The development of efficient algae cryopreservation methods is pivotal to the establishment of long-term culture collections as well as algae breeding and genetic modification programs. However, the unpredictable responses of distinct algal species to cryopreservation agents and protocols have hampered the standardization of universal methods so far. The results presented in this study indicate that intrinsic biological factors (i.e.: cell morphology and phylogenetic origin) play a role in the definition of which culture age and cryoprotectant agent type and concentration should be used to achieve successful cryopreservation. Through the use of Central Composite Rotatable Design (CCRD) it was possible to define optimized protocols for cryopreserving three major morphotypic groups found among chlorophytes (i.e.: coenobium, coccoid or palmella forming strains). These optimized protocols were then validated upon fifteen strains from Sphaeropleales, Chlamydomonadales and Chlorelalles clades. Analysis reveals that the use of DMSO as the sole cryoprotectant provided the highest post-freezing cell viability recovery rates for 80% of the coccoid strains tested, while the combination of glycerol and PEG400 allowed efficient cryopreservation of 100% of the coenobium and palmella forming strains evaluated. In addition, results suggest that coenobium-forming strains have higher resistance to freeze/thawing when frozen at mid-log phase of growth, while coccoid and plamelloid strains present higher survival rates when cryopreserved at late-log phase. Such information is of valuable practical use since a simple visual inspection of algae strain's predominant morphotype can guide the choice of the most suitable cryopreservation protocol.

Little is known about the anatomical development of the reproductive system of the American Oil Palm (Elaeis oleifera); an species known in Brazil as Caiaue, that holds a series of characteristics not found in the African Oil palm (Elaeis guineensis), rendering it as an important source of genetic variability to be explored by African Oil palm breeding programs in Brazil and elsewhere. Among those characteristics are its small stature, resistance to the Bud Rot and high oil quality. The main objective of this study was to conduct a histo-anatomical analysis of the complete development of the reproductive system of three E. oleifera individuals collected at different localities in the Brazilian Amazon forest. Besides that, rachillas collected from different leaf axils were analyzed to determine the meiotic stage of microsporocytes useful for future cytogenetic and cloning studies. The microscopy analysis of the American Oil Palm individuals BR174, Coari and Manicore started with the youngest phase of the inflorescence until it reached its full development. Flowers were collected from inflorescences in different stages of development and from different parts of a rachilla, fixed and embedded in Technovit 7100 degrees resin and/or paraffin. Ultrathin longitudinal and cross sections of the inflorescences were performed for comparison between individuals with light or phase contrast microscopy. For Scanning Electron Microscopy, flowers were dissected from different parts of a rachilla, fixed, critical point dried, metalized with a nanolayer of gold/palladium and analyzed in a Scanning Electron Microscope Zeiss DSM 962. Results obtained showed the same pattern of floral development previously reported for the African oil palm; however, punctual and temporal variations occurred among the three individuals. The meiotic development in male flowers occurs in an acropetal way in this species, and leaf + 14 turned out to be the one were most meiotic stages were identified, rendering it as the ideal stage for collecting samples for cytogenetic studies as well as for tissue culture aiming the production of haploid plants.

The effect of temperature and petal wetness on the infection of Botrytis cinerea in rose flowers was studied by combining temperatures of 10, 15, 20 and 25°C with periods of petal wetness of 8, 16, 24 and 32 h. An increase in the severity of gray mold was observed when petal wetness period increased. Lower level of disease severity was verified at 10°C for all wetness periods as well as for the temperature of 25°C and wetness period of eight hours. In this wetness period, the lowest severity indexes were observed in all the temperatures tested. The maximum disease severity was observed at 20°C with 24 h of petal wetness. A model of multiple regression analysis was tested to associate temperature and wetness period. A quadratic effect of temperature was observed, which was overcome by free water time. The results show that infection on the rose flower petals depends on the period of wetness of the petals; maximum estimated severity occurred at 25°C with 32 h of petal wetness; the temperature of 10°C can reduce the severity of gray independently of the petal wetness period; at higher temperatures high disease severity is dependent on the wetness on the petals. Key words: Disease severity, flowers, gray mold, Rosa hybrid.

