ITS rDNA Sequences Research Articles

Untargeted Metabolomics Approach for the Differentiation between Panax vietnamensis var. vietnamensis and Panax vietnamensis var. fuscidiscus.

Panax vietnamensis var. vietnamensis (PVV) and Panax vietnamensis var. fuscidiscus (PVF) both belong to Panax vietnamensis species and are chemically and morphologically similar, making it hard to distinguish for the consumer. Herein, 42 PVF and 12 PVV samples were collected in Quang Nam and Lai Chau Province, respectively, and subsequently characterized by ITSr-DNA sequence data to verify their origins. Next, untargeted metabolomics combined with multivariate statistical analysis was developed to differentiate PVV and PVF. The metabolic profiles of PVV and PVF were found to be distinct and classified well using Partial Least-Squares Discriminant Analysis (PLS-DA) in the training set. Among them, seven ginsenosides were of high abundance in PVV, while six were of high abundance in PVF. Next, the test set was used to validate 13 putative differential markers found in the training set, illustrating a complete match with the expression patterns of these ginsenosides in the training set. Finally, PLS-DA and linear Support Vector Machine models both indicated distinct ginsenoside profiles of PVV and PVF without misclassification in the test set. Conclusively, the developed untargeted metabolomics approach might serve as a powerful tool for the authentication of PVV and PVF at the metabolome level.

Cylindromonium dirinariae sp. nov. (Ascomycota, Hypocreales), a new nectrioid lichenicolous species on Dirinaria applanata in Japan.

A nectrioid fungus forming a pinkish colony with mainly solitary phialides producing ellipsoid, aseptate conidia in mucoid packets was isolated from Dirinaria applanata. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA sequences revealed that the isolates represented a member of the genus Cylindromonium. Based on further morphological examination, nucleotide sequence comparison, and phylogenetic analysis based on LSU rDNA, tef1, and rpb2 in addition to the phylogenetic analysis using the ITS rDNA sequences, the fungus from Dirinaria represents a new species, which is described here as Cylindromonium dirinariae sp. nov. Furthermore, inoculation experiments revealed that this species can also produce perithecia when inoculated on the host lichen in laboratory environments. Citation: Ohmaki A, Okane I, Crous PW, Verkley GJM (2023). Cylindromonium dirinariae sp. nov. (Ascomycota, Hypocreales), a new nectrioid lichenicolous species on Dirinaria applanata in Japan. Fungal Systematics and Evolution 11: 1-10. doi: 10.3114/fuse.2023.11.01.

Isolation and identification of carotenoid-producing Rhodotorula sp. from soils collected in the coastal environment of Kien Giang and Tra Vinh provinces

Rhodotorula is a genus of marine yeasts that is considered the most suitable for carotenoid production. In this study, collected soils at Kien Giang and Tra Vinh provinces were cultured on yeast extract peptone dextrose (YEPD) agar for 72 hours at room temperature. Twenty-three isolates exhibiting yellow, orange, and pink pigments were tested for physiological and biochemical characteristics such as carbohydrate fermentation and assimilation, nitrate assimilation, urease production, and growth temperature. Their oval dimensions were 4.24x3.64 μm for isolate 5S1-T and 3.88x3.17 μm for isolate 5S4-T. The two isolates could assimilate carbohydrate and nitrate sources and produce urease. The ITS rDNA sequence of isolates 5S1-T and 5S4-T both had 100% matches to Rhodotorula sphaerocarpaCBS:5940 and Rhodotorula sphaerocarpa CBS:5939, respectively. The maximum absorbance wavelengths of the solution extractions from the 5S1-T and 5S4-T were 496 and 487 nm, respectively. The total carotenoid content of strain 5S1-T was found to be the highest (1.81 mg/l) followed by strain 5S4-T (1.16 mg/ml). The antioxidant abilities of the 5S1-T and 5S4-T were demonstrated by scavenging the free radical DPPH and ferric reducing capacity. The free radical scavenging activity of the extract from isolate 5S1-T (96.15%) was higher than that of isolate 5S4-T (88.61%) at 0.14 mg/ml. The absorbance at 700 nm of the extracts of both 5S1-T and 5S4-T significantly increased from 0.05 to 0.3 mg/ml. The two strains 5S1-T and 5S4-T of Rhodotorulasphaerocarpa discovered in this study could contribute to carotenoid and antioxidant-producing yeasts for industries in the future.

