Fungi associated with Paspalum guenoarum seeds: their impact on physiology and control / Fungos associados com sementes de Paspalum guenoarum: seus impactos na fisiologia e controle

Of the natural pastures grown in southern Brazil, those of the genus Paspalum are the most important. One of the factors that hinder their cultivation is the availability of quality seeds, that are often compromised by the presence of fungi. This study determined the in vitro sensitivity of Paspalum guenoarum ecotype azulão seed-associated fungus to certain fungicides and to measure the efficiency of chemical treatments for fungal control and seed physiological performance. Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata and Fusarium incarnatum associated with seeds were tested in vitro against Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram and Tiofanato-metílico at 0, 1, 2.5, 5, 10 and 30 µg/mL. This experiment was conducted in a completely randomized design (factorial 4 × 4 × 6). Qualitative variables were compared using the Tukey test (P ≤ 0.05) and quantitative variables were subjected to regression analysis. Carbendazim; Tiram and Fludioxonil; Metalaxil-M had the best fungicidal performances, each inhibiting three of the four fungi with LD50s below 1 µg/mL. Subsequently, these two fungicides were used alone or in combination in the in vivo test. A completely randomized design was used and the means were compared using the Tukey test (P < 0.05). The chemical treatment of the seeds resulted in improvement of five of the six evaluated physiological parameters. The identification of the primary fungi associated with Paspalum seeds reported in this research, as well as damage done to them, can be diminished using appropriate measures such as seed treatments.(AU)

Das pastagens naturais cultivadas no sul do Brasil, as do gênero Paspalum são as mais importantes. Um dos fatores que dificultam o cultivo é a disponibilidade de sementes de qualidade, muitas vezes comprometidas pela presença de fungos. Os objetivos deste estudo foram determinar a sensibilidade in vitro de fungos associados a sementes de Paspalum guenoarum ecótipo azulão a determinados fungicidas, medir a eficiência de tratamentos químicos para controle desses fungos e o desempenho fisiológico das sementes. Os fungos associados às sementes, Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata e Fusarium incarnatum foram testados in vitro contra Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram e Tiofanato-metílico em 0, 1, 2,5, 5, 10 e 30 µg/mL. Este experimento foi conduzido em um delineamento inteiramente casualizado (fatorial 4 × 4 × 6). As variáveis ​​qualitativas foram comparadas pelo teste de Tukey (P ≤ 0,05) e as variáveis ​​quantitativas foram submetidas à análise de regressão. Carbendazim; Tiram e Fludioxonil; Metalaxil-M tiveram os melhores desempenhos fungicidas, cada um inibindo três dos quatro fungos com LD50 abaixo de 1 µg/mL. Posteriormente, esses dois fungicidas foram utilizados isoladamente ou em combinação no teste in vivo. O delineamento experimental foi inteiramente casualizado e as médias comparadas pelo teste de Tukey (P < 0,05). O tratamento químico das sementes resultou na melhoria de cinco dos seis parâmetros fisiológicos avaliados. A identificação dos fungos primários associados às sementes de Paspalum relatadas neste trabalho, bem como os danos causados ​​por eles, podem ser diminuídos usando medidas apropriadas, como tratamento de sementes.(AU)

Fungi associated with Paspalum guenoarum seeds: their impact on physiology and control / Fungos associados com sementes de Paspalum guenoarum: seus impactos na fisiologia e controle

