Interaction between N-fertilizer and water availability on borer-rot complex in sugarcane

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Binding analysis of Bacillus thuringiensis Cry1 proteins in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribution of the root system of peach palm under drip irrigation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Testis morphology and dichotomic spermatogenesis in the sugarcane borer Diatraea saccharalis (F.) (Lepidoptera: Crambidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Carbohydrate concentration in 'BRS Rubimel' peach trees during the annual cycle

The carbohydrate storage is necessary to support the plant growth in periods of stress, during the dormancy, in the beginning of the vegetative development and during the fruiting time. In this context, this work intended to evaluate the carbohydrate concentrations of the peach (Prunus persica (L.) Batsch) tree ‘BRS Rubimel’, cultivated under subtropical conditions. The experiment was performed at the experimental farm Lageado, of the Faculty of Agricultural Sciences of UNESP at Botucatu/SP. The evaluated peach trees were two years old and were cultivated in the spacing of 6.0 x 4.0 m. The adopted experimental delineation was in randomized blocks, making use of four plants per parcel with four repetitions. The treatments corresponded to the period of the collection of leaves and branches, during the annual cycle, corresponding to January to May and July to December 2012. The sample collection of the roots was performed in January, April, August, November and December 2012. Four fruits per plant were collected. The concentration of starch in the roots of the peach tree were superior to the ones from the branches, from August to December. The carbohydrate with the higher storage level in the peach tree ‘BRS Rubimel’ was starch.

Agronomic performance of peach trees grown in subtropical region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A Potential Plan of Action for Emerald Ash Borer in Nebraska

Abstract Emerald Ash Borer (Agrilus planipennis) (EAB) is an invasive insect pest. It feeds on the cambium tissues of ash tree species. It was first discovered in the United States in 2002 in Detroit, Michigan. Their effects on ash trees are deadly, and it is quickly spreading across the Midwest. Nebraska has not yet been invaded, but confirmed findings continue getting closer and closer. The major problem facing Nebraskans, with regards to EAB, is how to begin preparations to prevent a dramatic economic loss when an infestation does occur. So, to address this problem, I have conducted street and park tree inventories, to determine the amount of ash trees that are contained in Nebraska’s community forests; and with that data I have attempted to create a possible EAB action plan for Nebraska communities. Based on inventory findings, I have calculated that 6% of Nebraska’s community trees are ash, which is a large percentage. Then, I proposed a plan of action for communities that involve planting a diverse landscape, and a combination of ash replacement programs, and treatment for ash that are less valuable or damaged.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

The First Occurrence in the Fossil Record of an Aquatic Avian Twig-Nest with Phoenicopteriformes Eggs: Evolutionary Implications

Background: We describe the first occurrence in the fossil record of an aquatic avian twig-nest with five eggs in situ (Early Miocene Tudela Formation, Ebro Basin, Spain). Extensive outcrops of this formation reveal autochthonous avian osteological and oological fossils that represent a single taxon identified as a basal phoenicopterid. Although the eggshell structure is definitively phoenicopterid, the characteristics of both the nest and the eggs are similar to those of modern grebes. These observations allow us to address the origin of the disparities between the sister taxa Podicipedidae and Phoenicopteridae crown clades, and traces the evolution of the nesting and reproductive environments for phoenicopteriforms. Methodology/Principal Findings: Multi-disciplinary analyses performed on fossilized vegetation and eggshells from the eggs in the nest and its embedding sediments indicate that this new phoenicopterid thrived under a semi-arid climate in an oligohaline (seasonally mesohaline) shallow endorheic lacustine environment. High-end microcharacterizations including SEM, TEM, and EBSD techniques were pivotal to identifying these phoenicopterid eggshells. Anatomical comparisons of the fossil bones with those of Phoenicopteriformes and Podicipediformes crown clades and extinct palaelodids confirm that this avian fossil assemblage belongs to a new and basal phoenicopterid. Conclusions/Significance: Although the Podicipediformes-Phoenicopteriformes sister group relationship is now well supported, flamingos and grebes exhibit feeding, reproductive, and nesting strategies that diverge significantly. Our multi-disciplinary study is the first to reveal that the phoenicopteriform reproductive behaviour, nesting ecology and nest characteristics derived from grebe-like type strategies to reach the extremely specialized conditions observed in modern flamingo crown groups. Furthermore, our study enables us to map ecological and reproductive characters on the Phoenicopteriformes evolutionary lineage. Our results demonstrate that the nesting paleoenvironments of flamingos were closely linked to the unique ecology of this locality, which is a direct result of special climatic (high evaporitic regime) and geological (fault system) conditions.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. Key message Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Mating Behavior of the Coffee Berry Borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae)

