clover (*Trifolium* 

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

*repens* L.) growth and nitrogen fixation. New Zealand Journal of Agricultural Research. 1972;**15**(4):653-666. DOI: 10.1080/00288233.1972.10421622

[30] Caradus JR. The structure and function of white clover root systems. In: Brady NC, editor. Advances in Agronomy. Vol. 43: Academic Press; 1990. pp. 1-46. DOI: 10.1016/ S0065-2113(08)60475-7

[31] Chapman DF. Growth and demography of *Trifolium repens* Stolons in Grazed Hill pastures. Journal of Applied Ecology. 1983;**20**(2):597-608. DOI: 10.2307/2403529

[32] Janssen PWL, Hoekstra NJ, van der Schoot JR, van Eekeren N. White clover (*Trifolium repens*) population dynamics are partly dependent on timing of seminal taproot death. Grass and Forage Science. 2022:1-11. DOI: 10.1111/ gfs.12598

[33] Brock JL, Tilbrook JC. Effect of cultivar of white clover on plant morphology during the establishment of mixed pastures under sheep grazing. New Zealand Journal of Agricultural Research. 2000;**43**(3):335-343. DOI: 10.1080/00288233.2000.9513432

[34] Gibson PB, Beinhart G, Halpin JE, Hollowell EA. Selection and evaluation of white clover clones. I. Basis for selection and a comparison of two methods of propagation for advanced evaluations. Crop Science. 1963;**3**(1):83- 86. DOI: 10.2135/cropsci1963.0011183X0 00300010025x

[35] Harris SL, Clark DA. Effect of high rates of nitrogen fertiliser on white clover growth, morphology, and nitrogen fixation activity in grazed dairy pasture in northern New Zealand. New Zealand Journal of Agricultural Research. 1996;**39**(1):149-158. DOI: 10.1080/00288233.1996.9513173

[36] Woodfield DR, Baird IJ, PTP C. Genetic control of white clover seed yield potential. Proceedings of the New Zealand Grassland Association. 2004;**66**:111-117. DOI: 10.33584/ jnzg.2004.66.2567

[37] Thomas RG. Growth of the white clover plant in relation to seed production. In: Lancashire JA, editor. Herbage Seed Production. Vol. 1. Lincoln, New Zealand: Grassland Research and Practice Series; 1979. pp. 56-63. DOI: 10.33584/rps.1.1979.3286

[38] Widdup KHW, Woodfield DR, Baird IJ, Clifford PTP. Response to selection for seed yield in six white clover cultivars. Proceedings of the New Zealand Grassland Association. 2004;**66**:103-110. DOI: 10.33584/jnzg.2004.66.2566

[39] Woodfield DR, Caradus JR. Factors affecting white clover persistence in New Zealand pastures. Proceedings of the New Zealand Grassland Association. 1996;**58**:229-235. DOI: 10.33584/ jnzg.1996.58.2196

[40] Bradshaw AD. Evolutionary significance of phenotypic plasticity in plants. In: Caspari EW, Thoday JM, editors. Advances in Genetics. Vol. 13. Academic Press; 1965. pp. 115-155. DOI: 10.1016/S0065-2660(08)60048-6

[41] West-Eberhard MJ. Phenotypic plasticity and the origins of diversity. Annual Review of Ecology and Systematics. 1989;**20**(1):249-278. DOI: 10.1146/annurev.es.20.110189.001341

[42] Caradus JR, Hay MJM, Mackay AD, Thomas VJ, Dunlop J, Lambert MG, et al. Variation within white clover (*Trifolium repens* L.) for phenotypic plasticity of morphological and yield related characters, induced by phosphorus supply. New Phytologist. 1993;**123**(1):175-184. DOI: 10.1111/j.1469- 8137.1993.tb04543.x

[43] Hill J. Plasticity of White Clover Growth in Competition with Perennial Ryegrass. Aberystwyth, UK: University College of Wales; 1977

[44] Horikawa Y. Plastic allocation of photosynthetic product in white clover *(Trifolium repens* L.). Journal of Japanese Society of Grasslands Science. 1986;**32**:225-234

[45] Nölke I, Tonn B, Isselstein J. Seasonal plasticity is more important than population variability in effects on white clover architecture and productivity. Annals of Botany. 2021;**128**(1):73-82. Epub 2021/03/14. DOI: 10.1093/aob/mcab040

[46] Crawford-Sidebotham TJ. The role of slugs and snails in the maintenance of the cyanogenesis polymorphisms of *Lotus corniculatus* and *Trifolium repens*. Heredity. 1972;**28**(3):405-411. DOI: 10.1038/hdy.1972.52

[47] Dirzo R, Harper JL. Experimental studies on slug-plant interactions: IV. The performance of cyanogenic and *Acyanogenic morphs* of *Trifolium repens* in the field. Journal of Ecology. 1982;**70**(1):119-138. DOI: 10.2307/2259868

[48] Saucy F, Studer J, Aerni V, Schneiter B. Preference for acyanogenic white clover (*Trifolium repens*) in the vole Arvicola terrestris: I. Experiments with two varieties. Journal of Chemical Ecology. 1999;**25**(6):1441-1454. DOI: 10.1023/A:1020943313142

