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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.

Leaf rust caused by

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.

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.

Although lupin anthracnose caused by

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.

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.

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.