Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection Rust caused by the fungus Puccinia psidii is currently the most important disease of eucalyptus. It is widely disseminated in Brazil, and causes serious damage in nurseries and plantation areas. The identification of resistant germplasm along with knowledge of the genetic basis of resistance heredity are the first requirements for the success of breeding programs aiming to develop resistant varieties. Earlier studies carried out under controlled conditions suggested a monogenic control as well as the participation of at least two genes promoting resistance to rust. The goal of this study was to evaluate the resistance to P. psidii under field conditions in fourteen progenies from controlled crosses and self-crosses among four hybrid clones of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla ST Blake that contrast for resistance to the fungus. Results indicated that resistance could be explained by one locus with main effects and at least three different alleles. However, loci with minor effects may influence the resistance, since variation on severity classes was observed. Differences in segregation of resistance between reciprocal crosses were not observed, indicating absence of cytoplasmic effects.

Effect of acibenzolar-S-methyl and Saccharomyces cerevisiae on the activation of Eucalyptus defences against rust

Tree defence mechanisms against the fungus Puccinia psidii were examined by comparing the activities of defence-related enzymes (chitinase, peroxidase and phenylalanine ammonia-lyase) of two Eucalyptus grandis x E. urophylla (urograndis) hybrids, previously classified as either susceptible to rust (VR hybrid) or moderately resistant to rust (C0 hybrid). Furthermore, the potential of disease control by artificial activation of host defences using either acibenzolar-S-methyl (ASM) or Saccharomyces cerevisiae extract was also investigated. Greenhouse inoculation trials revealed that the C0 hybrid had lower disease severity than the VR hybrid but following foliar applications of either ASM or S. cerevisiae extract treatment, disease severity (evaluated at 15 days after inoculation) was reduced in both hybrids. This enhanced resistance was associated with the induction of a hypersensitive reaction which appeared to be effective in controlling rust in both clones. The activity of all enzymes differed between clones and inducer treatment. The role of the defence-related enzymes in imparting resistance to eucalypt hybrids against rust is discussed.

Zn uptake, physiological response and stress attenuation in mycorrhizal jack bean growing in soil with increasing Zn concentrations

The influence of arbuscular mycorrhizal fungi (AMF) inoculation on Canavalia ensiformis growth. nutrient and Zn uptake, and on some physiological parameters in response to increasing soil Zn concentrations was studied. Treatments were applied in seven replicates in a 2 x 4 factorial design, consisting of the inoculation or not with the AMF Glomus etunicatum, and the addition of Zn to soil at the concentrations of 0, 100, 300 and 900 mg kg(-1). AMF inoculation enhanced the accumulation of Zn in tissues and promoted biomass yields and root nodulation. Mycorrhizal plants exhibited relative tolerance to Zn up to 300 mg kg(-1) without exhibiting visual symptoms of toxicity, in contrast to non-mycorrhizal plants which exhibited a significant growth reduction at the same soil Zn concentration. The highest concentration of Zn added to soil was highly toxic to the plants. Leaves of plants grown in high Zn concentration exhibited a Zn-induced proline accumulation and also an increase in soluble amino acid contents; however proline contents were lower in mycorrhizal jack beans. Plants in association or not with the AMF exhibited marked differences in the foliar soluble amino acid profile and composition in response to Zn addition to soil. In general, Zn induced oxidative stress which could be verified by increased lipid peroxidation rates and changes in catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase activities. In summary, G. etunicatum was able to maintain an efficient symbiosis with jack bean plants in moderately contaminated Zn-soils, improving plant performance under those conditions, which is likely to be due to a combination of physiological and nutritional changes caused by the intimate relation between fungus and plant. The enhanced Zn uptake by AMF inoculated jack bean plants might be of interest for phytoremediation purposes. (C) 2009 Elsevier Ltd. All rights reserved.

Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches` broom pathogen Moniliophthora perniciosa

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches` broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabollite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Dormancy as exaptation to protect mimetic seeds against deterioration before dispersal

Mimetic seeds simulate the appearance of fleshy fruits and arilled seeds without producing nutritive tissues as a reward for seed dispersers. In this strategy of seed dispersal, seeds may remain attached to the mother plant for long periods after maturity, increasing their availability to naive seed dispersers. The hypothesis that seed coat impermeability in many tropical Fabaceae with mimetic seeds serves as an exaptation to protect the seeds from deterioration and rotting while awaiting dispersal was investigated. Seed coat impermeability was evaluated in five mimetic-seeded species of tropical Fabaceae in south-eastern Brazil (Abarema langsdorffii, Abrus precatorius, Adenanthera pavonina, Erythrina velutina and Ormosia arborea) and in Erythrina speciosa, a `basal` species in its genus, which has monochromatic brown seeds and no mimetic displays. Seed hardness was evaluated as a defence against accelerated ageing (humid chamber at 41 degrees C for 144 h). Seed development and physiological potential of O. arborea was evaluated and the effect of holding mature seeds in pods on the mother plant in the field for a period of 1 year under humid tropical conditions was compared with seeds stored under controlled conditions (15 degrees C and 40 % relative air humidity). All five mimetic-seeded species, and E. speciosa, showed strong coat impermeability, which protected the seeds against deterioration in accelerated ageing. Most O. arborea seeds only became dormant 2 months after pod dehiscence. Germination of seeds after 1 year on the plant in a humid tropical climate was 56 %, compared with 80 % for seeds stored in controlled conditions (15 degrees C, 45 % relative humidity). Seedling shoot length after 1 year did not differ between seed sources. Dormancy acts in mimetic-seeded species as an exaptation to reduce seed deterioration, allowing an increase in their effective dispersal period and mitigating the losses incurred by low removal rates by naive avian frugivores.