[49] Ellsbury MM, Pederson GA, Fairbrother TE. Resistance to foliarfeeding hyperine weevils (Coleoptera: Curculionidae) in cyanogenic white clover. Journal of Economic Entomology. 1992;**85**(6):2467-2472. DOI: 10.1093/ jee/85.6.2467

[50] Wilkinson HT, Millar RL. Cyanogenic potential of *Trifolium repens* L. in relation to pepper spot caused by *Stemphylium sarciniforme*. Canadian Journal of Botany. 1978;**56**(20):2491- 2496. DOI: 10.1139/b78-300

[51] Goldson SL, Rowarth JS, Caradus JR. The impact of invasive invertebrate pests in pastoral agriculture: A review. New Zealand Journal of Agricultural Research. 2005;**48**(4):401-415. DOI: 10.1080/00288233.2005.9513673

[52] Gerard P, Ferguson C, Van Amsterdam S. Comparison of New Zealand perennial clovers for resilience against common pasture pests. New Zealand Plant Protection. 2017;**70**:241- 249. DOI: 10.30843/nzpp.2017.70.57

[53] Ferguson CM, Barratt BIP, Bell N, Goldson SL, Hardwick S, Jackson M, et al. Quantifying the economic cost of invertebrate pests to New Zealand's pastoral industry. New Zealand Journal of Agricultural Research. 2019;**62**(3):255- 315. DOI: 10.1080/00288233.2018.1478860

[54] Watson RN, Yeates GW, Littler RA, Steele KW. Responses in nitrogen fixation and herbage production following pesticide applications on temperate pastures. Proceedings of the Australasian Conference on Grassland Invertebrate Ecology. 1985;**4**:103-113

[55] Watson RN, Mercer CF. Pasture nematodes: The major scourge of white clover. Proceedings of the New Zealand Grassland Association. 2000;**62**:195-199

[56] Watson RN, Bell NL, Neville FJ, Aalders LT, Rohan TC. Breeding white clover for tolerance to nematodes: Overview of process and progress. Proceedings of the New Zealand Grassland Association. 2007;**69**:193-196. DOI: 10.33584/jnzg.2007.69.2661

[57] Mercer CF, Watson RN, Woodfield DR. Performance of *White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

nematode-resistant white clover in field trials. Proceedings of the New Zealand Grassland Association. 2005;**67**:29-34

[58] Van den Bosch J, Caradus JR, Lane GA, Gaynor DL, Dymock JJ. Screening white clover for resistance to grass grub in a controlled environment. New Zealand Journal of Agricultural Research. 1995;**38**(3):329-336. DOI: 10.1080/00288233.1995.9513134

[59] Daday H. Gene frequencies in wild populations of *Trifolium repens* L. I. Distribution by latitude. Heredity. 1954;**8**(3):61-78. DOI: 10.1038/ hdy.1954.40

[60] Daday H. Gene frequencies in wild populations of *Trifolium repens* L. II. Distribution by altitude. Heredity. 1954;**8**:377-384. DOI: 10.1038/hdy.1954.40

[61] Caradus JR, Mackay AC, Bosch JVD, Greer DH, Wewala GS. Intraspecific variation for frost hardiness in white clover. The Journal of Agricultural Science. 1989;**112**(2):151-157. Epub 2009/03/27. DOI: 10.1017/S002185960008504X

[62] Caradus JR, Forde MB, Wewala S, Mackay AC. Description and classification of a white clover (*Trifolium repens* l.) germplasm collection from southwest europe. New Zealand Journal of Agricultural Research. 1990;**33**(3):367-375. DOI: 10.1080/00288233.1990.10428433

[63] Sutka J. Genetic studies of frost resistance in wheat. Theoretical and Applied Genetics. 1981;**59**(3):145- 152. Epub 1981/03/01. DOI: 10.1007/ bf00264968

[64] Yang SC. Studies on the relationship between seedling tolerance to low temperature and glabrous hull in longgrain *indica* rice. Journal of Agricultural Research of China. 1982;**31**:102-107

[65] R⊘snes K, Junttila O, Ernstsen A, Sandli N. Development of cold tolerance in White clover (*Trifolium repens* L.) in relation to carbohydrate and free amino acid content. Acta Agriculturae Scandinavica, Section B—Soil and Plant Science. 1993;**43**(3):151-155. DOI: 10.1080/09064719309411233

[66] Brock J, Caradus J. Influence of grazing management and drought on white clover population performance and genotypic frequency. In: Woodfield D, editor. White Clover: New Zealand's Competitive Edge Grassland Association Grassland Research and Practice. 1995. pp. 79-82

[67] Lane LA, Ayres JF, Lovett JV. The pastoral significance, adaptive characteristics, and grazing value of white clover (*Trifolium repens* L.) in dryland environments in Australia: A review. Australian Journal of Experimental Agriculture. 2000;**40**(7):1033-1046. DOI: 10.1071/ EA99141

[68] Caradus JR, Woodfield DR. Genetic control of adaptive root characteristics in white clover. Plant and Soil. 1998;**200**(1):63-69. DOI: 10.1023/A:1004296707631

[69] Bouton JH, Woodfield DR, Hoveland CS, McCann MA, Caradus JR. Enhanced survival and animal performance from ecotype derived white clover cultivars. Crop Science. 2005;**45**(4):1596-1602. DOI: 10.2135/cropsci2004.0335

