White Clover Plants Research Articles

In Vitro Screening of Endophytic Micromonospora Strains Associated with White Clover for Antimicrobial Activity against Phytopathogenic Fungi and Promotion of Plant Growth

Bacteria belonging to the genus Micromonospora are recognized as microorganisms with the potential to be used in biotechnology processes, given their beneficial influence on plant growth and the biocontrol of phytopathogens. In this study, nineteen Micromonospora isolates originating from the root nodules of white clover plants were taxonomically assigned based on the phylogenetic analysis of the 16S rRNA gene and four housekeeping genes. The antifungal properties of the bacteria against phytopathogenic Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Sclerotinia sclerotiorum, and Verticillium albo-atrum were tested with the agar plug test and the dual culture test. The ability to produce various metallophores was determined with the agar plug diffusion test on modified chrome azurol S (CAS) agar medium. International Streptomyces Project-2 medium (ISP2) broth amended with 0.2% L-tryptophan was used to indicate the bacterial ability to produce auxins. The strains belonging to M. tulbaghiae, M. inaquosa, and M. violae showed in vitro potential as antimicrobial agents against the tested fungi. M. inaquosa strain 152, M. violae strain 126, M. violae strain 66, and M. violae strain 45 were recognized as the most efficient metallophore producers. M. alfalfae strain 55 and M. lupini strain 5052 were identified as the most promising auxin compound producers and, therefore, show potential as plant-growth-promoting bacteria.

ASSESSMENT OF HEAVY METALS ACCUMULATION IN PERENNIAL LEGUMES (WHITE CLOVER AND RED CLOVER) IN THE COPȘA MICĂ AREA, ROMANIA

The impact of heavy metals on perennial legumes, just like on other plant species, can be detrimental and has several ecological and agricultural implications. The ability of perennial legumes to fix atmospheric nitrogen through symbiotic interactions with nitrogen-fixing bacteria in their root nodules makes them a significant group of plants. The present study aims to evaluate the total concentration of heavy metals (cadmium, lead, copper, and zinc) in red and white clover plants harvested from grasslands located in heavily polluted areas (Copșa Mică, Axente Sever, Valea Viilor, Micăsasa and Târnava). White clover (Trifolium repens L.) recorded Cd content values between 0.06 - 3.53 mg/kg dry weight, Cu had values between 4.09 - 33.4 mg/kg dry weight, Pb content was between 0.18 - 4.77 mg/kg dry weight and Zn had values between 38 - 186 mg/kg dry weight. Regarding red clover (Trifolium pratense L.) the heavy metal content values were as follows: for Cd (0.08 - 3.38 mg/ kg d.w), for Cu (6.03 - 13.5 mg/kg d.w), for Pb (0.13 – 2.34 mg/kg d.w), for Zn (33.1 - 169 mg/kg d.w). Our results show that clover species harvested from grasslands in these geographical areas have high levels of heavy metals due to metallurgical activities. Grazing in these areas affected by heavy metal pollution can pose serious health problems for animals and later for humans through consumption of their products.

Urban spatial heterogeneity shapes the evolution of an antiherbivore defense trait and its genes in white clover

Urbanization is a global threat to biodiversity due to its large impact on environmental changes. Recently, urban environmental change has been shown to impact the evolution of many species. However, much remains unknown about how urban environments influence evolutionary processes and outcomes due to the non‐linearity and discontinuity of environmental variables along urban–rural gradients. Here, we focused on the evolution of hydrogen cyanide (HCN) production and its components (presence/absence of cyanogenic glycosides and the hydrolytic enzyme linamarase) in the herbaceous plant white clover Trifolium repens, which thrive in both urban and rural areas. To comprehensively elucidate how plants evolve and adapt to heterogenous urban environments, we collected 3299 white clover plants from 122 populations throughout Sapporo, Japan. We examined the spatial variation in environmental factors, such as herbivory, sky openness, impervious surface cover, snow depth, and temperature, and how variation in these factors was related to the production of HCN, cyanogenic glycosides, and linamarase. Environmental factors showed complex spatial variation due to the heterogeneity of the urban landscape. Among these factors, herbivory, sky openness, and impervious surface cover were highly related to the frequency of plants producing HCN in populations. We also found that impervious surface cover was related to the frequency of plants producing cyanogenic glycosides, while herbivory pressure was not. As a result, the cyanogenic glycoside frequency showed a clearer trend along urban–rural gradient rather than HCN frequency, and thus, the predicted spatial distributions of HCN and cyanogenic glycosides were inconsistent. These results suggest that urban landscape heterogeneity and trait multifunctionality determines mosaic‐like spatial distribution of evolutionary traits.

