ITS rDNA Research Articles

Taxonomic revision of the genus Sarcosphaera (Ascomycota: Pezizales) in Europe and North America revealed unexpected diversity

Sarcosphaera (Ascomycota: Pezizales) is a genus of rare spring fungi, currently encompassing a single species, S. coronaria, which is red-listed in many European countries. The genus is characterized by large bright-colored fruit-bodies and high arsenic content. This study investigates the genetic diversity across Europe and North America. Using integrated molecular techniques, including ITS and 28S rDNA sequencing, we aimed to resolve the taxonomic ambiguities and assess their arsenic hyperaccumulation capabilities, a rare trait among fungi. We identified the presence of two species in the Western Palearctic (S. coronaria, S. crassa) and at least 12 additional lineages in North America, indicating a more complex taxonomic structure than previously recognized. To consolidate the taxonomy and nomenclature of these fungi, we selected reference specimens for S. coronaria and S. crassa and discussed their morphological and ecological differences. Two genera, Caulocarpa (C. montana) and Hydnotryopsis (H. setchellii), a genus comprising hypogeous fungi, are newly placed in the genus Sarcosphaera. Sarcosphaera is thus another genus where the rapid evolution of hypogeous forms from their above-ground ancestors took place. Furthermore, two new Sarcosphaera species, S. columbiana and S. pacifica, are described from the Pacific Northwest. Investigation of arsenic content of Sarcosphaera species revealed that arsenic mass fractions in ascocarps vary significantly between species and even within species across different geographical locations. Our findings have important implications for the taxonomy and conservation of the S. coronaria species complex and also point to future directions for further research in the taxonomy, geomycology, and toxicology of this group of fungi.

Morphological and molecular characterisation of two new species of Rhipidocotyle (Digenea: Bucephalidae Poche, 1907) from Sphyraena putnamae Jordan & Seale in Mozambique.

Species-level delineation of digenetic trematodes is complex and can be best achieved by integrative taxonomy using both genetic characterisation and morphological analysis. Two new Bucephalidae species of the genus Rhipidocotyle Diesing, 1858 are described here based on specimens collected from the intestine of Sphyraena putnamae Jordan & Seale following this approach. Adults of R. siphonyaka n. sp. and R. nolwe n. sp. possess tentacles and a tegument with scales. They are distinguished from their congeners by the arrangement of the digestive structures, the extent of the uterus relative to vitelline fields, and the arrangement of the reproductive structures. Rhipidocotyle siphonyaka n. sp. differs from R. nolwe n. sp. in having the pharynx and mouth positioned in the pre-uterine field, tandem testes, longer body length, and shorter pre-vitelline and post-testicular distance. Rhipidocotyle siphonyaka n. sp. differs from its congeners in having a tube-like intestinal caecum, pharynx and mouth opening positioned in the pre-vitelline field. Rhipidocotyle nolwe n. sp. appears to be similar, morphologically and morphometrically, to Rhipidocotyle khalili (Nagaty, 1937). Despite their similarities, R. nolwe n. sp. has a shorter body length and egg size. Moreover, the molecular analysis of 28S and ITS rDNA fragments indicate that R. siphonyaka n. sp. and R. nolwe n. sp. are closely related phylogenetically but distinct from one another and other Bucephalidae for which molecular data are available.

Additions to the genus Kirschsteiniothelia (Dothideomycetes); Three novel species and a new host record, based on morphology and phylogeny

During a survey of microfungi associated with forest plants, four specimens related to Kirschsteiniothelia were collected from decaying wood in Guizhou, Hainan and Yunnan Provinces, China. Kirschsteiniothelia species have sexual and asexual forms. They are commonly found as saprophytes on decaying wood and have been reported as disease-causing pathogens in humans as well. In this study, we introduce three novel Kirschsteiniothelia species (K. bulbosapicalis, K. dendryphioides and K. longirostrata) and describe a new host record for K. atra, based on morphology and multi-gene phylogenetic analyses of a concatenated ITS, LSU and SSU rDNA sequence data. These taxa produced a dendryphiopsis- or sporidesmium-like asexual morph and detailed descriptions and micromorphological illustrations are provided. Furthermore, we provide a checklist for the accepted Kirschsteiniothelia species, including detailed host information, habitat preferences, molecular data, existing morphological type, country of origin and corresponding references.

