Internal Transcribed Spacer Region Research Articles

Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Genetic diversity of noble crayfish in Finland based on ITS1 microsatellite-like repeat variation: implications to the conservation and management

During the last two centuries, native noble crayfish (Astacus astacus) has been introduced and re-introduced into many waters in Finland, to widen the area of distribution, and to re-establish the collapsed populations. Recent studies have revealed narrowed genetic diversity in A. astacus populations, especially in North Europe, due to crayfish plagues and due to the past translocation policies with numerous introductions also with small number of individuals. However, preliminary data based on the microsatellite like-repeat variation in ITS1 (Internal Transcribed Spacer 1) have indicated also genetic heterogeneity among Swedish and Finnish populations. In this study, the genetic diversity of 38 Finnish A. astacus populations was analysed based on variation in ITS1 region. Stocking histories of populations were obtained from official records and from local fishermen. Eighth out of 38 populations showed divergence in paired comparison with other analysed populations (Population Divergence Test, p < 0.05) indicating either possibility of autochthonous origin or donor. Potential autochthonous populations, as well as refugee areas within original distribution range and within designated protection area, should be further distinguished and monitored to maintain the remaining genetic diversity of the populations.

Meningoencephalitis with malacia caused by Sarcocystis calchasi in a rock pigeon in Japan.

Sarcocystis spp. cause pigeon protozoan encephalitis, a neuronal disease. A female pigeon exhibiting torticollis had a necrotic area in the cerebral hemisphere surrounded by lesions with perivascular cuffing, gliosis, granulomatous foci, and meningitis. Non-necrotic lesions were also observed in the brainstem. Intact and degenerative schizonts were observed within the neuropils and neurons in the lesions. Deoxyribonucleic acid (DNA) was extracted from paraffin-embedded brain tissues and genetically analyzed after gel electrophoresis to determine Sarcocystis spp. using specific primer sets for 28S ribosomal ribonucleic acid and internal transcribed spacer region-1. DNA sequencing confirmed a significant homology with S. calchasi. This is the first report of meningoencephalitis with malacia caused by S. calchasi in a rock pigeon in Japan.

First Report of Fusarium tricinctum Causing Root Rot on Chinese dwarf cherry (Cerasus humilis) in China.

Chinese dwarf cherry (Cerasus humilis) is a perennial small shrub indigenous to northern China, highly regarded for its calcium-rich fruit known as 'calcium fruit'. These fruits have a remarkable capacity to aid in human calcium absorption. Additionally, they contain beneficial flavonoids that hold promise for applications in the healthcare industry (Wang et al., 2018). In July 2020, a concerning development occurred on the farms in Jingtai County (37.48o N, 103.82o E), Baiyin City, Gansu Province in China. Approximately 20 to 30% of C. humilis at the full culture stage exhibited symptoms of root rot, including brownish leaves, rotten roots, and plant mortality, leading to a decrease of over 30% in calcium fruit yield. To identify the pathogen, the surface of symptomatic roots was sterilized with 3% NaOCl for 2 minutes, followed by 70% ethanol for 30 seconds, and rinsed three times with sterile water. Tissue sections (5×5mm) from the margin of the necrotic lesion were cut and cultured on potato dextrose agar (PDA) medium, and incubated for 7 days at 25℃. Five pure culture isolates were obtained from individual spores. Initially, the isolates exhibited abundant white aerial mycelia that turned light pink on the third day. Macroconidia were falciform, two to five septate, straight or slightly curved, and measuring 20.1 to 32.5×2.2 to 3.8 μm (n=50). Napiform microconidia were oval-ellipsoid, non-septate, and measuring 6.2 to 9.3×4.2 to 5.8 μm (n=50). Based on these morphological characteristics, the fungus was tentatively identified as Fusarium species (Leslie and Summerell, 2006). To confirm the identification, the internal transcribed spacer region (ITS) and the translation elongation factor (EF1α) of the isolate CH-2 were partially amplified and sequenced using the primers ITS1/ITS4 and EF2T/EF3 (Li et al., 2013, Yang et al., 2022). Upon comparison with the sequences in GenBank, 857 bp ITS sequence showed 100% homology to Fusarium tricinctum isolate QY3-1 (GenBank accessions no. MZ572963.1), and 665 bp EF1α sequence showed 99.7% homology to F. tricinctum strain TQC-C2 (GenBank accessions no. KF939493.1). The resulting sequences were deposited into GenBank with accession nos. OQ581576 and OQ848462 respectively. A maximum likelihood (ML) phylogenetic analysis based on combined partial ITS and EF1α data set was conducted via ML bootstrapping using MEGA 11. According to morphology and phylogenetic analysis, the isolate was identified as F. tricinctum (Wang et al., 2022). For a pathogenicity test, a pot experiment was conducted in a greenhouse with a temperature range of 20-27℃ and 60% relative humidity. Roots of C. humilis were immersed in a spore suspension (1×107 conidia/ml) of isolate F. tricinctum CH-2 for approximately 5 minutes. Subsequently the treated roots were planted in pots filled with sterilized field soil, while roots dipped in sterilized water were used as the control. The experiment considered of ten pots for the inoculation treatment and six pots for the control treatment. All pots were maintained in the greenhouse. After 15 days, it was observed that 80% of the inoculated plants displayed symptoms consistent with the field observations, indicating successful infection. In contrast, plants in the control treatment did not exhibit any symptoms. The same fungal pathogen as F. tricinctum CH-2 was reisolated from the diseased root tissue and confirmed through morphological and molecular assays, thereby satisfying Koch's postulates. This is the first documented report of F. tricinctum causing root rot in C. humilis in China. This disease has the potential to become one of the most significant diseases affecting C. humilis in China.

