Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa

Xylella fastidiosa causes Pierce??s disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.     

Assessment of Pierce's disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce's disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.     

The growth of a plant-parasitic bacterium,Clavibacter xyli subsp. cynodontis,is enhanced by xylem fluid components

Clavibacter xyli subspecies cynodontis is a gram-positive coryneform bacterium, originally isolated from Bermuda grass. The bacterium is xylem-limited, slow-growing and nutritionally fastidious. We found that extracts of Bermuda grass and maize, as well as xylem fluid collected from decapitated maize plants, enhanced the growth of the bacteria in vitro. The growth was enhanced even with 1% (v/v) maize xylem sap in a rich culture medium, giving both an increased growth rate and a higher stationary phase cell density. The relative increase in growth was inversely correlated with the size of bacterial inoculum. When liquid medium was stored for more than 2 months, the bacteria did not grow at all unless the medium was conditioned with a plant exudate. We fractionated maize xylem fluid by ultrafiltration and chromatographic methods and tested all the fractions for bacterial growth induction. The xylem sap was found to contain two distinct growth-enhancing compounds: a small hydrophilic compound and a proteinaceous compound of more than 10 kDa.     

A soluble carbohydrate elicitor from Blumeria graminis f. sp. tritici is recognized by a broad range of cereals

Wheat plants rapidly recognize pathogenic and non-pathogenic conidia of the powdery mildew fungusBlumeria (syn. Erysiphe)graminis on their leaf surfaces. This suggests that a chemical signal emanates from conidia at the pre-penetration stage of infection. Conidia of B. graminis f. sp. tritici were found to contain an elicitor that was easily washed off their surface. The elicitor activity is heat stable and could not be removed by phenol extraction. By contrast, elicitor activity is sensitive to periodate oxidation and partial acid hydrolysis suggesting that the elicitor activity resides in a carbohydrate moiety. Analysis of carbohydrates revealed mostly glucose, with smaller amounts of xylose and mannose. The glucosyl residues of the B. graminis elicitor were found to be linked (1 → 2)-, (1 → 4), and (1 → 6)-, with (1 → 4, 1 → 6)- branch point residues, and no 3-linked glucose residues were detected. As treatment with β -mannanase significantly reduced elicitor activity, mixed-linkage (1 → 4), (1 → 6)-mannosyl residues appeared to be important for elicitor activity. The B. graminis elicitor induced the expression of all defence-related genes tested in wheat and also induced resistance to subsequent attack by B. graminis f. sp. tritici. In contrast, a hypersensitive response was not induced by the elicitor in the absence or the presence of a challenging inoculum of B. graminis f. sp. tritici. The elicitor also induced the accumulation of thaumatin-like proteins in barley, oat, rye, rice and maize, but did not induce necrosis in any of these species. This suggests that the B. graminis elicitor represents a host non-specific determinant of non-self recognition in cereals activating general defence responses other than the hypersensitive reaction.     

Biochemical aspects of reactive oxygen species formation in the interaction between Lycopersicon spp. and Oidium neolycopersici

Three Lycopersicon spp. accessions differing in the level of resistance to Oidium neolycopersici L. Kiss (tomato powdery mildew) were studied. Defence reactions occurring in tissue of Lycopersicon esculentum cv. Amateur (susceptible control), Lycopersicon hirsutum f. glabratum (LA 2128) (highly resistant) and Lycopersicon chmielewskii (LA 2663) (moderately resistant) were investigated during the first 120 h post-inoculation (hpi). A hypersensitive reaction was detected after 48 hpi in both resistant tomato accessions. Changes in accumulation of hydrogen peroxide and enzymes involved in its metabolism (catalase, peroxidases, superoxide dismutase) were monitored. In resistant accessions, intensive H₂O₂ production correlated with increased activity of cytosolic guaiacol peroxidase, syringaldazine peroxidase and ascorbate peroxidase. Catalase activity increased especially in moderately resistant L. chmielewskii. A similar degree of lipid peroxidation occurred in all Lycopersicon accessions. An increase in the concentration of free phenols but no change in the level of cell-wall-bound phenols were observed during the first 120 hpi in all Lycopersicon spp. accessions. Spermidine represented the major part of the total polyamine content. Pathogen-induced lignification was not observed in any of the studied accessions.     