Development of Ramulosis Disease of Cotton Under Controlled Environment and Field Conditions

Colletotrichum gossypii var. cephalosporioides, the fungus that causes ramulosis disease of cotton, is widespread in Brazil and can cause severe yield loss. Because weather conditions greatly affect disease development, the objective of this work was to develop weather-based models to assess disease favorability. Latent period, incidence, and severity of ramulosis symptoms were evaluated in controlled environment experiments using factorial combinations of temperature (15, 20, 25, 30, and 35 degrees C) and leaf wetness duration (0, 4, 8, 16, 32, and 64 h after inoculation). Severity was modeled as an exponential function of leaf wetness duration and temperature. At the optimum temperature of disease development, 27 degrees C, average latent period was 10 days. Maximum ramulosis severity occurred from 20 to 30 degrees C, with sharp decreases at lower and higher temperatures. Ramulosis severity increased as wetness periods were increased from 4 to 32 h. In field experiments at Piracicaba, Sao Paulo State, Brazil, cotton plots were inoculated (10(5) conidia ml(-1)) and ramulosis severity was evaluated weekly. The model obtained from the controlled environment study was used to generate a disease favorability index for comparison with disease progress rate in the field. Hourly measurements of solar radiation, temperature, relative humidity, leaf wetness duration, rainfall, and wind speed were also evaluated as possible explanatory variables. Both the disease favorability model and a model based on rainfall explained ramulosis growth rate well, with R(2) of 0.89 and 0.91, respectively. They are proposed as models of ramulosis development rate on cotton in Brazil, and weather-disease relationships revealed by this work can form the basis of a warning system for ramulosis development.

Use of growth regulators to overcome seed dormancy in Xylopia aromatica (Annonaceae)

Xylopia aromatica is a species of the Annonaceae family, native to the Brazilian ""Cerrado"". Seeds of this species usually possess morphophysiological dormancy which makes propagation more difficult. The objective of the present study was to evaluate the efficiency of removing the aril and sarcotesta and applying plant growth regulators to overcome dormancy in X. aromatica seeds. Seeds were separated into two groups: one consisting of seeds with aril and sarcotesta and another without these two seed coat appendices. Seeds with and without these appendices were soaked for 48 hours in distilled water or Promalin (R) (gibberellin 4 [GA(4)] + gibberellin 7 [GA(7)] and cytokinin [6-Benziladenine]) solutions of 250, 500 and 1,000 mg.L(-1), and sown in ""Cerrado"" soil. Later, seeds without the aril and sarcotesta were soaked for 48 hours in distilled water. Promalin (R) or GA(4) + GA(7) solutions at same concentrations and sown in sand or ""Cerrado"" soil. The removal of the aril and sarcotesta had a positive effect on the seed germination. Application of plant growth regulators helped to overcome dormancy in X. aromatica, with the greatest percentage of seedling emergence being observed in seeds treated with Promalin at 250 and 500 mg.L(-1) then sown in sand.

Evaluation of Automotive Varnish as a Coating for Density Measurements of Soil Clods

Saran F-310 resin (Dow Chemical Co, Midland, MI) has been widely used to coat soil clods for density and size measurements; however, the manufacturer has recently stopped producing this resin and supplies are difficult to obtain. Hence, we evaluated the feasibility of using Lazzudur 7502 (Sherwin-Williams, Cleveland, OH) automotive varnish to coat soil clods for density measurement. Preliminary evaluations showed that immersion of clods in the varnish did nor affect clod cohesion and that a single immersion in Lazzudur with 30 min of post-immersion drying produced density results nor significantly (P < 0.05) different to those obtained using saran. This technique was tested across seven soils and no significant (P < 0.05) difference was found in the density of the clods measured using the two coating methods. This work suggests that automotive varnish can he used as an alternative to saran resin for clod density measurements.

A new set of microsatellite markers for the genetic characterization of Ceratocystis fimbriata, an economically important plant pathogen

Ceratocystis fimbriata is a fungal pathogen which attacks several economically important plants, but occurs in host-associated, morphologically indistinguishable forms. In Brazil, this fungus seriously attacks mango trees (Mangifera indica), causing severe loss of yield. This work aimed to develop and characterize a novel set of microsatellite markers for this important pathogen, providing researchers with new molecular tools for the characterization of isolates. Twenty polymorphic primer pairs were designed from a microsatellite-enriched library. We tested the usefulness of these markers through genotyping thirteen isolates of the fungus. On average, 6.65 alleles per locus were detected, revealing the ability of this set of markers to characterize C. fimbriata isolates associated to mango and to other plant species.