First Report of Fruit Rot Caused by Fusarium incarnatum-equiseti species complex on Greenhouse Bell Pepper in Malaysia.

In Malaysia, bell pepper (Capsicum annuum var. grossum), also known as sweet pepper or paprika, is one of the highly imported vegetable crops. In 2021 alone, Malaysia imported nearly 74 thousand metric tons of its chilies, including bell peppers, from other countries (DOSM, 2022). Often, farmers grow the bell peppers in moderate to cool conditions within highland regions for local commercial purposes. In June 2022, the Malaysian Agricultural Research and Development Institute (MARDI) in Serdang, Selangor, conducted a research study to grow lowland bell peppers under a glasshouse rain protection system. A disease inspection carried out found fruit rot on approximately 30% of mature bell pepper fruits in the greenhouse. Symptoms appeared as firm and sunken black lesions covered with white to light pink spore masses on the outer surface, which eventually fell off. Infected fruit parts were disinfected with 10% hypochlorite (NaOCl) for 2 min, followed by double washing with sterile distilled water, air-dried, and placed onto potato dextrose agar (PDA). After 3 days of incubation, the fungal colonies that grew from the symptomatic tissue pieces were transferred onto new PDA to obtain pure cultures. The pure fungal colony appeared dense, whitish aerial mycelium that slowly became cream to pinkish-orange after 7 days of incubation at room temperature (25±2 °C). To examine the morphology features, the pure cultures were subbed onto carnation leaf agar (CLA) and incubated at 25±2°C for 14 days. Macroconidia were abundant, slightly curved with tapered apical cells, 3- to 5-septate, and ranged between 21.8 and 34.0 x 3.0 and 5.1 μm. Microconidia were single-celled, often 1-septate, and ranged between 10.0 and 12.6 x 2.1 and 3.4 μm. Chlamydospores were globose and in chains. The fungus was identified as Fusarium sp. according to Fusarium key by Leslie and Summerell (2006). PCR amplification and DNA sequencing were performed using primers EF1F/EF2R and ITS1/ITS4 (O'Donnell et al., 1998; White et al., 1990) to amplify the partial elongation factor 1-alpha (TEF1-α) gene and internal transcribed spacer region (ITS), respectively. The TEF1-α and ITS sequences of this isolate were deposited in GenBank as OQ672911 and OR349657. BLAST analysis with TEF1-α gene sequences revealed 99.74% and 99.33% sequence identity with F. pernambucanum (accession no. ON330424) and Fusarium isolate NRRL 25134 (accession no. JF740755), respectively; both belonged to the Fusarium incarnatum-equiseti species complex (FIESC). BLAST search of the TEF1-α sequence in the database of the International Mycological Association (www.mycobank.org) showed 99.18% identity with FIESC (NRRL 36548). The ITS sequences were 100% identical to those of F. incarnatum (MT563420, MT563419, and MT563418). Pathogenicity test was conducted on three unwounded and three wounded mature red bell pepper fruits (SP299 Red Masta variety). Two healthy bell peppers were used as controls for each treatment. Prior to inoculation, the fruits were surface-sterilized by dipping in 70% ethanol and rinsed twice with sterile distilled water. Unwounded fruits were inoculated with fungal mycelium disks (5 mm diameter), whereas control fruits were inoculated with sterile PDA agar disks. For wound method, 6 µl of spore suspension (1x106 spores/ml) was obtained from 7-day-old cultures and injected (1 mm depth) into the fruit wall using a sterile syringe needle. Control fruits were inoculated with sterile distilled water only. Each fruit was inoculated with the inoculum at three distinct spots and kept in a humid chamber at a temperature of 25±2 °C. The pathogenicity test was done twice. Five days post-inoculation, the control fruits showed no symptoms, whereas all inoculated wounded and non-wounded fruits developed necrotic lesions with white mycelium growing on the inoculation points. The pathogen was successfully re-isolated from the infected fruits and morphologically identified as FIESC, fulfilling Kochs postulates. It has been reported previously that the members of FIESC are responsible for the fruit rot of bell peppers under greenhouse conditions (Ramdial et al., 2016). To the best of our knowledge, this is the first report of FIESC causing fruit rot on greenhouse bell peppers in Malaysia. This fruit rot disease may impose significant constraints on bell pepper production in Malaysia; hence, effective strategies to control the pathogen and prevent disease dispersal should be implemented.

