rDNA Region Research Articles

Action Mechanism of Marine Endophytic Fungi Aspergillus terreus as Antibacterial Agent Against Vibrio harveyi

Mangrove ecosystems are among the most diverse marine environments, housing a variety of vegetation, such as shrubs, plants, trees, palms, and other adaptable species that thrive in both freshwater and saline conditions. These mangrove habitats serve as a rich source of secondary metabolites, contributing to various pharmacological activities and playing a crucial ecological role. Endophytic fungi constitute a substantial and measurable portion of fungal biodiversity, known to influence plant community structure and diversity. The purpose of this study is to evaluate the cytotoxic and antibacterial qualities of endophytic fungi that were isolated from Sonneratia alba mangrove leaves that were gathered in Buton, Southeast Sulawesi, Indonesia. The fungi were isolated using surface sterilization techniques and identified based on their morphological characteristics and ITS regions of rDNA. Isolate WB 1-2, obtained from the leaves, was identified as Aspergillus terreus. The marine endophytic isolate, Aspergillus terreus, exhibited antibacterial activity against Vibrio harveyi. Scanning Electron Microscope observations conducted after 24 hours revealed inhibited cell growth in Vibrio harveyi test cells, indicating damage to the cells caused by the hyphae of marine endophytic fungi.

First Report of Botrytis cinerea Causing Gray Mold on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis (L.) O. Kuntze) is an important economic crop cultivated in China. In March 2019, leaf blight symptoms were observed on tea trees (C. sinensis cv. Fuding-dabaicha) in Enshi City (30º02'66" S, 109º01'56" W), Hubei Province (Fig. 1A). The disease occurred on the senescent flowers and mature leaves. The average disease incidence was approximately 10% of plants in the surveyed 30 ha tea garden. Initial symptoms consisted of brown lesions, which expanded and became covered with gray mold, followed by abscission. To identify the pathogen, 20 symptomatic leaves were collected and 0.5 cm2 diseased leaf pieces were excised and surface sterilized by immersion in 1.5% sodium hypochlorite (NaOCl) for 2 min, and rinsed three times in sterilized distilled water. The leaf pieces were allowed to dry, placed on potato dextrose agar (PDA) plates, and incubated at 20°C under 12h fluorescent light. The fungus formed gray to grayish brown colonies (Fig. 1B) and produced conidia after 1 week. The isolate was purified by single spore isolation. The conidia were one-celled, ellipsoid or ovoid, almost colorless, with a size range of 5.3 to 10.24 × 5.2 to 8.1 μm (n = 50) (Fig. 1C). The sclerotia produced in culture were black, round or irregular in shape and 1.3 to 2.2 × 2.0 to 3.3 mm (average 1.8 × 2.3 mm) in size. Morphological characteristics of these isolates matched the description of Botrytis spp. (Hong et al. 2001). Representative isolate LCHM was selected for molecular identification based on DNA sequencing of the ITS region of rDNA and three nuclear protein-coding genes (G3PDH, HSP60 and RPB2) (Staats et al. 2005). BLAST analysis showed that isolate LCHM (GenBank Acc. Nos. MN448502, MN448500, MN433708 and MN448501 for ITS, G3PDH, HSP60 and RPB2, respectively) shared 99 to 100% identity with B. cinerea (GenBank Acc. Nos. MH316147.1, MG846500.1, MG846504.1 and MG846510.1, respectively), which suggested that isolate LCHM belongs to B. cinerea. This identification was further confirmed by phylogenetic analysis based on combined DNA sequence data of G3PDH, HSP60 and RPB2 (Fig. 2). For the pathogenicity test, needle-wounded, attached leaves on 1-year-old C. sinensis cv. Fuding-dabaicha plants were inoculated with mycelial plugs (6 mm diameter from 2-day-old PDA cultures) or a conidial suspension (20 μL, 1×106 conidia/ml in half strength potato dextrose broth) of isolate LCHM on three plants per treatment (six leaves). The control treatments were also wounded, but only treated with agar plugs or half strength PDB. All inoculated plants and controls were incubated in a growth chamber (20°C, 90 ± 10% RH). Leaves inoculated with mycelial plugs, whether wounded or not, showed brown necrotic lesions around the agar plugs after 2 days (Fig. 1D, E), whereas conidial inoculations of wound sites showed necrotic lesions after 6 days (Fig. 1 F, G). Non-wounded leaves inoculated with conidia and all the control treatments remained symptomless. B. cinerea was reisolated from the inoculated leaves and isolates were morphologically similar to the original cultures. Gray mold of C. sinensis caused by B. cinerea has been recorded in Japan, Turkey, and Brazil (Hamaya 1981, Aziz and Harun 2010, Pereira and Mio, 2020). To our knowledge, this is the first report of gray mold caused by B. cinerea on C. sinensis in China. B. cinerea may cause economic losses of tea, therefore, more surveys in other tea-growing regions should be done to specifically search for this disease.

