Internal Transcribed Spacer rDNA Research Articles

Molecular Identification of Microsporum canis Isolated from Infected Children with Tinea corporis and Tinea capitis in Baghdad

Microsporum canis is considered one of the filamentous fungi that cause surface fungal contagion in the humans and animals. The present study aimed to diagnose M. canis via the molecular method and differentiating its local Iraqi isolates from global isolates. Microscopic examination showed 55 specimens with M. canis from 130 specimens collected from children aged between 4-10 years suspected of dermatophytes who attended Medical City Laboratories and Baghdad Hospital in Baghdad city from 1/12/2022 to 1/3/2023. The results showed that the frequency of M. canis infections was 55/130 (42.31%). The results demonstrated significant differences in the animals' contact (p <0.0001), lesions (0.03) and habitation area (p =0.002). Whilst the ages appeared with non-significant differences (p =0.6). In order to confirm the microscopic examination and compare the Iraqi isolates with other global ones, the 55 positive results with M. canis were further diagnosed by using internal transcribed spacer (ITS) 1 and 4 universal primers with a size of 550 bp for PCR amplicons. PCR amplicons sequencing showed only one isolate of M. Canis that differed from global isolates registered in the database of NCBI. The Iraqi local isolate of M. canis was registered with accession number: OM185328. In conclusion, the PCR technique using ITS rDNA aided in confirming the detection of dermatophytes.

Identification and Characterization of Bipolaris secalis and B. setariae Associated with Brown Stripe Disease on Saccharum officinarum in Yunnan, China.

Sugarcane (Saccharum officinarum L.) is the most important sugar crop that belongs to the Poaceae family and is mainly cultivated in tropical and subtropical regions worldwide. Brown stripe disease is a common and serious foliar fungal disease of sugarcane and has become a serious threat to sugarcane production in China. Yunnan Province is the second-largest sugar base in China. From 2022 to 2023, brown stripe-like symptoms from different sugarcane variety were observed in Yunnan. Thirty-nine Bipolaris-like isolates were obtained from symptomatic leaves of different sugarcane varieties and characterized by means of morphological identification combined with multilocus phylogeny comprising internal transcribed spacer rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the translation elongation factor-1α (TEF-1α) gene sequence date. The analysis provided strong support for the delineation and identification of two Bipolaris species associated with sugarcane brown stripe, including Bipolaris secalis and B. setariae. Pathogenicity text in potted sugarcane plants confirmed that these two Bipolaris species can cause brown stripe of sugarcane, thus fulfilling Koch's postulates. This study confirmed that B. secalis and B. setariae were the pathogens of brown stripe in Yunnan Province. To the best of our knowledge, this is the first to report B. secalis as causal agents of brown stripe of sugarcane. Collectively, this finding provides a basis for sugarcane brown stripe disease accurate diagnosis and would be helpful to the development of effective management strategies.

Cadophora arctica (Ploettnerulaceae, Helotiales), a new species from Franz Josef Land

A novel species in genus Cadophora is described as Cadophora arctica. The new species was isolated from, Franz Josef Land, Russia and is represented by ex-type strain VKM F-5011. To characterize the species, we used a polyphasic taxonomic approach. A description based on morphological characters is given and it is shown that the new species is morphologically different from closely related species (to C. caespitosa, C. fastigiata, C. melinii and C. rotunda). Partial sequences of internal transcribed spacer rDNA region (ITS1-5.8S-ITS2), D1/D2 region of 28S rDNA (LSU), the loci encoding β-tubulin (BenA) and partial translation elongation factor 1-α gene (TEF1) were analyzed as well. Sequences data, macro- and micromorphological characteristics distinguish C. arctica from all known species in genus Cadophora. C. arctica, isolated from Arctic drift wood, is able to grow at 2°C and is considered a psychrotolerant species.

Metal tolerance of Río Tinto fungi.

