Draft Genomic Resources for the Brown Rot Fungal Pathogen Monilinia laxa.

Monilinia laxa is the causal agent of brown rot on stone fruit, and it can cause heavy yield losses during field production and postharvest storage. This article reports the draft genome assembly of the M. laxa Mlax316 strain, obtained using a hybrid genome assembly with both Illumina short-reads and PacBio long-reads sequencing technologies. The complete draft genome consists of 49 scaffolds with total size of 42.81 Mb, and scaffold N50 of 2,449.4 kb. Annotation of the M. laxa assembly identified 11,163 genes and 12,424 proteins which were functionally annotated. This new genome draft improves current genomic resources available for M. laxa and represents a useful tool for further research into its interactions with host plants and into evolution in the Monilinia genus.

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits.

BACKGROUND: Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. RESULTS: Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. CONCLUSIONS: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena.

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology.

The Mycovirome in a Worldwide Collection of the Brown Rot Fungus Monilinia fructicola.

The fungus Monilinia fructicola is responsible for brown rot on stone and pome fruit and causes heavy yield losses both pre- and post-harvest. Several mycoviruses are known to infect fungal plant pathogens. In this study, a metagenomic approach was applied to obtain a comprehensive characterization of the mycovirome in a worldwide collection of 58 M. fructicola strains. Deep sequencing of double-stranded (ds)RNA extracts revealed a great abundance and variety of mycoviruses. A total of 32 phylogenetically distinct positive-sense (+) single-stranded (ss)RNA viruses were identified. They included twelve mitoviruses, one in the proposed family Splipalmiviridae, and twelve botourmiaviruses (phylum Lenarviricota), eleven of which were novel viral species; two hypoviruses, three in the proposed family Fusariviridae, and one barnavirus (phylum Pisuviricota); as well as one novel beny-like virus (phylum Kitrinoviricota), the first one identified in Ascomycetes. A partial sequence of a new putative ssDNA mycovirus related to viruses within the Parvoviridae family was detected in a M. fructicola isolate from Serbia. The availability of genomic sequences of mycoviruses will serve as a solid basis for further research aimed at deepening the knowledge on virus-host and virus-virus interactions and to explore their potential as biocontrol agents against brown rot disease.

A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits.

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy µL-1 of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R 2 = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.

Surface Dielectric Barrier Discharge plasma: a suitable measure against fungal plant pathogens.

Fungal diseases seriously affect agricultural production and the food industry. Crop protection is usually achieved by synthetic fungicides, therefore more sustainable and innovative technologies are increasingly required. The atmospheric pressure low-temperature plasma is a novel suitable measure. We report on the effect of plasma treatment on phytopathogenic fungi causing quantitative and qualitative losses of products both in the field and postharvest. We focus our attention on the in vitro direct inhibitory effect of non-contact Surface Dielectric Barrier Discharge on conidia germination of Botrytis cinerea, Monilinia fructicola, Aspergillus carbonarius and Alternaria alternata. A few minutes of treatment was required to completely inactivate the fungi on an artificial medium. Morphological analysis of spores by Scanning Electron Microscopy suggests that the main mechanism is plasma etching due to Reactive Oxygen Species or UV radiation. Spectroscopic analysis of plasma generated in humid air gives the hint that the rotational temperature of gas should not play a relevant role being very close to room temperature. In vivo experiments on artificially inoculated cherry fruits demonstrated that inactivation of fungal spores by the direct inhibitory effect of plasma extend their shelf life. Pre-treatment of fruits before inoculation improve the resistance to infections maybe by activating defense responses in plant tissues.

Global transcriptome analysis and differentially expressed genes in grapevine after application of the yeast-derived defense inducer cerevisane.

BACKGROUND: Cerevisane, made up of cell wall derivatives from the Saccharomyces cerevisiae strain LAS117, is proposed as a resistance inducer in plants. The mode of action of cerevisane was investigated through transcriptome analysis (RNA-Seq) carried out on leaves of potted vines cv. Italia grown in the greenhouse and sprayed at 1-week intervals with cerevisane. Analyses were performed at three time points after one and three sprays as well as on vines challenged with artificial inoculation with Plasmopara viticola, Erysiphe necator and Botrytis cinerea. RESULTS: Cerevisane proved effective against downy mildew and caused an increase in expression levels of several genes related to defense responses to fungal pathogens and other stresses and down-regulation of genes involved in several processes related to plant growth and development. Up-regulated genes included genes encoding (i) enzymes involved in hormone metabolism (i.e. salicylic acid, jasmonate, ethylene) and related plant responses, (ii) defense compounds (i.e. pathogenesis-related proteins, phenylalanine ammonia-lyase, stilbene synthases, lipoxygenase, leucine-rich repeat receptor-like protein kinases, non-specific plant lipid transfer proteins, serine-threonine protein kinases involved in signal transduction, superoxide dismutase and glutathione S-transferase involved in response to oxidative stress), (iii) secondary metabolites (i.e. phenylpropanoids, terpenoids, lignin), and (iv) photosynthetic processes (light harvesting chlorophyll A/B-binding proteins and components of the photosystems). CONCLUSION: Cerevisane can be a useful tool in protection schedules against downy mildew on grapevine aimed at reducing the usage of synthetic fungicides and preventing fungicide resistance. The results provide the first basic knowledge on the mode of action of yeast-derived elicitors effective against P. viticola on grapevine. © 2019 Society of Chemical Industry.

