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Maize (Zea mays) is one of the most important crops worldwide, and fungal diseases are responsible for major losses in food production. Anthracnose caused by Colletotrichum graminicola can infect all maize tissues, although stalk rot and seedling blight cause more significant economic damage (Munkvold and White, 2016). Anthracnose stalk rot is characterized by a distinctive external blackening of the lower stalks resulting in large black streaks, while the pith turns dark brown and has a shredded appearance. Like most stalk rots, the most obvious symptom is a sudden death of plants before grain maturity, and plant lodging. Symptoms commonly appear late in the season, suspicious maize stems of cultivar Tuy exhibiting symptoms of anthracnose stalk rot were collected from a field in Pontevedra, Galicia, Spain (Geographical coordinates: 42°23'27.1" N - 8°30'46.3" W) between June and December of 2022. Stem samples, approximately 50 mm2, were dissected and surface-disinfected for 90 seconds in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one half-strength acidified potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and lactic acid 90% (1.5 mL/L) and incubated for 5 days at 25 ºC (Sukno et al. 2008). Single spores were transferred to fresh PDA plates to obtain pure culture isolates. A total of six isolates were obtained, and among them, two were selected for further characterization (SP-36820-1 and SP-36820-3). Colonies grown on PDA have dark gray aerial mycelium with orange-colored spore masses. Conidia are falcate, slightly curved, tapered toward the tips, and are produced in acervuli with setae, measuring 37.65 to 24.84 x 8.02 to 4.67 µm, respectively (n = 100). These morphological characteristics are in agreement with C. graminicola previously described by Bergstrom and Nicholson (1999). Isolates were grown in potato dextrose broth (PDB) for 3 days at 25 ºC and total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The internal transcribed spacer region of rDNA and the manganese-type superoxide dismutase gene (SOD2) were amplified using primers ITS4/ITS5 (White et al. 1990) and SOD625/SOD507 (Fang et al. 2002) and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to strains of C. graminicola. All sequences were deposited in GenBank (see e-Xtra 1 for accession numbers). To confirm Koch’s postulates, plants of a derivative of maize inbred line Mo940 (developmental stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µL) of a suspension of 3 x 105 conidia per milliliter were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23ºC. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25ºC with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After four days inoculated leaves presented brown elongated lesions with necrotic centers consistent with C. graminicola infection, whereas control plants remained asymptomatic. The strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of Colletotrichum graminicola causing maize anthracnose in Spain. Recently, maize anthracnose was also reported in Bosnia and Herzegovina and China (Duan et al. 2019; Cuevas-Fernández et al. 2019), suggesting the pathogen’s geographic range is increasing, which may be a threat to maize cultivation in locations with optimal humid conditions for disease development.

Colletotrichum is one of the most diverse and destructive plant pathogenic fungi containing genus, responsible for significant losses in agriculture and forest plants. In the present study, we present the draft whole-genome sequence of two closely related species belonging to the Colletotrichum acutatum species complex: C. abscissum, the causal agent of citrus post-bloom fruit drop and C. filicis, a rare species described to accommodate an isolate obtained from an identified fern in Costa Rica. The data resources presented here will provide insights into genetic elements associated with citrus post-bloom fruit drop and into the evolution of Colletotrichum.