Biological and Molecular Characterization of Clover Yellow Vein Virus Infecting Trifolium repens in China

White clover (Trifolium repens L.) is an important perennial legume forage and ornamental plant, and is widely distributed and cultivated in the world. Recently, white clover plants showing symptoms of leaf mosaic and redding were observed in Hohhot, Inner Mongolia of China. In this work, flexuous filamentous viral particles of about 700 × 13 nm in size were observed in the symptomatic leaf samples. The infection of clover yellow vein virus (ClYVV) was confirmed by small RNA sequencing and RT-PCR validation. Mechanical inoculation assays showed that this ClYVV isolate (ClYVV-IM) can infect a range of herbaceous species, including Nicotiana benthamiana, N. occidentalis, Chenopodium quinoa, C. amaranticolor, Vicia faba, Vigna unguiculata, and Solanum lycopersicum, causing various symptoms. The complete genome sequence of ClYVV-IM consists of 9565 nt and shared sequence identities, ranging from 83.05% to 96.30%, with those of the other ClYVV isolates published in GenBank. Phylogenetic analyses based on the polyprotein nucleotide and amino acid sequences clustered 15 ClYVV isolates into two groups and ClYVV-IM located in Group I. Two potential recombination events located at 914–2970 nt and 5153–5694 nt were detected in the genome of ClYVV-IM. To our knowledge, this is the first report of occurrence and complete genome of ClYVV infecting white clover in China.

White clover (Trifolium repens) population dynamics are partly dependent on timing of seminal taproot death

AbstractThe expanded usage of white clover has increased the importance of understanding white clover dynamics in pastures. It is assumed that clover plants have a higher tolerance for moisture and nutrient deficiencies when the taproot is still present. Therefore, the survival of the seminal taproot can influence the dynamics of clover. Past breeding efforts in countries like New Zealand have focussed on increasing the taproot longevity through hybridisation with a close relative of white clover. However, there is no direct evidence whether increased survival of the taproot results in increased performance of white clover. In this study, we aimed to (i) assess the relationship between taproot volume and taproot survival, and (ii) whether the timing of death of the seminal taproot influences the population dynamics of white clover varieties. In a two‐year field experiment with 18 white clover varieties grown in monoculture and in mixture with Lolium perenne L, the taproot characteristics and population dynamics were studied. It was shown that taproot volume was positively correlated to both leaf size and taproot presence during autumn 2017, 1 year after sowing. The combination of the timing of death of the seminal tap root and the development of stolons seems to play a more important role in increasing the persistence of white clover than the absolute survival of the seminal taproot. Future research should focus on understanding the transition from a taprooted white clover to a stolonous white clover plant in relation to specific weather events such as winter frost conditions.

First Report of Powdery Mildew Caused by Erysiphe polygoni on Trifolium repens in China.

