Gene expression analysis of ABC- and MFS-transporters in the fungal biocontrol agent Clonostachys rosea

The aim of this work was to provide a validation of 6 putative reference genes that could be included in a gene expression study related to the beneficial isolate Clonostachys rosea IK726. No data are available about validation of reference genes in C. rosea. In the present work the selection of these genes was based both on published data referred to other biological systems and on the available Expressed Sequence Tags (ESTs) from C. rosea IK726. Several growth conditions were taken into account for C. rosea IK726, whose mRNA was extracted and retro-transcribed into cDNA and then used as template for Real Time quantitative PCR (RT-qPCR). Two different excel based algorithms – ‘NormFinder’ and ‘BestKeeper’ – were chosen in order to analyze expression data and find the ‘best’ reference gene among chosen ones. From statistical analysis, actin was chosen and used as reference gene for further expression analysis. In the latter part of this work, the expression level of 8 putative genes encoding ABC- and MFS-transporters – which were selected according to C. rosea IK726 ESTs data – was evaluated on different treatments such as fungal-fungal interaction, zearalenone (ZEA, mycotoxin) and four different fungicides. ‘Delta-delta method’ was adopted as relative quantification method using actin as reference gene

Draft Whole-Genome Sequence of Trichoderma gamsii T6085, a Promising Biocontrol Agent of Fusarium Head Blight on Wheat

Trichoderma gamsii T6085 is a promising beneficial isolate whose effects consist of growth inhibition of the main agents causing Fusarium head blight, reduction of mycotoxin accumulation, competition for wheat debris, and reduction of the disease in both the lab and the field. Here, we present the first genome assembly of a T. gamsii isolate, providing a useful platform for the scientific community

Draft Whole-Genome Sequence of the Biocontrol Agent Trichoderma harzianum T6776

Trichoderma harzianum T6776 is a promising beneficial isolate whose effects consist of growth promotion, positive response of photosynthetic activity, hormonal signaling, and carbon partitioning in tomato, coupled with biocontrol of plant pathogens. Here, we present the first genome assembly of T6776, providing a useful platform for the scientific community

Molecular diversity of anthracnose pathogen populations associated with UK strawberry production suggests multiple introductions of three different Colletotrichum species.

Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production

Insights on the Evolution of Mycoparasitism from the Genome of Clonostachys rosea

Peer reviewe

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum

Background: Many species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi. Results: We sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc. Conclusions: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content

The cross-talk between Trichoderma gamsii and Fusarium graminearum at distance: a genome-wide transcriptome analysis

The present thesis reported on the cross-talk at distance between the beneficial fungus Trichoderma gamsii T6085 and the mycotoxigenic fungal pathogen Fusarium graminearum ITEM 124 (one of the main causal agents of Fusarium Head Blight disease, FHB). The cross-talk was investigated both at the physiological and the molecular levels. Previous studies have already reported on the mycoparasitic behaviour showed by T. gamsii T6085 against F. graminearum ITEM 124 and on its ability to reduce both the incidence and severity of FHB in field conditions. Furthermore, T. gamsii T6085 is able to compete for substrate possession with the pathogen and to reduce deoxynivalenol (DON) production. We carried out a genome-wide transcriptomic analysis to elucidate the molecular cross-talk at the sensing phase, i.e. before contact, between the two fungi. To this end, their genomes were sequenced and used as platforms for transcriptomics. The gene expression patterns of the two fungi were regulated in opposite ways. While an overall up-regulation of gene expression was recorded in F. graminearum ITEM 124 (‘buzzing mode’), gene expression in T. gamsii T6085 was predominantly down-regulated (‘stealth mode’). Moreover, F. graminearum ITEM 124 accelerated in presence of T. gamsii T6085 before contact, supporting the observed increased metabolic activity. The annotation and functional characterization of genes differentially expressed during the sensing phase revealed that competition for iron occurred. In fact, while T. gamsii T6085 up-regulated a ferric reductase involved in iron uptake, F. graminearum ITEM 124 down-regulated several genes involved in stress responses having iron as cofactor. Furthermore, while F. graminearum ITEM 124 up-regulated its entire repertoire of killer toxin-like chitinases, T. gamsii T6085 decreased the degree of interaction at distance by down-regulating six extracellularly secreted chitinases, supporting the ‘stealth mode’. This study showed how the communication between Trichoderma and Fusarium is definitely regulated before contact. The study of gene expression profiles unravelled a strongly different behaviour of the two fungi when they face each other. The ‘buzzing’ and ‘stealth’ modes showed by F. graminearum ITEM 124 and T. gamsii T6085, respectively, at distance opened new fields of investigation towards the successful development of T. gamsii T6085 as biocontrol agent

First report of Colletotrichum godetiae causing grape (Vitis vinifera) berry rot in Italy