[70] Ayres JF, Caradus JR, Murison RD, Lane LA, Woodfield DR. Grasslands trophy: A new white clover ('*Trifolium repens* L.') cultivar with tolerance of summer moisture stress. Australian Journal of Experimental Agriculture. 2007;**47**(1):110-115. DOI: 10.1071/ EA04029

[71] Jahufer MZZ, Ford JL, Widdup KH, Harris C, Cousins G, Ayres JF, et al. Improving white clover for Australasia. Crop and Pasture Science. 2012;**63**(9):739- 745. DOI: 10.1071/CP12142

[72] Jackman RH, Mouat MCH. Competition between grass and clover for phosphate. II. Effect of root activity, efficiency of response to phosphate,and soil moisture. New Zealand Journal of Agricultural Research. 1972;**15**(4):667-675. DOI: 10.1080/00288233.1972.10421623

[73] Caradus J. Distinguishing between grass and legume species for efficiency of phosphorus use. New Zealand Journal of Agricultural Research. 1980;**23**(1):75-81. DOI: 10.1080/00288233.1980.10417847

[74] Mackay AD, Caradus JR. White clover cultivar responses to phosphate fertilisers of differing solubility. Proceedings New Zealand Grassland Association. 1988;**49**:151-156. DOI: 10.33584/jnzg.1988.49.1845

[75] Mackay AD, Caradus JR, Dunlop J, Wewala GS, MCH M, Lambert MG, et al., editors. Response to phosphorus of a world collection of white clover cultivars. In: Proceedings 3rd International Symposium on Genetic Aspects of Plant Mineral Nutrition 19-24 June 1988. Braunschweig, F.R.G; 1990

[76] Dunlop J, Lambert MG, Van Den Bosch J, Caradus JR, Hart AL, Wewala GS, et al. A programme to breed a cultivar of *Trifolium repens* L. for more efficient use of phosphate. In: El Bassam N, Dambroth M, Loughman BC, editors. Genetic Aspects of Plant Mineral Nutrition. Dordrecht: Springer Netherlands; 1990. p. 547-552.

[77] Caradus JR, Mackay AD, Wewala S, Dunlop J, Hart A, Van Den Bosch J, et al. Inheritance of phosphorus response

in white clover (*Trifolium repens* L.). Plant and Soil. 1992;**146**(1):199-208. DOI: 10.1007/BF00012013

[78] Caradus JR. Selection for improved adaptation of white clover to low phosphorus and acid soils. Euphytica. 1994;**77**(3):243-250. DOI: 10.1007/ BF02262637

[79] Caradus JR, Dunn A. Adaptation to low fertility hill country in New Zealand of white clover lines selected for differences in response to phosphorus. New Zealand Journal of Agricultural Research. 2000;**43**(1):63-69. DOI: 10.1080/00288233.2000.9513409

[80] Caradus JR. Selection for root hair length in white clover (*Trifolium repens* L.). Euphytica. 1979;**28**(2):489-494. DOI: 10.1007/BF00056609

[81] Caradus JR. Genetic differences in the length of root hairs in white clover and their effect on phosphorus uptake. In: Scaife A, editor. Plant Nutrition Proceedings of 9th International Plant Nutrition. Plant Nutrition. England: Colloquium, Warwick University; 1982. pp. 84-88

[82] Crush JR, Caradus JR. Effect of mycorrhizas on growth of some white clovers. New Zealand Journal of Agricultural Research. 1980;**23**(2):233-237. DOI: 10.1080/00288233.1980.10430791

[83] Caradus JR, Crush JR, Ouyang L, Fraser W. Evaluation of aluminiumtolerant white clover (*Trifolium repens*) selections on East Otago upland soils. New Zealand Journal of Agricultural Research. 2001;**44**(2-3):141-150. DOI: 10.1080/00288233.2001.9513470

[84] Williams WM, Hussain SW. Development of a breeding strategy for interspecific hybrids between Caucasian clover and white clover.

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

New Zealand Journal of Agricultural Research. 2008;**51**(2):115-126. DOI: 10.1080/00288230809510441

[85] Williams WM. *Trifolium* interspecific hybridisation: Widening the white clover gene pool. Crop and Pasture Science. 2014;**65**(11):1091-1106. DOI: 10.1071/ CP13294

[86] Lloyd DC, Vale JE, Sizer-Coverdale EM, Marshall AH, editors. Interspecific hybridisation of white clover and Caucasian clover confers grazing tolerance. Grassland resources for extensive farming systems in marginal lands: major drivers and future scenarios. In: Proceedings of the 19th Symposium of the European Grassland Federation; 7-10 May 2017; Alghero, Italy. 2017

[87] Gerard PJ, Aalders LT, Hardwick S, Wilson DJ. Investigation into the contrasting production of eight perennial clover cultivars in the first two years at field sites in in Waikato and Canterbury. New Zealand Journal of Agricultural Research. 2022;**65**(4-5):271-289. DOI: 10.1080/00288233.2020.1775657

[88] Widdup KH, Hussain SW, Williams WM, Lowther WL, Pryor HN, Sutherland BL. The development and plant characteristics of interspecific hybrids between white and caucasian clover. In: Moot DJ, editor. Legumes for Dryland Pastures. 11. NZ Grassland Association Research and Practice Series: Lincoln University; 2003. pp. 143-148

