Infection by Moniliophthora perniciosa reprograms tomato Micro-Tom physiology, establishes a sink, and increases secondary cell wall synthesis.

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

Moniliophthora perniciosa, the causal agent of witches' broom disease of cacao, interferes with cytokinin metabolism during infection of Micro-Tom tomato and promotes symptom development.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.

Stigma structure and receptivity in papaya (Carica papaya L.).

The objective of this study was to evaluate the stigma morphoanatomy and receptivity in ten promising papaya accessions, to expand knowledge useful for genetic improvement of the culture. The morphoanatomy was analyzed by light and scanning electron microscopy, and the stigma receptivity was investigated by application of hydrogen peroxide and α-naphthyl-acetate with fast blue B salt, in pre-anthesis, anthesis and post-anthesis. The papaya accessions presented dry stigma surface, presence of one to six erect stigmatic lobes, with structures joined to the upper base of the style and covered with numerous elongated unicellular tubular papillae, distributed on both faces of the epidermis. The morphoanatomy had a similar pattern in all the accessions, differing only in the timing of floral development. The stigma receptivity in some accessions occurred even before floral opening, continuing with greater intensity in anthesis and post-anthesis. Pre-anthesis is the stage least propitious for controlled hybridizations due to the weak or absent stigmatic receptivity. The results obtained provide information on the floral stage that is most propitious for fertilization, as well as supporting future investigations of the botanical morphology of the species.

Post-seminal development and cryopreservation of endemic or endangered bromeliads.

Vriesea bahiana, Hohenbergia castellanosii and Encholirium spectabile are endemic Brazilian species that are considered endemic or endangered. Development of strategies to conserve these species is important to prevent irreversible genetic erosion. The objective of this study was to evaluate the post-seminal development and seed cryopreservation of three endemic or in danger of extinction bromeliad species in Brazil, to obtain a protocol that can safeguard the genetic variability of these species. In the seed cryopreservation assay, we evaluated five desiccation periods. The seeds in the cryotubes were taken from the desiccator and immediately plunged into liquid nitrogen. For the analysis of post-seminal development, seeds in different germination stages were collected and evaluated by light and scanning electron microscopy. Vriesea bahiana seeds frozen in liquid nitrogen presented almost 100% germination, indicating dormancy break of this species. Vriesea bahiana can be cryopreserved with 5.9% water content after being dried for 24 hours. Hohenbergia castellanosii and E. spectabile seeds did not need to be desiccated before being cryopreserved. The most relevant morphological traits for differentiation of genera and subfamilies of Bromeliaceae are the shape and type of seed appendages. In this study, all three species presented well-differentiated size and shape of their structures.

Morphoanatomical aspects of the starting material for the improvement of pineapple cryopreservation by the droplet-vitrification technique.

Cryopreservation of pineapple shoot tips has been established from various protocols, including droplet vitrification. Thus, this work aimed to evaluate the morphoanatomical conditions of the starting material over different times (30, 45 and 60 days) of culture before freezing and its correlation with the survival percentage of the cryopreserved shoot tips. Four accessions, Ananas comosus var. comosus (BGA-009); var. bracteatus (BGA-119); var. parguazensis (BGA-376), var. erectifolius (BGA-750) from the Pineapple Active Germplasm Bank (BGA Pineapple) and two hybrids from the Embrapa Genetic Breeding Program, FIB-ROX1 (var. bracteatus X var. erectifolius) and FIB-ROX2 ( var. erectifolius X var. bracteatus), recently introduced in the field from in vitro storage, were used. Histological sections before freezing and the percentages of survival after freezing were obtained taking into account the different times of cultivation of the donor plants. The results showed a significative interaction between genotypes (accessions and hybrids) and the culture period. The accessions BGA-009 and BGA-119 showed the highest survival rates, with 95% and 90% respectively for the 30-day culture time. Different results were obtained for each genotype, showing the need for improvements in the standardization of starting material, which would allow better repeatability of the protocol.

