Arbuscular mycorrhizal fungi enhance monoterpene production in red clover (Trifolium pratense L.): a potential tool for pest control.

The possibility of modifying terpene production in plants is a defensive strategy that has been studied in conjunction with their biosynthetic pathways. A biotic factor such as Arbuscular Mycorrhizal Fungi (AMF) could modify terpene production in Trifolium pratense L. In this work, the enzymatic production of monoterpenes in Superqueli INIA cultivar with two AMF was evaluated via HeadSpace-Gas Chromatography (HS-GC). A significant increase of (S)-limonene was found in plants inoculated with Claroideoglomus claroideum as well as with the AMF mix (genera Scutellospora, Acaulospora and Glomus). Moreover, significant increases in other monoterpenes such as (-)-ß-pinene, myrcene, linalool, were observed. Results showed higher monoterpene production capacities in the Superqueli-INIA cultivar, suggesting the participation of monoterpene synthases (MTS). The significant rise of (S)-limonene in red clover plants inoculated with AMF suggests this strategy could be implemented in an agronomical manage for controlling the H. obscurus, the primary pest.

Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology.

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Multi-species pastures for grazing dairy cows in small-scale dairy systems in the highlands of Mexico.

Grazing in small-scale dairy systems reduces costs and enhances sustainability. However, binary pastures (BP) have low persistency, which may be increased by including more species given their flexibility to withstand different agroecological and management situations. The objective was to assess a multi-species (MS) pasture of perennial ryegrass (RG), tall fescue (TF), bromegrass (BG), red clover (RC) and white clover (WC) in comparison to a BP of perennial ryegrass (RG) - WC grazed by six Holstein dairy cows during the dry season in an on-farm double cross-over experiment, with three 14-day each experimental periods for animal variables, and a split-plot design for pasture variables at 3 cow/ha. There were differences (P < 0.05) between pastures for sward height (MS 5.0 vs. BP 4.0 ± 0.10 cm, both MS and BP) and net herbage accumulation (MS 902 vs BP 228 ± 172.4 kg DM/ha, both MS and BP) and differences (P < 0.05) for chemical composition among periods. There were differences (P < 0.01) among periods for estimated DM intake, but no differences (P > 0.05) for milk yield (mean 16.8 kg/cow/day), milk fat or protein content (mean 31.8 and 28.8 g/kg). Multi-species pastures are a viable alternative for SSDS in the highlands of central Mexico, being more resistant to harsh environment and to weed and kikuyu grass invasion.

Effect of diet and type of pregnancy on plasma metabolic response in sheep and its further effect on lamb performance.

This trial evaluated the individual and interactional effects of diet and type of pregnancy (twin or single) on plasma metabolic response in ewes and their lambs from late pre-partum to late post-partum. Thus, a flock of 18 Ile de France breed sheep, consisting of 8 twin-bearing and 10 single-bearing ewes, were allocated to one of two groups according to their diet, either based on ad libitum naturalized pasture hay (NPH) or red clover hay (RCH), from d 45 pre-partum to d 60 post-partum. Plasma samples were collected at different times to determine albumin, cholesterol, total protein and urea, plus glucose and ß-hydroxybutyrate (BHB) concentration in ewes. The data was processed using the lme4 package for R, and SPSS Statistics 23.0 for Windows. The results showed that both diet and type of pregnancy influenced the metabolic profile in ewes, showing an inverse relationship between single- and twin-bearing ewes regarding glucose and especially BHB proportions from pre-partum to birth. During post-partum, higher urea concentrations were observed in twin- and single-bearing ewes fed RCH in contrast to those fed NPH, as a result of the higher-quality forage offered to ewes. Regarding lambs, the diet and type of pregnancy influenced the total protein and urea levels, where an inverse relationship at birth and early post-partum between albumin and cholesterol vs. total protein and urea was detected, reflecting a trend (P value between 0.06 and 0.07) to a better performance by groups of single lambs, especially those from single-bearing ewes fed RCH. Finally, under the conditions of this study, the maternal diet and type of pregnancy influenced the plasma metabolic response in ewes and their lambs, affecting the lamb performance especially at birth.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

OBJECTIVES: The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. RESULTS: We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days. CONCLUSION: This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Trifolium pratense: Friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.