Termitomyces assamicus (Lyophyllaceae)—A new species of Termitomyces from India

Based on morphological and molecular phylogenetic analysis (ITS-rDNA sequence), a new species, Termitomyces assamicus, is described after the Indian state of Assam. Termitomyces assamicus is characterized by a medium-sized pileus, brownish orange to grey or greyish red with a conical, pointed perforatorium; white to pale grey lamellae with a serrated edge; an orange grey to greyish orange stipe measuring 50–100 mm long; absence of annulus; greyish brown to yellowish brown pseudorhiza measuring 70–100 mm long; ellipsoid basidiospores with a mean of 8.3 × 5.0 μm; clavate cheilocystidia measuring 21–36 × 8–16 µm; and broadly clavate to pyriform pleurocystidia (23–32 × 12–23 µm). The new species was compared with closely related taxa using morpho-molecular data. A detailed morphological description with field images, micromorphology illustration, and a phylogenetic tree to show the position of the new species are also provided.

First report of leaf blight caused by Alternaria alternata on Platanus acerifolia in China.

Platanus acerifolia Willd. is widely planted in cities in China due to its strong adaptability to different environmental conditions. In August 2021, light brown, oval to circular, sunken spots were observed on leaves of P. acerifolia trees with 8-35% incidence, leading to severe necrosis and abscission of leaves on a street in Haidian district of Beijing (116°29'84''E, 39°95'93''N). Small pieces (5 mm×5 mm) were taken from the margin of diseased tissues, disinfected with 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed with sterile water, then plated on potato dextrose agar (PDA) and incubated at 28°C. After 4 days, representative isolates were transferred to new PDA plates. Four isolates with similar morphological characteristics were obtained and deposited in the culture collection (ID: DAA3, DAA5, DAA6 and DAA7) of our laboratory. Colonies on PDA were dense, fluffy, and light to dark gray, with a prominent white margin. Conidia formed in chains on the branched conidiophores, and were obpyriform to ellipsoid, 19.5-32.3×5.5-10.2 μm (average=26.4×7.1 µm, n=30) in size, with 3 to 5 transversal and 1 to 3 longitudinal septa. These morphological characteristics matched those of Alternaria spp. (Simmons 2007). Genomic DNA was extracted with modified CTAB method and the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB2), glyceraldehyde 3 - dehydrogenase (GPD), endopolygalacturonase (EndoPG), as well as Alternaria major allergen (Alt a1) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), gpd1/gpd2 (Berbee et al. 1999), PG3/PG2b (Andrew et al. 2009), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained sequences were deposited in GenBank (accession numbers: OM228653-OM228656, OM221523-OM221542). In a BLAST search, the sequences were 100% identical with corresponding sequences of A. alternata. Phylogenetic analysis based on combined sequences using maximum parsimony method showed that the four isolates clustered together with the type strain CBS 916.96 of A. alternata. For pathogenicity test, three healthy leaves of three one-year-old P. acerifolia plants were wounded with a sterile needle and inoculated with 20 µl of spore suspension (106 conidia/ml). Plants inoculated with sterile water were treated as control. The pathogenicity test was also conducted on the unwounded leaves. After 8 days of inoculation at 25°C and 90% RH with a 12-h photoperiod, the symptoms on spore suspension-inoculated leaves were similar to those observed on trees in the street, whereas the control leaves remained symptomless. Lesions on wounded leaves were larger than those on unwounded leaves. The assay was repeated twice with consistent results. The pathogen was re-isolated from symptomatic leaf tissues and identified based on morphological and rDNA-ITS sequencing, thus, fulfilling Koch's postulates. Examples of other tree species where Alternaria alternata has been reported to cause leaf blight were Ophiopogon japonicas in China (Wang et al. 2021) and Pistacia terebinthus in Spain (López-Moral et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf blight on P. acerifolia in China. The identification could provide information for developing effective disease management strategies.