Of the natural pastures grown in southern Brazil, those of the genus Paspalum are the most important. One of the factors that hinder their cultivation is the availability of quality seeds, that are often compromised by the presence of fungi. This study determined the in vitro sensitivity of Paspalum guenoarum ecotype azulão seed-associated fungus to certain fungicides and to measure the efficiency of chemical treatments for fungal control and seed physiological performance. Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata and Fusarium incarnatum associated with seeds were tested in vitro against Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram and Tiofanato-metílico at 0, 1, 2.5, 5, 10 and 30 µg/mL. This experiment was conducted in a completely randomized design (factorial 4 × 4 × 6). Qualitative variables were compared using the Tukey test (P ≤ 0.05) and quantitative variables were subjected to regression analysis. Carbendazim; Tiram and Fludioxonil; Metalaxil-M had the best fungicidal performances, each inhibiting three of the four fungi with LD50s below 1 µg/mL. Subsequently, these two fungicides were used alone or in combination in the in vivo test. A completely randomized design was used and the means were compared using the Tukey test (P < 0.05). The chemical treatment of the seeds resulted in improvement of five of the six evaluated physiological parameters. The identification of the primary fungi associated with Paspalum seeds reported in this research, as well as damage done to them, can be diminished using appropriate measures such as seed treatments.

Das pastagens naturais cultivadas no sul do Brasil, as do gênero Paspalum são as mais importantes. Um dos fatores que dificultam o cultivo é a disponibilidade de sementes de qualidade, muitas vezes comprometidas pela presença de fungos. Os objetivos deste estudo foram determinar a sensibilidade in vitro de fungos associados a sementes de Paspalum guenoarum ecótipo azulão a determinados fungicidas, medir a eficiência de tratamentos químicos para controle desses fungos e o desempenho fisiológico das sementes. Os fungos associados às sementes, Bipolaris micropus, Epicoccum sorghinum, Curvularia geniculata e Fusarium incarnatum foram testados in vitro contra Carbendazim; Tiram, Fludioxonil; Metalaxil-M, Carboxina; Tiram e Tiofanato-metílico em 0, 1, 2,5, 5, 10 e 30 µg/mL. Este experimento foi conduzido em um delineamento inteiramente casualizado (fatorial 4 × 4 × 6). As variáveis ​​qualitativas foram comparadas pelo teste de Tukey (P ≤ 0,05) e as variáveis ​​quantitativas foram submetidas à análise de regressão. Carbendazim; Tiram e Fludioxonil; Metalaxil-M tiveram os melhores desempenhos fungicidas, cada um inibindo três dos quatro fungos com LD50 abaixo de 1 µg/mL. Posteriormente, esses dois fungicidas foram utilizados isoladamente ou em combinação no teste in vivo. O delineamento experimental foi inteiramente casualizado e as médias comparadas pelo teste de Tukey (P < 0,05). O tratamento químico das sementes resultou na melhoria de cinco dos seis parâmetros fisiológicos avaliados. A identificação dos fungos primários associados às sementes de Paspalum relatadas neste trabalho, bem como os danos causados ​​por eles, podem ser diminuídos usando medidas apropriadas, como tratamento de sementes.

Seed germination and antioxidant enzyme activity in seedlings of diploid and tetraploid bahiagrass under water restriction / Germinação de sementes e atividade de enzimas antioxidantes em plântulas de grama forquilha diplóide e tetraploide sob restrição hídrica

The objective of the following research was to investigate the physiological responses of two bahiagrass genotypes (Paspalum notatum Flügge) submitted to water restriction, induced by polyethylene glycol (PEG6000), during seed germination. Seeds of cv. Pensacola (diploid) and Bagual ecotype (tetraploid) were placed on paper moistened with osmotic solutions at potentials of 0.0; -0.1; -0.2 and -0.3 MPa. Composed of two subsamples of 100 seeds and four experimental units per treatment, the germination test was conducted in a germination chamber under alternating temperatures of 30 °C (8 hours) and 20 °C (16 hours), with presence of light during the higher temperature. Final germination percentage and germination speed index of the seeds, as well as early growth, activity and expression of antioxidant enzymes of seedlings were evaluated. The water restriction induced by PEG6000 reduced the percentage of germination in Pensacola (from 74% to 59%) and Bagual (34% to 10%). For the latter, the evaluation of seedlings showed higher dry mass in relation to cv. Pensacola (1.7 times higher in root and 1.6 times in shoot). In response to moderate water restriction, the genotypes showed increases in root growth (1.7 times higher in -0.1 compared to 0.0 MPa), both in expression and enzymatic activity (superoxide dismutase and catalase).(AU)