Background Although (Elaeis guineensis) is planted on only 5% of the total world vegetable oil acreage, it accounts for more than 30% of vegetable oil produced worldwide [1]. In Brazil, the Federal Government launched a plan to boost Oil Palm production as a way to meet the biofuels market increasing demand for vegetable oil. Nevertheless, the Oil Palm expansion areas in Brazil coincide with the area of occurrence of bud rot [2], a major disease that is decimating plantations already established in the area. In a way to circumvent such problem, breeders are now using E. oleifera germplasm in Oil Palm breeding programs, generating inter-specific hybrids not only resistant to bud rot, but with higher unsaturated fatty acid content, lower height [3]. E. oleifera, however, lack the genomic resources currently available for Oil Palm [1], hampering many possible studies that could potentially help breeding. Based on the foregoing, we started a project to develop a large set of molecular markers for the species based on the DArTSeq platform [4].

Aiming at generating a comprehensive genomic database on Elaeis spp., our group is leading several R&D initiatives with Elaeis guineensis (African oil palm) and Elaeis oleifera (American oil palm), including the whole-genome sequencing of the last. Genome size estimates currently available for this genus are controversial, as they indicate that American oil palm genome is about half the size of the African oil palm genome and that the genome of the interspecific hybrid is bigger than both the parental species genomes. We estimated the genome size of three E. guineensis genotypes, five E. oleifera genotypes, and two interspecific hybrids genotypes. On average, the genome size of E. guineensis is 4.32 ± 0.173 pg, while that of E. oleifera is 4.43 ± 0.018 pg. This indicates that both genomes are similar in size, even though E. oleifera is in fact bigger. As expected, the hybrid genome size is around the average of the two genomes, 4.40 ± 0.016 pg. Additionally, we demonstrate that both species present around 38% of GC content. As our results contradict the currently available data on Elaeis spp. genome sizes, we propose that the actual genome size of the Elaeis species is around 4 pg and that American oil palm possesses a larger genome than African oil palm.

We started a project to explore American Oil Palm genetic resources through a combination of genetic and genomic approaches.

Objetivou-se neste trabalho avaliar a dissimilaridade de 206 acessos do Banco Ativo de Germoplasma de E. oleifera mantido pela Embrapa, como forma de investigar a extensao e organizacao da diversidade genetica amostrada nesta colecao.

Pfaffia glomerata is a medicinal plant widely distributed in Brazil, which is considered the world's greatest supplier of P glomerata roots. Among active ingredients contained in this plant, the steroid beta-ecydisone (20E) is the most important compound extracted from roots. This steroid presents therapeutic properties for the treatment of diabetes and haemorrhoids, besides having bioenergy, tonic and aphrodisiac effects. The root-knot nematode Meloidogyne spp. is a major limiting factor in root production. Recent studies showed resistance of accessions of P. glomerata to Meloidogyne incognita. The aims of this work were: i) to correlate the concentration of 20E with resistance and susceptibility of P. glomerata accessions to M. incognita in inoculated and non-inoculated plants; ii) to study the effect of the parasitism of M. incognita on the concentration of the steroid 20E in the roots; and iii) to clarify resistance mechanisms by comparing the response of a highly resistant UFV with a highly susceptible accession (Farm) to nematode infection. The concentration of 20E in the healthy susceptible Farm accession was significantly higher than in the healthy resistant UFV accession, showing that the resistance mechanism was not related to 20E concentrations. Plants of the Farm accession infected with M. incognita showed higher levels of 20E than the non-infected control. A positive and significant Pearson correlation coefficient was observed between 20E concentrations and gall indexes. Resistance of UFV to the root-knot nematode M. incognita was associated with unidentified factors that limited nematode penetration or emigration of second-stage juveniles and with post-penetration responses, including the hypersensitive response. Giant cells were sometimes found in the resistant cultivar, but displayed a highly vacuolated and degraded cytoplasm with thinner cell walls than those induced in the susceptible accession. Microscope observations under UV light showed a strong autofluorescence, suggesting that phenolic compounds may be involved in ginseng UFV resistance.

