Direct evidence of pseudogamy in apomictic Brachiaria brizantha (Poaceae)

Brachiaria brizantha is a forage grass that has several apomictic accessions. B. brizantha cv. Marandu is a natural tetraploid aposporous apomict widely cultivated in Brazil. Pseudogamy was detected in this species by observation that seed set is suppressed in plants that have had the stigmas excised from the flowers. The egg cell develops parthenogenetically in the apomictic plants, meaning that fertilisation is necessary for the formation of the endosperm. A thorough knowledge of all the events of seed formation in natural apomictic plants is essential for a complete understanding of this mode of reproduction. In this paper, we show direct evidence of pseudogamy in B. brizantha through the cytological analysis of polar nucleus fertilisation and the determination of triploid level of the endosperm tissue. The development of the male gametophyte gives rise to a reduced tri-celled pollen, the viability of which varies throughout the year, reaching 88% in the peak of the flowering period. Discharge of the male gamete takes place around 10 h after pollination and monospermy is the predominant system observed. Precocious embryony was also observed in these plants; embryos arise from egg cells. Endosperm development followed the free nucleus model and was associated with the presence of an embryo. Cellularisation and reserve uptake occurred 2 days after pollination (DAP) and mature endosperm was observed 8 DAP. The triploid level of the endosperm in the apomictic accession confirmed the 2:1 maternal:paternal ratio of genome contribution in the tissue.     

Microsporogenesis in Brachiaria brizantha (Poaceae) as a selection tool for breeding

The genus Brachiaria comprises more than 100 species and is the single most important genus of forage grass in the tropics. Brachiaria brizantha, widely used in Brazilian pastures for beef and dairy production, is native to tropical Africa. As a subsidy to the breeding program underway in Brazil, cytological studies were employed to determine the chromosome number and to evaluate microsporogenesis in 46 accessions of this species available at Embrapa Beef Cattle (Brazil). Thirty-four accessions presented 2n = 36; seven had 2n = 45, and five had 2n = 54 chromosomes. Based on the higher level of chromosome association observed in diakinesis, in tetra-, penta-, and hexavalents, respectively, it was concluded that they are derived from x = 9; consequently, these accessions are tetra- (2n = 4x = 36), penta- (2n = 5x = 45), and hexaploids (2n = 6x = 54). The most common meiotic abnormalities were irregular chromosome segregation due to polyploidy. Chromosome stickiness, abnormal cytokinesis, non-congressed bivalents in metaphase I and chromosomes in metaphase II, and chromosome elimination were recorded at varying frequencies in several accessions. The mean percentage of meiotic abnormalities ranged from 0.36 to 95.76%. All the abnormalities had the potential to affect pollen viability by generating unbalanced gametes. Among the accessions, only the tetraploid ones with less than 40% of abnormalities are suitable as pollen donors in intra- and interspecific crosses. Currently, accessions with a high level of ploidy (5 and 6n) cannot be used as male genitors in crosses because of the lack of sexual female genitors with the same levels of ploidy.     

Plant growth promoting bacteria in Brachiaria brizantha

Brachiaria brizantha is considered one of the preferred fodders among farmers for having high forage yield and large production of root mass. The association of beneficial bacteria with these grasses can be very valuable in the recovery of the pasture areas with nutritional deficiency. With the aim of studying this possibility, we carried out the sampling of soil and roots of B. brizantha in three areas (Nova Odessa-SP, S?o Carlos-SP and Campo Verde-MT, Brazil). Seventy-two bacterial strains were isolated and used in tests to evaluate their biotechnological potential. Almost all isolates presented at least one positive feature. Sixty-eight isolates produced analogues of indole-3-acetic acid, ten showed nitrogenase activity when subjected to the method of increasing the concentration of total nitrogen (total N) in the culture medium and sixty-five isolates showed nitrogenase activity when subjected to acetylene reduction technique. The partial sequencing of 16S rRNA of these isolates allowed the identification of seven main groups, with the prevalence of those affiliated to the genus Stenotrophomonas (69?%). At the end, this work elected the strains C4 (Pseudomonadaceae) and C7 (Rhodospirillaceae) as promising organisms for the development of inoculants due to their higher nitrogenase activity.     

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae)

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.     

