Uncovering the hidden leaf mycobiota of native juniper (Juniperus seravschanica) in Oman's arid highlands

Internal transcribed spacer or ITS region of nuclear ribosomal DNA (rDNA) has been used to evaluate genetic assortment and phylogenetic relationship in nine sugarcane genotypes including Saccharum species and another related genus as Erianthus, Narenga and hybrid. DNA was extracted from selected genotypes and ITS (ITS-1 and ITS-2) regions were amplified using specific primers. The sequence lengths ITS-1 showed 205- 207 bp, while ITS2 was ranged from 211- 218 bp. However, G+C content (%) 65.2% - 67% in ITS-1 and in ITS-2 68.4% - 99.7%. The sequence lengths of fragment and GC content of ITS-1 and ITS-2 regions showed variable. To evaluate the phylogenetic association of both the region of ITS (ITS-1 and ITS-2) neighbor-joining (NJ) method was employed. The cluster A of ITS-1 and cluster B for ITS-2 and cluster C combined between ITS1+ ITS2 sequences gave two distinct groups A and B. The group A represented the ITS1 sequences which showed two subgroups I and II. The A-I subgroup consisted of wild species of sugarcane; Erianthus, Narenga and S. robustum, whereas the A-II subgroup consisted of the Saccharum species and hybrid. The ITS2 sequences in the group B showed better correlation amongst each other. The sequences ITS-1 & ITS-2 combined and compared with some selected sequences from NCBI database using NJ method. The results have confirmed that ITS region can be used for evaluating the genetic assortment in Saccharum and its closely related genes.

Fig (Ficus carica L.) is widely grown in Oman mainly for its nutritional value and health benefits. In May 2020, survey was conducted for fig trees which were showing symptoms of decline in three farms where more than 1500 fig trees were grown. The incidence of the disease was 3 to 4 % of fig trees showing symptoms of dieback of the twigs, death of entire branch and discoloration of vascular tissues were observed in Sohar, North Al-Batinah governorate of Oman. The disease severity was range between 25 to 40 % in most of the infected tree. The death of severely affected trees was observed in about 3 % of the orchards. Symptomatic samples were collected and isolation of the causal agent was performed. Infected tissues were cut into small pieces and surface-sterilized with 1 % NaOCl for 3 min. The tissues were rinsed with sterile distilled water 3-4 times, blotted dry on sterile filter paper and placed on potato dextrose agar (PDA) medium under aseptic conditions. After 2-3 days of incubation of plates at 27°C, pure culture of the fungus was obtained by hyphal tip isolation technique. Two identical isolates were grown in the PDA medium. The fungal colony was dark grey to black colored on PDA medium and the fungus produced dense aerial mycelium and numerous dark colored pycnidia in 25 days at ambient temperature range between 23-25 ℃ and nature photoperiod. Pycnidia were raptured and 20 emerged conidia were measured. The mature conidia were 1-septate, brown, ellipsoid, with thick cell wall and having longitudinal striation. The average measurement of the length of 20 conidia was 22.5-25 (24.9 µm) however the width measured was 12.5-15 (15 µm). To identify the fungus, DNA was extracted from the mycelium using phenol-chloroform-isoamyl alcohol method, following Al-Mahmooli et al. (2015). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α (TEF) gene were amplified using ITS-4 and ITS-5 primers for ITS (White et al. 1990), EF1-983F and EF1-2218R and EF1-728F and EF1-986R primers for TEF (Alves et al. 2008), respectively and the amplified products were sequenced. BLASTn analysis of the ITS sequence of the fungal isolate revealed 100% identity with reference sequences of Lasiodiplodia theobromae (Pat.) Griffon & Maubl (MT075438.1). The sequence of TEF gene was 99% identical to the sequences of L. theobromae (XM_035519539.1 and MN461169.1). The nucleotide sequences were submitted to GenBank and accessioned with (MW590660) for ITS and (MZ159970, MZ159971, OM654917) for TEF gene. For phylogenetic analysis, we constructed a combined ITS-TEF dataset, following (Alves et. 2008). Using Maximum Likelihood phylogenetic analysis of the combined ITS-TEF dataset, our specimen falls in a clade formed by the members of L. theobromae (Supplementary materials). Pathogenicity of the fungus of three replicates (three fig seedlings) was established by artificial inoculation of 2- to 3-month-old potted fig seedlings with the fungus by making pin prick injuries on the stem at 5 cm above the soil level with a sterile inoculation needle, placing mycelial discs containing spores and pycnidia (15 × 15 mm) from 20-days old colonies grown on PDA on the injured surface and wrapping with parafilm which removed 48 hrs after establishing of infection. The three inoculated plants showed withering and drying of leaves 14 days after inoculation (DAI) begins near to the inoculation site expend to the younger leaves. Eventually, seedlings were showing dieback and death of entire seedling occurs in 25-28 DAI. The mock-inoculated plants remained symptomless. The fungus re-isolated from the diseased plants was confirmed by sequencing of ITS regions. The prevalence of dieback could be a potential threat to cultivation of fig in Oman. To the best of our knowledge, this is the first report of dieback disease caused by L. theobromae on fig in Oman.

