Morpho-molecular Characterization Research Articles

Morpho-molecular characterization of fungi associated with seeds of hybrid rice varieties in bangladesh

Rice, as the dominant crop in Bangladesh, is vulnerable to various fungal pathogens. Many of these pathogens infect the seeds, which can later transmit diseases and serve as an efficient means for spreading seed-borne pathogens over long distances. In this study, seven hybrid rice varieties, specifically BRRI Hybrid Dhan 101 through BRRI Hybrid Dhan 107, were collected from the Bangladesh Rice Research Institute (BRRI) to detect and identify seed-borne fungi using both morphological and molecular techniques. Fourteen fungal species were isolated from the selected rice varieties using “Tissue planting method” and “Blotter method”, of which 4 fungal isolates were identified by molecular techniques. Molecular characterization of the isolates was conducted based on the sequence information of internal transcribed spacer (ITS) regions. The sequences obtained using the ITS1 and ITS4 primers were compared with those in the NCBI GenBank using BLAST analysis. Hybrid rice is comparatively more advantageous than inbreed rice because of its high yield, shorter plant life, and higher assimilation translocation. Therefore, it is crucial to identify and manage the pathogenic fungi associated with the seeds to ensure good seed quality, which can contribute to improving the economy of Bangladesh. Bangladesh J. Bot. 53(3): 511-518, 2024 (September)

Morpho-molecular characterization of phoma-like fungi from Morus alba in northern Thailand; a novel species (Boeremia albae) and a new host record (B. maritima)

Boeremia was established to accommodate phoma-resembling fungi. Its species occur in terrestrial ecosystems as endophytes, saprobes and pathogens, except one species reported from a marine ecosystem. Boeremia species are characterized by hyaline, thin-walled, and aseptate (occasionally 1(–2)-septate) conidia that are variable in shape, and hyaline, straight or slightly curved, thick-walled, and 1-septate ascospores that are usually constricted at the septum. In the past, host associations were used to delimit Boeremia species. However, since Boeremia taxa have overlapping morphological characters and are cryptic, it renders taxonomic identification arduous. Therefore, the use of other approaches including multi-gene phylogenetic analyses are imperative. Recommended DNA markers for species delineation are the internal transcribed spacer (ITS, nuclear rDNA consisting of ITS1-5.8S-ITS2) and large subunit (28S, D1–D2 domains of nuclear 28S rDNA) loci, and the genes for actin (ACT1), beta-tubulin (TBB1), RNA polymerase 2 (RPB2) and translation elongation factor 1α (TEF1). Here, we applied morphological and molecular phylogenetic analyses to establish a new taxon (B. albae), and a new host and geographical record for B. maritima associated with leaf spots of Morus alba (Moraceae) in northern Thailand. By providing sequence data for three additional gene regions, our phylogenetic analyses impart a stable phylogenetic placement of the ex-type strain of B. maritima, as illustrated. This is the first study that reports Boeremia species from M. alba, and B. maritima from a terrestrial habitat.

Morphological and phylogenetic appraisal of a new Kirschsteiniothelia (Dothideomycetes, Kirschsteiniotheliales) species from Yunnan Province, China

Kirschsteiniothelia is a saprotrophic genus that occurs in both terrestrial and aquatic habitats. This study introduces a novel species of Kirschsteiniothelia, discovered on the bark of Abies fabri, collected from Yunnan Province, China, and named K. laojunensis. The identification was established through a combination of molecular analyses and morphological characteristics. Kirschsteiniothelia laojunensis is characterized by its large subglobose to globose, black perithecia, clavate, 8-spored asci, fusiform asymmetric ascospores, which are hyaline when immature and become greenish to brown at maturity. This discovery marks the first occurrence of Kirschsteiniothelia on tree bark, with some lichen thallus tissue surrounding the base of the perithecia. Detailed morpho-molecular characterization along with a synopsis table for the genus are provided.

