Morphological characterization of Hypnaceae (Bryopsida,Hypnales): Investigating four genera from Western Himalayas by using LM and SEM techniques

The Hypnaceae is one of the largest and diversified family among the pleurocarpous mosses which consists of nearly 60 genera and 1,000 species around the world. In Pakistan, it is represented by 15 species and 8 genera. The current research article provides the detail morphological investigation of four different species (genera) of the family Hypnaceae that is, Gollania clarescens, Hypnum revolutum, Homomallium andoi, and Taxiphyllum taxirameum in the Western Himalayas, Pakistan. The research article reports the first SEM study of the leaf surfaces of the studied taxa, along with comprehensive morphological characters of the four species. The main objective of the research project is to present the comparative light and scanning electron microscopic study to discuss the morphology in detail because previously the family is just reported in different bryophyte checklists of the Western Himalayas. Based on results, morphological characters, micromorphological observations, qualitative and quantitative attributes, taxonomic keys for the studied taxa, distribution, and habitat are described. Stereoscope was used to analyze different qualitative characters, and light microscope was used to observe and measure the laminal and alar cells of the leaf. The SEM study reveals many important surface features like cell shape and cell wall. The laminal cells were mostly linear and elongated with thin cell walls. The specimens used for the SEM were air dried, so the laminal cells were somewhat constricted and concaved. The current study project will help to make the contribution in the taxonomy and morphology of this family.     

Isolation and species diversity of arbuscular mycorrhizal fungi in the rhizosphere of <i>Puccinellia tenuiflora</i> of Songnen saline-alkaline grassland,China

Salinization has led to the deterioration of the ecological environment, affected the growth of plants, and hindered the development of agriculture and forestry. Arbuscular mycorrhizal (AM) fungi, as important soil microorganisms, play significant physiological and ecological roles in promoting plant nutrient absorption and improving soil structure. Puccinellia tenuiflora (Turcz.) Scribn. et Merr. in Songnen saline-alkaline grassland was selected as the research object to observe AM fungal colonization of the roots and explore the species and diversity of AM fungi in symbiotic association with P. tenuiflora. This study showed that AM fungi colonized in P. tenuiflora roots and formed a typical Arum–type mycorrhizal structure. A significant correlation was observed between vesicular abundance and the colonization intensity of mycorrhiza. Isolation and identification revealed 40 species of AM fungi in the rhizosphere of P. tenuiflora, belonging to 14 genera, of which two species could not be identified. The richness of the genus Glomus was the highest, accounting for 30% of the total species. Funneliformis mosseae and Rhizophagus intraradices were isolated from all the samples and were the species with the widest distribution in the rhizosphere of P. tenuiflora. Correlation analysis showed that pH only had a significant impact on the distribution of a few species, such as Glomus pustulatum, Diversispora spurca, Glomus aggregatum, Rhizophagus clarum, and Acaulospora foveata. The present study provides a theoretical basis for further exploring the resources of AM fungi in saline-alkaline soil.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Morpho-anatomical characterization of some members of euglenophycota (algae) of north-east Punjab,Pakistan based on light microscopy (LM) and scanning electron microscopy (SEM)

The study was carried out in four districts, that is, Gujranwala, Gujarat, Narowal and Sialkot of Punjab, Pakistan. The sampling was carried out randomly in different seasons from the water bodies especially from wastewater. Twenty-one species belonging to Euglenophycota were identified using light microscopy and scanning electron microscopy from which 04 species belong to genus Phacus, 02 species belonging to Trachelomonas and Euglena based on light microscopy. It was observed that Euglena was the most diverse genus and it is supposed to be the indicator species of the polluted water. It was observed that E. oblonga was found in maximum pH range, that is, 7.0–11.0. Similarly, E. brevicaudatus was found in maximum EC, that is, 169 ± 1.5 ms/cm these outcomes indicated that for internal examination along with LM, SEM was necessary for correct identification of algal sample up to specie level.     

