Virulence factors of the genus Fusarium with targets in plants.

Fusarium spp. comprise various species of filamentous fungi that cause severe diseases in plant crops of both agricultural and forestry interest. These plant pathogens produce a wide range of molecules with diverse chemical structures and biological activities. Genetic functional analyses of some of these compounds have shown their role as virulence factors (VF). However, their mode of action and contributions to the infection process for many of these molecules are still unknown. This review aims to analyze the state of the art in Fusarium VF, emphasizing their biological targets on the plant hosts. It also addresses the current experimental approaches to improve our understanding of their role in virulence and suggests relevant research questions that remain to be answered with a greater focus on species of agroeconomic importance. In this review, a total of 37 confirmed VF are described, including 22 proteinaceous and 15 non-proteinaceous molecules, mainly from Fusarium oxysporum and Fusarium graminearum and, to a lesser extent, in Fusarium verticillioides and Fusarium solani.

The biological relevance of the FspTF transcription factor, homologous of Bqt4, in Fusarium sp. associated with the ambrosia beetle Xylosandrus morigerus.

Transcription factors in phytopathogenic fungi are key players due to their gene expression regulation leading to fungal growth and pathogenicity. The KilA-N family encompasses transcription factors unique to fungi, and the Bqt4 subfamily is included in it and is poorly understood in filamentous fungi. In this study, we evaluated the role in growth and pathogenesis of the homologous of Bqt4, FspTF, in Fusarium sp. isolated from the ambrosia beetle Xylosandrus morigerus through the characterization of a CRISPR/Cas9 edited strain in Fsptf. The phenotypic analysis revealed that TF65-6, the edited strain, modified its mycelia growth and conidia production, exhibited affectation in mycelia and culture pigmentation, and in the response to certain stress conditions. In addition, the plant infection process was compromised. Untargeted metabolomic and transcriptomic analysis, clearly showed that FspTF may regulate secondary metabolism, transmembrane transport, virulence, and diverse metabolic pathways such as lipid metabolism, and signal transduction. These data highlight for the first time the biological relevance of an orthologue of Bqt4 in Fusarium sp. associated with an ambrosia beetle.

Microorganisms Associated with the Ambrosial Beetle Xyleborus affinis with Plant Growth-Promotion Activity in Arabidopsis Seedlings and Antifungal Activity Against Phytopathogenic Fungus Fusarium sp. INECOL_BM-06.

Plants interact with a great diversity of microorganisms or insects throughout their life cycle in the environment. Plant and insect interactions are common; besides, a great variety of microorganisms associated with insects can induce pathogenic damage in the host, as mutualist phytopathogenic fungus. However, there are other microorganisms present in the insect-fungal association, whose biological/ecological activities and functions during plant interaction are unknown. In the present work evaluated, the role of microorganisms associated with Xyleborus affinis, an important beetle species within the Xyleborini tribe, is characterized by attacking many plant species, some of which are of agricultural and forestry importance. We isolated six strains of microorganisms associated with X. affinis shown as plant growth-promoting activity and altered the root system architecture independent of auxin-signaling pathway in Arabidopsis seedlings and antifungal activity against the phytopathogenic fungus Fusarium sp. INECOL_BM-06. In addition, evaluating the tripartite interaction plant-microorganism-fungus, interestingly, we found that microorganisms can induce protection against the phytopathogenic fungus Fusarium sp. INECOL_BM-06 involving the jasmonic acid-signaling pathway and independent of salicylic acid-signaling pathway. Our results showed the important role of this microorganisms during the plant- and insect-microorganism interactions, and the biological potential use of these microorganisms as novel agents of biological control in the crops of agricultural and forestry is important.

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq.

