Modelling of Reactive Black 5 decolourization in the presence of heavy metals by the newly isolated Pseudomonas aeruginosa strain Gb30.

AIM: The effect of heavy metals presence on the decolourization of Reactive Black 5 by Pseudomonas aeruginosa was evaluated. METHODS AND RESULTS: In the current study, a newly isolated strain identified as P. aeruginosa strain Gb 30 was selected for its ability to remove high concentration of Reactive Black 5 and resistance to several heavy metals (Cu2+ >Zn2+ >Cd2+ >Cr6+ ). Strain Gb30 was used to assess the effect of heavy metals presence on RB5 decolourization. The strain growth exhibited different responses at a fixed concentration of EC50 (10 h) for each heavy metal. The addition of Zn2+ and Cd2+ had no effect on decolourization yield after 24 h of incubation, whereas Cr6+ and Cu2+ ions reduced decolourization up to 17%. In order to understand the relationship between heavy metals contamination and decolourization, experimental data relating the initial decolourization rate of RB5 to the concentrations of single and associated heavy metals were fitted to three different inhibition kinetic models. CONCLUSIONS: In this study, we showed that P. aeruginosa strain Gb30 could be used for dye removal even at high concentrations of heavy metals. The developed models could provide basic information that may help for the best management of the bacteria-mediated decolourization process at the industrial scale. SIGNIFICANCE AND IMPACT OF THE STUDY: This study opens new directions for the management of textile industry wastewaters containing dyes and heavy metals using bioaugmentation by P. aeruginosa strain Gb30.

First Report of Root Rot Caused by Pythium spiculum Affecting Cork Oaks at Doñana Biological Reserve in Spain.

Cork oaks (Quercus suber L.) are key tree species at Doñana Biological Reserve (DBR), Huelva, Spain. Sampling was conducted on a total of 13 trees exhibiting symptoms of decline (foliar wilting and defoliation, branch dieback, and root necrosis). In 2008. Phytophthora cinnamomi was isolated from feeder roots of one tree and Pythium spiculum from two additional oaks. In 2011, both pathogens were isolated from six oaks, only P. cinnamomi from three oaks, and only Py. spiculum from one oak. This expansion was associated with high winter rainfall levels since 2009 that led to long periods of soil flooding. While P. cinnamomi is well known to cause a root disease on Q. suber (2), P. spiculum is a newly described species isolated from Quercus, Vitis, Prunus, Castanea, and Celtis species, but its pathogenicity was demonstrated only on Q. ilex (syn. Q. rotundifolia) (1). Pathogenicity tests were conducted on 4-year-old Q. suber plants. Inocula consisted of two isolates of Py. spiculum from DBR (DO8 and DO36 from Q. suber). For comparison with these, three isolates previously tested on Q. ilex (1) were included: two isolates of Py. spiculum, PA54 (from Q. suber) and PE156 (from Q. ilex); and one isolate of P. cinnamomi, PE90 (from Q. ilex). All these isolates came from the Andalucía region, stored at the oomycete collection of the University of Córdoba, and showed a 99 to 100% homology with their expected ITS sequences in GenBank (DQ196131 for Py. spiculum and AY943301 for P. cinnamomi). Inoculum was prepared by shaking and mixing propagule-bearing mycelium produced in carrot broth petri dishes (20°C, 4 weeks) in sterile water, to produce a concentration of 3 × 104 oospores × ml-1 (Py. spiculum) or 3 × 104 chlamydospores × ml-1 (P. cinnamomi). One hundred milliliters of inoculum was applied to each root (1). There were 10 inoculated plants per isolate and 10 non-inoculated control plants. All plants were waterlogged 2 days per week to favor root infection and maintained in an acclimatised greenhouse (12-28°C). Three months later, the inoculated plants showed symptoms of root necrosis that resulted in foliar wilting followed occasionally by defoliation. Control plants did not develop foliar symptoms nor root necrosis. Root damage severity assessed on a 0 to 4 scale (3) exhibited significant differences (P < 0.05) in relation to the control plants for all the isolates tested, with isolate PE90 (P. cinnamomi) and isolates PA54, DO8, and DO36 (P. spiculum) all averaging a root necrosis value of 2.5. Isolate PE156 of P. spiculum produced values of root necrosis (1.6 in average) significantly lower (P < 0.05) than the rest. This isolate belongs to the low virulence group of P. spiculum described on Q. ilex (1). The inoculated oomycete was always reisolated from necrotic roots and never from roots of control plants. To the best of our knowledge, this is the first report of P. spiculum as the cause of root rot of Q. suber. References: (1) Romero et al. J. Phytopathol. 155:289, 2007. (2) Sánchez et al. For. Pathol. 32:5, 2002. (3) Sánchez et al. For. Pathol. 35:115, 2005.