Southwest Spain's Río Tinto is a stressful acidic microbial habitat with a noticeably high concentration of toxic heavy metals. Nevertheless, it has an unexpected degree of eukaryotic diversity in its basin, with a high diversity of fungal saprotrophs. Although some studies on the eukaryotic diversity in Rio Tinto have been published, none of them used molecular methodologies to describe the fungal diversity and taxonomic affiliations that emerge along the river in different seasons. The aim of the present study was to isolate and describe the seasonal diversity of the fungal community in the Río Tinto basin and its correlation with the physicochemical parameters existing along the river's course. The taxonomic affiliation of 359 fungal isolates, based on the complete internal transcribed spacer DNA sequences, revealed a high degree of diversity, identifying species belonging primarily to the phylum Ascomycota, but representatives of the Basidiomycota and Mucoromycota phyla were also present. In total, 40 representative isolates along the river were evaluated for their tolerance to toxic heavy metals. Some of the isolates were able to grow in the presence of 1000 mM of Cu2+, 750 mM of As5+ and Cd2+, and 100 mM of Co2+, Ni2+, and Pb2+.

Pseudoperonospora cubensis Causing Downy Mildew on Luffa aegyptiaca in Korea

In November 2021, sponge gourd (<i>Luffa aegyptiaca</i>) plants with typical downy mildew symptom were observed in Korea. Based on morphological characteristics of sporangiophores and sporangia, combined with molecular phylogenetic analysis of the internal transcribed spacer rDNA and <i>cox</i>2 mtDNA regions, the causal pathogen was identified as <i>Pseudoperonospora cubensis</i> (Oomycota). This is the first report of downy mildew caused by <i>P. cubensis</i> on <i>L. aegyptiaca</i> in Korea. Given that <i>P. cubensis</i> exhibits high disease incidence and severity on many Cucurbitaceae crops, it could pose a significant threat to sponge gourd cultivation.

First Report of Rugonectria rugulosa Causing Dieback of Prunus salicina "Banbianhong" in China.

Banbianhong (Prunus salicina) is a valuable fruit crop cultivated in the southwestern regions of China for its high quality fruit and substantial economic benefits (Wang et al. 2021). In July 2023, dieback disease was observed in Banbianhong in Suijiang County, Zhaotong, Yunnan Province, with an incidence of up to 10%. The disease caused brownish nodules in the cortex of the infected plant, leading to dry cracking, and exposing grayish-brown xylem, which caused the plant to wither and die. Upon longitudinal cutting of twigs, lesions were evident and characterized by browning and necrosis of the cauline bundle and pith within the tissue. The diseased tissues were sectioned into 2 mm2 pieces and surface disinfected with 70% ethanol and 1% NaClO and rinsed with double distilled water. The tissue fragments were plated onto potato dextrose agar (PDA), and incubated for 7 days at 28°C. A total of six isolates were obtained, and two isolates (WG2-1 and WG2-3) were selected for morphological, molecular identification and pathogenicity testing. On PDA, the colony diameter of WG2-1 and WG2-3 were recorded as 5.50±0.56 mm/day and 6.08±0.37 mm/day, respectively. The microconidia of both isolates were zero-septate, smooth, greenish, subglobose to ellipsoidal, and measured 5 to 10 × 1.5 to 3 µm (n = 50) in size. However, macroconidia were not observed. The internal transcribed spacer rDNA (ITS) and β-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/4 (White et al. 1989) and Bt2a/2b (Glass and Donaldson 1995). BLAST analysis of the obtained ITS sequences (accession Nos. PP581792) and TUB2 sequences (accession Nos. PP2815212) sequences showed 100% identity with Rugonectria rugulosa (accession nos. MG991753 for ITS and KM232007 for TUB2). Based on these characteristics, the isolates have been identified as R. rugulosa and have been deposited in the Agricultural Environment and Resource Research Institute plant pathogen lab at Yunnan Academy of Agricultural Sciences. In this study, Pathogenicity tests were conducted on one-year-old Prunus salicina "Banbianhong" twigs to complete Koch's postulates. The twigs were wounded using a 1-mm sterile corking borer and 14-day-old mycelium plugs of isolate (WG2-1) were inoculated and covered with sealing films. Sterile PDA plugs were placed into the wounds of control twigs. Eighteen healthy twigs were inoculated with isolates or PDA plugs (n = 6 twigs/treatment, the size of the twigs: 8 cm length and diameter about 0.7 cm, with three replications) and were placed in a closed sterile tray with the wet filter papers in a thermostatic incubator (type, HYC-260) at 28°C, respectively. The twigs inoculated with the isolates exhibited brown lesions site of inoculation, whereas the controls remained asymptomatic. R. rugulosa was successfully reisolated from the lesions but not from these control wounds and was identified by the methods described above. R. rugulosa has been reported to be associated with the die-back disease of Falcataria moluccana in China (Wang et al. 2019). The occurrence and spread of R. rugulosa in growing areas of Prunus salicina "Banbianhong" in Suijiang county have caused severe economic losses. To our knowledge, this is the first report of the die-back disease of Prunus salicina "Banbianhong" caused by R. rugulosa. These results confirm the pathogenicity of R. rugulosa in Prunus salicina "Banbianhong" and provide valuable insights for developing disease management and prevention strategies.