Leaf rust caused by Cerotelium fici (Cast.) Arth. is the main disease affecting Moraceae family plants, such as Ficus and Morus species (Galleti and Rezende 2016; Srikantaswamy et al. 2006). In August 2020, rust symptoms were observed in 100% of mulberry (Morus nigra L.) trees in an experimental orchard (Piracicaba, SP, Brazil; 22°42’28”S, 47°37’42”W). Mulberry leaves with high rust severity became yellowish and fell-off prematurely. Pustules were light brown with yellowish halo and presented mean size of 0.9 mm2. Uredinial paraphyses (n = 50) measured 42.2 ± 0.67 µm long with wall uniformly ca 0.6-1.1 μm thick. Urediniospores were brownish, echinulate, globoid to broadly ellipsoid, and measured 27.1 ± 0.29 × 21.0 ± 0.27 µm with a wall thickness of 0.6 ± 0.01 µm (n = 100). The morphology of the urediniospores observed in this study was similar to that reported in the literature for C. fici on Morus alba and Ficus spp. (Gupta et al. 1994; McKenzie 1986; Hennen et al. 2005). We used a low-coverage genome-skimming approach to retrieve genetic information of the rRNA cluster and the mtDNA. Genomic DNA was extracted from 3-4 mg of stored urediniospores at -80 °C, macerated in liquid nitrogen, using a modified cetyl trimethylammonium bromide extraction procedure (Lo Piccolo et al. 2012), and sequenced with 150-bp paired-end reads on Illumina NovaSeq 6000 System. Raw data, (45,761,957 X 2 reads) were assembled with SPAdes v3.15.1 (Bankevich et al., 2012) and the output used to create a custom BLAST database. Loci used for the phylogenetic analyses were identified by BLASTn using, as a query, sequences of C. fici from Ficus sp. from Australia publicly available: Accession No. MH047210.1 for the rRNA and MW036502.1 for COX3. The retrieved sequences were deposited in GenBank under accession numbers OM296992 and OP797407 for the partial rRNA cluster and COX3, respectively. The Bayesian inference phylogenetic analysis of the three concatenate loci (18S, 28S, and COX3) revealed that the isolate obtained in this study (MN1) was clustered in a well-supported clade with C. fici type species. Pathogenicity tests were conducted using mulberry potted plants under greenhouse conditions (25 ± 5 °C). The urediniospores suspension (5 × 104 urediniospores ml-1) with 0.05% Tween 20 was sprayed with an airbrush on fully expanded leaves until run-off. As a control, mulberry plants were sprayed with distilled water and kept under the same conditions. Inoculated and mock-inoculated plants were kept in a dark moist chamber at 23 °C (± 2 °C) for 24 h. After this period, plants were moved to the greenhouse. The experimental design was completely randomized with five replicates, each replicate consisted of one potted plant and the experiment was performed twice. At 12 days post-inoculation, all inoculated plants showed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. The first symptoms were small pustules on the abaxial surface of fully expanded leaves. Small chlorotic lesions were observed on the adaxial leaf surface, which evolved into necrotic lesions. The pathogen was re-inoculated into potted plants, where it was maintained through monthly inoculations. To our knowledge, this is the first report of mulberry rust on M. nigra in Brazil. As mulberry leaves are the only natural food for silkworm (Bombyx mori L.), rust poses a significant threat to the sericulture industry because the disease can decrease production and quality of mulberry foliage.

Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing.

Coniothyrium minitans (synonym, Paraphaeosphaeria minitans) is a highly specific mycoparasite of the wide host range crop pathogen Sclerotinia sclerotiorum. The capability of C. minitans to destroy the sclerotia of S. sclerotiorum has been well recognized and it is available as a widely used biocontrol product Contans WG. We present the draft genome sequence of C. minitans Conio (IMI 134523), which has previously been used in extensive studies that formed part of a registration package of the commercial product. This work provides a distinctive resource for further research into the molecular basis of mycoparasitism to harness the biocontrol potential of C. minitans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

The fungal genus Alternaria is a relevant pathogen for several commodities including citrus and pomegranate fruits. On citrus, it mainly causes brown spots on fruits and leaves, whereas on pomegranate, it mostly causes a fruit heart rot. In the present study the presence of Alternaria rots on citrus and pomegranate fruits cultivated in Albania was assessed. Representative fruits were collected from different regions. Nineteen and thirteen Alternaria spp. isolates were obtained from pomegranate and citrus samples, respectively. The isolates were identified at species and morphotype level. Micro and macroscopic features separated isolates into four morphotypes. BLAST and phylogenetic analysis using the SCAR Marker OPA1-3 confirmed the isolate identity. All 32 isolates proved to be Alternaria alternata and belonged mainly to morphotype alternata, followed by limoniasperae and tenuissima. All Alternaria strains proved to possess the pksI gene of alternariol biosynthesis. Citrus isolates were tested for the presence of genes of the biosynthesis of the phytotoxins ACT and ACR, but none of them proved to possess them. Concluding, Alternaria spp. might represent a treat to pomegranate and citrus production in Albania, and thus effective control means are needed.