Post-seminal development and morphoanatomy of vegetative and reproductive organs in Stevia rebaudiana (Bert.) Bertoni (Asteraceae).

Stevia rebaudiana belongs to the Asteraceae family with high economic and medicinal potential. This article describes and illustrates morphological and histological aspects of leaves and reproductive organs, and the germination process, to provide detailed information on this species and to contribute to taxonomic, phylogenetic and pharmacobotanical projects. The fruit is a cypsela, small, simple, dry, indehiscent, monospermic, light or dark colored, with aristate pappus, and the seed presents a spatulate axile embryo. Germination is phaneroepigeal with a pivotal root system and many absorbing root hairs. The leaves are simple, elliptical to obovate, with two types of trichomes (glandular and tector), with a short petiole, exhibiting an opposite decussate phyllotaxy. Our results showed 37.5% germination after 12 days, only in the dark cypsela, the light colored being considered unviable. The inflorescence is paniculate and the florets are grouped in capitula with isomorphic ends, monoclinous (bisexual), dichlamydeous, heterochlamydeous, pentamerous calyx and corolla, gamossepalous and gamopetalous. The androecium is gamostemone comprised of five stamens with free filaments, isodynamous and epipetalous stamens, synandrous and rimose anthers. The flower presents an inferior ovary, bicarpelar, unilocular and ovules with a basal placentation. The pollen grains are small, isopolar, radial symmetry, tricolporate, with echinate ornamentation.

Comparative pollen morphological analysis in the subgenera Passiflora and Decaloba.

The genus Passiflora is the most diversified of the Passifloraceae, and its palynology presents wide morphological variability. The objective of the study was to evaluate the pollen morphology of 18 Passiflora species in order to identify informative pollinic characteristics to contribute to the taxonomic classification of the genus. The morphology of the pollen grains and the exine structure were investigated using light microscopy and scanning electron microscopy. Differences in the pollen morphology were found in the studied species, mainly in terms of shape, pollen aperture and ornamentation pattern of the exine. Most of the species belonging to the subgenus Passiflora presented ornamented 6-syncolpate pollen grains with an oblate-spheroidal shape and an exine with large lumens. In the subgenus Decaloba the pollen grains were 6-colporate, 12-colpate and 12-colporate, with subprolate to prolate-spheroidal shape, as well as an exine with smaller lumen size and few ornamented. The pollen morphology of the species of the subgenera Passiflora and Decaloba has shown palynological characteristics that have specific diagnostic value, thus allowing a better understanding of the taxonomy of the genus Passiflora.

Pollen morphology and viability in Bromeliaceae.

Pollen morphology characterization is important in taxonomy, conservation and plant breeding, and pollen viability studies can support breeding programs. This study investigated pollen morphology and male fertility in 18 species of Bromeliaceae with ornamental potential. For morphological characterization, pollen grains were acetolyzed and characterization of exine was done using scanning and transmission electron microscopy. Pollen viability was investigated by in vitro germination and histochemical tests. Species belonging to Aechmea and Ananas genera presented medium size pollen, except for Ae. fasciata, with large pollen. Al. nahoumii, P. sagenarius and the Vriesea species analyzed showed large pollen, except for V. carinata, with very large pollen. Pollen of Aechmea, Ananas and P. sagenarius presented bilateral symmetry, diporate, exine varying from tectate to semitectate. Al. nahoumii and Vriesea species presented pollen with bilateral symmetry, monocolpate; exine was semitectate, reticulate and heterobrochate. Germination percentage and tube growth were greater in SM and BKM media. Histochemical tests showed pollen viability above 70% for all species, except for Ananas sp. (40%). Pollen morphology is important for the identification of species, especially in this family, which contains a large number of species. High rates of viability favor fertilization and seed production, essential for efficient hybrid production and conservation.

Anther development in Brachiaria brizantha (syn. Urochloa brizantha) and perspective for microspore in vitro culture.

Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa brizantha) is of great agronomical importance due to the large areas cultivated with this species. This cultivar is apomictic and tetraploid. Sexual diploid genotype is available for this species. The difference in levels of ploidy among sexual and apomictic plants contributes to hindering Brachiaria breeding programs. The induction of haploids and double haploids is of great interest for the generation of new genotypes with potential use in intraspecific crosses. A key factor for the success of this technique is identifying adequate microspore developmental stages for efficient embryogenesis induction. Knowledge of the morphological changes during microsporogenesis and microgametogenesis and sporophytic tissues composing the anther is critical for identifying the stages in which microspores present a higher potential for embryogenic callus and somatic embryo through in vitro culture. In this work, morphological markers were associated with anther and pollen grain developmental stages, through histological analysis. Anther development was divided into 11 stages using morphological and cytological characteristics, from anther with archesporial cells to anther dehiscence. The morphological characteristics of each stage are presented. In addition, the response of stage 8 anthers to in vitro culture indicates microspores initiating somatic embryogenic pathway.

Biochemical and histological characterization of tomato mutants.

Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT), we observed that the malondialdehyde (MDA) content was enhanced in the diageotropica (dgt) and lutescent (l) mutants, whilst the highest levels of hydrogen peroxide (H(2)O(2)) were observed in high pigment 1 (hp1) and aurea (au) mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT) activity when compared to MT. Guaiacol peroxidase (GPOX) was enhanced in both sitiens (sit) and notabilis (not) mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX). Based on PAGE analysis, the activities of glutathione reductase (GR) isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD) isoform III was reduced in leaves of sit, epi, Never ripe (Nr) and green flesh (gf) mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.

Description of the immatures of Linepithema humile Mayr (Hymenoptera: Formicidae).

Linepithema humile Mayr is an ant species originally native to South America that has been spread accidently throughout the globe through international trade. It is a serious urban and crop pest. Despite its economic importance, little is known about the larvae of this species apart from a brief description based on a few specimens. The present investigation is aimed at describing every immature stage of L. humile. Three larval instars were determined through the frequency distribution of the maximum width of head capsules from a sample of 525 larvae. The morphological descriptions were based on 150 eggs, 70 larvae, and 90 pupae examined by light and scanning electron microscopy. Some morphological characteristics reported to be typical of Linepithema Mayr larvae were confirmed--dolichoderoid body shape, presence of dorsal protuberance, sparse simple body hairs, presence of nine pairs of spiracles and dolichoderoid mandibles. We concluded that an earlier published description was based on queen larvae, and that the protuberance is only present in worker larvae. The information provided in this study may aid ant systematics and phylogenetics, as well provide a better understanding of the biology of this species.

Abscisic acid-deficient sit tomato mutant responses to cadmium-induced stress.

There is a very effective cross-talk between signals triggered by reactive oxygen species and hormonal responses in plants, activating proteins/enzymes likely to be involved in stress tolerance. Abscisic acid (ABA) is known as a stress hormone that takes part in the integration of signals. This work aimed to characterize the biochemical response and ultrastructural changes induced by cadmium (Cd) in the Micro-Tom (MT) sitiens ABA-deficient mutant (sit) and its wild-type (MT) counterpart. MT and sit plants were grown over a 96-h period in the presence of Cd (0, 10, and 100 µM CdCl2). The overall results indicated increases in lipid peroxidation, hydrogen peroxide content and in the activities of the key antioxidant enzymes such as catalase, glutathione reductase, and ascorbate peroxidase in both genotypes. On the other hand, no alteration was observed in chlorophyll content, while the activity of another antioxidant enzyme, superoxide dismutase, remained constant or even decreased in the presence of Cd. Roots and shoots of the sit mutant and MT were analyzed by light and transmission electron microscopy in order to characterize the structural changes caused by the exposure to this metal. Cd caused a decrease in intercellular spaces in shoots and a decrease in cell size in roots of both genotypes. In leaves, Cd affected organelle shape and internal organization of the thylakoid membranes, whereas noticeable increase in the number of mitochondria and vacuoles in MT and sit roots were observed. These results add new information that should help unravel the relative importance of ABA in regulating the cell responses to stressful conditions induced by Cd apart from providing the first characterization of this mutant to oxidative stress.

