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Didymella vitalbina (CBS 123707) a. Colony on OA after 14 d; b. colony on MEA after 14 d; c. longitudinal section through a perithecium; d. ascus; e. ascospores; f. pycnidial wall with conidiogenous cells; g. pycnidia; h. longitudinal section through a pycnidium; i. conidia.-Scale bars: c, h = 50 µm; d, f = 5 µm; e, i = 10 µm; g = 100 µm.
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The fungal pathogen Phoma clematidina is used as a biological agent to control the invasive plant species Clematis vitalba in New Zealand. Research conducted on P. clematidina as a potential biocontrol agent against C. vitalba, led to the discovery of two perithecial-forming strains. To assess the diversity of P. clematidina and to clarify the tele...
Citations
... The isolation was carried out by the direct method in PDA medium (19 g per liter of commercial PDA extract water-Himedia ® ) and the purification by monosporic culture. The identification of the pathogen species was performed by partial sequencing of the actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (β-tub2) nuclear genes using the primers ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF/GDR (Templeton et al. 1992), and BT2F/BT4R (Woudenberg et al. 2009), respectively. The pathogenicity was verified by Koch's postulates. ...
Among the various diseases occurring in the pecan tree crop, anthracnose, caused by species of the genus Colletotrichum, stands out. Biocontrol using Trichoderma presents as a promising measure to be used in disease control because it has a broad spectrum of action on phytopathogens, enables the activation of the defense system, and the promotion of plant growth, contributes to environmental sustainability and food security. This study aimed to investigate the in vitro antagonistic action of Trichoderma species on Colletotrichum, the etiological agent of anthracnose in pecan. The dual-culture assay and the inhibition test by volatile metabolites were performed with five species of Trichoderma (T. harzianum, T. koningiopsis, T. asperellum, T. tomentosum, and T. virens). Mycelial growth was evaluated daily until the seventh day. In the dual-culture assay, all species showed antagonist potential, inhibiting the mycelial growth of the pathogens from the third day onward. Trichoderma virens and T. tomentosum showed greater antagonist potential and stood out in the volatile metabolites.
... The strains were grown for 14-21 d on malt extract agar (MEA) were used for DNA extraction using the Wizard® Genomic DNA purification Kit (Promega Corp., Madison, WI, USA), according to the manufacturer's instructions. Three gene regions, ITS, LSU and TUB, were amplified using the primers ITS4-ITS5 (White et al. 1990, Ward andAdamas 1998), LR0R-LR5 (Vilgalys and Hester 1990) and TUB2Fd-TUB4Fd (Woudenberg et al. 2009 microphotographs were taken by Nikon Eclipse80i equipped with a Nikon DSRi2 camera. ...
The genus Sporendonema (Gymnoascaceae, Onygenales) was introduced in 1827 with the type species S. casei for a red mould on cheese. Cheese is a consistent niche for this species. Sphaerosporium equinum is another species classified in Gym-noascaceae and has also been reported from cheese. Recently, other habitats have been reported for both Sporendonema casei and Sphaerosporium equinum. The present study aimed to investigate the taxonomy of Sporendonema and Sphaerosporium, as well as a close neighbour, Arachniotus. Two strains of Hormis-cium aurantiacum, another related cheese-associated species were also included in the analyses. Strains were evaluated in terms of macro-and micromor-phology, physiology including salt tolerance, growth rate at different temperatures, casein degradation, cellulase activity, lipolytic activity, and multi-locus phylogeny with sequences of the nuclear riboso-mal internal transcribed spacer region, the D1-D2 region of the large subunit and partial β-tubulin locus sequences. The results showed that the analysed species were congeneric, and the generic names Arach-niotus and Sphaerosporium should be reduced to the synonymy of Sporendonema. Therefore, four new combinations as well as one lectotype and one epitype were designated in Sporendonema. Two strains attributed to Sphaerosporium equinum from substrates other than cheese were found to be phylogenetically and morphologically deviant and were introduced as a new species named Sporendonema isthmoides. Supplementary Information The online version contains supplementary material available at https:// doi.
... Total genomic DNA was extracted using the BioTeke Fungus Genomic DNA Extraction kit (DP2032, BioTeke) following the manufacturer's instruction. Primer combinations such as ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Wang et al. 2022a), EF1-728F/EF2 (O'Donnell et al. 1998;Carbone and Kohn 1999), CAL-228F/CAL-2Rd (Carbone and Kohn 1999;Lombard et al. 2015), rpb2-5F2/rpb2-7CR (Sung et al. 2007;O'Donnell et al. 2007) and T1/TUB4Rd (O'Donnell and Cigelnik 1997;Woudenberg et al. 2009) were used for amplification of the internal transcribed spacers (ITS), the 28S nrRNA locus (LSU), translation elongation factor 1-alpha gene region (tef1), calmodulin gene (cmdA), RNA polymerase II second largest subunit gene (rpb2) and beta-tubulin gene (tub2), respectively. The PCR products were sent to Quintarabio (Wuhan, China) for purification and sequencing. ...
