Evaluation of Phoma sp. Biomass as an Endophytic Fungus for Synthesis of Extracellular Gold Nanoparticles with Antibacterial and Antifungal Properties.

The aim of our study was to examine the different concentrations of AuNPs as a new antimicrobial substance to control the pathogenic activity. The extracellular synthesis of AuNPs performed by using Phoma sp. as an endophytic fungus. Endophytic fungus was isolated from vascular tissue of peach trees (Prunus persica) from Baft, located in Kerman province, Iran. The UltraViolet-Visible Spectroscopy (UV-Vis spectroscopy) and Fourier transform infrared spectroscopy provided the absorbance peak at 526 nm, while the X-ray diffraction and transmission electron microscopy images released the formation of spherical AuNPs with sizes in the range of 10-100 nm. The findings of inhibition zone test of Au nanoparticles (AuNPs) showed a desirable antifungal and antibacterial activity against phytopathogens including Rhizoctonia solani AG1-IA (AG1-IA has been identified as the dominant anastomosis group) and Xanthomonas oryzae pv. oryzae. The highest inhibition level against sclerotia formation was 93% for AuNPs at a concentration of 80 µg/mL. Application of endophytic fungus biomass for synthesis of AuNPs is relatively inexpensive, single step and environmentally friendly. In vitro study of the antifungal activity of AuNPs at concentrations of 10, 20, 40 and 80 µg/mL was conducted against rice fungal pathogen R. solani to reduce sclerotia formation. The experimental data revealed that the Inhibition rate (RH) for sclerotia formation was (15, 33, 74 and 93%), respectively, for their corresponding AuNPs concentrations (10, 20, 40 and 80 µg/mL). Our findings obviously indicated that the RH strongly depend on AuNPs rates, and enhance upon an increase in AuNPs rates. The application of endophytic fungi biomass for green synthesis is our future goal.

The Green Synthesis of MgO Nano-Flowers Using Rosmarinus officinalis L. (Rosemary) and the Antibacterial Activities against Xanthomonas oryzae pv. oryzae.

Recently, the use of herbs in the agriculture and food industry has increased significantly. In particular, Rosmarinus officinalis L. extracts have been reported to have strong antibacterial properties, which depend on their chemical composition. The present study displayed a biological method for synthesis of magnesium oxide (MgO) nano-flowers. The nano-flowers are developed without using any catalyst agent. Aqueous Rosemary extract was used to synthesize MgO nano-flowers (MgONFs) in stirring conditions and temperature at 70°C for 4 h. The mixture solution was checked by UV-Vis spectrum to confirm the presence of nanoparticles. The MgO nano-flowers powder was further characterized in this study by the X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. In addition, bacteriological tests indicated that MgO nano-flowers significantly inhibited bacterial growth, biofilm formation, and motility of Xanthomonas oryzae pv. oryzae, which is the causal agent of bacterial blight disease in rice. The electronic microscopic observation showed that bacterial cell death may be mainly due to destroy of cell integrity, resulting in leakage of intracellular content. As recommended, the use of Rosemary extract is an effective and green way to produce the MgO nano-flowers, which can be widely used in agricultural fields to suppress bacterial infection.

Low-iron conditions induces the hypersensitive reaction and pathogenicity hrp genes expression in Xanthomonas and is involved in modulation of hypersensitive response and virulence.

