First report of grapefruit rot caused by Colletotrichum gloeosporioides and C. karsti in France.

The grapefruit (Citrus paradisi) is a citrus hybrid tree (C. maxima & C. sinensis). Due to nutritional value and its bioactive compounds, the fruits are recognized as a functional food, valued as promoting health. French grapefruit production is low (7.5 Kt/year) but is confined to a restricted area in Corsica and benefits from a quality label, the economic impact of its cultivation being therefore locally significant. Since 2015 previously unreported symptoms have been repeatedly observed on grapefruits in more than half of the orchards in Corsica, with an incidence of 30% of fruits altered. Brown to black circular spots were observed on fruits and leaves, surrounded by chlorotic halos on the latter. On the mature fruit, lesions were round, 4 to 10 mm in diameter, brown and dry (e-Xtra 1). Although the lesions are superficial, the fruits cannot be marketed due to constraints linked to the quality label. 75 fungal isolates were obtained from symptomatic fruits or leaves collected in Corsica (in 2016, 2017, and 2021). Cultures obtained after 7 days on PDA at 25°C, were white to light grey in colour, forming concentric rings or dark spots on the agar surface. We did not observe any notable difference among the isolates except some evolved towards a more marked grey. Colonies tend to form a cottony aerial mycelium and orange conidial masses appear with age. The conidia were hyaline, aseptate, cylindrical with ends rounded, and measured 14.9 ± 0.95 µm length and 5.1 ± 0.45 µm width (n = 50). Cultural and morphological characteristics were similar to those described for C. gloeosporioides s. lat. or C. boninense s. lat. (Weir et al. 2012 ; Damm et al. 2012). Total genomic DNA was extracted from all isolates, and the ITS region of rDNA was amplified with ITS 5 & 4 primers, then sequenced (GenBank Accession Nos. OQ509805-808). For 90% of isolates GenBank BLASTn results were 100% identical to C. gloeosporioides isolates sequences, whereas for other isolates the resulting sequences were 100% identical to C. karsti or C. boninense isolates sequences. Four strains (three C. gloeosporioides with light colour differences, in order to see if there was diversity among isolates of C. gloeosporioides s. lato ; and one C. karsti) were further characterized by sequencing partial actin [ACT], calmodulin [CAL], chitin synthase [CHS-1], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ß-tubulin 2 [TUB2], for all strains ; glutamine synthetase [GS], Apn2-Mat1-2-1 intergenic spacer and partial mating type (Mat1-2) gene [ApMAT] for C. gloeosporioides s. lat., and HIS3 for C. boninense s. lat. (Weir et al. 2012 ; Silva et al, 2012) (GenBank Accession Nos. OQ509805-808 & OQ507698-724). Multilocus phylogenetic analyses carried out with the obtained and Genbank available sequences confirmed that 3 isolates (UBOCC-A-116036, -116038, & -116039) clustered within C. gloeosporioides s. s., while the other (UBOCC-A-116037) clustered within C. karsti (e-Xtra 2) 'Star ruby' grapefruits were surface sterilized then wound-inoculated with 20 µl of a conidial suspension (105 conidia ml-1) of UBOCC-A-116036 & 116037 isolates or 20 µl sterile water for control (ten fruits for each isolate or control). After 10 days incubation at 20°C, symptoms, identical to those initially observed, developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies re-isolated from the lesions were morphologically like the original isolates. Recently, various infections caused by some Colletotrichum sp. have strongly compromised citrus production in different Mediterranean countries: ie Italy (Aiello et al. 2015), Portugal (Ramos et al. 2016), Tunisia (Ben Hadj Daoud et al. 2019), Turkey (Uysal et al. 2022). In these studies, C. gloeosporioides s. s. and C. karsti were identified as the causal agents. These two species were the predominant Colletotrichum sp. associated with Citrus and allied genera in Europe (Guarnaccia et al. 2017). To our knowledge, our study is the first report of C. gloeosporioides and C. karsti causing anthracnose on grapefruit in France, which confirms the incidence of these two pathogens on the Mediterranean rim. Given the economic importance of citrus cultivation in the Mediterranean region, the presence of Colletotrichum spp. should deserves to be monitored, and a control strategy should be considered.

Phylogenetic Diversity and Effect of Temperature on Pathogenicity of Colletotrichum lupini.