First report of Root rot in Cerasus subhirtella Caused by Fusarium solani and Fusarium oxysporum in China.

Cerasus subhirtella (Miq.) Sok. is a widely used ornamental tree in urban areas around China and has a high ornamental value. From 2018 to 2020, a root rot disease was observed in C. subhirtella in Meitan County, Weng'an County, and Guiyang city of Guizhou, China (106.71 E, 26.57 N). Diseased C. subhirtella trees exhibited wilting with leaf chlorosis accompanied by brown to black root discoloration. In an area of 100 ha in total, with disease incidence ranging from 60 to 80%. Six symptomatic plants with root rot were randomly collected from three locations where disease symptoms were observed for pathogen isolation. Fifty fragments of diseased roots (5×5mm) were disinfected in 3% sodium hypochlorite for 30 s and 75% alcohol for 60 s, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA; BoWei, Shanghai), and incubated at 28 °C in the dark for 7 days. Eighteen isolates were purified by single spore culturing. Typical Fusarium spp. colonies were obtained from all root samples. On PDA, the colonies showed white and the hyphae were dense, while the colony of isolate YH15 showed pale yellow on the back, radial growth and produced chlamydospores. The macrospores (YH15) were straight to subarcuate, measured 15.3 to 25.1 × 2.5 to 6.2 μm (n=50). The microconidia (YH26) were ellipsoid to ovoid, measured 8.6 to 12.7 × 1.6 to 5.1 μm (n=50). These morphological characteristics were consistent with Fusarium spp., as described recently in Vitullo et al. (2014). To confirm the morphological diagnosis, genomic DNA from the isolates was extracted. The internal transcribed spacer (ITS) (White et al, 1990) region of rDNA and a β-tubulin (Varga et al, 2011) gene fragment were amplified with the primers ITS1/ITS4 and Bt2a/Bt2b, respectively, and were subsequently sequenced. Maximum likelihood analysis was carried out using MEGA 11.0. BLAST analysis revealed that the ITS and β-tubulin sequences of isolate YH15 were 100% homologous with F. oxysporum, and the isolate YH26 had a 99.69~100% homology with F. solani. Sequences of isolate YH15 and YH26 were deposited in GenBank (ITS: OQ363005 and OQ363049; β-tubulin: OQ398187 and OQ398180). The isolate YH15 was thus identified as F. oxysporum by the morphological characteristics and sequences analysis, and the isolate YH26 was identified as F. solani. A reconstructed phylogenetic tree also confirmed their phylogenetic position. The healthy 2-year-old C. subhirtella plants grown in autoclaved acid yellow soil were used for the pathogenicity tests. Then, 50 mL of conidial suspension (2.0×105 conidia/mL, in medium) of 7-day-old isolates YH15 and YH26 were gently applied to the soil in each of the 10 pots as the treatment. A sterilized fungal culture matrix (PDB; BoWei, Shanghai) was applied to each of 10 pots as a control. All pots (30 cm high, 25 cm upper diameter, 15 cm base diameter) were placed in a greenhouse (25 °C, 12 h photoperiod). After 30 days of inoculation, all plants inoculated with the isolates showed wilting symptoms, and the roots showed light-brown to dark-brown lesions. No symptoms were observed in the controls. The pathogen was reisolated from all symptomatic roots and identified as F. oxysporum and F. solani as described above. The pathogenicity test was repeated twice with similar results. Although this fungus was previously reported to cause root disease in many hosts (Li et al., 2020; Gibert et al., 2022), this is the first report of F. oxysporum and F. solani causing root rot in C. subhirtella in China.