White clover (Trifolium repens L.) belongs to the Fabaceae family legume and is cultivated in China for its medicinal properties and ornamental value. White clover is grown around the world for forage, turf , green manure and soil conservation purposes (Zhang el al. 2016). In October 2021, an investigation of a 1,000 m2 plant nursery in Lanzhou, China (36°06'N, 103°83'E) found that 80% of White clover plants were infected, and powdery mildew covered 95% of the leaf area. The disease had seriously destroyed the forage quality and reduced the ornamental value. Initially, thin, radial, irregular white colonies appeared on leaves and gradually spread to stems. The white colonies then expanded and thickened to cover upper surface of the leaf, and microscopic hyphae appeared on the bottom of the leaf. In severe cases, the infection resulted in dieback of the leaf. A small area of sporulating fungus was stripped off from the leaf surface with tape and mounted in sterile water for microscopic examination (Mukhtar et al. 2017). Conidiophores were cylindrical, consisting of a foot cell followed by three to four short cells, measuring 75 to 160 × 7 to 10 μm. Conidiophores had straight, cylindric foot cells ranging from 25 to 40 µm long. Singly produced conidia were hyaline and ranged in shape from oblong to cylindrical. Conidia lacked distinct fibrotic bodies and measured 30 to 45 × 15 to 25 μm in length. Long, unbranched germ tubes formed from the ends of the conidia and nipple-shaped appressoria developed on epiphytic mycelia. Based on these morphological characteristics, the pathogen was initially identified morphologically as Erysiphe polygoni (Braun and Cook 2012). To validate the identity, the internal transcribed spacer (ITS) region of the pathogen (SY77) rDNA was amplified by PCR and sequenced using the ITS1/ITS4 primers (White et al. 1990). The resulting sequences were registered to GenBank (GenBank Accession No.OM280998). The ITS sequence of the SY77 was 100% (640/640) identical to E. polygoni (LC009892) on Polygonum aviculare in the United Kingdom and 99% (638/640) identical to E. polygoni (MK685172) on Antigonon leptopus in Taiwan. MEGA 7.0 was used to conduct the neighbor-joining phylogenetic analysis using the ITS sequences from GenBank. The data indicated that the strain SY77 and E. polygoni clustered together on the same branch. Pathogenicity tests were conducted by gently pressing the infected leaves onto five healthy potted White clover plants, while five non-inoculated plants were used as controls (Michael et al. 2021). The plants were maintained in a growth chamber (25 ℃, 14 h light, and 10 h dark period, RH > 80%). After 10 days, the inoculated plants developed powdery mildew symptoms, whereas the control plants remained symptom-free. The fungus on the inoculated plants was re-isolated, re-identified, and confirmed as E. polygoni based on morphological observations and molecular identification. There is no previous report on E. polygoni causing powdery mildew on White clover in China. The powdery mildew caused by E. polygoni on Red clover has been reported in China and Bulgaria, respectively (Yuan el al.1991; Galina el al. 2017). To our knowledge, this is the first report of powdery mildew caused by E. polygoni on White clover in China.

Metabolic Regulation and Lipidomic Remodeling in Relation to Spermidine-induced Stress Tolerance to High Temperature in Plants.

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 μM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsis thaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsis thaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsis thaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

Arbuscular mycorrhizal fungi reduce potassium, cadmium and ammonium losses but increases nitrate loss under high intensity leaching events

BackgroundNutrients and heavy metals can be lost from soils via leaching, and arbuscular mycorrhizal fungi (AMF) can influence these events. Soil column experiments were carried out to examine whether leaching intensity and AMF can alter nutrient and Cd uptake in white clover plants and the extent of their losses through leaching.ResultsThe presence of AMF significantly increased shoot and total biomass, as well as increased N, P, Cu and Zn uptake independent of water amount applied; while root P and Cu uptakes were promoted by AMF at any water amount treatments. Higher water amounts led to reductions in total N, K and Zn uptake for AMF-colonized plants in comparison to moderate water amount treatments. In the absence of AMF, white clover at low water amount treatment exhibited maximal root Cd uptake. At high water amount treatments, the presence of AMF significantly decreased leachate volumes and the amount of leached NH4+, K and Cd while AMF significantly increased the amounts of leached NO3−.ConclusionsOverall we found that AMF-colonized white clover plants reduced NH4+, K and Cd loss from soils but increased the risk of NO3− loss under high intensity leaching conditions.

Global urban environmental change drives adaptation in white clover.

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

First report of Soybean dwarf virus infecting white clover (Trifolium repens) in Finland.