The mating behavior of the coffee berry borer, (Ferrari), was observed under laboratory conditions. Pairs of adult virgin male and female beetles were placed in the wells of a polystyrene microtiter plate, one pair per well. The mating activity of each pair was recorded for 24 h. The mating behavior of the coffee berry borer was similar to that of other Scolytinae and was clearly divided into precopulatory, copulatory and postcopulatory phases. The beetles started to mate within a few hours of emergence. Repeated mating occurred during the 24-hour period and increased in frequency with age. However, we cannot address multiple matings in , since we did not simulate the female-biased sex ratio of this species and the experimental design did not allow females to avoid additional mating attempts by males. In addition, further studies are necessary that focus on the effectiveness of sperm transmission and direct and indirect effects of multiple matings on the ei females and their offspring.

Light energy management in peach: utilization, photoprotection , photodamage and recovery. Maximizing light absorption in orchard is not always the best solution

The relation between the intercepted light and orchard productivity was considered linear, although this dependence seems to be more subordinate to planting system rather than light intensity. At whole plant level not always the increase of irradiance determines productivity improvement. One of the reasons can be the plant intrinsic un-efficiency in using energy. Generally in full light only the 5 – 10% of the total incoming energy is allocated to net photosynthesis. Therefore preserving or improving this efficiency becomes pivotal for scientist and fruit growers. Even tough a conspicuous energy amount is reflected or transmitted, plants can not avoid to absorb photons in excess. The chlorophyll over-excitation promotes the reactive species production increasing the photoinhibition risks. The dangerous consequences of photoinhibition forced plants to evolve a complex and multilevel machine able to dissipate the energy excess quenching heat (Non Photochemical Quenching), moving electrons (water-water cycle , cyclic transport around PSI, glutathione-ascorbate cycle and photorespiration) and scavenging the generated reactive species. The price plants must pay for this equipment is the use of CO2 and reducing power with a consequent decrease of the photosynthetic efficiency, both because some photons are not used for carboxylation and an effective CO2 and reducing power loss occurs. Net photosynthesis increases with light until the saturation point, additional PPFD doesn’t improve carboxylation but it rises the efficiency of the alternative pathways in energy dissipation but also ROS production and photoinhibition risks. The wide photo-protective apparatus, although is not able to cope with the excessive incoming energy, therefore photodamage occurs. Each event increasing the photon pressure and/or decreasing the efficiency of the described photo-protective mechanisms (i.e. thermal stress, water and nutritional deficiency) can emphasize the photoinhibition. Likely in nature a small amount of not damaged photosystems is found because of the effective, efficient and energy consuming recovery system. Since the damaged PSII is quickly repaired with energy expense, it would be interesting to investigate how much PSII recovery costs to plant productivity. This PhD. dissertation purposes to improve the knowledge about the several strategies accomplished for managing the incoming energy and the light excess implication on photo-damage in peach. The thesis is organized in three scientific units. In the first section a new rapid, non-intrusive, whole tissue and universal technique for functional PSII determination was implemented and validated on different kinds of plants as C3 and C4 species, woody and herbaceous plants, wild type and Chlorophyll b-less mutant and monocot and dicot plants. In the second unit, using a “singular” experimental orchard named “Asymmetric orchard”, the relation between light environment and photosynthetic performance, water use and photoinhibition was investigated in peach at whole plant level, furthermore the effect of photon pressure variation on energy management was considered on single leaf. In the third section the quenching analysis method suggested by Kornyeyev and Hendrickson (2007) was validate on peach. Afterwards it was applied in the field where the influence of moderate light and water reduction on peach photosynthetic performances, water requirements, energy management and photoinhibition was studied. Using solar energy as fuel for life plant is intrinsically suicidal since the high constant photodamage risk. This dissertation would try to highlight the complex relation existing between plant, in particular peach, and light analysing the principal strategies plants developed to manage the incoming light for deriving the maximal benefits as possible minimizing the risks. In the first instance the new method proposed for functional PSII determination based on P700 redox kinetics seems to be a valid, non intrusive, universal and field-applicable technique, even because it is able to measure in deep the whole leaf tissue rather than the first leaf layers as fluorescence. Fluorescence Fv/Fm parameter gives a good estimate of functional PSII but only when data obtained by ad-axial and ab-axial leaf surface are averaged. In addition to this method the energy quenching analysis proposed by Kornyeyev and Hendrickson (2007), combined with the photosynthesis model proposed by von Caemmerer (2000) is a forceful tool to analyse and study, even in the field, the relation between plant and environmental factors such as water, temperature but first of all light. “Asymmetric” training system is a good way to study light energy, photosynthetic performance and water use relations in the field. At whole plant level net carboxylation increases with PPFD reaching a saturating point. Light excess rather than improve photosynthesis may emphasize water and thermal stress leading to stomatal limitation. Furthermore too much light does not promote net carboxylation improvement but PSII damage, in fact in the most light exposed plants about 50-60% of the total PSII is inactivated. At single leaf level, net carboxylation increases till saturation point (1000 – 1200 μmolm-2s-1) and light excess is dissipated by non photochemical quenching and non net carboxylative transports. The latter follows a quite similar pattern of Pn/PPFD curve reaching the saturation point at almost the same photon flux density. At middle-low irradiance NPQ seems to be lumen pH limited because the incoming photon pressure is not enough to generate the optimum lumen pH for violaxanthin de-epoxidase (VDE) full activation. Peach leaves try to cope with the light excess increasing the non net carboxylative transports. While PPFD rises the xanthophyll cycle is more and more activated and the rate of non net carboxylative transports is reduced. Some of these alternative transports, such as the water-water cycle, the cyclic transport around the PSI and the glutathione-ascorbate cycle are able to generate additional H+ in lumen in order to support the VDE activation when light can be limiting. Moreover the alternative transports seems to be involved as an important dissipative way when high temperature and sub-optimal conductance emphasize the photoinhibition risks. In peach, a moderate water and light reduction does not determine net carboxylation decrease but, diminishing the incoming light and the environmental evapo-transpiration request, stomatal conductance decreases, improving water use efficiency. Therefore lowering light intensity till not limiting levels, water could be saved not compromising net photosynthesis. The quenching analysis is able to partition absorbed energy in the several utilization, photoprotection and photo-oxidation pathways. When recovery is permitted only few PSII remained un-repaired, although more net PSII damage is recorded in plants placed in full light. Even in this experiment, in over saturating light the main dissipation pathway is the non photochemical quenching; at middle-low irradiance it seems to be pH limited and other transports, such as photorespiration and alternative transports, are used to support photoprotection and to contribute for creating the optimal trans-thylakoidal ΔpH for violaxanthin de-epoxidase. These alternative pathways become the main quenching mechanisms at very low light environment. Another aspect pointed out by this study is the role of NPQ as dissipative pathway when conductance becomes severely limiting. The evidence that in nature a small amount of damaged PSII is seen indicates the presence of an effective and efficient recovery mechanism that masks the real photodamage occurring during the day. At single leaf level, when repair is not allowed leaves in full light are two fold more photoinhibited than the shaded ones. Therefore light in excess of the photosynthetic optima does not promote net carboxylation but increases water loss and PSII damage. The more is photoinhibition the more must be the photosystems to be repaired and consequently the energy and dry matter to allocate in this essential activity. Since above the saturation point net photosynthesis is constant while photoinhibition increases it would be interesting to investigate how photodamage costs in terms of tree productivity. An other aspect of pivotal importance to be further widened is the combined influence of light and other environmental parameters, like water status, temperature and nutrition on peach light, water and phtosyntate management.