EDX measurements and SEM examination of surface of some imported woods inoculated by three mold fungi

Wood samples of Pinus rigida, Juglans nigra, and Fagus sylvatica were superficially inoculated with three mold fungi (Penicillium selerotigenum, Paecilomyces variotii, and Aspergillus niger). The changes in wood surfaces and the elemental composition were observed by scan electron microscope (SEM) and the electron dispersive X-ray spectroscopy (EDX), respectively. Wood elemental composition was an indication of the growth of mold fungi, in which P. selerotigenum, and A. niger have consumed high amount of carbon (C) of F. sylvatica wood compared to control treatment. Also, P. selerotigenum consumed high amount of C of J. nigra wood compared to the control. While, the three mold fungi showed little changes in C content in P. rigida wood. The nitrogen element in the studied woods was completely consumed by the three mold fungi. Cl element increased in the three woods as inoculated by the three fungi compared to untreated woods. Other elements such as S, Si, Ca, Zn, Cu, and Fe varied in their content in accordance to the interaction among wood species and fungi. The growth of the three mold fungi showed distribution and structures of spores and hyphae covering deteriorated wood surfaces. The study suggests that the three mold fungi metabolize the carbon-rich constituents of the investigated wood species in a proportional manner and produce large fruiting structures that release vast numbers of spores in nature.     

Leaf epidermal micromorphology and its implications in systematics of certain taxa of the fern family Pteridaceae from Northern Pakistan

The present study is insights into foliar epidermal anatomy for characterizing clades, and their utility in taxonomic segregation of certain species of Pteridaceae from Northern Pakistan. The leaf epidermal anatomy of 10 species of Pteridaceae representing four genera were examined using light and scanning electron microscope. A micromorphological matrix was constructed for eight qualitative and 12 quantitative characters. unweighted pair group method with arithmetic means and principal components analysis statistical analysis were performed to test the validity of foliar epidermal anatomical features as method of separating species and genera, and phylogenetic clusters among species are constructed using qualitative and quantitative traits. The qualitative characters described here are shape of epidermal cells, stomata, guard cell and subsidiary cells, anticlinal wall pattern, and trichomes types which is helpful in defining groups within Pteridaceae. In addition, the size of stomata, guard cells, subsidiary cells, stomatal pore epidermal cells, and trichomes are quantitatively analyzed. All species have hypostomatic leaves. Two types of stomata were observed in studied species, anomocytic and polocytic. Anomocytic stomata were observed in three genera namely: Adiantum, Onychium, and Chielanthes whereas Pteris can be discriminated from other genera by its polocytic stomata. On the basis of multivariate analysis present study does provides sufficient information on the taxonomic importance of foliar anatomy which validate its efficacy in species and genera discrimination. From result obtained here it is further possible to use leaf micromorphologic data in ferns phylogeny and providing basis for future taxonomic delimitation in other taxa.     

Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) and its taxonomic significance

The Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) from different phytogeographical region of Pakistan has been evaluated. In this research, 16 species belong to 6 genera of subfamily Caryophylloideae have been studied using light (LM) and scanning electron microscopy (SEM) for both qualitative and quantitative characters. Different palyno‐morphological features were observed including; pollen ornamentation, pore ornamentation, echini arrangement, echinidensity, number of pori, size of polar and equatorial diameter, P/E ratio, exine thickness, and size of pore were studied. The palyno‐morphological characters of subfamily Caryophylloideae have taxonomically significant in identification and delimitation of species. Two pollen types, i.e., subspheroidal (15 species) and prolate (one species Vaccaria hispanica) were observed. Microechinate‐punctate and microechinate‐perforate pollen ornamentation were examined. Two pollen types of pori, i.e., prominent (5 species) and sunken (11 species) ornamentation have been reported. Three types of echini arrangement have been reported irregular, regular and rather regular, while three type of echinidensity (i.e., medium, dense and sparse) were observed. Pori numbers were found different in different species range from 5 in Silene apetala to 19‐35 in Silene vulgaris. Based on different palyno‐morphological characters, taxonomic key was develop for quick and accurate identification. The quantitative data were processed using SPSS software for average, minimum, maximum, and standard error.     