Soybean dwarf virus (SbDV, genus Luteovirus) is a single-stranded positive-sense RNA virus able to infect several legume species. SbDV was first reported in Japan where it was associated with significant yield losses in soybean (Tamada, 1969). Since then the virus has been detected worldwide. In Europe, the virus has only been reported from Germany (Abraham et al. 2007; Gaafar et al. 2020). In July 2018, several white clover plants (Trifolium repens L.) with leaf discoloration were observed in different locations in Oulu region in northern Finland. Individual plants were collected and analysed for the presence of viruses using small-RNA (sRNA) sequencing (Kreuze et. al. 2009) and reverse transcription-PCR (RT-PCR). Total RNA was extracted using EZNA micro RNA kit (Omega Bio-Tek, GA, USA). For sRNA analysis, sequencing libraries were constructed using the TruSeq small RNA library prep kit (Illumina, CA, USA) and sequenced on Illumina MiSeq platform. On average, 1.3 million single-end reads were obtained per sample, of which 27% were 18-25 nt long and used for the subsequent analysis. Contig assembly and virus identification with VirusDetect software (Zheng et al. 2017) detected SbDV in five out of six white clover samples analysed. Depending on the sample, 26-39 contigs (with lengths up to 301-469 nt) aligned to complete genome of a SbDV isolate previously described from white clover in USA (accession no. JN674402). The cumulative alignment coverage ranged from 35.5 % to 65.3 % with nucleotide identities between 94.4 % and 97.3 %. Additionally, two samples seemed to contain an unidentified closterovirus and one contained White clover cryptic virus 2. No additional viruses were detected from two of the samples.To confirm the presence of SbDV, the samples were tested by RT-PCR using primers MDF, MYF and MUR in multiplex (Schneider et al. 2011) together with SuperScript III One-Step RT-PCR System with the Platinum Taq DNA polymerase kit (Thermo Fisher Scientific, USA), essentially as instructed by the manufacturer. RT-PCR product of approximately 400 bp was produced from each of the five samples previously tested SbDV positive by sRNA analysis. No products were produced from the sample that was SbDV negative in sRNA analysis. Direct sequencing of two of the PCR products produced 347 and 361 bp sequences (GenBank: MZ355392 and MW929169) that were 95.7 % and 95.2 % identical, respectively, to a SbDV isolate (accession no. AB038148) that causes yellowing on soybean and is transmitted by Acyrthosiphon pisum (Terauchi et al. 2003). To our knowledge this is the first report of SbDV in Finland. SbDV is transmitted only by aphids (neither mechanical nor seed transmission occurs). In siRNA analysis all the isolates from Finland formed contigs that aligned almost perfectly (100 % coverage with ≥ 99 % nucleotide identity) to the coat protein (accession no. EF466131) of an SbDV isolate transmittable from white clover to faba bean by A. pisum (Abraham et al. 2007), an aphid common in Finland. Although significant yield losses by SbDV have only been reported on soybean (Tamada, 1969), the virus also causes symptoms in other legume crops, such as growth reduction on pea (Tian et al. 2017) and faba bean (Abraham et al. 2007), both of which are cultivated in Finland.

Alterations of Endogenous Hormones, Antioxidant Metabolism, and Aquaporin Gene Expression in Relation to γ-Aminobutyric Acid-Regulated Thermotolerance in White Clover.

Persistent high temperature decreases the yield and quality of crops, including many important herbs. White clover (Trifolium repens) is a perennial herb with high feeding and medicinal value, but is sensitive to temperatures above 30 °C. The present study was conducted to elucidate the impact of changes in endogenous γ-aminobutyric acid (GABA) level by exogenous GABA pretreatment on heat tolerance of white clover, associated with alterations in endogenous hormones, antioxidant metabolism, and aquaporin-related gene expression in root and leaf of white clover plants under high-temperature stress. Our results reveal that improvement in endogenous GABA level in leaf and root by GABA pretreatment could significantly alleviate the damage to white clover during high-temperature stress, as demonstrated by enhancements in cell membrane stability, photosynthetic capacity, and osmotic adjustment ability, as well as lower oxidative damage and chlorophyll loss. The GABA significantly enhanced gene expression and enzyme activities involved in antioxidant defense, including superoxide dismutase, catalase, peroxidase, and key enzymes of the ascorbic acid–glutathione cycle, thus reducing the accumulation of reactive oxygen species and the oxidative injury to membrane lipids and proteins. The GABA also increased endogenous indole-3-acetic acid content in roots and leaves and cytokinin content in leaves, associated with growth maintenance and reduced leaf senescence under heat stress. The GABA significantly upregulated the expression of PIP1-1 and PIP2-7 in leaves and the TIP2-1 expression in leaves and roots under high temperature, and also alleviated the heat-induced inhibition of PIP1-1, PIP2-2, TIP2-2, and NIP1-2 expression in roots, which could help to improve the water transportation and homeostasis from roots to leaves. In addition, the GABA-induced aquaporins expression and decline in endogenous abscisic acid level could improve the heat dissipation capacity through maintaining higher stomatal opening and transpiration in white clovers under high-temperature stress.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

First report of Leaf Spot Associated with Boeremia exigua on White Clover in China.