First detection of the obligate biotroph Phytophthora cyperi causing disease on yellow nutsedge in the United States, and establishment of molecular barcoding sequences for the species

A pathogen causing disease on yellow nutsedge (Cyperus esculentus) growing in and near irrigated areas of onion seed and bulb fields in Idaho and Oregon during July and August, 1996 was morphologically and molecularly characterized. Plant symptoms included necrotic spots, blotches and blighting of leaves, stems (culms) and bracts. Symptomatic tissue contained glistening, white masses of mycelium and oval, semi-papillate sporangia emerging from stomata on sporangiophores. Later, subspherical oogonia with smooth oospores were found embedded in older, infected tissue. Phytophthora cyperi was identified as the causal agent and pathogenicity was confirmed by inoculating healthy nutsedge plants with sporangia from infected plants. Infected samples tested negative using Agdia’s Phytophthora ELISA kit and all efforts to culture the organism on semi-selective or general fungal media failed. To aid future efforts to detect this species, sequences of the two primary barcoding loci, ITS rDNA and mitochondrial cox1 were obtained from the organism and analyzed, finding an unexpectedly large ITS sequence characterized by a ~1300 bp insertion.

Sarocladium kiliense: A first report of phytopathogenic fungus causing maize stalk rot in India

Post-flowering stalk rot (PFSR) disease drastically affects maize yields, leading to substantial economic losses. Fusarium verticillioides was widely recognized as a major pathogen responsible for maize PFSR disease. Astonishingly, apart from F. verticillioides, another pathogen was also observed infecting the stalk rot and thus, further pathogen purification was initiated. In this study, we revealed the first report of Sarocladium kiliense, as a maize stalk rot pathogen. This research study was validated by isolation, purification, and morphological examination, which revealed the variability among S. kiliense isolates in traits such as pigmentation, colony color, mycelial characteristics, and conidial morphology. Notably, isolates exhibited distinct pigmentation patterns: 3 were orange and 3 were yellow, with consistent white to dirty white colony colors. Pathogenicity assays using toothpick inoculation demonstrated that all the S. kiliense isolates caused significant damage to the maize stalks, with brown discoloration, conversion of pith into a powdery texture, and subsequent stalk lodging. Isolate PKSK55 exhibited the highest pathogenicity, producing the longest lesions among tested isolates. Molecular characterization using ITS rDNA and RPB-2 genes confirmed the identity of S. kiliense across all isolates. Conclusively, this research uncovered and documents the first report of S. kiliense causing stalk rot in maize in India.

Gyrodactylus serrai n. sp. (Gyrodactylidae), from the Near-Threatened Clanwilliam Sawfin, Cheilobarbus serra (Peters) (Cyprinidae, Smilogastrinae), in the Cape Fold Ecoregion, South Africa.

A new species of Gyrodactylus is described from the gills of the near-threatened Clanwilliam sawfin, Cheilobarbus serra (Smiliogastrinae) collected from the Matjies River, Cape Fold Ecoregion, Western Cape Province, South Africa. Morphometry and morphology of the haptoral hard parts (hamuli, bars and marginal hooks) of Gyrodactylus serrai n. sp. differ from the other known species of the genus in the smaller size of hamuli and the shape and size of marginal hooks. Furthermore, ITS rDNA for the new species is unique among available Gyrodactylus spp. data in GenBank. Based on the uncorrected p-distances, G. serrai n. sp. is genetically most closely related to Gyrodactylus moroccensis Rahmouni, 2023 and Gyrodactylus pseudomoroccensis Rahmouni, 2023 from two species of Luciobarbus (Barbinae) from northern Africa, with interspecific divergence of 8.7% and 8.8%, respectively. The presence of a median ridge in the terminal part of the ventral bar membrane at G. serrai n. sp. most probably represents a morphological link to the North African Gyrodactylus spp. that suggests a morphogenetic association across the African continent as a result of ancient waterways that facilitated the dispersion of cyprinids and their parasite fauna or an independent evolution event retaining similarities from a common ancestor. The description of Gyrodactylus serrai n. sp. represents only the second species of Gyrodactylus described from an endemic South African cyprinid host, underscoring the need for focused research on this group of fishes to provide a sound understanding of the parasitic communities of these highly threatened and poorly studied hosts.