Morphological and Molecular Characterization of the Pathogen Associated with Infected Leaf and Fruits of Tomato

This investigation describes the morpho-molecular characterization of the pathogen associated with infected leaf and fruits of tomato (Solanum lycopersicum L.). The fungal pathogen was identified based on their cultural morphology, microscopic features and analysis of Internal Transcribed Spacer (ITS) region sequences of nuclear ribosomal DNA (nrDNA). Cultural morphology including colour of the young and mature colony, growth pattern, concentric rings etc. and microscopic features including shape and size of microconidia, macroconidia, phialide, shape of apical and basal cells of macroconidia, septation in conidia, inter-septal distance and formation of chlamydospores corroborated the identity with the pathogenic fungi Fusarium oxysporum. The phylogenetic analysis based ITS regions sequences of nrDNA of the fungal isolate showed 100% identity with Fusarium oxysporum. Thus the ITS of nrDNA sequences validated the morphological data. Pathogenicity test also reconfirmed the identity of the test isolate of Fusarium oxysporum. The Chittagong Univ. J. B. Sci.,Vol. 10(1 &2):31-40, 2020.

Morphomolecular characterization of invasive fruit borer infesting apple

Apple, a vital fruit crop worldwide and a major crop grown in northern parts of India, acts as a backbone for the survival and livelihood of the farming community. However, it is prone to severe damage from insect pests and diseases. In the past few years, due to erratic weather patterns, there has been an alarmingly increased infestation of different insect pests, both invasive and non-invasive, resulting in substantial economic losses to this industry. One similar case was seen in the Himalayan region of India, where the apple crop in Karewas was heavily damaged by some invasive fruit borer, feeding on pulp and making galleries to reach the seed locules, thereby destroying the seed material. To ascertain the true identity of this pest, a study based on morpho-molecular characterization of this pest was carried out in the Faculty of Agriculture, Wadura, SKUAST Kashmir, India, during the years 2021 and 2022. The invasive fruit samples were collected from apple orchards at different locations (experimental sites) in North India by installing delta sticky traps @ 5 traps/ha for moth collection. The fruit-boring larvae and pupa were also collected and reared in confined chambers of the laboratory with controlled temperature and humidity. All the laboratory investigations were conducted at the Division of Entomology, FoA, Wadura, SKUAST Kashmir. During the investigation, it was confirmed that the invasive borer is a codling moth (Cydia pomonella L.), a member of the family Tortricidae, order Lepidoptera, which was earlier having quarantine importance in India, as it was only present in the Ladakh region of India. From the phylogenetic analysis of sample sequences, the species of codling moth present at all experimental sites was more identical to the codling moth sequence from Leh (the northernmost arid region), India. Further, the study of life cycle and voltinism revealed that codling moth completes three generations per year in Kashmir, with a single cycle lasting up to 2.5 months. However, the timing of various generations varies, depending on prevailing weather conditions at specific locations and times. The number of generations completed by codling moth in Kashmir conditions during apple growing season was three full and a half overwintering generations. The study of the life cycle of this important pest was necessary to know the weak link for its effective management and to prevent the economic loss in apple fruit.

Morpho-molecular characterization revealed distinct diversity among rice blast (Pyricularia oryzae) isolates

Morpho-molecular characterization revealed distinct diversity among rice blast (Pyricularia oryzae) isolates

Morpho-molecular characterization reveals some interesting macrofungi from the cold arid region of Kargil, India

Morpho-molecular characterization reveals some interesting macrofungi from the cold arid region of Kargil, India

Morpho-Molecular Characterization Reveals a New Genus, Three Novel Species and Two New Host Records in Xylariomycetidae.

Xylariomycetidae comprises extremely diverse taxa that are widespread on decaying wood worldwide. An investigation of the diversity of microfungi on oil tree plantations in Sichuan Province was conducted during 2020-2021. Twelve saprobic taxa representing five species were identified as members of Amphisphaeriales and Xylariales through morphological comparisons. Phylogenetic analyses of combined ITS, LSU, rpb2, tub2 and tef1 sequence data indicated a distinct clade formed by three strains within Xylariomycetidae, unrelated to any currently recognized families. Thus, a novel anthostomella-like genus, Bicellulospora, is proposed and treated as Xylariales genera incertae sedis. Bicellulospora is characterized by dark brown to black, immersed, subglobose ascomata with a clypeus, cylindrical asci, and hyaline to yellowish brown, inequilaterally ellipsoidal ascospores with a large upper cell and a dwarf lower cell. Two new species of Amphisphaeria, namely A. oleae and A. verniciae, are introduced based on multi-gene phylogenetic analyses (ITS, LSU, rpb2 and tub2) coupled with morphological characteristics. Amphisphaeria micheliae and Endocalyx ptychospermatis are reported as new host records.