First Report of Root Rot Caused by Pythium dissotocum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbeć and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.

First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China.

In April 2022, leaf rust disease of Parthenocissus semicordata was discovered in the urban greenbelt in Guangzhou city, China (23.06°N, 113.16°E). The disease incidence was 70% and disease severity was 75%. Chlorotic spots and red-brown necrotic flecks were present on the upper surface of infected leaves, and orange uredinia were distributed on the lower surface (Fig. 1 a-c). Two representative disease plants were collected as voucher specimens and dried, then deposited in Mycological Herbarium of the Zhongkai University of Agriculture and Engineering (MHZU GR0413, MHZU GR0414). Microscopic examination of the pustules of the samples revealed the presence of uredial paraphyses and urediniospores (Fig.1 d-f). Uredial paraphyses were hyaline, incurved, length 20-25 μm and dorsal wall 4.5-8.5 μm thick. Urediniospores were subglobose to ovoid, 16-24.5 × 10.5-17 μm. The wall of the urediniospore was hyaline or pale yellow, echinulate, and 1.0-2.0 μm thick. Telial structures were not observed. The morphological characteristics of uredial paraphyses and urediniospores were consistent with Yoshitaka's description of Neophysopella vitis (Yoshitaka 2000). Ten uredinia were picked using a sterile tweezer and the FlaPure Plant DNA Extraction Kit (Genesand Biotech Co., Ltd) was used to extract genomic DNA. Primers ITS4-rust (5'-CAGATTACAAATTTGGGCT-3') /ITS 5u (5'-CAAGGTTTCTGTAGGTG-3') were used to amplify internal transcribed spacer (ITS) and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') /LR6 (5'-CGCCAGTTCTGCTTACC-3') to amplify large subunit (LSU) rDNA regions (Zhao et al. 2021). The sequences were submitted to NCBI (ITS: OQ991182 OQ991183, LSU: OQ979612 OQ979613), and blastn analyses of ITS and LSU showed 99.81% and 99.84% identity with the sequences from N. vitis (ITS: OQ304336, LSU: OM420266), respectively, found in GenBank. ITS combined with LSU sequences from the current study and reference sequences from Zhao et al. (2021) were used to construct Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees using CIPRES website. ML and BI phylogenetic trees showed that the sequences from the current study grouped with N. vitis, with bootstrap support and posterior probability of 98% and 1.0, respectively (Fig. 2). Morphological and molecular analyses confirmed that the rust fungus was N. vitis. In the pathogenicity test, urediniospores were picked from fresh diseased leaves with sterile tweezers, prepared into spore suspensions (1.0 × 106 urediniospores/ml), and sprayed on a one-week-old leaf from three healthy of potted plants of P. semicordata, while three other healthy leaves were sprayed with sterile water as control. Each treated leaf was wrapped in a plastic bag and incubated in the dark at 25℃ for 48 h after which plastic bags were removed and the plant were moved to a greenhouse at 25℃. At 15 days after inoculation, symptoms and the morphology of urediniospores were confirmed to be the same as those observed in the field collection and no symptoms or uredinia were observed in the control group (Fig.1 g, h). Yoshitaka first discovered leaf rust on P. semicordata caused by N. vitis in Nepal(Yoshitaka 2000). To our knowledge, this is the first report of N. vitis causing leaf rust on P. semicordata in China. P. semicordata is often used as an ornamental plant for exterior wall decoration in urban landscaping and court. The occurrence of this disease can lead to the decline of the P. semicordata leaves and the impairment of the plants aesthetically.

