ReDivia - Repositorio Digital de l'Instit Valencià d'Investigacions Agràries
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Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime
Get PDFP>Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs
Extracellular proteases inhibit protein-dependent biofilm formation in Staphylococcus aureus
No full textIn Staphylococcus aureus, biofilm formation can be associated with the production of surface-anchored proteins, including Bap, SasG, FnBPs or Spa. By mutational analysis, and using a model strain in which biofilm formation was Bap-dependent, we found that sigma(B) was essential for protein-dependent biofilm development. Non-polar mutations of sigma(B) in genetically unrelated S. aureus strains lowered the Bap expression and completely impaired biofilm development. Although Northern blot analysis showed a slight reduction in bap, transcription, we demonstrated that Aur and SspA, two proteases that are overexpressed in the sigB mutant strain and are capable of degrading Bap, inhibit biofilm formation. Interestingly, a double sigB-agr mutant, which showed a diminished capacity to express extracellular proteases, was able to restore biofilm formation. Since the vast majority of the S. aureus global regulators control the expression of the extracellular proteases, the results of this work demonstrate the existence of a new pathway controlling protein-mediated biofilm formation in which different global regulators modulate biofilm formation by controlling the expression of S. aureus extracellular proteases
Tracking medfly predation by the wolf spider, Pardosa cribata Simon, in citrus orchards using PCR-based gut-content analysis
No full textThe Mediterranean fruit fly, Ceratitis capitata (Wiedemann), which is often controlled chemically, is a major citrus pest in Spain; however, alternative biological control strategies such as those based on the conservation of polyphagous predators should be developed. The wolf spider, Pardosa cribata Simon, is an abundant predator found in citrus orchards in eastern Spain. In this study, we have evaluated polymerase chain reaction (PCR)-based techniques as a means of detecting C. capitata DNA remains in P. cribata specimens. To do so, two pairs of C. capitata species-specific primers were designed and tested. Primer specificity was tested on species closely related to C. capitata and with other pests and natural enemies present in citrus orchards. Medfly DNA was detectable in 100% of P. cribata from 0 to 12 h post ingestion for both primer pairs, decreasing to 37% at 96 h after prey ingestion for one pair of primers. DNA detectability half-lives were of 78.25 h and 78.08 h for each pair of primers but no statistical differences were found between them. Pardosa cribata specimens were field-collected daily after sterile C. capitata pupae had been deployed in the citrus orchard. Afterwards, the wolf spiders were analyzed and DNA remains of C. capitata were detected in 5% of them, with a peak of 15% coinciding with maximum C. capitata emergence. This study is the first to reveal the potential use of DNA markers to track medfly predation by P. cribata in citrus orchards and provides a new tool to estimate the potential role of this spider in biological-control conservation programs
Identification of a GCC transcription factor responding to fruit colour change events in citrus through the transcriptomic analyses of two mutants
Get PDFBackground: External ripening in Citrus fruits is morphologically characterized by a colour shift from green to orange due to the degradation of chlorophylls and the accumulation of carotenoid pigments. Although numerous genes coding for enzymes involved in such biochemical pathways have been identified, the molecular control of this process has been scarcely studied. In this work we used the Citrus clementina mutants 39B3 and 39E7, showing delayed colour break, to isolate genes potentially related to the regulation of peel ripening and its physiological or biochemical effects. Results: Pigment analyses revealed different profiles of carotenoid and chlorophyll modification in 39B3 and 39E7 mutants. Flavedo from 39B3 fruits showed an overall delay in carotenoid accumulation and chlorophyll degradation, while the flavedo of 39E7 was devoid of the apocarotenoid beta-citraurin among other carotenoid alterations. A Citrus microarray containing about 20,000 cDNA fragments was used to identify genes that were differentially expressed during colour change in the flavedo of 39B3 and 39E7 mutants respect to the parental variety. The results highlighted 73 and 90 genes that were respectively up-and down-regulated in both mutants. CcGCC1 gene, coding for a GCC type transcriptional factor, was found to be down-regulated. CcGCC1 expression was strongly induced at the onset of colour change in the flavedo of parental clementine fruit. Moreover, treatment of fruits with gibberellins, a retardant of external ripening, delayed both colour break and CcGCC1 overexpression. Conclusions: In this work, the citrus fruit ripening mutants 39B3 and 39E7 have been characterized at the phenotypic, biochemical and transcriptomic level. A defective synthesis of the apocarotenoid beta-citraurin has been proposed to cause the yellowish colour of fully ripe 39E7 flavedo. The analyses of the mutant transcriptomes revealed that colour change during peel ripening was strongly associated with a major mobilization of mineral elements and with other previously known metabolic and photosynthetic changes. The expression of CcGCC1 was associated with peel ripening since CcGCC1 down-regulation correlated with a delay in colour break induced by genetic, developmental and hormonal causes