Verticillium species are known as plant pathogens responsible for wilt diseases in a large variety of dicotyledon plants and crops in many parts of the world. Here we present the draft genome sequence of Verticillium dahliae Kleb. (strain VdGL16) isolated in Italy from the invasive alien species Ailanthus altissima (Mill.; commonly known as tree-of-heaven) showing Verticillium wilt symptoms. The comparison between the newly sequenced genome with those publicly available revealed candidate genes putatively involved in pathogenicity. The genome represents a new useful source for future research on Verticillium genetics and biology as well as research on novel approaches in the control of A. altissima.

The genus Stagonosporopsis is classified within the Didymellaceae family and has around 40 associated species. Among them, several species are important plant pathogens responsible for significant losses in economically important crops worldwide. Stagonosporopsis vannaccii is a newly described species pathogenic to soybean. Here, we present the draft whole-genome sequence, gene prediction, and annotation of S. vannaccii isolate LFN0148 (also known as IMI 507030). To our knowledge, this is the first genome sequenced of this species and represents a new useful source for future research on fungal comparative genomics studies.

Colletotrichum is a large genus of plant pathogenic fungi comprising more than 200 species. In this work, we present the genome sequences of four Colletotrichum species pathogenic to soybean: C. truncatum, C. plurivorum, C. musicola, and C. sojae. While C. truncatum is globally considered the most important pathogen, the other three species have been described and associated with soybean only recently. The genome sequences will provide insights into factors that contribute to pathogenicity toward soybean and will be useful for further research into the evolution of Colletotrichum.

Soybean (Glycine max L.) is one of the most important crops worldwide as a source of protein-rich foods and animal feeds. Anthracnose, one of the major limiting factors to soybean production (Dias et al. 2016), is caused by species such as Colletotrichum truncatum, C. sojae, and C. plurivorum (Damm et al. 2009, 2019). In December 2016 and 2017, soybean plants of cultivars Monsoy 8768 and Pioneer y-70 with typical symptoms of anthracnose (necrotic and irregular brown lesions on stems, leaves, and pods) were collected in Mato Grosso, Brazil. Commercial fields sampled showed 10 to 15% incidence of anthracnose in 1 ha in each sampled area. In total, 10 different geographic locations were sampled. Colletotrichum strains were isolated and cultured on potato dextrose agar at 25°C with a 12-h light photoperiod from surface-disinfected (70% alcohol followed by 0.5% sodium hypochlorite) plant tissues. Among others, three single-spore isolates (LFN0048 from Sinop, LFN0074 and LFN0090 from Lucas do Rio Verde) showed different morphology; isolates LFN0048 and LFN0074 were selected for further characterization. Total DNA was extracted and partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), and β-tubulin (TUB2) genes were amplified and sequenced. The sequences were deposited in GenBank (accession numbers MN604249 and MK163893 for HIS3, MN604248 and MK142674 for GAPDH, and MN604250 and MK142675 for TUB) and were compared with most similar reference sequences of Colletotrichum (Damm et al. 2019). Both isolates clustered with Colletotrichum musicola epitype (CBS 132885), showing 100 and 98.5% similarity in GAPDH, 99.5 and 98.9% in HIS3, and 99.2% in TUB2. On PDA, colonies showed dark-gray aerial mycelium with entire margins, reverse violaceous-black. Conidia and ascospore size and shape match those previously described by Damm et al. (2019): 12.12 to 15.86 × 4.93 to 6.95 µm and 15.5 to 19.34 × 5 to 7.84 µm, respectively (n = 100). Appressoria (n = 50) were single or in loose groups, violaceous-black with predominant obovoid, truncated, and cylindrical shapes, with smooth, undulate, or lobate margin, and 9.25 to 29.79 × 7.22 to 21.06 µm. Perithecia, paraphyses; and unitunicate eight-spored asci were also observed. Asci were cylindrical to clavate, smooth-walled, and 48.12 to 68.78 × 9.59 to 14.47 µm (n = 50). Soybean anthracnose is seed-borne (Dias et al. 2018; Rogério et al. 2017); therefore, pathogenicity tests were carried out on pregerminated seeds. Five seeds of Brasmax 8579 cultivar were inoculated with 10 µl of a conidial suspension (106 conidia/ml) that was placed in the emerging radicle, and five mock-inoculated seeds were used as a control. Seedlings were planted in vermiculite and incubated at 25°C with a 12-h photoperiod. After 7 days, inoculated plants showed necrotic lesions on the cotyledons, leaflets, and hypocotyl, whereas control plants remained asymptomatic. The experiment was repeated three times. C. musicola was reisolated from the symptomatic tissues, and the identity was confirmed by morphology and multilocus phylogeny. Until now, C. musicola has been reported to be associated with Musa sp. (Damm et al. 2019) and Colocasia esculenta (Vásquez-López et al. 2019) in Mexico, and with Phaseolus lunatus in Brazil (Cavalcante et al. 2018). To our knowledge, this is the first report of C. musicola joining a group of new and emergent species of Colletotrichum causing anthracnose in soybean-producing regions around the world.

