A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.

Citrus Sudden Death (CSD), a new, graft-transmissible disease of sweet orange and mandarin trees grafted on Rangpur lime rootstock, was first seen in 1999 in Brazil, where it is present in the southern Triângulo Mineiro and northwestern São Paulo State. The disease is a serious threat to the citrus industry, as 85% of 200 million sweet orange trees in the State of São Paulo are grafted on Rangpur lime. After showing general decline symptoms, affected trees suddenly collapse and die, in a manner similar to trees grafted on sour orange rootstock when affected by tristeza decline caused by infection with Citrus tristeza virus (CTV). In tristeza-affected trees, the sour orange bark near the bud union undergoes profound anatomical changes. Light and electron microscopic studies showed very similar changes in the Rangpur lime bark below the bud union of CSD-affected trees: size reduction of phloem cells, collapse and necrosis of sieve tubes, overproduction and degradation of phloem, accumulation of nonfunctioning phloem (NFP), and invasion of the cortex by old NFP. In both diseases, the sweet orange bark near the bud union was also affected by necrosis of sieve tubes, and the phloem parenchyma contained characteristic “chromatic” cells. In CSD-affected trees, these cells were seen not only in the sweet orange phloem, but also in the Rangpur lime phloem. Recent observations indicated that CSD affected not only citrus trees grafted on Rangpur lime but also those on Volkamer lemon, with anatomical symptoms similar to those seen in Rangpur lime bark. Trees on alternative rootstocks, such as Cleopatra mandarin and Swingle citrumelo, showed no symptoms of CSD. CSD-affected trees did recover when they were inarched with seedlings of these rootstocks, but not when inarched with Rangpur lime seedlings. These results indicate that CSD is a bud union disease. In addition, the bark of inarched Rangpur lime and Volkamer lemon seedlings showed, near the approach-graft union, the same anatomical alterations as the bud union bark from the Rangpur lime rootstock in CSD-affected trees. The dsRNA patterns from CSD-affected trees and unaffected trees were similar and indicative of CTV. CSD-affected trees did not react by immunoprinting-ELISA using monoclonal antibodies against 11 viruses. No evidence supported the involvement of viroids in CSD. The potential involvement of CTV and other viruses in CSD is discussed.

Observations of cells of axenic peach palm (Bactris gasipaes) microplants by light microscopy revealed movements of small particles within the cells. The phenomenon was characterized initially as Brownian movement, but electron microscopy revealed the presence of an intracellular bacterial community in these plants. Microscopy observations revealed the particular shapes of bacterial cells colonizing inner tissues of analyzed plants. Applying a molecular characterization by polymerase chain reaction and denaturing gradient gel electrophoresis, it was revealed the existence of bacterial rRNA within the plants. Sequencing of the rRNA identified three different phylogenetic groups; two bands had a high degree of similarity to sequences from Moraxella sp. and Brevibacillus sp., and a third sequence was similar to a non-cultivated cyanobacterium. The presence of those endosymbionts, called bacteriosomes, in axenic peach palm microplants raises the question of whether these stable endosymbionts were acquired in the process of evolution and how could they benefit the process of plants micropropagation.

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.

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.

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.

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.