Transcriptome sequence resource for the cucurbit powdery mildew pathogen Podosphaera xanthii.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew in Southern Italy. Illumina sequencing of mRNA from two P. xanthii isolates of opposite mating types (MAT1-1 and MAT1-2) and their sexual cross was used to obtain a detailed de novo Trinity-based assembly of the transcriptome of the fungus. Over 60 million of high-quality paired-end reads were obtained and assembled into 71,095 contigs corresponding to putative transcripts that were functionally annotated. More than 55% of the assembled transcripts (40,221 contigs) had a significant hit in BLASTx search and included sequences related to sexual compatibility and reproduction, as well as several classes of transposable elements and putative mycoviruses. The availability of these new transcriptomic data and investigations on potential source of genetic variation in P. xanthii will promote new insights on the pathogen and its interactions with host plants and associated microbiome.

New High-Quality Draft Genome of the Brown Rot Fungal Pathogen Monilinia fructicola.

Brown rot is a worldwide fungal disease of stone and pome fruit that is caused by several Monilinia species. Among these, Monilinia fructicola can cause severe preharvest and postharvest losses, especially for stone fruit. Here, we present a high-quality draft genome assembly of M. fructicola Mfrc123 strain obtained using both Illumina and PacBio sequencing technologies. The genome assembly comprised 20 scaffolds, including 29 telomere sequences at both ends of 10 scaffolds, and at a single end of 9 scaffolds. The total length was 44.05 Mb, with a scaffold N50 of 2,592 kb. Annotation of the M. fructicola assembly identified a total of 12,118 genes and 13,749 proteins that were functionally annotated. This newly generated reference genome is expected to significantly contribute to comparative analysis of genome biology and evolution within Monilinia species.

Use of biocontrol agents and botanicals in integrated management of Botrytis cinerea in table grape vineyards.

BACKGROUND: There is increasing interest in the use of biological control agents (BCAs) and botanicals (BOTs) due to increasing awareness of the environmental and human health risks associated with synthetic plant protection products. The BCAs Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain D747 and Aureobasidium pullulans strains DSM14940 and DSM14941, and the BOTs Melaleuca alternifolia and terpenic extracts are proposed for the control of grey mould in vineyards. This study was aimed at evaluating their effectiveness in integrated crop management strategies and their outcomes in terms of the management of fungicide resistance and residues. RESULTS: In field trials carried out on table grapes in southern Italy, use of BCAs or BOTs alternately or mixtures of BCAs or BOTs with the succinate dehydrogenase inhibitor fungicide fluopyram showed efficacy of up to 96% against grey mould on bunches, comparable with the chemical reference strategy (up to 87%). By contrast, use of BCAs or BOTs (up to 11 sprays) alone was not effective (< 30%) under high disease pressure. The integrated use of BCAs or BOTs reduced the spread of succinate dehydrogenase inhibitor-resistant conidia, as well as fungicide residues in grapes. CONCLUSIONS: Spray schedules based on integration of BCAs or BOTs with fungicides are effective against grey mould and reduce the risk of fungicide resistance in B. cinerea and fungicide residues in grapes. © 2017 Society of Chemical Industry.

Occurrence of fungicide resistance in populations of Botryotinia fuckeliana (Botrytis cinerea) on table grape and strawberry in southern Italy.

BACKGROUND: Botryotinia fuckeliana (Botrytis cinerea) is a pathogen with a high risk of development of resistance to fungicides. Fungicide resistance was monitored during 2008-2011 in B. fuckeliana populations from both table-grape vineyards and greenhouse-grown strawberries in southern Italy. RESULTS: Isolates showing different levels of resistance to anilinopyrimidines (APs) were detected at high frequency (up to 98%) in fields treated intensively with APs (4-7 sprays season(-1) ). A slight decrease in sensitivity to fludioxonil, always combined with AP resistance, was generally found at lower frequencies. The repeated use of fenhexamid on grapevine (3-8 sprays season(-1) ) led to a strong selection of highly resistant isolates (up to 100%). Boscalid-resistant mutants were detected at very variable frequencies (0-73%). Occurrence of resistance to quinone outside inhibitors (QoIs) was also ascertained. Multiple fungicide resistance to 2-6 different modes of action were frequently recovered. Single nucleotide polymorphisms (SNPs) in the target genes Erg27, SdhB and cytb were associated with resistance to fenehexamid, boscalid and QoIs respectively. CONCLUSION: Resistance to the fungicides commonly used against grey mould on table grape and strawberry is quite common in southern Italy. This is an outcome of the incorrect use of fungicides, often because of the maximum number of detectable residues of plant protection products imposed by big international retailers, and underlines the crucial role of antiresistance strategies in integrated pest management.

Molecular characterisation and detection of resistance to succinate dehydrogenase inhibitor fungicides in Botryotinia fuckeliana (Botrytis cinerea).

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations. RESULTS: By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity (H272R), hypersensitivity (H272Y) or moderate resistance (H272V) to fluopyram. Allele-specific (AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes. CONCLUSION: Fluopyram-resistant mutants are currently rarely detected in the field sprayed with boscalid, but this may change with intensive exposure of the fungal population to fluopyram. PCR assays/methods developed in the study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventive measures.

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides.

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1-3 mg L(-1) trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.