Novel Approaches to Mortierella alpina Identification and Arachidonic Acid Production Optimization.

Arachidonic acid (ARA) is an integral constituent of cell structures and is instrumental for the nervous, muscular, and immune systems' functions. The sore need for this nutrient may be fulfilled via production based on the fungus Mortierella alpina. The identity of the M. alpina culture obtained from Assiut University, Egypt, was confirmed based on internal transcribed spacer DNA barcoding and elongation enzyme RNA sequencing. Liquid media glucose and peptone as carbon and nitrogen sources, respectively, and diverse micronutritional factors were adjusted for optimal biomass and ARA production. Shake flask cultivation at 25 °C for 7 days produced around 0.570 g of ARA per liter of culture. M. alpina treatment using mutagen 5-fluorouracil and octyl gallate-supplemented glucose-yeast-agar screening plates and shake-flask incubation at 25 °C, then at 20 °C, followed by aging at 10 °C, led to >3 g ARA/liter culture, a yield considered suitable for potential commercial production.

Development of PCR-Multiplex Assays for Identification of the Herpotrichiellaceae Family and Agents Causing Chromoblastomycosis.

The Herpotrichiellaceae family is an important group of dematiaceous filamentous fungi, associated with a variety of pathogenic fungal species causing chromoblastomycosis (CBM) and phaeohyphomycosis (PHM), both with polymorphic clinical manifestations and worldwide incidence. Currently, the identification of this family and determination of the causative agent is challenging due to the subjectivity of morphological identification methods, necessitating the use of molecular techniques to complement diagnosis. In this context, genetic sequencing of the Internal Transcribed Spacer (ITS) has become the norm due to a lack of alternative molecular tools for identifying these agents. Therefore, this study aimed to develop PCR-Multiplex methodologies to address this gap. Sequences from the ITS and Large Subunit (LSU) of ribosomal DNA were used, and after manual curation and in vitro analyses, primers were synthesized for the identification of the targets. The primers were optimized and validated in vitro, resulting in two PCR-Multiplex methodologies: one for identifying the Herpotrichiellaceae family and the bantiana clade, and another for determining the species Fonsecaea pedrosoi and Fonsecaea monophora. Ultimately, the assays developed in this study aim to complement other identification approaches for these agents, reducing the need for sequencing, improving the management of these infections, and enhancing the accuracy of epidemiological information.