The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively breeders must better understand the genetic differences of the potential crossing parents for introgression hybridization, but extensive genetic information is lacking. As an alternative to achieve information on genetic collinearity we make use of modern chromosome map technology known as high-resolution fluorescent in situ hybridization (FISH). This article presents the technical aspects and applications of such a technology in Musa species. The technique deals with BAC clone positioning on pachytene chromosomes of Calcutta 4 (Musa acuminata ssp. burmanicoides, A genome group, section Eumusa) and M. velutina (section Rodochlamys). Pollen mother cells digestion with pectolytic enzymes and maceration with acetic acid were optimized for making cell spread preparations appropriate for FISH. As an example of this approach we chose BAC clones that contain markers to known resistance genes and hybridize them for establishing their relative positions on the two species. Technical challenges for adapting existing protocols to the banana cells are presented. We also discuss how this technique can be instrumental for validating collinearity between potential crossing parents and how the method can be helpful in future mapping initiatives, and how this method allows identification of chromosomal rearrangements between related Musa species and cultivars.

The neotropical stink bugs, Euschistus heros, Piezodorus guildinii and Edessa meditabunda, are important pests of soybean and other crops throughout Central America and in South America from Northern Argentina to Brazil. Mate finding and host plant location in these species depend largely on their chemical communication, and semiochemicals are important mediators of these behaviors. In this study scanning electron microscopy was used to examine the external morphology, distribution and abundance of antennal sensilla on males, females and 5th instar nymphs of these species. Nine morphologically different sensilla types were found: trichod sensilla, type 1 and 2 (ST1 and ST2), long and short basiconic sensilla (SB1, SB2, and SB3), slit-tipped and knob-shaped basiconic sensilla, long chaetic sensilla (Sch) and coeloconic sensilla (Sco). Differences were detected in the abundance and arrangement of the sensilla over the antennal segments in individuals of the same species and among the species studied. The Sch, Sco and the slit-tipped and knob-shaped basiconic sensilla accounted for the major difference in sensilla types among the species. The ST1 was the most abundant type and was restricted to the flagellum. The pedicel of E. heros differs from the pedicels of P. guildinii and E. meditabunda mainly by the absence of Sch. There was a sexual dimorphism in ST1, SB1 and SB2, and this may be an indicative of their roles in detection of male-produced sex pheromone and odors derived from the host plants. The SB2 was lacking in the antennal tip of both sexes and 5th instar nymphs, but was abundant on the second flagellar segment of females of the three species. The same types of sensilla were found on 5th instar nymphs, but always in significantly lower numbers. The morphology and putative functions of each sensilla were compared and discussed.

Harpin is a protein produced by Erwinia amylovora that is involved in its pathogenesis of apple. We studied cellular correlates of harpin-induced resistance in apple fruit to blue mold caused by Penicillium expansum using Light (LM), scanning (SEM) and transmission (TEM) electron microscopy. SEM Study of wound surfaces showed the fungus colonizing the wounds within 48 h, and profuse colonization of the wound and penetration of the tissue occurred within 72 h after inoculation of control samples. In harpin-treated samples, spore germination and wound colonization were not evident until 96 h. LM showed that intramural mycelial growth appeared as early as 72 h, and intense tissue colonization occurred by 96 h after inoculation in the control samples. In the harpin-treated fruit, spore germination and wound colonization occurred only by 144 h in the treated specimens. Numerous putative tannin vacuoles and appositions were observed in the epidermal and hypodermal cells of inoculated harpin-treated samples, but only a few appeared in control samples. TEM showed that intramural growth of the fungus occurred frequently in the controls. In harpin-treated specimens, wall depositions and appositions Occurred very frequently. The results Suggest that harpin may trigger or intensify cellular responses in harpin-treated apples.