Morpho-molecular characterization of sand-dwelling dinoflagellate communities from the German Wadden Sea and insights into their spatiotemporal distribution

ABSTRACT Benthic protist communities, including sand-dwelling dinoflagellates, are still poorly characterized worldwide and there is a lack of knowledge on their diversity and spatiotemporal dynamics. In this study, sediments were obtained from several coastal locations of the German Wadden Sea during the summer months of 2017 and 2018 to determine the diversity and spatiotemporal distribution of sand-dwelling dinoflagellates. The analyses consisted of light microscopy and the metabarcoding of small subunit (SSU) rRNA gene V4 region amplicons. Additionally, SSU rRNA sequences were newly obtained for 15 observed taxa included in a reference database and were used to taxonomically assign the obtained amplicon sequence variants (ASVs). The combination of morphological and molecular approaches allowed a deep characterization of dinoflagellate communities. Seventy-three morphospecies were detected by microscopy, while metabarcoding ASVs (amplicon sequence variants) were assigned to 116 species, resulting in a more comprehensive biodiversity inventory than achieved with either method alone. Pfiesteriaceae, Togula compacta and Thecadinium yashimaense predominated in the different sampling locations. Communities from Wilhelmshaven diverged from the others, consistent with its muddier sediment. A high level of resemblance was determined for the communities from each location, regardless of the sampling month and year, indicating a high summertime stability of benthic communities. Further efforts will be needed to characterize benthic protist diversity, as a first step in obtaining a complete picture of the habitat characteristics and physico-chemical conditions that influence benthic community composition.

Morpho-molecular characterization of golden potato cyst nematode population from Bauko, Mountain Province, Philippines

Morpho-molecular characterization of golden potato cyst nematode population from Bauko, Mountain Province, Philippines

Genetic relatedness among interspecific hybrids in the genus Capsicum and their implications in breeding King chilli

The present study explores the potentiality of interspecific crosses using three different Capsicum species, comprising of two C. annuum (Krishna and Capsicum), five C. chinense (King chilli A1, A5, A18, A19, A20) and one C. frutescens (Mem) genotypes, with the objective to identify the incompatibility barriers, accomplish the morpho-molecular characterization of parents and hybrids and assess the genetic relatedness between the genotypes.Amongst the three species used for the crossing programme, 'Krishna' (C. annuum) and 'Mem' (C. frutescens) are local cultivars of Assam (India) and possess excellent adaptability. Guinness World Records certified King chilli, or 'Bhut Jolokia' (C. chinense), as the world's hottest chilli (10,001,304 SHU) in September 2007. King chilli has tremendous export potential but is prone to viral diseases, thereby necessitating the transfer of desirable disease resistance from local cultivars.It was observed from the study that the reciprocal crosses involving C. chinense and C. frutescens were compatible, but C. frutescens (Mem) was crossable with only accession A5, suggesting genotype specificity in the interspecific crosses. The usual Euclidean distance between Mem and A5 was 716.605Similarly reciprocal crosses involving C. annuum and C. chinense were partially incompatible. Both the C. annuum (Krishna, Capsicum) genotypes successfully crossed with King chilli accession A18 thereby exhibiting genotype specificity. It was observed that in the cross A18 x Krishna the F1 seeds failed to germinate and in the cross A18 x Capsicum the F1 plants exhibited dwarfism. Hence, unilateral incompatibility was observed when King chilli accession A18 was used as the female parent. In the reciprocal crosses, germination and plant development was normal.The pollen viability ranged from 72.64 to 94.92 % for parents and 23.55 to 81.02 % for the hybrids. Thus, hybrids exhibited lower pollen viability compared to their parents. Significant morphological differences were visible between the parents and their combinations; some features were similar to the male or female parent, and others were either intermediate or novel.Parental polymorphism was detected using thirty random SSR primers, and the markers showing polymorphic bands were used to confirm the hybridity of F1s. Therefore, an attempt was made in this study to assess the possibility of interspecific crosses to transfer disease resistance from the related species to enhance the quality of King chilli, thus improving its commercial valve.

