Light Microscopy Research Articles

Micromorphological and Phytochemical Evaluation of Heliotropium rariflorum Stocks From Karak, Pakistan

Abstract Micromorphological and phytochemical studies play a major role in quality control and standardization of traditional or herbal medications. In the present research, micromorphological assessment of Heliotropium rarifloum stocks was performed through light and scanning electron microscopies (LM & SEM). The anatomy of leaves, stem and root showed salient histological features. Both surfaces of the leaves had setose glandular trichomes measuring 20–38 × 6–15 µm. The lower epidermis had comparatively a maximum anomocytic stomata (16–35) and stomatal index (12–33). The mature pollen grains were small (74 µm) and spheroidal shaped, with psilate exine (2 μm) sculpturing. Vein termination and vein islet number of the upper epidermis were 5–20 and 5–15, respectively. The palisade ratio of the leaf lamina for the upper and lower epidermis was 2–10 and 2–8. LM and SEM of the powdered samples displayed crystals, phloem fibers, xylem, vessels, sieve tube elements, companion cells, and tracheids. Extractive values determination, fluorescence, and phytochemical analysis were employed for quality control according to the World Health Organization (WHO) guidelines. Phytochemical screening revealed various secondary metabolites. It is suggested that H. rariflorum might be a reliable source of nutrients and secondary metabolites and might be more medically effective. The current findings confirm its standardization and validation.

Deciphering the spatial distribution of Gli1-lineage cells in dental, oral, and craniofacial regions

Abstract The craniofacial bone, crucial for protecting brain tissue and supporting facial structure, undergoes continuous remodeling through mesenchymal (MSCs) or skeletal stem cells (SSCs) in their niches. Gli1 is an ideal marker for labeling MSCs and osteoprogenitors in this region, and Gli1-lineage cells are identified as pivotal for bone growth, development, repair, and regeneration. Despite its significance, the distribution of Gli1-lineage cells across the dental, oral, and craniofacial (DOC) regions remains to be systematically explored. Utilizing tissue-clearing and light sheet fluorescence microscopy (LSFM) with a Gli1CreER; tdTomatoAi14 mouse model, we mapped the spatial distribution of Gli1-lineage cells throughout the skull, focusing on calvarial bones, sutures, bone marrow, teeth, periodontium, jaw bones, and the temporomandibular joint (TMJ). We found Gli1-lineage cells widespread in these areas, underscoring their significance in DOC regions. Additionally, we observed their role in repairing calvarial bone defects, providing novel insights into craniofacial biology and stem cell niches and enhancing our understanding of stem cells and their progeny’s behavior in vivo.

Bioinformatic analysis reveals the association between bacterial morphology and antibiotic resistance using light microscopy with deep learning

Although it is well known that the morphology of Gram-negative rods changes on exposure to antibiotics, the morphology of antibiotic-resistant bacteria in the absence of antibiotics has not been widely investigated. Here, we studied the morphologies of 10 antibiotic-resistant strains of Escherichia coli and used bioinformatics tools to classify the resistant cells under light microscopy in the absence of antibiotics. The antibiotic-resistant strains showed differences in morphology from the sensitive parental strain, and the differences were most prominent in the quinolone-and β-lactam-resistant bacteria. A cluster analysis revealed increased proportions of fatter or shorter cells in the antibiotic-resistant strains. A correlation analysis of morphological features and gene expression suggested that genes related to energy metabolism and antibiotic resistance were highly correlated with the morphological characteristics of the resistant strains. Our newly proposed deep learning method for single-cell classification achieved a high level of performance in classifying quinolone-and β-lactam-resistant strains.