[89] Naeem M, Verry IM, Kemp PD, Millner JP, Williams WM. Comparing mating designs to restore seed production of interspecific hybrids between *Trifolium repens* (white clover) and *Trifolium uniflorum*. Plant Breeding. 2017;**136**(3):420- 426. DOI: 10.1111/pbr.12481

[90] Jaramillo DM, Sheridan H, Soder K, Dubeux JCB. Enhancing the sustainability of temperate pasture systems through more diverse swards. Agronomy. 2021;**11**(10):1912. DOI: 10.3390/agronomy11101912

[91] Stewart TA. Utilising clover in grass based animal production systems. Occasional Symposium of the British Grassland Society. 1984;**16**:93-103

[92] Carlton AJ, Cameron KC, Di HJ, Edwards GR, Clough TJ. Nitrate leaching losses are lower from ryegrass/white clover forages containing plantain than from ryegrass/white clover forages under different irrigation. New Zealand Journal of Agricultural Research. 2019;**62**(2):150-172. DOI: 10.1080/00288233.2018.1461659

[93] Hoglund J, Crush J, Brock J, Ball R, Carran R. Nitrogen fixation in pasture. New Zealand Journal of Experimental Agriculture. 1979;**7**:45-51

[94] Ledgard SF. Nitrogen cycling in low input legume-based agriculture, with emphasis on legume/grass pastures. Plant and Soil. 2001;**228**(1):43-59. DOI: https://link.springer.com/article/10. 1023/A:1004810620983

[95] Nesheim L, Boller BC. Nitrogen fixation by white clover when competing with grasses at moderately low temperatures. Plant and Soil. 1991;**133**(1):47-56. DOI: 10.1007/ BF00011898

[96] Seresinhe T, Hartwig UA, Kessler W, Nosberger J. Symbiotic nitrogen fixation of white clover in a mixed sward is not limited by height of repeated cutting. Journal of Agronomy and Crop Science. 1994;**172**(4):279-288. DOI: 10.1111/j.1439-037X.1994.tb00179.x

[97] Chu ACP, Robertson AG. The effects of shading and defoliation on nodulation and nitrogen fixation by white clover.

Plant and Soil. 1974;**41**(3):509-519. DOI: 10.1007/BF02185812

[98] Ledgard SF, Sprosen MS, Steele KW. Nitrogen fixation by nine white clover cultivars in grazed pasture, as affected by nitrogen fertilization. Plant and Soil. 1996;**178**(2):193-203. DOI: 10.1007/ BF00011583

[99] Ledgard SF, Sprosen MS, Penno JW, Rajendram GS. Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization. Plant and Soil. 2001;**229**(2):177-187. DOI: 10.1023/A:1004833804002

[100] Enriquez-Hidalgo D, Gilliland TJ, Hennessy D. Herbage and nitrogen yields, fixation and transfer by white clover to companion grasses in grazed swards under different rates of nitrogen fertilization. Grass and Forage Science. 2016;**71**(4):559-574. DOI: 10.1111/ gfs.12201

[101] Kessler W, Boller BC, Nösberger J. Distinct influence of root and shoot temperature on nitrogen fixation by white clover. Annals of Botany. 1990;**65**(3):341-346. DOI: 10.1093/ oxfordjournals.aob.a087942

[102] Gierus M, Kleen J, Loges R, Taube F. Forage legume species determine the nutritional quality of binary mixtures with perennial ryegrass in the first production year. Animal Feed Science and Technology. 2012;**172**(3):150-161. DOI: 10.1016/j.anifeedsci.2011.12.026

[103] Louarn G, Pereira-Lopès E, Fustec J, Mary B, Voisin A-S, de Faccio Carvalho PC, et al. The amounts and dynamics of nitrogen transfer to grasses differ in alfalfa and white clover-based grass-legume mixtures as a result of rooting strategies and rhizodeposit quality. Plant and Soil.

2015;**389**(1):289-305. DOI: 10.1007/ s11104-014-2354-8

[104] Ulyatt MJ, Lancashire JA, Jones WT. The nutritive value of legumes. Proceedings of the New Zealand Grassland Association. 1976;**38**:107-118. DOI: 10.33584/jnzg.1976.38.1475

[105] Rogers GL, Porter RHD, Robinson IB. Comparison of perennial ryegrass and white cloverfor milk production. In: Proceedings Conference "Dairy Production from Pasture". New Zealand Society of Animal Production; 1982. pp. 213-214

[106] Gibb MJ, Treacher TT. The performance of weaned lambs offered diets containing different proportions of fresh perennial ryegrass and white clover. Animal Science. 1984;**39**(3):413- 420. Epub 2010/09/02. DOI: 10.1017/ S0003356100032141

[107] Dewhurst RJ, Delaby L, Moloney A, Boland T, Lewis E. Nutritive value of forage legumes used for grazing and silage. Irish Journal of Agricultural and Food Research. 2009;**48**(2):167- 187. DOI: https://www.jstor.org/ stable/20720367

[108] Guy C, Hennessy D, Gilliland TJ, Coughlan F, McClearn B, Dineen M, et al. Comparison of perennial ryegrass, *Lolium perenne* L., ploidy and white clover, *Trifolium repens* L., inclusion for herbage production, utilization and nutritive value. Grass and Forage Science. 2018;**73**(4):865-877. DOI: 10.1111/ gfs.12366

[109] Brink GE, Sanderson MA, Casler MD. Grass and legume effects on nutritive value of complex forage mixtures. Crop Science. 2015;**55**(3):1329- 1337. DOI: 10.2135/cropsci2014.09.0666

[110] Harris SL, Auldist MJ, Clark DA, Jansen EBL. Effects of white clover

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

content in the diet on herbage intake, milk production and milk composition of New Zealand dairy cows housed indoors. Journal of Dairy Research. 1998;**65**(3):389-400. DOI: 10.1017/ S0022029998002969

[111] Harris S, Clark D, Jansen E. Optimum white clover content for milk production: New Zealand Society of Animal Production; 1997. 169-71 p.