Taxonomic importance of spore morphology in Thelypteridaceae from Northern Pakistan

Spore morphology of Thelypteridaceae species growing in Malakand Division, Northern Pakistan, was studied using both light microscopy and scanning electron microscopy. The taxa are Christella dentata and Glaphyropteridopsis erubescens in the subfamily Thelypteridoideae, and Phegopteris connectilis, Pseudophegopteris pyrrhorhachis, and Pseudophegopteris levingei in the subfamily Phegopteridoideae. The studied species exhibit differences in spore size, exospore thickness, color, and ornamentation. Spores of the studied species are monolete and medium‐sized, and shape is ellipsoidal in both polar and equatorial views. The average measurement of the polar diameter ranges from 27 μm to 31 μm, whereas in the equatorial direction it varied from 20 μm to 40 μm. The exospore thickness ranges from 1.2 μm to 2.4 μm. Reticulate, laevigate with microgranules, cristate, and coarsely echinate surface ornamentation are observed among the species. Multivariate analysis including unweighted pair group method with arithmetic mean and principal component analysis was used for the grouping and discrimination of species and genera.     

Variations in fungal spore dimensions in relation to preparatory techniques for light microscopy and scanning electron microscopy

The volumes of urediospores of Uromyces viciae-fabae and ascospores of Sordaria humana in the critical point-dried, freeze-dried, osmium vapour-fixed/freeze-dried, frozen-hydrated and ‘wet’ states were measured. Dried spores shrank and were significantly smaller than frozenhydrated and ‘wet’ spores. Urediospores exhibited a greater range of dimensional changes in relation to the preparatory treatment than did ascospores. Ascospores, dried in different ways, were not significantly different from each other but were significantly smaller than frozenhydrated ones. Both urediospores and ascospores dehydrate naturally and collapse. On wetting they imbibe water rapidly and swell. Low temperature scanning electron microscopy may be suitable for investigating fungal spores in their ‘natural states' on the leaf surface.     

Scanning electron microscopy of rust haustoria

The three-dimensional structure of rust haustoria has been studied by scanning electron microscopy (SEM). Rust infected leaves were fixed and embedded in Spurr's resin as for transmission electron microscopy (TEM). The cut surface of blocks containing embedded material was etched with sodium ethoxide and examined in the SEM.     

Low-temperature SEM for detection of fungicide activity

Low-temperature scanning electron microscopy (LTSEM) combined with cryopreparation methods provided images of well-preserved biological surfaces and structures on a routine basis. Fractures of wheat leaves revealed epidermal and parenchymatous cells and masses of fungal hyphae growing in intercellular spaces. Freeze-fractured plant cells contained haustoria of the brown rust fungus Puccinia triticina. Extrahaustorial matrices were clearly distinguishable and at infection sites granular material was found. Activity of the triazole fungicide cyproconazole was mainly directed towards fungal hyphae and sporogenic tissue, resulting in a stronger branching and swelling of hyphal tips and collapse of fungal cells. Cryofixation methods combined with the use of a cryopreparation unit (Balzers SCU 020) were more reliable in interpreting the observed biological events through easier discrimination between evidence and artefacts. More advantages in the use of the cryopreparation unit are described in detail.     

Systematic approach to the correct identification of Asplenium dalhousiae (Aspleniaceae) with their medicinal uses

In present study, multiple microscope techniques were used for the systematics identification of the species Asplenium dalhousiae. The plant was collected from different phytogeographical and its natural habitat of Pakistan, where it shows higher diversity. Morphology, foliar epidermal anatomy, and spore morphological characters of the species were studied in detailed using multiple microscopic techniques through light microscopy (LM) and scanning electron microscopy (SEM). LM and SEM were used for the systematics identification of the species. Traditionally, the species is used in the ailment of many diseases, so the spore morphology, anatomical features, and morphological characters are relevant to describe the species taxonomy. The importance of multiple methods of taxonomic study (e.g., documentation and morphological characteristics) for characterizing herbs are important step in systematic certification to maintain the efficacy of herbal medicines. The aim of the present study is to examine the morphological, anatomical, and spore morphology of the species A. dalhousiae in more detailed for the correct taxonomic identification and their medicinal validation from Pakistan.     