Successive responses of three coral holobiont components (coral hosts, symbiotic algae, and bacteria) to daily temperature fluctuations

Corals and their associated microbiota form a “holobiont,” which includes symbiotic algae and other associated microbiota dominated by bacteria. The composition of holobiont is influenced by various environmental factors, such as increasing sea water temperatures. Previous studies of the effects of temperature changes on coral physiology and associated bacterial communities have been conducted based on stable water temperatures set by mean temperatures, or by slowly increasing/decreasing temperatures. However, the daily temperature fluctuations that corals experience in nature are not stable. The current understanding of the effects of large daily temperature fluctuations on coral and associated bacterial community dynamics is limited. Hence, we conducted a four-week tank experiment using different large daily temperature fluctuations (±5 °C and ±7 °C) accompanied by continuous warming conditions (26 °C to 29 °C) to investigate the effects on two common reef-building corals, Stylophora pistillata and Pocillopora acuta. During the experiment, the activity of coral host catalase and superoxide dismutase were measured, the photosynthetic ability of symbiotic algae was recorded, and the variation in bacterial communities was analyzed using the V6-V8 region of 16S rDNA. According to the results, different parts of the holobionts of two coral species exhibited varying response rates to the continuous warming conditions and diurnal temperature fluctuations. Additionally, it was found that diurnal temperature fluctuations may mitigate the heat stress on the host and reduce the changes in bacterial response to warming. Therefore, it is suggested that the holobionts of different coral species may adopt different adaptation and survival strategies in response to diurnal temperature fluctuations and warming. Finally, based on the response of these two coral species under the conditions of diurnal temperature fluctuations and continuous warming, Acinetobacter and Rhodobacteraceae were identified as potential indicator coral-associated bacteria. This is the first study to investigate the tripartite dynamic response of coral, symbiotic algae and bacteria to daily temperature fluctuations.

Reduced genetic diversity and the success of the invasive peacock bass (Cichliformes: Cichlidae).

Several species of Cichla successfully colonized lakes and reservoirs of Brazil, since the 1960's, causing serious damage to local wildlife. In this study, 135 peacock bass were collected in a reservoir complex in order to identify if they represented a single dominant species or multiple ones, as several Cichla species have been reported in the basin. Specimens were identified by color pattern, morphometric and meristic data, and using mitochondrial markers COI, 16S rDNA and Control Region (CR). Overlapping morphological data and similar coloration patterns prevented their identification using the taxonomic keys to species identification available in the literature. However, Bayesian and maximum likelihood from sequencing data demonstrated the occurrence of a single species, Cichla kelberi. A single haplotype was observed for the 16S and CR, while three were detected for COI, with a dominant haplotype present in 98.5% of the samples. The extreme low diversity of the transplanted C. kelberi evidenced a limited number of founding maternal lineages. The success of this colonization seems to rely mainly on abiotic factors, such as increased water transparency of lentic environments that favor visual predators that along with the absence of predators, have made C. kelberi a successful invader of these reservoirs.

Taxonomy and phylogeny of three rust fungi parasitic on Vitaceae in Korea, with Neophysopella viticola sp. nov. on Vitis vinifera

Rust fungi are important pathogens of trees, ornamental plants, and food crops. Field research targeting rust diseases across Korea uncovered three distinct species of Neophysopella,affecting different members of the family Vitaceae. In our examination of both wild and cultivated grapevine (Vitis) species, including V. amurensis, V. coignetiae, and V. vinifera, we found N. meliosmaemyrianthae as well as a previously undescribed rust species, which we describe here as N. viticola sp.nov. Neophysopella ampelopsidis, which was previously identified as a rust pathogen of Vitis spp., has been observed to specifically infect Ampelopsis brevipedunculata. Through molecular sequenceanalysis of the ITS, LSU rDNA, and cox3 mtDNA regions, we determined the phylogenetic placements of these three species. Our morphological studies revealed the distinguishing features of N. viticola, particularly its unique teliospore layer and urediniospore dimensions. The emergence of N. viticola oneconomically valuable grapevines raises concerns about potential agricultural losses, highlighting the urgency for developing specialized management approaches in viticulture.

