Morphoanatomical Changes in Eucalyptus grandis Leaves Associated with Resistance to Austropuccinia psidii in Plants of Two Ages.

The fungus Austropuccinia psidii infects young tissues of Eucalyptus plants until they are two years old in the nursery and field, causing Myrtaceae rust. The characteristics making older eucalypt leaves resistant to A. psidii and the reason for the low levels of this pathogen in older plants need evaluations. The aim of this study was to evaluate the morphological differences between Eucalyptus grandis leaves of different growth stages and two plant ages to propose a visual phenological scale to classify E. grandis leaves according to their maturation stages and to evaluate the time of leaf maturation for young and adult plants. A scale, based on a morphological differentiation for E. grandis leaves, was made. The color, shape and size distinguished the leaves of the first five leaf pairs. Anatomical analysis showed a higher percentage of reinforced tissue, such as sclerenchyma-like tissue and collenchyma, greater leaf blade thickness, absence of lower palisade parenchyma in the mature leaves and a higher number of cavities with essential oils than in younger ones. Changes in anatomical characteristics that could reduce the susceptibility of older E. grandis leaves to A. psidii coincide with the time of developing leaf resistance. Reduced infection of this pathogen in older plants appears to be associated with a more rapid maturation of their leaf tissues.

Initial Studies of the Response of Rubber Tree Seedlings Treated with Saprobic Fungi from the Semiarid Region of Northeast Brazil to Anthracnose.

Anthracnose, caused by fungi of the genus Colletotrichum, is present in the main areas where rubber trees (Hevea brasiliensis) are planted. Thus, considering that biological agents can be an alternative for disease control, the present study aimed to carry out initial studies to investigate the response of rubber tree seedlings inoculated with Colletotrichum and treated with saprobes fungi from the semiarid region of Northeast Brazil (Curvularia eragrostidis, Memnoniella levispora, Myrothecium roridum and Phialomyces macrosporus). Seedlings of the rubber tree clone RRIM600 were sprayed with biocontrol agents as preventive and curative treatments seven days before and after C. tamarilloi inoculation, respectively. Assessments included plant response to disease expression based on the percentage of symptomatic area on treated leaves, percentage of graft death, and percentage of apical death in seedlings 30 days after inoculation with C. tamarilloi. In addition, the enzymes peroxidase and phenylalanine ammonia lyase (PAL) had their activity quantified by their association with plant resistance to pathogens. The fungus C. eragrostidis had the best result in controlling anthracnose when applied as a preventive treatment, showing 10% less disease than the untreated plant. The same was observed for the fungus P. macrosporus when used in the curative form. These fungi also reduced the graft death. In these cases, PAL activity was higher and may be linked to the induction of resistance against the pathogen. The peroxidase activity was not expressive for treatments with saprobic fungi in the periods studied. Therefore, among the tested fungi, C. eragrostidis and P. macrosporus are promising for the control of anthracnose, deserving further studies.

Monitoring Colletotrichum Colonization and Reproduction in Different Rubber Tree Clones.

Anthracnose, caused by fungi of the genus Colletotrichum, is present in the major rubber tree crop areas in Brazil, especially in São Paulo, Mato Grosso do Sul, Paraná, Minas Gerais, Espírito Santo, and northern states. This disease can affect different tissues of the rubber tree, leading to production losses. Thus, a better understanding of the pathosystem Colletotrichum x rubber tree can provide evidence to subsequent epidemiological research and phytosanitary management studies of this disease in the field. The present study aimed to investigate C.tamarilloi colonization and reproduction steps in resistant clones (IAC 502, IAC 507, RRIM 937) and in one susceptible clone (RRIM 600) of the rubber tree, verifying the influence of temperature up to 48 h after inoculation of the fungus, under 24 h wetness. Samples were analyzed under a light, a UV and a scanning electron microscope. Data indicated that the fungus had a delay in its development in resistant clones and, although colonization was expressive 48 h after inoculation, the new spore formation rate in the analyzed samples was lower in resistant clones. For RRIM 600, rapid colonization and intensive sporulation could be observed.

Structural and Biochemical Aspects Related to Resistance and Susceptibility of Rubber Tree Clones to Anthracnose.