Anastomosis Grouping and Phylogenetic analysis of Rhizoctonia Isolates on Wheat in Türkiye

The aim of this study is to determine the species and to evaluate the genetic diversity of the pathogenic and nonpathogenic Rhizoctonia spp. and anastomosis groups (AG) from wheat plants and rhizosphere soils in Turkey. Rhizoctonia species were isolated from plants and rhizosphere soils in wheat fields in 5 provinces in the Central Anatolian Region of Türkiye. As a result of the isolations, a total of 88 multinucleate (MN) and binucleate (BN) Rhizoctonia isolates were obtained. Identifications of the isolates were determined by rDNA-ITS sequence analyzes. The identified isolates belonged to MN Waitea circinata var. zeae, W. circinata var. oryzae, W. circinata var. circinata, MN Rhizoctonia solani AG 2-1, 2-2, 3, 4 HGII, 4 HGIII, 5, 8, 11 and BN AG A, DI, E, G, H, I, I-like, K. The most isolated group was W. circinata var. circinata. In pathogenicity studies, the most virulent group was determined as R. solani AG 4. Among the binucleate isolates, groups other than R. cerealis AG DI were not found to be pathogenic. Neighbor-joining phylogenetic trees of isolates were constructed from rDNA-ITS sequences. As a result of this study, the regional distribution of MN and BN Rhizoctonia AG isolates in important wheat production areas in the Central Anatolia Region, Turkey was determined. In addition, this study is the first comprehensive study in which the genetic diversity of Rhizoctonia AGs isolates obtained from wheat and rhizosphere soils in the region was evaluated with a molecular approach.

Root associated Fusarium solani Species Complex (FSSC) in epiphytic and terrestrial orchids

Abstract. Sukarno N, Mursidawati S, Listiyowati S, Nugraha NH, Fadillah WN, Waite M. 2023. Root associated Fusarium solani Species Complex (FSSC) in epiphytic and terrestrial orchids. Biodiversitas 24: 2577-2586. Members of the Fusarium solani Species Complex (FSSC) are ecologically diverse, covering human and plant pathogens, saprobes, and endophytic fungi of economically important plants. The role of FSSC on tropical orchids, however, has received little attention. This research aimed to isolate and identify the FSSC found as endophytes in roots of the epiphytic orchids Cymbidium finlaysonianum Lindl. and Vanda tricolor Lindl., and the terrestrial orchids Calanthe triplicata (Willemet) Ames and Phaius tankervilleae (Banks) Blume. Fungal isolation was obtained from peloton structures within the root. Fungal identification was done using combined morphological and molecular characteristics using ITS rDNA sequences. Four isolates of Fusarium were identified based on morphological characteristics. The BLAST analysis showed that all the isolates were in the Fusarium solani Species Complex (FSSC). Further phylogenetic relationship analysis indicated that all the FSSC isolates belonged to FSSC5 lineage or Fusarium solani sensu stricto, which is nested in FSSC clade 3 as a subgroup. The fungi F. solani sensu stricto V2 and CF34 were isolated from the epiphytic orchids, and F. solani sensu stricto C5 and P44 were isolated from the terrestrial orchids. It is interesting that the FSSC5 isolated from the epiphytic orchids form different groups than those of the terrestrial orchids. This is the first report showing the tropical orchids are the host of the FSSC5 as endophytes and this broadens the known host range and ecological behavior of the FSSC5.