On Seedling Morphoanatomy of Asteraceae Campestral Species: Some Adaptative and Taxonomic Considerations

Theoretical Framework: The vegetation of Parana State fields is composed mainly of woody grassy savanna, and one of the largest families in specific richness is Asteraceae. The campestral Asteraceae occurs in three microhabitats as dry fields (steppes “stricto sensu”), rocky outcrops (rupestrian vegetation refuges) and humid fields (higrophyllous steppes). Morphology and structure of Asteraceae seedlings can be useful for species recognition in the field at the juvenile stage, and to understand how seedlings establish themselves in different microhabitats. Objective: The aim of this paper was to analyze the seedling structure of 17 Asteraceae species, and to summarize some considerations on anatomical characters of the seedlings, which may be variations environmentally induced and they have some taxonomic or evolutionary value. Method: Cipselae of 17 species of Asteraceae were collected in the state parks of Paraná, Brazil, and placed to germinate in Petri plates. The seedling development occurred in greenhouse. Root, hypocotyl, cotyledons, epicotyl and eophylls were sectioned in rotation microtome, and analyzed in light microscope. Cotyledons and eophylls were investigated in a scanning electronic microscope. Results: Roots are diarch or tetrarch. Hypocotyl may have the root structure and partially or wholly as transition root-stem structure. Epicotyl shows the stem structure. Cotyledons and eophylls are dorsiventral. Trichome density and amphistomaty are xeromorphic characters that occur in the cotyledons and eophylls of the species. Conclusion: Some structural characters have diagnostic value for separation of investigated species, such as hypocotyl structure, root/stem transition type, epidermis, mesophyll tissues and midrib saliency of the cotyledons and eophylls, and presence of secretory ducts.

Synthesis, Structure, and Light Absorption of Octanoyl Amino Acids

Amino acid lanthanide complexes are a promising class of environmentally friendly and non-toxic optical organic materials that find extensive applications in biomedicine, molecular probes, and optical devices. In this study, three amino acid lanthanide complexes, octanoyl glycine gadolinium (Gd(oct-ala)3×H2O), octanoyl serine gadolinium (Gd(oct-ser)3×H2O), and octanoyl phenylalanine gadolinium (Gd(oct-phe)3×H2O), were synthesized by reacting octanoyl amino acid ligands with gadolinium chloride. The coordination structure of the gadolinium complexes was determined by elemental analysis and infrared spectroscopy. XRD and polarized light microscopy confirmed the amorphous structure of the complexes. DSC and TG-DTA were used to investigate the thermodynamic stability of the gadolinium complexes, and their solubility and UV-visible light absorption properties were also evaluated. Our results demonstrate that all three gadolinium complexes exhibit excellent UV-visible light absorption performance, with the Gd(oct-phe)3×H2O containing a phenyl ring showing the highest light absorption efficiency.

Retinal ischemia-reperfusion injury and pretreatment with Lycium barbarum glycopeptide.

To investigate the antioxidant protective effect of Lycium barbarum glycopeptide (LbGP) pretreatment on retinal ischemia-reperfusion (I/R) injury (RIRI) in rats. RIRI was induced in Sprague Dawley rats through anterior chamber perfusion, and pretreatment involved administering LbGP via gavage for 7d. After 24h of reperfusion, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (CREA) levels, retinal structure, expression of Caspase-3 and Caspase-8, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) in the retina were measured. The pretreatment with LbGP effectively protected the retina and retinal tissue from edema and inflammation in the ganglion cell layer (GCL) and nerve fiber layer (NFL) of rats subjected to RIRI, as shown by light microscopy and optical coherence tomography (OCT). Serum AST was higher in the model group than in the blank group (P=0.042), but no difference was found in ALT, AST, and CREA across the LbGP groups and model group. Caspase-3 expression was higher in the model group than in the blank group (P=0.006), but no difference was found among LbGP groups and the model group. Caspase-8 expression was higher in the model group than in the blank group (P=0.000), and lower in the 400 mg/kg LbGP group than in the model group (P=0.016). SOD activity was lower in the model group than in the blank group (P=0.001), and the decrease was slower in the 400 mg/kg LbGP group than in the model group (P=0.003). MDA content was higher in the model group than in the blank group (P=0.001), and lower in the 400 mg/kg LbGP group than in the model group (P=0.016). The pretreatment with LbGP did not result in any observed liver or renal toxicity in the model. LbGP pretreatment exhibits dose-dependent anti-inflammatory, and antioxidative effects by reducing Caspase-8 expression, preventing declines of SOD activity, and decreasing MDA content in the RIRI rat model.

