Mature Colonies Research Articles

Quantifying the fractal complexity of nutrient transport channels in Escherichia coli biofilms under varying cell shape and growth environment.

Recent mesoscopic characterization of nutrient-transporting channels in Escherichia coli has allowed the identification and measurement of individual channels in whole mature colony biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of colony biofilms formed by cell shape mutants of E. coli shows that channels have high fractal complexity, regardless of cell phenotype or growth medium. In particular, colony biofilms formed by the mutant strain ΔompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentrations in the medium. Osmotic stress leads to a dramatic reduction in the ΔompR cell size but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli colony biofilms.

Agreement between AST from minute colony (8-10h growth) and mature colony (16-18h/overnight incubation)

Agreement between AST from minute colony (8-10h growth) and mature colony (16-18h/overnight incubation)

The impact of methylparaben and chlorine on the architecture of Stenotrophomonas maltophilia biofilms

The biofilm architecture is significantly influenced by external environmental conditions. Biofilms grown on drinking water distribution systems (DWDS) are exposed to environmental contaminants, including parabens, and disinfection strategies, such as chlorine. Although changes in biofilm density and culturability from chemical exposure are widely reported, little is known about the effects of parabens and chlorine on biofilm morphology and architecture. This is the first study evaluating architectural changes in Stenotrophomonas maltophilia colony biofilms (representatives of bacterial communities presented in DWDS) induced by the exposure to methylparaben (MP) at environmental (15 μg/L) and in-use (15 mg/L) concentrations, and chlorine at 5 mg/L, using widefield epi-fluorescence mesoscopy with Mesolens. The GFP fluorescence of colony biofilms allowed the visualization of internal structures and Nile Red fluorescence permitted the inspection of the distribution of lipids. Our data show that exposure to MP triggers physiological and morphological adaptation in mature colony biofilms by increasing the complexity of internal structures, which may confer protection to embedded cells from external chemical molecules. These architectural modifications include changes in lipid distribution as an adaptive response to MP exposure. Although chlorine exposure affected colony biofilm diameter and architecture, the colony roundness was completely affected by the simultaneous presence of MP and chlorine. This work is pioneer in using Mesolens to highlight the risks of exposure to emerging environmental contaminants (MP), by affecting the architecture of biofilms formed by drinking water (DW) bacteria, even when combined with routine disinfection strategies.

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.

Evaluating Linear Coral Growth Estimation Using Photogrammetry and Alternative Point Cloud Comparison Methods

Abstract. Corals are critical reef-building organisms, providing essential habitat and ecosystem services. Tracking coral growth over time indicates coral reef health, which can be measured using various established techniques. Several coral growth-related studies have successfully applied photogrammetry to a particular coral of various types. While the focus of previous work was on standardised data processing and, to a certain degree, on the assessment of different point cloud comparison methods (Lange et al. 2022), little attention has been given to the impact of camera calibration. This study measured the annual linear extension of five Acropora spp. colonies using photogrammetry and evaluated all stages of imagery processing. A high focus was given to the analysis of the camera calibration method and the validation of camera parameters derived using an in-situ calibration of coral images with scale bars placed in the camera's field of view. We demonstrate that this method is as reliable as the calibration using a calibration frame. This study also examined the impact of the different point cloud comparison methods for Acropora spp. More specifically, the derived point clouds are compared by applying the point-to-point and point-to-model methods and manually selecting 12 coral branch tips. Histograms derived from the comparison methods were analysed and deemed a suitable and efficient alternative approach for measuring the maximum growth rate of mature colonies over shorter time periods (1 year or less).