Genetic Diversity and Population Structure of the Brachiaria brizantha Germplasm

Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. (syn. Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster) is a species used primarily as forage in tropical America and Southeast Asia. B. brizantha has been extensively researched since the 1980s with the initiation of the Tropical Forages Breeding Program conducted by the Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária; EMBRAPA), holding one of the largest germplasm collections in the world. This work has identified 15 new microsatellite markers for this species, which have been used in addition to five previously reported markers, to estimate the genetic similarities among 172 accessions and six cultivars of this species. Similarity index values ranged from 0.40 to 1.00. Two duplications were found in the germplasm. A Bayesian analysis performed using the STRUCTURE 2.3.3 program revealed the presence of three clusters with different allelic pools. This analysis is valuable for the performance of crosses to explore heterosis; however, the mode of reproduction of the accessions and ploidy barriers must be observed for effective exploration. A grouping analysis using the neighbor-joining method was consistent with the STRUCTURE analysis, and a combination approach suggested that this germplasm collection does not exhibit considerable genetic variability despite the presence of three distinct allelic pools. The lack of correlation between the genetic and geographic distances is also discussed.     

Silicon distribution and accumulation in shoot tissue of the tropical forage grass Brachiaria brizantha

Silicon (Si) accumulation in organs and cells is one of the most prominent characteristics of plants of the family Poaceae. Many species from this family are used as forage plants for animal feeding. The present study investigates in Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. cv. Marandu: (1) the dry matter production and Si content in shoot due to soil Si fertilizations; (2) the Si distribution among shoot parts; and (3) the silica deposition and localization in leaves. Plants of B. brizantha cv. Marandu were grown under contrasting Si supplies in soil and nutrient solution. Silica deposition and distribution in grass leaf blades were observed using light microscope and scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDXS). Silicon concentration in the B. brizantha shoot increased according to the Si supply. Silicon in grass leaves decreased following the order: mature leaf blades > recently expanded leaf blades > non-expanded leaf blades. Silicon accumulates mainly on the upper (adaxial) epidermis of the grass leaf blades and, especially, on the bulliform cells. The Si distribution on adaxial leaf blade surface is non uniform and reflects a silica deposition exclusively on the cell wall of bulliform cells.     

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.     

Cytological evidence of natural hybridization in Brachiaria brizantha Stapf (Gramineae)

Two accessions of Brachiaria brizantha under cytological analysis showed 2 n  = 5x = 45 chromosomes. Pentaploidy probably resulted from natural hybridization between two species that were not closely related: an apomictic tetraploid male (2 n  = 4x = 36), and a sexual diploid female (2 n  = 2x = 18). The lack of affinity between genomes was clearly indicated by asynchrony during meiosis. The haploid genome ( n  = 9) showed unique behaviour, remaining univalent during prophase I and metaphase I, and undergoing sister-chromatid segregation and lagging at anaphase I. The laggard genome did not always reach the poles in time to be included in the telophase nucleus. However, when the inclusion was effective, this genome was distributed peripherally, changing the otherwise spherical nucleus shape. In the second division, the haploid genome behaved similarly, but as there was sister-chromatid segregation during the first division, the chromatids were slow to reach the poles, forming several micronuclei at telophase II. The two accessions were characterized as allo-autopentaploids, with the tetraploid genome (2 n  = 4x = 36) designated as B (from B. brizantha ) and the haploid genome as X, representing a species with a distinct genome having little affinity with the B genome. Thus, the hybrids' genome composition is represented by BBBBX. By comparing their meiotic behaviour with that observed in synthetic hybrids between B. brizantha and B. ruziziensis analysed previously, B. ruziziensis is the putative diploid sexual parent species in these pentaploid accessions.  © 2006 The Linnean Society of London , Botanical Journal of the Linnean Society , 2006, 150 , 441–446.     

Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.     

Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis

In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.     

Abnormal meiosis in tetraploid genotypes ofBrachiaria brizantha (Poaceae) induced by colchicine: its implications for breeding

Meiotic behavior was analyzed in 6 progenies from 3 artificially induced tetraploid (2n = 4x = 36) sexual genotypes (C31, C41, and C48) of the normally apomicticBrachiaria brizantha (Hochst. ex A. Rich.) Stapf., syn.Urochloa brizantha (Hochst. ex A. Rich.) R. Webster. These are key plants to allow intraspecific hybridization of this important forage species, widely used for pastures in the tropics. The percentage of abnormal cells among the plants ranged from 39.8% to 63.2%. In the single plant derived from C48, only the common meiotic abnormalities typical of polyploids were observed, while in plants derived from C31 and C41, a distinct behavior was found. In the majority of cells of those plants, the chromosomes remained scattered in the cytoplasm in the first division, without forming a metaphase plate. This abnormality blocked chromosome movements at anaphase I. Several micronuclei of various sizes were formed and, after the occurrence of an irregular first cytokinesis, the meiocytes progressed normally to the second division, generating polyads with unbalanced microspores. Pollen viability was not correlated with meiotic abnormalities. The importance of these findings to theBrachiaria breeding program is discussed. The sexual progeny of C48 seems most suitable as female parents to be used in intra-and interspecific hybridization.     

Effect of the tropical grass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf on microbial population and activity in petroleum-contaminated soil

The effect of the tropical pasture grass Brachiaria brizantha on numbers of bacteria, fungi and degraders of alkanes, aromatics, cycloalkanes and crude oil in petroleum hydrocarbon contaminated and uncontaminated savannah soil was evaluated. Substrate induced soil respiration and soil pH were compared between planted and unplanted soil. B. brizantha had a mostly increasing effect on microbial numbers. As an exception, growth of bacteria was not or negatively affected. Microbial respiration and pH were always lower in planted than in unplanted soil. Low pH may result from enhanced oil degradation in planted soil leading to an accumulation of organic acids. A comparable stimulation of crude oil degraders and fungi in planted soil points to the importance of fungi. Since they tolerate lower pH values than bacteria, they are considered to play a central role in oil degradation. Given that the enhancement of crude oil degradation under the influence of B. brizantha could not clearly be correlated to microbial numbers and activity, other factors like oxygen availability, plant enzymes and synergistic degradation by microbial consortia have to be considered.     

Low phosphorus tolerance mechanisms: phosphorus recycling and photosynthate partitioning in the tropical forage grass, Brachiaria hybrid cultivar Mulato compared with rice

The Brachiaria hybrid cv. Mulato is well adapted to low-fertility acid soils deficient in phosphorus (P). To study the grassy forage's mechanisms for tolerating low P supply, we compared it with rice (Oryza sativa L. cv. Kitaake). We tested by using nutrient solution cultures, and quantified the effects of P deficiency on the enzymatic activities of phosphohydrolases and on carbon metabolism in P-deficient leaves. While P deficiency markedly induced activity of phosphohydrolases in both crops, the ratio of inorganic phosphorus to total P in leaves was greater in Brachiaria hybrid. Phosphorus deficiency in leaves also markedly influenced the partitioning of carbon in both crops. In the Brachiaria hybrid, compared with rice, the smaller proportion of (14)C partitioned into sugars and the larger proportion into amino acids and organic acids in leaves coincided with decreased levels of sucrose and starch. Hence, in P-deficient leaves of the Brachiaria hybrid, triose-P was metabolized into amino acids or organic acids. Results thus indicate that the Brachiaria hybrid, compared with rice, tolerates low P supply to leaves by enhancing sugar catabolism and by inducing the activity of several phosphohydrolases. This apparently causes rapid P turnover and enables the Brachiaria hybrid to use P more efficiently.     

Domestication, cultivation and utilization of two small millets, brachiaria ramosa and Setaria glauca (Poaceae), in South India

Brachiaria ramosa is cultivated in pure stands. Its grains are used in preparing at least nine traditional foods in South India. Setaria glauca is cultivated in mixed stands along with little millet (Panicum sumatrense). In Orissa state and in Southern India the grains are used to make at least six traditional foods as a supplementary ingredient. The weedy forms of these were found growing with upland rice and some millets in diverse agro-ecological niches, and are identified by various vernacular names by local farmers. Explorations were conducted to gather information on the evolutionary trends, domestication and ethnobotany of these minor food crops. These findings substantiate the belief that domestication process may have passed through three distinct phases: (i) an associated mimic weed with upland rice and some millets; (ii) a secondary crop mixed with little millet or kodo millet; and (iii) as an independent crop. Also, this process has been promoted as a means of imparting adaptation to an arid climate and in turn related to the diversification of the agriculture basic complex.     

Influence of fertilizer levels on phytoremediation of crude oil-contaminated soils with the tropical pasture grass Brachiaria brizantha (hochst. ex a. rich.) stapf

Determination of fertilizer levels in phytoremediation of petroleum hydrocarbons is a complex issue, since nutrient demands of the plant and of degrading microorganisms in the rhizosphere have to be considered In the present work, three fertilizer levels were tested in a greenhouse experiment with the aim of optimizing growth of the tropical pasture grass Brachiaria brizantha and enhance microbial degradation of heavy crude oil in soil Fertilizer was applied twice in a concentration of 200, 300, and 400 mg each of N, P, and K per kg soil before and after the first sampling (14 wk). The medium fertilizer concentration resulted in best root growth and highest absolute oil dissipation (18.4%) after 22 wk The highest concentration produced best shoot growth and highest relative oil dissipation after 14 wk (10.5% less than unplanted control). In general, degradation of total oil and grease was higher in planted than in unplanted soil, but differences diminished toward the end of the experiment. Next to fertiizer quantity, its composition is an important factor to be further studied, including the form of available nitrogen (N-NO3- vs. N-NH4+). Field trials are considered indispensable for further phytoremediation studies, since greenhouse experiments produce particular water and nutrient conditions.     

Availability and toxicity of cadmium to forage grasses grown in contaminated soil

It is important to know the mechanisms for forage development, especially those related to the tolerance of potentially toxic elements, when considering their use in phytoremediation in heavy metal contaminated areas. In this study, we evaluated plant growth, concentration, and the availability of cadmium (Cd) for forage grasses (Panicum maximum Jacq. cv. Aruana and cv. Tanzânia; Brachiaria decumbens cv. Basilisk; Brachiaria brizantha cv. Xaraés and cv. Marandu) cultivated in Cd contaminated soils. The experiments were performed under greenhouse conditions over a 90-day evaluation period, and the Cd rates were 2, 4, and 12 mg/kg of soil. The relative growth rate of the forage grasses decreased as Cd rates increased, and the following descending order of susceptibility was observed: Marandu > Xaraés > Aruana > Tanzânia > Basilisk, with regard to phytotoxicity in these plants. The forage Cd concentration increased in line with increases in the Cd rates. Cd contents extracted by Mehlich-1 and by diethylenetriaminepentaacetic acid presented high positive correlation with forage relative growth. The forage plants did not block Cd entry into the food chain because they were not capable of limiting Cd absorption.     

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.     

Importance of the nitrogen source in the grass species Brachiaria brizantha responses to sulfur limitation

Background and aims

Plant responses to S supply are highly dependent on N nutrition. We investigated the effect of S status on metabolic, nutritional, and production variables in Brachiaria brizantha treated with different N forms. Additionally, ¹⁵N and ³⁴S root influx were determined in plants under short- and long-term S deprivation.

Methods

Plants were submitted to soil fertilization treatments consisted of combinations of N forms [without N, ammonium (NH₄ ⁺), nitrate (NO₃ ^?) or NH₄ ⁺+NO₃ ^?] at S rates (0, 15, 30, or 45 mg dm^?3). N and S influx capacity was determined in hydroponically-grown plants.

Results

Shoot production due to S supply increased 53, 145 and 196 % with NH₄ ⁺, NH₄ ⁺+NO₃ ^? and NO₃ ^? treatments, respectively. No or low S impaired protein synthesis and led to high accumulation of N-NO₃ ^? and asparagine in NO₃ ^?-fed plants, both alone and with NH₄ ⁺. Proline accumulation was observed in NH₄ ⁺-fed plants. Short- and long-term S deprivation did not promote considerable changes in ¹⁵N influx. ³⁴S absorption decreased depending on the N form provided: NH₄ ⁺+NO₃ ^? > only NH₄ ⁺ > only NO₃ ^? > low N.

Conclusions

Including both NH₄ ⁺ and NO₃ ^? forms in fertilizer increases N and S intake potential and thereby enhances plant growth, nutritional value and production.