A new small thecate dinoflagellate Azadinium anteroporum sp. nov. (Amphidomataceae, Dinophyceae) isolated from the Asian Pacific

ABSTRACT The marine thecate dinoflagellate Azadinium includes species known to produce the diarrhetic shellfish toxins known as azaspiracids (AZAs). In this study, the morphology of a thecate dinoflagellate isolated from Mutsu Bay, Japan, was examined by LM and SEM, and its phylogenetic position was inferred from ITS and LSU rDNA sequences. The production of AZAs was examined by liquid chromatography-triple quadrupole mass spectrometer (LC-MS/MS). Cells were elliptical, 15.9–22.4 µm long and 11.1–17.1 µm wide. A chloroplast was positioned peripherally, with a pyrenoid near the cingulum, and the nucleus was placed in the hyposome. The hyposome was smaller than the episome, with an antapical spine on plate 2''''. Thecal tabulation was Po, cp, X, 4', 3a, 6'', 6C, 5S, 6''', 2''''. The apical pore complex consisted of a symmetrical apical pore plate (Po), a cover plate (cp) and a small canal plate (X), and was surrounded by a prominent collar formed by the apical plates. A ventral pore was located on the mid-anterior edge of plate 1' and contacted with the X-plate and/or the ventral left of plate Po. Plate 1'' was in contact with plate 1a. AZA was not detected in the cultures. Molecular phylogeny showed that the species was related to Azadinium species that possess the ventral pore on the left side of plate 1', i.e. A. cuneatum, A. dalianense, A. obesum, A. poporum, A. spinosum and A. trinitatum, but that it was not included in these species. The new species Azadinium anteroporum sp. nov. differs in the ventral pore position (mid-anterior edge of plate 1') from these related species (which have the ventral pore on the left side of plate 1'), and from A. polongum (with ventral pore on the posterior left side of 1') and all other Azadinium species (with ventral pore on the right side of 1').

Screening Trichoderma strains isolated from soil growing Curcuma longa in Hung Yen province for treating waste after turmeric processing

The cultivation and processing of turmeric are developing, resulting in a large amount of turmeric waste causing serious environmental pollution. Trichodermafungi are potential microbial sources for agricultural waste treatment thanks to their ability to synthesise several extracellular enzymes. In this study, 15 strains of Trichoderma were isolated from turmeric-growing soil in Hung Yen province. Among them, 4 strains, which were named HYT11, HYT12, HYT13, and HYT14, were found to have a high potential for synthesising cellulase, amylase, and protease enzymes. Based on morphological characteristics and ITS rDNA sequence analysis, 3 strains (HYT11, HYT12, and HYT13) were identified as Trichoderma asperellum and the strain HYT14 was identified as Trichoderma harzianum. All four strains showed the ability to grow and form conidia on turmeric waste. The study also determined the optimal conditions for the conidia formation of these strains, including a nutrient medium containing potato extract (200 g/l) supplemented with 2% glucose, 1% peptone, and 2% agar, and the cultivation of 7 days at 30oC. In summary, the research has selected four Trichodermastrains, HYT11, HYT12, HYT13, and HYT14, which are promising for application in treating turmeric waste.

Heterochlamydomonas uralensis sp. nov. (Chlorophyta, Chlamydomonadaceae), New Species Described from the Mountain Tundra Community in the Subpolar Urals (Russia)

This paper describes a new species from the class Chlorophyceae, Heterochlamydomonas uralensis sp. nov., isolated from the soil of the carex-moss-lichen tundra in the Subpolar Urals Mountains, Russia. The taxon is studied using morphological methods and DNA identification. Heterochlamydomonas uralensis differs from other Heterochlamydomonas spp. by cellular morphology, detected on light and ultrastructural levels, and 18S and ITS2 rDNA sequences. These results support the separation of the species Heterochlamydomonas uralensis sp. nov. This study is important for connecting data from DNA metabarcoding with species described on the basis of morphological characteristics.

First Report of Stem Canker on Dangshen (Codonopsis pilosula) Caused by Binucleate Rhizoctonia AG-K in China.