Virulence Bioassay of Entomopathogenic Fungi against Adults of Atta mexicana under Controlled Conditions

Leafcutter ants (Atta spp.) are one of the mos t economically harmful pests in agriculture, considered dominant in the Neotropics and South America. Mature colonies of A. mexicana have a great economic impact on Mexico’s agriculture. Microbial agents in the form of biopesticides are an effective component of integrated pest management (IPM) strategies and may present a better alternative to synthetic insecticides. Among the fungi most used as biological insecticides there are Beauveria bassiana and Metarhizium anisopliae. The objective of this research was to evaluate the effect of the entomopathogenic fungi B. bassiana and M. anisopliae of commercial origin and a native strain of B. bassiana from México (MA-Bb1) on adults of Atta mexicana under controlled conditions. In the bioassay, five formulations and a control group were tested (B. bassiana MA-Bb1, B. bassiana MA-Bb1+ Diatomin®, B. bassiana®, M. anisopliae®, Diatomin®, and Tween 80 (0.01%). The MA-Bb1+ Diatomin® biopreparation induced the highest mortality (100%) in four-week-old A. mexicana, followed by the MA-Bb1, M. anisopliae®, and B. bassiana® biopreparations, which caused mortality of 83.33%, 73.98%, and 68.70%, respectively. Treatments containing B. bassiana and M. anisopliae were efficient in controlling A. mexicana under controlled conditions. The most efficient biological control was achieved with the B. bassiana fungus and Diatomin®, which presented the highest total death rate in A. mexicana 96 h post infection, in contrast to the control group (Tween 80), which attained the lowest speed of death in the present investigation.

Antimicrobial and antibiofilm activities of Inga cylindrica trypsin inhibitor.

Inga cylindrica, a tropical fruit tree of the Fabaceae family (subfamily Mimosoideae), is native to South America. The seeds from this family are an essential source of trypsin inhibitors, which display promising bioactivity for increasing host defense against pathogens. The aim of the present study was to characterize the antimicrobial and antibiofilm activities of the trypsin inhibitor extracted from I. cylindrica seeds, ICTI. ICTI demonstrated antifungal activity with a minimum inhibitory concentration (MIC) of 32.11 μmol.L-1, and a minimum fungicidal concentration (MFC) of 32.1 μmol.L-1, against Cryptococcus gattii, Candida albicans, Candida glabrata and Candida guilliermondii. Combining ICTI with Amphotericin B had a significant synergistic effect, reducing the concentration of the antibiotic by 75% for C. albicans and 94% for C. gatti. The significant increase (16 x) in activity observed with ergosterol (1.01 mol.L-1) for C. albicans and C. gatti, and the lack of activity against bacterial strains, suggests that ICTI interferes with the integrity of the fungal cell membrane. Treatment with ICTI at 10 x MIC resulted in a 51% reduction in biofilm formation and a 56% decrease in mature biofilm colonies for C. albicans. Finally, ICTI displayed no toxicity in the in vivo Galleria mellonella model. Given its antifungal and antibiofilm properties, ICTI could be a promising candidate for the development of new antimicrobial drug prototypes.

Social administration of juvenile hormone to larvae increases body size and nutritional needs for pupation.

Social insects often display extreme variation in body size and morphology within the same colony. In many species, adult morphology is socially regulated by workers during larval development. While larval nutrition may play a role in this regulation, it is often difficult to identify precisely what larvae receive from rearing workers, especially when larvae are fed through social regurgitation. Across insects, juvenile hormone is a major regulator of development. In the ant Camponotus floridanus, this hormone is present in the socially regurgitated fluid of workers. We investigated the role the social transfer of juvenile hormone in the social regulation of development. To do this, we administered an artificial regurgitate to larvae through a newly developed handfeeding method that was or was not supplemented with juvenile hormone. Orally administered juvenile hormone increased the nutritional needs of larvae, allowing them to reach a larger size at pupation. Instead of causing them to grow faster, the juvenile hormone treatment extended larval developmental time, allowing them to accumulate resources over a longer period. Handfeeding ant larvae with juvenile hormone resulted in larger adult workers after metamorphosis, suggesting a role for socially transferred juvenile hormone in the colony-level regulation of worker size over colony maturation.

Does the distance between the nest and the food source affect the foraging behavior of Nasutitermes corniger (Termitidae)?

