Characterization of Pathogenic and Nonpathogenic Fusarium oxysporum Isolates Associated with Commercial Tomato Crops in the Andean Region of Colombia.

In Colombia, tomato production under protected conditions represents an important economic contribution to the agricultural sector. Fusarium wilt diseases, caused by pathogenic formae speciales of the soil-borne fungus Fusarium oxysporum Schltdl., cause significant yield losses in tomatoes throughout the world. Investigation of the F. oxysporum-tomato pathosystem in Colombia is required to develop appropriate alternative disease management. In this study, 120 fungal isolates were obtained from four different departments in the Central Andean Region in Colombia from tomato crops with symptoms of wilt disease. A molecular characterization of the fungal isolates was performed using the SIX1, SIX3, and SIX4 effector genes of Fusarium oxysporum f. sp. lycopersici W.C. Snyder & H.N. Hansen (Fol). Additionally, we developed a new specific marker to distinguish between Fusarium oxysporum f. sp. radicis-lycopersici Jarvis & Shoemaker (Forl) and Fol isolates. Furthermore, a phylogenetic analysis using the Translation Elongation Factor 1-alpha (EF1a) gene was performed with the collected isolates. Two isolates (named Fol59 and Fol-UDC10) were identified as Fol race 2, four isolates were identified as Forl, six isolates were identified as F. solani, and most of the isolates were grouped within the F. oxysporum species complex. The phylogenetic tree of EF1a showed that most of the isolates could potentially correspond to nonpathogenic strains of F. oxysporum. Additional pathogenicity assays carried out with Fol59 and Fol-UDC10 confirmed that both isolates were highly virulent strains. This study represents a contribution to the understanding of the local interaction between tomatoes and F. oxysporum in Colombia.

Putative Novel Effector Genes Revealed by the Genomic Analysis of the Phytopathogenic Fungus Fusarium oxysporum f. sp. physali (Foph) That Infects Cape Gooseberry Plants.

The vascular wilt disease caused by the fungus Fusarium oxysporum f. sp. physali (Foph) is one of the most limiting factors for the production and export of cape gooseberry (Physalis peruviana) in Colombia. A transcriptomic analysis of a highly virulent strain of F. oxysporum in cape gooseberry plants, revealed the presence of secreted in the xylem (SIX) effector genes, known to be involved in the pathogenicity of other formae speciales (ff. spp.) of F. oxysporum. This pathogenic strain was classified as a new f. sp. named Foph, due to its specificity for cape gooseberry hosts. Here, we sequenced and assembled the genome of five strains of F. oxysporum from a fungal collection associated to the cape gooseberry crop (including Foph), focusing on the validation of the presence of SIX homologous and on the identification of putative effectors unique to Foph. By comparative and phylogenomic analyses based on single-copy orthologous, we found that Foph is closely related to F. oxysporum ff. spp., associated with solanaceous hosts. We confirmed the presence of highly identical homologous genomic regions between Foph and Fol that contain effector genes and identified six new putative effector genes, specific to Foph pathogenic strains. We also conducted a molecular characterization using this set of putative novel effectors in a panel of 36 additional stains of F. oxysporum including two of the four sequenced strains, from the fungal collection mentioned above. These results suggest the polyphyletic origin of Foph and the putative independent acquisition of new candidate effectors in different clades of related strains. The novel effector candidates identified in this genomic analysis, represent new sources involved in the interaction between Foph and cape gooseberry, that could be implemented to develop appropriate management strategies of the wilt disease caused by Foph in the cape gooseberry crop.

Evidence for horizontal gene transfer and separation of effector recognition from effector function revealed by analysis of effector genes shared between cape gooseberry- and tomato-infecting formae speciales of Fusarium oxysporum.