Paraphaeosphaeria genus includes plant pathogens or biocontrol agents as well as bioremediators and endophytic fungi. Paraphaeosphaeria sporulosa 10515 was isolated in 2013 as an endophyte of Festuca spp. collected on Mount Etna at 1,832 meters above sea level. Here, we present the first-draft whole-genome sequence of a P. sporulosa endophytic isolate. This data will be useful for future research on understanding the genetic bases of endophytism.

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease. References: (1) R. Baroncelli. Colletotrichum acutatum sensu lato: From diversity study to genome analysis. Coventry, United Kingdom, PhD thesis, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012.

International audience; Bitter rot is one of the prevalent diseases of apple (Malus pumila Mill.) worldwide. The disease affects the fruit preharvest in orchards and/or postharvest in storage, resulting in considerable economic losses. Until recently the reported causal agents in Europe belong to the Colletotrichum acutatum species complex (Baroncelli et al. 2014; Nodet et al. 2016); however, species belonging to C. gloeosporioides species complex were reported in the United States (Munir et al. 2016), South America (Velho et al. 2018), Korea (Park et al. 2018), and recently in Belgium (Grammen et al. 2019). In September 2017, bitter rot symptoms were observed on apple fruit (cultivars Joya Cripps Red, Granny Smith, and Pink Lady) in four orchards in the region of Occitanie in France. The rot began as circular brown spots, 1 to 2 mm in diameter, which enlarged rapidly. Sixteen isolates were obtained from symptomatic apples by culturing pieces of necrotic tissue on potato dextrose agar. Cultures showed light-gray, cottony mycelium that became darker with age, with the reverse color being brownish and becoming black with age. Conidia were produced in small orange masses and were mainly cylindrical, with rounded ends. For all isolates, the production of perithecia was observed in culture, and asci and ascospores were observed under the microscope. The width and length of 50 conidia were examined and ranged from 3.1 to 4 µm (average 3.5 µm) and from 8 to 13 µm (average 10.5 µm), respectively. Based on these morphological characteristics, those isolates correspond to teleomorph of species belonging to C. gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from the 16 isolates, and the internal transcribed spacer region of rDNA was amplified using the universal primers ITS4 and ITS5 and then sequenced. For all isolates, the resulting sequences were 100% identical to C. fructicola sequences obtained by a BLAST search in GenBank. Three other loci (partial GAPDH, TUB2, and ApMat genes) were amplified and sequenced to further characterize two isolates (UBOCC-A-118064 and UBOCC-A-118065; GenBank accession nos. MK114103 to MK114110, respectively). Multilocus phylogenetic analysis carried out with the obtained and reference sequences (Da Lio et al. 2018) revealed that the isolates clustered within C. fructicola, as suggested by the BLAST results; this is also consistent with their initial identification as C. gloeosporioides. To confirm Koch’s postulates, for the two characterized isolates, 10 ‘Golden Delicious’ apples were surface sterilized and then wound inoculated with 20 μl of a conidial suspension (105 conidia/ml). After 10 days of incubation at 20°C, symptoms identical to those initially observed developed around the inoculation point, whereas controls inoculated with water remained symptomless. Fungal thalli reisolated from the lesions were morphologically similar to the original isolate. To our knowledge, this is the first report in Europe of C. fructicola causing bitter rot on apple. According to Munir et al. (2016), C. fructicola was reported to be more aggressive than species belonging to the C. acutatum species complex such as C. fioriniae previously identified in France (Nodet et al. 2016); moreover, this species is associated with Glomerella leaf spot, an emerging leaf disease never described in Europe (Velho et al. 2018). All those points encourage the development of species-specific management strategies for this pathogen in European countries.