Abstract. Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots?Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multi‐species samples.Results: The technique successfully identified species present in multi‐species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each species.Conclusions: This method is the first to identify the species composition of mixed root samples. It should be applicable to most plant species because the ITS region is flanked by universal primers and most species have unique ITS sequences. The ability to determine species‐specific rooting distributions has broad applications in vegetation science.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

In April 2018, damping-off of rice (Oryza sativa L.) seedlings at the 2-to-3-leaf stage was observed in three fields in the counties of Wharton and Matagorda of Texas and Jefferson-Davis Parish of Louisiana. All affected areas were 1 ha or greater, with 10 to 20% of the seedlings showing the symptoms. Infected seedlings showed dark-brown necrotic lesions on the roots and/or mesocotyls where white superficial mycelium was usually present. Symptomatic tissues excised from 10 diseased seedlings of each field were surface sterilized with 1% NaOCl, double rinsed in sterilized distilled water, and plated on potato dextrose agar (PDA). The plates were incubated at 25°C with a 12-h photoperiod in a growth chamber. After 48 h, hyphal tips of fungal colonies were transferred onto PDA and 12 isolates were obtained. Clamp connections and dolipore septa were observed in young hyphae, indicating that these isolates were a basidiomycete fungus. Young hyphal cells were binucleate based on safranin O stain (Bandoni 1979). No fruiting bodies or sclerotia produced on PDA after one month of incubation. Based on these morphological characteristics, these isolates were identified as belonging to sterile white basidiomycetes (SWB) (Howard et al. 1977). To further identify the isolates into the species level, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of a representative isolate was sequenced with primer ITS1 and ITS4 (Vinnere et al. 2005). The ITS sequence (GenBank acc. no. MT524457) had more than 97% sequence similarity with known Marasmius graminum strains from Denmark (JN943595) (Schoch et al. 2012) and Sweden (MH857692) (Vu et al. 2019). Pathogenicity was tested with three representative isolates in a growth chamber using a modified method (Carling and Leiner 1990). Pots (6.5 cm in diameter x 7.5 cm height) were filled with 100 g of sterilized sand and watered to field capacity. Five PDA plugs (4 mm in diameter) from 5-day-old growing culture were placed on the sand surface of each pot. Pots inoculated with PDA plugs without fungus served as the controls. Five seeds of rice cv. Presidio were planted into each pot and covered with 10 g of sterilized sand. Pots were maintained at 25±2°C in a growth chamber with a 12-h photoperiod for 14 days. There were four replicated pots for each treatment and the experiment repeated twice. After 2 weeks, severe damping-off and associated symptoms similar to those observed in the field appeared in the inoculated pots. No symptoms developed in the control pots. The same fungus was consistently re-isolated from infected plants. Based on morphological characteristics and rDNA-ITS sequencing, these isolates were identified as M. graminum. The SWB fungus was first reported as a causal agent of stem rot of snap bean in Florida (Howard et al. 1977) and Nebraska (Harveson 2002), root or hypocotyl rot of corn, snap bean, squash and peanut in Georgia (Sumner et al. 1979; Bell and Sumner 1984), and crown rot of pigeon pea (Cajanus cajan) in Puerto Rico (Kaiser et al. 1987). Later, the SWB strain (ATCC 28344) causing stem rot of snap bean in Florida was further identified as M. graminum based on nuclear large subunit rRNA gene (Vinnere et al. 2005). Comparing the ITS region of this isolate (AY445120) with our isolate revealed a 99% similarity. To our knowledge, this is the first report that the SWB fungus M. graminum causes seedling blight in rice. Identification of this new disease will help to develop management strategies for control of stand loss in rice.