PHYLOGENETIC STUDY OF TEN SPECIES FROM CENTAUREA (ASTERACEAE) IN DUHOK CITY, KURDISTAN REGION-IRAQ

The current research aimed to estimate the evolutionary relationships of ten Centaurea L. species growing naturally in the Duhok City, Kurdistan region of Iraq. The combing Start Codon Targeted (SCoT) markers with Internal Transcribed Spacer (ITS) gene region barcode were performed. To detect the DNA sequence variations and phylogenetic tree reconstruction, the Dice similarity matrix, the unweighted pair group method with arithmetic mean (UPGMA) clustering and Maximum Likelihood (ML) methods were applied. 104 polymorphic bands were scored with an average of 10.4. The Polymorphic Information Content (PIC) and Resolving Power (Rp) values ranged between (0.24 to 0.36) and 3.4 in primer (SCoT1) to 12 in primer (SCoT53) with an average of 0.319 and 5.74 respectively. The lowest similarity value was 0.52 between C. behen L. and C. solstitialis L., while the highest was 0.82 between C. balsamita and C. rigida. The reconstructed polytomous dendrogram was as follows: clade one; C. solstitialis L.; clade 2, C. balsamita Lam. and C. virgata Lam.; clade three subdivide into two subclades: C. iberica Trev. ex Spreng., C. hayalolepis Boiss., C. brugueriana (DC) Hand. Mazz. and C. gigantea Sch. Bip. Ex Boiss., C. regia Boiss., C. rigida Banks & Sol., C. behen Lam. Furthermore, C. brugueriana (DC) Hand. Mazz., C. iberica Trev. ex Spreng, C. behen L., C. solstiotialis L. and C. balsamita Lam. were nested with National Center for Biotechnology Information (NCBI). In contrast, the remaining taxa were mixed with other closely related species. Thus, ScoT markers and ITS DNA barcode were considerably effective for investigating the evolutionary relationships of Centaurea taxa.

Pythium aphanidermatum as a Novel Oomycete Pathogen Causing Leak on Potato (Solanum tuberosum) in Korea

Potato (Solanum tuberosum L.) is one of the most important food crops in Korea. In July 2021 and 2022, dark black-rot symptoms with pink tinges were observed on field-grown potato tubers in Hongsung and Chuncheon, Korea, respectively. We obtained four isolates (HSv05 and HSv10 from Hongsung, and CCp03 and CCp05 from Chuncheon) from diseased tubers and identified these isolates as Pythium aphanidermatum by analyzing the sequences of internal transcribed spacer rDNA region and mitochondrial cytochrome c oxidase subunit II (COX2) mtDNA gene. Additionally, we compared the cultural and morphological characteristics of these four isolates with those of the reference isolate KACC 48066 of P. aphanidermatum and the literature. Further, we tested the pathogenicity of all these isolates against potato tubers. The cultural and morphological characteristics of the four test isolates were similar to those of the reference isolate and the literature; all four test isolates proved pathogenic to potato tubers. Therefore, we concluded that P. aphanidermatum is the causal agent of potato leak and this is the first report of the disease on potato in Korea.

Evaluation of ITS1 rDNA primers for the detection and identification of African trypanosomes in mammalian hosts and tsetse flies.

Although several primers targeted to the internal transcribed-spacer 1 (ITS1) of the ribosomal DNA (rDNA) have been designed to improve the detection of African trypanosomes, no study tried to compare their agreement level and ability to amplify different trypanosome species in tsetse flies and mammals in various epidemiological settings. This study was designed to fill this gap, by targeting tsetse-infested areas of Cameroon. For this, archived DNA samples reporting at-least one trypanosome species with species-specific PCR primers were reviewed. Ten sets of primers targeting different ITS1 rDNA sequences of trypanosomes were selected for assessment using single-round and nested-PCR method. Amplification rates (sensitivity) and agreement level of different ITS1 assays were compared using Cohen's-Kappa and McNemar's x2 statistic. Little agreement level (k = 0.05–0.52) were observed between different ITS1-primers PCRs detection of African trypanosome species despite significant (X2=54.3, p = 0.0001) high amplification rate 91.6 % (339/370). This sensitivity varied from quite low for T. simiae (11.9 %) and T. vivax (27.3 %) to fairly good for T. congolence (51.9 %), Trypanozoon (32.4 %) and T. theileri (40.3 %). Primers set targeting ITS1-A sequence of trypanosome species recorded the highest sensitivity (50.5 %) with fairly good agreement compared to 39.2 % for ITS1-C (k = 0.52), 32.4 % for ITS1-R (k = 0.47), 29.7 % for ITS1-N (k = 0.48) and 23.0 % for ITS1-KIN (k = 0.43) respectively. This study revealed a diversity in the sensitivity of different trypanosome species with different sets of ITS-primers enhancing the need to use the same sets of primers in different bio-ecological settings. The use of nested-PCR instead of single-round PCR enabled improvement of trypanosome infections detection in both tsetse and mammals. Among the sets of ITS1-primers tested, those designed by to amplify ITS1-A can be considered as the most appropriate for the detection of trypanosome infections in mammals and tsetse flies.