Retraction notice to “Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus” [Pesticide Biochemistry and Physiology 94 (2009) 5-9]

A total of 185 hexanic, dichloromethanic, ethanolic and hydroethanolic extracts from 24 species of Cerrado plants, were tested against Zabrotes subfasciatus, Acanthoscelides obtectus, and human saliva α-amylases. Twelve crude extracts presented inhibition rates greater than 80% against digestive α-amylases of the insect pest Z. subfasciatus, at a concentration of 1 mg mL⁻¹. These extracts were also tested against A. obtectus and human saliva α-amylases to verify their affinity and specificity of action. The hydroethanolic Kielmeyera coriacea stem bark extract presented a strong inhibitory potential, with IC₅₀ values of 110 μg mL⁻¹ for Z. subfasciatus and 272.12 μg mL⁻¹ for A. obtectus, in addition to a 97.09% reduction in enzyme activity of human saliva α-amylases at 125 μg mL⁻¹. The hexanic Aspidosperma macrocarpon root wood extract totally inhibited the activity of Z. subfasciatus α-amylases, reduced the enzyme activity of A. obtectus by 14.69% at 1 mg mL⁻¹, but did not alter the activity of human saliva α-amylases, thus characterizing greater inhibition affinity and specificity. The results suggest that the application of plant extracts against insect α-amylases represent a promising biotechnological tool for development of new insect pest control strategies, with noticeable affinity and specificity of action against different target enzymes.     

Agrobacterium induces plant cell death in wheat (Triticum aestivum L.)

The symptoms of gall or hairy root do not occur in the interactions between wheat (Triticum aestivum L.) and other monocotyledonous plants, with Agrobacterium tumefaciens or Agrobacterium rhizogenes. However, both bacteria colonized wheat root surfaces at similar levels (2.0 × 10⁷ colony forming U g⁻¹ root) and grew without inhibition in suspension with intact or wounded wheat embryos or root segments present. Suspension-cultured wheat embryo cells grown in 7.4 m M O₂ displayed 23% cell death after 1 h exposure to Agrobacterium cells, while the extent of cell death with 2.1 m M O₂ averaged 8%. Cultured wheat embryo and root cells rapidly produced hydrogen peroxide (H₂O₂) when contacted with A. tumefaciens or A. rhizogenes. Production of H₂O₂ was lower at 2.1 m M O₂ than 7.4 mM O₂. Browning and autofluorescence of epidermal cells of callus derived from wheat embryos and wheat roots was observed after inoculation with Agrobacterium. An increase in ferulic acid was detected in the walls of roots exposed to Agrobacterium. However, neither lignin nor callose was detected by diagnostic staining methods. These findings suggest that Agrobacterium induced a resistance-like response in wheat that may reduce the efficacy of transformation and limit the normal symptom formation.     