Question: Is it possible to determine the species composition of root samples containing multiple species, without first disentangling individual roots? Methods: The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified and sequenced from four California annual grassland species (two Poaceae and two Asteraceae). Restriction enzymes that cut the ITS region of each species into uniquely sized fragments were identified based on DNA sequence variation of the ITS regions. Mixed root samples were analysed to test the ability of the method to identify the presence or absence of each species in multi-species samples. Results: The technique successfully identified species present in multi-species samples. ITS regions were shorter in Poaceae than in Asteraceae, so size differences alone were sufficient to distinguish these taxonomic groups. At the species level, digestion of ITS regions with the appropriate restriction enzymes yielded at least one uniquely sized fragment for each ...

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR- amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms (565-700 base pairs (bp)), substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in ITS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxa- ceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150- 1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the ITS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population-level phylogenetic analysis in many non-flowering seed plants.

Cucumber (Cucumis sativus L.), an important vegetable with high economic value, has become one of the most economically important domestic vegetables in China. In April 2019, a previously undescribed crown rot with about 80% incidence was observed in many vegetable greenhouses in Huaian city, Jiangsu Province. Typical symptoms of the infected fruits appeared to be that young fruit withered and were covered with abundant white mycelia. The symptoms were similar to fruit rot caused by Botrytis cinerea. It is possible that this similarity has caused this disease to be overlooked. The pathogen was isolated as follows: diseased tissues were washed with sterile water, and potato dextrose agar (PDA) plates were inoculated with mycelium from diseased fruit. After incubation for 7 days at 25°C in the dark, the spores were isolated from each colony, and a single spore was isolated for pure culture. After single-spore isolation, 56 isolates with similar morphological characteristics were obtained. After incubation for 7 days at 25°C, the colony with abundant whitish to yellowish aerial mycelia produced pale brown to dark brown pigment and numerous falciform macroconidia (20.4 to 59 × 2.9 to 5.1 μm) in PDA plates. Moreover, macroconidia (three to five septa) exhibited pronounced dorsiventral curvature, with a tapered and elongated apical cell and prominent foot-shaped basal cell. These morphological characteristics of the isolates are consistent with Fusarium incarnatum described in previous studies (Gai et al. 2016; Ramdial et al. 2016). For molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA, a partial elongation factor-1 alpha (EF-1α) gene, Histone 3 gene (H3) and β-tubulin gene of 16 isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (Lu et al. 2014), EF-1/EF-2 (O’Donnell et al. 1998), H3-1a/1b, and Bt2a/2b (Glass and Donaldson 1995), respectively. The sequences of each gene for 16 isolates were completely consistent and deposited in NCBI GenBank nucleotide database (MN227262 for the ITS region, MN233576 for β-tubulin gene, MN233577 for EF-1α gene, and MN233578 for H3 gene) and showed 99.65, 100, 100, and 100% identity to F. incarnatum species (MH979697 for ITS region, KJ020861 for β-tubulin gene, JX269001 for EF-1α gene, and MH979696 for H3 gene), respectively. To confirm the pathogenicity of the isolates, 10 μl of conidial suspension (10⁶ conidia/ml) of each isolate was inoculated on nine healthy fruits wounded with a sterilized needle, and another nine fruits were inoculated with sterilized water as controls. Six isolates and two cucumber cultivars were tested. All the inoculated fruits were placed under 25°C and 90% relative humidity. After about 5 to 7 days, they were similar to the symptoms observed on cucumber fruit in the greenhouses, but no symptoms were observed on the control fruits. The experiment was performed three times. Furthermore, F. incarnatum was reisolated from the infected cucumber fruits, fulfilling Koch’s postulates. To our knowledge, this is the first report of F. incarnatum causing crown rot on cucumber in China. Although cucumber crown rot is reported as an emerging vegetable disease, it is serious in cucumber greenhouses, causing severe economic losses to the cucumber industry in China.