First report of Sphaerulina azaleae causing leaf spot on Rhododendron pulchrum Sweet in China.

Rhododendron pulchrum Sweet is a popular ornamental evergreen shrub, renowned for its exquisite flowers. In November 2020, leaf spot disease was observed at Qujing Normal University (25.527°N; 103.744°E), Qujing, Yunnan, China. Symptoms were observed on 25-35 % leaves. This led to early leaf drop, significantly reducing its ornamental value. The infected leaves exhibited irregular, dark brown to blackish spots, with random distribution. To investigate the causal agent, ten symptomatic leaves were collected for pathogen isolation. Leaf spots bearing ascomata were picked with a sterile needle, placed on water agar, and then incubated at 26 ℃ for 24 h. Individual germinated spores were transferred onto PDA for further purification and morphological study. Two pure isolates (chl01 and chl02) were obtained. The colonies on PDA were circular, raised, pink-white at the center, dark brown at the margin, and dark brown to black from reverse after four weeks at 26 °C. Moreover, colonies on PDA sporulated following a 5-hour UV light exposure in a laminar flow hood and a subsequent 14 h light: 10 h dark (14:10) cycle for 5 days at 26 °C. The conidiophores are reduced to conidiogenous cells which are 1.5-3 × 1.5-2.5 µm (n = 10), integrated, hyaline, proliferating sympodially near the apex, cylindrical, and widest at the base. Conidia are hyaline, slightly verruculose, guttulate, cylindrical, straight or irregularly curved, subobtuse at the apex, truncate at the base, and 19-28 × 3-4 µm (n= 30), with 1-3 septa. The morphological features were similar to the genus Sphaerulina (Quaedvlieg et al. 2013). To confirm the candidate pathogen, we performed PCR and gene sequencing using specific primers ACT-512F/ACT2Rd, EF1-728F/EF2, ITS4/ITS5, LSU1Fd/LR5 and fRPB2-5F/fRPB2-414R for actin (ACT), the partial translation elongation factor 1-alpha (TEF1), internal transcribed spacer region (ITS), large subunit ribosomal RNA gene (LSU), and RNA polymerase II subunit (RPB2) genes, respectively (Choi et al. 2020). Sequences were deposited in GenBank with accession numbers OR359635 (ACT), OR359637 (TEF1), OR335339 (ITS), OR335299 (LSU) and OR359636 (RPB2). BLAST searches showed 97.34% (ACT), 99.47% (TEF1), 100% (ITS), 100% (LSU) and 100% (RPB2) identity with those of Sphaerulina azaleae CBS 128605 from GenBank, respectively. A multilocus phylogenetic tree was generated using maximum likelihood (ML) based on the concatenated ACT-TEF-ITS-LSU-RPB2 sequence, placing our isolates within the S. azaleae group with high confidence. To fulfill Koch's postulates, three R. pulchrum plants in the landscape (three leaves per plant) were inoculated by pipetting 0.2 ml spore suspension (1 × 106 conidia/ml) onto the surface of each leaf (Khoo et al. 2022). Three additional plants in the landscape were inoculated with sterile water on similar aged leaves. The leaves were enclosed in plastic bags for 72 hours. At 7 days postinoculation, infected leaves exhibited the symptoms as described above, whereas the controls showed no symptoms. This experiment was repeated twice. The same fungus was reisolated from the infected leaves and identified based on morphology and Sanger sequencing. It is worth noting that S. azaleae can cause leaf spot on Rhododendron yedoense f. poukhanense (Choi et al. 2020). To our knowledge, this is the first report of S. azaleae causing leaf spot on R. pulchrum Sweet in China, indicating that appropriate management strategies are needed.