Although lupin anthracnose caused by Colletotrichum lupini is a significant threat for spring and winter lupin crops, it has been poorly studied so far. This study aimed at characterizing the (i) phylogenetic, (ii) morphological, and (iii) physiological diversity of collected isolates from anthracnose-affected lupins. The genetic identification of representative isolates (n = 71) revealed that they were all C. lupini species, further confirming that lupin anthracnose is caused by this species. However, multilocus sequencing on these isolates and 16 additional reference strains of C. lupini revealed a separation into two distinct genetic groups, both of them characterized by a very low genetic diversity. The diversity of morphological characteristics of a selected subset of C. lupini isolates was further evaluated. To the best of our knowledge, microsclerotia production observed for some isolates has never been reported so far within the Colletotrichum acutatum species complex. Finally, the modeling of growth responses of a subset of C. lupini strains revealed the capacity of some strains to grow in vitro at 5°C. This ability was also evidenced in planta, because C. lupini DNA was detectable in plants from 14 days postinoculation at 5°C onward, whereas symptoms began to appear a week later, although at a very low level. Since lupin crops are planted during winter or early spring, growth studies in vitro and in planta demonstrated the capability of the species to grow at temperatures ranging from 5 to 30°C, with an optimum close to 25°C. In this study, C. lupini-specific primers were also designed for real-time quantitative PCR on fungal DNA and allowed the detection of C. lupini in asymptomatic field samples. These results open perspectives to detect earlier and limit the development of this pathogen in lupin crops.

Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates.

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi.

Isolation, Identification and Enzymatic Activity of Halotolerant and Halophilic Fungi from the Great Sebkha of Oran in Northwestern of Algeria.

The Great Sebkha of Oran is a closed depression located in northwestern of Algeria. Despite the ranking of this sebkha among the wetlands of global importance by Ramsar Convention in 2002, no studies on the fungal community in this area have been carried out. In our study, samples were collected from two different regions. The first region is characterized by halophilic vegetation and cereal crops and the second by a total absence of vegetation. The isolated strains were identified morphologically then by molecular analysis. The biotechnological interest of the strains was evaluated by testing their ability to grow at different concentration of NaCl and to produce extracellular enzymes (i.e., lipase, amylase, protease, and cellulase) on solid medium. The results showed that the soil of sebkha is alkaline, with the exception of the soil of cereal crops that is neutral, and extremely saline. In this work, the species Gymnoascus halophilus, Trichoderma gamsii, the two phytopathogenic fungi, Fusarium brachygibbosum and Penicillium allii, and the teleomorphic form of P. longicatenatum observed for the first time in this species, were isolated for the first time in Algeria. The halotolerance test revealed that the majority of the isolated are halotolerant. Wallemia sp. and two strains of G. halophilus are the only obligate halophilic strains. All strains are capable to secrete at least one of the four tested enzymes. The most interesting species presenting the highest enzymatic index were Aspergillus sp. strain A4, Chaetomium sp. strain H1, P. vinaceum, G. halophilus, Wallemia sp. and Ustilago cynodontis.

Development and application of MALDI-TOF MS for identification of food spoilage fungi.

Filamentous fungi are frequently involved in food spoilage and cause important food losses and substantial economic damage. Their rapid and accurate identification is a key step to better manage food safety and quality. In recent years, MALDI-TOF MS has emerged as a powerful tool to identify microorganisms and has successfully been applied to the identification of filamentous fungi especially in the clinical context. The aim of this study was to implement a spectral database representative of food spoilage molds. To this end, after application of a standardized extraction protocol, 6477 spectra were acquired from 618 fungal strains belonging to 136 species and integrated in the VITEK MS database. The performances of this database were then evaluated by cross-validation and ∼95% of correct identification to the species level was achieved, independently of the cultivation medium and incubation time. The database was also challenged with external isolates belonging to 52 species claimed in the database and 90% were correctly identified to the species level. To our best knowledge, this is the most comprehensive database of food-relevant filamentous fungi developed to date. This study demonstrates that MALDI-TOF MS could be an alternative to conventional techniques for the rapid and reliable identification of spoilage fungi in food and industrial environments.

Combined Metabarcoding and Multi-locus approach for Genetic characterization of Colletotrichum species associated with common walnut (Juglans regia) anthracnose in France.

Juglans regia (walnut) is a species belonging to the family Juglandaceae. Broadly spread in diverse temperate and subtropical regions, walnut is primarily cultivated for its nuts. In France, Colletotrichum sp. on walnut was detected for the first time in 2007; in 2011 the disease led to 50-70% losses in nut production. A combined approach of metabarcoding analysis and multi-locus genetic characterization of isolated strains has been used for taxonomic designation and to study the genetic variability of this pathogen in France. Evidence indicates that four Colletotrichum species are associated with walnut in France: 3 belong to the C. acutatum species complex and 1 to the C. gloeosporioides species complex. Results also show that C. godetiae is the most abundant species followed by C. fioriniae; while C. nymphaeae and another Colletotrichum sp. belonging to the C. gloeosporioides complex are found rarely. Representative isolates of detected species were also used to confirm pathogenicity on walnut fruits. The results show a high variability of lesion's dimensions among isolates tested. This study highlights the genetic and pathogenic heterogeneity of Colletotrichum species associated with walnut anthracnose in France providing useful information for targeted treatments or selection of resistant cultivars, in order to better control the disease.