In October 2016, rotting grape berries were detected on grapevine (Vitis vinifera) in Livorno (Nugola, Tuscany, Italy). Symptoms on grape berries skins varied from circular brown spots to rotting fruits. Both berries and petioles were covered with creamy salmon-colored masses of conidia. Rotten grape berries loss turgor and turn into ‘mummies’ (e-Xtra 1) over time. Symptoms suggested that a member of the genus Colletotrichum could be involved. Single spore cultures were obtained from conidial masses and grown in the laboratory at 25°C with a 12 hour light period on potato dextrose agar (PDA). Monoconidial isolates had light grey cottony aerial mycelium with colony color ranging from whitish to dark grey, while the reverse ranged from whitish to salmon-pink. Conidia were hyaline and unicellular, cylindrical or clavate and often with a light median constriction. However, Colletotrichum spp. are often difficult to distinguish morphologically. Total genomic DNA was extracted from monoconidial isolate SS354. The ITS region of rDNA and partial GAPDH, CHS-1, HIS3, ACT and TUB2 genes were amplified and sequenced according to Damm et al. (2012). Sequences were deposited in GenBank (Accession No. KY293406 for ITS, KY293407 for TUB, KY293403 for CHS, KY293405 for HIS3, KY293402 for ACT and KY293404 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed that the SS354 isolate clustered within C. godetiae (e-Xtra 2). Pathogenicity tests were performed in laboratory by inoculating detached grape berries with or without petioles at the petiole insertion point with 20 µL of a conidial suspension (105 conidia/mL) of the isolate SS354. Grape berries without petiole developed symptoms similar to those observed in the field. Fungal colonies re-isolated from the lesions on berries were morphologically identical to isolate SS354. Control grape berries inoculated with sterile water remained healthy as well as grape berries with petioles inoculated with the pathogen. This suggests that C. godetiae is able to infect wounded grape berries. However, information regarding other infection routes were not searched, as this was not the aim of this work. This is the first report of C. godetiae causing grape berry rot in Italy. The phylogenetic analysis reveals that C. godetiae SS354 is closely related to C. godetiae RB118, the causal agent of anthracnose on grapevine in UK (Baroncelli et al. 2014). Since C. godetiae is polyphagous, cross-infections between grape and other crops are possible. Remarkably, Cacciola et al. (2012) reported C. clavatum (syn. C. godetiae) as the prevalent Colletotrichum species associated with epidemic outbreaks of olive anthracnose in Italy. However, at present no information regarding cross-infection of C. godetiae between grapevine and olive are available. Due to the high economic and social value of wine production in Italy (in 2013 only in Tuscany the production of grapes accounted for 8 million tons), a monitoring plan based on simple molecular identification tools should be advisable.Total genomic DNA was extracted from one monoconidial isolate (SS354) and the ITS region of rDNA was amplified, using the universal primers ITS4 and ITS5, then sequenced. The resulting sequence was 100% identical to those of C. acutatum species complex obtained by a BLAST search in GenBank. Based on Damm et al. (2012) five other loci were used to further characterise the isolate: partial GAPDH, CHS-1, HIS3, ACT and TUB2 gene sequences were amplified and sequenced. Sequences were deposited in GenBank (Accession No. KY293406 for ITS, KY293407 for TUB, KY293403 for CHS, KY293405 for HIS3, KY293402 for ACT and KY293404 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained sequences and reference sequences (Damm et al. 2012) revealed that the SS354 clustered within C. godetiae (e-Xtra 2). Pathogenicity tests were performed in laboratory. 20 µL of a conidial suspension (105 conidia/mL) of C. godetiae SS354 was inoculated at the petiole insertion point on detached grape berries with or without petioles. Grape berries without petiole developed primary symptoms similar to those observed in the field. Fungal colonies re-isolated from lesions were morphologically identical to C. godetiae SS354. Control grape berries inoculated with sterile water remained healthy as well as grape berries with petioles inoculated with the pathogen. This latter evidence suggests that C. godetiae is able to infect grape berries from wounded tissues. However, information regarding other infection routes were not searched, as this was not the aim of this work. This is the first report of C. godetiae causing anthracnose on grapevine in Italy. The phylogenetic analysis reveals that C. godetiae SS354 is very close to C. godetiae RB118, the causal agent of anthracnose on grapevine in UK (Baroncelli et al., 2014). Since C. godetiae is a polyphagous pathogen, cross-infections between grape and other crops are possible. Remarkably, Cacciola and colleagues (2012) reported C. clavatum (synonymous to C. godetiae) as the prevalent Colletotrichum species associated with epidemic outbreaks of olive anthracnose in Italy. However, at present no information regarding cross-infection of C. godetiae between grapevine and olive are available. Due to the high economic and social value of wine production in Italy, in 2013 grapes production accounted for 8 million tonnes with 2.6 million hectoliters of wine production only in Tuscany, a monitoring plan based on simple molecular identification tools should be advisable