Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants.These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogenPhytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundantPex(Phytophthoraextracellular protein) cDNAs. Of these, 78 (55%)PexcDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein ofP. infestans. To identify which of thePexcDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using anAgrobacterium tumefaciensbinary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs,crn1andcrn2, encoding extracellular proteins that belong to a large and complex protein family inPhytophthora. Further characterization of thecrngenes indicated that they are both expressed inP. infestansduring colonization of the host plant tomato and thatcrn2induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.

To overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans-potato and -tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coveragethe P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pa...

In this study we investigated the genetic control of avirulence in the diploid oomycete pathogen Phytophthora infestans, the causal agent of late blight on potato. The dominant avirulence (Avr) genes matched six race-specific resistance genes introgressed in potato from a wild Solanum species. AFLP markers linked to Avr genes were selected by bulked segregant analysis and used to construct two high-density linkage maps, one containing Avr4 (located on linkage group A2-a) and the other containing a cluster of three tightly linked genes, Avr3, Avr10, and Avr11 (located on linkage group VIII). Bulked segregant analysis also resulted in a marker linked to Avr1 and this allowed positioning of Avr1 on linkage group IV. No bulked segregant analysis was performed for Avr2, but linkage to a set of random markers placed Avr2 on linkage group VI. Of the six Avr genes, five were located on the most distal part of the linkage group, possibly close to the telomere. The high-density mapping was initiated to facilitate future positional cloning of P. infestans Avr genes.

Hemp (Cannabis sativa L.) is a multipurpose plant attracting increasing interest as a source for the production of natural fibers, paper, bio-building material and food. In this research we studied the agronomical performance of Cannabis sativa cv. Eletta Campana irrigated with saline water. Under those conditions, we tested the effect of protein hydrolysate (PH) biostimulant application in overcoming and/or balancing deleterious salinity effects. The results of the diverse treatments were also investigated at the physiological level, focusing on photosynthesis by means of a chlorophyll a fluorescence technique, which give an insight into the plant primary photochemical reactions. Four salinity levels of the irrigation solution (fresh water–EC0, and NaCl solutions at EC 2.0, 4.0 or 6.0 dS m⁻¹, EC2, EC4 and EC6, respectively) were combined with 2 biostimulant treatments (untreated (control) or treated with a commercial legume-derived protein hydrolysate (LDPH)). The increasing salinity affected plant photochemistry resulting in lower plant growth and seed production, while the LDPH biostimulant showed a protective effect, which improved crop performance both in control and in salinity conditions. The LDPH treatment improved seeds yield (+38.6% on average of all treated plants respect to untreated plants), as well as residual biomass, relevant in fiber production.

The use of beneficial microbes as inoculants able to improve fitness, growth and health of plants also in stress conditions is an attractive low-cost and eco-friendly alternative strategy to harmful chemical inputs. Thirteen potential plant growth-promoting bacteria were isolated from the rhizosphere of wheat plants cultivated under drought stress and nitrogen deficiency. Among these, the two isolates TL8 and TL13 showed multiple plant growth promotion activities as production of indole-3-acetic acid (IAA), siderophores, ammonia, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production, the ability to solubilize phosphate as well as exerted antimicrobial activity against plant pathogens as Botrytis spp. and Phytophthora spp. The two selected strains were identified as Kosakonia pseudosacchari by sequencing of 16S rRNA gene. They resulted also tolerant to abiotic stress and were able to efficiently colonize plant roots as observed in vitro assay under fluorescence microscope. Based on the best PGP properties, the strain K. pseudosacchari TL13 was selected to develop a new microbial based formulate. A sustainable and environmentally friendly process for inoculant production was developed using agro-industrial by-products for microbial growth. Moreover, the application of K. pseudosacchari TL13- based formulates in pot experiment improved growth performance of maize plants.