Rich and diverse fungal species occur in different habitats on the earth. Many new taxa are being reported and described in increasing numbers with the advent of molecular phylogenetics. However, there are still a number of unknown fungi that have not yet been discovered and described. During a survey of fungal diversity in different habitats in China, we identified and proposed two new species, based on the morphology and multi-gene phylogenetic analyses. Herein, we report the descriptions, illustrations and molecular phylogeny of the two new species, Bisifusarium keratinophilum sp. nov. and Ovatospora sinensis sp. nov.
... Isolates within the boninense species complex were further identified using internal transcribed spacer and intervening 5.8S nrDNA gene (ITS), βtubulin (tub2), actin (act) and histone (his3) gene sequences. The gapdh, ApMat, gs, ITS, tub2, act and his3 gene sequences were amplified and sequenced with primers GDF1 and GDR1 (Guerber et al., 2003), AMF1 and AMR1 (Silva et al., 2012), GSF1 and GSR1 (Stephenson et al., 1997), ITS-1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990), Btub2Fd and Btub4Rd (Woudenberg et al., 2009), ACT-512F and ACT-783R (Carbone & Kohn, 1999) and CYLH3F and CYLH3R (Crous et al., 2004), respectively (Table S1). PCR amplifications were performed in a 2720 thermal cycler (Applied Biosystems) using 25 μL reaction mixtures that contained 1 × PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTP, 1 U Taq DNA polymerase (MangoTaq DNA polymerase; Bioline), 0.4 μM of each primer and 6-10 ng template DNA. ...
Up to 32 Colletotrichum species have been reported to be associated with pre‐ or postharvest diseases of citrus globally, while in Australia, six species have been reported to cause citrus leaf and fruit disease. Twig or shoot dieback has recently been observed as an emerging disease in citrus orchards in Western Australia. Colletotrichum species were isolated from diseased twigs showing dieback (withertip) or lesions, with or without gummosis, collected from 12 varieties of orange, mandarin and lemon. Colletotrichum gloeosporioides sensu stricto, Colletotrichum karstii and Colletotrichum novae‐zelandiae were identified using a polyphasic approach that included multigene phylogenetic analysis using sequences of internal transcribed spacer and intervening 5.8S nrDNA (ITS), glyceraldehyde‐3‐phosphate dehydrogenase ( gapdh ), β‐tubulin ( tub2 ), actin ( act ) and histone ( his3 ) for isolates in the boninense species complex, and Apn2–Mat1–2 intergenic spacer and partial mating type (Mat1–2) ( ApMat ) and glutamine synthetase ( gs ) for isolates in the gloeosporioides species complex, as well as morphological characteristics. C. gloeosporioides was the most prevalent species associated with twig dieback in Western Australia, while C. novae‐zelandiae was reported for the first time in Australia. Pathogenicity tests on shoot twigs from lemon and orange trees confirmed C. gloeosporioides , C. karstii and C. novae‐zelandiae as the cause of twig dieback, with C. gloeosporioides being the most aggressive species. Knowledge of the species causing twig dieback and their lifestyle will assist the development of integrated control methods.
... The genomic DNA from 18 selected isolates was extracted from colonies grown in a PDA medium for seven days, using the CTAB method [21]. The actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (β-tub2) regions were amplified for each sample using the primers ACT-512 F/ACT-783R [22], GDF/GDR [23], and BT2F/BT4R [24], respectively. The PCR reaction contained approximately 30 ng of DNA, 1x buffer, 200 µM of each dNTP, 1.5 mM MgCl 2 , 200 nM of each primer, and 1 U of GoTaq DNA polymerase (Promega). ...
Pecan (Carya illinoinensis) is one important exotic forest crop cultivated in South America, specifically in Brazil, Uruguay, and Argentina. However, diseases such as anthracnose, favored by high humidity conditions and high summer temperatures, make its cultivation difficult, causing important loss to pecan farmers. This study used morphological and molecular approaches to identify the Colletotrichum species causing anthracnose in pecan plantations in Southern Brazil. The isolates obtained from pecan fruits with anthracnose symptoms were grouped through quantitative morphological characteristics into three distinct morphotypes. Molecular analysis of nuclear genes allowed the identification of six species of Colletotrichum causing anthracnose in pecan: C. nymphaeae, C. fioriniae, C. gloeosporioides, C. siamense, C. kahawae, and C. karsti. Three of these species are reported for the first time as causal agents of anthracnose in pecan. Therefore, these results provide an important basis for the adoption and/or development of anthracnose management strategies in pecan orchards cultivated in southern Brazil and neighboring countries.