Morphological characteristics and phylogenetic analyses revealed two new species from China and a new record from Jilin Province of Agaricales.

In this study, we have found two new species-Strophariasubrugosoannulata and Strophariamicroaeruginosa. Phylogenetic analyses, based on the internal transcribed spacer regions (ITS) and the nuclear ribosomal RNA gene (nrLSU), suggest that the two new species are distinct and monophyletic. S.subrugosoannulata is distinguished from other species of the genus Stropharia by the pileus covered with greyish-orange squamules in the centre, stipe light brown and surface covered with white triangular squamules. S.microaeruginosa differs from other species in its pileus bluish-grey when young becoming lighter towards margins, later greyish-turquoise lightens towards the edges and surface radially striate when young, lamellae adnate to subdecurrent, stipe with white squamules at the base, acanthocytes absent. The new record species from Jilin Province, Clitolyophyllumumbilicatum was also confirmed, based on morphological and molecular study. Here, we have given full descriptions of each species, colour images, illustrations and two phylogenetic trees that show the placement and relationship of the two new species and the new record are provided.

Microbial fungicides can positively affect aubergine photosynthetic properties, soil enzyme activity and microbial community structure.

This study examined the effects of microbial agents on the enzyme activity, microbial community construction and potential functions of inter-root soil of aubergine (Fragaria × ananassa Duch.). This study also sought to clarify the adaptability of inter-root microorganisms to environmental factors to provide a theoretical basis for the stability of the microbiology of inter-root soil of aubergine and for the ecological preservation of farmland soil. Eggplant inter-root soils treated with Bacillus subtilis (QZ_T1), Bacillus subtilis (QZ_T2), Bacillus amyloliquefaciens (QZ_T3), Verticillium thuringiensis (QZ_T4) and Verticillium purpureum (QZ_T5) were used to analyse the effects of different microbial agents on the inter-root soils of aubergine compared to the untreated control group (QZ_CK). The effects of different microbial agents on the characteristics and functions of inter-root soil microbial communities were analysed using 16S rRNA and ITS (internal transcribed spacer region) high-throughput sequencing techniques. The bacterial diversity index and fungal diversity index of the aubergine inter-root soil increased significantly with the application of microbial fungicides; gas exchange parameters and soil enzyme activities also increased. The structural and functional composition of the bacterial and fungal communities in the aubergine inter-root soil changed after fungicide treatment compared to the control, with a decrease in the abundance of phytopathogenic fungi and an increase in the abundance of beneficial fungi in the soil. Enhancement of key community functions, reduction of pathogenic fungi, modulation of environmental factors and improved functional stability of microbial communities were important factors contributing to the microbial stability of fungicide-treated aubergine inter-root soils.

Starmerella kisarazuensis f.a., sp. nov., a novel yeast isolated from Trifolium pratense flowers.

Three yeast isolates, NBRC 115909T, NBRC 115910 and NBRC 116270, were isolated from Trifolium pratense (red clover) flowers collected from Kisarazu, Chiba, Japan. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions revealed that these isolates represent a single novel species within the genus Starmerella. Also, no ascospore formation was observed. The yeast isolates were closely related to Starmerella vitae UWOPS 00-107.2T and Starmerella bombi NRRL Y-17081T. They differed from S. vitae, the most closely related species with a validly published name, by ten nucleotide substitutions with two gaps in the D1/D2 domains and 20 nucleotide substitutions in the ITS region. Moreover, the three isolates exhibited distinct phenotypic characteristics from the closely related species. Therefore, we suggest that these three isolates represent a novel species, designated as Starmerella kisarazuensis f.a., sp. nov. The holotype is NBRC 115909T (isotype: CBS 18485T).

First report of Fusarium cugenangense causing root rot of tea plants (Camellia sinensis) in China.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.