Estudos morfológicos e moleculares sobre um parasita nematoide, Syphacia obvelata, que infecta camundongos de laboratório (Mus musculus) na Arábia Saudita

ABSTRACT During screening laboratory mice (Mus musculus) at the Zoology Department of the College of Science, King Saud University, some tiny oxyurid nematodes were detected from the large intestine of these rodents. Worms were identified using morphological and morphometric description. Furthermore, DNA was extracted from worms and subjected to polymerase chain reaction to amplify 18S rDNA and ITS (ITS1, 5.8S and ITS2) regions. Worms detected from caecum and colon of mice were detected in 80% of mice investigated. Morphologically, worms showed elongated body with tapered anterior end and narrow posterior part with cuticular annulations. Male worms measured 0.71-1.12 (0.90) long and 0.01-0.12 (0.11) wide. Female worms measured 3.12-5.011 (4.30) long and 0.13-0.29 (0.17) wide. Esophagus followed by intestine which opens at the posterior end via anal opening in females and via cloacal opening in males. Males have a single spicule with a gubernaculum and an accessory hook. Females’ uteri were pack with eggs. Data from 18S rDNA revealed a sequence which was identical to Syphacia obvelata in NCBI GenBank. Similarly, sequences from ITS regions grouped with sequences from S. obvelata confirming the morphological identity of the worm. However, it showed 3 mutations at the ITS2 region from related sequences from S. obvelata at NCBI GenBank.

First Report of Nigrospora hainanensis Causing Leaf Blight on Brachiaria Griseb in China.

Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 μL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.

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.

Lophium arboricola (Mytilinidiales, Ascomycota) from conifer resins

Lophium arboricola is known as a saprotrophic fungus from different substrates, but it was mainly reported from conifers. In this study, two strains of fungi found growing on resin were molecularly identified as Lophium arboricola. The species was isolated from the resin of Picea abies and Abies alba in Poland. It is the first report of L. arboricola from resin substrate and, simultaneously, the first indication of a resinicolous lifestyle of this species. Isolated strains were morphologically characterized. The phylogenetic analysis was conducted based on ITS and LSU rDNA regions. Moreover, it is the first published report of L. arboricola from Poland.

Two novel species and a new host record of Alternaria (Pleosporales, Pleosporaceae) from sunflower (Compositae) in Myanmar.

Sunflower (Helianthusannuus L.) is a widely cultivated, fast-growing crop known for its seeds and oil, with substantial ecological and economic importance globally. However, it faces challenges from leaf diseases caused by Alternaria species, which threaten its yield. Three small-spored Alternaria species were isolated from leaf spot and blight symptoms on sunflower in Myanmar. All the species were determined based on morphological characterization and a multi-locus phylogenetic assessment of seven genes, including the internal transcribed spacer of rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-α (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG), and an anonymous gene region (OPA10-2). The results introduced two new Alternaria species, A.myanmarensis sp. nov. and A.yamethinensis sp. nov., and a known species of A.burnsii, firstly reported from sunflower.

Replication stress response in fission yeast differentially depends on maintaining proper levels of Srs2 helicase and Rrp1, Rrp2 DNA translocases.

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome.

Coniochaeta fodinicola (Fungi: Sordariomycetes) from a sulphurous spring in Poland

Coniochaeta fodinicola is an acidophilic fungus which has been isolated only several times from extremely acidic environments. In this study, Coniochaeta fodinicola was isolated from a sulphurous spring with slightly acidic, nearly neutral pH water (pH = 6.8) in Poland. The identification of this fungus was conducted based on sequencing and phyloge- netic analyses of ITS and LSU rDNA regions. Detailed morphological characteristics were provided for the isolated strain. The finding of C. fodinicola in a slightly acidic environment indicates that the species may occur in a broader range of conditions than previously known.

Prevalence and Intensity of Larvae of the Genus Anisakis sensu lato (Nematoda, Anisakidae) in Bigeye Scad, Selar crumenophthalmus (Bloch 1793), from the Indian Ocean off Java, Indonesia