... The primers EF1-728F and EF1-986R (Carbone and Kohn 1999) were used to amplify partial region of translation elongation factor-1α (tef1) in isolates of Botryosphaeriaceae and Diaporthe species (Guarnaccia et al. 2020(Guarnaccia et al. , 2022aAiello et al. 2022). The partial β-tubulin (tub2) gene was amplified with primers T1-Bt2b (Glass and Donaldson 1995;O'Donnell and Cigelnik 1997) for isolates belonging to Botryosphaeriaceae family and Diaporthe genus (Guarnaccia et al. 2020(Guarnaccia et al. , 2022a, whilst primers Tub2fd-Tub4fd (Woudenberg et al. 2009) were used to amplify the same region in isolates of the genus Nothophoma (Chen et al. 2015). The partial RNA polymerase second largest subunit (rpb2) gene was amplified with the primers: Rpb2-5f2-Rpb2-7cr (Liu et al. 1999;Reeb et al. 2004) for isolates identified as Nothophoma sp. ...
Italy is the second largest hazelnut producer worldwide and Piedmont is one of the most productive regions in the country. The changing climatic condition and fungal trunk diseases (FTD) can have a severe impact on this crop. Particularly, the considerable spread of Cytospora cankers (‘Mal dello stacco’) and dieback represent a serious concern for producers. Thus, considering the limited studies on the causal agents, different surveys were conducted in seven hazelnut orchards during 2021 and 2022. Eight fungal species were identified: Anthostoma decipiens, Botryosphaeria dothidea, Diaporthe eres, Dia. rudis, Diplodia seriata, Dip. subglobosa, Dothiorella parva and Nothophoma brennandiae . Species identification was achieved through multilocus phylogeny and morphology assessment. All the fungal species were pathogenic on healthy hazelnut plants (cultivar Tonda Gentile) and A. decipiens and Dia. eres were the most aggressive. The present study is the first report of B. dothidea and Dia. eres as causal agents of FTD on hazelnut in Italy and of Dia. rudis, Dip. subglobosa and N. brennandiae worldwide. Moreover, the study provides clarification of the fungal pathogens associated with FTD on this crop in Piedmont, thus laying the base for further studies on epidemiology, ecology and management strategies.
... For instance, De Gruyter et al. [2] established the family Didymellaceae with Didymella as type genus, but the initial Didymella species had only SSU and LSU sequences. Woudenberg et al. [47] and Thambugala et al. [48] introduced D. clematidis and D. eriobotryae using ITS, LSU, and TUB2. Liu et al. [49] introduced D. cirsii using ITS and LSU. ...
Didymella contains numerous plant pathogenic and saprobic species associated with a wide range of hosts. Over the course of our mycological surveys of plant pathogens from terrestrial plants in Jiangxi Province, China, eight strains isolated from diseased leaves of four host genera represented three new species of Didymella, D. bischofiae sp. nov., D. clerodendri sp. nov., and D. pittospori sp. nov. Phylogenetic analyses of combined ITS, LSU, RPB2, and TUB2 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), revealed their taxonomic placement within Didymella. Both morphological examinations and molecular phylogenetic analyses supported D. bischofiae, D. clerodendri, and D. pittospori as three new taxa within Didymella. Illustrations and descriptions of these three taxa were provided, along with comparisons with closely related taxa in the genus.
... Genomic DNA of the isolates was extracted using the protocol in a previous study [10], with slight modifications. In polymerase chain reaction (PCR) experiments, partial large subunit nuclear ribosomal DNA (LSU), internal transcribed spacer regions 1 & 2 including 5.8S nrDNA (ITS), β-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) gene regions were investigated with the primer sets of LR0R [11] and LR7 [12] for LSU, V9G [13] and ITS4 [14] for ITS, Btub2Fd and Btub4Rd [15] for TUB2, and RPB2-5f2 [16] and fRPB2-7cR [17] for RPB2. Conditions of PCR amplification for all the genes were followed as in the previous studies [6,7]. ...
During a disease survey in October 2019, leaf spot symptoms with a yellow halo were observed on Korean angelica (Anglica gigas) plants grown in fields in Pyeongchang, Gangwon Province, Korea. Incidence of diseased leaves of the plants in the investigated fields ranged from 10% to 60%. Morphological and cultural characteristics of two single-spore isolates from the leaf lesions indicated that they belonged to the genus Didymella. Molecular phylogenetic analyses using combined sequences of LSU, ITS, TUB2, and RPB2 regions showed distinct clustering of the isolates from other Didymella species. In addition, the morphological and cultural characteristics of the isolates were somewhat different from those of closely related Didymella spp. Therefore, the novelty of the isolates was proved based on the investigations. Pathogenicity of the novel Didymella species isolates was confirmed on leaves of Korean angelica plants via artificial inoculation. This study reveals that Didymella gigantis sp. nov. causes leaf spot in Korean angelica.
... Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/jof9121167/s1, Figure S1: The procedure for purification of the fraction of Fr. 4.4 and determination of purity of the sub-fraction Fr.4.4.; Figure S2: A Bayesian phylogenetic tree of 38 fungal taxa.The tree was constructed using concatenated sequences of ITS and LSU; Figure S3: Isolation of D. macrostoma P2 and L. biglobosa Lb20 in rapeseed cotyledons; Figure S4: Two leaves of rapeseed treated with the culture filtrate (CF) of D. macrostoma P2 and PDB alone; Figure S5: 13 C NMR (125 MHz, DSMOMeOH-d 6 , NMR) spectrum for penicillither from Didymella macrostoma P2; Figure S6: 1 H NMR (600 MHz, DSMO-d 6 , NMR) spectrum for penicillither A from Didymella macrostoma P2; Table S1: PCR primers used in this study for amplification of different genes; Table S2: PCR thermal programs for amplification of different fungal genes; Table S3: Origin of the fungal isolates used in this study and GenBank accession numbers; Refs [65][66][67][68][69] ...
Eight fungal strains (P1 to P8) were isolated from rapeseed stems (Brassica napus) infected with the blackleg pathogen Leptosphaeria biglobosa (Lb). They formed pycnidia with similar morphology to those of Lb, and thus were considered as Lb relatives (LbRs). The species-level identification of these strains was performed. Their virulence on rapeseed and efficacy in the suppression of Lb infection were determined, and the biocontrol potential and biocontrol mechanisms of strain P2 were investigated. The results showed that the LbRs belong to two teleomorphic genera in the family Didymellaceae, Didymella for P1 to P7 and Boeremia for P8. Pathogenicity tests on rapeseed cotyledons and stems indicated the LbRs were weakly virulent compared to L. biglobosa. Co-inoculation assays on rapeseed cotyledons demonstrated that P1 to P7 (especially P1 to P4) had a suppressive effect on Lb infection, whereas P8 had a marginal effect on infection by L. biglobosa. Moreover, D. macrostoma P2 displayed a more aggressive behavior than L. biglobosa in the endophytic colonization of healthy rapeseed cotyledons. Cultures of P2 in potato dextrose broth (PDB) and pycnidiospore mucilages exuded from P2 pycnidia showed antifungal activity to L. biglobosa. Further leaf assays revealed that antifungal metabolites (AM) of strain P2 from PDB cultures effectively suppressed infection by L. biglobosa, Botrytis cinerea (gray mold), and Sclerotinia sclerotiorum (white mold). An antifungal metabolite, namely penicillither, was purified and identified from PDB cultures and detected in pycnidiospore mucilages of strain P2. This study suggests that the LbRs are a repertoire for screening biocontrol agents (BCAs) against rapeseed diseases, and D. macrostoma P2 is a multi-functional BCA, a penicillither producer, and an endophyte.
... DNA was extracted after mechanical lysis in CTAB buffer according to the protocol described by Gerrits van den Ende and de Hoog [25]. The following genes were amplified and Sanger sequenced for identification: internal transcribed spacer 1 and 2 including the 5.8S rDNA (ITS; primer pair ITS1/ ITS4 [26]), partial large ribosomal subunit rDNA including its D1/D2 domains (LSU; primer pair NL1/NL4 [26]), partial small ribosomal subunit (SSU; primer pair NS1/ NS24 [27]), partial sequences of genes encoding β-tubulin 2 (TUB2; primer pair Btub2Fd/Btub2Rd [28]) and the largest subunit of RNA polymerase II (RPB1; primer pair RPB1-Af/RPB1-Cr [29] ...
The huge amounts of keratin-rich waste generated daily by various industries, slaughterhouses, and processing plants need to be properly managed. Most keratin degradation-related research focuses on keratin from bird feathers, but a vast minority focuses on keratin from sheep wool, which also presents a serious environmental problem. In this article, we describe the isolation, identification, and characterization of new keratinolytic microorganisms capable of sheep wool degradation from sheep wool and soil enriched with wool keratin. We isolated four bacterial species from the genus Bacillus (B. subtilis, B. altitudinis, B. mycoides, and B. wiedmannii), one streptomycete species Streptomyces coelicoflavus identified by whole genome sequencing, and a fungal species Aphanoascus reticulisporus. In some of the isolated microorganisms, we detected keratinolytic activity for the first time, and for most of them, the ability to degrade sheep wool has not been previously demonstrated. The keratinases of the new isolates are active in a wide range of temperatures (25–85 °C) and pH (6.0–10.0), so all isolates show great potential for further biotechnological use in industry and in various environmental and agricultural applications to reduce and recycle keratin-rich wastes such as sheep wool and waste woollen textiles.
