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.

The Response of Red Clover (Trifolium pratense L.) to Separate and Mixed Inoculations with Rhizobium leguminosarum and Azospirillum brasilense in Presence of Polycyclic Aromatic Hydrocarbons.

This study aimed to evaluate the impact of co-inoculation Rhizobium sp. and Azospirillum sp. on plant (Trifolium pratense L.) growth in the presence of polycyclic aromatic hydrocarbon (PAH) contamination (anthracene, phenanthrene, and pyrene). Eight strains from the genus Rhizobium leguminosarum bv. trifolii were selected for biotest analysis. Two methods of inoculation were used in the chamber experiment: (1) R. leguminosarum alone and (2) a combined inoculant (R. leguminosarum and Azospirillum brasilense). For comparison, non-contaminated controls were also used. The results demonstrated that co-inoculation of plants with Rhizobium and Azospirillum resulted in more root and shoot biomass than in plants inoculated with R. leguminosarum alone. The results indicated that application of a co-inoculation of bacteria from Rhizobium and Azospirillum species had a positive effect on clover nodulation and growth under the condition of PAH contamination.

Canopy structure and morphogenesis of Italian ryegrass intercropped with red clover under cutting intervals determined by thermal sum / Estrutura do dossel e morfogênese do azevém anual consorciado com trevo vermelho em intervalos de corte determinados por soma térmica

The canopy structure and morphogenesis of ryegrass (Lolium multiflorum Lam.) was investigated after it was over seeded with Tifton 85 (Cynodon spp.) and intercropped with red clover (Trifolium pratense L.). Time intervals between cuts were determined by different thermal sums (125, 250, 375, and 500 degree-days [DD]). Cuts were conducted at a height equivalent to 50% of the average canopy height. Morphogenic and structural data were collected from July to October of 2016 and 2017, from a completely randomized block experimental design. Canopy height, extended tiller height, pseudostem length, and forage mass were fitted to linear equations according to the thermal sums. Interaction of ryegrass with red clover was the highest (12.1%) for longer cut intervals (375 and 500 DD). The leaf appearance rate (0.0080 leaf/DD), phyllochron value (128.8 DD), and senescence rate (0.10 cm/DD) of ryegrass were comparable between the different intervals. The slowest elongation rate (0.06 cm/DD) with the shortest interval duration between cuts (171.6 DD) was observed in the 125 DD interval. According to the findings from this 2-year experiment, the use of a 250 DD cut interval was recommended between grazing events, taking into account the efficiency of forage use, ryegrass leaf, stem ratio, and interactions with red clover in the canopy under rotational stocking.(AU)

A estrutura do dossel e a morfogênese do azevém (Lolium multiflorum Lam.) foram investigadas após a sobressemeadura em Tifton 85 (Cynodon spp.) e consorciado com trevo vermelho (Trifolium pratense L.). Os intervalos de tempo entre os cortes foram determinados por diferentes somas térmicas (125, 250, 375 e 500 graus-dia [GD]). Os cortes foram realizados a uma altura equivalente a 50% da altura média do dossel. Os dados morfogênicos e estruturais foram coletados no período de julho a outubro de 2016 e 2017, em um delineamento experimental de blocos ao acaso. A altura do dossel, a altura estendida do perfilho, o comprimento do pseudocolmo e a massa de forragem se ajustaram a equações lineares de acordo com a soma térmica. A consorciação de azevém e trevo vermelho foi maior (12,1%) para intervalos de corte mais longos (375 e 500 GD). A taxa de aparecimento foliar (0,0080 folha/GD), filocrono (128,8 GD) e a taxa de senescência (0,10 cm /GD) do azevém não foram diferentes entre os intervalos de corte. A menor taxa de elongação (0,06 cm/GD) e duração de elongação (171,6 GD) foi observada no menor intervalo entre cortes, 125 GD. De acordo com os achados deste experimento de dois anos, recomenda-se o uso de um intervalo de corte de 250 GD entre os eventos de pastejo, levando em consideração a eficiência do uso de forragem, proporção de folhas e colmo de azevém e trevo vermelho no dossel, sob lotação rotativa.(AU)

Canopy structure and morphogenesis of Italian ryegrass intercropped with red clover under cutting intervals determined by thermal sum / Estrutura do dossel e morfogênese do azevém anual consorciado com trevo vermelho em intervalos de corte determinados por soma térmica