In spring 2019, an anthracnose-like disease was observed on fruit in pomegranate orchards of cv. Wonderful in the Fier region of Albania. Disease prevalence in the assayed area ranged from 8 to 10%. Symptoms appeared as light to dark brown circular, sunken lesions with increasing diameter, which then merged. This study aimed to characterize the causal agent on the basis of morphology, molecular phylogeny, and pathogenicity. Micro- and macro-morphological features revealed that the disease-associated fungus belonged to Colletotrichum gloeosporioides species complex. A multi-locus molecular phylogeny using partial sequences of barcoding genes/regions (ITS, ACT, CHS-1, GAPDH, TUB2, CAL, GS, and ApMat) ascribed strain CG1 to C. gloeosporioides sensu stricto (s.s.). Pathogenicity tests, performed by inoculating healthy fruit, confirmed C. gloeosporioides s.s. as causal agent of anthracnose on pomegranate fruit in Albania.

Colletotrichum orchidophilum is a plant-pathogenic fungus infecting a wide range of plant species belonging to the family Orchidaceae. In addition to its economic impact, C. orchidophilum has been used in recent years in evolutionary studies because it represents the closest related species to the C. acutatum species complex. Here, we present the first-draft whole-genome sequence of C. orchidophilum IMI 309357, providing a resource for future research on anthracnose of Orchidaceae and other hosts.

The ability of Trichoderma spp. to antagonize most phytopathogenic fungiand to interact with plants by inducing their resistance and promotingtheir growth has been widely described. Endopolygalacturonases(endoPGs) produced by Trichoderma spp. can assist root penetration andplay a pre-eliciting role in induced systemic resistance (ISR), a beneficialeffect detected in plants colonized by Trichoderma. In this study, twoendoPG genes (Tvpg1 and Tvpg2) have been identified and partially char-acterized in a T. virens isolate, previously investigated for its antagonisticability in several biological systems. An extensive in silico analysis showedrelatively low sequence similarities (53%) between TvPG1 and TvPG2proteins and underscored interesting phylogenetic relationships in thecomparison of endoPG genes among fungal genomes. The phylogeny ofendoPGs suggests a differentiation in action mechanisms associated withbiological functions as one cluster shows only genes from plant-interact-ing organisms. A gene expression analysis demonstrated a different regu-lation pattern for those genes. The Tvpg1 gene was induced both when thefungus was grown in liquid cultures supplemented with pectin or plantcell walls and when it was applied to tomato roots in a growth chamber.Expression times were comparable in both systems. On the contrary, theTvpg2 gene displayed a constitutive expression in all conditions tested,including controls. The molecular communication between tomato rootsand T. virens was investigated by checking the expression of a tomato PGIP(Lepgip1) in roots colonized by fungal mycelium. The Lepgip1 transcriptwas induced at times coincident with Tvpg1 expression, suggesting a corre-lation between the two gene products. This paper reports the results offirst investigations of T. virens endoPG genes performed on a structural,phylogenetic and functional level.IntroductionFungi belonging to the genus Trichoderma areubiquitous and typically soilborne micro-organisms(Esposito and Da Silva 1998), which display a consid-erable range of lifestyles that have been recentlyinvestigated by means of genomics (Martinez et al.2008; Druzhinina et al. 2011; Kubicek et al. 2011;Gruber et al. 2013). Trichoderma spp. have beenwidely studied as biocontrol agents of fungal plantpathogens due to the frequent discovery of isolateswith antagonistic abilities against plant-pathogenicfungi (Kubicek et al. 2001; Benitez et al. 2004). Onemeans by which Trichoderma spp. can control orreduce plant disease is through induced resistanceinvolving a direct interaction with plants (De Meyer