Simple sequence repeat genotyping of Colletotrichum species associated with soybean anthracnose in Uttarakhand and other states of India

AbstractAnthracnose is one of the most devastating diseases affecting soybean (Glycine max L. Merril) in India and is incited by the Colletotrichum truncatum species complex. In this study, 33 isolates of Colletotrichum spp. isolates were collected from major soybean‐growing states of India. The cultural, morphological and molecular characteristics (Internal Transcribed Spacer rDNA region) of all isolates were demonstrated. Twelve simple sequence repeat (SSR) markers were selected to amplify the genomic DNA of all isolates, to assess the genetic diversity. On the basis of morpho‐cultural and molecular characterization of three different Colletotrichum species, C. truncatum, C. cliviicola and C. cholorophyti were identified. Representative Colletotrichum isolates (Ct‐Pant, Ct‐Gur and Ct‐Gag) from each species group were found pathogenic to susceptible soybean variety (PS 1092) and fulfilled Koch's postulates. The SSR genotyping of 33 isolates revealed that all examined SSR markers exhibited a high degree of polymorphism, as indicated by the mean values of 0.51 for polymorphic information content, 1.97 for marker index and 1.99 for resolving power. For each primer, the average number of alleles per locus was 3.58. The results of Jaccard similarity coefficient‐based UPGMA (Un‐weighted Pair‐Group Method with Arithmetic Mean) cluster analysis and principal component analysis showed that isolates of the same species were clustered together, but no clear‐cut grouping was obtained on the basis of geographical distribution. Thus, it can be inferred from the study that C. truncatum accessions obtained from various geographical locations of India exhibit substantial genetic variation and a significant level of relatedness.

Five novel Curvularia species (Pleosporaceae, Pleosporales) isolated from fairy circles in the Namib desert

The Namib Desert (Namibia) is home to fairy circles which are barren, circular to almost-circular patches of land surrounded by grasses. During a survey of the fungi associated with the most common grass species, Stipagrostis ciliata (Poaceae), and its rhizospheric soils associated with these fairy circles, Curvularia was commonly isolated (80 strains). Curvularia is a cosmopolitan fungal genus that occurs in diverse geographical locations and on a wide range of substrates, but particularly on foliar plants. Curvularia strains were identified based on multilocus sequence comparisons of their internal transcribed spacer rDNA region (ITS), and the partial gene regions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1). The strains belonged to 13 species, including the discovery of five novel Curvularia species. The aim of this paper was to report on the identified species and to formally describe and name the new species as C. deserticola, C. gobabebensis, C. maraisii, C. namibensis, and C. stipagrostidicola.

First Report of Rust Disease on Alcea rosea caused by Puccinia modiolae in China.

Alcea rosea, belonging to the Alcea genus in the Malvaceae family, originated from China, but it is now grown worldwide. A. rosea has been widely used in traditional Chinese medicine to alleviate constipation, pain, swelling, and sores. In February 2023, typical symptoms of fungal infection were observed on A. rosea at Guizhou Normal University in Guiyang, Guizhou Province, China. The disease incidence was over 90% (n = 100) for the surveyed A. rosea plants, and the disease severity range from 30% to 90%. The initial symptoms of A. rosea rust were the appearance of chlorotic spots on the leaves. Subsequently, numerous reddish to dark-brown erumpent pustules (telia) were observed. Gradually, the entire plant was covered by rust and the center of each lesion turned brown, necrotic, and ruptured over times, eventually causing defoliation. Voucher specimens of infected A. rosea leaves as representative samples have been deposited at Guizhou Normal University (GNU2023LS008). Telia are round in shape, mostly aggregated in mass, with a diameter of 0.28-0.78 mm (0.46 mm, n = 20). They range in color from reddish-brown to dark brown, and are mainly hypophyllous but occasionally formed on the adaxial leaf surface. The teliospores are fusoid with dimensions of 31.3-93.8 × 10.9-21.3 μm (57.5 × 15.1 µm average, n = 50), hyaline or yellowish to light-brown in color, mostly two-celled, with a smooth wall (1.5-3.0 µm) and a thickened apex (3.0-9.0 µm). However, teliospores which are one-, three-, or four-celled with a notch at the apex, are rarely observed. The morphological characteristics of host symptoms and teliospores were similar to those of Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). For phylogenetic analysis, genomic DNA was extracted from the teliospores of infected leaves. To confirm the species-level identification, PCR was performed on the extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS) and large subunit (LSU) regions using primer pairs ITS1/ITS4 (Schoch et al. 2012) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The resulting ITS DNA sequence (GenBank accession no. OR607960) showed 100% identity with P. modiolae sequences (OP369291.1), when the query coverage was 100%. The LSU DNA sequence obtained (OR607961.2) shared 99.85% similarity with P. modiolae (MK458702.1). A phylogenetic tree was constructed using MEGA7.0 and the maximum likelihood method based on the ITS and LSU sequences. The fungal isolates collected in this study and several reference sequences of P. modiolae were grouped within a clade that included the isolates reported on A. rosea in Korea (Ryu et al. 2023), with 100% bootstrap support. Pathogenicity testing was conducted by gently pressing spore powder of naturally diseased leaves onto young leaves of three healthy A. rosea plants, with three noninoculated healthy plants serving as controls. The inoculated and noninoculated plants were kept in a growth chamber at the 26°C with a 12 hour light/dark cycle and 80% humidity. After 2 weeks, all inoculated A. rosea plants showed characteristic disease symptoms of rust infection and telia of P. modiolae, while control plants remained symptomless. The pathogen was identical to that observed on the original diseased leaves. The study results indicate that the causal fungus responsible for the disease is P. modiolae, which has been previously reported on Malvaceae plants (Farr and Rossman 2022). To the best of our knowledge, this is the first report of P. modiolae on A. rosea in China. This study will contribute to an increased understanding of the host range of Puccinia modiolae.

First report of leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

Gastropod invasions in anthropogenically impacted impoundments in South Africa: Tracing their origins and exploring field evidence of parasite spillback and amplification