Genetic Transformation of Passionflower and Evaluation of R(1) and R(2) Generations for Resistance to Cowpea aphid borne mosaic virus

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for `IAC 275` and three for `IAC 277`. Transgenic lines were clonally propagated and evaluated for resistance to CABMV After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the RI generation was evaluated together with the RI generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R(1) plants became infected, whereas 13 of 279 T16R(1) remained asymptomatic after four successive inoculations. A TI6R(2) generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant TI6R(2) plants which contained the capsid protein gene are being crossed for further analyses.

Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean

Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean Asian rust, caused by the fungus Phakopsora pachyrhizi, is one of the most serious phytosanitary problems of soybean in Brazil, especially because no cultivars with satisfactory resistance levels as yet exist. The objective of this study was to evaluate the influence of luminosity and of leaf epicuticular wax on the infection of soybean by P. pachyrhizi. The adaxial and abaxial leaflet surfaces of the first trifoliate leaf from cultivar BRS 154, phenological stage V2, were inoculated with a suspension of 105 uredospores/mL. The plants were kept for 24 hours in a humid chamber at temperature of 23 degrees C, in light or dark conditions, using a factorial design. Subsequently, the plants were maintained for 14 days under a 12-hour photoperiod. The disease severity and density were evaluated. For in vitro experiments, in light or dark conditions, the evaluation was done in terms of uredospore germination and appressorium formation. The wax content of adaxial and abaxial leaflets was analyzed quantitatively using chloroform extraction and ultrastructurally using scanning electron microscope. Higher density and severity were observed when the adaxial surface was inoculated, with later incubation of the plants in the dark, with no significant interaction between these factors. Spore germination in the dark (40.7%) was statistically different from spore germination in the light (28.5%). The same effect was observed with appressorium formation, in the dark (24.7%) and in the light (12.8%). The quantity and the ultrastructural aspects of epicuticular wax content did not show differences between the adaxial and abaxial surfaces; nor did they show any effect on infection by Phakopsora pachyrhizi in the soybean cultivar studied.

Identification and partial characterization of a Carica papaya-infecting isolate of Alfalfa mosaic virus in Brazil

A Carica papaya plant with severe yellow leaf mosaic, leaf distortion, and systemic necrosis was found in the municipality of Piracicaba, state of So Paulo, Brazil. Transmission electron microscopy (TEM) analysis revealed the presence of potyvirus-like particles and bacilliform particles similar to those of the Alfamovirus genus. The potyvirus was identified as Papaya ringspot virus-type P (PRSV-P). Biological, serological, and molecular studies confirmed the bacilliform virus as an isolate of Alfalfa mosaic virus (AMV). Partial nucleotide and amino acid sequences of the coat protein gene of this AMV isolate shared 97-98% identity with the AMV isolates in the GenBank database. This report is the first of the natural infection of papaya plants by AMV.

Evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene

The variability of a fragment of the nucleocapsid gene of orchid fleck virus (OFV) was investigated by single-strand conformational polymorphism (SSCP) analysis and nucleotide sequencing. Forty-eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis yielded six different band patterns, and phylogenetic analysis based on the nucleotide fragment sequence obtained in this work and six available in GenBank showed two different groups, one with isolates 023Germany and So-Japan, and other with the rest of the isolates. None of the analyses showed geographic correlation among the Brazilian strains. The data obtained in this study showed a low genetic variation in this region of the genome; the d(N)/d(S) ratio of 0.251-0.405 demonstrated a negative selective pressure that maintains the stability of the analyzed fragments.

Isoflavones and antioxidant capacity of Peruvian and Brazilian lupin cultivars

Seed coats, cotyledons and hypocotyls from six Peruvian (Lupinus mutabilis Sweet) and two Brazilian (Lupinus albus and Lupinus angustifolius) lupin cultivars were assessed regarding their content of isoflavones and antioxidant capacity. Genistein and a genistein derivative were detected in seed coats and cotyledons from Peruvian cultivars. Total isoflavones ranged from 9.8 to 87, 16.1 to 30.8 and 1.3 to 6.1 mg/100 g of sample in fresh weight (expressed as genistein) in seed coat, cotyledon and hypocotyl fractions, respectively, from mutabilis species, whereas no isoflavones were detected in L. angustifolius and L. albus. A significant correlation (r = 0.99) was found between the total isoflavone levels and the antioxidant capacity measured by the 2,2-diphenyl-1-picrylhydrazyl radical-scavenging method in all fractions of Peruvian samples. No condensed tannins were detected in any of the lupin cultivars. The H-6 Andean cultivar is promising for its high isoflavone content and antioxidant capacity. Insights from this study indicate that lupin cultivars of the mutabilis species have similar isoflavone profiles and that isoflavones are more concentrated in the cotyledon seed fraction than in the seed coat or hypocotyl fractions. (C) 2008 Elsevier Inc. All rights reserved.