Two new species of Phylloporus (Boletaceae) from the montane cloud forest of Eastern Mexico

Phylloporus represents a group of fungi belonging to the lamellate Boletineae, which despite not having been exhaustively documented worldwide, most of its species have been recognized in tropical regions. The present research proposes the description of Phylloporus montanus and P. xalapensis as new species to science. Both were discovered in Mexico through a sampling focused on the study of the genus in the montane cloud forest from the east region of the country. The new species were recognized based on a morpho-anatomic study and supported by a phylogeny inferred from internal transcribed spacer (ITS rDNA), nuclear large ribosomal subunit (nLSU) and translation elongation factor 1-alpha (TEF1) sequences. Descriptions of each species are detailed, accompanied by illustrations and photographs.

Distribution and environmental preferences of Saprolegnia and Leptolegnia (Oomycota) species in fish hatcheries in Hungary

We studied for the first time the abundance and species distribution of water molds (Oomycota, Saprolegniales) in fish hatcheries in Hungary, with a focus on Saprolegnia and Leptolegnia spp. A total of 182 water mold isolates were obtained from different developmental stages of fish (eggs, fry, and adults) and water samples collected during the spawning season of cyprinids and salmonids. Of the ten water mold species identified based on their ITS–rDNA sequences, S. parasitica, S. ferax, and S. australis were the most abundant. Statistical analyses of species proportions in relation to the sample source and host preference revealed that S. parasitica was significantly more abundant in the host than in water samples, whereas the opposite trend was observed for S. ferax and S. australis. In addition, our results confirmed previous findings that S. australis and S. ferax prefer warm water (+21–24 °C) and that S. parasitica has a wide temperature tolerance (+3–24 °C). The DNA sequence identity analysis showed that the species demarcation criterion for Saprolegnia spp. was approximately 98 % identity, based on ITS–rDNA sequences. Furthermore, our phylogenetic analyses suggest that taxonomic revision of Leptolegnia spp. is necessary.

Three novel Ascomycota (Saccharomycetes, Saccharomycetales) yeast species derived from the traditional Mexican alcoholic beverage Pulque.

The abundant variety of yeasts and their diverse applications have essential roles in traditional Mexican alcoholic beverage fermentation processes. During our investigation of yeast diversity associated with Pulque, 41 yeast strains were characterized. Among them, 31 strains were eight known species belonging to seven genera. According to morphological and phylogenetic analyses (ITS and LSU rDNA), ten unidentified yeast strains were identified to be three novel species and proposed: Starmerellaelongatum sp. nov., Kazachstaniaparagamospora sp. nov. and Pichiateotihuacanensis sp. nov. Our study has resulted in the isolation of yeast species not previously detected in Pulque, expanding our knowledge of the diversity of its associated yeast communities.

Aspergillus capibaribensis (Eurotiales, Trichocomaceae), a new species in section Cremei

In this study we describe a new species of Aspergillus in section Cremei deposited in the URM culture collection (Micoteca URM Profa. Maria Auxiliadora Cavalcanti) of the Departamento de Micologia, Universidade Federal de Pernambuco (Recife, Pernambuco, Brazil). Another strain was obtained from samples taken from pollen pots inside nests of Melipona scutellaris (stingless bee). Morphological and phylogenetic evidence based on sequencing of ITS rDNA, BenA, CaM and rpb2 markers supported the description of taxonomic novelty. Aspergillus capibaribensis sp. nov. is morphologically similar and phylogenetically close to A. flaschentraegeri - the distinctive features are uniseriate and hyaline conidiophores, short and smooth conidia and smaller vesicles compared to the micromorphological structures of A. flaschentraegeri. This record also expanded the knowledge of species in Aspergillus section Cremei in a tropical country such as Brazil.

Molecular identification of Hypomyces chrysospermus mycoparasitic fungus isolated from mushrooms in a local market of Iraq.