Morpho-Molecular Characterization and Seasonality analysis of Greenidea psidii Van Der Goot on Guava in Terai Zone of West Bengal, India

Greenidea psidii, Guava aphid comes under the subfamily Greenideinae within the Aphididae and was first discovered in the World in 1916 on guava. In terai region of West Bengal these species are regarded as potential pest of guava (Psidium guajava). Therefore, quick and precise species identification is immediately needed for early detection and risk analysis followed by integrated management of the aphid pests. During morpho- taxonomy study we found that apterous aphids have long sized siphunculi, alongwith long setae present on the body. Moreover, the identity of the aphid species was confirmed through DNA barcoding of the mitochondrial cytochrome c oxidase sub unit I (COX-I) gene, another analytical tool for quick and precise identification. The COX-I sequence was successfully blasted for similarity search and uploaded in NCBI software (Accession No. OR085959). Maximum likelihood method for phylogenetic analysis reveals that all the G. psidii sequences were clustered in the same clade and confirmed the identity of the species of the present study. The seasonal fluctuation studies reveals that the maximum population of 21.85 aphids/ twigs and 17.45 aphids/ twigs were observed on 10th SW during 2021 and on 11th SW during 2022, respectively.

Morpho-molecular characterization based diversity analysis of Bipolaris isolates infecting graminaceous hosts

Morpho-molecular characterization based diversity analysis of Bipolaris isolates infecting graminaceous hosts

Morpho-molecular characterization and bioecology of leaf folder, Pycnarmon cribrata (Fabricius) on nirgundi (Vitex negundo): an aromatic medicinal shrub from India

The occurrence of leaf folder, Pycnarmon cribrata (Fabricius, 1794) (Lepidoptera: Crambidae) on the medicinal plant nirgundi, Vitex negundo Linn. has been observed in West Bengal, India. The pest damaged the foliage by folding the leaflets, feeding within and ultimately making the foliage dry and papery. However, the identity of the pest species was confirmed through morphological characteristics of male and female genitalia as well as sequence amplified product of Cytochrome c oxidase subunit I (COI) gene (GenBank No. ON545929). Biological studies revealed that the entire life cycle of the moth completed in 29.90 ± 2.42 days on average, while the range was between 25-34 days. The peak larval incidence was noticed in 16th SMW (Standard Meteorological Week) and 24th SMW with population of 12 and 15 larvae per plant during 2021 and 2022, respectively. The percent plant incidence levels ranged from 15 to 100% during the first year i.e. 2021 and 30 to 100% during the second year i.e. 2022. The correlation of mean larval population (r = −0.66) and percent plant incidence (r = −0.71) with weather parameters indicated a highly significant negative association with the maximum temperature and a significant positive correlation with maximum and minimum relative humidity. The parasitization of Choeras sp. (Braconidae: Hymenoptera) (GenBank No. OP293230) was observed on old age larvae of P. cribrata and the natural parasitization was recorded as 26% during the study period. This study provides a comprehensive information on the occurrence, nature of damage, biology, morphometry, seasonal incidence and natural enemy fauna associated with P. cribrata which will be helpful for implementation of Integrated Pest Management (IPM) strategies to curtail the pest problem on V. negundo, a valuable medicinal plant.

