Effects of Deficit Irrigation and Huanglongbing on Sweet Orange Trees.

This study addresses the interactive effects of deficit irrigation and huanglongbing (HLB) infection on the physiological, biochemical, and oxidative stress responses of sweet orange trees. We sought to answer: (i) What are the causes for the reduction in water uptake in HLB infected plants? (ii) Is the water status of plants negatively affected by HLB infection? (iii) What are the key physiological traits impaired in HLB-infected plants? and (iv) What conditions can mitigate both disease severity and physiological/biochemical impairments in HLB-infected plants? Two water management treatments were applied for 11 weeks to 1-year-old-trees that were either healthy (HLB-) or infected with HLB (+) and grown in 12-L pots. Half of the trees were fully irrigated (FI) to saturation, whereas half were deficit-irrigated (DI) using 40% of the water required to saturate the substrate. Our results demonstrated that: reduced water uptake capacity in HLB+ plants was associated with reduced root growth, leaf area, stomatal conductance, and transpiration. Leaf water potential was not negatively affected by HLB infection. HLB increased leaf respiration rates (ca. 41%) and starch synthesis, downregulated starch breakdown, blocked electron transport, improved oxidative stress, and reduced leaf photosynthesis (ca. 57%) and photorespiration (ca.57%). Deficit irrigation reduced both leaf respiration (ca. 45%) and accumulation of starch (ca.53%) by increasing maltose (ca. 20%), sucrose, glucose, and fructose contents in the leaves, decreasing bacterial population (ca. 9%) and triggering a series of protective measures against further impairments in the physiology and biochemistry of HLB-infected plants. Such results provide a more complete physiological and biochemical overview of HLB-infected plants and can guide future studies to screen genetic tolerance to HLB and improve management strategies under field orchard conditions.

High-Quality Draft Genome Sequence Resources of Eight Xylella fastidiosa Strains Isolated from Citrus, Coffee, Plum, and Hibiscus in South America.

Xylella fastidiosa subsp. pauca, once confined to South America and infecting mainly citrus and coffee plants, has been found to be associated with other hosts and in other geographic regions. We present high-quality draft genome sequences of X. fastidiosa subsp. pauca strains J1a12, B111, U24D, and XRB isolated from citrus plants in Brazil, strain Fb7 isolated from a citrus plant in Argentina and strains 3124, Pr8x, and Hib4 isolated, respectively, from coffee, plum, and hibiscus plants in Brazil. Sequencing was performed using Roche 454-GS FLX, MiSeq-Illumina or Pacific Biosciences platforms. These high-quality genome assemblies will be useful for further studies about the genomic diversity, evolution, and biology of X. fastidiosa.

High-quality draft genome sequence resources of eight Xylella fastidiosa strains isolated from citrus, coffee, plum and hibiscus in South America.

Xylella fastidiosa subsp. pauca , once confined to South America and infecting mainly citrus and coffee plants, has been found to be associated with other hosts and in other geographic regions. We present high-quality draft genome sequences of X. fastidiosa subsp. pauca strains J1a12, B111, U24D and XRB isolated from citrus plants in Brazil, strain Fb7 isolated from a citrus plant in Argentina and strains 3124, Pr8x and Hib4 isolated, respectively, from coffee, plum and hibiscus plants in Brazil. Sequencing was performed using Roche 454-GS FLX, MiSeq-Illumina or Pacific Biosciences platforms. These high-quality genome assemblies will be useful for further studies about the genomic diversity, evolution, and biology of X. fastidiosa.

High-quality draft genome sequence resources of eight Xylella fastidiosa strains isolated from citrus, coffee, plum and hibiscus in South America.

Xylella fastidiosa subsp. pauca , once confined to South America and infecting mainly citrus and coffee plants, has been found to be associated with other hosts and in other geographic regions. We present high-quality draft genome sequences of X. fastidiosa subsp. pauca strains J1a12, B111, U24D and XRB isolated from citrus plants in Brazil, strain Fb7 isolated from a citrus plant in Argentina and strains 3124, Pr8x and Hib4 isolated, respectively, from coffee, plum and hibiscus plants in Brazil. Sequencing was performed using Roche 454-GS FLX, MiSeq-Illumina or Pacific Biosciences platforms. These high-quality genome assemblies will be useful for further studies about the genomic diversity, evolution, and biology of X. fastidiosa.

Distribution and Genetic Diversity of Xylella fastidiosa subsp. pauca Associated with Olive Quick Syndrome Symptoms in Southeastern Brazil.

In Brazil, the host expansion of Xylella fastidiosa subsp. pauca was recently demonstrated with the report of diseased olive trees (Olea europaea), whose symptoms were associated with olive quick decline syndrome previously described in southern Italy. We employed both polymerase chain reaction-based techniques and culture medium isolation to investigate the geographic distribution of X. fastidiosa as well as the genetic signatures of 21 strains isolated from 11 olive orchards in both São Paulo and Minas Gerais States in Brazil. X. fastidiosa subsp. pauca was detected in 83% of the orchards examined in the region, and was positively diagnosed in 43.7% of all sampled plants with typical scorching symptoms. Of the 21 strains characterized with fast-evolving microsatellite (single sequence repeat [SSR]) markers, 20 different multilocus microsatellite genotypes were observed with the overall allelic diversity of HNei = 0.38. Principal component analysis using the SSR markers clustered all strains, except for three, in one cluster demonstrating a limited range of genetic diversity. Multilocus sequence typing analysis showed the prevalence of a sequence type (ST16) in 75% of the samples; three other novel STs (84, 85, and 86), were detected, all belonging to the X. fastidiosa subsp. pauca cluster. These results show that genetically diverse strains of X. fastidiosa subsp. pauca are widely present in olive orchards in southeastern Brazil, which is consistent with the long history of this bacterium in that region.