Lima bean production has been an economically valuable staple in Delaware agriculture for almost a century, with annual revenue approaching 8 million dollars (USDA-NASS, 2019; Evans et al. 2007). From 2019-2021, lima beans displaying symptoms of brown discoloration, referred to as "brown bean" were observed in the green baby lima variety 'Cypress' across multiple commercial and research fields. Symptoms were present in approximately 1-5% of beans and not visible until pods were opened for harvest. Thirty-seven symptomatic beans were collected and surface disinfested in 0.85% sodium hypochlorite for 30 s, rinsed in sterile deionized water for 30 s, sectioned into four pieces and plated onto potato dextrose agar (PDA) amended with 50 µg/ml penicillin G and streptomycin sulfate. Petri dishes were incubated at 23ºC and observed for colony morphology. Pure cultures were obtained with tan colonies that had mycelia with right angle branching and septations near the branch, consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). DNA extraction and pathogen identification was confirmed by sequencing of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA using primers ITS4/ITS5 (White et al. 1990) for thirty-seven isolates collected in 2019 and 2020. Isolates were identified as Rhizoctonia solani AG 4 (99.9% sequence identity with GenBank Accession [MN106359.1].) A representative isolate was selected to complete Koch's postulates and the sequence was deposited in GenBank as accession number MW560551. To observe colonization ability, 10 detached pods were sterilized in 75% EtOH for 60 s, then rinsed in Milli-Q water. The detached pods were divided among two 150 mm Petri dishes containing a single 150 mm filter paper saturated with Milli-Q water. Five 1 mm2 agar plugs colonized with the representative R. solani isolate were placed 0.5 cm apart along the length of the pod. Plates were sealed with parafilm and left at room temperature. Control pods were kept in identical conditions but inoculated using clean agar plugs. The trial was repeated and a second trial was conducted on 12 attached asymptomatic pods from C-Elite Select lima bean plants at the succulent seed stage to complete Koch's Postulates. Pods were surface disinfested with 70% ethanol. Three attached pods were wounded with the tip of a sterile scalpel blade where a colonized agar plug was placed and loosely wrapped with a thin parafilm layer to maintain contact. Three attached pods not wounded were also inoculated with a colonized agar plug and wrapped by parafilm. Three wounded and non-wounded pods received clean agar plug controls. Both attached and detached pods were kept at room temperature for one week until symptoms began to manifest on the pod surfaces, at which point the beans from infected pods were removed and placed on PDA, three to a plate. In the attached assay, all beans of both wounded and non-wounded pods developed symptoms. The plates were stored in identical conditions and monitored for 5 days until tan colonies were observed. Culture morphology was consistent with the original isolate in all beans. Sequencing of the ITS region confirmed identity as R. solani AG-4. No symptoms were observed on control pods or seeds. Rhizoctonia solani is most frequently associated with symptoms of root rot (Sharma-Poudyal et al. 2015), but no stem symptoms are associated with the late season "brown bean" that has been observed throughout production in recent years. To our knowledge, this is the first report of Rhizoctonia solani AG 4 causing symptoms of brown bean of lima bean in Delaware. In preliminary observations, symptoms seem to be worse in pods that could have had contact with the soil directly or via rain splash. This disease cannot be detected until pods are split open, which has potential to reduce lima bean quality at harvest. Further monitoring should be conducted to quantify yield impacts and develop appropriate preventative and curative techniques.

Downy mildew, caused by Peronosclerospora sorghi [Weston and Uppal (Shaw)], is one of the most serious diseases of sorghum and corn worldwide. Pathogenic variability among various isolates of P. sorghi has been reported. However, the molecular relationships between the isolates from sorghum and corn are unknown. In the present study DNA was extracted from P. sorghi isolates from sorghum and corn collected from different locations of Tamil Nadu, India, and their genetic variability was investigated using restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA. PCR amplification of the ITS regions of the P. sorghi isolates from sorghum and corn gave products of approximately 550 base pairs in length with slight variations in a few isolates. Restriction fragment length polymorphism analysis of the ITS regions of nuclear rDNA of P. sorghi with HhaI, EcoRI and MspI confirmed the heterogeneity among the P. sorghi isolates from sorghum and corn.

DNA sequence variation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from Arabis holboellii, A. drummondii, and its putative hybrid A. divaricarpa was analyzed to study hybrid speciation in a species system geographically covering nearly the entire North American continent. Based on molecular systematics the investigated species are better combined under the genus Boechera. Multiple intraindividual ITS copies were detected in numerous accessions of A. divaricarpa, and, to a minor extent, in the parental taxa. Comparative phylogenetic analysis demonstrates that reticulate evolution is common. Consequently, concerted evolution of ITS regions resulted in different types of ITS fragments not only in hybrid populations but also in one of the parental taxa, A. holboellii. Hybrid formation often occurred independently at different sites and at different times, which is reflected by ITS copies resampling the original parental sequence variation in different ways. Some biogeographic structuring of genetic diversity is apparent and mirrors postglacial migration routes. Hybridization, reticulation, and apomixis are assumed to be the major forces driving speciation processes in this species complex. Analysis of conserved regions and secondary structures of the ITS region provided no evidence that, in this system, hybrid ITS evolution is predominantly driven in a particular direction. However, two regions in the ITS1 and ITS2, respectively, show higher mutation rates than expected from outgroup comparisons. Strong evidence for the occurrence of apomixis in A. holboellii and A. divaricarpa has come from pollen size measurements and estimations of pollen quality, which favor the hypothesis that A. drummondii served as paternal hybridization partner more frequently than A. holboellii.