Dangshen (Codonopsis pilosula) is a well-known medicinal and food homologous plant in China, which is widely used as a tonic agent and has good immunomodulatory effects (Bai et al. 2020; Luan et al. 2021). To retain the best medicinal properties, growers imitated the original ecological planting method for cultivating C. pilosula in hillside fields in Wutai county, Shanxi province, China. In July and August 2021 and 2022, stem canker disease was observed in C. pilosula. The basal part of the stems showed slightly sunken brown lesions, and the disease incidence was up to 20% in the investigated fields (6.67 ha). To identify the causal agents of stem canker, 12 small pieces (approximately 5 mm long) from 12 diseased samples (one piece per sample) were cut from the border of the lesions, surface-sterilized (70% ethanol for 30 s, 0.5% NaClO for 3 min), washed three times with sterile water, and then incubated on water agar (WA) at 25 °C for 24 h. Isolates with right-angle branching, a septum near the branch, and a slight constriction at the branch base were selected, and their hyphal tips were transferred onto potato dextrose agar (PDA) plates. After incubation at 25 °C, 12 Rhizoctonia-like isolates (Dcp-19 to Dcp-30) with white colonies were obtained. White monilioid cells in aerial mycelia formed as they aged but did not produce sclerotia. Based on nuclear fluorescence staining with 1 μg·mL-1 4'-6-diamidino-2-phenylindole as described by Ahvenniemi et al. (2009), there were two nuclei per hyphal cell for all the 12 isolates. Moreover, the sequences of internal transcribed spacer region of ribosomal DNA (rDNA-ITS) of all the 12 isolates were amplified using the primers ITS1/ITS4 (White et al. 1990). For identical sequences, only the rDNA-ITS sequence (674 bp) of Dcp-19 has been deposited in GenBank (accession no. ON004932) and BLASTn analyses showed 100% homology with Rhizoctonia AG-K (MF070696). Maximum likelihood phylogenetic analysis further confirmed the identification. Healthy C. pilosula plants grown for two years in hillside fields were transplanted into sterile soil for pathogenicity testing. And the 12 isolates were all done in this test. Sterilized wheat seeds were placed on a 2-day-old colony of the isolate and incubated for 7 days. One fungus-infested seed was placed at the base of the stem and covered with sterilized soil. Control plants were inoculated with sterilized wheat seeds. Tests were performed on three plants for each isolate. The experiment was repeated twice. All the plants were cultivated at 22 °C and 50% relative humidity. After three weeks, the basal stems of the control plants were still healthy and did not have lesions, but the treated plants exhibited sunken brown canker lesions. The mean disease incidence of all the 12 isolates was 58.33%. The AG-K isolates re-isolated from the lesions of treated plants were confirmed by the morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. To our knowledge, this is the first report of stem canker on C. pilosula caused by binucleate Rhizoctonia AG-K in China.

Low genetic heterogeneity of Leishmania major in different geographical regions of Iran.

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Evidence for two morphologically cryptic species of Hysterolecitha Linton, 1910 (Trematoda: Lecithasteridae) infecting overlapping host ranges in Moreton Bay, Australia

Integration of morphological and molecular approaches to species delineation has become an essential part of digenean trematode taxonomy, particularly when delimiting cryptic species. Here, we use an integrated approach to distinguish and describe two morphologically cryptic species of Hysterolecitha Linton, 1910 (Trematoda: Lecithasteridae) from fishes of Moreton Bay, Queensland, Australia. Morphological analyses of Hysterolecitha specimens from six fish species demonstrated a complete overlap in morphometric data with no reliable differences in their gross morphological characters that suggested the presence of more than one species. Distinctions in ITS2 rDNA and cox1 mtDNA sequence data for corresponding specimens suggested the presence of two forms. A principal component analysis on an imputed dataset showed clear separation between the two forms. These two forms are partially separated on the basis of their host’s identity. Therefore, we describe two morphologically cryptic species: Hysterolecitha melaen. sp. from three species of Abudefduf Forsskål and one species of Parma Günther (Pomacentridae), with the Bengal sergeant, Abudefduf bengalensis (Bloch), as the type-host; and Hysterolecitha phisonin. sp. from species of Pomacentridae (including A. bengalensis), Pomatomidae and Siganidae, with the black rabbitfish, Siganus fuscescens (Houttuyn), as the type-host.