Role of sucrose in the development of Fusarium wilt in lupine embryo axes

The response of embryo axes of Lupinus luteus L. cv. Polo to invasion by Fusarium oxysporum Schlecht f.sp. lupini was studied 84, 96 and 108 h after inoculation and under various trophic conditions. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). The exogenous sucrose caused an accumulation of sucrose, glucose and fructose in +Sn embryo axes. Pathogen invasion generally resulted in a sharp decline in soluble carbohydrate content of +Si embryo axes. Symptoms of Fusarium wilt included growth inhibition of the embryo axes and a decrease in their fresh weight, which was particularly strong in −Si axes. While the addition of exogenous sucrose to the medium 24 h prior to inoculation significantly alleviated the disease, its application 48 h after inoculation did not restrict disease development. In +Si embryo axes, the activity of peroxidases (EC 1.11.1.7), covalently and ionically bound to the cell walls, increased considerably, mainly up to 96 h after inoculation. This increase was significantly higher than that seen in +Sn embryo axes. These results are in contrast to those of an earlier study [Morkunas I, Marczak Ł, Stachowiak J, Stobiecki M. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005;43:363–73], which showed a pronounced decrease in the flavonoid glycoside content of +Si tissues. Furthermore, starting 96 h after inoculation, a decrease in phenylalanine ammonialyase activity (PAL, EC 4.3.1.5) was observed in inoculated tissues, in comparison to non-inoculated tissues. The decrease was especially pronounced in +Si tissues. However, a significant increase in the free isoflavone aglycones content of +Si embryo exes, i.e. genistein, 2′-hydroxygenistein, wighteone and luteone. Simultaneously, a strong increase in the activity of β-glucosidase (EC 3.2.1.21), hydrolyzing isoflavone glycosides, was observed in inoculated tissues. The activity of these enzymes was much higher in −Si embryo axes than in +Si embryo axes. The results suggest that soluble carbohydrates may play a role in the restriction of Fusarium wilt development, as they may be related both to intensified lignification and to the antimicrobial role of free aglycones, which may be products of the hydrolysis of isoflavone glycosides generated prior to the invasion stage.     

Effects of three esca-associated fungi on Vitis vinifera L.: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines

Secondary metabolites and host defense compounds were shown to occur in xylem sap, and leaves of Vitis vinifera cv. Italia and cv. Matilde naturally infected by the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme). Samples of xylem sap and leaves were collected from healthy vines and from vines showing severe symptoms of brown wood-streaking caused by Pch and Tmi, or from vines with symptoms of both brown wood-streaking and white rot caused by Fme. Xylem sap collection was carried out during the early spring of 2003 and 2004, corresponding to the phenological phases: (A) cotton bud; (B) green tip; (C) leaves out; (D) stretched out leaves; and (E) visible clusters. In the present work we have studied the accumulation of biomolecules (pentaketides and α-glucans), host defense compounds (benzaldehydes, benzoic acid and cinnamic acid derivatives, flavonols, flavanols, flavan-3-ol derivatives and stilbenes) at different stages of grapevine development. Accumulation and changes in total phenolics and recurring phenolics, and of three phytotoxic secondary metabolites (scytalone, isosclerone and pullulan) were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that phenolic concentrations are strongly related to the cv.     

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants

A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod‐shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.     

Effects of trichlorfon and sodium dodecyl sulphate on antioxidant defense system and acetylcholinesterase of Tilapia nilotica in vitro

The effects of organophosphorus insecticide trichlorfon, surfactant sodium dodecyl sulphate (SDS), and the mixture of trichlorfon and SDS on the antioxidant defense system and acetylcholinesterase (AChE) in Tilapia nilotica were assessed in vitro. Various concentrations of trichlorfon (0, 0.0001, 0.001, 0.01, 0.1 and 1 g/L) and SDS (0, 0.0625, 0.125, 0.25, 0.5, 1 g/L) were incubated with homogenate of liver and muscle, respectively, at 25 °C for 0, 30, 60 and 90 min. Two concentrations of mixture of trichlorfon and SDS (0.0001 g/L trichlorfon + 0.5 g/L SDS, 0.1 g/L trichlorfon + 0.5 g/L SDS) and 0.0001 g/L trichlorfon, 0.1 g/L trichlorfon, 0.5 g/L SDS and control, were incubated simultaneously with homogenate of liver and muscle, respectively, at 25 °C for 60 min. After incubation, the content of reduced-glutathione (GSH) and the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in homogenate of liver were determined, and the activities of AChE in homogenate of muscle were also measured.Treatment with trichlorfon caused a significant concentration-dependent and time-related inhibition of AChE activity at all treatment concentrations and times since trichlorfon is a cholinesterase inhibitor. For the same trichlorfon treatment, an apparent decrease in GSH content was found in concentration of 0.01, 0.1, 1 g/L, whereas no significant alteration in antioxidant enzyme activity were found at all experiment concentrations and times, which might indicate that antioxidant enzymes have not involved in the metabolism of trichlorfon. The depletion of GSH might indicate that ROS could be involved in the toxic effects of trichlorfon. Exposure of SDS can inhibit activities of AChE, GST and CAT at concentrations of 0.5 and/or 1 g/L, which could be due to the denaturing process of SDS to the enzymes. For the mixture exposure of trichlorfon and SDS, the effect of the mixture of 0.0001 g/L trichlorfon and 0.5 g/L SDS on inhibition of AChE shows synergistic other than simple additive of trichlorfon and SDS. The combined effects of chemicals and detergents deserve to be particularly noted. It should be noted that the toxicity experiments were made in tissue homogenates instead of whole organisms. The responses against the toxic compounds will not be the same in both systems.     

Differential colonization patterns of Xylella fastidiosa infecting citrus genotypes

Xylella fastidiosa is a phytopathogenic bacterium that causes disease in many different crops worldwide. In Brazil, X. fastidiosa subsp. pauca causes citrus variegated chlorosis (CVC), which is a disease responsible for economic losses in the citrus agribusiness. Variable host responses to bacterial colonization and disease development have been observed. This work studies the colonization processes of a pathogenic GFP‐labelled X. fastidiosa citrus strain in sweet orange (susceptible) and tangor (resistant) parents and two resulting hybrids that exhibited contrasting responses to CVC. Xylella fastidiosa showed increased populations and movement in the susceptible genotypes, but slower compared to other hosts such as grapevine. Scanning electron microscopy revealed that the predominant pitted stem morphology in citrus makes the bacterial movement difficult. In susceptible genotypes X. fastidiosa can move from the primary to the secondary xylem, whilst it is confined to the primary xylem in resistant plants. Associated with this is an induction of lignification that occurs earlier in the resistant genotypes when in the presence of the pathogen, and represents a genetic mechanism that leads to formation of a physical barrier, impairing bacterial colonization.     

Genetic analysis of abamectin resistance in Tetranychus cinnabarinus

The carmine spider mite is the most serious crop mite pests in China. Abamectin has been used to control insects and mites worldwide but carmine spider mites, Tetranychus cinnabarinus, had developed resistance to it. Genetic research on insecticide resistance has been fundamental for understanding the resistance development, studying resistance mechanisms, and designing appropriate resistance management strategies to control insect pests. A resistant colony of T. cinnabarinus, RRG42, was established to examine the inheritance of abamectin resistance in T. cinnabarinus. The females of T. cinnabarinus were selected for bioassay using a slide dip method. After 42 generations of selection, the RRG42 strain was 8.7-fold resistant to abamectin compared with the susceptible strain (SS). The logarithm (log) concentration–probit response curve for F₁s from reciprocal crosses, of F₁RS and F₁SR, were inclined to that for SS and the degree of dominance (D) values for F₁s were −0.81 and −0.17. There was a significant difference in values of LC₅₀ and slope of log concentration–probit lines between F₁RS and F₁SR. The observed mortalities of BC1 (F₁RS♀ × RRG42♂) and BC1′ (F₁SR♀ × SS♂) were significantly different from the expected mortalities based on a monogenic resistance in the chi-square tests. The inheritance of abamectin resistance in T. cinnabarinus is incompletely recessive and may be controlled by more than one gene. The maternal or cytoplasmic effect may exist in the inheritance of resistance to abamectin in T. cinnabarinus.     