This research investigates the prevalence and mean intensity of Anisakis larvae on the bigeye scad, Selar crumenophthalmus (Bloch, 1793), along the Indian Ocean off Java. In total, 498 fish samples were collected from four locations: the Indian Ocean off East Java, Yogyakarta Special Territory, Central Java, and West Java. Each fish sample was measured for length and weight, then examined for Anisakis larvae in the abdominal cavity, liver, gonads, digestive tract, and muscle. Morphological characterisation was done using scanning electron microscope (SEM). The ITS rDNA region was amplified using PCR, then used to perform RFLP for identification and sequenced to form a phylogenetic tree. The results demonstrated that the bigeye scad was vulnerable to infection by Anisakis nematodes with varying prevalence and mean intensity at each location. The bigeye scad from the Indian Ocean off East Java had the highest prevalence and mean intensity (P = 75.44 %; MI = 10.38 larvae per-host), while from West Java had the lowest prevalence (P = 1.45 %; MI = 3.33 larvae per-host). The prevalence of Anisakis in the bigeye scad tends to decrease as one moves to the west from the sampling locations. Most larvae were found in the abdominal cavity (70 %–100 %), whereas a relatively low prevalence was found in other organs. The direct sequencing in the ITS rDNA region showed that Anisakis larvae found in the bigeye scad were A. typica (Diesing, 1860), which was indicated as A. typica var. indonesiensis (A. typica sp. B). Anisakis infection can be utilised as a biological indicator for movement patterns and fish stock differentiation.

MOLECULAR GENETIC CHARACTERIZATION OF UKRAINIAN REPRESENTATIVES OF THE COMPLEX SPECIES ACONITUM ANTHORA BASED ON THE ITS1-5.8S- ITS2 REGION OF THE NUCLEAR GENOME

Problem. The question of determining the boundaries of a species, in addition to the fundamental aspects, is of practical importance for nature conservation activities. One of the taxonomically problematic groups is the complex species A. anthora s. l. In the flora of Ukraine, four taxa with controversial status are distinguished within its borders, A. anthora L., A. eulophum Rchb., A. jacquinii Rchb. and A. pseudanthora Błocki ex Pacz. The last two species are included in the Red Book of Ukraine and the IUCN list. Determining the taxonomic position of these closely related forms requires the use of molecular genetic approaches. The ITS1-5.8S-ITS2 region of 35S rDNA is considered the most popular and informative marker for plant taxonomy and barcoding. Aim. To clarify the taxonomic status and phylogenetic relationships of representatives of A. anthora s. l. using the molecular marker ITS1-5.8S-ITS2 of 35S rDNA. Methods. Herbarium specimens of four representatives of the group A. anthora s. l. were collected on the territory of Western Ukraine. DNA was isolated by a modified CTAB method and then used for PCR amplification of the ITS1-5.8S-ITS2 region. PCR products were purified and Sanger sequenced. The sequences obtained were aligned with the sequences deposited in the GenBank database for representatives of two subgenera of the genus Aconitum, i.e. the nominative subgenus and the subgenus Lycoctonum. The resulting matrix was used for principal component analysis (PCA) and phylogenetic analysis using the Maximum Likelihood method. Main results. The sequences of ITS1-5.8S-ITS2 that we obtained for A. anthora, A. eulophum, A. jacquinii, and A. pseudanthora samples were found to be almost identical. They differed from each other in only five of the 641 alignment positions. Among them, four out of five positions appeared to be polymorphic even within individual genomes. According to the results of PCA analysis, all four samples of A. anthora s. l. formed a dense cluster separated by significant distances from the clusters formed by representatives of the subgenera Aconitum and Lycoctonum. In the generated phylogenetic tree, three main monophyletic clades are present, which correspond to the subgenera Aconitum and Lycoctonum and the A. anthora s. l. group. The Lycoctonum clade was sister to other members of the genus, and the Aconitum and A. anthora s. l. clades were sister to each other. Conclusions. The use of the ITS1-5.8S-ITS2 region of 35S rDNA for phylogenetic analysis confirms the need to grant the A. anthora s. l. group the status of a separate subgenus within the genus Aconitum. The high genetic affinity between samples of A. anthora s. l. from the territory of Ukraine indicates that the species names A. eulophum, A. jacquinii, and A. pseudanthora should be considered synonyms of A. anthora.