Erysiphe machilicola: a new powdery mildew species on Duthie’s Bay Tree

A new species of Erysiphe infecting Duthie’s bay tree in Azad Jammu & Kashmir, Pakistan, is described and illustrated based on molecular and morphological analyses. Erysiphe machilicola sp. nov. is morphologically distinct from other Erysiphe species on hosts of the Lauraceae by having longer conidiophores, larger chasmothecia with longer appendages, above all, it is distinguished from E. machiliana, the only so far known Erysiphe species on Machilus, by lacking bristle-like appendages (remnant of anchor hyphae) and a different phylogenetic position, confirmed by sequence analyses of the type material of the latter species. The phylogenetic relationship of the new species to other closely related species is discussed based on ITS rDNA sequences, which phylogenetically supports its identity as a new species within the genus Erysiphe. A comprehensive description along with photographs and a phylogram showing its exact phylogenetic position and a comparison with closely related taxa is provided.

Biosynthesis of Silver Nanoparticles and Exploring Their Potential of Reducing the Contamination of the In Vitro Culture Media and Inducing the Callus Growth of Rumex nervosus Explants.

Among biological methods, green synthesis of the nanomaterials using plant extracts was shown to be an environmentally friendly, economical, and simple approach. In the current study, the biogenic synthesis of silver nanoparticles (AgNPs) was achieved using the leaf extract of Hibiscus tiliaceus, in order to prevent the contamination of the tissue culture media and induce callus growth. The nanostructures of the fabricated AgNPs were characterized using UV-visible spectroscopy, Fourier transform infra-red spectra (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), zeta size, and zeta potential techniques. Our results indicate that The UV-vis spectrum of AgNPs exhibited an absorption band at 415 nm. The FTIR analysis identified the functional groups which could involve in the reduction of silver ions to AgNPs, this was also confirmed by the (hkl) diffraction peaks in the XRD diffractogram. Moreover, the TEM analysis showed a spherical nanoparticle with a size ranging from 21 and 26 nm. Thereafter, the potential antibacterial and antifungal activity of the biogenic AgNPs was evaluated against Bacillus pumilus and Alternaria alternata which were isolated from the in vitro culture media and identified based on 16S rDNA and ITS rDNA sequences, respectively. The results showed that the AgNPs significantly inhibited the growth of Alternaria alternata and Bacillus pumilus at all applied concentrations (5, 10, 20 and 40 mg/L). Compared to the control more fungal radial growth reduction (42.59%,) and bacterial inhibition (98.12%) were registered in the plates containing high doses of AgNPs (40 mg/L). Using Rumex nervosus explants, the biosynthesized AgNPs were tested for their impact to promote callus growth. The obtained results showed a significant effect of AgNPs on callus fresh weight at all applied doses. Moreover, AgNPs treatments showed a polymorphism of 12.5% which was detected by RAPD markers. In summary, the results revealed that AgNPs (40 mg/L) can be effectively added to the in vitro culture media for reducing microbial contamination and improving callus growth while greatly maintaining its genetic stability.

Isolation, Characterization and Phylogenetic Analysis of Stagonospora tainanensis, the Pathogen Causing Sugarcane Leaf Blight in China