[112] Papadopoulos YA, Charmley E, McRae KB, Farid A, Price MA. Addition of white clover to orchardgrass pasture improves the performance of grazing lambs, but not herbage production. Canadian Journal of Animal Science. 2001;**81**(4):517-523. DOI: 10.4141/ a97-061

[113] Cranston L, Kenyon PR, Morris ST, Kemp PD. A review of the use of chicory, plantain, red clover and white clover in a sward mix for increased sheep and beef production. Journal of New Zealand Grasslands. 2015;**77**:89-94. DOI: 10.33584/jnzg.2015.77.475

[114] Turner KE, Belesky DP, Fedders JM, Solomon MB. Autumngrazed Orchardgrass-White clover pasture: Nutritive value of herbage and lamb performance. Journal of Production Agriculture. 1998;**11**(1):85- 91. DOI: 10.2134/jpa1998.0085

[115] Cramer DA, Barton RA, Shorland FB, Czochanska Z. A comparison of the effects of white clover (*Trifolium repens*) and of perennial ryegrass (*Lolium perenne*) on fat composition and flavour of lamb. The Journal of Agricultural Science. 1967;**69**(3):367-373. Epub 2009/03/27. DOI: 10.1017/S0021859600019031

[116] Vipond JE, Swift G, Noble RC, Horgan G. Effects of clover in the diet of grazed lambs on production and

carcass composition. Animal Science. 1993;**57**(2):253-261. Epub 2010/09/02. DOI: 10.1017/S0003356100006863

[117] Boufaïed H, Chouinard PY, Tremblay GF, Petit HV, Michaud R, Bélanger G. Fatty acids in forages. I. Factors affecting concentrations. Canadian Journal of Animal Science. 2003;**83**(3):501-511. DOI: 10.4141/ a02-098

[118] Scales GH. Carcass fatness in lambs grazing various forages at different rates of liveweight gain. New Zealand Journal of Agricultural Research. 1993;**36**(2):243-251. DOI: 10.1080/00288233.1993.10417760

[119] Waghorn G, Caradus JR. Screening white clover cultivars for improved nutritive value—development of a method. Proceedings of the New Zealand Grassland Association. 1994;**56**:49-53. DOI: 10.33584/jnzg.1994.56.2117

[120] Husse S, Huguenin-Elie O, Buchmann N, Lüscher A. Larger yields of mixtures than monocultures of cultivated grassland species match with asynchrony in shoot growth among species but not with increased light interception. Field Crops Research. 2016;**194**:1-11. DOI: 10.1016/j. fcr.2016.04.021

[121] Prieto I, Violle C, Barre P, Durand JL, Ghesquiere M, Litrico I. Complementary effects of species and genetic diversity on productivity and stability of sown grasslands. Nat Plants. 2015;**1**(4):15033. Epub 2015/01/01. DOI: 10.1038/nplants.2015.33

[122] Tonn B, Koops D, Schweneker D, Heshmati S, Feuerstein U, Isselstein J. Temporal synchrony of white clover populations with perennial ryegrass affects yield and yield stability of grass-clover mixtures. In: Proceedings of the Joint 20th Symposium of the European Grassland Federation and the 33rd Meeting of the EUCARPIA Section 'Fodder Crops and Amenity Grasses' Zürich, Switzerland 24-27 June 2019. Vol. 24. The Netherlands: Wageningen Academic Publishers; 2019. pp. 81-83

[123] Egan M, Lynch MB, Hennessy D. Including white clover in nitrogen fertilized perennial ryegrass swards: Effects on dry matter intake and milk production of spring calving dairy cows. The Journal of Agricultural Science. 2017;**155**(4):657-668. Epub 2016/11/28. DOI: 10.1017/S0021859616000952

[124] McClearn B, Shalloo L, Gilliland TJ, Coughlan F, McCarthy B. An economic comparison of pasture-based production systems differing in sward type and cow genotype. Journal of Dairy Science. 2020;**103**(5):4455-4465. DOI: 10.3168/ jds.2019-17552

[125] Bouton J. The economic benefits of forage improvement in the United States. Euphytica. 2007;**154**(3):263-270. DOI: 10.1007/s10681-006-9220-6

[126] Caradus JR, Woodfield DR, Stewart AV. Overview and vision for white clover. In: Woodfield D, editor. White Clover: New Zealand's Competitive Edge. NZ Grassland Association Grassland Research and Practice Series 6:1-6 6. New Zealand: Lincoln University; 1995

[127] Nixon C. How valuable is that plant species? Application of a method for enumerating the contribution of selected plant species to New Zealand's GDP. NZIER report to the Ministry for Primary Industries. MPI Technical Paper No: 2016/62. 2016:227