International audience; Bitter rot is a common disease mainly affecting apples (Malus domestica) worldwide, and resulting in considerable economic losses, but pears can also be affected (Ivic et al. 2013). Most of the reports refer to bitter rot of Chinese and Asian pears (Pyrus bretschneideri and P. pyrifolia) caused by C. acutatum sensu lato or C. fructicola (Jiang et al. 2014; Kim et al. 2007; Li et al. 2013). To our knowledge, the only report of bitter rot on European pears (P. communis) was done by Ivic et al. (2013) in Croatia and the causal agent was identified as C. fioriniae. In the last 4 to 5 years, anthracnose symptoms have been repeatedly observed on the pear cultivar Beurré Hardy in September during harvest in an orchard near Brest, France. Lesions were round, 1 to 4 cm in diameter, brown, and dry, with acervuli, producing orange spore masses in concentric rings. Two fungal isolates were obtained (in 2015 and 2016, respectively) from symptomatic pears by culturing necrotic tissue pieces on potato dextrose agar (PDA) at 25°C in the dark. Cultures were light gray, with cottony aerial mycelium becoming darker with age and reverse ranging from brownish pink to dark gray with black spots. Cultures have dark melanized structures similar to acervuli that oozed orange-colored conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and measured 10.0 to 14.0 μm × 3.0 to 3.5 μm. Both cultural and morphological characteristics were similar to those described for C. acutatum sensu lato (Damm et al. 2012). Total genomic DNA was extracted and the rDNA ITS region was amplified using universal primers ITS4 and ITS5 then sequenced. For both isolates (UBOCC-A-116033 and UBOCC-A-116034), resulting sequences were 100% identical to C. acutatum species complex sequences. Based on Damm et al. (2012), strains were further characterized by sequencing partial ACT, CHS-1, GAPDH, HIS3, and TUB2 genes (GenBank accession nos. KY344741–52). Multilocus phylogenetic analyses carried out with the obtained and reference sequences (Damm et al. 2012) revealed that both isolates clustered within C. fioriniae, as observed using BLAST; this result was consistent with their initial identification as belonging to the C. acutatum species complex. Koch’s postulates were performed on 10 ‘Comice’ pears for each isolate. Surface sterilized fruits were wound-inoculated with 20 μl of a conidial suspension (105 conidia ml–1). 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 reisolated from the lesions were morphologically similar to the original isolates. To our knowledge, this is the first report of pear bitter rot caused by C. fioriniae in France. Moreover, to date, bitter rot was not considered as a major problem for pear growing, but as pears are the eighth largest fruit crop in production yield worldwide, pear bitter rot caused by Colletotrichum species may become a major problem in the future and require further investigation.

Colletotrichum graminicola is the causal agent of maize (Zea mays) anthracnose. The most common symptoms are leaf blight, top dieback, and stalk rot (Munkvold and White 2016). Owing to its negative impact on maize production, it is important to study the distribution and propagation of this disease. In September 2018, maize stem samples exhibiting symptoms of anthracnose stalk rot (glossy, black, and irregularly shaped lesions) were collected from a field in the Odžak (Posavska Mahala) region of northeastern Bosnia and Herzegovina. Stem samples, approximately 50 mm2, were dissected and surface disinfected for 90 s in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one-half-strength acidified potato dextrose agar (PDA) Petri dishes supplemented with ampicillin (100 µg/ml) and incubated for 4 to 6 days at 25°C (Sukno et al. 2008). Single-spore isolates from three distinct stem pieces were cultured on PDA. The aerial mycelium was dark gray with orange-colored spore masses, and conidia were falcate, slightly curved, tapered toward the tips, and produced in acervuli with setae. The length and width of 50 conidia were measured and ranged from 25 to 35 µm (average, 29.8 µm; standard deviation, ± 2.0 µm) and from 7 to 11 µm (average, 9.0 µm; standard deviation, ± 0.7 µm), respectively. These morphological characteristics are consistent with C. graminicola (Bergstrom and Nicholson 1999). Two isolates were grown in potato dextrose broth for 3 days at 25°C, and total genomic DNA was extracted following the protocol of Baek and Kenerley (1998). The internal transcriber spacer (ITS) region of rDNA was amplified using universal fungal primers ITS4 and ITS5 and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to ITS rDNA sequences of C. graminicola isolates. One sequence was deposited in GenBank under accession number MK955539. These results were consistent with the initial identification of C. graminicola. To confirm Koch’s postulates, maize plants (vegetative stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µl) of a suspension of 3 × 105 conidia/ml were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23°C. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25°C with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After 6 days, the leaves of the inoculated plants presented brown elongated lesions with chlorotic margins and necrotic centers consistent with C. graminicola infection. Controls inoculated with water did not exhibit any symptoms. Microscopic analysis of the surface of infected leaves revealed acervuli, setae, and conidia that were identical to the original isolates. To our knowledge, this is the first report of C. graminicola causing maize anthracnose in Bosnia and Herzegovina (Arsenijević et al. 1996). Previous reports have shown that maize anthracnose is an important disease in Croatia (Palaversic et al. 2009), and it has been reported in Slovenia. Because it may also be a threat for neighboring countries such as Serbia, Romania, and Hungary, where the cultivation of maize is an important component of the economy, continued surveillance is needed.