In this study, mycoparasitic fungus was identified by using the rDNA internal transcribed spacer barcode marker, i.e. ITS rDNA gene. Amplicons were sequenced and identified by NCBI - BLAST (Basic local alignment search tool). The BLAST results revealed that NOOR strain matched 100% with accession numbers [MH854754.1]. The phylogenetic tree shows close relationship between NOOR strain and H. chrysospermus [MH854754-1], H. chrysospermus [MZ389105-1], H. chrysospermus [MK605327- 1], H. chrysospermus [MT595227-1], H. [EU816370-1], H. chrysospermus [MG685885-1]. The Iraqi isolate was recorded as NOOR strain in the National Centre for Biotechnology Information for the first time in Iraq.

Discovery of adults of the gorgoderid trematode Cercaria duplicata with first morphological description, molecular identification and notes on host specificity

Rhopalocercous Cercaria duplicata von Baer, 1827 develops in an intermediate host, the unionid bivalve Anodonta anatina (L.), but its adult form has been unknown. We examined eight fish species occurring in the presence of a highly infested population of A. anatina in the Zesławice reservoir (S Poland). Gravid Phyllodistomum specimens were obtained from the ureters of ide, Leuciscus idus (L.) and common rudd, Scardinius erythrophthalmus (L.). One of the rudd specimens was doubly infected, a trematode was also found in the urinary bladder. In addition, a gravid Phyllodistomum specimen was found in the ureter of a tench Tinca tinca (L.), caught in Lake Ilmėdas (Lithuania). In order to clarify the phylogenetic position of larval and adult gorgoderids and to establish their life cycle, ITS2 and 28S rDNA sequences were analysed. The analysis showed that adult Phyllodistomum specimens located in the ureters are conspecific with C. duplicata. The trematode found in the urinary bladder of S. erythrophthalmus was P. folium (Olfers, 1816). It is suggested that adult stages of C. duplicata should be referred to as Phyllodistomum duplicatum n. comb. The intercaecal position of the uterus and the deeply-lobed ovary are the main features distinguishing it from other Phyllodistomum species. Host specificity and ecology are discussed.

Competitive antagonistic action of laccase between Trichoderma species and the newly identified wood pathogenic Ganoderma camelum.

Ganoderma, a well-known genus in the Ganodermataceae family, has caused the extinction of several tree species due to its pathogenicity. This study explored the pathogenic effect of a newly identified Ganoderma species on trees and its competitive efficiency against Trichoderma species. Ganoderma camelum sp. nov. is characterized by small sessile basidiomata and a velvety, soft, camel-brown pileus. Phylogenetic analysis and ITS rDNA sequences indicated that the species were Trichoderma and Ganoderma camelum. Both fungal species competed antagonistically by secreting laccase. The laccase activity of G. camelum, with a value of 8.3 ± 4.0 U/mL, demonstrated the highest competitive activity against Trichoderma species. The laccase produced by T. atroviride (2.62 U/mL) was most effective in countering the pathogenic action of the novel G. camelum. The molecular weights of laccase were determined using SDS-PAGE (62.0 kDa for G. camelum and 57.0 kDa for T. atroviride). Due to the white rot induced by this Ganoderma species in the host tree, G. camelum showed the highest percentage inhibition of radial growth (76.3%) compared to T. atroviride (28.7%). This study aimed to evaluate the competitive antagonistic activity of Ganoderma and Trichoderma on malt extract agar media in the context of white rot disease in the host tree. This study concluded that the laccase from G. camelum caused weight loss in rubber wood blocks through laccase action, indicating tissue injury in the host species. Therefore, it was also concluded that G. camelum was more effective in pathogenic action of the host and resisted the biological action of T. atroviride. In principal components analysis (PCA), all the species associated with laccase exhibited a very strong influence on the variability of the system. The PIRG rate (percentage inhibition of radial growth) was strongly and positively correlated with laccase activity.

Revision of the Most Primitive Taxa of the Family Gyrodactylidae (van Beneden et Hesse, 1864) (Platyhelminthes, Monopisthocotyla) Based on ITS rDNA Phylogeny.