Low-dose pro-resolving mediators temporally reset the resolution response to microbial inflammation

BackgroundSpecialized pro-resolving mediators (SPMs) promote resolution of inflammation, clear infections and stimulate tissue regeneration. These include resolvins, protectins, and maresins. During self-resolving acute inflammation, SPMs are produced and have key functions activating endogenous resolution response for returning to homeostasis. Herein, we addressed whether infections initiated with ongoing inflammation alter resolution programs, and if low-dose repetitive SPM regimen re-programs the resolution response.MethodsInflammation was initiated with zymosan (1 mg/mouse) followed by E. coli (105 CFU/mouse) infections carried out in murine peritonitis, and exudates collected at 4-72 h. Leukocytes were enumerated using light microscopy, percentages of PMN, monocytes and macrophages were determined using flow cytometry, and resolution indices calculated. Lipid mediators and SPM profiles were established using mass spectrometry-based metabololipidomics. Repetitive dosing with a SPM panel consisting of RvD1, RvD2, RvD5, MaR1 and RvE2 (0.1 ng/mouse each, i.p.) was given to mice, followed by zymosan challenge. Leukocyte composition, resolution indices and RNA-sequencing were carried out for the repetitive SPM treatments.ResultsE. coli infections initiated acute inflammation-resolution programs with temporal SPM production in the infectious exudates. Zymosan-induced inflammation prior to E. coli peritonitis shifted exudate resolution indices and delayed E. coli clearance. Lipid mediator metabololipidomics demonstrated that E. coli infection with ongoing zymosan-induced inflammation shifted the time course of exudate SPMs, activating a SPM cluster that included RvD1, RvD5 and MaR1 during the initiation phase of infectious inflammation (0-4 h); RvD5 and MaR1 were present also in the resolution phase (24-48 h). To emulate daily SPM regimens used in humans, a repetitive subthreshold dosing of the SPM panel RvD1, RvD2, RvD5, MaR1 and RvE2 each at 0.1 ng per mouse was administered. This low-dose SPM regimen accelerated exudate PMN clearance following zymosan-induced inflammation, and shortened the resolution interval by > 70%. These low-dose SPMs regulated genes and pathways related to immune response, chemokine clearance and tissue repair, as demonstrated by using RNA-sequencing.ConclusionsInfections encountered during ongoing inflammation in mice reset the resolution mechanisms of inflammation via SPM clusters. Low-dose SPMs activate innate immune responses and pathways towards the resolution response that can be reprogrammed.

What to Check on Your Light Microscope Before Collecting Images

What to Check on Your Light Microscope Before Collecting Images

A century of andrology in Cell & Tissue Research: looking back while moving forward.

This article commemorates the 100th anniversary of the first issue of Cell & Tissue Research (CTR), the longest-running active journal dedicated to cell biology. Reflecting the significant contributions of spermatology and embryology to the early days of cell biology, the majority of articles in CTR's inaugural issue centered on plant and animal sperm cells. A brief synopsis of these articles provides a launching point for revisiting 100years of research on the male germ cells and fertility in humans and animals and offers a perspective on the current state and future directions of the andrology field. Early technological advances in light and electron microscopy enabled descriptive studies that ushered in the era of mechanistic, biochemistry-based inquiry focused on the understanding of physiological sperm processes such as sperm capacitation, acrosomal exocytosis, and sperm-egg interactions. In the last 20years, progress in flow cytometry, cell imaging, and omics revealed new information on sperm proteome, transcriptome, metabolome, and overall phenome of fertile and infertile spermatozoa. Going back to the journal's roots, recent advances in male germ cell isolation, transplantation, modification, and cryopreservation have been discussed on the pages of CTR. Newest trends such as gene editing and artificial intelligence/machine learning are now making inroads into andrological inquiry and assisted reproductive therapy of male infertility.

