High Temperature Enhances the Resistance of Cultivated African Rice, Oryza glaberrima, to Bacterial Blight.

Rice bacterial blight (BB) is caused by Xanthomonas oryzae pv. oryzae and is responsible for substantial yield loss worldwide. Host resistance remains the most feasible control measure. However, pathogen variability leads to the failure of certain resistance genes to control the disease, and climate change with high amplitudes of heat predisposes the host plant to pathogen invasion. Due to pressure in natural selection, landrace species often carry a wide range of unique traits conferring tolerance of stress. Therefore, exploring their genetic background for host resistance could enable the identification of broad-spectrum resistance to combined abiotic and biotic stresses. Nineteen Oryza glaberrima accessions and O. sativa rice variety SUPA were evaluated for BB resistance under high temperature (35 and 31°C day and night, respectively) using 14 X. oryzae pv. oryzae strains originated from the Philippines. Under normal temperature, most of the accessions showed resistance to 9 strains (64.3%) and accession TOG6007 showed broad-spectrum resistance to 12 strains (85.7%). Under high temperature, most accessions showed a reduction in BB disease, whereas, accession TOG5620 showed disease reduction from all the X. oryzae pv. oryzae strains under high temperature. Molecular characterization using gene-based and linked markers for BB resistance genes Xa4, xa5, Xa7, xa13, and Xa21 revealed the susceptible alleles of Xa4, xa5, xa13, and Xa21 in O. glaberrima. However, no allele of Xa7 was detected among O. glaberrima accessions. Our results suggest that O. glaberrima accessions contain a BB resistance different from the Xa gene type. Genome-wide association mapping could be used to identify quantitative trait loci that are associated with BB resistance or combined BB resistance and high-temperature tolerance.

Population Structure, Pathogenicity, and Mating Type Distribution of Magnaporthe oryzae Isolates from East Africa.

Rice blast, caused by Magnaporthe oryzae, is one of the emergent threats to rice production in East Africa (EA), where little is known about the population genetics and pathogenicity of this pathogen. We investigated the genetic diversity and mating type (MAT) distribution of 88 isolates of M. oryzae from EA and representative isolates from West Africa (WA) and the Philippines (Asia) using amplified fragment length polymorphism markers and mating-type-specific primer sets. In addition, the aggressiveness of each isolate was evaluated by inoculating on the susceptible Oryza sativa indica 'Co39', scoring the disease severity and calculating the disease progress. Hierarchical analysis of molecular variance revealed a low level of genetic differentiation at two levels (FST 0.12 and FCT 0.11). No evidence of population structure was found among the 65 isolates from EA, and gene flow among EA populations was high. Moreover, pairwise population differentiation (GST) in EA populations ranged from 0.03 to 0.04, suggesting that >96% of genetic variation is derived from within populations. However, the populations from Asia and WA were moderately differentiated from EA ones. The spatial analysis of principal coordinates and STRUCTURE revealed overlapping between individual M. oryzae isolates from EA, with limited distinctness according to the geographic origin. All the populations were clonal, given the positive and significant index of association (IA) and standardized index of association (rd), which indicates a significant (P<0.001) departure from panmixia (IA and rd=0). Both MAT1-1 and MAT1-2 were detected. However, MAT1-1 was more prevalent than MAT1-2. Pathogenicity analysis revealed variability in aggressiveness, suggesting a potential existence of different races. Our data suggest that either M. oryzae populations from EA could be distributed as a single genetic population or gene flow is exerting a significant influence, effectively swamping the action of selection. This is the first study of genetic differentiation of rice-infecting M. oryzae strains from EA, and may guide further studies on the pathogen as well as resistance breeding efforts.

Pectobacterium aroidearum sp. nov., a soft rot pathogen with preference for monocotyledonous plants.

Several pectolytic bacterial strains, mainly isolated from monocotyledonous plants and previously identified as Pectobacterium carotovorum, were thought to belong to a novel species after several taxonomic analyses including DNA-DNA hybridization. In 16S rRNA gene sequence analyses, these strains had a similarity of >97.9 % to the 16S rRNA gene sequence of strains representing six other pectobacterial species and subspecies. These strains, represented by strain SCRI 109(T), also showed some unique chemotaxonomic features and quantitative differences in polar lipids, lipoquinones and fatty acids. A specific feature of strain SCRI 109(T) was the presence of DMK-8 lipoquinone, while the dominant fatty acids were the summed feature 3 (iso-C15 : 0 2-OH/C16 : 1ω7c), the unsaturated fatty acid C18 : 1ω7c and straight chain fatty acids, mainly C16 : 0. The DNA G+C content of strain SCRI 109(T) was 50.2 mol%. The taxonomic status of strain SCRI 109(T) and related strains in 16S rRNA gene sequence, chemotaxonomic, and physiological analyses was corroborated by the distinct clustering of these strains in multi-locus sequence analyses. It is proposed that these strains represent a novel species for which the name Pectobacterium aroidearum sp. nov. is proposed; the type strain is SCRI 109(T) ( = NCPPB 929(T) = LMG 2417(T) = ICMP 1522(T)).