Si-Accumulation In Artemisia annua Glandular Trichomes Increases Artemisinin Concentration, but Does Not Interfere In the Impairment of Toxoplasma gondii Growth.

Artemisia annua is used as a source of artemisinin, a potent therapeutic agent used for the treatment of infectious diseases, chiefly malaria. However, the low concentration (from 0.01 to 1.4% of dried leaf matter) of artemisinin in the plant obtained with the traditional cropping system makes it a relatively expensive drug, especially in developing countries. Considering that artemisinin and silicon (Si) are both stored in A. annua glandular trichomes, and that Si accumulation has never been investigated, this study aimed to look into Si effects on A. annua trichome artemisinin concentration, and whether leaf infusion from Si-treated A. annua plants is able to control Toxoplasma gondii growth. T. gondii is the etiologic agent of toxoplasmosis, a zoonotic parasitic disease whose traditional treatment shows significant side effects. The experimental design consisted of A. annua seedlings randomly planted in soil treated with different doses of calcium/magnesium silicate (0, 200, 400, 800, and 1600 kg ha-1). Analysis of foliar macronutrients showed significant increases of nitrogen content only at the highest dose of silicate. Foliar micronutrients, Si concentrations, and plant height were not affected by any of the silicate doses. However, the dose of 400 kg ha-1 of silicate increased the trichome size, which in turn raised artemisinin concentration in leaves and the infusion. In contrast, the 800 and 1600 kg ha-1 doses dramatically decreased artemisinin concentration. HeLa cell treatment with the infusion of A. annua grown in soil treated with 400 kg ha-1 of silicate decreased parasite proliferation in a dose-dependent manner when the treatment was carried out after or along with T. gondii infection. However, this effect was similar to A. annua grown in soil without silicate treatment. Thus, it can be concluded that, even though Si applied to the soil at 400 kg ha-1 has a positive effect on the A. annua glandular trichome size and the artemisinin concentration, this outcome cannot be directly associated with the efficiency of A. annua infusion on T. gondii growth, suggesting that other components from A. annua leaves could be acting in synergy with artemisinin.

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues.

BACKGROUND: Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. RESULTS: Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. CONCLUSIONS: Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Pollen morphology and viability in Bromeliaceae

ABSTRACT Pollen morphology characterization is important in taxonomy, conservation and plant breeding, and pollen viability studies can support breeding programs. This study investigated pollen morphology and male fertility in 18 species of Bromeliaceae with ornamental potential. For morphological characterization, pollen grains were acetolyzed and characterization of exine was done using scanning and transmission electron microscopy. Pollen viability was investigated by in vitro germination and histochemical tests. Species belonging to Aechmea and Ananas genera presented medium size pollen, except for Ae. fasciata, with large pollen. Al. nahoumii, P. sagenarius and the Vriesea species analyzed showed large pollen, except for V. carinata, with very large pollen. Pollen of Aechmea, Ananas and P. sagenarius presented bilateral symmetry, diporate, exine varying from tectate to semitectate. Al. nahoumii and Vriesea species presented pollen with bilateral symmetry, monocolpate; exine was semitectate, reticulate and heterobrochate. Germination percentage and tube growth were greater in SM and BKM media. Histochemical tests showed pollen viability above 70% for all species, except for Ananas sp. (40%). Pollen morphology is important for the identification of species, especially in this family, which contains a large number of species. High rates of viability favor fertilization and seed production, essential for efficient hybrid production and conservation.