Biosystematic studies of genus Withania Pauquy in Egypt

Withania (Solanaceae, Solanoideae) is a widespread genus. Comparative macro-, micro-morphological, anatomical, and molecular features of this genus in Egypt were examined using light and scanning electron microscopy to reassess the conflicted taxonomic relationships between the two studied species. The most significant morphological differences that have been found were: the shape of the lamina, apex, anther, and stigma, and the ratio of calyx tube/lobe; anatomical examination of taxonomic interest are as follows: number of vascular bundles, presence of ears and distribution of accessory vascular bundles in petiole and shape of spongy cells, and number of lower parenchyma in the midrib region of the leaf; trichomes of both species showed no significant differences; pollen, and seed characters are of taxonomic significance in differentiation and characterization between them. Protein profiling revealed that W. somnifera has only conserved proteins, while W. obtusifolia possessed both conserved and additional proteins in their SDS-PAGE banding patterns. Eleven starts codon-targeted (ScoT) primers were applied and produced 96 amplicons with an average of 70.83% polymorphism/primer. W. obtusifolia generated more polymorphic bands and maintained monomorphic ones. SDS-PAGE disclosed that both Withania species were 50% related. While Scot-Dendrogram revealed that both Withania species were poorly related. So, protein and molecular analyses showed considerable genetic variations between these two species.

In Situ Microscopy of Fatigue-Loaded Embedded Transverse Layers of Cross-Ply Laminates: The Role of an Inhomogeneous Fiber Distribution

Composites with continuous fiber reinforcement offer excellent fatigue properties but are tedious to characterize due to anisotropy and the interplay of fatigue properties, processing conditions, and the constituents. The global fiber volume content can affect both monotonic and fatigue strength. This dependence can increase the necessary testing effort even when processing conditions and constituents remain identical. This work presents an in situ edge observation method, enabling light microscopy during loading. As a result, digital image correlation can be employed to study local strains at cracking sites on the scale of fiber bundles. The geometric influence on fatigue damage is examined in non-crimp fabrics of glass and carbon fibers. Two epoxy resins (one modified by irradiation) are investigated to verify the geometric influence under changed polymer properties. The microscopy-based image correlation revealed that damage forms at very low global strains of only 0.2–0.3% in glass fiber-reinforced epoxy laminates. For carbon fiber-reinforced epoxy, laminate cracking was found to emanate mainly from regions containing stitching fibers. Across both reinforcements, irradiation treatment led to delayed cracks, emanating from interfaces. This detailed analysis of the damage formation is used as a basis for proposed applications of the in situ strain information.

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

An archaeological and archaeometric study of Late Punic–Roman Pottery from the Tas-Silġ Sanctuary and the Żejtun Villa, Malta

Abstract This interdisciplinary study contributes to the understanding of the use of raw materials and pottery production techniques in Late Punic–Late Republican Malta, focusing on the Tas-Silġ sanctuary and the Żejtun Villa. Plates, bowls and cooking vessels were described typologically, and their fabrics were characterised using polarised light microscopy and Energy Dispersive X-Ray Fluorescence. The aims were to classify these vessels into integrated and coherent fabric groups based on all analyses, to better understand the local production of vessels and to assess a possible local provenance. Four integrated fabric groups were identified and represent local productions using distinct raw materials or production techniques. These groups can be distinguished typologically, macroscopically, petrographically and chemically. Multivariate techniques, including the chemical analysis of Maltese clays, were produced to enhance the fabric classification and discuss their raw materials. The raw materials identified are consistent with what is known in Maltese geology. One group is distinctive, and the results suggest the possible use of a previously unidentified raw material, Terra Rossa, found over the Upper Coralline Limestone. This new classification provides the basis for further studies of Late Punic–Roman sites in the Maltese islands and the future identification of imports and exports from the Maltese islands.

