Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Irrigation requirements and transpiration coupling to the atmosphere of a citrus orchard in Southern Brazil

Crop evapotranspiration (ETc) was measured as evaporative heat flux from an irrigated acid lime orchard (Citrus latifolia Tanaka) using the aerodynamic method. Crop transpiration (T) was determined by a stem heat balance method. The irrigation requirements were determined by comparing the orchard evapotranspiration (ETc) and T with the reference evapotranspiration (ETo) derived from the Penman-Monteith equation, and the irrigation requirements were expressed as ETc/ETo (Kc) and T/ETo (Kcb) ratios. The influence of inter-row vegetation on the ETc was analyzed because the measurements were taken during the summer and winter, which are periods with different regional soil water content. In this study, the average Mc values obtained were 0.65 and 0.24 for the summer and winter, respectively. The strong coupling of citrus trees to the atmosphere and the sensitivity of citrus plants to large vapor pressure deficits and air/leaf temperatures caused variations in the Kcb in relation to the ETo ranges. During the summer, the Kcb value ranged from 0.34 when the ETo exceeded 5 mm d(-1) to 0.46 when the ETo was less than 3 mm d(-1). (C) 2011 Elsevier B.V. All rights reserved.

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of Sao Paulo, Brazil, to investigate the incidence of `Candidatus Liberibacter asiaticus` and `Ca. L. americanus`, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft-transmissibility of the two species, and their DNA relatedness to citrus-associated liberibacters, were also investigated. Quantitative PCR was applied to PCR-positive orange jasmine and HLB-positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR-positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR-negative trees. `Candidatus Liberibacter americanus` was more common in 2005/2006 (96 center dot 6%) and `Ca. L. asiaticus` in 2009 (84 center dot 8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including `Ca. L. americanus` (2/10) from/to orange jasmine and `Ca. L. americanus` (5/18) and `Ca. L. asiaticus` (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of `Ca. L. americanus` and `Ca. L. asiaticus` in orange jasmine averaged 4 center dot 3 and 3 center dot 0 log cells g-1 tissue, compared with 5 center dot 5 and 7 center dot 3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree-eradication programme or measures for insect control.

Natural infection of Nicandra physaloides by Tomato severe rugose virus in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in So Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (espinheira-santa) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Coll and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

Occurrence of phytoplasma associated with sugarcane yellow leaf syndrome in three regions of Sao Paulo State, Brazil

Sugarcane yellow leaf syndrome caused serious damage to crops in the Sao Paulo State, Brazil, in the 1990`s. The syndrome was also reported in other countries and investigations into the etiology revealed an association between the disease and virus and/or phytoplasma. The disease is potentially important and occurs endemically in that State, and for this reason the present study was conducted in order to demonstrate the occurrence of phytoplasma in three traditional sugarcane-producing areas, in Sao Paulo State, through molecular detection From naturally infected plants. Symptomatic plants belonging to varieties SP71-6163, SP71-6180 and SP89-1115 were sampled from Piracicaba, Jau and Ribeirao Preto, and total DNA was extracted from foliar tissues. Nested PCR was conducted with primer pairs P1/ Tint-16F2n/R2, and the amplified products were analyzed by electrophoresis on agarose gels. Amplified DNA fragments of 1.2 kb evidenced the presence of phytoplasma in 36% of symptomatic plants and revealed its Occurrence in all sampled regions. The results demonstrated that phytoplasma is associated with the disease and that it is important to keep a safe inspection of nurseries and monitoring plants in the field, as well as to Select Sugarcane genotypes with a good level of resistance in breeding programs.

Yield loss caused by huanglongbing in different sweet orange cultivars in So Paulo, Brazil

Citrus huanglongbing (HLB) reduces an affected orchard`s economic life. This work aimed to characterize yield loss due to HLB for different sweet orange cultivars and determine the relationship between disease severity and yield. Disease severity and yield were assessed on 949 individual trees distributed in 11 different blocks from sweet orange cultivars Hamlin, Westin, Pera and Valencia. In each block, plants showing a range of HLB severity levels and asymptomatic plants were selected. Total yield (weight of harvested fruit), mean weight of asymptomatic and symptomatic fruit, relative yield (symptomatic tree yield/mean yield of asymptomatic trees from the same block) and relative number of fruits (fruit number from symptomatic tree/mean number of fruits from asymptomatic trees from the same block) were determined. The weight of symptomatic fruit was lower than the weight of asymptomatic fruit, but the weights of asymptomatic and symptomatic fruit were not correlated with disease severity, indicating that the effects of HLB were restricted to symptomatic branches. The relationship of the relative yield with HLB severity can be satisfactorily described by a negative exponential model. The rates of yield decrease as a function of disease severity were similar for all assessed cultivars. A relative yield (up to 19%) was observed even for trees where disease severity was 100%. The strong linear relationship between relative number of fruits per tree and the relative yield per tree suggested that the yield reduction was due primarily to early fruit drop or lack of fruit set on affected branches.

Role of healthy nursery plants in orange yield during eight years of Citrus Variegated Chlorosis epidemics

The production and commercialization of citrus seedlings inspected and produced in protected screen-houses has become mandatory in Sao Paulo State, Brazil since January 2003. This law was intended to avoid the dispersion of Citrus Variegated Chlorosis (CVC), disease caused by Xylella fastidiosa. Our objective was to compare the yield over 8 years of `Natal` sweet orange trees grafted onto Rangpur lime obtained from healthy nursery plants and from plants artificially inoculated with X. fastidiosa. Yield was evaluated in an orchard planted in February 1999 with two treatments: (i) trees from healthy nursery plant, and (ii) trees from plants artificially inoculated with X. fastidiosa. The mean yield was 21% higher in trees from healthy nursery plants, as compared to trees from inoculated nursery plants. This difference represents a gain of approximately 203 boxes of 40.8 kg each, considering a planting density of 550 plants per hectare. (C) 2011 Elsevier B.V. All rights reserved.