White clover (Trifolium repens L.) is an important perennial legume forage widely cultivated in China (Zhang et al. 2016). In April 2018, severe necrotic lesions on leaves were observed in a cultivated white clover field in Chongqing, China. Approximately 90% of plants in the field were affected. Leaf spots were amphigenous, dark-brown, elliptic to subcircular, with diameter ranging between 1 to 12 mm, well defined by brown margins and yellow halos. Severely infected leaves became withered and abscised. Stems and flowers were not affected by the disease. Symptomatic leaves were surface sterilized with 70% ethanol for 30 s followed by 0.1% HgCl2 treatment for 3 min, and rinsed in sterile water three times. Thereafter, tissue samples from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/L of chloramphenicol and incubated at 25℃ in the dark. An olivaceous gray fungal colony was consistently isolated (90.5% isolation frequency). After 15 days of incubation, subglobose, black pycnidia developed in the cultural medium. Conidia were hyaline, ellipsoid to oblong, nonseptate(n = 50), ranging from 4.0 to 7.5 μm long (5.6 ± 2.3µm) × 2.0 to 3.8 μm wide(2.8 ± 1.0 µm). On the basis of its morphological characteristics, the fungus was identified as Boeremia sp. (Aveskamp et al. 2010). To confirm the identity, the internal transcribed spacer region (ITS), large subunit ribosomal RNA (LSU), partial actin (ACT), RNA polymerase II second largest subunit (rpb2) and beta-tubulin (tub2) genes were amplified with primers ITS1/ITS4, LR0R/LR7, ACT-512F/ACT-783R, RPB2-5F2/fRPB2-7cR, and Btub2Fd/Btub4Rd, respectively, in eight representative isolates and sequenced(Aveskamp et al. 2009; Chen et al. 2015). BLAST results showed 100% identity of the ITS (506/506 nucleotides), LSU (966/966 nucleotides), ACT (244/244 nucleotides) and tub2 (297/297 nucleotides) sequences with those of B. exigua (KY419536, MK398746, EU880878, and MK514090) and 99.83% identity of those of the rpb2 (593/594 nucleotides) sequence with B. exigua (KT389572).Based on morphology and DNA sequence analysis, the associated fungus was identified as B. exigua. Representative sequences of one isolate (BT2-1) were deposited in GenBank (MN826339, MN836592, MT265217, MT265218 and MT265219). In a pathogenicity test, ten 2-month-old potted white clover plants were spray-inoculated with a spore and mycelial suspension (approximately 105 CFU/mL) and the control plants were inoculated with sterile distilled water. Plants were incubated in a greenhouse at 20 to 24°C under natural light and enclosed in plastic bags for the first 3 days to maintain high humidity. After 10 days, typical dark-brown lesions similar to those seen in naturally infected leaves developed on the inoculated leaves and not on the control plants. B. exigua was reisolated from the lesions, thus completing Koch's postulates. There is some evidence that B exigua is capable of invading seedling root tissue of white clover and causing necrotic lesions on roots (Skipp and Christensen,1982). However, to our knowledge, this is the first report of leaf spot on T. repens caused by B. exigua. This disease severely reduces forage quality and yield. Proper identification of the causal organism is essential in formulating management strategies.

Greenotyper: Image-Based Plant Phenotyping Using Distributed Computing and Deep Learning.

Image-based phenotype data with high temporal resolution offers advantages over end-point measurements in plant quantitative genetics experiments, because growth dynamics can be assessed and analysed for genotype-phenotype association. Recently, network-based camera systems have been deployed as customizable, low-cost phenotyping solutions. Here, we implemented a large, automated image-capture system based on distributed computing using 180 networked Raspberry Pi units that could simultaneously monitor 1,800 white clover (Trifolium repens) plants. The camera system proved stable with an average uptime of 96% across all 180 cameras. For analysis of the captured images, we developed the Greenotyper image analysis pipeline. It detected the location of the plants with a bounding box accuracy of 97.98%, and the U-net-based plant segmentation had an intersection over union accuracy of 0.84 and a pixel accuracy of 0.95. We used Greenotyper to analyze a total of 355,027 images, which required 24–36 h. Automated phenotyping using a large number of static cameras and plants thus proved a cost-effective alternative to systems relying on conveyor belts or mobile cameras.