Seven years of negative detection results confirm that Xylella fastidiosa,the causal agent of CVC,is not transmitted from seeds to seedlings

Knowledge about the mechanism of transmission of systemic pathogens of citrus species is highly important for the safe movement of citrus germplasm, management of citrus mother trees, and also production of young plants. Among systemic pathogens of citrus, Xylella fastidiosa the causal agent of the citrus variegated chlorosis (CVC), is one the most important pathogens causing decline in tree vigour and productivity. Seven-year experiments were conducted to evaluate the hypothesis of seed-to-seedling transmission of X. fastidiosa. This bacterium was found colonizing the fruit (exocarp, central axis and mesocarp) and the seed parts (seed coat and endosperm plus embryo). After 7 years of PCR assay, no positive PCR detection of X. fastidiosa was confirmed in seedlings propagated from the seeds infected with X. fastidiosa. This result demonstrates the lack of seed-to-seedling transmission of this bacterium.     

The chemotaxis regulator <Emphasis Type="Italic">pilG</Emphasis> of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> is required for virulence in <Emphasis Type="Italic">Vitis vinifera</Emphasis> grapevines

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce’s disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce’s Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.     

Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus

Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10–14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors.     

Oxygen consumption and ammonia excretion of the freshwater crab Trichodactylus borellianus exposed to chlorpyrifos and endosulfan insecticides

The effects of lethal and sublethal concentrations of chlorpyrifos and endosulfan on oxygen consumption and ammonia excretion rate of the crab Trichodactylus borellianus were evaluated. Oxygen consumption and energy expenditure had significant effect in relation to exposure times. Regarding endosulfan, a significant difference in consumption among times of exposure was registered in 625 μg L⁻¹. Moreover, at the highest concentration, energy expenditure rate was observed stabilized during 1–3 h. A significant increase in ammonia excretion was evidenced in 150 and 300 μg L⁻¹ of chlorpyrifos. The O:N ratio showed a decrease in chlorpyrifos and in 2500 μg L⁻¹ of endosulfan. This indicated a shift towards protein primary metabolism. An increment in the O:N ratio was observed in the lower endosulfan solutions. The relation oxygen:nitrogen showed a shift towards lipid and carbohydrate primary metabolism. This work indicated the complexity of the metabolism in the freshwater crab affected by xenobiotic elements.     

Nile tilapia (Oreochromis niloticus), liver morphology,CYP1A activity and thyroid hormones after Endosulfan dietary exposure

Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g⁻¹ of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g⁻¹ for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g⁻¹, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g⁻¹ of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant.     

Mining new resources for grape resistance against Botryosphaeriaceae: a focus on Vitis vinifera subsp. sylvestris

Botryosphaeria dieback is an important grapevine trunk disease with global impact. Susceptibility differences between grape varieties manifest as different expression of canopy symptoms in the field. However, the cause of these dieback symptoms and their relation with wood necrosis remain only partially understood. As a first step towards future strategies for resistance breeding, wood necrosis was investigated over a large selection of the Vitaceae family members following artificial inoculation of the Botryosphaeriaceae fungi Neofusicoccum parvum and Diplodia seriata into woody internodes. Large variation of resistance levels was found, with good performance in several accessions from V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. To get insight into the mechanisms of this apparent resistance, expression of defence genes was studied in V. vinifera cv. Chardonnay, Gewürztraminer and different V. vinifera subsp. sylvestris genotypes, in both green and necrotic areas of inoculated woods. Resistance to Botryosphaeriaceae in V. vinifera subsp. sylvestris correlated with earlier and higher induction of some defence genes, both in green and necrotic wood. Moreover, leaves of several V. vinifera subsp. sylvestris accessions were also less susceptible to necrosis induced by treatment with a culture filtrate of Botryosphaeriaceae, compared to commercial cultivars of V. vinifera. The results show that V. vinifera subsp. sylvestris provides interesting genetic resources for breeding new varieties with enhanced resistance to botryosphaeria dieback.