Description of the immatures of Linepithema humile Mayr (Hymenoptera: Formicidae)

Linepithema humile Mayr is an ant species originally native to South America that has been spread accidently throughout the globe through international trade. It is a serious urban and crop pest. Despite its economic importance, little is known about the larvae of this species apart from a brief description based on a few specimens. The present investigation is aimed at describing every immature stage of L. humile. Three larval instars were determined through the frequency distribution of the maximum width of head capsules from a sample of 525 larvae. The morphological descriptions were based on 150 eggs, 70 larvae, and 90 pupae examined by light and scanning electron microscopy. Some morphological characteristics reported to be typical of Linepithema Mayr larvae were confirmed - dolichoderoid body shape, presence of dorsal protuberance, sparse simple body hairs, presence of nine pairs of spiracles and dolichoderoid mandibles. We concluded that an earlier published description was based on queen larvae, and that the protuberance is only present in worker larvae. The information provided in this study may aid ant systematics and phylogenetics, as well provide a better understanding of the biology of this species.

Efeito da aplicação de silício em plantas de milho no desenvolvimento biológico da lagarta-do-cartucho Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) / Effect of silicon application on corn plants upon the biological development of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

The effect of the application of silicon to corn plants on the development of the fall armyworm Spodoptera frugiperda (J.E. Smith) was evaluated. Silicon treatment consisted in applying 3.2 ml of a sodium silicate solution [25-28 percent (w/v) SiO2] plus 96.8 ml of water into the soil at 5, 10, 15, 20 and 25 days after emergence of corn plants kept in pots. Another group of plants were grown free of silicon. Larvae were fed on leaves obtained from silicon treated plants and their mortality at the end of the 2nd larval instar, length of the larval and pupal period, and pupal weight were compared to those of larvae fed on normal leaves. No effect of silicon was observed on the length of larval and pupal phase, pupal weight and mortality of pupae. However, increased mortality and cannibalism were registered in groups of fall armyworm at the end of the 2nd instar and greater mortality of the larvae was seen at the 2nd and 6th instars fed on silicon-treated corn plant leaves in comparison with the control. The jaws mandibles of fall armyworm at the 6th instar showed marked wear on the incisor region when in contact with leaves with higher silicon content. Silicon application can make fall armyworm feeding difficult, causing higher mortality and cannibalism, thus increasing plant resistance to this species.

Este trabalho, objetivou avaliar o efeito da aplicação de silício em plantas de milho no desenvolvimento da lagarta-do-cartucho Spodoptera frugiperda (J.E. Smith). O tratamento com silício consistiu na aplicação de 3,2 ml de solução de silicato de sódio [25-28 por cento (p/v) de SiO2], mais 96,8 ml de água, via solo aos cinco dias, 10, 15, 20 e 25 dias após a emergência de plantas de milho mantidas em vasos. Outro grupo de plantas foi cultivado sem aplicação de silício. Avaliou-se o desenvolvimento de lagartas alimentadas com folhas de milho retiradas de plantas tratadas com silício, em comparação com lagartas alimentadas com folhas de milho não tratadas, registrando-se a mortalidade ao final do 2° ínstar larval, a duração do período larval e pupal, a mortalidade larval e pupal e o peso de pupa. Não se observou efeito do silício na duração da fase larval e pupal, peso de pupa e na mortalidade de pupas. Entretanto, verificou-se maior mortalidade e aumento de canibalismo em grupos de lagartas ao final do 2° ínstar, e maior mortalidade de lagartas individualizadas no 2° e 6° ínstares, quando foram alimentadas com folhas de plantas de milho tratadas com silício, em comparação com a testemunha. Observou-se que as mandíbulas das lagartas, nos seis ínstares, apresentaram desgaste acentuado na região incisora quando em contato com folhas com maior teor de silício. A aplicação de silício pode dificultar a alimentação de lagartas, causando aumento de mortalidade e canibalismo e, portanto, tornando as plantas de milho mais resistentes à lagarta-do-cartucho.