Early diagnosis of Cladosporium fulvum in greenhouse tomato plants based on visible/near-infrared (VIS/NIR) and near-infrared (NIR) data fusion.

Plant diseases can inflict varying degrees of damage on agricultural production. Therefore, identifying a rapid, non-destructive early diagnostic method is crucial for safeguarding plants. Cladosporium fulvum (C. fulvum) is one of the major diseases in tomato growth. This work presents a method of data fusion using two hyperspectral imaging systems of visible/near-infrared (VIS/NIR) and near-infrared (NIR) spectroscopy for the early diagnosis of C. fulvum in greenhouse tomatoes. First, hyperspectral images of samples at health and different times of infection were collected. The average spectral data of the image regions of interest were extracted and preprocessed for subsequent spectral datasets. Then different classification models were established for VIS/NIR and NIR data, optimized through various variable selection and data fusion methods. The principal component analysis-radial basis function neural network (PCA-RBF) model established using low-level data fusion achieved optimal results, achieving accuracies of 100% and 99.3% for calibration and prediction, respectively. Moreover, both the macro-averaged F1 (Macro-F1) values reached 1, and the geometric mean (G-mean) values reached 1 and 1, respectively. The results indicated that it was feasible to establish a PCA-RBF model by using the hyperspectral technique with low-level data fusion for the early detection of C. fulvum in greenhouse tomatoes.

Exploring the Antifungal Activity of Moroccan Bacterial and Fungal Isolates and a Strobilurin Fungicide in the Control of Cladosporium fulvum, the Causal Agent of Tomato Leaf Mold Disease.

The causal agent of tomato leaf mold, Cladosporium fulvum, is prevalent in greenhouses worldwide, especially under high humidity conditions. Despite its economic impact, studies on antifungal agents targeting C. fulvum remain limited. This study evaluates biocontrol agents (BCAs) as alternatives to chemical controls for managing this disease, alongside the strobilurin fungicide azoxystrobin. From a Moroccan collection of potential BCAs, five bacterial isolates (Alcaligenes faecalis ACBC1, Pantoea agglomerans ACBC2, ACBP1, ACBP2, and Bacillus amyloliquefaciens SF14) and three fungal isolates (Trichoderma spp. OT1, AT2, and BT3) were selected and tested. The in vitro results demonstrated that P. agglomerans isolates reduced mycelial growth by over 60% at 12 days post-inoculation (dpi), while Trichoderma isolates achieved 100% inhibition in just 5 dpi. All bacterial isolates produced volatile organic compounds (VOCs) with mycelial inhibition rates ranging from 38.8% to 57.4%. Likewise, bacterial cell-free filtrates significantly inhibited the pathogen's mycelial growth. Greenhouse tests validated these findings, showing that all the tested isolates were effective in reducing disease incidence and severity. Azoxystrobin effectively impeded C. fulvum growth, particularly in protective treatments. Fourier transform infrared spectroscopy (FTIR) analysis revealed significant biochemical changes in the treated plants, indicating fungal activity. This study provides valuable insights into the efficacy of these BCAs and azoxystrobin, contributing to integrated management strategies for tomato leaf mold disease.

Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.

Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.

Mapping of the Cladosporium fulvum resistance gene Cf-16, a major gene involved in leaf mold disease in tomato.

Tomato (Solanum lycopersicum) is widely cultivated and consumed worldwide. Tomato leaf mold, caused by Cladosporium fulvum, is one of the most devastating diseases in tomato production. At present, some tomato leaf mold resistance (Cf series) genes used in production gradually lose resistance due to the continuous and rapid differentiation of C. fulvum physiological races. The Cf-16 gene derived from the "Ontario7816" tomato cultivar has shown effective resistance in field trials for many years, but few studies have reported on the mapping of the Cf-16 gene, which has not been cloned, limiting its utilization in tomato breeding. Here, we mapped Cf-16 using a novel comprehensive strategy including bulk segregation analysis (BSA), genome resequencing and SSR molecular markers. A genetic analysis revealed that Cf-16 resistance in "Ontario7816" is controlled by one major dominant locus. The Cf-16 gene was mapped in a region of 2.6 cM at chromosome 6 between two markers, namely, TGS447 and TES312, by using an F2 population from a cross between the resistant cultivar "Ontario7816" and susceptible line "Moneymaker." Two nucleotide-binding-site-leucine-rich repeat (NBS-LRR) resistance genes, namely, XM_004240667.3 and XM_010323727.1, were identified in this interval. They are strong candidates for the Cf-16 gene. The mapping of Cf-16 may speed up its utilization for breeding resistant tomato varieties and represents an important step forward in our understanding of the mechanism underlying resistance to tomato leaf mold.

Integrating omics reveals that miRNA-guided genetic regulation on plant hormone level and defense response pathways shape resistance to Cladosporium fulvum in the tomato Cf-10-gene-carrying line.

Invasion of C. fulvum causes the most serious diseases affecting the reproduction of tomatoes. Cf-10-gene-carrying line showed remarkable resistance to Cladosporium fulvum. To exploit its defense response mechanism, we performed a multiple-omics profiling of Cf-10-gene-carrying line and a susceptible line without carrying any resistance genes at non-inoculation and 3 days post-inoculation (dpi) of C. fulvum. We detected 54 differentially expressed miRNAs (DE-miRNAs) between the non-inoculation and 3 dpi in the Cf-10-gene-carrying line, which potentially regulated plant-pathogen interaction pathways and hormone signaling pathways. We also revealed 3,016 differentially expressed genes (DEGs) between the non-inoculated and 3 dpi in the Cf-10-gene-carrying line whose functions enriched in pathways that were potentially regulated by the DE-miRNAs. Integrating DE-miRNAs, gene expression and plant-hormone metabolites indicated a regulation network where the downregulation of miRNAs at 3 dpi activated crucial resistance genes to trigger host hypersensitive cell death, improved hormone levels and upregulated the receptors/critical responsive transcription factors (TFs) of plant hormones, to shape immunity to the pathogen. Notably, our transcriptome, miRNA and hormone metabolites profiling and qPCR analysis suggested that that the downregulation of miR9472 potentially upregulated the expression of SAR Deficient 1 (SARD1), a key regulator for ICS1 (Isochorismate Synthase 1) induction and salicylic acid (SA) synthesis, to improve the level of SA in the Cf-10-gene-carrying line. Our results exploited potential regulatory network and new pathways underlying the resistance to C. fulvum in Cf-10-gene-carrying line, providing a more comprehensive genetic circuit and valuable gene targets for modulating resistance to the virus.

Physiological and transcriptome profiling revealed defense networks during Cladosporium fulvum and tomato interaction at the early stage.

Tomato leaf mold caused by Cladosporium fulvum (C. fulvum) is a serious fungal disease which results in huge yield losses in tomato cultivation worldwide. In our study, we discovered that ROS (reactive oxygen species) burst was triggered by C. fulvum treatment in tomato leaves. RNA-sequencing was used to identify differentially expressed genes (DEGs) induced by C. fulvum inoculation at the early stage of invasion in susceptible tomato plants. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate functions of DEGs in tomato plants. Based on our comparative analysis, DEGs related to plant-pathogen interaction pathway, plant hormone signal transduction pathway and the plant phenylpropanoid pathway were further analyzed. Our results discovered that a number of core defense genes against fungal invasion were induced and plant hormone signal transduction pathways were impacted by C. fulvum inoculation. Further, our results showed that SA (salicylic acid) and ABA (abscisic acid) contents were accumulated while JA (jasmonic acid) content decreased after C. fulvum inoculation in comparison with control, and quantitative real-time PCR to detect the relative expression of genes involved in SA, ABA and JA signaling pathway further confirmed our results. Together, results will contribute to understanding the mechanisms of C. fulvum and tomato interaction in future.

Transcriptome Analysis of the Cf-13-Mediated Hypersensitive Response of Tomato to Cladosporium fulvum Infection.

Tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum) is one of the most common diseases affecting greenhouse tomato production. Cf proteins can recognize corresponding AVR proteins produced by C. fulvum, and Cf genes are associated with leaf mold resistance. Given that there are many physiological races of C. fulvum and that these races rapidly mutate, resistance to common Cf genes (such as Cf-2, Cf-4, Cf-5, and Cf-9) has decreased. In the field, Ont7813 plants (carrying the Cf-13 gene) show effective resistance to C. fulvum; thus, these plants could be used as new, disease-resistant materials. To explore the mechanism of the Cf-13-mediated resistance response, transcriptome sequencing was performed on three replicates each of Ont7813 (Cf-13) and Moneymaker (MM; carrying the Cf-0 gene) at 0, 9, and 15 days after inoculation (dai) for a total of 18 samples. In total, 943 genes were differentially expressed, specifically in the Ont7813 response process as compared to the Moneymaker response process. Gene ontology (GO) classification of these 943 differentially expressed genes (DEGs) showed that GO terms, including "hydrogen peroxide metabolic process (GO_Process)", "secondary active transmembrane transporter activity (GO_Function)", and "mismatch repair complex (GO_Component)", which were the same as 11 other GO terms, were significantly enriched. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many key regulatory genes of the Cf-13-mediated resistance response processes were involved in the "plant hormone signal transduction" pathway, the "plant-pathogen interaction" pathway, and the "MAPK signaling pathway-plant" pathway. Moreover, during C. fulvum infection, jasmonic acid (JA) and salicylic acid (SA) contents significantly increased in Ont7813 at the early stage. These results lay a vital foundation for further understanding the molecular mechanism of the Cf-13 gene in response to C. fulvum infection.

The Diversity of Passalora fulva Isolates Collected from Tomato Plants in U.S. High Tunnels.

High tunnels extend the growing season of high value crops, including tomatoes, but the environmental conditions within high tunnels favor the spread of the tomato leaf mold pathogen, Passalora fulva (syn. Cladosporium fulvum). Tomato leaf mold results in defoliation, and if severe, losses in yield. Despite substantial research, little is known regarding the genetic structure and diversity of populations of P. fulva associated with high tunnel tomato production in the United States. From 2016 to 2019, a total of 50 P. fulva isolates were collected from tomato leaf samples in high tunnels in the Northeast and Minnesota. Other Cladosporium species were also isolated from the leaf surfaces. Koch's postulates were conducted to confirm that P. fulva was the cause of the disease symptoms observed. Race determination experiments revealed that the isolates belonged to either race 0 (six isolates) or race 2 (44 isolates). Polymorphisms were identified within four previously characterized effector genes: Avr2, Avr4, Avr4e, and Avr9. The largest number of polymorphisms were observed for Avr2. Both mating type genes, MAT1-1-1 and MAT1-2-1, were present in the isolate collection. For further insights into the pathogen diversity, the 50 isolates were genotyped at 7,514 single-nucleotide polymorphism loci using genotyping-by-sequencing. Differentiation by region but not by year was observed. Within the collection of 50 isolates, there were 18 distinct genotypes. Information regarding P. fulva population diversity will enable better management recommendations for growers, as high tunnel production of tomatoes expands.

Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance.

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance (Hcr) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.

Genes Encoding Recognition of the Cladosporium fulvum Effector Protein Ecp5 Are Encoded at Several Loci in the Tomato Genome.

The molecular interactions between tomato and Cladosporium fulvum have been an important model for molecular plant pathology. Complex genetic loci on tomato chromosomes 1 and 6 harbor genes for resistance to Cladosporium fulvum, encoding receptor like-proteins that perceive distinct Cladosporium fulvum effectors and trigger plant defenses. Here, we report classical mapping strategies for loci in tomato accessions that respond to Cladosporium fulvum effector Ecp5, which is very sequence-monomorphic. We screened 139 wild tomato accessions for an Ecp5-induced hypersensitive response, and in five accessions, the Ecp5-induced hypersensitive response segregated as a monogenic trait, mapping to distinct loci in the tomato genome. We identified at least three loci on chromosomes 1, 7 and 12 that harbor distinct Cf-Ecp5 genes in four different accessions. Our mapping showed that the Cf-Ecp5 in Solanum pimpinellifolium G1.1161 is located at the Milky Way locus. The Cf-Ecp5 in Solanum pimpinellifolium LA0722 was mapped to the bottom arm of chromosome 7, while the Cf-Ecp5 genes in Solanum lycopersicum Ontario 7522 and Solanum pimpinellifolium LA2852 were mapped to the same locus on the top arm of chromosome 12. Bi-parental crosses between accessions carrying distinct Cf-Ecp5 genes revealed putative genetically unlinked suppressors of the Ecp5-induced hypersensitive response. Our mapping also showed that Cf-11 is located on chromosome 11, close to the Cf-3 locus. The Ecp5-induced hypersensitive response is widely distributed within tomato species and is variable in strength. This novel example of convergent evolution could be used for choosing different functional Cf-Ecp5 genes according to individual plant breeding needs.

Comparative transcriptome analysis reveals the response mechanism of Cf-16-mediated resistance to Cladosporium fulvum infection in tomato.

BACKGROUND: Leaf mold disease caused by Cladosporium fulvum is a serious threat affecting the global production of tomato. Cf genes are associated with leaf mold resistance, including Cf-16, which confers effective resistance to leaf mold in tomato. However, the molecular mechanism of the Cf-16-mediated resistance response is largely unknown. RESULTS: We performed a comparative transcriptome analysis of C. fulvum-resistant (cv. Ontario7816) and C. fulvum-susceptible (cv. Moneymaker) tomato cultivars to identify differentially expressed genes (DEGs) at 4 and 8 days post inoculation (dpi) with C. fulvum. In total, 1588 and 939 more DEGs were found in Cf-16 tomato than in Moneymaker at 4 and 8 dpi, respectively. Additionally, 1350 DEGs were shared between the 4- and 8-dpi Cf-16 groups, suggesting the existence of common core DEGs in response to C. fulvum infection. The up-regulated DEGs in Cf-16 tomato were primarily associated with defense processes and phytohormone signaling, including salicylic acid (SA) and jasmonic acid (JA). Moreover, SA and JA levels were significantly increased in Cf-16 tomato at the early stages of C. fulvum infection. Contrary to the previous study, the number of up-regulated genes in Cf-16 compared to Cf-10 and Cf-12 tomatoes was significantly higher at the early stages of C. fulvum infection. CONCLUSION: Our results provide new insight into the Cf-mediated mechanism of resistance to C. fulvum, especially the unique characteristics of Cf-16 tomato in response to this fungus.

Transcriptome profiling reveals the response process of tomato carrying Cf-19 and Cladosporium fulvum interaction.

BACKGROUND: During tomato cultivation, tomato leaf mould is a common disease caused by Cladosporium fulvum (C. fulvum). By encoding Cf proteins, which can recognize corresponding AVR proteins produced by C. fulvum, Cf genes provide resistance to C. fulvum, and the resistance response patterns mediated by different Cf genes are not identical. Plants carrying the Cf-19 gene show effective resistance to C. fulvum in the field and can be used as new resistant materials in breeding. In this study, to identify key regulatory genes related to resistance and to understand the resistance response process in tomato plants carrying Cf-19, RNA sequencing (RNA-seq) was used to analyse the differences between the response of resistant plants (CGN18423, carrying the Cf-19 gene) and susceptible plants (Moneymaker (MM), carrying the Cf-0 gene) at 0, 7 and 20 days after inoculation (dai). RESULTS: A total of 418 differentially expressed genes (DEGs) were identified specifically in the CGN18423 response process. Gene Ontology (GO) analysis revealed that GO terms including "plasma membrane (GO_Component)", "histidine decarboxylase activity (GO_Function)", and "carboxylic acid metabolic process (GO_Process)", as well as other 10 GO terms, were significantly enriched. The "plant hormone signal transduction" pathway, which was unique to CGN18423 in the 0-7 dai comparison, was identified. Moreover, ten key regulatory points were screened from the "plant hormone signal transduction" pathway and the "plant pathogen interaction" pathway. Hormone content measurements revealed that the salicylic acid (SA) contents increased and peaked at 7 dai, after which the contents deceased and reached minimum values in both CGN18423 and MM plants at 20 dai. The jasmonic acid (JA) content increased to a very high level at 7 dai but then decreased to nearly the initial level at 20 dai in CGN18423, while it continued to increase slightly during the whole process from 0 to 20 dai in MM. CONCLUSIONS: The initial responses are very different between the resistant and susceptible plants. The "plant hormone signal transduction" pathway is important for the formation of Cf-19-mediated immunity. In addition, both JA and SA play roles in regulating the Cf-19-dependent resistance response.

A conserved GH17 glycosyl hydrolase from plant pathogenic Dothideomycetes releases a DAMP causing cell death in tomato.

To facilitate infection, pathogens deploy a plethora of effectors to suppress basal host immunity induced by exogenous microbe-associated or endogenous damage-associated molecular patterns (DAMPs). In this study, we have characterized family 17 glycosyl hydrolases of the tomato pathogen Cladosporium fulvum (CfGH17) and studied their role in infection. Heterologous expression of CfGH17-1 to 5 by potato virus X in different tomato cultivars showed that CfGH17-1 and CfGH17-5 enzymes induce cell death in Cf-0, Cf-1 and Cf-5 but not in Cf-Ecp3 tomato cultivars or tobacco. Moreover, CfGH17-1 orthologues from other phytopathogens, including Dothistroma septosporum and Mycosphaerella fijiensis, also trigger cell death in tomato. CfGH17-1 and CfGH17-5 are predicted to be ß-1,3-glucanases and their enzymatic activity is required for the induction of cell death. CfGH17-1 hydrolyses laminarin, a linear 1,3-ß-glucan with 1,6-ß linkages. CfGH17-1 expression is down-regulated during the biotrophic phase of infection and up-regulated during the necrotrophic phase. Deletion of CfGH17-1 in C. fulvum did not reduce virulence on tomato, while constitutive expression of CfGH17-1 decreased virulence, suggesting that abundant presence of CfGH17-1 during biotrophic growth may release a DAMP that activates plant defence responses. Under natural conditions CfGH17-1 is suggested to play a role during saprophytic growth when the fungus thrives on dead host tissue, which is in line with its high levels of expression at late stages of infection when host tissues have become necrotic. We suggest that CfGH17-1 releases a DAMP from the host cell wall that is recognized by a yet unknown host plant receptor.

Molecular mapping of the Cf-10 gene by combining SNP/InDel-index and linkage analysis in tomato (Solanum lycopersicum).

BACKGROUND: Leaf mold, one of the major diseases of tomato caused by Cladosporium fulvum (C. fulvum), can dramatically reduce the yield and cause multimillion dollar losses annually worldwide. Mapping the resistance genes (R genes) of C. fulvum and devising MAS based strategies for breeding new cultivars is an effective approach to improve the resistance in tomato. Up to now, many C. fulvum genes or QTLs have been mapped using different genetic materials, but few studies focused on Cf-10 gene positioning. RESULTS: In this study, we investigated the genetic rules for Cf-10 and used a novel combinatorial strategy to rapidly map the Cf-10 gene. Initially, the performance of F1, F2 and BC1F1 individuals after infection, demonstrated that the resistance against C. fulvum was controlled by a single dominant gene. Two pools of resistant and susceptible individuals from F2 population were investigated, using mapping by sequencing approach and Cf-10 was found to be localized to 3.35 Mb and 3.74 Mb on chromosome 1, employing SNP/InDel index methods, respectively. After accounting for overlapping regions, these two algorithms yielded a total length of 3.29 Mb, narrowing down the target region. We further developed five serviceable KASP markers for this region based on sequencing data and conducted local QTL mapping using individuals from the F2 population, except for mapping by sequencing as mentioned above. Finally Cf-10 gene was mapped spanning a region of 790 kb, where only one gene (Solyc01g007130.3) was annotated as probable receptor protein kinase TMK1 with a LRR motif, a common R gene characteristic. The RT-qPCR analysis further confirmed the localization and the relative expression of Solyc01g007130.3 in Ontario 792 and was found to be significantly higher than that in Moneymaker at 9 dpi and 12 dpi, respectively. CONCLUSION: This study proposed a novel combinatorial strategy by combining SNP-index, InDel-index analyses and local QTL mapping using KASP genotyping approach to rapidly map genes responsible for specific traits and provided a robust base for cloning the Cf-10 gene. Furthermore, these analyses suggest that Solyc01g007130.3 is a potential candidate to be regarded as Cf-10 gene.

Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

Expression of strong antimicrobial peptides in plants is of great interest to combat a wide range of plant pathogens. To bring the Dermaseptin B1 (DrsB1) peptide to the intimate contact of the plant pathogens cell wall surface, the DrsB1 encoding sequence was fused to the C-terminal part of the two copies of the chitin-binding domain (CBD) of the Avr4 effector protein and used for Agrobacterium rhizogenes-mediated transformation. The expression of the recombinant protein in the tobacco hairy roots (HRs) was confirmed by molecular analysis. Antimicrobial activity analysis of the recombinant protein purified from the transgenic HRs showed that the (CBD)2-DrsB1 recombinant protein had a significant (p < 0.01) antimicrobial effect on the growth of different fungal and bacterial pathogens. The results of this study indicated that the recombinant protein had a higher antifungal activity against chitin-producing Alternaria alternata than Pythium spp. Scanning electron microscopy images demonstrated that the recombinant protein led to fungal hypha deformation, fragmentation, and agglutination of growing hypha, possibly by dissociating fungal cell wall components. In vitro evidences suggest that the expression of the (CBD)2-DrsB1 recombinant protein in plants by generating transgenic lines is a promising approach to produce disease-resistant plants, resistance to chitin-producing pathogenic fungi.

Physiological and RNA-seq analyses provide insights into the response mechanism of the Cf-10-mediated resistance to Cladosporium fulvum infection in tomato.

KEY MESSAGE: Based on the physiological and RNA-seq analysis, some progress has been made in elucidating the Cf-10-mediated resistance responses to C. fulvum infection in tomato. GO and KEGG enrichment analysis revealed that the DEGs were significantly associated with defense-signaling pathways like oxidation-reduction processes, oxidoreductase activity and plant hormone signal transduction. Leaf mold, caused by the fungus Cladosporium fulvum, is one of the most common diseases affecting tomatoes worldwide. Cf series genes including Cf-2, Cf-4, Cf-5, Cf-9 and Cf-10 play very important roles in resisting tomato leaf mold. Understanding the molecular mechanism of Cf gene-mediated resistance is thus the key to facilitating genetic engineering of resistance to C. fulvum infection. Progress has been made in elucidating two Cf genes, Cf -19 and Cf -12, and how they mediate resistance responses to C. fulvum infection in tomato. However, the mechanism of the Cf-10- mediated resistance response is still unclear. In the present study, RNA-seq was used to analyze changes in the transcriptome at different stages of C. fulvum infection. A total of 2,242 differentially expressed genes (DEGs) responsive to C. fulvum between 0 and 16 days post infection (dpi) were identified, including 1,501 upregulated and 741 downregulated genes. The majority of DEGs were associated with defense-signaling pathways including oxidation-reduction processes, oxidoreductase activity and plant hormone signal transduction. Four DEGs associated with plant-pathogen interaction were uniquely activated in Cf-10 tomato and validated by qRT-PCR. In addition, physiological indicators including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were measured at 0-21 dpi, and hormone expression [Jasmonic acid (JA) and salicylic acid (SA)] was estimated at 0 and 16 dpi to elucidate the mechanism of the Cf-10-mediated resistance response. C. fulvum infection induced the activities of POD, CAT and SOD, and decreased ROS levels. JA was determined to participate in the resistance response to C. fulvum during the initial infection period. The results of this study provide accountable evidence for the physiological and transcriptional regulation of the Cf-10-mediated resistance response to C. fulvum infection, facilitating further understanding of the molecular mechanism of Cf-10-mediated resistance to C. fulvum infection.