For the past 25 years, the ITS rDNA (ITS1-5.8S-ITS2) of Gyrodactylidae has been crucial for species identification, description, and phylogeny. This family includes 25 genera parasitizing marine and freshwater fish, initially distinguished by morphological differences in attachment and/or male copulatory organs. Gyrodactylus Nordmann, 1832, the most species-rich genus, has approximately 500 described species and an additional 25,000 species suspected. The genus is not monophyletic, and the functionally adaptive nature of morphological diagnostic characters complicates the delimitation of new genera. A phylogeny based on ITS rDNA was proposed to address these challenges, using only complete sequences of primitive taxa. Fifty-four sequences were aligned with the MUSCLE v5.1 algorithm, creating a 1590 ps long matrix. Maximum Likelihood (ML) and Bayesian Inference (BI) methods with the models TVM+F+G4 and SYM+G4 for ITS1-ITS2 and 5.8S, respectively, were inferred using IQ-TREE v2.3.5 and BEAST v2.7.6.0. The findings revealed eleven main lineages. Four of them are proposed for classification into new genera: Cichlidarus gen. nov., Iraqemembranatus gen. nov., Macracanthus gen. nov., and Rysavyius gen. nov. Elevating the subgenus G. (Gyrodactylus) to genus rank was supported. The presented phylogeny provides a foundation for developing a classification system within Gyrodactylidae that is both reasonable and comprehensive.

Root Endophytic Microorganisms Contribute to the Attribute of Full-Year Shooting in Woody Bamboo Cephalostachyum pingbianense.

Cephalostachyum pingbianense (Hsueh & Y.M. Yang ex Yi et al.) D.Z. Li & H.Q. Yang is unique among bamboo species for its ability to produce bamboo shoots in all seasons under natural conditions. Apart from the physiological mechanism, information regarding the effects of endophytic microorganisms on this full-year shooting characteristic is limited. We hypothesize that root endophytic microorganisms will have a positive impact on the full-year bamboo shooting characteristic of C. pingbianense by increasing the availability or supply of nutrients. To identify the seasonal variations in the root endophytic bacterial and fungal communities of C. pingbianense, and to assess their correlation with bamboo shoot productivity, the roots of C. pingbianense were selected as research materials, and the 16S rRNA and ITS rDNA genes of root endophytic microorganisms were sequenced using the Illumina platform. Following this sequencing, raw sequencing reads were processed, and OTUs were annotated. Alpha and beta diversity, microbial composition, and functional predictions were analyzed, with correlations to bamboo shoot numbers assessed. The results showed that seasonal changes significantly affected the community diversity and structure of root endophytic microbes of C. pingbianense. Bacterial communities in root samples from all seasons contained more nitrogen-fixing microorganisms, with members of the Burkholderiales and Rhizobiales predominating. The relative abundances of ectomycorrhizal and arbuscular mycorrhizal fungi in the autumn sample were significantly higher than in other seasons. Correlation analysis revealed that the bamboo shoot productivity was significantly and positively correlated with bacterial functions of nitrogen fixation, arsenate detoxification, and ureolysis, as well as with symbiotrophic fungi, ectomycorrhizal fungi, and arbuscular mycorrhizal fungi. At the genus level, the bacterial genus Herbaspirillum and the fungal genera Russula, unclassified_f_Acaulosporaceae, and unclassified_f_Glomeraceae were found to have a significant positive correlation with bamboo shoot number. Our study provides an ecological perspective for understanding the highly productive attribute of C. pingbianense and offers new insights into the forest management of woody bamboos.

Unveiling the Hidden Enemies: Morphological and Molecular Characterization of Pod Rot Pathogens in Groundnut (Arachis hypogea L.) in India

Pod rot of groundnut is a complex disease caused by multiple pathogens causes significant economic losses. Present study was undertaken to know the pathogens associated with the disease. In Chittoor, Andhra Pradesh, roving survey against pod rot of groundnut revealed association of three fungal species: Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium spp. Molecular analysis with modified CTAB method and performing PCR using ITS1 and ITS4 primers yielded DNA bands of 650-700 bp, 550-600 bp, and 500-600 bp for Sclerotium, Rhizoctonia and Fusarium species, respectively. Sequencing of the rDNA ITS region confirmed the identities of the pathogens as Sclerotium rolfsii, Rhizoctonia bataticola, Fusarium solani, Fusarium oxysporum, and Fusarium keratoplasticum, based on similarity with NCBI reference sequences. This study confirms that Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium spp. are the primary causal agents of pod rot disease in Andhra Pradesh.