Comparative morpho-molecular characterization of elite walnut variety Parbat (JWSP-06) with local selections of north-western Himalayan region of Jammu and Kashmir, India

In the present study, we compared our previously identified elite walnut selection JWSP06 (Parbat) with other walnut selections from the region. Based on DUS (distinctiveness, uniformity, and stability) characterization, Parbat is high yielding late maturing selection with superior quality nut and kernel traits. This selection is also resistant to walnut anthracnose. Recently the Parbat was notified as an elite walnut selection by the Government of Jammu and Kashmir. DNA profiling authenticate that “Parbat” is distinct from regional walnut selections such as Hamdan, Sulaiman, and Bhushan. The comparative study revealed that the quality and yield capacity of the Parbat are higher than those of local walnut selections. Regardless of the walnut selection, the marker WSSR-8 efficiently separates all genotypes. Breeding walnuts through conventional methods require genetically distinct parents. Our comparative study has identified Parbat as distinct from regional selections. This study has opened a door to breed quality walnut cultivars by crossing Parbat with Sulaiman and Hamdan. Being located at different geographic areas, the walnut selections need to be planted side by side for an effective hybridization-based breeding program.

Identification of Eight High Yielding Strains via Morpho-Molecular Characterization of Thirty-Three Wild Strains of Calocybe indica.

Calocybe indica, generally referred as milky mushroom, is one of the edible mushroom species suitable for cultivation in the tropical and sub-tropical regions of the world. However, lack of potential high yielding strains has limited its wider adaptability. To overcome this limitation, in this study, the germplasms of C. indica from different geographical regions of India were characterized based on their morphological, molecular and agronomical attributes. Internal transcribed spacers (ITS1 and ITS4)-based PCR amplification, sequencing and nucleotide analysis confirmed the identity of all the studied strains as C. indica. Further, evaluation of these strains for morphological and yield parameters led to the identification of eight high yielding strains in comparison to the control (DMRO-302). Moreover, genetic diversity analysis of these thirty-three strains was performed using ten sequence-related amplified polymorphism (SRAP) markers/combinations. The Unweighted Pair-group Method with Arithmetic Averages (UPGMA)-based phylogenetic analysis categorized the thirty-three strains along with the control into three clusters. Cluster I possesses the maximum number of strains. Among the high yielding strains, high antioxidant activity and phenol content was recorded in DMRO-54, while maximum protein content was observed in DMRO-202 and DMRO-299 as compared with the control strain. The outcome of this study will help the mushroom breeders and growers in commercializing C. indica.

Identification and morpho-molecular characterization of low spore strain in oyster mushroom

Sporocarps of oyster mushroom liberate enormous spores and cause allergic reactions to workers involved in its cultivation. These spore-related allergies include stiffness or pain in the forearms, limbs, itchy throat, grogginess, and respiratory problems and are major problems during oyster mushroom cultivation. In this study, we have generated seven hybrids using single-spore isolates (SSIs) of Pleurotus ostreatus var. florida (DMRP-49) and P. ostreatus (DMRP-30). Chimera was observed during cultivation trial of these hybrids and led to the development of low spore-producing/sporeless strain (DMRP-395) as evident from spore print and microscopic analysis. Further, the cultivation trial of this sporeless strain revealed a bunchy fruiting pattern and required 20-24°C temperature for fruiting. At par yield was observed in sporeless strain. Notably, a prominent infundibuliform-shaped pileus along with central attachment of stipe was observed in the sporeless strain. Moreover, genetic diversity and principal component biplot analysis revealed resemblance of sporeless strain with one of the parental strain, i.e., P. ostreatus var. florida (DMRP-49). The developed sporeless strain (DMRP-395) contains high protein and at par yield as compared with the control (DMRP-136). This sporeless strain will be helpful to reduce spore-related allergic responses in mushroom growers.

Morpho-molecular characterization of Clonostachys rosea and deciphering its biomolecules untangles the anti-fungal action against Fusarium oxysporum f.sp. lycopersici