Pollen diversity and its implications to the systematics of mimosaceous species by LM and SEM

In this study we have discussed pollen morphology of 14 mimosaceous species belonging to five genera by using light microscopy (LM) and scanning electron microscopy (SEM). The palynological features of species were determined by both qualitative and quantitative characters. The qualitative characters include pollen shape, colpi arrangement, and exine sculpturing. Oblate spheroidal shaped pollens were noted in Albizia procera, Albizia lebbeck, Acacia tortilis, Acacia ampliceps, and Acacia modesta, subprolate shaped pollen in Leucaena leucocephala, Prosopis julifera, and Acacia nilotica, prolate pollen in Acacia farnesiana and Prosopis glandulosa while spheroidal, sub spheroidal and sub oblate pollen grains were observed in Acacia catechu, Mimosa himalayana, Prosopis cineraria, respectively. Sparsely foveolate, reticulate, scabrate, and scrobiclate exine sculpturing were observed. Colpi with sunken ornamentation were seen in P. cineraria, P. glandulosa, P. juliflora, and L. leucocephala while colpi were absent in rest of the studied species. The quantitative characters were statistically studied using SPSS software. The current study gives important morpho‐palynological characters for identifying and validation of close related and similar taxa which will aid to the phylogenetic analysis of Mimosaceae family.     

Pollen morphological investigations of family Cactaceae and its taxonomic implication by light microscopy and scanning electron microscopy

The family Cactaceae is the diversified group of angiosperm plants whose pollen statistics has been used for taxonomic identification. In this article, we present the pollen morphology of eight species belong to seven taxonomically complex genera of Cactaceae including Astrophytum, Cylindropuntia, Echinocereus, Echinopsis, Mammillaria, Opuntia, and Thelocactus using light and scanning electron microscopy. The pollen grains were acetolyzed, measured, described, and electron photomicrographs were taken. Cactaceae can be characterized by presenting different palynomorphological features including pollen type, sculpturing, polar and equatorial diameter, aperture orientation, exine thickness, P/E ratio, and echini features. Four types of pollen shapes, that is, prolate spheroidal (three species), subprolate (two species), prolate (two species), and oblate spheroidal in Echinocereus reichenbachii were observed. The polar and equatorial diameter observed maximum in O. ficus indica 116.95 and 112.27 μm while minimum in M. compressa 38.42 and 21.05 μm. Pollen of two types, tricolpate in members of subfamily Cactioideae and pantoporate in the Opuntioideae were examined. The fertility percentage has been observed maximum in Opuntia macrocentra (83.84%) and minimum in Opuntia ficus‐indica (57.89%). Exine sculpturing showing great variations such as granulate, reticulate, granulate perforate and micro‐echinate foveolate ornamentation was examined only in Echinopsis eyriesii. A key to species, based on pollen micromorphological attributes, has been constructed for correct identification of complex cactus species.     

Taxonomic study of subfamily Nepetoideae (Lamiaceae) by polynomorphological approach

The present study is insight into pollen morphology for characterizing species and their utility in the taxonomic separation of certain taxa of subfamily Nepetoideae (Lamiaceae) from Pakistan. The pollen micromorphology of 11 species of the Nepetoideae was analyzed and documented using light microscopy and scanning electron microscopy (SEM) for both qualitative and quantitative characteristics. Most species have hexazonocolpate pollen grains but trizonocolpate and tetrazonocolpate pollen with circular and oval amb were also rarely observed in Mentha spicata. The basic pollen shape in most of the studied species was subspheroidal but prolate grains were also observed in M. spicata, S. coccinea, and S. plebeia. The exine sculpturing of Nepetoideae pollen was taxonomically very informative particularly at subfamily level. Observations of exine sculpturing with SEM revealed various types of pollen grains: reticulate, bireticulate, microreticulate, perforate, aerolate, and gammate. The bireticulate type further subdivided into three subtypes based on the number of secondary lumina in each primary lumen and is characterized by varying characteristics of the secondary reticulum and primary muri. A significant variation was observed in colpus surface ornamentation. The maximum polar diameter was found in O. americanum (58 ± 5.8 μm) and the maximum equatorial diameter observed in O. basilicum (50.25 ± 1.37 μm). Pollen features of the studied species were discussed and compared based on the current taxonomical concepts. The results showed that pollen traits of the subfamily Nepetoideae was found significant to classify the taxa. Furthermore, pollen features provide additional evidence to distinguish macromorphologically similar taxa from each other.     