[128] Dairy industry payout history. https://www.interest.co.nz/ruraldata/dairy-industry-payout-history [Internet]. Available from: https:// www.interest.co.nz/rural-data/dairyindustry-payout-history [Accessed: 2 December 2022]

[129] Dodd M, Tozer K, Vogeler I, Greenfield R, Stevens D, Rhodes T, et al. Quantifying the value proposition for white clover persistence on a New Zealand summer-dry hill-country farm. Journal of New Zealand Grasslands. 2020;**82**:199-209. DOI: 10.33584/ jnzg.2020.82.2973

[130] Woodfield DR, Brummer EC, editors. Integrating Molecular Techniques to Maximise the Genetic Potential of Forage Legumes. Molecular Breeding of Forage Crops. Dordrecht: Springer Netherlands; 2001

[131] Jahufer MZZ, Cooper M, Ayres JF, Bray RA. Identification of research to improve the efficiency of breeding strategies for white clover in Australia—A review. Australian Journal of Agricultural Research. 2002;**53**(3):239-257. DOI: 10.1071/ ar01110

[132] Zhang Y, Sledge MK, Bouton JH. Genome mapping of white clover (*Trifolium repens* L.) and comparative analysis within the Trifolieae using cross-species SSR markers. Theoretical and Applied Genetics. 2007;**114**(8):1367-1378. DOI: 10.1007/s00122-007-0523-3

[133] Ehoche GAS, Larking A, Jauregui R, Cousins G, O'Connor J, Jahufer Z, et al. Developing genomic selection for dry matter yield in white clover. Journal of New Zealand Grasslands. 2021;**83**:79-90. DOI: 10.33584/jnzg.2021.83.3502

[134] Ehoche OG. Assessing the potential of genomic selection to improve yield and persistence in white clover: a thesis presented in the partial

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Biology at Massey University, Manawatu, New Zealand [Doctoral]: Massey University; 2020

[135] Lukjanová E, Řepková J. Chromosome and genome diversity in the genus Trifolium (Fabaceae). Plants (Basel). 2021;**10**(11). Article ID: 2518. Epub 2021/11/28. DOI: 10.3390/ plants10112518

[136] Barrett BA, Baird IJ, Woodfield DR. A QTL analysis of White clover seed production. Crop Science. 2005;**45**(5):1844-1850. DOI: 10.2135/ cropsci2004.0679

[137] Barrett BA, Baird IJ, Woodfield DR. Genetic tools for increased white clover seed production. Proceedings of the New Zealand Grassland Association. 2004;**66**:119-126

[138] Ballizany WL. Novel markers for drought resistance in white clover [doctoral dissertation]. Lincoln University; 2011

[139] Casler MD, Brummer EC. Theoretical expected genetic gains for among-and-within-family selection methods in perennial forage crops. Crop Science. 2008;**48**(3):890-902. DOI: 10.2135/cropsci2007.09.0499

[140] Goddard ME, Hayes BJ. Genomic selection. Journal of Animal Breeding Genetics. 2007;**124**:323-330

[141] Knight R. Quantitative genetics, statistics and plant breeding. In: A 'Course Manual in Plant Breeding'. Australia: Australian Vice-Chancellors' Commitee; 1979

[142] Voisey CR, White DWR, Dudas B, Appleby RD, Ealing PM, Scott AG. Agrobacterium-mediated transformation of white clover using direct shoot organogenesis. Plant Cell Reports. 1994;**13**(6):309-314. DOI: 10.1007/bf00232627

[143] McManus MT, Laing WA, Watson LM, Markwick N, Voisey CR, White DWR. Expression of the soybean (Kunitz) trypsin inhibitor in leaves of white clover (*Trifolium repens* L.). Plant Science. 2005;**168**(5):1211-1220. DOI: 10.1016/j.plantsci.2004.12.020

[144] Panter S, Chu PG, Ludlow E, Garrett R, Kalla R, Jahufer MZZ, et al. Molecular breeding of transgenic white clover (*Trifolium repens* L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene. Transgenic Research. 2012;**21**(3):619- 632. DOI: 10.1007/s11248-011-9557-z

[145] Ludlow EJ, Mouradov A, Spangenberg GC. Post-transcriptional gene silencing as an efficient tool for engineering resistance to white clover mosaic virus in white clover (*Trifolium repens*). Journal of Plant Physiology. 2009;**166**(14):1557-1567. Epub 2009/08/08. DOI: 10.1016/j. jplph.2009.07.001

[146] Woodfield DR, White DWR. Breeding strategies for developing transgenic white clover cultivars. In: Woodfield DR, editor. White Clover: New Zealand's Competitive Edge. 6. NZ Grassland Association Grassland Research and Practice Series: Lincoln University New Zealand; 1995. pp. 125-130

[147] Ma XF, Wright E, Ge Y, Bell J, Xi Y, Bouton JH, et al. Improving phosphorus acquisition of white clover (*Trifolium repens* L.) by transgenic expression of plant-derived phytase and acid phosphatase genes. Plant Science. 2009;**176**(4):479-488. DOI: 10.1016/j. plantsci.2009.01.001

[148] Hancock KR, Collette V, Fraser K, Greig M, Xue H, Richardson K, et al. Expression of the R2R3-MYB transcription factor TaMYB14 from T*rifolium arvense* activates proanthocyanidin biosynthesis in the legumes *Trifolium repens* and *Medicago sativa*. Plant Physiology. 2012;**159**(3):1204- 1220. DOI: 10.1104/pp.112.195420