Clonostachys rosea, a promising mycoparasite, has great biological control abilities against a variety of fungal plant diseases, insects and nematode pests. In the present study, three isolates of Clonostachys rosea were isolated from carrot, undisturbed forest and black gram rhizosphere soils respectively, out of 50 soil samples taken from different field and horticultural crops. Out of 15 onion plant samples from various locations, one isolate of C. rosea was isolated from root samples of onion. Two isolates of C. rosea from mycoparasitized sclerotia on cabbage head rot infected sample and naturally deceased whitefly in cotton was isolated respectively. They were morphologically and molecularly confirmed as C. rosea based on the fungal taxonomical keys and PCR amplification with ITS, translation elongation factor 1 alpha (TEF-1α), species specific primer sets respectively. The antagonistic ability of C. rosea against Fusarium oxysporum f.sp. lycopersici (Fol) has been shown using confrontation assay. Molecular docking of biomolecules of C. rosea using AutoDock vina module in PyRx 0.8 software reported dl-Glyceraldehyde dimer and 2(3H)-Furanone, dihydro-4-hydroxy as best hits with highest binding energy in the range of – 7.1 to −5.6 kcal/mol against target protein Global transcription regulator sge1 (SIX Gene Expression 1) of Fol. We also observed comparatively less binding energy for commercially available fungicide on docking against the target protein of Fol.

Screening of castor germplasm for wilt reaction and morpho-molecular characterization of resistant genotypes

Castor (Ricinus communis L.) is an important industrial versatile non-edible oilseed C3 crop belongs to spurge family. Its oil has exceptional properties which provides an industrial importance to this crop. The present investigation was aimed to evaluate the genotypes of castor for Fusarium wilt reaction in pot followed by characterization of resistant genotypes for yield related traits in field and inter-genotype genetic diversity at DNA level. The percent disease incidence (PDI) among 50 genotypes ranged from 0 to 100%. A total of 36 genotypes were found wilt resistant (28 highly resistant and 8 resistant). ANOVA revealed that the genotypes MSS was significant for each trait studied, indicating the existence of plentiful variability in the experimental material. The morphological characterization showed that DCS-109 (73.30 cm) had a dwarf stature The genotype RG-1954 was superior for oil content (50.29%) with moderate for shelling out (67.63). RG-1673 was outstanding for seed boldness as 100 seed weight for this genotype was maximum (38.98 gm). JI-403 had maximum seed yield per plant (SYPP; 354.88 gm). SYPP positively associated with all traits except oil and seed length:breadth ratio. The path analysis revealed that the direct effects of NPR (0.549), TLFP (0.916), and CPP on SYPP are quite significant. A total of 38 alleles from 18 SSR markers were amplified in 36 genotypes. The NJ tree could divide 36 genotypes into three main clusters. AMOVA revealed 15% and 85% variance among and within subpopulations, respectively. Both morphological and SSR data demonstrated to be effective tools for discerning inter-genotypes diversity and categorizing high-yielding and disesae-tolerant castor genotypes lines.

Morpho-molecular characterization of ethnic Bora rice for conservation and breeding

Glutinous Bora rice plays an important role in socio-economic and cultural livelihood of Boro tribes of Assam. However, neither systematic survey had been done, nor databanks are available on their detailed morpho-molecular characterization essential for breeding programme. Present study attempted to prepare a morphological database with molecular signature using a total of 22 Bora rice lines collected from upper Brahmaputra basin of Assam, which were phenotyped with 27 popular agro-morphological traits followed by marker based genotyping, sequence diversity study and expression profiling for selected genetic loci associated with low amylose content. Long day flowering and less number of tillers with panicles are the main discouraging traits of Bora rice that showed negative association with culm length. Among the 8 different phenotypic markers established, anthocyanin pigment in different parts of the plant and availability of awn may be used as identifying criteria for selection of the hybrid lines introgress with “Komal” trait. In marker based genotyping, all the studied marker loci were reported to be polymorphic with variable number of allelic forms and RM241 showed highest PIC value. In case of expression study, 5 days (after anthesis) old developing grains of selected Komal genotype (Aghani Bora) showed 90.9-fold down regulation of GBSSI (loci associated with amylose deposition in rice grain) with respect to the commonly grown popular rice (IR36). Phylogenetic relationship study with 5 different rice genomes (O. rufipogon, O. glaberrima, O. sativa japonica group, O. sativa indica group and IR36) based on sequence diversity of six major genetic loci (associated with starch deposition in endosperm) showed distinct relationship. Selected morpho-molecular markers (polymorphic between Bora and improved rice) were subjected to validation on hybrid population developed from selected Bora rice (Vogali Bora) and improved rice (IR36), which showed a promising response for use in marker assisted breeding program.