The aim of the present study was to evaluate structural and biochemical aspects related to the interaction of resistant (RRIM 937, IAC 502 and 507) and susceptible (RRIM 600) rubber tree clones with C. tamarillo. For such analysis, ultrathin sections of the leaf limb were embedded in historesin and differently stained to verify structural alterations and presence of starch grains, arginine, lipids, tannins and lignins. The total proteins and activity of the enzymes peroxidase and (PAL) were quantified. Stomatal density was also analyzed under a scanning electron microscope. Data indicated alterations in the cell content of resistant clones inoculated with the pathogen, as well as greater lignin and lipid accumulation in these samples. For tannins, there was no difference between inoculated and non-inoculated clones. Arginine was found at greater quantities in IAC 502 and 507. Starch grains were not detected in any of the analyzed samples. Protein level and stomatal density were lower in resistant clones. Peroxidase activity was more expressive in resistant clones. PAL activity, there was no significant difference between clones. The lignin and lipids, total protein, peroxidase activity and stomatal density may be related to the resistance of rubber tree clones to anthracnose.

Influence of Mycosphaerella and Teratosphaeria leaf diseases on chemical composition of essential oils of Eucalyptus globulus and effect of these essential oils on ascospores germination.

In a first step, essential oils were extracted from Eucalyptus globulus leaves, healthy and with symptoms and signs of Mycosphaerella leaf disease (MLD) and Teratosphaeria leaf disease (TLD), in two leaf stages. Stage 1: sessile, oval leaves covered by a waxy layer of a bluish colour, with opposite phyllotaxis, inserted along stems of quadrangular section. Stage 2: narrow and sickle leaves with a greyish green surface, mainly on the abaxial surface, inserted in alternating pairs along rounded stems. The essential oils were extracted by hydrodistillation and were analyzed by gas chromatography coupled with mass spectrometry. Chemical composition data and percentages of essential oil constituents were submitted to cluster analysis and principal component analysis. In a second step, under in vitro conditions, was evaluated the germination of Teratosphaeria nubilosa (one of the causal agents of TLD) ascospores in contact with the four types of essential oils extracted. The evaluations were performed at 24, 48 and 72 h after the experiments were assembled. The present study made it possible to distinguish and identify the chemical composition of essential oils from the eucalypt leaves used, and allowed 1,8-cineole to be identified as the major component for the essential oils investigated. The contact between essential oils and T. nubilosa spores allowed to prove the inhibition of the ascospores germination, being more efficient for the essential oils extracted from materials with the disease, which presented high amounts of 1,8-cineole.

Thousand cankers disease in Juglans: Optimizing sampling and identification procedures for the vector Pityophthorus juglandis, and the causal agent Geosmithia morbida.

Lindgren funnel traps were used to monitor Pityophthorus juglandis occurrence. Traps were placed directly on walnut trees, with the top tied to one of the lower branches (about 2m high). An 8-funnel model was used instead of a 4-funnel trap, with the specific pheromone bait positioned between the fourth and the fifth funnel. Traps were customized with a 5mm metal mesh which was placed inside the bottom funnel so that debris (mainly foliage) and larger non-target insects would not end up inside the collecting jar. Geosmithia morbida was isolated from beetle adults, larvae and necrotic woody tissue around beetle galleries. Contaminant-free colonies were subcultured in purity and identified by: a) colony phenotyping [morphology, texture and pigmentation; margin type (regular/irregular; lobed/non-lobed); mycelium compactness; surface bumpiness; growth/temperature relationships]; b) micromorphology: type, morphology and ontogeny of conidiophores, metulae and phialides; conidiogenesis; shape, dimension and pigmentation of conidia; c) DNA fingerprinting.•Our protocol was customized to prevent traps from swinging in the wind and to optimize beetle catches by transversely fixing the bottom of funnel traps to the tree trunk with wooden shafts for stability.•To enhance fungus isolation in purity, a semi-selective Potato Dextrose Agar (PDA) medium, enriched with the antibiotics Ampicillin (Policillin-N) and Rifampicin (Rifamycin), was devised to prevent contamination by Gram-positive and Gram-negative bacteria and by mycobacteria.

Ocorrência de epidemia do mal das folhas em regiões de "escape" do Brasil / Occurrence of South American leaf blight epidemic in escape zones of the Brazil

Entre janeiro e fevereiro de 2013 ocorreu uma epidemia de mal das folhas em regiões consideradas como de "escape" à doença, causando preocupações e indagações nos produtores de borracha e ao setor heveícola como um todo. Uma possível adaptação do fungo a essas zonas foi levantada e a consistência do controle da doença pela utilização do binômio plantas com troca uniforme das folhas + período seco, típicos de regiões de "escape", foi questionada. O objetivo deste trabalho foi trazer informações importantes aos heveicultores sobre a interação clima-patógeno-hospedeiro e comparar variáveis de normais climatológicas (média histórica) e dados climáticos deste período de ocorrência da epidemia (janeiro e fevereiro de 2013). Concluiu-se que não foi o patógeno que se adaptou às plantas de seringueira em clima mais seco, nas regiões de "escape", mas que o clima mais úmido ocorreu nessas regiões mais secas, nesse período.(AU)