Antifeedant Effects of Essential Oil, Extracts, and Isolated Sesquiterpenes from Pilgerodendron uviferum (D. Don) Florin Heartwood on Red Clover Borer Hylastinus obscurus (Coleoptera: Curculionidae).

The beetle Hylastinus obscurus Marsham (Coleoptera: Curculionidae), endemic to Europe and Northern Africa, is one of the most important red clover pests in Chile. As commercial insecticides are less effective against this pest, plant secondary metabolites have been considered as an alternative for its control. Here, we have investigated the chemical composition of essential oil (EO), petroleum ether extract (PEE), and dichloromethane extract (DCME) from Pilgerodendron uviferum heartwood. Additionally, the effects of EO and extracts on the feeding behavior (% of weight shift) of H. obscurus have been evaluated. The composition of EO, PEE, and DCME were analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The results showed the presence of a similar mixture of sesquiterpenes in the essential oil and in both of the extracts, which accounted for circa 60% of the total mixture. Sesquiterpenes were further isolated using chromatographic methods and were structurally characterized by optical rotation, GC⁻MS, FTIR, and 1D and 2D NMR experiments. The physicochemical properties of the isolated sesquiterpenes, including lipophilicity and vapor pressure, were also determined. The sesquiterpenes were identified as the following: (-)-trans-calamenene (1), cadalene (2), (-)-cubenol (3), (-)-epi-cubenol (4), (-)-torreyol (5), and (-)-15-copaenol (6). The antifeedant activity of EO, extracts, and isolated sesquiterpenes were evaluated using artificial diets in a non-choice test. Relative to the control, the EO, DCME extract, and the isolated sesquiterpenes, namely, (-)-trans-calamenene (1), cadalene (2), and (5) torreyol, were found to be the most effective treatments against H. obscurus. Our study showed that the compounds occurring in P. uviferum heartwood were effective in reducing the adult growth of H. obscurus. The physicochemical properties of the isolated sesquiterpenes might have been associated with antifeedant effects.

Methane production from four forages at three maturity stages in a ruminal in vitro system / Producción de metano de cuatro forrajes en tres estados de madurez en un sistema ruminal in vitro / Produção de metano de quatro forragens em três estágios de maturidade em um sistema ruminal in vitro

Abstract Background: Forage characteristics can modify in vitro methane production. There is little information about in vitro methane production of legumes and grasses at different maturity stages in tropical highland grazing systems. Objective: To evaluate the effect of species and forage maturity on in vitro methane production. Methods: Four forage species grown in tropical highlands of Colombia, two grasses: Kikuyu (Cenchrus clandestinus, previously named Pennisetum clandestinum) and ryegrass (Lolium perenne var. Samsum), and two legumes: Lotus (Lotus uliginosus var. Maku) and red clover (Trifolium pratense) were harvested in two paddocks at three maturity stages (young, intermediate, and mature). In vitro 48 h gas production was measured and methane proportion in gas was quantified by gas chromatography. Data were analysed as a randomized complete block (paddocks) design with a factorial arrangement 4×3 (4 species × 3 maturity stages) using the GLM procedure of SAS®. Results: Lotus produced less methane (p<0.01) than ryegrass, clover, and kikuyu (35.5 vs 64.7, 55.7 or 51.4 mL/g degraded organic matter, respectively). Younger forages produced less methane than intermediate and mature forages (42.8 vs 56.3 and 56.4 mL/g degraded organic matter, respectively). Cellulose concentration and organic matter degradability explained 67% (p<0.01) of methane production. Conclusion: Forage composition, presence of condensed tannins, and changes in fermentation patterns may explain the differences observed in in vitro methane production among species and maturity stages.