Morphological aspects of Sticholecitha serpentis (Plagiorchiida: Plagiorchiidae) in a new Viperidae host in the Brazilian Amazon

Introduction: Sticholecitha serpentis is a poorly studied parasite of the snakes’ esophagus, with an unclear taxonomy. Currently, there are few records of S. serpentis in snakes in Brazil. Although it has been recorded in snakes of the family Viperidae, it has not yet been reported in Bothrops atrox. Objectives: To present new morphological characters, as well as ecological and parasitic relationships of S. serpentis with B. atrox. Methods: The samples were obtained in a dryland area in the Tapajós National Forest, Pará State, Brazil. Ten specimens of B. atrox were examined manually with the aid of tweezers, and three were positive for trematodes identified as S. serpentis in the oral cavity. Results: We provide scanning electron microscopy images, with detailed images of the S. serpentis tegument and its spines, as well as details of the dorsal keel using light microscopy methods. Moreover, we report the first record of S. serpentis parasitizing an Amazonian viperid, with infection frequency data of 30 %. Conclusion: We emphasize that the new morphological data presented in this manuscript provide additional information to facilitate the identification of this trematode in future studies.

Intravitreal chlorhexidine for sterilizing the vitreous cavity in an animal model of bacterial endophthalmitis

Purpose To evaluate the efficacy and safety of intravitreal chlorhexidine (CHX) for sterilising the vitreous cavity in bacterial endophthalmitis. Methods For in-vitro experiments, full-thickness retina explants were harvested from freshly enucleated pig eyes. Six-millimeter circular sensory retina patches were then incubated in varying concentrations of CHX (0.625-800 µg/mL) for 24 hours. Retinal cell viability was determined at the end of the incubation period with a live-dead assay. The bactericidal effects of the tested CHX concentrations were determined using a quantitative suspension test on Staphylococcus epidermidis. The safety of CHX was also tested by injecting varying doses of CHX (50-400 µg/mL) into the vitreous cavity of albino rabbits followed by flash electroretinography (ERG) and light microscopy. The bactericidal effect of the non-toxic CHX doses was determined using the rabbit model of endophthalmitis created by injecting 3000 CFU/0.1 mL of Staphylococcus epidermidis. Results In vitro concentrations of CHX greater than 6.25 µgr/mL exerted a bactericidal effect, while concentrations of CHX less than 200 µg/mL did not impair retinal cell viability. Intravitreal concentrations of CHX between 20-100 µg/mL were adequate to sterilise the infected rabbit vitreous cavity in the animal model. No significant functional or anatomical deleterious effect was observed with ERG or light microscopy. Conclusion CHX can sterilise the vitreous cavity in an animal model of bacterial endophthalmitis without impairing retinal cell viability. Our results encourage further research for clinical use of chlorhexidine in treatment of bacterial endophthalmitis.

A cross-sectional analysis identifies a low prevalence of Plasmodium ovale species infections in symptomatic and asymptomatic individuals in Kilifi county, Kenya.

Background The focus on P. falciparum diagnosis has led to an underestimation of the global burden of malaria resulting from neglected Plasmodium species. However, there is still scarce data on the prevalence of P. ovale species (spp) globally. To address this knowledge gap, data collected from cross-sectional studies in Kilifi county were used to: 1) determine the prevalence of P. ovale spp infections; and 2) determine the sensitivity of different diagnostic assays in detecting P. ovale spp infections. Methods A total of 531 individuals were sampled across three study sites in Kilifi County, Kenya between 2009 and 2020. Blood smears were prepared from peripheral blood and screened for Plasmodium parasite stages using light microscopy. Molecular screening involved DNA extraction of dried blood spots and blood in ethylenediaminetetraacetic acid, polymerase chain reaction (PCR) using primers targeting the 18 small ribosomal subunit and sequencing. Results Microscopy screening revealed that the most prevalent species was P. falciparum (32.0%) followed by P. malariae (9.0%) and then P. ovale spp( 1.5%). PCR screening identified additional P. ovale spp positives cases. Overall PCR results indicate that43 (8.1%) out of the 531 individuals harbored P. ovale spp infection with the highest prevalence reported in the tertiary health facility, (14.6%, 95% CI 8-23.6%), followed by the primary health facility (8.3%, 95% CI 5.4-11.9%), and the community from a cross-sectional blood survey, (3.6%, 95% CI 1.2-8.2%). Microscopy screening for P. ovale spp had a low sensitivity of 7% (95% CI 1-19-30%) and a high specificity of 99% (95% CI 98-100%). Sequencing results confirmed the presence of P.ovale curtisi. Conclusions This study provides baseline data for P.ovale spp surveillance in Kilifi County, primarily using PCR to improve diagnosis. These results suggest that malaria elimination and eradication efforts should not only concentrate on P. falciparum but should embrace a holistic approach towards elimination of all Plasmodium spp.

A new species of Craterium (Myxomycetes, Physarales, Physaraceae) with a mottled peridium

A new species of Craterium, described herein as С. guttatum, was recovered in Russia, during intensive field work in the Kaluzhskiye Zaseki Nature Reserve (Kaluga Oblast’) and in the Sudogodskiy District of the Vladimir Oblast’. Morphological details of sporocarps and spores were examined by light and scanning electron microscopy. The new species is characterized by a unique combination of morphological characters, such as a beaker-shaped sporotheca with a mottled peridium and a prominent line of dehiscence separating the upper part of the sporotheca in the form of a convex white lid, as well as spores densely ornamented with small warts grouped in clusters. The partial sequences of the 18S rRNA, EF1α and mtSSU genes of C. guttatum differ significantly from all Craterium species sequences available to date.

Variability in oil droplet numbers and proportions in the retinas of anole lizards

Colored oil droplets are a common feature in the cones of almost all diurnal lizards. Using white light microscopy, three chromotypes of droplet can be identified in anoles – yellow, green and colorless. These droplets have been associated with different cone classes using microspectrophotometry. The principal member of double cones contain a yellow droplet while the accessory member contains a diffuse yellow pigment. Both members contain the LWS visual pigment. One class of large single cone contains a yellow droplet and the LWS visual pigment. The two remaining classes of large single cone contain the green droplet and either the LWS or MWS visual pigment. As such, by noting the distribution and numbers of the different droplet chromotypes, information about cone distribution, type, and number can be deduced microscopically. Retinas from three anole species (A. cristatellus, A. sagrei, and A. carolinensis) were isolated, flattened and oriented using the two foveae as landmarks. A 19-gauge needle was used to punch out six full-thickness retinal disks from identical retinal regions from both eyes of the three species. The different oil droplets were microscopically identified by color and counted. In all three species at all six retinal areas, the colorless droplets associated with the UVS and SWS single cones represented approximately 10% of the total droplets counted. However, the proportions of the yellow and green droplets were significantly different between the species. For A. cristatellus, 80% of the droplets were green while 10% were yellow. For A. sagrei approximately 85% of the droplets were yellow while only 5% were green. For A. carolinensis 50% of the droplets were yellow while 40% were green. The possible significance of these variable proportions is discussed in terms of possible effects on color vision and ecotype of the three anoles.

The yeast Wickerhamomyces anomalus acts as a predator of the olive anthracnose-causing fungi, Colletotrichum nymphaeae, C. godetiae, and C. gloeosporioides

Olive tree anthracnose is caused by infection with Colletotrichum fungi, which in Portugal are mostly C. nymphaeae, C. godetiae, and C. gloeosporioides s.s. Severe economic losses are caused by this disease that would benefit from a greener and more efficient alternative to the present agrochemical methods. Yeasts are serious candidates for pre-harvest/in field biocontrol of fungal infections. This work identified the yeast Wickerhamomyces anomalus as a strong antagonizer of the three fungi and studied in vitro this ability and its associated mechanisms. Antagonism was shown to not depend on the secretion of volatile compounds (VOCs), or siderophores or any other agar-diffusible compound, including hydrolytic enzymes. Rather, it occurred mostly in a cell-to-cell contact dependent manner. This was devised through detailed microscopic assessment of yeast-fungus cocultures. This showed that W. anomalus antagonism of the three Colletotrichum proceeded through (i) the adhesion of yeast cells to the phytopathogen hyphae, (ii) the secretion of a viscous extracellular matrix, and (iii) the emptying of the hyphae. Yeasts ultimately putatively feed on hyphal contents, which is supported by light microscopy observation of MB and PI co-culture-stained samples. Accordingly, numerous W. anomalus cells were observed packing inside C. godetiae emptied hyphae. This behaviour can be considered microbial predation and classified as necrotrophic mycoparasitism, more explicitly in the case of C. godetiae. The results support the prospect of future application of W. anomalus as a living biofungicide/BCA in the preharvest control of olive anthracnose.