Expression of hrp (hypersensitive reaction and pathogenicity) genes inside the host is crucial for virulence of phytopathogenic bacteria. The hrp genes encode components of type3 secretion system (T3SS), HR elicitors and several regulators, which are involved in the co-ordinated expression of hrp genes in the host environment and in hrp inducing chemically defined medium. However, little is known about specific host or environmental factors which may play a role in the induction of hrp gene expression. In this study, we show that iron-limiting condition elicits induced expression of hrp genes, including type3 secretion system (T3SS) and effectors (T3E). Expression analysis using qRT-PCR and promoter probe strains suggest significant induction in the expression of Hrp and T3S-associated genes of Xanthomonas campestris pv. campestris (Xcc) under low-iron condition, and is suppressed by exogenous supplementation of iron. Furthermore, we show that with exogenous iron supplementation, wild type Xcc exhibited reduced disease symptoms in host-plant, and exhibited significant reduction in HR and callose deposition in the non-host plants. Xanthomonas oryzae and oryzicola pathovars also exhibited the iron affect, albeit to a lesser extend compared with the Xcc. Overall, our results suggest that low-iron condition inside the host may play a crucial role in pathogenicity.

The Effect of Citrus Essential Oils and Their Constituents on Growth of Xanthomonas citri subsp. citri.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc), is the most devastating of the citrus diseases worldwide. During our study, we found that Essential oils (EOs) of some citrus cultivars are effective on Xcc. Therefore, it prompted us to determine the plant metabolites responsible for the antibacterial properties. We obtained EOs from some locally cultivated citrus by using a Clevenger apparatus and their major constituents were identified by gas chromatography/mass spectrometry (GC-MS). The effect of Citrus aurantium, C. aurantifolia, Fortunella sp. EOs and their major constituents were evaluated against Xcc-KVXCC1 using a disk diffusion assay. Minimal inhibitory and bactericidal concentration of the EOs and their constituents were determined using the broth microdilution method. C. aurantium, C. aurantifolia Eos, and their major constituents including citral, linalool, citronellal, geraniol, α-terpineol, and linalyl acetate indicated antibacterial effects against Xcc. The C. aurantifolia EO and citral showed the highest antibacterial activity among the tested EOs and constituents with inhibition zones of 15 ± 0.33 mm and 16.67 ± 0.88 mm, respectively. Synergistic effects of the constituents were observed between α-terpineol-citral, citral-citronellal, citral-geraniol, and citronellal-geraniol by using a microdilution checkerboard assay. Transmission electron microscopy revealed that exposure of Xcc cells to citral caused cell wall damage and altered cytoplasmic density. We introduced C. aurantifolia and C. aurantium EOs, and their constituents citral, α-terpineol, citronellal, geraniol, and linalool as possible control agents for CBC.

Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots.

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.

XocR, a LuxR solo required for virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, a serious bacterial disease of rice in Asia and parts of Africa. The virulence mechanisms of Xoc are not entirely clear and control measures for BLS are poorly developed. The solo LuxR proteins are widespread and shown to be involved in virulence in some plant associated bacteria (PAB). Here, we have cloned and characterized a PAB LuxR solo from Xoc, named as XocR. Mutation of xocR almost completely impaired the virulence ability of Xoc on host rice, but did not alter the ability to trigger HR (hypersensitive response, a programmed cell death) on non-host (plant) tobacco, suggesting the diversity of function of xocR in host and non-host plants. We also provide evidence to show that xocR is involved in the regulation of growth-independent cell motility in response to a yet-to-be-identified rice signal, as mutation of xocR impaired cell swimming motility of wild-type Rs105 in the presence but not absence of rice macerate. We further found that xocR regulated the transcription of two characterized virulence-associated genes (recN and trpE) in the presence of rice macerate. The promoter regions of recN and trpE possessed a potential binding motif (an imperfect pip box-like element) of XocR, raising the possibility that XocR might directly bind the promoter regions of these two genes to regulate their transcriptional activity. Our studies add a new member of PAB LuxR solos and also provide new insights into the role of PAB LuxR solo in the virulence of Xanthomonas species.

Cell-cell signalling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice.

