Impact of winter pruning of pomegranate trees on Aphis punicae (Hemiptera,Aphididae) and its natural enemies in Tunisia

Winter pruning is a cultural practice used to modify vegetative growth, which is likely to affect the development of pests. However, it has been poorly defined as a cultural method for diminishing the population levels of the pomegranate aphid Aphis punicae (Hemiptera, Aphididae) in pomegranate orchards. This study aimed to evaluate the impact of winter pruning of pomegranate on A. punicae population and their natural enemies. The results showed that winter pruning significantly decreased the population of A. punicae (p < 0.05) in pomegranate. Also, number of natural enemies decreased in pomegranate trees after winter pruning. This technical practice affects the number of winged and wingless individuals, the mean relative growth rate, the specific age of the population and the infestation rate. Winter pruning for A. punicae control in pomegranate orchards is highly recommended.     

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10⁻² to 1.4 × 10⁻² pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.     

Ca2+-dependent lipid binding and membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in tobacco

PopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipid-binding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca2+ for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion-conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.     

Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: <Emphasis Type="Italic">Pediococcus parvulus</Emphasis> and <Emphasis Type="Italic">P. damnosus</Emphasis>

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.     

Evolution of the cutinase gene family: evidence for lateral gene transfer of a candidate Phytophthora virulence factor

Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases -- well documented virulence factors in fungi -- between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.     

Malachite green decolourization and detoxification by the laccase from a newly isolated strain of Trametes sp.

The decolourization and detoxification of the triarylmethane dye Malachite green (MG) by laccase from Trametes sp. were investigated. The laccase decolorized efficiently the dye down to 97% of 50 mg L^?1 initial concentration of MG when only 0.1 U mL^?1 of laccase was used in the reaction mixture. The effects of different physicochemical parameters were tested and optimal decolourization rates occurred at pH 6 and at temperatures between 50 and 60 °C. Decolourization of MG occurred in the presence of metal ions which could be found in textile industry effluent. 1-hydroxybenzotriazole (HBT) affected positively the decolourization of MG. The presence of some phenolic compounds namely ferulic, coumaric, gallic, and tannic acids was found to be inhibiting for the decolourization at a concentration of 10 mM.The effect of laccase inhibitors in the decolourization of MG was tested with l-cysteine, and ethylene diamine tetra-acetic acid (EDTA) at concentrations of 0.1, 1 and 10 mM. It was demonstrated that l-cysteine and EDTA inhibited the decolourization starting from 1 mM concentration. However, for NaCl a concentration of 100 mM was needed for the inhibition of laccase. The decolourization of MG resulted in the removal of its toxicity against Phanerochaete chrysosporium.The stability of the laccase toward temperature and HBT free radicals was also assessed during MG decolourization. It was shown that laccase was stable at 50 °C but in the presence of the laccase mediator HBT, the stability of the enzyme was severely affected resulting in a loss of 50% of the activity after 3 h incubation.     

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.