Tarhan method in the chemical properties and determinations of carbonatoblastites

Up to now, in geology/earth science literature, carbonatites (carbonatic rocks) have been accepted as magmatic-origin, rarely seen in nature, depth rocks (plutonic). As a result of our scientific studies (field, petrography, mineralogical etc.), it has been definitely determined that carbonatites are not magmatic-origin intrusion, carbonatic dyke, pegmatitic, stock and lens-type depth rocks. On the other hand, carbonatites develop in the regional dynamothermal Tarhan metamorphism cycle, in the second phase, under the changing physical conditions (P/T) of the Abukuma type reversed regional regressive dynamothermal metamorphism facies and sub-facies, where temperatures are effective compared to the developed pressures (T>P, temperatures have left their mark on metamorphism). The previously existing (pre-exiting) pure-impure primary source rock units, carbonate/limestones and primary source rock units develop within pure-impure marbles, which are metamorphic equivalents of Barrow type regional progressive dynamothermal metamorphism, where pressures are effective compared to temperatures in the first phase of the metamorphism cycle (P>T, pressures mark the metamorphism). They were derived from the rock units in question in solid phase and in-situ (autochthonous where they are). Therefore, carbonatites, which have been known as magmatic-originated depth rocks until today, are metamorphic-originated, rootless, metablastic rock series and derivatives, which are a type of new modern metamorphic rocks. It has been determined that they are carbonatoblastite type metablastic rocks with different primary rocks of origin (pure-impure carbonate/limestone). Up to now, carbonatites have been determined by a very expensive method according to O/C isotope ratios. However, carbonatites accepted to be of magmatic origin correspond to carbonatoblastites of metamorphic origin. Therefore, it has been suggested to apply the Tarhan Method, which is much cheaper and more practical in the determination of carbonatoblastites. According to the Tarhan method, petrographic thin sections are made from pure-impure carbonate/limestone, pure-impure limestones and pure-impure carbonatoblastites. In the polarized light microscope, optical signals/marking of single optical axis minerals (uniax minerals) of different carbonate type minerals in petrographic thin sections of different carbonate rocks are determined (quartz, calcite, calcite-dolomite, dolomite, apatite, nepheline, siderite, aragonite, witherite, stronsionite etc.). Single optical axis rock-forming main minerals of different carbonate origin and other rock-forming main single optical axis silicate minerals (quartz, orthoclase, foid, etc.) found together with the minerals in question often give a single optical axis optically positive (+) sign (ne > no). The petrographic thin section giving optically positive (+) sign shows that the rock sample belonging to it is carbonatoblastite (known as rare carbonatites of magmatic origin) type, 3rd generation metamorphic rock. If the main different carbonate minerals with rock-forming minerals and the main silicate minerals with single optical axis rock-forming minerals found together with them mostly give single optical axis optically negative (-) sign (ne < no), the mentioned petrographic thin sections indicate that they belong to limestone/carbonate which are 1st generation pure-impure primary source rocks of sedimentary origin and 2nd generation pure-impure marbles of metamorphic origin. However, the single optical negative (-) sign of different carbonate type minerals of marbles is much more effective than the optically negative (-) signs of different carbonate minerals that form limestones. As a result; Carbonatoblastic rock series and their derivatives/carbonatoblastites (known as carbonatites, which are rare depth rocks of magmatic origin) generally have a positive (+) sign (ne > no) with a single optical axis. In other words, all carbonate-based carbonatoblast and silicate-based rock-forming main-secondary-trace anhydrous, light-colored, expanding cage/structured, tectosilicate/framework crystalloblast neominerals are generally single-axis positive (+) sign. However, the carbonate-dominated rock-forming main carbonate minerals within the vertical and lateral gradual transition zones of carbonatoblastites and marbles (metamixitic metamorphic transition zone) have a single optical axis with optically negative-positive (±) and optically positive-negative (±) signs.