Invasive snails are associated with ecological problems in freshwater bodies worldwide. However, their impact on the transmission of digenean infections remain underreported. In the present study, 1708 specimens representing four snail species were sampled from four impoundments in the Limpopo River system in South Africa. Gyraulus chinensis (Planorbidae), Physella acuta (Physidae) and Pseudosuccinea columella (Lymnaeidae), which are invasive, were found in all the sampling sites. In contrast, the native lymnaeid Radix natalensis occurred at only one study site. Digeneans were observed only from R. natalensis (prevalence = 49%) and Ps. columella (prevalence = 23%). Morphological and genetic analyses revealed four digeneans: Fasciola nyanzae, Orientocreadium sp., Petasiger sp. and Patagifer vioscai. Pseudosuccinea columella was infected by the four digeneans while R. natalensis harboured only Orientocreadium sp. and Petasiger sp. Partial sequences of Orientocreadium sp. from the current study differed from congeners whose DNA data are available on GenBank, by p-distances of at least 1.84 and 2.2% for 28S and the internal transcribed spacer (ITS) rDNA, respectively. Phylogenetic analyses demonstrated that the present species is sister to Orientocreadium batrachoides. Genetic and phylogenetic data based on 28S and ITS rDNA suggested that Petasiger sp. from the present study and isolates of three unidentified Petasiger spp. from Kenya, Hungary and Australia, were representatives of the same species. This is the first known report of Orientocreadium, Petasiger and Patagifer from Ps. columella. The occurrence of F. nyanzae in Ps. columella indicates spillback from R. natalensis. These findings echo the concerns raised in previous studies about the potential role of Ps. columella in the amplification of digenean diseases in its introduced range. Phylogenetic analyses of partial sequences of the cytochrome c oxidase subunit 1 mitochondrial gene (cox1) showed multiple lineages of Ps. columella in North and South America. Pseudosuccinea columella specimens from the present study belong to an invasive genotype that has spread globally and has been reported from Zimbabwe, Egypt, Portugal, Australia, Argentina, Colombia and New Mexico (USA). Physella acuta from the current study had a stronger genetic relationship with isolates from Canada and Iceland, than with isolates from other parts of Africa, suggesting several invasion routes into Africa. This is the first known DNA characterisation of G. chinensis from Africa. Phylogenetic reconstruction indicated multiple exit events of G. chinensis from Asia into Europe and Africa. South African isolates clustered in a recent branch containing isolates from the Czech Republic and Hong Kong, China. Considering the presence of invasive snails in all the sampling sites in the present study, it is necessary to investigate the factors that enhance their establishment and to monitor their effects on the native snail populations.

Leaf Spot of Hedychium coronarium Caused by Colletotrichum gloeosporioides in China

Hedychium coronarium (white ginger) is widely cultivated for garden decoration and folk medicine. Since 2020, symptomatic leaves showed brown necrosis and yellow borders on H. coronarium in the field (approximately 200 m2) at Southwest University, Rongchang District, Chongqing City. Small brown-yellow spots gradually enlarged and caused withering in severe cases with a mortality rate of around 10%. Disease incidence and severity varied from 55 to 65% and from 30 to 40%, respectively. Infected tissues (5 mm in diameter) were cut from lesion margins, surface sterilized in 70% ethanol for 10 s and 0.1% acidic mercuric chloride for 3 min, followed by rinsing in sterile water three times, and then were cultured onto potato dextrose agar (PDA) at 25 °C. Five isolates were transferred onto fresh PDA and purified by single-spore culturing. The colonies were initially white and turned hoary, and the diameter reached 32.95 to 38.37 mm × 32.42 to 38.61 mm after 3 days of incubation. Pale gray abundant fluffy aerial mycelia were arranged irregularly and densely. Hyphae were septate and branched, 2 to 5 µm in width. Conidiophores were pale brown, septate, branched, cylindrical to ampulliform. Conidia were hyaline, smooth-walled, cylindrical with obtuse ends, and 8.1 to 13.3 μm × 2.4 to 5.8 μm (n = 50) in size. Appressoria were medium brown to dark brown, aseptate, in irregular shape, solitary or in groups, and measured 1.5 to 12.5 μm × 2.1 to 13.3 μm. Morphological characteristics of isolates agreed with the description of Colletotrichum (Liu et al. 2015). Genomic DNA was extracted from fungal colonies incubated on PDA for 7 days following the instructions from the PlantGen DNA Kit (CWBIO, China). The internal transcribed spacer (ITS) region, and fragments of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2) and large subunit ribosomal DNA (LSU rDNA) genes were amplified by primer pairs ITS1/ ITS4, GDF1/GDR1, ACT512F/ACT783R (Naz et al. 2017), T1/Bt2b (Glass et al. 1995) and LROR/LR7 (Castlebury et al. 2002), respectively. The sequence of representative isolate CG-H (GenBank accession nos. OM010355, OM238213, OM238214, OM045778 and OM010358 for ITS, GAPDH, ACT, TUB2 and LSU rDNA, respectively) exhibited 99 to 100% identity to Colletotrichum gloeosporioides. A multi-locus phylogenetic tree with concatenated sequences of ITS, GAPDH, ACT and TUB regions was constructed using the maximum likelihood method by MEGA7, which revealed that strain CG-H was grouped with C. gloeosporioides. To confirm the pathogenicity, six healthy H. coronarium plants were surface sterilized, and conidial suspension (1 × 106 conidia/mL) was sprayed onto the leaves. Six plants were inoculated with sterile distilled water as controls. All the plants grew in a greenhouse at 25 °C under 12 h/12 h photoperiod. The experiment was repeated four times. Yellow lesions appeared after 7 days of inoculation, irregular-shaped brown spots were formed and slightly sunken within 14 days, and the whole leaf gradually became withered in 50 days. All inoculated plants exhibited leaf spot symptoms while the control plants remained asymptomatic. C. gloeosporioides was re-isolated from lesions of leaves and identified by morphology and sequence analysis, fully confirming Koch’s postulates. This is the first report of C. gloeosporioides associated with H. coronarium leaf spot in China and worldwide. Further studies will be conducted on the sensitivity of C. gloeosporioides to various fungicides.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Molecular Verification of Trichophyton in the Brazilian URM Culture Collection.