Analysis of cell wall proteins regulated in stem of susceptible and resistant tomato species after inoculation with Ralstonia solanacearum: a proteomic approach.

Proteomics approach was used to elucidate the molecular interactions taking place at the stem cell wall level when tomato species were inoculated with Ralstonia solanacearum, a causative agent of bacterial wilt. Cell wall proteins from both resistant and susceptible plants before and after the bacterial inoculation were extracted from purified cell wall with salt buffers and separated with 2-D IEF/SDS-PAGE and with 3-D IEF/SDS/SDS-PAGE for basic proteins. The gels stained with colloidal Coomassie revealed varied abundance of protein spots between two species (eight proteins in higher abundance in resistant and six other in susceptible). Moreover, proteins were regulated differentially in response to bacterial inoculation in resistant (seven proteins increased and eight other decreased) as well as in susceptible plants (five proteins elevated and eight other suppressed). Combination of MALDI-TOF/TOF MS and LC-ESI-IonTrap MS/MS lead to the identification of those proteins. Plants responded to pathogen inoculation by elevating the expression of pathogenesis related, other defense related and glycolytic proteins in both species. However, cell wall metabolic proteins in susceptible, and antioxidant, stress related as well as energy metabolism proteins in resistant lines were suppressed. Most of the proteins of the comparative analysis and other randomly picked spots were predicted to have secretion signals except some classical cytosolic proteins.

Rice pyramided line IRBB67 (Xa4/Xa7) homeostasis under combined stress of high temperature and bacterial blight.

Rice bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) implies substantial yield loss to rice. In times of climate change, increasing temperatures are observed and further acceleration is expected worldwide. Increasing temperature often turns into inhibition of host plant defense to pathogens. Recently, a reduced resistance in rice IRBB4 carrying Xa4, but an increase in resistance in IRBB7 carrying Xa7 resistance by increasing temperature has been reported. Influence of high temperature on both R genes (Xa4+Xa7) combined in IRBB67 was analyzed under growth chamber conditions and transcriptomic analysis performed. The pyramided line IRBB67 showed no differences in lesion length between both temperature regimes, demonstrating that non-effectiveness of Xa4 at high temperature did not affect IRBB67 resistance. Moreover, Xa4 complements Xa7 resistance with no Xoo spread in planta beyond the symptomatic area under both temperature regimes in IRBB67. Time course transcriptomic analysis revealed that temperature enhanced IRBB67 resistance to combined heat and Xoo. Our findings highlight altered cellular compartments and point at a role of the cell wall involved in Xoo resistance and heat stress tolerance in both susceptible (IR24) and the resistant (IRBB67) NILs. Interestingly, up-regulation of trehalose-6-phosphatase gene and low affinity cation transporter in IRBB67 suggest that IRBB67 maintained a certain homeostasis under high temperature which may have enhanced its resistance. The interplay of both heat stress and Xoo responses as determined by up-regulated and down-regulated genes demonstrates how resistant plants cope with combined biotic and abiotic stresses.

Structure of the oligosaccharide chain of the SR-type lipopolysaccharide of Ralstonia solanacearum Toudk-2.

Aiming at improving classification and taxonomy of Gram-negative phytopathogenic bacteria, we studied the structure of the lipopolysaccharide of Ralstonia solanacearum. Mild acid hydrolysis of the lipopolysaccharide of strain Toudk-2 followed by gel chromatography resulted in an O-polysaccharide and two oligosaccharide fractions. The smallest-size oligosaccharide fraction was studied by sugar analysis, high-resolution electrospray ionization mass spectrometry, and, after fractionation by anion-exchange chromatography on HiTrap Q, by one- and two-dimensional (1)H and (13)C NMR spectroscopy. It was found that the isolated oligosaccharides consist of the lipopolysaccharide core with one O-polysaccharide repeat (O-unit) attached. The core exists in two major glycoforms differing from each other in a lateral octulosonic acid residue, which is either D-glycero-D-talo-oct-2-ulosonic acid or 3-deoxy-D-manno-oct-2-ulosonic acid. A peculiar feature of the core is the occurrence of 4-amino-4-deoxy-L-arabinose nonstoichiometrically linked to a heptose residue. The full structures of the core and the biological O-unit as well as the site of the attachment of the O-unit to the core were established.