In January and February 2013 occurred a South American leaf blight epidemic in regions considered as escape zones, causing concern to rubber tree growers and throughout the rubber industry. A possible adaptation of the fungus to escape zones was raised and the consistency of disease control by the use of the binomial: plants with uniform renewal of leaves + dry season, typical of the escape zones, was questioned. The aim of this work was to bring relevant information to the rubber growers about the interaction climate-pathogen-host and to compare normal climate (historical average) and climatic data of the occurrence period of epidemic (January and February 2013). We conclude that was not the pathogen that has adapted to rubber tree in drier conditions, typical in escape regions, but a more humid climate occurred in these drier regions during this period.(AU)

Biocompatibility studies of natural rubber latex from different tree clones and collection methods.

Natural rubber latex (NRL) has several features that make it an excellent biomaterial to promote the growth and repair of tissues, skin and bones. Most of the research with NRL membranes uses a mixture of different clones and chemical preservatives in the collection process. In this study, we compared five clones that produce NRL, seeking to identify their differences in biocompatibility. The clones studied were RRIM 600, PB 235, GT1, PR 255 and IAN 873 commonly found in plantations in Brazil. We did also study the effect of ammonia used during latex collection. NRL membranes were prepared aseptically and sterilized. In the in vitro tests, the membranes remained in direct contact with mouse fibroblasts cells for three periods, 24, 48 and 72 h. In the in vivo tests, the membranes were implanted subcutaneously in rabbits. The results indicated the biocompatibility of the membranes obtained from all clones. Membranes from the clones RRIM 600 and IAN 873 induced greater cell proliferation, suggesting greater bioactivity. It was found that the membranes made from latex that was in contact with ammonia during collection, showed cytotoxic and genotoxic effects in cultures, as well as necrosis, and increased inflammatory cells in the rabbit's tissues close to the implant.

Comprehensive two-dimensional gas chromatography combined to multivariate data analysis for detection of disease-resistant clones of Eucalyptus.

In this paper it is reported the use of the chromatographic profiles from volatile fractions of plant clones - in this case, hybrids of Eucalyptus grandis×Eucalyptus urophylla - to determine specimens susceptible to rust disease. The analytes were isolated by headspace solid phase microextraction (HS-SPME) and analyzed by comprehensive two-dimensional gas chromatography combined to fast quadrupole mass spectrometry (GC×GC-qMS). Parallel Factor Analysis (PARAFAC) was employed for estimate the correlation between the chromatographic profiles and resistance against Eucalyptus rust, after preliminary variable selection performed by Fisher ratio analysis. The proposed method allowed the differentiation between susceptible and non-susceptible clones and determination of three resistance biomarkers. This approach can be a valuable alternative for the otherwise time-consuming and labor-intensive methods commonly used.

Determination of disease biomarkers in Eucalyptus by comprehensive two-dimensional gas chromatography and multivariate data analysis.

In this paper is reported the use of the chromatographic profiles of volatiles to determine disease markers in plants - in this case, leaves of Eucalyptus globulus contaminated by the necrotroph fungus Teratosphaeria nubilosa. The volatile fraction was isolated by headspace solid phase microextraction (HS-SPME) and analyzed by comprehensive two-dimensional gas chromatography-fast quadrupole mass spectrometry (GC×GC-qMS). For the correlation between the metabolic profile described by the chromatograms and the presence of the infection, unfolded-partial least squares discriminant analysis (U-PLS-DA) with orthogonal signal correction (OSC) were employed. The proposed method was checked to be independent of factors such as the age of the harvested plants. The manipulation of the mathematical model obtained also resulted in graphic representations similar to real chromatograms, which allowed the tentative identification of more than 40 compounds potentially useful as disease biomarkers for this plant/pathogen pair. The proposed methodology can be considered as highly reliable, since the diagnosis is based on the whole chromatographic profile rather than in the detection of a single analyte.

Selection of fungi for accelerated decay in stumps of Eucalyptus spp.

The objective of this study was to select decay fungi for biological degradation of stumps and roots from Eucalyptus spp. in forest stands. Five fungal isolates were cultured. Subsequently, inoculated in accelerated-decay tests and field tests with tree stumps of Eucalyptus spp. present in a stand belonging to Votorantim Company were conducted. The results showed that: (1) the decay of stumps by Pycnoporus sanguineus and Ganoderma applanatum increased by up to 49.2% and 48.9%, respectively; (2) the enclosing stumps in a polyninyl chloride resulted in a reduced on the resistance due to enhanced decay as measured by an inpact penetrometer in the forest stands at Luiz Antônio, Capão Bonito, and Jacareí, SP, Brazil; and (3) the selected fungi did not show any pathogenic potential toward seedlings and adult trees. This study presents the foundation for the development of a biological system for stump removal in forest stands comprising Eucalyptus spp.

Decay resistance of thermally-modified Eucalyptus grandis wood at 140 degrees C, 160 degrees C, 180 degrees C, 200 degrees C and 220 degrees C.

The objective of this study was to evaluate the effect of thermal treatment on the biological resistance of Eucalyptus grandis wood to the decay fungus Picnoporus sanguineus. Boards from 5 years and 11 months old E. grandis trees, taken from the stock possessed by the Duratex-SA company, were thermally-modified at 140 degrees C, 160 degrees C, 180 degrees C, 200 degrees C and 220 degrees C in the Laboratory of Wood Drying and Preservation from UNESP, Botucatu, SP, Brazil. Samples of each treatment were treated according to ASTM D-2017 (1994). The experiment tested the accelerated decay caused by the decay fungus P. sanguineus on a system of soil-block wood. The results of thermal treatment showed that an increase of temperature of 180-220 degrees C caused reductions of between 15.7% and 82.4% in the weight loss in the samples from E. grandis incubated with P. sanguineus.

Boron transport in Eucalyptus. 2. Identification in silico of a putative boron transporter for xylem loading in eucalypt

Boron (B) is a low mobility plant micronutrient whose molecular mechanisms of absorption and translocation are still controversial. Many factors are involved in tolerance to Boron excess or deficiency. Recently, the first protein linked to boron transport in biological systems, BOR1, was characterized in Arabidopsis thaliana. This protein is involved in boron xylem loading and is similar to bicarbonate transporters found in animals. There are indications that BOR1 is a member of a conserved protein family in plants. In this work, FORESTS database was used to identify sequences similar to this protein family, looking for a probable BOR1 homolog in eucalypt. We found five consensus sequences similar to BOR1; three of them were then used in multiple alignment analysis. Based on amino acid similarity and in silico expression patterns, a consensus sequence was identified as a candidate BOR1 homolog, helping deeper experimental assays that could identify the function of this protein family in Eucalyptus

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (https://forests.esalq.usp.br) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site

Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4) directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen), oxadiazole (oxadiazon and oxadiargyl), and aryl triazolinone (sulfentrazone and carfentrazone) herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project

Estudo dos fungos anemófilos da cidade de Botucatu e sua correlação com sensibilização em pacientes com doenças alérgicas respiratórias / Study of fungi in the air of Botucatu, Brazil and their correlation with sensitization in patients with respiratory allergic diseases

Objetivos: Estudar a prevalência de fungos anemófilos da cidade de Botucatu, através de um caça-esporos desenvolvido para essa finalidade, e correlacionar esses fungos com sensibilização alérgica em pacientes com asma e rinite. Métodos: Coletaram-se fungos do ar com caçaes-poros padrão volumétrico, no período de junho de 2001 a maio de 2002, semanalmente. Contaram-se as Unidades Formadoras de Colônias (UFC) e identificaram-se os gêneros encontrados; entrevistou-se 119 pacientes de dois serviços de Alergia Clínica da cidade de Botucatu, classificando-os de acordo com a gravidade de suas doenças respiratórias (asma e rinite). Também foram selecionados dez controles sem qualquer doença alérgica atual ou pregressa. Procedeu-se à realização de testes cutâneos de puntura com os aeroalérgenos mais comuns, sendo que para os fungos foi feito também o teste intradérmico. Resultados: O caça-esporos aqui apresentado foi comparativamente melhor do que o método gravitacional. Identificou-se 65 diferentes gêneros de fungos, sendo que os predominantes foram: Cladosporium, leveduras, Epicoccum, Trichoderma, Fusarium, Penicillium, Helminthosporium e Aspergillus; quanto aos testes cutâneos, obteve-se 83,5 per cent de positividade a fungos, sendo que o teste intradérmico mostrou-se muito superior para esse diagnóstico. Os fungos que causaram mais sensibilização não foram os mais freqüentes do ar. Foram eles: Aspergillus, Neurospora, Candida, Penicillium, Cladosporium, Mucor, Chaethomium, Rhizopus, Alternaria, Fuzarium e Metarhizium. Conclusões: Estudo de fungos ambientais pode ser realizado com um caça-esporos mais acessível aos pesquisadores brasileiros; tais fungos representam importantes sensibilizantes em pacientes com asma e rinite e o diagnóstico dessa sensibilização é muito mais eficaz através de testes intradérmicos.