DNA barcoding of some endangered plant species of the Altai Mountains based on five genetic markers

The analysis of DNA nucleotide sequences is an important source of information in the identification of plant taxa or plant components in the composition of medicines. In this work, we studied the possibility of using universal molecular markers for DNA barcoding of rare plant species distributed in the Altai Biosphere Reserve, located in the Altai Republic (South Siberia, Russia). In order to identify commercially-used endangered species, universal markers recommended for DNA barcoding were applied: ITS (Internal Transcribed Spacers) region of nuclear ribosomal DNA and four regions of chloroplast DNA: trnL-F, trnH-psbA rpl32-trnL, and rpl16. Among 61 species of rare flowering plants protected in the Altai Biosphere Reserve, ten species belonging to eight families were studied. The results of sequencing showed that ITS, rpl32-trnL and trnH-psbA core plant barcodes are the most effective markers for discriminating rare species from the Altai Biosphere Reserve.

Genetic and ecological differentiation of Caragana jubata (Pall.) Poir.

Caragana jubata (Pall.) Poir. is a cold-tolerant mountain shrub and important medicinal species of Fabaceae family with a disjunctive pan-Asian range. Sequences of the atpH-atpI and trnL-trnF intergenic spacers of chloroplast DNA and of the internal transcribed spacer region, ITS-1, of the nuclear ribosomal DNA were used to investigate the intraspecific evolution and phylogeography of this species. Eleven cpDNA haplotypes were identified. The population subdivision is very high (GST = 0.743, NST = 0.943), suggesting a distinct phylogeographic structure (NST > GST, P < 0.001). Phylogenetic analyses of the eleven haplotypes were clustered into four clades, consistent with their respective distributions in four separate regions: Tibet, Tien Shan, Buryatia and Magadan Region. This pronounced phylogeographic gap is also confirmed by nrITS data, however, there is some mixture of genotypes in the Magadan and Buryat populations. Genetic evidence suggests that the center of origin of C. jubata was Tibet and adjacent mountain systems, from where the species spread westwards along continuous mountain chains towards the Tien Shan and northeastwards to the coast of the Sea of Okhotsk and further northwards to Arctic. Divergence of Tien Shan populations of C. jubata may have been caused by climate change in the Pleistocene associated with increased aridity and expansion of deserts, while subsequent climate-induced cycles of range contraction/expansion increased geographic isolation and fragmentation of more northern populations. Marginal northern populations now occupy sites with the most favourable environmental conditions. Special attention is paid to the Okhotsk populations of this species preserved in Magadan Region.

Identification and antifungal sensitivity of Fusarium species isolated from piscine hosts.

Fusarium is a huge genus of filamentous fungi that has the potential to cause emerging diseases. Members of this genus can cause infections in plants, animals and humans. Here, we report the isolation of F. oxysporum and F. equiseti from 2 important fish species, Oncorhynchus mykiss (rainbow trout) and Tor putitora (golden mahseer), respectively. F. oxysporum has emerged as a significant fungal pathogen causing infection in many fish. However, F. equiseti has been isolated mainly from plants. As far as the available literatures are concerned, this is the first report on the isolation of F. oxysporum and F. equiseti from these hosts. The isolates were identified based on growth morphology and microscopic observation. F. oxysporum produced violet pigmentation on potato dextrose agar, while F. equiseti had yellow colouration. F. oxysporum produced 1- to 2-celled microconidia along with straight or curved macroconidia having 3 to 4 septa. F. equiseti produced abundant macroconidia with 4 or more septa. Species were further confirmed based on the nucleotide sequences of the internal transcribed spacer region. In a molecular phylogeny analysis, F. oxysporum and F. equiseti formed 2 different clades. In an antifungal sensitivity assay, F. oxysporum was found to be susceptible to clotrimazole with a minimum inhibitory concentration of 1.0 µg ml-1, whereas F. equiseti was susceptible to clotrimazole, ketoconazole and fluconazole. Overall, the main findings of this study are the infection of new hosts by Fusarium species and the limited activity of many antifungal drugs against these pathogens.

Mycodiversity in a micro-habitat: twelve Cladosporium species, including four new taxa, isolated from uredinia of coffee leaf rust, Hemileia vastatrix

During surveys in the centres of origin of the coffee leaf rust (CLR), Hemileia vastatrix in Africa, as well asin its exotic range in Brazil, 23 isolates of the genus Cladosporium were obtained from uredinial pustules. Using a phylogenetic analysis of all isolates involving a combination of partial sequences of the internal transcribed spacer region of rDNA (ITS) and two gene regions: actin (act) and translation elongation factor-1α (tef1), 12 species weredelimited; including four new species – Cladosporium chlamydosporiformans, C. hemileiicola, C. mucilaginosum and C. setoides. GCPSR criteria were employed for species recognition, supported by morphological and cultural characters. The potential of these purported mycoparasites is discussed in the context of biological control of CLR in Latin America.