Resumen Antecedentes: Las características de los forrajes pueden afectar la producción de metano. Hay poca información sobre la producción de metano de leguminosas y gramíneas en diferentes estados de madurez en los sistemas pastoriles de trópico alto. Objetivo: Evaluar el efecto de la especie y la madurez de los forrajes sobre la producción de metano in vitro. Métodos: Cuatro especies forrajeras de trópico alto colombiano, dos gramíneas: Kikuyo (Cenchrus clandestinus, anteriormente llamado Pennisetum clandestinum) y ryegrass (Lolium perenne var. Samsum) y dos leguminosas: Lotus (Lotus uliginosus var. Maku) y trébol rojo (Trifolium pratense), fueron cosechadas de dos parcelas en tres estados de madurez (joven, intermedio y maduro). Se midió la producción de gas in vitro a las 48 h y la proporción de metano en el gas por cromatografía de gases. Los datos se analizaron empleando un diseño de bloques completos al azar (parcelas) con un arreglo factorial 4×3 (4 especies × 3 estados de madurez) mediante el procedimiento GLM de SAS®. Resultados: El lotus produjo menos metano (p<0,01) que el ryegrass, trébol o kikuyo (35,5 vs 64,7, 55,7 o 51,4 mL/g materia orgánica degradada, respectivamente). Los forrajes jóvenes produjeron menos metano que aquellos de edad intermedia y madura (42,8 vs 56,3 y 56,4 mL/g materia orgánica degradada, respectivamente). La concentración de celulosa y la digestibilidad de la materia orgánica explicaron el 67% (p<0,01) de la producción de metano. Conclusión: La composición de la pastura, la concentración de taninos condensados y los cambios en los patrones de fermentación pueden explicar las diferencias en la producción de metano in vitro según la especie y madurez del forraje.

Resumo Antecedentes: As caraterísticas das forragens podem modificar a produção de metano. Existe pouca informação sobre a produção de metano de leguminosas e gramíneas em diferentes fases de maturidade nos sistemas de pastoreio de trópico alto. Objetivo: Avaliar o efeito da espécie e a maturidade das forragens sobre a produção de metano in vitro. Métodos: Quatro espécies de forragens de trópico alto colombiano, duas gramíneas: Capim quicuio (Cenchrus clandestinus), antigamente chamado Pennisetum clandestinum) e azevém (Lolium perenne var. Samsum) e duas leguminosas: Trevina (Lotus uliginosus var. Maku) y trevo (Trifolium pratense), foram colhidas de duas parcelas diferentes em três fases diferentes de maturidade (jovem, intermedia e madura). Foi avaliada a produção de gás in vitro às 48 h e a proporção de metano foi determinada por cromatografia de gases. Os dados foram analisados com um delineamento em blocos ao acaso (parcelas) com um arranjo fatorial 4×3 (4 espécies × 3 fases de maturidade) utilizando o procedimento GLM de SAS®. Resultados: Trevina produz menos metano (p<0,01) que azevém, trevo o capim quicuio (35,5 vs 64,7, 55,7 ou 51,4 mL/g matéria orgânica degradada, respetivamente). Forragens jovens produzem menos metano que forragens com idade intermedia e matura (42,8 vs 56,3 y 56,4 mL/g matéria orgânica degradada, respetivamente). A concentração de celulosa e a digestibilidade da matéria orgânica explicaram o 67% (p<0,01) da produção de metano. Conclusão: A composição da pastagem, a concentração de taninos condensados e as mudanças nos padrões de fermentação podem explicar as diferencias na produção de metano in vitro entre espécies e maturidade de forragens.