Culture of Staphylococcus xylosus in fish processing by-product-based media for lipase production.

AIMS: The objective of this study was to demonstrate that fish-processing by-products could be used as sole raw material to sustain the growth of Staphylococcus xylosus for lipase production. METHODS AND RESULTS: Bacterial growth was tested on supernatants generated by boiling (100 degrees C for 20 min) of tuna, sardine, cuttlefish and shrimp by-products from fish processing industries. Among all samples tested, only supernatants generated from shrimp and cuttlefish by-products sustained the growth of S. xylosus. Shrimp-based medium gave the highest growth (A(600) = 22) after 22 h of culture and exhibited the maximum lipase activity (28 U ml(-1)). This effect may be explained by better availability of nutrients, especially, in shrimp by-products. Standard medium (SM) amendments to sardine and tuna by-product-based media stimulated the growth of S. xylosus and the highest A(600) values were obtained with 75% SM. Lipase activity, however, remained below 4 U ml(-1) for both sardine and tuna by-product-based media. CONCLUSIONS: Fish by-products could be used for the production of highly valuable enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of fish by-products in producing S. xylosus-growth media can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of biomass and enzyme production.

First Report of Pythium intermedium Causing Root Rot on Rosa canina Rootstock in France.

Old cultivars of roses (Rosa spp.) established on Rosa canina as rootstock are grown in field collections in the repository at Gaillard in eastern France, close to the Swiss border. During June 2005 and July 2006, several rose plants in full vegetation were severely wilted, leading ultimately to plant death. Necrotic lesions were present on the roots, and marginal tissue was excised and incubated in the dark at 20°C on PARP V8 agar plates (1). Similar isolates were obtained in both years, which were inoculated within 4 days on V8 agar medium and incubated at 25°C. The pathogen colonies grew approximately 30 mm daily on V8 agar. Colonies on corn meal agar (CMA) (1) were submerged, with or without a vague radiate pattern on potato carrot agar. Main hyphae were as much as 7 µm wide. Sporangia and zoospores were not produced. Hyphal swellings as much as 25 µm in diameter were abundant, terminal, and sometimes intercalary, often forming regular, dense chains in basipetal succession at hyphal tips and readily liberated. The morphological characters matched those described for Pythium intermedium (2). DNA sequencing of the ITS rDNA (ITS1, ITS2, and 5.8S rDNA) was generated using primers ITS5 and ITS4 in primary PCR (35 cycles: 96°C for 1 min, 55°C for 1 min, and 72°C for 2 min) and sequencing reactions (1). All isolates yielded identical ITS sequences, which were identical to the ex-type strain of P. intermedium, CBS 266.38 (GenBank Accession No. AY598647). The ITS sequence was deposited as GenBank Accession No. EF078693 (National Center for Biotechnology Information, NCBI, Bethesda, MD). Pathogenicity testing was performed on 3-year-old Rosa canina rootstocks from clonal propagation in 25 cm in diameter and 40 cm deep pots of compost 297 (Ricoter, Aarberg, Switzerland). Five-day-old mycelial plugs of our isolates grown on CMA (1) were inoculated in two pots, and roots were mechanically wounded. Sterile plugs were used on two control plants set aside. All plants were kept for 2 weeks at 20°C in a glasshouse with sufficient watering. Extensive necrotic lesions developed on inoculated roots, and severe wilting and leaves drying out were also observed. The controls showed no symptoms. P. intermedium was reisolated from margins of root lesions, thus fulfilling the postulates of Koch. P. intermedium was originally isolated from dead plant material (2) but is known as a typical soil inhabitant. It also has been isolated from water and living material from numerous plant species, but never from the genus Rosa until now. To our knowledge, this is the first record of P. intermedium on Rosa spp. grafted on Rosa canina in France. References: (1) L. Belbahri et al. FEMS Microbiol Lett 255:209, 2006. (2) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

First Report of Downy Mildew on Greenhouse and Landscape Coleus Caused by a Peronospora sp. in Louisiana and New York.