Sugarcane leaf blight (SLB), a major fungal leaf disease of sugarcane (Saccharum spp.), has been attributed to Stagonospora tainanensis. In December 2020 and May 2021, signs of leaf blight were observed on sugarcane in the fields of Chongzuo City, in the Guangxi Province of China. Lesions on the leaves were characterized by yellow or dark red spots in the center. Fungal species were isolated, purified and subjected to pathogenicity evaluation on the sugarcane plants. An isolate that caused symptoms the same as those observed in the field was initially identified as S. tainanensis (Leptosphaeria taiwanensis, perfect state) based on its morphological characteristics both of asexual and sexual stages. Dark brown and nearly spherical pycnidia with conidia of long ellipsoidal, hyaline, one to four cells and 29.27 to 54.39 μm long and 9.03 to 16.12 μm wide were found on corn meal agar medium. Ascomata with asci of cylindrical to clavate, a short stipe and eight spores slightly constricted at the septum, with the size of the spore ranging from 36 to 44 μm long and 8.5 to 12 μm wide, were formed on the sugarcane-leaf-decoction saccharose agar medium. The identity of the species was further confirmed by rDNA ITS and TEF-1α sequencing. The optimal temperature for mycelial growth was 25 °C and the optimal pH was 6.0. The pathogen grew well in a medium with oats as the carbon source and yeast extract as the nitrogen source, but poorly in a medium with urea as the nitrogen source. This study is the first to identify the sugarcane leaf blight pathogen in Guangxi, and the first publication describing the biological characterization of S. tainanensis. The occurrence of sugarcane leaf blight should alert sugarcane breeders and plant pathologists to consider integrating control of this potentially important disease into the agenda of their breeding and disease control programs.

Short-term soil amendment by sewage sludge anaerobic digestate in a tomato monoculture suppresses Fusarium vascular wilt disease by changing the taxonomic characteristics of soil microbiota

Digestate incorporation into topsoil determines soil fertility improvement by changing composition and structure of soil microbiota. However, how microbiota responds to short-term soil amendment by sewage sludge anaerobic digestate (SSD) for suppressing Fusarium vascular wilt disease is unknown. This study compares the effects of three SSD-based treatments to suppress Fusarium oxysporum f. sp. lycopersici (Fol) in a long-term cherry tomato monoculture under field condition in a 3-year trial. Three sampling sites with two application times (two bulk soils at 3–12 weeks after amendment and one tomato rhizosphere soil at 12 weeks post-amendment) were chosen. Three digestate typologies (liquid, centrifuged, and dried) having physicochemical features and heavy metals content below the legal limits were tested at 50 l, 3.5 kg and 2.5 kg m−2y−1 dose, respectively. Fusarium wilt disease was measured for three consecutive years by severity index and Fol abundance in tomato vascular tissue was assessed by ITS rDNA gene sequencing. Fol abundance and taxonomic structure of Fusarium community in the rhizosphere were determined at specie level at the end of the trial by ITS rDNA and EF1α rDNA genes sequencing, respectively. The taxonomic structure (α-, β-diversity) of soil bacterial community (SBC) was characterized from phylum up to genus level at the end of the trial in all the sites by 16S rRNA gene sequencing. The results showed that dry SSD reduced disease severity in field up to 18 % and Fol abundance in tomato up to 0.9 ITS copies g−1 tissue. Fol abundance was decreased in the rhizosphere up to 0.0027 ng μl−1 DNA, while the Fusarium community shifted between treated-soils and un-amended. The SBC composition (α-diversity) changed in the rhizosphere by applying dry digestate, while the SBC structure (β-diversity) shifted either among treatments or sites. Bacterial members related to Fol suppression (Bacillus, Chitinophaga, Flavihumibacter, Flavobacterium, Pseudomonas and Terrimonas) increased in the rhizosphere (P < 0.01, P < 0.001) more than in the bulk soils by applying both dewatered-SSDs. Thus, digestate soil amendment carried out for three consecutive years has affected tomato Fusarium wilt severity by changing the taxonomic characteristics of fusaria and bacteria communities of the amended soil.

Candida species causing fungal keratitis: molecular identification, antifungal susceptibility, biofilm formation, and clinical aspects.