ABSTRACT: The canopy structure and morphogenesis of ryegrass (Lolium multiflorum Lam.) was investigated after it was over seeded with Tifton 85 (Cynodon spp.) and intercropped with red clover (Trifolium pratense L.). Time intervals between cuts were determined by different thermal sums (125, 250, 375, and 500 degree-days [DD]). Cuts were conducted at a height equivalent to 50% of the average canopy height. Morphogenic and structural data were collected from July to October of 2016 and 2017, from a completely randomized block experimental design. Canopy height, extended tiller height, pseudostem length, and forage mass were fitted to linear equations according to the thermal sums. Interaction of ryegrass with red clover was the highest (12.1%) for longer cut intervals (375 and 500 DD). The leaf appearance rate (0.0080 leaf/DD), phyllochron value (128.8 DD), and senescence rate (0.10 cm/DD) of ryegrass were comparable between the different intervals. The slowest elongation rate (0.06 cm/DD) with the shortest interval duration between cuts (171.6 DD) was observed in the 125 DD interval. According to the findings from this 2-year experiment, the use of a 250 DD cut interval was recommended between grazing events, taking into account the efficiency of forage use, ryegrass leaf, stem ratio, and interactions with red clover in the canopy under rotational stocking.

RESUMO: A estrutura do dossel e a morfogênese do azevém (Lolium multiflorum Lam.) foram investigadas após a sobressemeadura em Tifton 85 (Cynodon spp.) e consorciado com trevo vermelho (Trifolium pratense L.). Os intervalos de tempo entre os cortes foram determinados por diferentes somas térmicas (125, 250, 375 e 500 graus-dia [GD]). Os cortes foram realizados a uma altura equivalente a 50% da altura média do dossel. Os dados morfogênicos e estruturais foram coletados no período de julho a outubro de 2016 e 2017, em um delineamento experimental de blocos ao acaso. A altura do dossel, a altura estendida do perfilho, o comprimento do pseudocolmo e a massa de forragem se ajustaram a equações lineares de acordo com a soma térmica. A consorciação de azevém e trevo vermelho foi maior (12,1%) para intervalos de corte mais longos (375 e 500 GD). A taxa de aparecimento foliar (0,0080 folha/GD), filocrono (128,8 GD) e a taxa de senescência (0,10 cm /GD) do azevém não foram diferentes entre os intervalos de corte. A menor taxa de elongação (0,06 cm/GD) e duração de elongação (171,6 GD) foi observada no menor intervalo entre cortes, 125 GD. De acordo com os achados deste experimento de dois anos, recomenda-se o uso de um intervalo de corte de 250 GD entre os eventos de pastejo, levando em consideração a eficiência do uso de forragem, proporção de folhas e colmo de azevém e trevo vermelho no dossel, sob lotação rotativa.

Expression of seed vigor and initial growth of red clover under fungicide influence / Expressão do vigor de sementes e crescimento inicial de trevo vermelho sob influência de fungicida

The objective of this study was to evaluate the expression of seeds vigor and physiological performance of initial growth of red clover plants under the influence of fungicide doses of carboxin + thiram. Three lots of seeds were used (high; medium and low vigor) treated with 0; 150; 250 fungicide doses and 350 mL i.a./100 kg of seeds. Germination, first count, emergence, speed index of seedling emergence, length and dry matter of shoot and root, leaf area and shoot/root ratio were evaluated. The experimental design was completely randomized in a factorial 3 x 4 (three lots - high, medium and low vigor x four fungicide doses of carboxin + thiram). Data were submitted to analysis of variance and if significant at 5% probability, adjusted for orthogonal polynomials. It was possible to observe that there were significant differences for the means of the variables germination, first germination count, emergency, emergency speed index, shoot length, root length, aerial part dry mass, root dry mass, leaf area, leaf area ratio and shoot and root ratio. The fungicide doses of 150 to 350 mL i.a./100 kg of seeds result in greater seedling emergence, higher leaf area ratio and increased shoot/root ratio in plants from lots of medium vigor seeds.The fungicide consisted of carboxin + thiram enhances the expression of seed vigor and physiological performance of seedlings growth derived from lots of medium vigor seeds.(AU)

O objetivo deste trabalho foi avaliar a expressão do vigor de sementes e o desempenho fisiológico de crescimento inicial de plantas de trevo vermelho submetidas à influência de doses de fungicida à base de carboxin + thiram. Foram utilizados três lotes de sementes (alto; médio e baixo vigor) tratadas com as doses de fungicida 0; 150; 250 e 350 mL i.a./100 kg de sementes. Foram avaliados a germinação, primeira contagem, emergência, índice de velocidade de emergência de plântulas, comprimento e matéria seca de parte aérea e de raiz, área foliar, razão de parte aérea e raiz. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial 3 x 4 (três lotes - alto, médio e baixo vigor x quatro doses fungicidas de carboxin + tiram). Os dados foram submetidos à análise de variância e se significativos a 5% de probabilidade, foram ajustados para polinômios ortogonais. De acordo com os resultados foi possível observar que houve diferença significativa para as médias das variáveis germinação, primeira contagem de germinação, emergência, índice de velocidade de emergência, comprimento de parte aérea, comprimento de raiz, massa seca de parte aérea, massa seca de raiz, área foliar, razão de área foliar e razão de parte aérea e raiz. As doses de fungicida 150 a 350 mL i.a./100 kg de sementes resultam na maior emergência de plântulas, superior razão de área foliar e incremento na razão parte aérea e raiz em plântulas originadas de sementes de lotes com médio vigor. O fungicida a base de carboxin + thiram realça a expressão do vigor de sementes e o desempenho fisiológico de crescimento de plântulas originadas de lotes de sementes de médio vigor.(AU)

