Stefania Pollastro

Sexual Behaviour and Mating System of Botryotinia fuckeliana, Teleomorph of Botrytis cinerea

Summary: Analysis of 213 field isolates of Botrytis cinerea and 240 ascospore isolates of its sexual form Botryotinia fuckeliana indicated that sexual compatibility of this fungus is controlled by a single mating type gene with two alleles. Most isolates were heterothallic, that is, they were self-sterile and able to produce ascospore progeny when crossed with reference strains carrying the mating type gene MAT1-1 or MAT1-2. About 16% of the field isolates and 6% of the ascospore progeny were homothallic, that is, self fertile and compatible with both MAT1-1 and MAT1-2 strains. Both mating types are widespread in nature. The close association of MAT1-1 and MAT1-2 field isolates on various hosts in several regions of Italy shows that sexual reproduction and meiotic recombination might be an important source of genetic variation in this pathogenic fungus.

Characterization and genetic analysis of field isolates ofBotryotinia fuckeliana (Botrytis cinerea) resistant to dichlofluanid

Field isolates ofBotryotinia fuckeliana were collected from naturally infected plants. Their responses to the multisite fungicide dichlofluanid in mycelium growth test fell into three phenotypic classes, characterized by the following EC50 (and MIC) values inμg ml−1: sensitivity, 1–3 (6–10); low resistance, 3–10 (> 100); high resistance, 10–30 (> 100). The corresponding values obtained for these classes in a spore germination test were respectively: ≅ 0.05 (0.2), 0.05–0.1 (0.5), 0.5–1 (0.9–1.5). Resistant isolates were crossed with two sensitive and two resistant strains of appropriate mating type to determine the genetic basis of resistance. Distribution of resistance phenotypes in ascospore progeny indicated that a gene, namedDic1, was probably responsible for the low or high resistance of 14 mutants selectively collected from experimental plots of greenhouse-grown gerbera sprayed several times with dichlofluanid or tolyfluanid. A second gene, namedDic2, was probably responsible for the low resistance displayed by two isolates (from grapevine and from carnation) maintained in the laboratory collection. As a result of the investigation, the use of dichlofluanid in integrated management programmes against grey mould is discussed.

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.

RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.

Observations on the Fungi Associated with Esca and on Spatial Distribution of Esca-Symptomatic Plants in Apulian (Italy) Vineyards

The paper reports the results of observations on the fungi associated with deteriorated wood of esca affected vines and the spatial distribution of diseased plants in 21 vineyards located in Apulia (Southern Italy). Examination of over 43,000 plants revealed that the incidence of plants showing symptoms of esca was 12% (5-18%) in vineyards younger than 10 years and 22% (4-54%) in older ones. The most common deteriorations of the wood were a white rot of soft consistency and a dark brown discoloration of a hard consistency including longitudinal black streaks. Mycological analysis was carried out on 554 diseased vines from 5 vineyards. Phellinus sp. was frequently isolated from white rot, whereas Phaeoacremonium spp., Botryosphaeria spp. and Eutypa lata were more often isolated from dark brown tissues. Preliminary observations of maps of plants with symptoms of esca seem to indicate a tendency to aggregation, especially in young vineyards.

Usage of Molecular Markers (PCR-RAPD) for Studying Genetic Variability in "Phellinus" ("Fomitiporia") sp.

PCR-RAPD was used to explore the genetic variability in Phellinus (Fomitiporia) sp. isolates from escaaffected vines. The use of 20 random primers yielded 180 polymorphic markers. Cluster-analysis grouped isolates by their origin, geographical location and host plant, but not by their tentative identification as Phellinus sp. or F. punctata. This shows that all isolates tested belonged to a single species, probably F. punctata. Observations carried out on samples of isolates representative of the population of Phellinus sp. present in each of two vineyards indicated that clonal plant-to-plant propagation of the fungus did not occur and that infections were probably caused by basidiospores. RAPD markers common to all tested isolates of Phellinus sp. (F. punctata), but never observed with other grape-associated fungi, were identified and are now being exploited to set up diagnostic techniques based on PCR or molecular probes.

Specific SCAR Primers for Fungi Associated with Wood Decay of Grapevine

RAPD (Random Amplified Polymorphic DNA) analysis, a technique based on the polymerase chain reaction, was applied to explore variation in 178 isolates of Fomitiporia punctata, 94 of Phaeomoniella chlamydospora and 34 of Phomopsis viticola, selected as being representative of fungal populations from different vineyards and locations. The analysis showed a broad genetic variability in F. punctata and a very high genetic uniformity in P. chlamydospora. With P. viticola, isolates belonging to different vegetative compatibility groups were investigated; the analysis evidenced high genetic similarity among isolates within groups and broad inter-group variation. For each pathogen, specific RAPD markers were selected, cloned and sequenced. The obtained sequences were used to design sequence-characterised amplified region (SCAR) primers specific for each pathogen. These are being used to develop molecular diagnostic tools.

Global Transcriptome Analysis and Identification of Differentially Expressed Genes in Strawberry after Preharvest Application of Benzothiadiazole and Chitosan

The use of resistance inducers is a novel strategy to elicit defense responses in strawberry fruit to protect against preharvest and postharvest decay. However, the mechanisms behind the specific resistance inducers are not completely understood. Here, global transcriptional changes in strawberry fruit were investigated using RNA-Seq technology. Preharvest, benzothiadiazole (BTH) and chitosan were applied to the plant canopy, and the fruit were harvested at 6, 12, and 24 h post-treatment. Overall, 5,062 and 5,210 differentially expressed genes (fold change ≥ 2) were identified in these fruits under the BTH and chitosan treatments, respectively, as compared to the control expression. About 80% of these genes were differentially expressed by both elicitors. Comprehensive functional enrichment analysis highlighted different gene modulation over time for transcripts associated with photosynthesis and heat-shock proteins, according to elicitor. Up-regulation of genes associated with reprogramming of protein metabolism was observed in fruit treated with both elicitors, which led to increased storage proteins. Several genes associated with the plant immune system, hormone metabolism, systemic acquired resistance, and biotic and abiotic stresses were differentially expressed in treated versus untreated plants. The RNA-Seq output was confirmed using RT-qPCR for 12 selected genes. This study demonstrates that these two elicitors affect cell networks associated with plant defenses in different ways, and suggests a role for chloroplasts as the primary target in this modulation of the plant defense responses, which actively communicate these signals through changes in redox status. The genes identified in this study represent markers to better elucidate plant/pathogen/resistance-inducer interactions, and to plan novel sustainable disease management strategies.