Metallomics and chemical speciation: towards a better understanding of metal-induced stress in plants

Most metal ions are toxic to plants, even at low concentrations, despite the fact that some are essential for growth and play key roles in metabolism. The majority of metals induce the formation of reactive oxygen species, which require the synthesis of additional antoxidant compounds and enzymes for their removal. New techniques that have greatly improved the identification, localisation and quantification of metals within plant tissues have led to the science of metallomics. This advancement in knowledge should eventually allow the characterisation of plants used in the process of phytoremediation of soils contaminated with toxic metals.

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro-Tom) plants to cadmium-induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro-Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl(2), respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl(2), whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl(2). Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl(2) after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl(2), but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl(2) when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl(2), whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl(2). The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl(2). However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl(2) from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.

Viroid species associated with the bark-cracking phenotype of `Tahiti` acid lime in the State of Sao Paulo, Brazil

Viroids have been used as ""graft transmissible dwarfing agents"" (GTDA) in several countries, mainly to reduce growth of citrus trees, thus increasing their density in orchards. In the State of Sao Paulo, Brazil, plants of the acid lime `Tahiti` are usually grafted with a complex of GTDA, presumably viroids. The aim of the present work was the identification and molecular characterization of the viroids infecting trees of acid lime `Tahiti` displaying ""Quebra galho"" (bark-cracking). Viroids were identified and characterized by biological indexing in `Etrog` citron, Northern-blot hybridization, RT-PCR, cloning and complete sequencing of the RNA genomes. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus dwarfing viroid (CDVd) were found in different combinations. Although we have not been able to infer a direct relationship between the agronomical performance and symptom severity with the presence of a specific viroid or viroid combination, the differences in the severity of ""Quebra-galho"" symptoms among different trees is probably associated with the presence (or absence) of CEVd, with its interaction with other viroids perhaps determining the different phenotypes observed in the field.

Detection of Sourgrass (Digitaria insularis) Biotypes Resistant to Glyphosate in Brazil

Sourgrass is a perennial weed infesting annual and perennial crops in Brazil. Three biotypes (R1, R2, and R3) of sourgrass suspected to be glyphosate-resistant (R) and another one (S) from a natural area without glyphosate application, in Brazil, were tested for resistance to glyphosate based on screening, dose-response, and shikimic acid assays. Both screening and dose-response assays confirmed glyphosate resistance in the three sourgrass biotypes. Dose-response assay indicated a resistance factor of 2.3 for biotype RI and 3.9 for biotypes R2 and R3. The hypothesis of a glyphosate resistance was corroborated on the basis of shikimic acid accumulation, where the S biotype accumulated 3.3, 5.0, and 5.7 times more shikimic acid than biotypes R1, R2, and R3, respectively, 168 h after treatment with 157.50 g ae ha(-1) of glyphosate. There were no differences in contact angle of spray droplets on leaves and spray retention, indicating that differential capture of herbicide by leaves was not responsible for resistance in these biotypes. The results confirmed resistance of sourgrass to glyphosate in Brazil.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Greenhouse gases emission from soil contaminated with automobile industry residue in Brazil

Solid waste of the automobile industry containing large amounts of heavy metals might affect the emission of greenhouse gases (GHG) when applied to the soil. Accumulation of inorganic chemical elements in the environment generally occurs due to human activity (industry, agriculture, mining and waste landfills). Residues from human activities may release heavy metals to the soil solution, causing toxicity to plants and other soil organisms. Heavy metals may also be adsorbed to clay minerals and/or complexed by the soil organic matter, becoming a potential source of pollutants. Not much is known about the behavior of solid wastes in tropical soil as regarded as source of greenhouse gases (GHG). The emission of GHG (CO(2), CH(4) and N(2)O) was evaluated in incubated soil samples collected in an area contaminated with a solid residue from an automobile industry. Samples were randomly collected at 0 to 0.2 m (a mix of soil and residue), 0.2 to 0.4 m (only residue) and 0.4 to 0.6 m (only soil). A contiguous uncontaminated area, cultivated with sugarcane, was also sampled following the same protocol. Canonical Discriminant Analysis and Principal Component Analysis were applied to the data to evaluate the GHG emission rates. Emission rates of GHG were greater in the samples from the contaminated than the sugarcane area, particularly high during the first days of incubation. CO(2) emissions were greater in samples collected at the upper layer for both areas, while CH(4) and N(2)O emissions were similar in all samples. The emission rates of CH(4) were the most efficient variables to differentiate contaminated and uncontaminated areas.

LEAF AREA REDUCTION IN CORN GROWN IN A TROPICAL REGION OF BRAZIL AND ITS EFFECTS ON AGRONOMIC TRAITS

Global climate change may reduce leaf area in crop plants due to factors such as increasing occurrence of pests and diseases. The aim of this work was to estimate the impact of leaf area reduction on agronomic traits in corn. An experiment simulating leaf area reduction was carried out in a tropical region of Brazil. The agronomic performance of corn plants was evaluated at different percentages of leaf loss. It was observed that leaf area reductions over 41.01% significantly harm yield, mass of 1000 grains, cob density, and stem and root quality Crop improvement programs should take into account the development of genotypes resistant to factors that cause leaf area reduction in tropical crops.