Maximum diurnal trunk shrinkage is a sensitive indicator of plant water, stress in Diospyros kaki (Persimmon) trees
No full textPersimmon tree (Diospyros kaki Lf) is a deciduous fruit tree included in the so-called group of minor fruit tree species Worldwide It is not widely grown but nowadays Kaki culture is of some importance in the south-east of Spain because of the high fruit commercial value Currently neither it is known about Kaki trees water needs nor crop responses to the irrigation regime The objective of the present research was to assess the feasibility of using maximum diurnal trunk shrinkage (MDS) as a plant water stress indicator for Kaki trees During two drought cycles in trees under either full or deficit irrigation the MDS obtained by means of LVDT sensors was compared with a reference indicator of fruit trees water status the midday stem water potential (Psi(stern)) In addition stomatal conductance and fruit diameter variations were also. followed As water restrictions began there was an immediate increase in MDS in correspondence with a decrease in Psi(stern) Pooling data from both drought cycles and irrigation regimes MDS and Psi(stern) were linearly correlated (r(2) = 0 77***) The magnitude of differences between well watered and deficit irrigated trees was much larger in the case of MDS than for Psi(stern) However the tree-to-tree variability of the MDS readings was three times higher than for Psi(stern) average coefficient of variation of 14% and 38% for 'Poem and MDS respectively Overall results reported indicated that MDS is a sensitive indicator of Kaki water status and it can be further used as an irrigation scheduling indicator for optimum irrigation management of this crop However the large MDS tree-to-tree variability should be taken into account when selecting the number of trees to monitor within an orchard
Natural mortality of the California red scakem 'aonidiella aurantii' in citrus of eastern Spain and its natural enemies associated
No full textThe inclusion of ensiled citrus pulp in diets for growing pigs: Effects on voluntary intake, growth performance, gut microbiology and meat quality
No full textCitrus pulp was evaluated as an alternative feedstuff in diets for growing-finishing pigs. Three diets were formulated on an iso-nutrient basis to contain 0 (C), 50 (PM) or 100 (PH) g of ensiled citrus pulp per kg on a dry matter basis (DM). At 56.7 ± 7.1 kg of body weight, 72 pigs were allocated into 3 groups according to the citrus pulp level in the diet. Body weight and voluntary feed intake (VFI) were recorded on days 0, 29 and 55. Backfat thickness (BF) and loin depth (LD) were measured at P2 using ultrasounds. Faecal samples were taken directly from the rectum and cultured for Enterobacteriaceae and Lactobacillus counts. At slaughter, carcass weight, BF at gluteus medius and meat colour was recorded. Also fatty acid profile in subcutaneous fat was determined. Pigs fed citrus pulp showed a lower (P < 0.05) VFI on a DM basis and, consequently, a lower (P < 0.05) weight gain (ADG) compared to C pigs during the first 4 weeks of study. Thereafter, no differences were found between groups. At the end of the study, C pigs showed a greater level of BF at P2 than PM and PH. The inclusion of citrus pulp significantly reduced (P < 0.05) Enterobacteriaceae in faeces, not affecting Lactobacillus population. The inclusion of citrus pulp lead to no differences in BF at gluteus medius and meat colour but tended (P < 0.10) to decrease carcass yield and increase oleic acid percentage in subcutaneous fat. It was concluded that growing pigs can adapt their gastrointestinal tract to citrus pulp as a source of high-fermentable carbohydrates without detrimental effects on growth performance and meat quality, and potential benefits on gut microbiology
Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance
No full textCitrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies
Cyt1Aa protein from Bacillus thuringiensis (Berliner) serovar israelensis is active against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann)
No full textBACKGROUND: Ceratitis capitata (Wiedemann) is one of the world's most destructive fruit pests. The aim of this study was to ascertain insecticidal activity of Bacillus thuringiensis (Berliner) delta-endotoxins to C. capitata. RESULTS: Among 42 selected Bacillus strains, only B. thuringiensis serovar israelensis (Bti) solubilised protoxins showed biological activity against C. capitata neonate larvae, whereas Bti spore and crystal mixture was inactive. Insecticidal activity of Bti protoxins was significantly enhanced by incubation with Culex pipiens L. gut extracts. Overdigestion of Bti protoxins with Sesamia nonagrioides (Lefebvre) gut extracts suppressed biological activity against C. capitata, and this correlated with degradation of Cyt toxins. Cyt1Aa solubilised protoxin showed the highest toxicity, LC50 after 7 days of 4.93 mu g cm(-2), while proteolytical processing of Cyt1Aa protoxins by larval gut extracts did not enhance insecticidal activity. CONCLUSION: The present study provides evidence for the first time of the insecticidal activity of a B. thuringiensis strain against C. capitata and identifies a single delta-endotoxin with potential for controlling this pest. (C) 2010 Society of Chemical Industr