The interaction between Dicyma pulvinata and Fusicladium macrosporum was Studied by scanning electron microscopy. Spores of D. pulvinata germinated oil the Surface of F. macrosporum lesions induced oil artificially infected rubber plants were fixed 8 h after inoculation. D. pulvinata germ tubes seemed to elongate toward F. macrosporum. Close contact between the antagonistic Fungus and F macrosporum spores was verified 24 h after application of D. pulvinata. At the end of the process, spores of F. macrosporum seemed to have disintegrated and to be devoid of content. The hyperparasite grew completely over the pathogen. Six to seven days after application of the antagonistic Fungus, D. pulvinata conidiophores were observed emerging from E macrosporum structures with profuse sporulation. Studies have also shown the possibility of D. pulvinata producing hydrolytic enzymes, which could be associated with the control of plant pathogens. This information may help to elucidate some of the modes of action of D. pulvinata, a potential biological control agent for South American leaf blight of Hevea rubber plant.

Anthracnose, caused by Colletotrichum gloeosporioides, is a major post-harvest disease in papaya fruit. The major objectives of the present work were to isolate, select and test the in vitro and in vivo ability of epiphytic microorganisms, isolated from papaya fruit and leaf surfaces, in controlling anthracnose onset after harvest. A total of 75 bacteria, 67 yeasts and 22 mycelial fungi were isolated. Thirty yeast isolates were able to inhibit the mycelial growth of C. gloeosporioide in vitro and seven of those were used in in vivo assays, resulting in the identification of two very effective isolates. Isolate CEN63, identified molecularly as Cryptococcus magnus, was the most effective in controlling the disease and therefore was studied in more detail. The results of the assays with C. magnus provided evidence that when fruit were treated with the antagonists at concentrations of 107 to 108 cells/ml, as early as 24 h, preferentially 48 h, before inoculation with the pathogen, the development of disease was significantly reduced. C. magnus is a potential antagonist for the development of a commercial product. Additional studies on the modes of action of this yeast isolate, as on its ability to interact with fungicides are being conducted to generate solid basis for the development of an environmentally friendly control agent.

The objective of this work was to investigate possible modes of action of the yeast Cryptococcus magnus in controlling anthracnose (Colletotrichum gloeosporioides) on post harvested papaya fruits. Scanning electron microscopy was used to analyze the effect of the yeast on inoculations done after harvest. Results showed that C. magnus is able to colonize wound surfaces much faster than the pathogen, outcompeting the later for space and probably for nutrients. In addition, C. magnus produces a flocculent matrix, which affects hyphae integrity. The competition for space and the production of substances that affect hyphae integrity are among the most important modes of action of this yeast.

Sclerotinia sclerotiorum causes rot in a broad range of crops including lettuce, soybean, dry bean and tomato. Pathogenesis of Sclerotinia has been associated with the copious production of oxalic acid. Enzymes capable of degrading oxalic acid have been utilized to produce transgenic resistant plants. Transgenic lettuce lines containing the decarboxylase gene (oxdc) isolated from a Flammulina sp. were produced by Agrobacterium-mediated transformation. Out of 80 regenerated plants, PCR analysis revealed the presence of the oxdc gene in 34 lines. Except for eight lines, the primary transformants transferred the foreign gene to the first generation in a Mendelian fashion. In a detached-leaf assay inoculated with agar plugs of a 2-day-old S. sclerotiorum culture, two lines (P100 and P43) were symptomless, while line P57 showed a delay in symptom development when compared with a nontransgenic control line. RT-PCR analysis carried out with the resistant lines showed the expression of oxdc gene transcripts.