O presente estudo objetivou avaliar as respostas fisiológicas de dois genótipos de grama forquilha (Paspalum notatum Flügge) sob restrição hídrica induzida por polietileno glicol (PEG6000) durante a germinação de sementes. Sementes da cv. Pensacola (diplóide) e do ecótipo Bagual (tetraplóide) foram colocadas em papel umedecido com soluções osmóticas nos potenciais de 0,0; -0,1; -0,2 e -0,3 MPa. Composto de duas subamostras de 100 sementes e quatro unidades experimentais por tratamento, o teste de germinação foi conduzido em câmara de germinação sob temperatura alternada de 30-20 ºC, com presença de luz durante a maior temperatura. Foram avaliados a porcentagem final e o índice de velocidade de germinação das sementes, assim como o crescimento inicial, a atividade e a expressão de enzimas antioxidantes das plântulas. A restrição hídrica induzida por PEG6000 reduziu o percentual de germinação em Pensacola (de 74 para 59%) e Bagual (34 para 10%). Este último, na avaliação de plântulas, apresentou massa seca superior em relação à cv. Pensacola (1.7 vezes superior em raiz e 1.6 vezes superior em parte aérea). Em resposta à restrição hídrica moderada, os genótipos apresentaram aumentos tanto do crescimento radicular (1.7 vezes superior em -0.1 em comparação com 0.0 MPa), quanto da expressão e da atividade enzimática (superóxido dismutase e catalase).(AU)

Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.

BACKGROUND: Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. RESULTS: The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3-8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. CONCLUSIONS: This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

Genetic assessment of seed yield-related traits in superior hybrids of Paspalum plicatulum × Paspalum guenoarum

This study aimed to estimate the genetic parameters, genetic correlations, genetic dissimilarity, and predicted gains with the selection of interspecific apomictic Paspalum plicatulum × Paspalum guenoarum hybrids in relation to seed yield traits. Nineteen genotypes that showed increased forage yield in previous studies were planted in a randomized complete block design with ten blocks. Traits evaluated in this study were: number of tillers/plant, number of reproductive tillers/plant, number of racemes/inflorescence, number of seeds/inflorescence, weight of a thousand seeds, and seed yield. Data were analyzed using a mixed model approach. The genotypes evaluated in our study showed improvement potential in relation to high heritability (H2 g > 0.50), high precision (Acgen> 90%), and genetic dissimilarity (forming three groups) for seed yield traits. Seed yield showed high correlation to reproductive tillers that can be used as indirect selection tools for genetic improvement of seed yield.(AU)

Relative DNA content in diploid, polyploid, and multiploid species of Paspalum (Poaceae) with relation to reproductive mode and taxonomy.

It is generally accepted that polyploids have downsized basic genomes rather than additive values with respect to their related diploids. Changes in genome size have been reported in correlation with several biological characteristics. About 75 % of around 350 species recognized for Paspalum (Poaceae) are polyploid and most polyploids are apomictic. Multiploid species are common with most of them bearing sexual diploid and apomictic tetraploid or other ploidy levels. DNA content in the embryo and the endosperm was measured by flow cytometry in a seed-by-seed analysis of 47 species including 77 different entities. The relative DNA content of the embryo informed the genome size of the accession while the embryo:endosperm ratio of DNA content revealed its reproductive mode. The genome sizes (2C-value) varied from 0.5 to 6.5 pg and for 29 species were measured for the first time. Flow cytometry provided new information on the reproductive mode for 12 species and one botanical variety and supplied new data for 10 species concerning cytotypes reported for the first time. There was no significant difference between the mean basic genome sizes (1Cx-values) of 32 sexual and 45 apomictic entities. Seventeen entities were diploid and 60 were polyploids with different degrees. There were no clear patterns of changes in 1Cx-values due to polyploidy or reproductive systems, and the existing variations are in concordance with subgeneric taxonomical grouping.