Cell-cell communication mediated by diffusible signal factor (DSF) plays an important role in virulence of several Xanthomonas group of plant pathogens. In the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzicola, DSF is required for virulence and in planta growth. In order to understand the role of DSF in promoting in planta growth and virulence, we have characterized the DSF deficient mutant of X. oryzae pv. oryzicola. Mutant analysis by expression analysis, radiolabelled iron uptake studies and growth under low-iron conditions indicated that DSF positively regulates ferric iron uptake. Further, the DSF deficient mutant of X. oryzae pv. oryzicola exhibited a reduced capacity to use ferric form of iron for growth under low-iron conditions. Exogenous iron supplementation in the rice leaves rescued the in planta growth deficiency of the DSF deficient mutant. These data suggest that DSF promotes in planta growth of X. oryzae pv. oryzicola by positively regulating functions involved in ferric iron uptake which is important for its virulence. Our results also indicate that requirement of iron uptake strategies to utilize either Fe(3+) or Fe(2+) form of iron for colonization may vary substantially among closely related members of the Xanthomonas group of plant pathogens.

Ecological impact of solar ultraviolet-B (UV-B: 320-290 nm) radiation on Corynebacterium aquaticum and Xanthomonas sp. colonization on tea phyllosphere in relation to blister blight disease incidence in the field.

AIMS: To assess the effects of solar UV-B radiation on phyllosphere bacteria of tea leaves in relation to blister blight disease in the field. METHODS AND RESULTS: The effects of UV-B radiation on the phyllosphere microbiology of tea (Camellia sinensis) were studied in contrasting wet and dry seasons at a tropical site. Wavelength-selective filters were used to separate the effects of UV-B from those of other factors. Bacterial populations were quantified in relation to the incidence of blister blight disease. Attenuation of UV-B increased the survival of Xanthomonas sp. when populations were not water limited, and increased the incidence of blister blight, but had no effect on Corynebacterium aquaticum. CONCLUSIONS: The effects of solar UV-B on phyllosphere bacteria were substantial but depended on both species and interactions with other environmental variables. Xanthomonas sp. was more sensitive to UV-B than C. aquaticum, but this did not result in differences in population density under high radiation conditions (dry season), but only in the wet season when other factors were not limiting. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of UV-B on leaf surface microbiology in the tropics is marked but depends on other conditions, and the contrasting UV-B responses of different organisms can be masked by other limiting factors.

Growth deficiency of a Xanthomonas oryzae pv. oryzae fur mutant in rice leaves is rescued by ascorbic acid supplementation.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. A mutation was isolated in the ferric uptake regulator (fur) gene of X. oryzae pv. oryzae and it was shown to result in the production of siderophores in a constitutive manner. The fur mutant is hypersensitive to the metallo-antibiotic streptonigrin, a phenotype that is indicative of intracellular free-iron overload, and also exhibits a slow growth phenotype on rich medium. The fur mutant is virulence deficient, hypersensitive to hydrogen peroxide, and exhibits reduced catalase activity. Exogenous supplementation with ascorbic acid (an antioxidant) rescues the growth deficiency of the fur mutant in rice leaves. The virulence deficiency of the X. oryzae pv. oryzae fur mutant is proposed to be due, at least in part, to an impaired ability to cope with the oxidative stress conditions that are encountered during infection.

PhyA, a secreted protein of Xanthomonas oryzae pv. oryzae, is required for optimum virulence and growth on phytic acid as a sole phosphate source.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed beta-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.

Electron microscopic cytochemistry of polysaccharides in bacteria Xanthomonas fuscans, the cause of the bean fuscous blight.

High resolution cytochemical staining of polysaccharides in the cells of the bean fuscous blight bacteria Xanthomonas fuscans (Burkholder) Bur. revealed extensive deposits of a periodic acid-oxidizable compound in the cytoplasm, which accumulated at the onset of polysaccharide excretion and occupied a substantial part of the cell volume. In the course of subsequent culture growth the intracellular polysaccharides gradually disappeared and polysaccharide microfibrils were visualized in the medium. Our observations indicate that the exuded phytopathogenic polysaccharides may be synthetized in the cytoplasm of the bacteria.