Trichophyton species cause dermatophytosis in humans, with a high, worldwide frequency of reports and important public health relevance. We evaluated 61 Trichophyton strains from different sources deposited in the University Recife Mycology (URM) culture collection of the Universidade Federal de Pernambuco, Brazil. Strains were phenotypically identified and confirmed by sequencing Internal Transcribed Spacers rDNA and partial beta-tubulin 2-exon. Additionally, we evaluated their susceptibility to terbinafine and itraconazole. Physiological analyses included urease activity and growth in casein medium. Phenotypic methods allowed the reliable identification of T. rubrum only, whereas, for other species, molecular methods were mandatory. All Trichophyton species exhibited susceptibility profiles to itraconazole (0.04-5.33μg/mL) and terbinafine (0.17-3.33μg/mL). Our analyses revealed a heterogeneous distribution of T. mentagrophytes, which does not support the current distribution within the species complex of T. mentagrophytes and its genotypes.

Isolation, identification, and antibacterial activity of endophytic fungus from Acanthus ilicifolius

Abstract Antimicrobial-resistant bacteria pose serious public health risks, necessitating bioprospecting for novel antimicrobial drugs. The endophytic fungi of the mangrove ecosystem are hotspots for discovering new bioactive chemical compounds. In this context, an investigation was designed to determine the isolation of the major endophytic fungi inhabiting the leaves of Acanthus ilicifolius, a mangrove plant with a long history of traditional use in the Chinese and Indian medical systems. Based on the morphological characterizations and molecular analysis of internal transcribed spacer rDNA sequence data, the study identified three unique endophytic fungal species, namely, Periconia macrospinosa, Coprinopsis cinerea, and Alternaria sp. Of these, P. macrospinosa was identified as the most dominant one, with the highest relative frequency (35.22%). The antibacterial activity of P. macrospinosa isolate (CMFRI/fPM-01) was evaluated by the well and diffusion method against six human pathogens, viz., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. The results demonstrated a high and wide spectrum of antimicrobial action of the isolate against all the tested human pathogens, with no significant difference (P > 0.05) in the activity between the pathogens. The antibacterial activity was further confirmed by determining the fungal culture supernatant's minimum inhibitory concentration and minimum bactericidal concentration. Although the studied fungi are known from other sources, this is the first report of P. macrospinosa and C. cinerea as endophytes in A. ilicifolius leaves. The outcomes also showed that the P. macrospinosa isolate could be used to discover effective antibacterial drugs against various human diseases.