Nitrogen uptake and growth of white clover inoculated with indigenous and exotic Rhizobium strains

Little information is available on investigating the effectiveness of indigenous and exotic rhizobia in enhancing nitrogen uptake and growth of white clover. This study aimed to evaluate the nitrogen fixation effectiveness of native rhizobia isolated from white clover grown in Taiwan as compared to an exotic strain, Rhizobium leguminosarum bv. trifolii BCRC 14266. Indigenous rhizobia were isolated from nodules in roots of white clover, and 16S rRNA gene sequences were used to identify their taxonomy. To compare the symbiotic effectiveness of indigenous and exotic rhizobia, white clover plants were inoculated respectively with two indigenous isolates (CHB 1120 and CHB 1121), BCRC 14266, and a non-inoculated control. Based on 16S rRNA gene sequences, CHB 1120 and CHB 1121 could be identified as Rhizobium species. Compared with the non-inoculated control and BCRC 14266, CHB 1120 and CHB 1121 significantly increased the dry weight of shoots and roots at 75 days after planting (DAP). Additionally, CHB 1120 and CHB 1121 significantly enhanced the amount of chlorophyll present in leaves estimated by SPAD readings, increasing nitrogen contents in shoots by 38.1 and 49.2%, respectively. In comparison with BCRC 14266, CHB 1120 and CHB 1121 significantly increased the nodule number and nitrogenase activity at both 45 and 75 DAP. In conclusion, the two indigenous strains significantly outperform the exotic strain and non-inoculated control in promoting nitrogen uptake and growth of white clover.

Effects of coinoculation of Rhizobium with plant growth promoting rhizobacteria on the nitrogen fixation and nutrient uptake of Trifolium repens in low phosphorus soil

Low soil phosphorus (P) availability is a notable limitation for legume nodulation, nitrogen fixation and yield, and it is therefore important to improve legume growth under P-deficient conditions. This study aimed to determine the effects of coinoculation with Rhizobium and plant growth promoting rhizobacter (PGPR) on the nitrogen fixation and nutrient uptake of white clover (Trifolium repens) in a P-deficient soil. White clover plants were inoculated with one of the two Rhizobium strains CHB1120 and CHB1121 alone or coinoculated with either of the Rhizobium strains and one of the two PGPR strains, Bacillus aryabhattai strain Sb and Azotobacter vinelandii strain G31. White clover inoculated with either CHB1120 or CHB1121 alone and coinoculated with one of the two Rhizobium strains and the PGPR significantly enhanced nodule numbers compared with the non-inoculated control. The coinoculation of either CHB1120 or CHB1121 and Sb showed a significantly greater level of nitrogenase than the single inoculation of CHB1120 or CHB1121. In addition, the single inoculation of CHB1120 or CHB1121 and their coinoculation with PGPR significantly increased nitrogen contents in both shoots and roots under P-deficient conditions. The coinoculation of CHB1121 and G31 showed the greatest N content in shoots. Moreover, the single inoculation of the two Rhizobium strains and their coinoculation with G31 and Sb significantly increased the K, Ca, and Mg contents in shoots compared with the non-inoculated control. In summary, the PGPR used in this study can enhance the symbiotic potential of rhizobia under low P conditions.