In May 2005, two commercial greenhouse flower growers, one in Louisiana (LA) and one in New York (NY), submitted coleus, Solenostemon scutellarioides (L.) Codd, plants for diagnosis after observing stunted growth, inward curling and twisting of leaves, and leaf abscission on multiple cultivars. Downy mildew-like growth was observable with hand lens or a microscope on the abaxial leaf surfaces of affected plants. Irregular necrotic spotting was present on some, but not all, plants on which sporulation was evident. Microscopic examination of LA material led to tentative identification of the pathogen as Peronospora lamii A. Braun (2). The pale brown conidia ranged from 17 to 26 × 15 to 26 µm (average 23 × 19 µm). Conidiophores ranged from 345 to 561 × 9 to 15 µm. No oospores were found. Additional coleus plants with downy mildew were subsequently found in three retail nurseries in LA in early summer. In NY, infected coleus plants were observed in landscapes in Farmington, Rochester, Ithaca, and in two commercial greenhouses between August and October 2005. NY samples sent to the USDA/APHIS in Beltsville, MD were examined, and the fungus was found to have morphology consistent with P. lamii. Two pathogenicity trials were conducted in NY. Conidia were rubbed from an infected coleus leaf onto the leaves of six healthy potted coleus plants of five cultivars and two basil plants that were placed in a shaded plastic tent in the greenhouse where temperatures ranged from 17 to 22°C. A household humidifier was used to supply mist inside the tent for 5 h per day. Six noninoculated plants of each coleus cultivar and two basil plants, placed in the same environment, served as controls. Downy mildew sporulation and some curling and twisting of leaves were observed 14 days after inoculation on all inoculated plants for three of the five cultivars (Florida Rustic Orange, Aurora Peach, and Aurora Mocha). Cvs. Florida Sun Rose and Lava showed no symptoms or signs of downy mildew. An irregularly shaped brown lesion developed on one inoculated basil leaf, and downy mildew sporulation was evident on the abaxial surface 35 days after inoculation. All noninoculated control plants remained disease free. In a second trial, conidia were rinsed from infected coleus leaves and sprayed onto the abaxial leaf surfaces of three coleus cv. Aurora Mocha plants. Three noninoculated plants served as controls and all were placed in a humidity tent. Leaf twisting and downy mildew sporulation were observed 13 days later on all inoculated plants, and control plants showed no sporulation or symptoms. A downy mildew causing disease of greenhouse-grown basil in Europe, originally identified as P. lamii on the basis of morphology, has recently been reported to be taxonomically distinguishable from P. lamii when tested by molecular methods (1). ITS sequences of coleus downy mildew from NY and LA were nearly identical (99% homology) to those of basil downy mildew from Switzerland and Italy (1). To our knowledge, this is the first report of downy mildew occurrence on coleus. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) S. M. Francis. Peronospora lamii. Descriptions of Pathogenic Fungi and Bacteria. No. 688. CMI, Kew, England, 1981.

A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant-pathogen interaction incompatible.

Plants activate disease resistance responses when they recognize pathogen-derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro-organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen-inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1-PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.

Characterization of an Arabidopsis-Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling.

Arabidopsis accessions were screened with isolates of Phytophthora porri originally isolated from other crucifer species. The described Arabidopsis-Phytophthora pathosystem shows the characteristics of a facultative biotrophic interaction similar to that seen in agronomically important diseases caused by Phytophthora species. In susceptible accessions, extensive colonization of the host tissue occurred and sexual and asexual spores were formed. In incompatible combinations, the plants reacted with a hypersensitive response (HR) and the formation of papillae at the sites of attempted penetration. Defence pathway mutants such as jar1 (jasmonic acid-insensitive), etr1 (ethylene receptor mutant) and ein2 (ethylene-insensitive) remained resistant towards P. porri. However, pad2, a mutant with reduced production of the phytoalexin camalexin, was hyper-susceptible. The accumulation of salicylic acid (SA) and PR1 protein was strongly reduced in pad2. Surprisingly, this lack of SA accumulation does not appear to be the cause of the hyper-susceptibility because interference with SA signalling in nahG plants or sid2 or npr1 mutants had only a minor effect on resistance. In addition, the functional SA analogue benzothiadiazol (BTH) did not induce resistance in susceptible plants including pad2. Similarly, the complete blockage of camalexin biosynthesis in pad3 did not cause susceptibility. Resistance of Arabidopsis against P. porri appears to depend on unknown defence mechanisms that are under the control of PAD2.

Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA.

With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).

Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis.

Transformed callus cultures of Nicotiana tabacum were generated in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the tobacco callus cultures. Three distinct phenotypic classes were identified among the transgenic cell lines in relation to growth of the cells, structure of the calli, and level of SAM. Transgene silencing was observed in several cultivated transgenic calli and this phenomenon was correlated directly with a low level of SAM-1 mRNA accompanied by a decrease of the SAM-S activity. The transgenic calli overexpressing the SAM-1 gene accumulated a high SAM level. The modifications in SAM-S activity were reflected in the pattern of secondary products present in the different cell lines, thereby demonstrating that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering.