Cladosporium Fulvum Research Articles

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.

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases "Required for Cladosporium resistance-3" (Rcr3) and "Phytophthora-inhibited protease-1" (Pip1) of tomato (Solanum lycopersicum) are both inhibited by Avirulence-2 (Avr2) from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signaling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signaling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signaling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

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.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, −7, and −8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

First observation of the beetle Sericoderus lateralis consuming Cladosporium fulvum in a greenhouse tomato: potential for biological protection or risk to the crop?

For the first time, the mycophagous beetle Sericoderus lateralis (Gyllenhal, 1827) was documented feeding on tomato leaf mold ( Cladosporium fulvum Cooke, 1878) tissues. The phenomenon was observed during the years 2022 and 2023 in a hydroponic tomato greenhouse situated near the Czech-Polish border within the cadastre of Dolní Lutyně municipality in Czechia. Greenhouse and laboratory observations confirmed that adult and larvae feeding activity led to a reduction in tomato leaf mold lesions. In addition, there was a positive correlation between tomato leaf mold disease progression and increased populations of S. lateralis in the tomato crop. Petri dish observations confirmed egg laying occurred on a diet of tomato leaf mold. Further research is warranted to discern whether S. lateralis is a potential biological control agent for tomato leaf mold or if it acts to facilitate the spread of the disease by acting as a spore vector.

Chromosome-Scale Genome Sequence of Nothopassalora personata (syn. Cercosporidium personatum), a Devastating Fungal Pathogen of Peanut

Nothopassalora personata, the causal agent of late leaf spot, is an economically important fungus that poses a significant threat to peanut production worldwide. Currently, studies on N. personata are focused on epidemiology, host resistance, early environmental detection, and molecular studies based on a limited number of genes for classification. A high-quality whole-genome sequence can be a valuable tool for studying complex species and identifying molecular mechanisms in pathogenicity and adaptation. This study reports a high-quality, chromosome-scale genome assembly for N. personata with a total length of 54.6 Mb, an N50 of 2.54 Mb, a longest contig length of 4.47 Mb, and a BUSCO genome completeness score of 99.5% with mostly single-copy genes. Genome annotation revealed 11,295 protein-encoding genes and 12,121 transcripts in which 411 of the 995 predicted proteins were putative effectors. Genome comparison with the closely related fungi Dothistroma septosporum NZE10 and Fulvia fulva (syn . Passalora fulva, Cladosporium fulvum) isolate Race 5 revealed 13 chromosomes. This genome sequence resource can be used as a reference for aligning sequences of multiple isolates to provide relevant information on the diversity and population genetic structure of N. personata to further inform peanut resistance breeding programs and devise the best disease management strategies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes

BackgroundFungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes.ResultsHere, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum.ConclusionsOur results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

Autonecrotic Tomato (Solanum lycopersicum L.) Line as a Potential Model for Applications in Proximal Sensing of Biotic and Abiotic Stress

The autonecrotic tomato line V20368 (working code IGSV) spontaneously develops necrotic lesions with acropetal progression in response to an increase in temperature and light irradiation. The process is associated with the interaction between tomato and Cladosporium fulvum, the fungal agent of leaf mold. The contemporary presence of an in-house allele encoding the Rcr3lyc protein and the resistance gene Cf-2pim (from Solanum pimpinellifolium) causes auto-necrosis on the leaves even in the absence of the pathogen (hybrid necrosis). The aim of the work was (i) to examine the potential value of the necrotic genotype as a model system for setting up theoretical guidance for monitoring the phytosanitary status of tomato plants and (ii) to develop a predictive model for the early detection of pathogens (or other stresses) in the tomato or other species. Eighteen IGSV tomato individuals at the 4–5th true-leaf stage were grown in three rows (six plants per row) considered to be replicates. The healthy control was the F1 hybrid Elisir (Olter). A second mutant line (SA410) deriving from a cross between the necrotic mutant and a mutant line of the lutescent (l) gene was used during foliar analysis via microspectrometry. The leaves of the mutants and normal plants were monitored through a portable VIS/NIR spectrometer SCIOTM (Consumer Physics, Tel Aviv, Israel) covering a spectral range between 740 and 1070 nm. Two months after the transplant, the acropetal progression of the autonecrosis showed three symptomatic areas (basal, median, apical) on each IGSV plant: necrotic, partially damaged, and green, respectively. Significantly lower chlorophyll content was found in the basal and median areas of IGSV compared with the control (Elisir). A supervised classification/modelling method (SIMCA) was used. Applying the SIMCA model to the dataset of 162 tomato samples led to the identification of the boundary between the healthy and damaged samples (translational critical distance). Two 10 nm wavelength ranges centred at 865 nm and 1055 nm exhibited a stronger link between symptomatology and spectral reflectance. Studies on specific highly informative mutants of the type described may allow for the development of predictive models for the early detection of pathogens (or other stresses) via proximal sensing.

The race distribution of the Fulvia fulva populations and mutations in the Avr genes in Turkey

AbstractTomato leaf mould caused by Fulvia fulva (syn. Cladosporium fulvum, Passalora fulva) is an especially important disease of greenhouse grown tomato. In this study, F. fulva isolates were obtained from Antalya, Muğla, Mersin, Burdur, Bartın, and Zonguldak provinces of Turkey. Pathogenicity tests were carried out with 47 isolates using a differential set of tomato varieties containing Cf‐2, Cf‐4, Cf‐5, Cf‐6, Cf‐9, and Cf‐4/11 resistance genes and Cf‐0 with no resistance gene. As a result of pathogenicity tests, races 2, 2.4, 2.5, 2.9, 2.5.9, 2.4.11, and 2.4.5.9.11 were identified. The most frequent races were 2 and 2.5. In addition, mutations in the Avr2, Avr4, Avr4E, Avr5, and Avr9 genes were investigated. While mutations in Avr2 and Avr5 genes occurred within the coding region, Avr4E and Avr9 genes occurred outside the coding DNA sequence. Based on mutations in Avr genes and mating types, 14 genotypes of F. fulva were determined. This study revealed the race distribution of the F. fulva populations and mutations in the Avr genes in Turkey. It will contribute to studies on the population structure and control of the pathogen.

Molecular and genetic basis of tomato resistance to major fungal diseases

This review presents an analysis of the literature, which indicates the popularity of breeding methods in the fight against oomycete and fungal diseases of tomato: late blight (pathogen − Phytophthora infestans (Mont.) de Bary), fusarium wilt (pathogen − Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder and Hansen), early blight or alternariosis (pathogens − representatives of the genus Alternaria), cladosporiosis (pathogen−Cladosporium fulvum Cook.). Theoretical and practical achievements, the latest developments in genetics, molecular biology, plant physiology, microbiology are considered. Special attention is paid to modern information about the diversity of tomato pathogens, their morphological, genetic, physiological features, including racial composition; the presence of pathogen resistance genes in the gene pool of the culture under study, their mapping and the possibility of introduction into the genome from other sources; about the developed markers of target genes and loci of quantitative traits; the degree of associations between a molecular marker and a target gene; about the features of inheritance of the studied trait, as well as the possibility of pyramiding R-genes and QTL in one genotype. To accelerate and improve the efficiency of tomato breeding for resistance to pathogens, these issues are of great importance. Their study will strengthen the integration of modern biotechnology with the traditional breeding process, which is carried out by classical methods.

In vitro susceptibility test of Cladosporium cladosporioides isolates from Argentinian tomato crops against commercial fungicides

Tomato cultivation is an important agricultural activity in northeast of Argentina, and disease control is indispensable for its production. The purpose of the current study was to identify two fungi isolated from tomato plants cultivated in greenhouses, with symptoms of leaf mould disease and a strain of the genus Cladosporium from a culture collection, and evaluate their in vitro susceptibility to four commercial fungicides. Macro and microscopic examination, molecular characterisation and sequence analysis were applied for identification. Broth dilution and spread plate methods were used to determine the minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC). The active ingredients of the products were azoxystrobin+difenoconazole, trifloxystrobin+tebuconazole, chlorothalonil and metalaxyl-M+mancozeb. The results were processed using the Kruskal-Wallis method. The isolates were identified as Cladosporium cladosporioidesa-c; consequently, lesions found on tomato plants did not corrspond to Cladosporium fulvum. There was a significant statistical difference between the obtained values. Qualitatively, the three strains had a similar behaviour for chlorothalonil (MIC values: 0.25 - 0.5 μg/ml, MFC values: 4 μg/ml). In all cases, tests with metalaxyl-M+mancozeb yielded higher values than those achieved for chlorothalonil (MIC values: 8 μg/ml, MFC values: 8- 32 μg/ml). trobilurin-formulated fungicides were less effective against C. cladosporioidesa-b (MIC values: 16-256 μg/ml, MFC values: >64 μg/ml). C. cladosporioidesc was the most sensitive isolate. The information about the presence of a non-frequent fungus and its fungicide susceptibility, would be useful for establishing control strategies and enhance production.

Transcription factor network analysis of the Cf-19-mediated resistance response in tomato infected by Cladosporium fulvum

Tomato leaf mold, caused by Cladosporium fulvum, is a common disease in tomato cultivation. The Cf-19 gene is an effective resistance gene to C. fulvum. Current studies have shown that the Cf-19-mediated resistance response pathway is regulated by a variety of TFs. In this study, in order to better understand the role of TFs in the Cf-19-mediated immune response of tomato against C. fulvum, differentially expressed TFs in different response stages were identified based on RNA-seq data. The results showed that a total of 37 families with 171 TF members were identified. These differentially expressed TFs were enriched in “nucleus” in the component category, “DNA-binding transcription factor activity” in the function category and “Regulation of transcription” in the process category in the Gene Ontology (GO) analysis results. Four pathways, including plant hormone signal transduction, MAPK signaling, plant-pathogen interaction and circadian rhythm, were enriched in the KEGG analysis results. Further analysis showed that 45 TFs were involved in the above four major pathways, and some TFs in different pathways had interaction relationships, which might be the key nodes mediating cross-talk between different pathways. EREB was predicted to be the core of multigroup interactions, and its interaction with WRKY80 is the node connecting the MAPK pathway and plant-pathogen interaction pathway. The function of the EREB gene was analyzed by VIGS, and it was found that the downregulated expression of this gene decreased the expression of WRKY80 and the disease resistance mediated by Cf-19.

Sensitivity of tomato leaf mould–causing Fulvia fulva to seven succinate dehydrogenase inhibitor (SDHI) fungicides in Nara Prefecture, Japan and high efficacy of isofetamid in controlling SDHI‐resistant isolates

AbstractLeaf mould caused by Fulvia fulva is a major disease of tomato in Japan. Resistance to several types of fungicides, including succinate dehydrogenase inhibitors (SDHIs), has been reported in isolates from Japan. In this study, we evaluated the sensitivity of F. fulva to SDHI fungicides, SDHI fungicide efficacy in controlling sensitive and resistant isolates, and the presence of mutations in the Sdh B gene, the target site of SDHI fungicides. The minimum inhibitory concentration (MIC) of six SDHI fungicides, excluding isofetamid, ranged from 0.1 to >100 μg/mL against mycelial growth of 45 F. fulva isolates collected in Nara prefecture, Japan, whereas that of isofetamid ranged from 0.1 to only 10 μg/mL. The peak MIC distribution of fluopyram and isofetamid was 10 μg/mL, that of inpyrfluxam was 100 μg/mL and was >100 μg/mL for the remaining SDHI fungicides. In a greenhouse inoculation experiment, only isofetamid controlled the highly resistant isolates with an MIC of 10 μg/mL and the remaining fungicides had MIC >100 μg/mL. In addition, moderately and highly resistant isolates were controlled by the application of isofetamid 6 h or 7 days before inoculation or 3 days after inoculation. Mutations in the Sdh B gene were not observed among sensitive, moderately resistant and highly resistant isolates, suggesting that mutations in other Sdh genes and/or changes in efflux pump expression were involved. Our results indicate the presence of cross‐resistance and different levels of sensitivity reduction among SDHI fungicides. Isofetamid showed the lowest resistance development among the tested SDHI fungicides and is considered effective in controlling tomato leaf mould.

Evaluation of some bioactive compounds of <i>Azadirachta indica</i> extracts and its application as safe fungicide against different plant pathogenic fungi

Extracts of leaves, seeds and twigs of Azadirachta indica was carried out using Soxhlet extraction technique. These extracts were subjected to percent yield, in vitro phytochemical screening and antifungal activities (minimum fungicidal concentration (MFC), minimum inhibitory concentration (MIC), zone of inhibition and spore germination assay) against plant pathogenic fungi Cladosporium fulvum, Colletotrichum coccodes, Fusarium oxysporum and Rhizoctonia solani. The Soxhlet extractive solvents were methanol, ethanol, chloroform, ethyl acetate and n–hexane. The maximum extractive yield were calculated in ethanol leaves (26±1.5%), methanol leaves (22±01%), n–hexane seeds (32±0.8%) and ethanol seeds (24±01%), while twigs calculated minimum extractive yield. The phytochemicals screening findings confirmed the occurrence of terpenoids, phenols, flavonoids, glycosides, alkaloids, tannins and saponins. The antifungal activities observed that leaves and seeds extracts of neem might cause the inhibition of mycelium and spore germination of tested fungi, though the antifungal activities rate of under study fungal strains diverse with dissimilar concentration and nature of extracts. However the entire concentrations of extract and crude extract especially the methanol and ethanol extracts of neem seeds and leaves reduce the plant pathogenic fungi growth at noteworthy rate. It was concluded from our results that neem leaves and seeds extracts were effectual as antifungal agents against the entire under study fungal strains however the most sensitive fungi were Rhizoctonia solani and Fusarium oxysporum. The findings acquired from this study revealed that chemical constituents from neem seeds and leaves may be formulated as latent agrochemical fungicides.

AlphaFold-Multimer predicts cross-kingdom interactions at the plant-pathogen interface

Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Here, we used AlphaFold-Multimer (AFM) to screen 1879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato. This screen of 11,274 protein pairs identified 15 non-annotated SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal pathogens Cladosporium fulvum and Fusarium oxysporum, respectively. Together with a P69B inhibitor from the bacterial pathogen Xanthomonas perforans and Kazal-like inhibitors of the oomycete pathogen Phytophthora infestans, P69B emerges as an effector hub targeted by different microbial kingdoms, consistent with a diversification of P69B orthologs and paralogs. This study demonstrates the power of artificial intelligence to predict cross-kingdom interactions at the plant-pathogen interface.

Aislamiento, caracterización cultural y morfológica de aislados monospóricos del agente causal del moho gris de la hoja de Solanum lycopersicum L. en la provincia de Tungurahua.

Gray leaf mold, caused by the ascomycete fungus Passalora fulva (Cooke) U. Braun & Crous (anamorph: Cladosporium fulvum), is one of the most common fungal diseases affecting tomato cultivation in Ecuador. This study aimed to isolate monosporic cultures from tomato leaves exhibiting signs of gray leaf mold and characterize them culturally and morphologically. The investigation was conducted in the Tungurahua province, where samples were collected from eight municipalities. Eighty monosporic isolates of C. fulvum were obtained from leaves affected by gray mold. The fungal isolates exhibited olive-green colonies on the obverse and black on the reverse, with raised surfaces, felty texture, and irregular shape. The majority of isolates displayed lobed edges. No pigmentation or exudates were observed in the colonies. Mycelial growth was measured at 7 and 14 days, with average velocities of 0.93 mm/day and 1.23 mm/day, respectively. The average hyphal diameter was 5.41 µm, while conidiophores had an average size of 33.01 µm, and conidia measured 6 µm in length and 5 µm in width. These characteristics correspond to the reported morphological descriptions of C. fulvum. Keywords: Cladosporium fulvum, cultural characterization, gray mold, tomato, morphology

Characterization of bifunctional alginate lyase Aly644 and antimicrobial activity of enzymatic hydrolysates.

An alginate lyase gene aly644 encoding a member of polysaccharide lyase family 6 was obtained from a metagenome of Antarctic macroalgae-associated microbes. The gene was expressed heterologously in Escherichia coli, and the recombinant protein was purified using a Ni-NTA His Tag Kit. With sodium alginate as the substrate, recombinant Aly644 exhibited an optimum reaction temperature of 50°C and an optimum reaction pH of 7.0. The Vmax and Km values of Aly644 toward sodium alginate were 112.36 mg/mL·min and 16.75 mg/mL, respectively. Substrate specificity analysis showed that Aly644 was a bifunctional alginate lyase that hydrolyzed both polyguluronic acid and polymannuronic acid. The hydrolysis products of Aly644 with sodium alginate as the substrate were detected by thin-layer chromatography, and were mainly di- and trisaccharides. The oligosaccharides produced by degradation of sodium alginate by Aly644 inhibited the mycelial growth of the plant pathogens Phytophthora capsici and Fulvia fulva; the 50% maximal effective concentration (EC50) values were 297.45 and 452.89 mg/L, and the 90% maximal effective concentration (EC90) values were 1341.45 and 2693.83 mg/L, respectively. This highlights that Aly644 is a potential candidate enzyme for the industrial production of alginate oligosaccharides with low degree of polymerization. Enzyme-hydrolyzed alginate oligosaccharides could support the development of green agriculture as natural antimicrobial agents. KEY POINTS: • An alginate lyase was obtained from a metagenome of Antarctic macroalgae-associated microbes. • Aly644 is a bifunctional alginate lyase with excellent thermostability and pH stability. • The enzymatic hydrolysates of Aly644 directly inhibited Phytophthora capsici and Fulvia fulva.

Hexaconazole and binary mixtures of it plus fludioxonil efficiently control tomato leaf mold caused by Cladosporium fulvum

Leaf mold caused by Cladosporium fulvum is one of the most severe foliar diseases of tomato. We were interested in whether we could use a binary mixture of hexaconazole and fludioxonil for the management of tomato leaf mold. The results showed that hexaconazole with an EC50 value of 0.30 μg/ml could significantly inhibit the mycelial growth of C. fulvum. A total of 126 isolates collected from Shandong Province were used to determine their sensitivity to hexaconazole, and the data fit a bimodal curve with a mean EC50 value of 4.69 μg/ml. The optimal proportion of the binary mixture of hexaconazole and fludioxonil against C. fulvum was 1:2, and its synergistic ratio (SR) was 2.23. Field trials in 2018 and 2019 indicated that both hexaconazole and the binary mixture of hexaconazole and fludioxonil (1:2) had favorable protective and curative efficacy against tomato leaf mold. Applying hexaconazole at 67.50 g/ha resulted in higher efficacy than did flusilazole at 75.00 g/ha, which had been registered in China to control tomato leaf mold. In addition, a binary mixture of hexaconazole and fludioxonil (1:2) at 60.75 g/ha had an efficacy of 85%, which was significantly higher than that of a commercial binary mixture of fluopyram and trifloxystrobin at 150.00 g/ha. Even when applied at 20.25 g/ha, hexaconazole could also provide better efficacy than thiophanate-methyl could regardless of whether it was applied before or after the occurrence of tomato leaf mold. In summary, hexaconazole and its binary mixture with fludioxonil (1:2) could be favorable candidates for the management of tomato leaf mold.

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 2.6cM 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.