France is the third largest European producer of apples (Malus domestica), after Poland and Italy, with 2.1 million tons yearly (Eurostat 2015), and the largest producer of European cider apples, with 8,500 ha and 250,000 tons of fruit. Cider apples from Brittany represent over a quarter of the national tonnage (Anonymous 2012). Apple bitter rot symptoms have been observed on ‘Galeuse’ apples (a local variety in Brittany) for nearly 5 to 7 years and again in September 2015, during harvest at one apple orchard near Brest, France. Lesions were round, 1 to 4 cm in diameter, brown and dry with acervuli, producing orange spore masses in concentric rings. Fungal isolates were obtained from symptomatic apples by culturing necrotic tissue pieces on potato dextrose agar at 25°C in the dark. Cultures were light gray, with cottony aerial mycelium becoming darker with age and with reverse colors ranging from brownish pink to dark gray with black spots. Cultures had dark melanized structures similar to acervuli that oozed orange-colored conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and 10.0 to 14.0 μm × 3.0 to 3.5 μm. Both cultural and morphological characteristics were similar to those described for Colletotrichum acutatum sensu lato (Damm et al. 2012). Total genomic DNA was extracted and the ITS region of rDNA was amplified using the universal primers ITS4 and ITS5, then sequenced. The resulting sequence was 100% identical to C. acutatum species complex sequences obtained by a BLAST search in GenBank. Based on Damm et al. (2012), five other loci were used to further characterize the isolate: partial GAPDH, CHS-1, HIS3, ACT, and TUB2 genes sequences were amplified and sequenced. Sequences were deposited in GenBank (Accession Nos. KT887552 for ITS, KT887551 for TUB, KT887555 for CHS, KT887553 for HIS3, KT887556 for ACT, and KT887554 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained sequences and reference sequences (Damm et al. 2012) revealed that the isolate clustered within C. fioriniae, as also suggested by the BLAST results, and consistent with the initial identification as C. acutatum. Koch’s postulates were performed on 10 ‘Golden Delicious’ apples. Surface sterilized fruits were wound-inoculated with 20 μl of a conidial suspension (105 conidia/ml). After 10 days incubating at 20°C, symptoms identical to those initially observed developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies reisolated from the lesions were morphologically similar to the original isolate. Previous studies have demonstrated that this pathogen is present worldwide on Malus spp., and apple bitter rot is considered one of the most important diseases in the United States (Ivic et al. 2013). In Europe, there are increasing numbers of reports ofColletotrichum species on apples (Ivic et al. 2013; Baroncelli et al. 2014), but to our knowledge, this is the first report in France of anthracnose on apples caused by C. fioriniae. In Europe, bitter rot caused by C. acutatum species may become a major problem and require additional investigation.

In 2012, Colletotrichum isolates were collected from field-grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichumacutatum sensu lato-related isolates. Phylogenetic analysis revealed that all of the strains isolated from C.tinctorius belonged to the species described as C.chrysanthemi, whereas all of the strains belonging to C.carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C.chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C.chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C.chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C.tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.

In October 2016, rotting grape berries were detected on grapevine (Vitis vinifera) in Livorno (Nugola, Tuscany, Italy). Symptoms on grape berry 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 lost turgor and turned into ‘mummies’ 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-h light period on potato dextrose agar (PDA). Monoconidial isolates had light gray cottony aerial mycelium with colony color ranging from whitish to dark gray, 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. 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 reisolated 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 the U.K. (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.

In November 2009, twelve specimens of grapevine ( Vitis vinifera ) cv. Brant in the area of Penrith, Cumbria in the United Kingdom were showing necrotic black leaf spotting and abundant orange sporulation on the upper leaf surface. Infected…