Expression of seed vigor and initial growth of red clover under fungicide influence / Expressão do vigor de sementes e crescimento inicial de trevo vermelho sob influência de fungicida

The objective of this study was to evaluate the expression of seeds vigor and physiological performance of initial growth of red clover plants under the influence of fungicide doses of carboxin + thiram. Three lots of seeds were used (high; medium and low vigor) treated with 0; 150; 250 fungicide doses and 350 mL i.a./100 kg of seeds. Germination, first count, emergence, speed index of seedling emergence, length and dry matter of shoot and root, leaf area and shoot/root ratio were evaluated. The experimental design was completely randomized in a factorial 3 x 4 (three lots - high, medium and low vigor x four fungicide doses of carboxin + thiram). Data were submitted to analysis of variance and if significant at 5% probability, adjusted for orthogonal polynomials. It was possible to observe that there were significant differences for the means of the variables germination, first germination count, emergency, emergency speed index, shoot length, root length, aerial part dry mass, root dry mass, leaf area, leaf area ratio and shoot and root ratio. The fungicide doses of 150 to 350 mL i.a./100 kg of seeds result in greater seedling emergence, higher leaf area ratio and increased shoot/root ratio in plants from lots of medium vigor seeds.The fungicide consisted of carboxin + thiram enhances the expression of seed vigor and physiological performance of seedlings growth derived from lots of medium vigor seeds.

O objetivo deste trabalho foi avaliar a expressão do vigor de sementes e o desempenho fisiológico de crescimento inicial de plantas de trevo vermelho submetidas à influência de doses de fungicida à base de carboxin + thiram. Foram utilizados três lotes de sementes (alto; médio e baixo vigor) tratadas com as doses de fungicida 0; 150; 250 e 350 mL i.a./100 kg de sementes. Foram avaliados a germinação, primeira contagem, emergência, índice de velocidade de emergência de plântulas, comprimento e matéria seca de parte aérea e de raiz, área foliar, razão de parte aérea e raiz. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial 3 x 4 (três lotes - alto, médio e baixo vigor x quatro doses fungicidas de carboxin + tiram). Os dados foram submetidos à análise de variância e se significativos a 5% de probabilidade, foram ajustados para polinômios ortogonais. De acordo com os resultados foi possível observar que houve diferença significativa para as médias das variáveis germinação, primeira contagem de germinação, emergência, índice de velocidade de emergência, comprimento de parte aérea, comprimento de raiz, massa seca de parte aérea, massa seca de raiz, área foliar, razão de área foliar e razão de parte aérea e raiz. As doses de fungicida 150 a 350 mL i.a./100 kg de sementes resultam na maior emergência de plântulas, superior razão de área foliar e incremento na razão parte aérea e raiz em plântulas originadas de sementes de lotes com médio vigor. O fungicida a base de carboxin + thiram realça a expressão do vigor de sementes e o desempenho fisiológico de crescimento de plântulas originadas de lotes de sementes de médio vigor.

Ultrastructural changes of the egg apparatus associated with fertilisation of natural tetraploid Trifolium pratense L. (Fabaceae)

The aim of this study is to describe the ultrastructural changes of the egg apparatus associated with fertilisation of the natural tetraploid Trifolium pratense. The pollen tube enters one of the synergids through the filliform apparatus from the micropyle. Before the entry of the pollen tube into the embryo sac, one of the synergids begins to degenerate, as indicated by increased electron density and a loss of volume. This cell serves as the site of entry for the pollen tube. Following fertilization, the vacuolar organisation in the zygote changes; in addition to the large micropylar vacuole, there are several small vacuoles of varying size. Ribosomal concentration increases significantly after fertilisation. In T. pratense, ultrastructural changes between the egg cell and zygote stages are noticeable. Several marked changes occur in the egg cell because of fertilisation. The zygote cell contains ribosomes has many mitochondria, plastids, lipids, vacuoles. After fertilization, most of the food reserves are located in the integument in the form of starch. The zygote shows ultrastructural changes when compared to the egg cell and appears to be metabolically active.

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.