Produção, composição química e taxa de lotação de sistemas forrageiros constituidos por coastcross-1 sobressemeada com azevém e trevos / Production, chemical composition and stocking rate of forage systems compounds for coastcross-1 intercropped with ryegrass and clovers

O objetivo dessa pesquisa foi avaliar a produção de forragem, os teores de proteína bruta e fibra em detergente neutro, e taxa de lotação de três sistemas forrageiros constituídos por Coastcross-1 (Cynodon dactylon L. Pers.) + azevém (Lolium multiflorum Lam., cv. Comum); Coastcross-1 + azevém + trevo vermelho (Trifolium pratense L., cv. Estanzuela 116) e Coastcross-1 + azevém + trevo branco (Trifolium repens L., cv. Yi). O experimento foi conduzido entre 26 de abril de 2007 e 22 de abril de 2008, sendo conduzidos 11 pastejos, seis no período hibernal e cinco no estival. Para avaliação foram utilizadas vacas em lactação da raça Holandesa que receberam suplementação alimentar com concentrado à razão de 0,9% do peso corporal/dia. Avaliou-se a massa de forragem de pré e pós-pastejo, as composições botânica e morfológica e os teores de proteína bruta e fibra em detergente neutro. O delineamento experimental foi inteiramente casualizado com três tratamentos (sistemas forrageiros), duas repetições (piquetes) e avaliações independentes (ciclos de pastejos). No período hibernal foram observados resultados superiores (P<0,05) nos sistemas forrageiros consorciados com leguminosas para produção de forragem e taxa de acúmulo diário de matéria seca. Para a taxa de desaparecimento de matéria seca e taxa de lotação não foram observadas diferenças entre os sistemas forrageiros. Foi observado efeito residual do trevo vermelho sobre a Coastcross-1 + azevém, com valor superior (P<0,05) para o teor de proteína bruta e menor teor de fibra em detergente neutro.

ABSTRACT: The objective of this research was to evaluate the forage production, crude protein and neutral detergent fiber level, stocking rate of three forage systems compounds for Coastcross-1 (Cynodon dactylon L. Pers.) + ryegrass (Lolium multiflorum Lam., cv. Comum); Coastcross-1 + ryegrass + red clover (Trifolium pratense L., cv. Estanzuela 116) and Coastcross-1 + ryegrass + white clover (Trifolium repens L., cv. Yi). The experiment was carried out from April 2007 to April 2008, for 11 grazing cycles, six in winter season and five in summer season. Holstein cows receiving 0.9% of body weight/day feed supplement concentrate were used. The herbage mass of pre and postgrazing, botanical and morphological composition crude protein and neutral detergent fiber level were evaluated. A randomized complete design was used with three treatments (forage systems) two replications (paddocks) and independent evaluations (grazing cycles). Superior value (P<0.05) of forage production and daily dry matter accumulation rate were found in winter season for the forage systems with legume. No differences in dry matter disappearance rate and stocking rate averages were detected among pastures. Residual effects of red clover were observed on Coastcross-1 + ryegrass with superior result (P<0.05) on crude protein and lower neutral detergent fiber level.

Produção, composição química e taxa de lotação de sistemas forrageiros constituidos por coastcross-1 sobressemeada com azevém e trevos / Production, chemical composition and stocking rate of forage systems compounds for coastcross-1 intercropped with ryegrass and clovers

O objetivo dessa pesquisa foi avaliar a produção de forragem, os teores de proteína bruta e fibra em detergente neutro, e taxa de lotação de três sistemas forrageiros constituídos por Coastcross-1 (Cynodon dactylon L. Pers.) + azevém (Lolium multiflorum Lam., cv. Comum); Coastcross-1 + azevém + trevo vermelho (Trifolium pratense L., cv. Estanzuela 116) e Coastcross-1 + azevém + trevo branco (Trifolium repens L., cv. Yi). O experimento foi conduzido entre 26 de abril de 2007 e 22 de abril de 2008, sendo conduzidos 11 pastejos, seis no período hibernal e cinco no estival. Para avaliação foram utilizadas vacas em lactação da raça Holandesa que receberam suplementação alimentar com concentrado à razão de 0,9% do peso corporal/dia. Avaliou-se a massa de forragem de pré e pós-pastejo, as composições botânica e morfológica e os teores de proteína bruta e fibra em detergente neutro. O delineamento experimental foi inteiramente casualizado com três tratamentos (sistemas forrageiros), duas repetições (piquetes) e avaliações independentes (ciclos de pastejos). No período hibernal foram observados resultados superiores (P<0,05) nos sistemas forrageiros consorciados com leguminosas para produção de forragem e taxa de acúmulo diário de matéria seca. Para a taxa de desaparecimento de matéria seca e taxa de lotação não foram observadas diferenças entre os sistemas forrageiros. Foi observado efeito residual do trevo vermelho sobre a Coastcross-1 + azevém, com valor superior (P<0,05) para o teor de proteína bruta e menor teor de fibra em detergente neutro.(AU)

