Francisco André Ossamu Tanaka

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences.

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa

The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa.

Anatomical and Physiological Responses of Citrus Trees to Varying Boron Availability Are Dependent on Rootstock.

In Citrus, water, nutrient transport and thereby fruit production, are influenced among other factors, by the interaction between rootstock and boron (B) nutrition. This study aimed to investigate how B affects the anatomical structure of roots and leaves as well as leaf gas exchange in sweet orange trees grafted on two contrasting rootstocks in response to B supply. Plants grafted on Swingle citrumelo or Sunki mandarin were grown in a nutrient solution of varying B concentration (deficient, adequate, and excessive). Those grafted on Swingle were more tolerant to both B deficiency and toxicity than those on Sunki, as revealed by higher shoot and root growth. In addition, plants grafted on Sunki exhibited more severe anatomical and physiological damages under B deficiency, showing thickening of xylem cell walls and impairments in whole-plant leaf-specific hydraulic conductance and leaf CO2 assimilation. Our data revealed that trees grafted on Swingle sustain better growth under low B availablitlity in the root medium and still respond positively to increased B levels by combining higher B absorption and root growth as well as better organization of xylem vessels. Taken together, those traits improved water and B transport to the plant canopy. Under B toxicity, Swingle rootstock would also favor plant growth by reducing anatomical and ultrastructural damage to leaf tissue and improving water transport compared with plants grafted on Sunki. From a practical point of view, our results highlight that B management in citrus orchards shall take into account rootstock varieties, of which the Swingle rootstock was characterized by its performance on regulating anatomical and ultrastructural damages, improving water transport and limiting negative impacts of B stress conditions on plant growth.

Cellular and molecular changes associated with competence acquisition during passion fruit somatic embryogenesis: ultrastructural characterization and analysis of SERK gene expression

The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus.

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species.

Detection of isometric, dsRNA-containing viral particles in Colletotrichum gloeosporioides isolated from cashew tree

Fungi are disease-causing agents in plants and affect crops of economic importance. One control method is to induce resistance in the host by using biological control with hypovirulent phytopathogenic fungi. Here, we report the detection of a mycovirus in a strain of Colletotrichum gloeosporioides causing anthracnose of cashew tree. The strain C. gloeosporioides URM 4903 was isolated from a cashew tree (Anacardium occidentale) in Igarassu, PE, Brazil. After nucleic acid extraction and electrophoresis, the band corresponding to a possible double-stranded RNA (dsRNA) was purified by cellulose column chromatography. Nine extrachromosomal bands were obtained. Enzymatic digestion with DNAse I and Nuclease S1 had no effect on these bands, indicating their dsRNA nature. Transmission electron microscopic examination of extracts from this strain showed the presence of isometric particles (30-35 nm in diameter). These data strongly suggest the infection of this C. gloeosporioides strain by a dsRNA mycovirus. Once the hypovirulence of this strain is confirmed, the strain may be used for the biological control of cashew anthracnose.

Detection of the "Candidatus Liberibacter americanus" in phloem vessels of experimentally infected cataranthus roseus by scanning electron microscopy