White clover incorporation at high nitrogen application levels: results from a 3-year study

Context High nitrogen (N)-fertiliser application levels and high stocking rates have been shown to optimise herbage dry-matter (DM) production in grass-only systems. Conversely, swards including white clover (Trifolium repens L.) are commonly suited to production systems at low N-fertiliser application levels (<150 kg N/ha) and low stocking rates (fewer than two livestock units (LU) per hectare). Despite this, the use of N fertiliser on grass–clover swards has generally increased during the past few decades, particularly in more intensively managed swards under dairy farming. Increasing inorganic N decreases sward white clover content in grass–clover swards, and increasing stocking rate on grass–clover swards can result in damage to white clover plants. However, under high N-application levels, a high stocking rate can also potentially minimise the negative effects of increased grass growth on sward white clover content. Aims The objective of the present study was to investigate the persistency of white clover in an intensive animal-grazing system, under a high N-fertiliser application level (250 kg N/ha) and at a high stocking rate (2.75 LU per hectare) over a 3-year period (2014–2016). Methods The study was a 2 × 2 factorial design, consisting of two perennial ryegrass ploidies (diploid, tetraploid) and two white clover treatments (grass-only, grass–clover). Four sward treatments (diploid-only, tetraploid-only, diploid + clover, tetraploid + clover) were evaluated over a full grazing season at a system scale. Sward measurements were taken at each grazing occasion for 3 years. Key results Over the 3 years, grass–clover swards produced an additional 1468 kg DM/ha when compared with grass-only swards. Sward white clover content decreased by 17% over the three study years. Conclusions This decrease in sward white clover content led to a decrease in contribution to cumulative herbage DM production. Implications Although the present study showed that white clover inclusion can increase herbage DM production at high N-fertiliser application levels, in association with high stocking rates, the initial extra herbage DM production on grass–clover swards decreased each year. Further studies should be undertaken to investigate whether the decline in white clover persistence observed is progressive over a longer time period.

Characterization of olfactory sensory neurons in the red clover seed weevil, Protapion trifolii (Coleoptera: Brentidae) and comparison to the closely related species P. fulvipes

Protapion trifolii and P. fulvipes (Coleoptera: Brentidae) are major pests in European clover seed production. Previous studies have reported a high host plant fidelity of these weevils for red and white clover species, respectively, driven by host plant olfactory cues. Given the specific host preferences observed in these weevils, we aimed to elucidate to which extent such selectivity is reflected in their peripheral olfactory systems. Using an electrophysiological approach, we performed the first functional characterisation of olfactory sensory neurons (OSNs) in P. trifolii to a panel of volatile compounds emitted by red clover plants, and compared the results with the reported OSN types of P. fulvipes. Nineteen OSN classes were characterized in P. trifolii, with the majority of these neurons responding strongly to common volatiles released by the host plant. Based on response profiles, eight of these OSN classes have clear matches to OSN classes in P. fulvipes. The OSN colocalisation patterns and antennal frequency of these classes are similar in the two species. Additionally, the responses of these OSNs are generally highly conserved in the two species, with clear response shifts only revealed for two of the OSN classes. These response shifts in combination with additional response dissimilarities for compounds that vary in abundance between red and white clover plants may underlie the species-specific host preferences. Further behavioural and field experiments should focus on these differentially detected compounds to elucidate their potential role in host selection and use in semiochemical-based control of these pests.

Водоспоживання рослин буркуну білого сорту південний залежно від застосування препаратів Трефлан 480 та Пульсар 40 в умовах Південного Степу України

This article provides research materials indicating that the total water consumption of the white clover plants of the one-year-old Pivdenny variety depends mainly on precipitation. In such conditions, moisture was most effectively used in crops using the Treflan 480 pre-through herbicide at an application rate of 3,0 l/ha and a post-emergence pulsar 40 at an application rate of 1,0 l/ha. With these application rates, the coefficient of water consumption was 420 and 368 m³/s. According to the calculations of water consumption of moisture by the white clover plants, on average for 2015-2017. occurred due to the amount of precipitation - 1931 m³/ha, which is 62% of the total water consumption of the crop. The use of soil moisture reserves during the growing season depended on the dose of application of the studied herbicides. By the end of the growing season, the white clover plants on the studied variants without herbicides, where there were a large number of different weeds, the soil moisture reserves were less and amounted to 719 m³/ha in the control plots. On average, according to the results of studies in 2015-2017. it was found that in the control the coefficient of water consumption had the highest value - 670 m³/s. On average for the years 2015-2017. the maximum yield of white clover seeds of the annual variety Yuzhniy – 840,0 kg/ha was observed on the variant with the use of the Pulsar 40 herbicide at an application rate of 1,0 l/ha, where the total water consumption of plants during the growing season was 3095 m³/ha and the coefficient of water consumption 368 m³/s.