The study aimed to evaluate the clinical aspects, molecular identification, biofilm formation, and antifungal susceptibility profile of Candida species isolated from fungal keratitis. Thirteen Candida isolates from 13 patients diagnosed with Candida keratitis were retrieved and grown in pure culture. Species identification was performed by micromorphology analysis and ITS-rDNA sequencing. The broth microdilution method tested the minimum inhibitory concentration (MIC) of four antifungal drugs (fluconazole, amphotericin B, voriconazole, and anidulafungin). The biofilms were cultured and incubated with antifungal drugs for 24 h. The XTT reduction assay measured the biofilm activity. Biofilm MICs were calculated based on a 50% reduction in metabolic activity compared with the activity of the drug-free control. Among isolates, two were C. albicans, 10 were C. parapsilosis (sensu stricto), and one was C. orthopsilosis. All isolates were classified as susceptible or intermediate to all four antifungal drugs. Four isolates were very low biofilm producers (30%). Nine isolates were biofilm producers, and all biofilm samples were unsusceptible to all drugs tested. Previous ocular surgery was the most common underlying condition for fungal keratitis (84.6%), and C. parapsilosis was the most frequent Candida species (76.9%). Four patients (30.7%) needed keratoplasty, whereas two (15.3%) required evisceration. The biofilm formation ability of Candida isolates decreased antifungal susceptibility compared with planktonic cells. Despite in vitro antifungal susceptibility, almost half of the patients were unresponsive to clinical treatment and needed surgery.

GENETIC DIVERSITY OF MAJOR POLYPHAGOUS SPIDER MITE SPECIES ACROSS HOST PLANTS AND SPATIAL DISTRIBUTION

Different agroecosystems of southern Karnataka sampled for plant associated mites recorded six genera of tetranychid mites viz., Aponychus, Eutetranychus, Oligonychus, Petrobia, Schizotetranychus and Tetranychus. Neutrality test revealed a lowest haplotype diversity (0.993) of T. neocaledonicus population and Tajima’s D and Fu’s Fs test values were negatively significant for T. fijiensis, E. orientalis and S. baltazari populations, indicating the greater expansion of populations. Genetic analysis with ITS2 (rDNA) sequences revealed highest similarity between mite genera Petrobia and Aponychus showing lowest genetic distance of 0.51. Schizotetranychus has sister relationship with Eutetranychus which in turn clustered with Petrobia and Aponychus. Tetranychus and Oligonychus genera with lower genetic distance of 0.60 are clustered together. The study confirmed the clustering of morphologically related genera and lower genetic distances observed between related genera, expressed sister relationship. Molecular phylogenetic analysis of species under genus Oligonychus showed clustering in line with the morphology-based species taxonomic keys, while polyphagous species under the genus Tetranychus did not express form this grouping. Genetic diversity study of ten polyphagous mite species across their host plants and geographical occurrence revealed grouping according to locations in the phylogenetic tree. However, populations of Oligonychus tylus with a narrow host range showed close relationship for host plants than geographical locations which was evident in phylogenetic tree branching.

Molecular characterization of endophytic fungi-Daldinia eschscholtzii from Aloe vera plants in Bangladesh

Aloe vera (L.) Burm. f. is a plant of health, beauty and medicine that is widely used in Bangladesh with high demand. An experiment was performed to find out the association of endophytic fungus with Aloe vera leaves and to assess their growth behavior on different culture media, temperatures and pH. The endophytic fungus-Daldinia eschscholtzii (Ehrenb.) Rehm. was identified based on the morphological and molecular characterization using the ITS-rDNA sequence. Various solid culture media were used to assess the fungal growth, and temperature regimes i.e., 15℃, 20℃, 25℃, 30℃, 35℃ were investigated. The fungal endophyte showed maximum mycelial growth on Potato Sucrose Agar followed by Potato Dextrose Agar and Carrot Agar media. Optimum growth and development of the endophyte was recorded at temperature 25 to 35℃. We observed the effect of five different pH conditions, viz., 5, 6, 7, 8, 9 on the mycelial growth of the fungus. However, the fungal endophyte was very insensitive to the pH of the substrate. To the best of our knowledge, this is the first known occurrence of D. eschscholtzii as an endophyte in Aloe vera in Bangladesh.&#x0D; Jahangirnagar University J. Biol. Sci. 11(1 &amp; 2): 1-11, 2022 (June &amp; December)