[149] Jiang Q, Zhang J-Y, Guo X, Bedair M, Sumner L, Bouton J, et al. Improvement of drought tolerance in white clover (*Trifolium repens*) by transgenic expression of a transcription factor gene WXP1. Functional Plant Biology. 2010;**37**(2):157-165. DOI: 10.1071/FP09177

[150] Bibbee A. Green Growth and Climate Change Policies in New Zealand OECD Economics Department Working Papers. Vol. 893. 36 p. Paris [Internet]: OECD Publishing; 2011. DOI: 10.1787/5kg51mc6k98r-en

[151] Muller A, Jawtusch J, Gattinger A. Mitigating Greenhouse Gases in Agriculture. Research Institute of Organic Agriculture; 2011. p. 88

[152] Garnett T, Godde C, Muller A, Röös E, Smith P, de Boer IJM, et al. Grazed and Confused? Ruminating on Cattle, Grazing Systems, Methane, Nitrous Oxide, the Soil Carbon Sequestration Question – and What It All Means for Greenhouse Gas Emissions. Food Climate Research Network (FCRN). Environmental Change Institute, University of Oxford; 2017. p. 127

[153] Harrison MT, Cullen BR, Mayberry DE, Cowie AL, Bilotto F, Badgery WB, et al. Carbon myopia: The urgent need for integrated social, economic and environmental action in the livestock sector. Global Change Biology. 2021;**27**:5726-5761. DOI: 10.1111/ gcb.15816

[154] Reinsch T, Malisch C, Loges R, Taube F. Nitrous oxide emissions from grass–clover swards as influenced by sward age and biological nitrogen fixation. Grass and Forage Science. 2020;**75**(4):372-384. DOI: 10.1111/ gfs.12496

[155] Waghorn GC, Ulyatt MJ, John A, Fisher MT. The effect of condensed tannins on the site of digestion of amino acids and other nutrients in sheep fed on lotus *Corniculatus* L. British Journal of Nutrition. 1987;**57**(1):115-126. DOI: 10.1079/BJN19870015

[156] Waghorn G. Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production—Progress and challenges. Animal Feed Science and Technology. 2008;**147**(1-3):116-139

[157] Woodfield D, McNabb W, Kennedy L, Cousins G, Caradus J, editors. Floral and Foliar Tannin Content in White Clover. Proceedings of the Fifteenth Trifolium Conference; 1998; Madison, Wisconsin 1998

[158] Hancock K, Collette V, Chapman E, Hanson K, Temple S, Moraga R, et al. Progress towards developing bloat-safe legumes for the farming industry. Crop and Pasture Science. 2014;**65**(11): 1107-1113. DOI: 10.1071/CP13308

[159] Woodfield DR, Roldan MB, Voisey CR, Cousins GR, Caradus JR. Improving environmental benefits of white clover through condensed tannin expression. Journal of New Zealand Grasslands. 2019;**81**:195-202. DOI: 10.33584/jnzg.2019.81.382

[160] Roldan MB, Cousins G, Muetzel S, Zeller WE, Fraser K, Salminen J-P, et al. Condensed tannins in White clover (*Trifolium repens*) foliar tissues expressing the transcription factor TaMYB14-1 bind to forage protein

*White Clover (*Trifolium repens *L.) Benefits in Grazed Pastures and Potential Improvements DOI: http://dx.doi.org/10.5772/intechopen.109625*

and reduce ammonia and methane emissions in vitro. Frontiers in Plant Science. 2022;**12**. Article ID: 777354. DOI: 10.3389/fpls.2021.777354

[161] Caradus JR, Voisey CR, Cousin GR, Kaur R, Woodfield DR, Blanc A, et al. The hunt for the "holy grail": Condensed tannins in the perennial forage legume white clover (*Trifolium repens* L.). Grass and Forage Science. 2022;**77**(2):111-123. DOI: 10.1111/gfs.12567

[162] Widdup KH, Ford JL, Cousins GR, Woodfield DR, Caradus JR, Barrett BA. A comparison of New Zealand and overseas white clover cultivars under grazing in New Zealand. Journal of New Zealand Grasslands. 2015;**77**:51-56. DOI: 10.33584/jnzg.2015.77.483

[163] Woodfield DR, Clifford PTP, Baird IJ, Cousins GR, Miller JE, Widdup KH, et al. Grasslands tribute: A multi-purpose white clover for Australasia. Proceedings of the New Zealand Grassland Association. 2003;**65**:157-162. DOI: 10.33584/ jnzg.2003.65.2488

[164] Shi S, Villamizar L, Gerard E, Ronson C, Wakelin S, Ballard R, et al. Increasing biological nitrogen fixation by white clover-rhizobia symbiosis. Journal of NZ Grasslands. 2019;**81**:231-234. DOI: 10.33584/jnzg.2019.81.380

[165] Irisarri P, Cardozo G, Tartaglia C, Reyno R, Gutiérrez P, Lattanzi FA, et al. Selection of competitive and efficient rhizobia strains for white clover. Frontiers in Microbiology. 2019;**10**(768). DOI: 10.3389/fmicb.2019.00768