Fusarium kuroshium is a novel member of the Ambrosia Fusarium Clade (AFC) that has been recognized as one of the symbionts of the invasive Kuroshio shot hole borer, an Asian ambrosia beetle. This complex is considered the causal agent of Fusarium dieback, a disease that has severely threatened natural forests, landscape trees, and avocado orchards in the last 8 years. Despite the interest in this species, the molecular responses of both the host and F. kuroshium during the infection process and disease establishment remain unknown. In this work, we established an in vitro pathosystem using Hass avocado stems inoculated with F. kuroshium to investigate differential gene expression at 1, 4, 7 and 14 days post-inoculation. RNA-seq technology allowed us to obtain data from both the plant and the fungus, and the sequences obtained from both organisms were analyzed independently. The pathosystem established was able to mimic Fusarium dieback symptoms, such as carbohydrate exudation, necrosis, and vascular tissue discoloration. The results provide interesting evidence regarding the genes that may play roles in the avocado defense response to Fusarium dieback disease. The avocado data set comprised a coding sequence collection of 51,379 UniGenes, from which 2,403 (4.67%) were identified as differentially expressed. The global expression analysis showed that F. kuroshium responsive UniGenes can be clustered into six groups according to their expression profiles. The biologically relevant functional categories that were identified included photosynthesis as well as responses to stress, hormones, abscisic acid, and water deprivation. Additionally, processes such as oxidation-reduction, organization and biogenesis of the cell wall and polysaccharide metabolism were detected. Moreover, we identified orthologues of nucleotide-binding leucine-rich receptors, and their possible action mode was analyzed. In F. kuroshium, we identified 57 differentially expressed genes. Interestingly, the alcohol metabolic process biological category had the highest number of upregulated genes, and the enzyme group in this category may play an important role in the mechanisms of secondary metabolite detoxification. Hydrolytic enzymes, such as endoglucanases and a pectate lyase, were also identified, as well as some proteases. In conclusion, our research was conducted mainly to explain how the vascular tissue of a recognized host of the ambrosia complex responds during F. kuroshium infection since Fusarium dieback is an ambrosia beetle-vectored disease and many variables facilitate its establishment.

Design of a diagnostic system based on molecular markers derived from the ascomycetes pan-genome analysis: The case of Fusarium dieback disease.

A key factor to take actions against phytosanitary problems is the accurate and rapid detection of the causal agent. Here, we develop a molecular diagnostics system based on comparative genomics to easily identify fusariosis and specific pathogenic species as the Fusarium kuroshium, the symbiont of the ambrosia beetle Euwallaceae kuroshio Gomez and Hulcr which is responsible for Fusarium dieback disease in San Diego CA, USA. We performed a pan-genome analysis using sixty-three ascomycetes fungi species including phytopathogens and fungi associated with the ambrosia beetles. Pan-genome analysis revealed that 2,631 orthologue genes are only shared by Fusarium spp., and on average 3,941 (SD ± 1,418.6) are species-specific genes. These genes were used for PCR primer design and tested on DNA isolated from i) different strains of ascomycete species, ii) artificially infected avocado stems and iii) plant tissue of field-collected samples presumably infected. Our results let us propose a useful set of primers to either identify any species from Fusarium genus or, in a specific manner, species such as F. kuroshium, F. oxysporum, and F. graminearum. The results suggest that the molecular strategy employed in this study can be expanded to design primers against different types of pathogens responsible for provoking critical plant diseases.

Environmental pH modulates transcriptomic responses in the fungus Fusarium sp. associated with KSHB Euwallacea sp. near fornicatus.