Mycosphaerellaceae - Chaos or clarity?

The Mycosphaerellaceae represent thousands of fungal species that are associated with diseases on a wide range of plant hosts. Understanding and stabilising the taxonomy of genera and species of Mycosphaerellaceae is therefore of the utmost importance given their impact on agriculture, horticulture and forestry. Based on previous molecular studies, several phylogenetic and morphologically distinct genera within the Mycosphaerellaceae have been delimited. In this study a multigene phylogenetic analysis (LSU, ITS and rpb2) was performed based on 415 isolates representing 297 taxa and incorporating ex-type strains where available. The main aim of this study was to resolve the phylogenetic relationships among the genera currently recognised within the family, and to clarify the position of the cercosporoid fungi among them. Based on these results many well-known genera are shown to be paraphyletic, with several synapomorphic characters that have evolved more than once within the family. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected, and 32 additional genera are described as new. Based on phylogenetic data 120 genera are now accepted within the family, but many currently accepted cercosporoid genera still remain unresolved pending fresh collections and DNA data. The present study provides a phylogenetic framework for future taxonomic work within the Mycosphaerellaceae.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants.

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

Cladosporium fulvum Effectors: Weapons in the Arms Race with Tomato.

In this review, I recount my personal history. My drive to study host-pathogen interactions was to find alternatives for agrochemicals, which was triggered after reading the book "Silent Spring" by Rachel Carson. I reflect on my research at the Laboratory of Phytopathology at Wageningen University, where I have worked for my entire career on the interaction between Cladosporium fulvum and tomato, and related gene-for-gene pathosystems. I describe different methods used to identify and sequence avirulence (Avr) genes from the pathogen and resistance (R) genes from the host. The major genes involved in classical gene-for-gene interactions have now been identified, and breeders can produce plants with multiple R genes providing durable and environmentally safe protection against pathogens. In some cases, this might require the use of genetically modified plants when R genes cannot be introduced by classical breeding.

Transcriptome Analysis of the Cf-12-Mediated Resistance Response to Cladosporium fulvum in Tomato.

Cf-12 is an effective gene for resisting tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum). Unlike many other Cf genes such as Cf-2, Cf-4, Cf-5, and Cf-9, no physiological races of C. fulvum that are virulent to Cf-12 carrying plant lines have been identified. In order to better understand the molecular mechanism of Cf-12 gene resistance response, RNA-Seq was used to analyze the transcriptome changes at three different stages of C. fulvum infection (0, 4, and 8 days post infection [dpi]). A total of 9100 differentially expressed genes (DEGs) between 4 and 0 dpi, 8643 DEGs between 8 and 0 dpi and 2547 DEGs between 8 and 4 dpi were identified. In addition, we found that 736 DEGs shared among the above three groups, suggesting the presence of a common core of DEGs in response to C. fulvum infection. These DEGs were significantly enriched in defense-signaling pathways such as the calcium dependent protein kinases pathway and the jasmonic acid signaling pathway. Additionally, we found that many transcription factor genes were among the DEGs, indicating that transcription factors play an important role in C. fulvum defense response. Our study provides new insight on the molecular mechanism of Cf resistance to C. fulvum, especially the unique features of Cf-12 in responding to C. fulvum infection.