Caterina Rotolo

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.

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.

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.

A TaqMan-based qPCR assay for quantitative detection of the biocontrol agents Bacillus subtilis strain QST713 and Bacillus amyloliquefaciens subsp. plantarum strain D747

Biological control agents (BCAs) play an important role in crop protection. They can improve sustainability, prevent resistance to fungicides in target pathogens and reduce fungicide residues on produce. Bacillus subtilis strain QST713 (Serenade Max; Bayer CropScience, Leverkusen, Germany) and Bacillus amyloliquefaciens subsp. plantarum strain D747 [Amylo-X; CBC Europe S.r.l.—Biogard Division, Nova Milanese (MB), Italy] are two commercially available BCAs that are used against grey mould on table grape and other crops. TaqMan-based quantitative (q)PCR assays were developed and validated for quantitative and specific detection of the two BCAs on grape bunches following field sprays. Specific molecular markers were developed for each BCA, and TaqMan probes were designed and tested in qPCR on DNA extracted from washing water of grape berries. The assay proved specific and sensitive for both BCAs allowing detection at density as low as three colony-forming units (CFU) per gram of berries. The method will be useful to investigate the population dynamics of the two BCAs following their applications in vineyards.

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.

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

BackgroundBrown 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.ResultsIllumina 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.ConclusionsThis 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.