Variation in peach fruit susceptibility to Monilinia laxa and gene expression

Brown rot caused by Monilinia laxa and Monilinia fructigena is considered one of the most important diseases affecting Prunus species. Although some losses can result from the rotten fruits in the orchard, most of the damage is caused to fruits during the post-harvest phase. Several studies reported that brown rot incidence during fruit development highly varies; it was found that at a period corresponding to the the pit hardening stage, fruit susceptibility drastically decreases, to be quickly restored afterwards. However the molecular basis of this phenomenon is still not well understood. Furthermore, no difference in the rot incidence was found between wound and un-wound fruits, suggesting that resistance associated more to a specifc biochemical response of the fruit, rather than to a higher mechanical resistance. So far, the interaction Monilinia-peach was analyzed through chemical approaches. In this study, a bio-molecular approach was undertaken in order to reveal alteration in gene expression associated to the variation of susceptibility. In this thesis three different methods for gene expression analysis were used to analyze the alterations in gene expression occurring in peach fruits during the pit hardening stage, in a period encompassing the temporary change in Monilinia susceptibility: real time PCR, microarray and cDNA AFLP techniques. In 2005, peach fruits (cv.K2) were weekly harvested during a 19-week long-period, starting from the fourth week after full bloom, until full maturity. At each sampling time, three replicates of 5 fruits each were dipped in the M.laxa conidial suspension or in distilled water, as negative control. The fruits were maintained at room temperature for 3 hours; afterwards, they were peeled with a scalpel; the peel was immediately frozen in liquid nitrogen and transferred to -80 °C until use. The degree of susceptibility of peach fruit to the pathogen was determined on 3 replicates of 20 fruits each, as percentage of infected fruits, after one week at 20 °C. Real time PCR analysis was performed to study the variation in expression of those genes encoding for the enzymes of the phenylpropanoid pathway (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), cinnamate 4-hydroxylase (C4H), leucoanthocyanidine reductase (LAR), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and of the jasmonate pathway, such as lipoxygenase (LOX), both involved in the production of important defense compounds. Alteration in gene expression was monitored on fruit samples of a period encompassing the pit hardening stage and the corresponding temporary resistance to M.laxa infections, weekly, from the 6thto the 12th week after full bloom (AFB) inoculated with M. laxa or mock-inoculated. The data suggest a critical change in the expression level of the phenylpropanoid pathway from the 7th to the 8th week AFB; such change could be directly physiologically associated to the peach growth and it could indirectly determine the decrease of susceptibility of peach fruit to Monilinia rot during the subsequent weeks. To investigate on the transcriptome variation underneath the temporary loss of susceptibility of peach fruits to Monilinia rot, the microarray and the cDNA AFLP techniques were used. The samples harvested on the 8th week AFB (named S, for susceptible ones) and on the 12th week AFB (named R, for resistant ones) were compared, both inoculated or mock-inoculated. The microarray experiments were carried out at the University of Padua (Dept. of Environmental Agronomy and Crop Science), using the μPEACH1.0 microarray together with the suited protocols. The analysis showed that 30 genes (corresponding to the 0.6% of the total sequences (4806) contained in the μPeach1.0 microarray) were found up-regulated and 31 ( 0.6%) down regulated in RH vs. SH fruits. On the other hand, 20 genes (0.4%) were shown to be up-regulated and 13 (0.3%) down-regulated in the RI vs. SI fruit. No genes were found differentially expressed in the mock-inoculated resistant fruits (RH) vs. the inoculated resistant ones (RI). Among the up-regulated genes an ATP sulfurylase, an heat shock protein 70, the major allergen Pru P1, an harpin inducing protein and S-adenosylmethionine decarboxylase were found, conversely among the down-regulated ones, cinnamyl alcohol dehydrogenase, an histidine- containing phosphotransfer protein and the ferritin were found. The microarray experimental results and the data indirectly derived, were tested by Real Time PCR analysis. cDNA AFLP analysis was also performed on the same samples. 339 transcript derived fragments considered significant for Monilinia resistance, were selected, sequenced and classified. Genes potentially involved in cell rescue and defence were well represented (8%); several genes (12.1%) involved in the protein folding, post-transductional modification and genes (9.2%) involved in cellular transport were also found. A further 10.3% of genes were classified as involved in the metabolism of aminoacid, carbohydrate and fatty acid. On the other hand, genes involved in the protein synthesis (5.7%) and in signal transduction and communication (5.7%) were found. Among the most interesting genes found differentially expressed between susceptible and resistant fruits, genes encoding for pathogenesis related (PR) proteins were found. To investigate on the association of Monilinia resistance and PR biological function, the major allergen Pru P1 (GenBank accession AM493970) and its isoform (here named Pru P2), were expressed in heterologous system and in vitro assayed for their anti-microbial activity. The ribonuclease activity of the recombinant Pru P1 and Pru P2 proteins was assayed against peach total RNA. As the other PR10 proteins, they showed a ribonucleolytic activity, that could be important to contrast pathogen penetration. Moreover Pru P1 and Pru P2 recombinant proteins were checked for direct antimicrobial activity. No inhibitory effect of Pru P1 or Pru P2 was detected against the selected fungi.