Infection of the Red Palm Weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: A SEM study

The Red Palm Weevil (Rhynchophorus ferrugineus) is a devastating pest of palms in the Mediterranean, Middle East, and Eastern countries. No effective control measures are available. R. ferrugineus has been found naturally infected by the entomopathogenic fungus Beauveria bassiana, but its infection process in this host is unknown. We have studied the infection of R. ferrugineus larvae and adults by B. bassiana using dry conidia and conidia suspensions using scanning electron microscopy (SEM). In early stages, SEM revealed acquisition of B. bassiana conidia by cuticle ornamentation in legs, antennae, and elytra of R. ferrugineus adults. Subsequently, conidia germinated and frequent episodes of hyphal/conidial fusion were found. Appressoria, signs of adhesion and cuticle degradation led to penetration (even direct) and colonization of R. ferrugineus hosts by the fungus. B. bassiana conidiophores were found in a R. ferrugineus cuticle, which indicate the completion of the life cycle of the fungus in the insect host. SEM has proven that dry conidia of B. bassiana is an adequate inoculum for R. ferrugineus infection. SEM revealed that conidia of B. bassiana attached to the cuticle of R. ferrugineus can germinate and differentiate appressoria. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Foliar epidermal anatomy of some selected wild edible fruits of Pakistan using light microscopy and scanning electron microscopy

The present study is focused on the detailed foliar epidermal anatomy of some selected wild edible fruits (WEFs) from Pakistan using light microscopy (LM) and scanning electron microscopy (SEM). The studied species are Ficus racemosa L., Solanum nigrum L., Capparis spinosa L., Physalis divaricata D.Don, Rosa moschata Herrm. and Ribes orientale Desf. collected from various localities of Pakistan. The objective of the present study is to investigate qualitative and quantitative anatomical characters for the identification and differentiation of collected wild edible fruits. The characters studied are shape and size of epidermal cells, anticlinal wall pattern, trichome type and shape, average number of stomata, length and width of stomata and pore. The detailed microscopic investigation and variations in the characters recorded have a key role in the determination and authentication of wild edible fruits. This study possesses great potential for plant taxonomists to further evaluate the species at molecular and genetic levels.     

Intraspecific variation in seed morphology of tribe vicieae (Papilionoidae) using scanning electron microscopy techniques

Seed micromorphology of 12 species of tribe Vicieae (Papilionoidae) representing five genera were examined using Scanning Electron Microscope (SEM). The different seed types were described, illustrated, compared, and their taxonomic importance is discussed. Seeds exhibit great diversity in ultrastructure and a variety of novel morphological features have been determined among and within genera of the tribe. Seeds were characterized by oval to round shape, ovate to oblong outline and striate to papillate ornamentation. Radiate and wavy cell pattern has been observed as a characteristic exomorphological feature. In Vicia sativa, testa cells superficially seem to form peltate hairs. As a result, seed micromorphology with few exceptions showed variation and its taxonomic value was significant in distinguishing taxa at species level. The disparity in shape and density of papillae or protuberances and alignment of testa cells, may possibly give further insight at intraspecific level within tribe. The findings also illustrated that the use of SEM in seed morphology can reveal hidden morphological affinities among species and showed potential in delimitation of Vicieae members generally at tribe, genus, and particularly at species level.     

Scanning electron microscopy applied to seed-borne fungi examination

The aim of this study was to test the standard scanning electron microscopy (SEM) as a potential alternative to study seed-borne fungi in seeds, by two different conditions of blotter test and water restriction treatment. In the blotter test, seeds were subjected to conditions that enabled pathogen growth and expression, whereas the water restriction method consisted in preventing seed germination during the incubation period, resulting in the artificial inoculation of fungi. In the first condition, seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.), and cotton (Gossypium hirsutum L.) were submitted to the standard blotter test and then prepared and observed with SEM. In the second condition, seeds of cotton (G. hirsutum), soybean (Glycine max L.), and common bean (P. vulgaris L.) were, respectively, inoculated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum, and Colletotrichum lindemuthianum by the water restriction technique, followed by preparation and observation with SEM. The standard SEM methodology was adopted to prepare the specimens. Considering the seeds submitted to the blotter test, it was possible to identify Fusarium sp. on maize, C. gossypii var. cephalosporioides, and Fusarium oxysporum on cotton, Aspergillus flavus, Penicillium sp., Rhizopus sp., and Mucor sp. on common bean. Structures of C. gossypii var. cephalosporioides, C. truncatum, and C. lindemuthianum were observed in the surface of inoculated seeds. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.