Characterization and report of binucleate Rhizoctonia AG‐Fb affecting soybean in Brazil

AbstractRhizoctonia root rot is a disease that can affect soybean leading to plant death usually in bare patches. Frequently, Rhizoctonia solani has been reported as the main causal agent of the disease. However, due to the great genetic diversity of the genus Rhizoctonia, there are other species that could also attack soybean plants. Thus, the objective of this study was to determine the pathogenicity and characterize an isolate of binucleate Rhizoctonia obtained from a soybean plant with symptoms of root rot cultivated in soil from southern Brazil. The isolate was identified based on the morphology and phylogenetic analysis of the internal transcribed spacer region (ITS) of the rDNA. Pathogenicity tests were then performed on seeds and roots of soybean and the optimum temperature for mycelial growth was determined. The isolate was identified as binucleate Rhizoctonia AG‐Fb and it was pathogenic to soybean seeds and roots. In the pathogenicity assay in soybean seed, the isolate reduced seed germination by 20% and caused seed rot index of 69.3. In the pathogenicity assay in soybean root, the inoculation of binucleate Rhizoctonia AG‐Fb caused 67.3 root rot severity index, and reduced the emergence speed index by 19%, seedling height by 31%, root dry mass by 25% and root volume by 43% in soybean seedlings. The optimum temperature for the mycelial growth of the isolate was 26°C. To the best of our knowledge, this is the first report of binucleate Rhizoctonia AG‐Fb associated with soybean root rot in Brazil.

Identification of Invasive Filamentous Mold Isolates Using DNA Sequencing: Experience of a Clinical Laboratory in a Resource-limited Setting

Background: Correct identification of clinically significant mold is becoming critical and cannot be relied only on phenotypic methods; hence, there is a dire need to develop an algorithm of workflows and capacity for molecular identification. This study shares the experience of DNA sequencing for invasive mold identification isolated and compared with phenotypic identification. Methods: This study was conducted at the microbiology laboratory, Aga Khan University, Karachi, Pakistan, and the Mycotic Disease Branch, Centers for Disease Control and Prevention, USA. Filamentous molds isolated from clinical specimens during January 2012–April 2013 were initially identified through phenotypic characteristics. Pan-fungal polymerase chain reaction targeting the internal transcribed spacer region of the ribosomal cistron and the D1/D2 domains of the 28S ribosomal cistron was performed. Sequencer version was utilized to edit and align the DNA sequences, and then sequences were identified using BLAST. The correlation between phenotypic and molecular identification was evaluated. Results: Gene sequencing identified 50% of clinical isolates as one of the Aspergillus species, followed by Mucorales 29%, Fusarium species 17%, and Cladosporium spp. 4%. Overall, 50% of clinical isolates were identified correctly till the species level by conventional methods. Phenotypic correlation with genotype till genus was 42%, while two isolates were wrongly identified phenotypically. Excellent agreement (100%) till species level between phenotypic and molecular identification for Aspergillus, while Mucorales had an agreement of 71%. Conclusion: Traditional phenotypic identification methods for filamentous molds had a good correlation with sequencing up to genus level identification; however, were not reliable up to species level.

Isolation and Identification of Fungi Associated with Post-Harvest Spoilage of Apple (Pyrus Malus) Traded in Kano Metropolis, Nigeria