Control of peach phenolic compounds content

Phenolic compounds play a central role in peach fruit colour, flavour and health attributes. Phenolic profiles of several peaches and nectarines and most of the structural genes leading to the anthocyanin synthesis in peach fruit have been studied. Moreover, crosses of red and non-red peaches suggested that a major gene controls skin colour of the extreme phenotypes ‘highlighter’ and ‘full-red’. However, there is no data about either the ‘flavan-3-ols specific genes’ (ANR and LAR) or the regulation of the flavonoid metabolism in this crop. In the present study, we determined the concentration of phenolic compounds in the yellowfleshed nectarine Prunus persica cv. ‘Stark Red Gold’ during fruit growth and ripening. We examined the transcript levels of the main structural genes of the flavonoid pathway. Gene expression of the biosynthetic genes correlated well with the concentration of flavan-3-ols, which was very low at the beginning of fruit development, strongly increased at mid-development and finally decreased again during ripening. In contrast, the only gene transcript which correlated with anthocyanin concentration was PpUFGT, which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. These patterns of gene expression could be explained by the involvement of different transcription factors, which up-regulate anthocyanin biosynthesis (PpMYB10 and PpbHLH3), or repress (PpMYBL2) the transcription of the structural genes. These transcription factors appeared to be involved also in the regulation of the lightinduced anthocyanin accumulation in ‘Stark Red Gold’ nectarines, suggesting that they play a critical role in the regulation of flavonoid biosynthesis in peaches and nectarines in response to both developmental and environmental stimuli. Phenolic profiles and expression patterns of the main flavonoid structural and regulatory genes were also determined for the extreme phenotypes denominated ‘highlighter’ and ‘full-red’ and hypotheses about the control of phenolic compounds content in these fruit are discussed.

Organic fertilization of peach trees: implication on nitrogen availability, root growth and carbon distribution within plant

In the last years, sustainable horticulture has been increasing; however, to be successful this practice needs an efficient soil fertility management to maintain a high productivity and fruit quality standards. For this purpose composted organic materials from agri-food industry and municipal solid waste has been used as a source to replace chemical fertilizers and increase soil organic matter. To better understand the influence of compost application on soil fertility and plant growth, we carried out a study comparing organic and mineral nitrogen (N) fertilization in micro propagated plants, potted trees and commercial peach orchard with these aims: 1. evaluation of tree development, CO2 fixation and carbon partition to the different organs of two-years-old potted peach trees. 2. Determination of soil N concentration and nitrate-N effect on plant growth and root oxidative stress of micro propagated plant after increasing rates of N applications. 3. Assessment of soil chemical and biological fertility, tree growth and yield and fruit quality in a commercial orchard. The addition of compost at high rate was effective in increasing CO2 fixation, promoting root growth, shoot and fruit biomass. Furthermore, organic fertilizers influenced C partitioning, favoring C accumulation in roots, wood and fruits. The higher CO2 fixation was the result of a larger tree leaf area, rather than an increase in leaf photosynthetic efficiency, showing a stimulation of plant growth by application of compost. High concentrations of compost increased total soil N concentration, but were not effective in increasing nitrate-N soil concentration; in contrast mineral-N applications increased linearly soil nitrate-N, even at the lowest rate tested. Soil nitrate-N concentration influenced positively plant growth at low rate (60- 80 mg kg-1), whereas at high concentrations showed negative effects. In this trial, the decrease of root growth, as a response to excessive nitrate-N soil concentration, was not anticipated by root oxidative stress. Continuous annual applications of compost for 10 years enhanced soil organic matter content and total soil N concentration. Additionally, high rate of compost application (10 t ha-1 year-1) enhanced microbial biomass. On the other hand, different fertilizers management did not modify tree yield, but influenced fruit size and precocity index. The present data support the idea that organic fertilizers can be used successfully as a substitute of mineral fertilizers in fruit tree nutrient management, since they promote an increase of soil chemical and biological fertility, prevent excessive nitrate-N soil concentration, promote plant growth and potentially C sequestration into the soil.