RNA sequencing (RNAseq) reads from cape gooseberry plants (Physalis peruviana) infected with Fusarium oxysporumf. sp. physali (Foph) were mapped against the lineage-specific transcriptome of Fusarium oxysporumf. sp. lycopersici (Fol) to look for putative effector genes. Homologues of Fol SIX1(designated SIX1a and SIX1b), SIX7, SIX10, SIX12, SIX15 and Ave1were identified. The near identity of the Foph and Fol SIX7, SIX10 and SIX12genes and their intergenic regions suggest that this gene cluster may have undergone recent lateral transfer. Foph SIX1a and SIX1bwere tested for their ability to complement a SIX1 knockout mutant of Fol. This mutant shows reduced pathogenicity on susceptible tomato plants, but is able to infect otherwise resistant tomato plants carrying the I-3 gene for Fusarium wilt resistance (SIX1 corresponds to Avr3). Neither SIX1a nor SIX1b could restore full pathogenicity on susceptible tomato plants, suggesting that any role they may play in pathogenicity is likely to be specific to cape gooseberry. SIX1b, but not SIX1a, was able to restore avirulence on tomato plants carrying I-3.These findings separate the recognition of SIX1 from its role as an effector and suggest direct recognition by I-3. A hypervariable region of SIX1undergoing diversifying selection within the F. oxysporum species complex is likely to play an important role in SIX1 recognition. These findings also indicate that I-3could potentially be deployed as a transgene in cape gooseberry to protect this emerging crop from Foph.Alternatively, cape gooseberry germplasm could be explored for I-3homologues capable of providing resistance to Foph.

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

Development and characterization of microsatellite markers for the Cape gooseberry Physalis peruviana.

Physalis peruviana, commonly known as Cape gooseberry, is an Andean Solanaceae fruit with high nutritional value and interesting medicinal properties. In the present study we report the development and characterization of microsatellite loci from a P. peruviana commercial Colombian genotype. We identified 932 imperfect and 201 perfect Simple Sequence Repeats (SSR) loci in untranslated regions (UTRs) and 304 imperfect and 83 perfect SSR loci in coding regions from the assembled Physalis peruviana leaf transcriptome. The UTR SSR loci were used for the development of 162 primers for amplification. The efficiency of these primers was tested via PCR in a panel of seven P. peruviana accessions including Colombia, Kenya and Ecuador ecotypes and one closely related species Physalis floridana. We obtained an amplification rate of 83% and a polymorphic rate of 22%. Here we report the first P. peruviana specific microsatellite set, a valuable tool for a wide variety of applications, including functional diversity, conservation and improvement of the species.

LINKAGE MAPPING OF CANDIDATE GENES FOR INDUCE RESISTANCE AND GROWTH PROMOTION BY Trichoderma koningiopsis (Th003) IN TOMATO Solanum lycopersicum / Mapeo de genes candidatos relacionados con inducción de resistencia sistemica y promoción de crecimiento por Trichoderma koningiopsis (Th003) en tomate Solanum lycopersicum

Induced systemic resistance (ISR) is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum) is inoculated with several strains of Trichoderma ssp. This study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis Th003. Forty-nine candidate genes previously identified on tomato plants treated with Th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named “TOMATO EXPEN 2000”. The location of six unigenes was similar to the location of resistance gene analogs (RGAs), defense related ESTs and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL) regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.

La resistencia sistémica inducida (ISR) es un mecanismo mediante el cual las plantas aumentan sus defensas frente a cualquier condición de estrés. El objetivo de este trabajo fue localizar en el mapa genético de tomate, genes candidatos involucrados en ISR y promoción de crecimiento inducidos por la cepa colombiana nativa Th003 de Trichoderma koningiopsis. Se realizó una búsqueda de polimorfismos en cuarenta y nueve genes candidatos previamente identificados en plantas de tomate inoculadas con Th003 y la cepa T382 de T. hamatum. Diez y seis de estos genes candidatos fueron integrados en el mapa genético de tomate altamente saturado, llamado “TOMATO EXPEN 2000”. La ubicación de seis unigenes fue similar a la localización de genes análogos de resistencia (RGAs), ESTs relacionados con defensa y QTLs de resistencia previamente identificados, sugiriendo posibles nuevos candidatos para estas regiones de QTLs. Los genes candidatos o marcadores pueden ser usados en futuros programas de selección asistida relacionados con ISR o promoción de crecimiento en tomate.