A quantitative real-time PCR assay for rapid detection and quantification of Amyloodinium ocellatum parasites in seawater samples

Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.

Fungal secondary metabolite gliotoxin enhances enzymatic activity in soils by reshaping their microbiome

Gliotoxin (GT) is a sulfur-containing epidithiodioxopiperazine produced by various filamentous fungi, including those used in biological plant protection (Trichoderma virens). The pronounced antimicrobial effect of GT on a variety of fungi and bacteria makes it a promising agent for controlling phytopathogens in agricultural systems. In this study, we aim to investigate the microbiological properties of the soil microbiome after the introduction of GT. GT was applied at doses of 10, 25, 50, 100 and 500 μM kg−1 soil. Soil sampling was carried out after 1, 7, 14, 30, 60 and 90 days of incubation. It was found that GT significantly stimulated the respiratory activity of soil microorganisms and maintained this activity throughout the experiment. Carbon of microbial biomass, on the contrary, decreases under the influence of GT and is restored at the end of the experiment only in microcosms with 10 and 25 μM GT. Separate estimates of bacterial and fungal biomass showed that the bacterial community increased in biomass on day 14, while fungal biomass increased on day 30 after the treatment. Under the influence of GT, the activity of soil enzymes involved in the carbon (CB, βG, βX), nitrogen (NAG, LAP) and phosphate (AP) cycles significantly increased. High-throughput amplicon sequencing of the ITS and 16S rDNA markers revealed that the soil fungal community is more susceptible to GT than the bacterial community. This was reflected in changes in alpha-diversity indices and in the pattern of changes in the abundance of some microbial genera. Thus, on the one hand, the data obtained provides insight into the biological effects of GT on the soil microbial community. On the other hand, it sets the direction for further research into the ecological role of antibiotics produced by soil and rhizosphere microorganisms.

Isolation and Characterization of Pathogen causing Northern Corn Leaf Blight of Maize in Ri-Bhoi, Meghalaya, India

The present study was carried out to identify and characterize the Exserohilum turcicum causing northern corn leaf blight of maize based on the cultural, morphological and molecular characteristics. Diseased leaf samples from maize fields exhibiting typical cigar shaped symptoms of northern corn leaf blight were collected from the research fields of CPGS-AS (Umiam), College of Agriculture (Kyrdemkulai) and ICAR-RC for NEH region (Umiam) located in Ri-Bhoi district, Meghalaya. Through cultural, morphological, molecular characterization the fungal isolate was identified as Exserohilum sp. Phylogenetic analysis of ITS rDNA gene sequence indicates as Exserohilum turcicum is associated with the disease northern corn leaf blight of maize. The findings underscore the importance of pathogen Exserohilum turcicum causing northern corn leaf blight impact on the maize crop, highlighting potential threat to its production.

Assessing management strategies for mitigating Rhizoctonia damping-off in sugar beet cultivation

Rhizoctonia solani is recognized as one of the most economically significant pathogens that can cause both crown root rot (CRR) and damping-off (DO) in the sugar beet (SB) plant. This report details the first investigative study on R. solani as a fungus causing DO of SB in Morocco. The identification of the fungal pathogen was done both based on morphological features as well as on molecular tools utilizing internal transcribed spacer region of ribosomal DNA (ITS rDNA) and specific primers. In vitro evaluation of the antifungal effect of four commonly used fungicides, tebuconazole, azoxystrobin, trifloxystrobin, and difenoconazole, revealed their effectiveness in inhibiting the growth of pathogenic strains on potato dextrose agar (PDA) medium. Bacillus subtilis Y1336, available in the market, was tested as a potential biocontrol agent (BCA) against R. solani-induced DO. The effectiveness of the BCA was confirmed through double culture tests on PDA, in vitro seed inoculation with both B. subtilis and R. solani, and by employing the antagonist agent as a seed coat in greenhouse settings. This marks the first instance of the effectiveness of B. subtilis Y1336 against R. solani-induced DO in SB. B. subtilis Y1336 should be integrated into SB damping-off control strategies, in conjunction with the fungicides that have been shown to be more effective.