Storage of fruits at suboptimal condition provides fungi to grow and some produce mycotoxins, which can be dangerous/harmful for human consumption. This study was conducted to isolate and identify fungi associated with spoilage of apples sourced from Kano markets, Nigeria. A total of nine (9) fresh apple samples were collected randomly from different supermarkets on September of 2023 in Kano, Nigeria. Each apple was placed in a sterile plastic bag at room temperature (25–30°C) for fifteen days or until fungal growth was evident all over the samples. Growth of fungal colonies on Potato Dextrose Agar (PDA) was observed and molecular confirmation by Polymerase Chain Reaction (PCR) was conducted along with sequencing (sanger sequencing). Four fruit spoilage fungi were isolated, namely: Aspergillus fumigatus, Aspergillus niger, Curvularia spp, and Yeast spp. Out of the 9 samples that were analyzed, A. fumigatus and Curvularia spp were the most frequent isolates, A. fumigatus was seen in a total of 3 isolates (33.33%), Curvularia spp in 3 isolates (33.33%), A.niger in 2 isolates, and the least was Yeast spp seen in 1 isolate (11.11%). Sequence analysis of the Internal Transcribed Spacer regions of the nuclear encoded rDNA of the A.fumigatus isolate showed significant alignments for Penicillium griseofulvum. This is a potential refinement of the initial morphological characterization. The unexpected identification underscores the significance of incorporating molecular methods in fungal identification processes. The variation between initial morphological identification and molecular results emphasizes the need for precise and accurate identification techniques, particularly when dealing with closely related species. Most of these fungal isolates are pathogenic and can still produce severe illness in immune-compromised individuals, and sometimes otherwise healthy people may also become infected. The study highlights the limitations of traditional fungal identification techniques, as molecular analysis revealed Penicillium griseofulvum instead of the initially identified Aspergillus fumigatus in fungi from damaged apples. This underscores the importance of integrating molecular methods with conventional approaches for accurate fungal identification, vital for managing postharvest losses and ensuring food safety.

First report of anthracnose caused by Colletotrichum karstii on Dalbergia odorifera in China.

Dalbergia odorifera (Family: Fabaceae) is a national second-grade protected tree in China with high medicinal and economic value (Zhao et al., 2020). In July, 2022, a leaves spot disease on D. odorifera with typical anthracnose symptoms was observed in plantations in Haikou (110.319153°E, 19.072900°N), Dongfang (108.630297°E, 19.103838°N) and Qiongzhong (109.704460°E, 19.088440°N), Hainan Province, China. Disease incidence was 7.5% (n = 50 plants). Early symptoms of infected leaves were small and round dark brown spots, which developed into larger irregular necrotic lesions and leaves withered. Leaf tissues (5×5 mm) at the disease-health junction of spots from 19 leaves were sterilized with 2.5% sodium hypochlorite for 1 min, and rinsed with sterile distilled water three times. These sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 28 ℃ for 5 d. 7 strains of fungi with similar morphology were isolated, and 3 single-hyphal isolates (HHL01, HHL02 and HHL03) from each location were selected for further study. Colonies on PDA were fluffy orange-yellow mycelium. Conidia were aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, 11.82 to 15.77 × 3.87 to 6.71 μm (n = 100; average = 13.75 × 5.52 μm). Appressoria formed on slides, measured 5.54 to 10.64 × 4.19 to 7.41 μm (n = 30; average = 8.06 × 5.97 μm) were brown to black, elliptical to irregular. For molecular biological identification, the genomic DNA of three isolates was extracted by fungal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The partial sequences of internal transcribed spacer region (ITS; ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GDF1/GDR1), actin (ACT; ACT512F/ACT783R), β-tubulin (TUB2; TI/Bt2b) and calmodulin (CAL; CL1C/CL2C) were amplified and sequenced by Sangon Biotech (Shanghai) Co., Ltd (Carbone and Kohn, 1999; Weir et al., 2012). The sequences were deposited as GenBank Accession Nos. OR018110-OR018112 (ITS); OR050529-OR050537 (GAPDH, ACT and CAL) and OR192168-OR192170 (TUB). BLASTn results showed these sequences were more than 99% identity with the strain of C. karstii CORCK1 (GenBank Accession Nos. HM585406, HM585387, HM581991, HM585424 and HM582010, respectively). Multi-locus phylogenetic tree of Colletotrichum spp. showed that those three isolates were sister to C. karstii based on the maximum likelihood and bayesian inference methods. To verify pathogenicity, 2 mL spore suspension (1 × 106conidia/ml) of the isolates was sprayed on each leaves of 1-year-old D. odorifera plants, and sterile distilled water was similarly sprayed on other leaves as a negative control. The plants were incubated in a greenhouse under 90% ± 5% RH at 28 °C. Light brown small round necrotic patches developed 3 days after inoculation, while the control was asymptomatic. Photographs were taken on the fifth day after inoculation. The fungi were re-isolated from the diseased leaves and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. karstii has been reported causing leaf rot of Carissa grandiflora in Spain (Garcia-Lopez et al., 2021), and anthracnose caused by C.tropicale was reported on D. odorifera (Yi et al., 2023). To our knowledge, this is the first report of Dalbergia odorifera leaf spot disease caused by Colletotrichum karstii. This finding provides an important basis for further research on the control of the disease.