Presence of Huanglongbing (HLB) disease in citrus orchards in the state of Sao Paulo has been known since 2004. Its identification was made by molecular techniques which revealed the presence of two forms of the bacteria “Candidatus Liberibacter”. One previously described in Asia as Ca. L. asiaticus and the other a novel type, Ca. L. americanus (Coletta-Fo et al., Plant Dis. 88:1382. 2004; Teixeira et al., Plant Dis. 89:107. 2005). Transmission electron microscopy confirmed the presence of characteristic bacteria in the phloem vessels of affected sweet orange leaves (Tanaka et al., Fitopatol. Bras. 31:99. 2006). Attempts to observe these bacteria by scanning electron microscopy (SEM) to understand their three dimensional morphology were hindered by the extremely low concentration at which they usually occur in citrus plants. On the other hand, Ca. L. americanus was successfully transferred to the ornamental periwinkle (Cataranthus roseus (L.) G. Don.) using dodder (Cuscuta sp.) as previously reported for other Ca. Liberibacter (Garnier & Bove, Proc. Conf. IOCV 12: 212. 1993). Infected plants developed chlorotic symptoms and contained higher concentrations of the bacterium as evaluated by molecular techniques and transmission electron microscopy. For in situ observations of Ca. L. americanus under SEM, midribs from symptomatic leaves and young stems were fixed with aldehyde, infiltrated with glycerol, immersed in liquid nitrogen and fractured with a scalpel, and the pieces post-fixed in 0.1% OsO4 for 24 h, dehydrated in acetone, dried at the critical point, gold coated by sputtering and examined in a LEO 435 VP scanning electron microscope. In the vascular region of the infected leaves and stems, exposed by the fracture of frozen tissues, many phloem vessels contained bacterial cells, interpreted as Ca. L. americanus, in varied numbers. Some vessels were obliterated by aggregated bacterial cells (Fig. 1 A). Bacteria were typically baciliform and elongated, 2-3 μm long with rounded ends, 200-300 nm wide and with a smooth outer surface (Fig. 1 B). These values match those obtained in sectioned bacterial cells. Such bacterial cells were absent in the phloem vessels of healthy, uninoculated periwinkle plants. This report is the first description of Candidatus Liberibacter by scanning electron microscopy. RESUMO Deteccao de Candidatus Liberibacter americanus em vasos de floema de Catharantus roseus infectados experimentalmente, utilizando microscopia eletronica de varredura “Candidatus Liberibacter americanus”, agente causal do Huanglongbing (HLB) foi visualizado pela primeira vez ao microscopio eletronico de varredura infectando vasos do floema de plantas de vinca (Catharantus roseus) experimentalmente infectadas utilizando-se Cuscuta sp. no Brasil.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Loss of type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility

Glandular trichomes are structures with widespread distribution and deep ecological significance. In the Solanum genus, type-IV glandular trichomes provide resistance to insect pests. The occurrence of these structures is, however, poorly described and controversial in cultivated tomato (Solanum lycopersicum). Optical and scanning electron microscopy were used to screen a series of well-known commercial tomato cultivars, revealing the presence of type-IV trichomes on embryonic (cotyledons) and juvenile leaves. A tomato line overexpressing the microRNA miR156, known to promote heterochronic development, and mutants affecting KNOX and CLAVATA3 genes possessed type-IV trichomes in adult leaves. A re-analysis of the Woolly (Wo) mutant, previously described as enhancing glandular trichome density, showed that this effect only occurs at the juvenile phase of vegetative development. Our results suggest the existence of at least two levels of regulation of multicellular trichome formation in tomato: one enhancing different types of trichomes, such as that controlled by the WOOLLY gene, and another dependent on developmental stage, which is fundamental for type-IV trichome formation. Their combined manipulation could represent an avenue for biotechnological engineering of trichome development in plants.

Ontogênese de caneluras em pedúnculo de flores de laranjeira doce infectados pelo vírus da tristeza dos citros estirpe "Capão Bonito"

Um estudo da ontogenese das caneluras induzidas em ramos de laranjeiras doces suscetiveis por isolados severos do virus da tristeza dos citros (Citrus tristeza virus - CTV) foi feito usando-se como modelo pedunculos florais e de frutos. O menor calibre destes orgaos permite um melhor acompanhamento do processo. As observacoes foram feitas em laranjeira cv. Pera infetada pelo isolado severo Capao Bonito do CTV. Cinco fases do processo de formacao de caneluras puderam ser deduzidas pelas analises anatomicas. As primeiras alteracoes sao representadas pelo aparecimento de celulas adensadas, hipertrofia e hiperplasia no parenquima e câmbio do floema e uma desorganizacao generalizada desta area. Segue-se uma atividade intensa do câmbio do floema adjacente e sua expansao em direcao ao xilema. Esta invasao do xilema resulta na ruptura do anel do xilema pela massa celular do floema constituida de celulas recem formadas de parede celular delgada. Esta invasao do floema em direcao ao xilema inicia um processo de degeneracao dos vasos e parenquima do xilema. Finalmente ha um colapso completo da regiao do xilema invadida, que e substituida pela massa do floema, resultando na canelura, notada ao se remover a casca.