[166] Ferguson S, Major AS, Sullivan JT, Bourke SD, Kelly SJ, Perry BJ, et al. Rhizobium leguminosarum bv. Trifolii NodD2 enhances competitive nodule colonization in the clover-Rhizobium Symbiosis. Applied and Environmental

Microbiology. 2020;**86**(18):e01268-e01220. DOI: 10.1128/AEM.01268-20

[167] Young SD, van Koten C, Gray CW, Cavanagh JAE, Wakelin SA. Symbiosis between *Rhizobium leguminosarum* bv. *trifolii* strain TA1 and a white clover cultivar benefits clover tolerance to cadmium toxicity. New Zealand Journal of Agricultural Research. 2020;**63**(3):353-364. DOI: 10.1080/00288233.2019.1680394

[168] Weith SK, Jahufer MZZ, Hofmann RW, Anderson CB, Luo D, Ehoche OG, et al. Quantitative genetic analysis reveals potential to breed for improved white clover growth in symbiosis with nitrogen-fixing Rhizobium bacteria. Frontiers in Plant Science. 2022;**13**:953400. Epub 2022/10/11. DOI: 10.3389/ fpls.2022.953400

[169] Xie MM, Zou YN, Wu QS, Zhang ZZ, Kuča K. Single or dual inoculation of arbuscular mycorrhizal fungi and rhizobia regulates plant growth and nitrogen acquisition in white clover. Plant, Soil and Environment. 2020;**66**(6):287-294. DOI: 10.17221/234/2020-PSE

[170] Li X-L, George E, Marschner H. Extension of the phosphorus depletion zone in VA-mycorrhizal white clover in a calcareous soil. Plant and Soil. 1991;**136**(1):41-48. DOI: 10.1007/ BF02465218

[171] Baylis GTS. Experiments on the ecological significance of phycomycetous mycorrhizas. New Phytologist. 1967;**66**(2):231-243. DOI: 10.1111/j.1469- 8137.1967.tb06001.x

[172] Mosse B. Plant growth responses to vesicular-arbuscular mycorrhizas IV. In soil given additional phosphate. New Phytologist. 1973;**72**(1):127-136. DOI: 10.1111/j.1469-8137.1973.tb02017.x [173] Hall IR. Effects of endomycorrhizas on the competitive ability of white clover. New Zealand Journal of Agricultural Research. 1978;**21**(3):509-515. DOI: 10.1080/00288233.1978.10427441

[174] Tuo X-Q, He L, Zou Y-N. Alleviation of drought stress in white clover after inoculation with arbuscular mycorrhizal fungi. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2017;**45**(1):220-224. DOI: 10.15835/ nbha45110709

[175] Rogers JB, Scott Laidlaw A, Christie P. The role of arbuscular mycorrhizal fungi in the transfer of nutrients between white clover and perennial ryegrass. Chemosphere. 2001;**42**(2):153-159. DOI: 10.1016/ S0045-6535(00)00120-X

[176] Janarthanam L. Bioprotectant with multifunctional microorganisms: A new dimension in plant protection. Journal of Biopesticides. 2013;**6**(2):219-230

[177] Pole A, Srivastava A, Zakeel MCM, Sharma VK, Suyal DC, Singh AK, et al. 12— Role of microbial biotechnology for strain improvement for agricultural sustainability. In: Soni R, Suyal DC, Yadav AN, Goel R, editors. Trends of Applied Microbiology for Sustainable Economy. Academic Press; 2022. pp. 285-317

[178] Sharma B, Kumawat KC. Functional diversity of microbes in rhizosphere: A key player for soil health conservation under changing climatic conditions. In: Vaishnav A, Arya SS, Choudhary DK, editors. Plant Stress Mitigators: Action and Application. Singapore: Springer Nature Singapore; 2022. pp. 469-493

[179] Matse DT, Huang C-H, Huang Y-M, Yen M-Y. Effects of coinoculation of Rhizobium with plant growth promoting rhizobacteria on the nitrogen fixation and nutrient uptake of Trifolium repens

in low phosphorus soil. Journal of Plant Nutrition. 2020;**43**(5):739-752. DOI: 10.1080/01904167.2019.1702205

[180] Martin G, Durand J-L, Duru M, Gastal F, Julier B, Litrico I, et al. Role of ley pastures in tomorrow's cropping systems. A review. Agronomy for Sustainable Development. 2020;**40**(3):17. DOI: 10.1007/s13593-020-00620-9

[181] Stein S, Hartung J, Möller K, Zikeli S. The effects of leguminous living mulch intercropping and its growth management on organic cabbage yield and biological nitrogen fixation. Agronomy. 2022;**12**(5):1009. DOI: 10.3390/agronomy12051009

[182] Deguchi S, Uozumi S, Touno E, Kaneko M, Tawaraya K. Arbuscular mycorrhizal colonization increases phosphorus uptake and growth of corn in a white clover living mulch system. Soil Science and Plant Nutrition. 2012;**58**(2):169-172. DOI: 10.1080/00380768.2012.662697

[183] Deguchi S, Shimazaki Y, Uozumi S, Tawaraya K, Kawamoto H, Tanaka O. White clover living mulch increases the yield of silage corn via arbuscular mycorrhizal fungus colonization. Plant and Soil. 2007;**291**(1):291-299. DOI: 10.1007/s11104-007-9194-8

#### **Chapter 7**