Genetic Structure and Molecular Variability Analysis of Citrus sudden death-associated virus Isolates from Infected Plants Grown in Brazil.

Citrus sudden death-associated virus (CSDaV) is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD) disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR), the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π) was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5' end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies.

Assessment of the diagnostic potential of Immmunocapture-PCR and Immuno-PCR for Citrus Variegated Chlorosis.

Xylella fastidiosa causes significant losses in many economically important crops. An efficient pathogen detection system is critical for epidemiology studies, particularly when large sample size is involved. In this study we report the development of immunomolecular assays like Immmunocapture-PCR and Immuno-PCR for direct detection of X. fastidiosa without DNA isolation. Whereas the reactivity of ELISA and PCR ranged from 10(6) to 10(4) bacterial cells, the IC-PCR sensitivity was up to 10(3) and the detection limit of I-PCR was up to 10(1) bacterial cells. These methods can use either plant sample extracts or cultivated media, and show no cross reaction for any other endophytic citrus-bacteria. Therefore, IC-PCR and I-PCR assays provide an alternative for quick and very sensitive methods to screening X. fastidiosa, with the advantage of not requiring any concentration or DNA purification steps while still allowing an accurate diagnosis of CVC.

Absence of classical heat shock response in the citrus pathogen Xylella fastidiosa.

The fastidious bacterium Xylella fastidiosa is associated with important crop diseases worldwide. We have recently shown that X. fastidiosa is a peculiar organism having unusually low values of gene codon bias throughout its genome and, unexpectedly, in the group of the most abundant proteins. Here, we hypothesized that the lack of codon usage optimization in X. fastidiosa would incapacitate this organism to undergo quick and massive changes in protein expression as occurs in a classical stress response. Proteomic analysis of the response to heat stress in X. fastidiosa revealed that no changes in protein expression can be detected. Moreover, stress-inducible proteins identified in the closely related citrus pathogen Xanthomonas axonopodis pv citri were found to be constitutively expressed in X. fastidiosa. These proteins have extremely high codon bias values in the X. citri and other well-studied organisms, but low values in X. fastidiosa. Because biased codon usage is well known to correlate to the rate of protein synthesis, we speculate that the peculiar codon bias distribution in X. fastidiosa is related to the absence of a classical stress response, and, probably, alternative strategies for survival of X. fastidiosa under stressfull conditions.

A kinetic model for Xylella fastidiosa adhesion, biofilm formation, and virulence.

Xylella fastidiosa is the causal agent of citrus variegated chlorosis and Pierce's disease which are the major threat to the citrus and wine industries. The most accepted hypothesis for Xf diseases affirms that it is a vascular occlusion caused by bacterial biofilm, embedded in an extracellular translucent matrix that was deduced to be the exopolysaccharide fastidian. Fourier transform infrared spectroscopy analysis demonstrated that virulent cells which form biofilm on glass have low fastidian content similar to the weak virulent ones. This indicates that high amounts of fastidian are not necessary for adhesion. In this paper we propose a kinetic model for X. fastidiosa adhesion, biofilm formation, and virulence based on electrostatic attraction between bacterial surface proteins and xylem walls. Fastidian is involved in final biofilm formation and cation sequestration in dilute sap.

Geographical Genetic Structure of Xylella fastidiosa from Citrus in São Paulo State, Brazil.

ABSTRACT A total of 360 Xylella fastidiosa strains were isolated from sweet orange (Citrus sinensis) cv. Pera plants growing in five geographic regions in the Brazilian state of São Paulo. The genetic variation of these strains was determined by 15 variable number tandem repeat (VNTR) and 58 random amplified polymorphic DNA (RAPD) markers. The mean values of genetic diversity (H) of X. fastidiosa strains within each geographic region determined by RAPD (H(RAPD)) were substantially lower than H(VNTR) values. H(RAPD) values ranged from 0.00 to 0.095, whereas the H(VNTR) values ranged from 0.024 to 0.285. A highly significant value of Nei's coefficient of gene differentiation (G(ST) = 0.355; P = 0.000) was detected among all five populations. Analysis of the molecular variance (AMOVA) also revealed significant genetic differentiation among regions or populations ( phi(STAT) = 0.810; P< 0.001). In addition, genetic differentiation among subpopulations (plants) within the regions (phi(STAT) = 0.699; P < 0.001) and within each plant (phi(STAT) = 368; P < 0.001) were statistically significant. These high values of genetic differentiation among X. fastidiosa strains from different regions suggest a genetic structure according to region of host origin. However, no apparent correlation between genetic distance and region of origin of populations were supported statistically by Mantel analysis (r = 0.27; P = 0.22).