ABSTRACT: The objective of this research was to evaluate the forage production, crude protein and neutral detergent fiber level, stocking rate of three forage systems compounds for Coastcross-1 (Cynodon dactylon L. Pers.) + ryegrass (Lolium multiflorum Lam., cv. Comum); Coastcross-1 + ryegrass + red clover (Trifolium pratense L., cv. Estanzuela 116) and Coastcross-1 + ryegrass + white clover (Trifolium repens L., cv. Yi). The experiment was carried out from April 2007 to April 2008, for 11 grazing cycles, six in winter season and five in summer season. Holstein cows receiving 0.9% of body weight/day feed supplement concentrate were used. The herbage mass of pre and postgrazing, botanical and morphological composition crude protein and neutral detergent fiber level were evaluated. A randomized complete design was used with three treatments (forage systems) two replications (paddocks) and independent evaluations (grazing cycles). Superior value (P<0.05) of forage production and daily dry matter accumulation rate were found in winter season for the forage systems with legume. No differences in dry matter disappearance rate and stocking rate averages were detected among pastures. Residual effects of red clover were observed on Coastcross-1 + ryegrass with superior result (P<0.05) on crude protein and lower neutral detergent fiber level.(AU)

Genetic variability of isoflavones in the USDA red clover core collection

Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g-1 of dry material for daidzein, 163.69 µg g-1 for genistein, 11353.29 µg g-1 for formononetin and 6568.8 µg g-1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.

In vitro isoflavonoid production and analysis in natural tetraploid Trifolium pratense (red clover) calluses

Isoflavones are polyphenolic phytoestrogens, predominantly found in leguminous plants. Trifolium pratense L., Fabaceae (red clover), is rich in isoflavones that possess estrogenic activity due to their similar molecular structure and effectiveness in preventing health conditions such as menopause, osteoporosis, cardiovascular disease, hypertension and hormone-dependent cancers. In this study, presence and amount of various phytoestrogens in the tetraploid plant and in the calluses derived from the plants were investigated. Calluses were generated from explants obtained from natural tetraploid T. pratense seedlings. The best callus formation was obtained from hypocotyl explants cultured in Phillips Collins and Gamborg B5 media containing different plant growth regulators. Flowers of plants and calluses were analysed for formononetin, biochanin A, genistein and daidzein contents using HPLC. In HPLC analysis, high levels of formononetin (0.249 µg/mg) were determined in natural tetraploid T. pratense flowers in addition to genistein and biochanin A. In calluses, highest isoflavone content (1.15 µg/mg formononetin) was observed in modified Gamborg B5 medium. Biochanin A content of calluses and the plant were found to be nearly the same. But formononetin and genistein contents of the calluses in this medium were found to be respectively 4.62 and 21.39 folds higher than the tetraploid plant.

Plant regeneration of natural tetraploid Trifolium Hum pratense L

The regeneration of natural tetraploid T. pratense, originated from Erzurum-Turkey, is reported in this study. This plant has low seed setting and hard seed problems due to polyploidy. Hypocotyl, cotyledon, apical meristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing different concentrations of BAP and NAA as growth regulators. The best shoot formation has been observed on explants initiated from apical meristem placed on PC-L2 medium that includes 2 mg dm-3 BAP and 1 mg dm-3 NAA. 94.4 percent of the shoots originated from calli were rooted on PC-L2 medium with 1 mg dm-3 NAA. In vitro organogénesis has been accomplished in the natural tetraploid T. pratense regenerated plants successively transferred to the field.