Elevated temperature increases in planta expression levels of virulence related genes in Magnaporthe oryzae and compromises resistance in Oryza sativa cv. Nipponbare.

Temperature changes have the potential to alter the incidence and severity of plant disease epidemics and pressures, as well as to reshape the co-evolutionary relationships between plants and pathogens. However, the molecular basis of temperature modulation of pathogenicity of plant pathogens is still unclear. Here, we studied the effect of temperature on biomass of Magnaporthe oryzae in planta using qPCR. Additionally, the transcriptomes of M. oryzae and rice were analysed using RNA-seq. Rice seedlings were exposed to 35°C and 28°C for 7 days before pathogen inoculation. Inoculated plants were kept in the dark at 28°C for 24h and later re-exposed to 35°C and 28°C for an additional 24h before sample collection. Plants grown and predisposed to 35°C prior to inoculation exhibited accelerated tissue necrosis compared with plants grown and inoculated at 28°C. In accordance with the disease severity observed on infected leaves, in planta fungal biomass was significantly higher at 35°C than 28°C. Moreover, M. oryzae exhibited increased expression levels of putative fungal effector genes in plants exposed to 35°C compared with plants exposed to 28°C. Collectively, this study revealed that temperature elevation could favour M. oryzae infection by compromising plant resistance and accelerating plant tissue colonisation with the pathogen.

High temperature effects on Pi54 conferred resistance to Magnaporthe oryzae in two genetic backgrounds of Oryza sativa.

The global temperatures are predicted to rise due to climate change. However, knowledge on the mechanisms underlying the effect of high temperature (HT) on plant pathogen interaction is limited. We investigated the effect of elevated temperature on host phenotypic, biochemical and gene expression patterns in the rice-Magnaporthe oryzae (Mo) pathosystem using two genetic backgrounds, Co39 (Oryzae sativa-indica) and LTH (O. sativa-japonica) with (CO and LT) and without (Co39 and LTH) R gene (Pi54). After exposure to 28°C and 35°C the two genetic backgrounds showed contrasting responses to Mo. At 28°C, CO, Co39 and LTH displayed a more severe disease phenotype than LT. Surprisingly, CO became resistant to Mo after exposure to 35°C. CO and LT were used for further analysis to determine the defence related biochemical and transcriptome changes associated with HT induced resistance. Pre-exposure to 35°C triggered intense callose deposits and cell wall fluorescence of the attacked epidermal cells, as well as, increased hydrogen peroxide (H2O2) and salicylic acid (SA) levels. Transcriptional changes due to combined stress (35°C+Mo) were largely overridden by pathogen infection in both backgrounds, suggesting that the plants tended to shift their response to the pathogen. However, significant differences in global gene expression patterns occurred between CO and LT in response to both single (35°C and Mo) and double stress (35°C+Mo). Collectively, our results suggest that rice lines carrying Pi54 respond to Mo by rapid induction of callose and H2O2, and that these resistance mechanisms are amplified at HT. The relative difference in disease severity between CO and LT at 28°C suggests that the genetic background of japonica rice facilitates the function of Pi54 more than the background of indica rice. The phenotypic plasticity and gene expression differences between CO and LT reveal the presence of intricate background specific molecular signatures that may potentially influence adaptation to plant stresses.

Metallic iron for safe drinking water provision: Considering a lost knowledge.

Around year 1890, the technology of using metallic iron (Fe0) for safe drinking water provision was already established in Europe. The science and technology to manufacture suitable Fe0 materials were known and further developed in this period. Scientists had then developed skills to (i) explore the suitability of individual Fe0 materials (e.g. iron filling, sponge iron) for selected applications, and (ii) establish treatment processes for households and water treatment plants. The recent (1990) discovery of Fe0 as reactive agent for environmental remediation and water treatment has not yet considered this ancient knowledge. In the present work, some key aspects of the ancient knowledge are presented together with some contemporised interpretations, in an attempt to demonstrate the scientific truth contained therein. It appears that the ancient knowledge is an independent validation of the scientific concept that in water treatment (Fe0/H2O system) Fe0 materials are generators of contaminant collectors.

Structure of the O-polysaccharide of Xanthomonas cassavae GSPB 2437.

The following structure of the O-polysaccharide of the phytopathogenic bacterium Xanthomonas cassavae GSPB 2437 was determined by sugar analysis along with 1H and 13C NMR spectroscopy: [structure: see text].

Endo- and exopolygalacturonases of Ralstonia solanacearum are inhibited by polygalacturonase-inhibiting protein (PGIP) activity in tomato stem extracts.

Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the 's' into the 'rs' colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4-8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate-polyacrylamid-gel electrophoresis (FACE-PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction.

Pathogenesis and stress related, as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum.

A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650-690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.