Spider Fungi: New species of Marasmius and Pusillomyces in the aerial rhizomorph webmaker guild in Amazonia

Rhizomorphs are hair- or wire-like melanized structures with structural differentiation analogous to plant roots that help fungi spread over an area and find food resources. Some species of multiple groups of the Ascomycota and the Basidiomycota produce different types of rhizomorphs. In the Agaricales, the structures are largely found in Marasmiineae, particularly in the Omphalotaceae, Marasmiaceae, and Physalacriaceae. Many rhizomorph-forming species spread over the forest floor (ground level), while others form aerial litter-trapping rhizomorph systems that hang on living branches of the forest understorey. Here, we describe three new species that form aerial systems, two in Marasmius and one in Pusillomyces, all of which inhabit areas of the Amazon rainforest in Amazonas State, Brazil. Support for these new species is based on evidence derived from morphological and molecular phylogenetic analyses using nuclear ribosomal internal transcribed spacer region (nrITS) and nuclear ribosomal large subunit (nrLSU) markers. Marasmius foliauceps sp. nov., with a pileipellis made up of Rotalis-type broom cells, is different from all other species described to date and branched along with species of the monophyletic Marasmius sect. Marasmius subsect. Marasmius, being conspecific with specimens from Guyana. Also different from all other described Marasmius species, Marasmius arachnotropus sp. nov. has a pileipellis mostly composed of Siccus-type but in transition to Rotalis-type broom cells and branched along with species of the Marasmius crinis-equi species complex. Pusillomyces cuieirasensis is morphologicallyvery similar to Pusillomyces manuripioides to which it is sister, but it has a non-umbonate pileus and smaller basidiospores. They all form aerial litter-trapping rhizomorph systems that mimic spider webs, particularly those hanging on the understorey. Whether made of mycelia or silk, such webs serve to capture food. The aerial rhizomorph-forming ‘spider fungi’ make up a guild with hidden species diversity in tropical systems.

High species diversity in Diaporthe associated with citrus diseases in China.

Species in Diaporthe have broad host ranges and cosmopolitan geographic distributions, occurring as endophytes, saprobes and plant pathogens. Previous studies have indicated that many Diaporthe species are associated with Citrus. To further determine the diversity of Diaporthe species associated with citrus diseases in China, we conducted extensive surveys in major citrus-producing areas from 2017-2020. Diseased tissues were collected from leaves, fruits, twigs, branches and trunks showing a range of symptoms including melanose, dieback, gummosis, wood decay and canker. Based on phylogenetic comparisons of DNA sequences of the internal transcribed spacer regions (ITS), calmodulin (cal), histone H3 (his3), translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2), 393 isolates from 10 provinces were identified as belonging to 36 species of Diaporthe, including 32 known species, namely D. apiculata, D. biconispora, D. biguttulata, D. caryae, D. citri, D. citriasiana, D. compacta, D. discoidispora, D. endophytica, D. eres, D. fusicola, D. fulvicolor, D. guangxiensis, D. hongkongensis, D. hubeiensis, D. limonicola, D. litchii, D. novem, D. passifloricola, D. penetriteum, D. pescicola, D. pometiae, D. sackstonii, D. sennicola, D. sojae, D. spinosa, D. subclavata, D. tectonae, D. tibetensis, D. unshiuensis, D. velutina and D. xishuangbanica, and four new species, namely D. gammata, D. jishouensis, D. ruiliensis and D. sexualispora. Among the 32 known species, 14 are reported for the first time on Citrus, and two are newly reported from China. Among the 36 species, D. citri was the dominant species as exemplified by its high frequency of isolation and virulence. Pathogenicity tests indicated that most Diaporthe species obtained in this study were weakly aggressive or non-pathogenic to the tested citrus varieties. Only D. citri produced the longest lesion lengths on citrus shoots and induced melanose on citrus leaves. These results further demonstrated that a rich diversity of Diaporthe species occupy Citrus, but only a few species are harmful and D. citri is the main pathogen for Citrus in China. The present study provides a basis from which targeted monitoring, prevention and control measures can be developed. Citation: Xiao XE, Liu YD, Zheng F, et al. 2023. High species diversity in Diaporthe associated with citrus diseases in China. Persoonia 51: 229-256. doi: 10.3767/persoonia.2023.51.06.