BACKGROUND: The Ambrosia Fusarium Clade phytopathogenic Fusarium fungi species have a symbiotic relationship with ambrosia beetles in the genus Euwallacea (Coleoptera: Curculionidae). Related beetle species referred to as Euwallacea sp. near fornicatus have been spread in California, USA and are recognized as the causal agents of Fusarium dieback, a disease that causes mortality of many plant species. Despite the importance of this fungi, no transcriptomic resources have been generated. The datasets described here represent the first ever transcripts available for these species. We focused our study on the isolated species of Fusarium that is associated with one of the cryptic species referred to as Kuroshio Shot Hole Borer (KSHB) Euwallacea sp. near fornicatus. RESULTS: Hydrogen concentration is a critical signal in fungi for growth and host colonization, the aim of this study was to evaluate the effect of different pH conditions on growth and gene expression of the fungus Fusarium sp. associated with KSHB. An RNA-seq approach was used to compare the gene expression of the fungus grown for 2 weeks in liquid medium at three different pH levels (5.0, 6.0, and 7.0). An unbuffered treatment was included to evaluate the capability of the fungus to change the pH of its environment and the impact in gene expression. The results showed that the fungus can grow and modulate its genetic expression at different pH conditions; however, growth was stunted in acidic pH in comparison with neutral pH. The results showed a differential expression pattern in each pH condition even when acidic conditions prevailed at the end of the experiment. After comparing transcriptomics data from the three treatments, we found a total of 4,943 unique transcripts that were differentially expressed. CONCLUSIONS: We identified transcripts related to pH signaling such as the conserved PAL/RIM pathway, some transcripts related to secondary metabolism and other transcripts that were differentially expressed. Our analysis suggests possible mechanisms involved in pathogenicity in this novel Fusarium species. This is the first report that shows transcriptomic data of this pathogen as well as the first report of genes and proteins involved in their metabolism identifying potential virulence factors.

Plant growth-promoting rhizobacteria associated with avocado display antagonistic activity against Phytophthora cinnamomi through volatile emissions.

Rhizobacteria associated with crops constitute an important source of potentially beneficial microorganisms with plant growth promoting activity or antagonistic effects against phytopathogens. In this study, we evaluated the plant growth promoting activity of 11 bacterial isolates that were obtained from the rhizosphere of healthy avocado trees and from that of avocado trees having survived root rot infestations. Seven bacterial isolates, belonging to the genera Bacillus, Pseudomonas and Arthrobacter, promoted in vitro growth of Arabidopsis thaliana. These isolates were then tested for antagonistic activity against Phytophthora cinnamomi, in direct dual culture assays. Two of those rhizobacterial isolates, obtained from symptomatic-declining trees, displayed antagonistic activity. Isolate A8a, which is closely related to Bacillus acidiceler, was also able to inhibit P. cinnamomi growth in vitro by 76% through the production of volatile compounds. Solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS) allowed to tentatively identify the main volatiles emitted by isolate A8a as 2,3,5-trimethylpyrazine, 6,10-dimethyl-5,9-undecadien-2-one and 3-amino-1,3-oxazolidin-2-one. These volatile compounds have been reported to show antifungal activity when produced by other bacterial isolates. These results confirm the significance of rhizobacteria and suggest that these bacteria could be used for biocontrol of soil borne oomycetes through their volatiles emissions.

Draft Genome Sequence of the Phytopathogenic Fungus Fusarium euwallaceae, the Causal Agent of Fusarium Dieback.

Here, we report the genome of Fusarium euwallaceae strain HFEW-16-IV-019, an isolate obtained from Kuroshio shot hole borer (a Euwallacea sp.). These beetles were collected in Tijuana, Mexico, from elm trees showing typical symptoms of Fusarium dieback. The final assembly consists of 287 scaffolds spanning 48,274,071 bp and 13,777 genes.

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells.

Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone.

Iron distribution through the developmental stages of Medicago truncatula nodules.

Paramount to symbiotic nitrogen fixation (SNF) is the synthesis of a number of metalloenzymes that use iron as a critical component of their catalytical core. Since this process is carried out by endosymbiotic rhizobia living in legume root nodules, the mechanisms involved in iron delivery to the rhizobia-containing cells are critical for SNF. In order to gain insight into iron transport to the nodule, we have used synchrotron-based X-ray fluorescence to determine the spatio-temporal distribution of this metal in nodules of the legume Medicago truncatula with hitherto unattained sensitivity and resolution. The data support a model in which iron is released from the vasculature into the apoplast of the infection/differentiation zone of the nodule (zone II). The infected cell subsequently takes up this apoplastic iron and delivers it to the symbiosome and the secretory system to synthesize ferroproteins. Upon senescence, iron is relocated to the vasculature to be reused by the shoot. These observations highlight the important role of yet to be discovered metal transporters in iron compartmentalization in the nodule and in the recovery of an essential and scarce nutrient for flowering and seed production.