Phylogenetic relationships among all 18 species of the genus Glycine were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Pairwise sequence divergence values ranged from 0.2% (a single nucleotide) between Glycine max and Glycine soja to 8.6% between Glycine hirticaulis and Glycine falcata. The length of the ITS1 and ITS2 sequences ranged from 215 to 238 nucleotides and from 205 to 222 nucleotides, respectively, and that of 5.8S was 168 nucleotides across all the species. Phylogenetic analyses of the ITS region clearly resolved all the genomic groups that were established previously based on cytogenetic and biochemical studies. Based on this study, we assign new genome symbols: HH to Glycine arenaria, H1H1 to Glycine hirticaulis, H2H2 to Glycine pindanica, II to Glycine albicans, and I1I1 to Glycine lactovirens. Parsimony analysis of the entire ITS region, using subgenus Soja as outgroup, resulted in a trichotomy consisting of the clades: G. falcata (F genome), Glycine cyrtoloba and Glycine curvata (C genome), and all other species (A, B, D, E, H, and I genomes) of the subgenus Glycine.

Small indigenous species (SIS) of fishes are available source of animal protein, vitamin and minerals, not commonly available in other foods in Bangladesh. These fishes are eaten both in fresh and dried condition. Puntious sophore was collected from different regions of Baikka Beel at Sreemangal Upazila, Moulvibazar district, Bangladesh to investigate mycoflora association with dried condition. Fungus was isolated from dried fish using tissue planting methods for classical and molecular characterization. Aspergillus elegans was identified through classical taxonomy and molecular approach based on ITS region of ribosomal DNA (rDNA) of fungi. For analyzing ITS4 and ITS5 were used. Mycelium of the identified fungus septated, hyaline, abundant and branched. Long, straight or flexous conidiophore were present. The molecular phylogeny in morphologically identified dry fish fungi of P. sophore such as A. elegans was studied based on their internal transcribed spacer (ITS). The sequences of ITS 600 bp region of A. elegans had 5.8S of rDNA sequences were identical. The highest mycelial growth was recorded in potato dextrose agar (PDA) medium for the growth and development of A. elegans. Temperature 30°C was optimum, this fungus grew well in pH 7. So, tested fungi grew well in neutral condition. Jahangirnagar University J. Biol. Sci. 10(1 & 2): 39-47, 2021 (June & December)

보길도에서 자라고 있는 황칠나무(Dendropanax morbifera)를 구입하여, 캘러스로 유도한 후, ribosomal DNA(nrDNA)의 internal transcribed spacer (ITS) region의 염기서열을 결정하였다 보길도의 황칠나무(Dendropanax morbifera)의 ITS region의 염기서열을 분석한 결과, 총 689염기를 결정하였다. 결정된 689염기 중에서 ITS1은 222 개염기, 5.8S rDNA는 160염기, ITS2는 233염기인 것으로 판명되었다. GenBank의 BLAST 프로그램(http://www.ncbi.nlm.nih.BLAST)을 사용하여 GenBank/EMBL/DDBJ에 등록되어 있는 Dendropanax 속 33의 염기서열을 수집한 후 multiple alignment를 수행한 결과, 유사도는 99.7%(D. chevalieri)에서 92.6%(Dendropanax arboreus)로 나타났으며, 일본황칠나무(D. trifidus)와는 유사도가 99.4%로 판명되었다. Dendropanax morbifera is an endemic tree species of Korea, it is restricted to the southern parts of Korea. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for Dendropanax morbifera grown at Bogil-do, Korea was determined. We investigated the sequence-based phylogenetic relationships of plants related and clarified its taxonomical position. The determined sequences consisted of 689 residues. ITS1 was 222 bp long while ITS2 was 233 bp long. The 5.8S rDNA was 160 bp long. The ITS region sequences of the Dendropanax species included in this study were obtained from GenBank. Oreopanax polycephalus was used as the outgroup. A pairwise alignment was calculated using the Clustal X program. A phylogenetic tree was constructed by the neighbor-joining method using the Tree view program. Sequence similarities among species including D. morbifera Bogil-do isolate showed the range 92.6 to 99.7% in sequence-based phylogenetic analysis using total 615 base pairs of ITS1, 5.8S rDNA and ITS2. D. morbifera Bogil-do isolate exhibited the highest degree of relatedness to D. chevalieri, sharing 99.7% ITS region similarity. D. morbifera Bogil-do isolate also showed to D. trifidus, sharing 99.4% ITS region similarity.

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30°C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% - 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.