Complete asynapsis resulting in 2n pollen formation in Paspalum jesuiticum Parodi (Poaceae).

Cytological investigation revealed complete asynapsis during microsporogenesis in 2 wild accessions of Paspalum jesuiticum collected in distinct Brazilian regions. Both accessions were hexaploid (2n = 6x = 60) and 60 univalents could be counted at diakinesis. In this phase, the majority of meiocytes exhibited univalents with both chromatids. After alignment at the metaphase plate, the chromatids segregated to the poles. Only 1 meiotic division (equational) occurred, and after cytokinesis, a dyad with 2n microspores was formed. The genetic constitution of the 2n gametes was equivalent to that of first division restitution (FDR). Since recombination did not occur, 100% transmission of parental heterozygosity could be expected from the FDR 2n gametes. The meiotic behavior of both accessions suggested that they resulted from a recent natural hybridization event. The potential use of the 2n gametes in Paspalum breeding programs has been discussed.

Microsporogenesis in Paspalum conspersum Schrad. (Virgata group) with different ploidy levels.

Knowledge about the cytology and reproductive behavior of a species is indispensable for hybridization programs. This is especially true for species belonging to the genus Paspalum, among which apomixis and a wide range of ploidy levels are frequently found. Paspalum conspersum Schrad. is a robust and warm-season perennial bunchgrass native to South America. Previous studies have indicated that both tetraploid and hexaploid races exist in this species; however, only information related to tetraploids has been applied to another taxon. In this study, a cytological investigation in two Brazilian accessions collected in different regions revealed tetraploidy in the accession BRA-012823 (2n = 4x = 40), with chromosome pairing in bivalents and normal meiosis and tetrad formation, and pentaploidy (2n = 5x = 50) in the accession BRA-022748, which presented total asynapsis. In this latter accession, 50 univalents could be scored at diakinesis. After alignment at the metaphase plate, sister chromatids segregated to the poles. Only one meiotic division (equational) occurred, and after cytokinesis, 100% of the dyads that formed had 2n microspores. The meiotic behavior during microsporogenesis, which showed 10 delayed univalents to reach the metaphase plate, suggests that this accession is a recent natural hybrid constituted by a parental genome with 40 chromosomes and another with 10 chromosomes. The potential usage of these accessions in Paspalum breeding has been discussed.

Harnessing apomictic reproduction in grasses: what we have learned from Paspalum.

BACKGROUND: Apomixis is an alternative route of plant reproduction that produces individuals genetically identical to the mother plant through seeds. Apomixis is desirable in agriculture, because it guarantees the perpetuation of superior genotypes (i.e. heterotic hybrid seeds) by self-seeding without loss of hybrid vigour. The Paspalum genus, an archetypal model system for mining apomixis gene(s), is composed of about 370 species that have extremely diverse reproductive systems, including self-incompatibility, self-fertility, full sexual reproduction, and facultative or obligate apomixis. Barriers to interspecific hybridization are relaxed in this genus, allowing the production of new hybrids from many different parental combinations. Paspalum is also tolerant to various parental genome contributions to the endosperm, allowing analyses of how sexually reproducing crop species might escape from dosage effects in the endosperm. SCOPE: In this article, the available literature characterizing apomixis in Paspalum spp. and its use in breeding is critically reviewed. In particular, a comparison is made across species of the structure and function of the genomic region controlling apomixis in order to identify a common core region shared by all apomictic Paspalum species and where apomixis genes are likely to be localized. Candidate genes are discussed, either as possible genetic determinants (including homologs to signal transduction and RNA methylation genes) or as downstream factors (such as cell-to-cell signalling and auxin response genes) depending, respectively, on their co-segregation with apomixis or less. Strategies to validate the role of candidate genes in apomictic process are also discussed, with special emphasis on plant transformation in natural apomictic species.

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants.

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3' UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.