Deteksi dan Identifikasi Molekuler Kapang Berpotensi sebagai Penghasil Patulin pada Buah Tropis

Production and export of tropical fruits in Indonesia have an increasing trend every year. The important factor in the production and export of fruits is food safety. One of the contaminants that may be found in fruits is mycotoxin, especially patulin. Patulin mainly found in fruits such as apple, orange, grape, and pear. This study aimed to detect molds from tropical fruits and to identify potentially patulin-producing molds. Detection of potentially patulin-producing molds obtained from tropical fruits was carried out using the isoepoxydon dehydrogenase (idh) gene. Species identification was carried out using internal transcribed spacer (ITS) region. The mold isolate sequences of ITS rDNA region were analyzed for their homology using both BLAST search and phylogenetic tree. A total of 26 molds were isolated from four types of fruit (malang apple, ambon banana, medan orange, and avocado) obtained from five different places in Bogor including fruit stalls, traditional markets, and supermarkets. The positive results of idh gene were mold isolates that have an amplicon at 620 bp. The result showed that 5 of 26 (19,2%) isolates were positive for idh gene, namely A11, A34, A43, A51 and B23. The positive isolates for idh gene were identified as Aspergillus aculeatus, Aspergillus niger, Cladosporium anthropophilum, Cladosporium tenuissimum, and Talaromyces verruculosus.

Two Intra-Individual ITS1 rDNA Sequence Variants Identified in the Female and Male Rotylenchulus reniformis Populations of Alabama.

Around 300 different plant species are infected by the plant-parasitic reniform nematode (Rotylenchulus reniformis), including cotton. This is a devasting nematode with a preference for cotton; it is commonly found in Alabama farms and causes severe reduction in yields. Its first internal transcribed spacer (ITS1) region can be sequenced, and potential mutations can be found in order to study the population dynamics of the reniform nematode. The goal of our study was to sequence the ITS1 rDNA region in male and female RNs that were collected from BelleMina, Hamilton, and Lamons locations in Alabama. After separating the single male and female RNs from the samples collected from the three selected listed sites above, the ITS1 region was amplified selectively using specific primers, and the resulting products were cloned and sequenced. Two distinct bands were observed after DNA amplification of male and female nematodes at 550 bp and 730 bp, respectively. The analysis of sequenced fragments among the three populations showed variation in average nucleotide frequencies of female and male RNs. Singletons within the female and male Hamilton populations ranged from 7.8% to 10%, and the variable sites ranged from 13.4% to 26%. However, female and male BelleMina populations had singletons ranging from 7.1% to 19.7% and variable regions in the range of 13.9% to 49.3%. The female and male Lamons populations had singletons ranging from 2.5% to 8.7% and variable regions in the range of 2.9% to 14.2%. Phylogenetic (neighbor-joining) analysis for the two ITS1 fragments (ITS-550 and ITS-730) showed relatively high intra-nematode variability. Different clone sequences from an individual nematode often had greater similarity with other nematodes than with their own sequences. RNA fold analysis of the ITS1 sequences revealed varied stem and loop structures, suggesting both conserved and variable regions in the variants identified from female and male RNs, thus underscoring the presence of significant intra- and inter-nematodal variation among RN populations in Alabama.

Morphological, Molecular Identification and Pathogenicity of Rhizoctonia solani Kuhn: A Seed and Soil Borne Pathogen Infecting French Bean

French bean (Phaseolus vulgaris L.), holds significant importance as a both vegetable and pulse crop in India. Root rot disease has become threat for successful french bean cultivation. Fusarium spp., Sclerotium rolfsii, Rhizoctonia sp., Macrophomina, Pythium spp. and Thielaviopsis sp. may incite root rot disease in french bean. The root rot caused by Rhizoctonia soalni Kuhn is one among the root rot causing pathogens, is a major devasticating disease in this crop. The objective of this investigation was to perform the morphological, molecular identification and pathogenicity of R. solani Kuhn infecting french bean root rot. The pathogen was isolated from the naturally infected french bean field with characteristic symptoms like yellowing, drying at the soil level, stunting, poor seedling establishment, uneven growth, chlorosis, and premature defoliation in severely affected cases. It was isolated using standard tissue isolation technique and identified based on morphological traits. The fungus was molecular identified by sequencing ITS region of rDNA by using ITS1 and ITS4 primers. Further, pathogenicity of R. solani was proved according to Koch’s postulates.