Research on food finding by pest termites can be used to inform the development of techniques to control their population; however, there is a paucity of information available on the foraging behavior of Nasutitermes corniger, an urban pest in South America. In the present study, we analyzed the effect of the distance between the nest and food on the exploration and recruitment of N. corniger during foraging behavior under laboratory conditions. Nests containing mature colonies were collected in the field and placed in a glass cube connected to a test arena (50.0 × 40.0 cm) in which Eucalyptus grandis blocks were supplied at three different distances: 10, 20 and 30 cm. In each test, the occurrence of the following events were recorded: initial exploitation, initial recruitment, and mass worker recruitment. Individuals in the blocks were counted at the end of each test and divided into the total number of recruited termites, recruited workers, consuming workers and recruited soldiers. Each test lasted 60 minutes and was repeated with 20 colonies. Nasutitermes corniger foragers showed the three behavioral events of interest at all three distances. The occurrences of initial exploitation and initial recruitment, the latency of the three events and the number of foragers were not affected by the distance between the nest and food. The occurrence of mass worker recruitment was the only event affected by this distance, with higher recruitment at shorter distances.

First report of Curvularia geniculata causing leave spot disease of Bletilla striata in Zhejiang of China.

Bletilla striata, a perennial herbaceous plant belonging to the family Orchidaceae, is native to China and is widely distributed in the Yangtze River basin. In China, B. striata is a popular medicinal plant that is typically used to reduce wound bleeding and inflammation. In September 2021, distinct leaf spot symptoms were observed in more than 50% of B. striata plants in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. Small, round, pale brown, necrotic spots were first observed on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins and eventually enlarged to 5-8 mm on the leaves. Over time, the small spots enlarged and coalesced into necrotic streaks (1-2 cm). Leaves with symptoms of disease were cut, surface-sterilized, and plated on potato dextrose agar (PDA). Fungal colonies (28×28 mm) with grayish-black mycelia from all tissues were produced after 3 days of incubation at 26 °C. The mature colonies eventually turned black in the center, with obvious rings appearing after 10 days of culture. Basal conidia ranged from pale to dark brown, whereas apical ones were pale brown, with central cells being larger and darker than basal cells. Conidia were smooth and either fusiform, cylindrical, or slightly curved with rounded tips. They ranged in length from 22.34 to 36.82 (mean = 28.63) μm with 2-4 septations and slight septal constrictions. Monospore isolation was performed to obtain a pure culture. Strain BJ2Y5 was subsequently stored in the strain Preservation Center of Wuhan University (Wuhan, China) and the strain preservation number CCTCC M 2023123 was obtained. Fresh mycelia and conidia that grew at 26 ℃ for 7 days were collected from PDA plates. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China). The phylogenetic position of isolate BJ2-Y5 was clarified based on DNA sequence analysis of three loci, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Berbee et al. 1999), the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of the second largest subunit of RNA polymerase II (RPB2; O'Donnell et al. 2007). A BLAST search (GenBank accession nos. OP913168, OP743380, and OP913171) showed 99% homology to the reference isolate CBS 220.52. Based on the morphological and molecular information presented in this study these isolates were identified as C. geniculata (Hosokawa et al. 2003). Furthermore, we evaluated the pathogenicity of B. striata leaves by smearing a conidial suspension (106 conidia/mL) on both sides of leaves with and without wounds. Five inoculated and three non-inoculated leaves (smeared with sterile distilled water as a negative control) were kept in a greenhouse at 26 °C under natural sunlight and covered with plastic bags for 72 h to maintain humidity. After 7 days, small round spots appeared on the wounds. Fifteen days later, the symptoms of disease on the wounded inoculated leaves were similar to those observed in the original sample, whereas the control plants remained healthy. No symptoms of infection were observed in unwounded inoculated leaves. C. geniculata was successfully re-isolated from all five inoculated leaves and was confirmed based on Koch's postulates. To the best of our knowledge, C. geniculata infection has not been previously reported in B. striata.

Analysis of the emerging physical network in young mycelia

Filamentous fungi develop intricate hyphal networks that support mycelial foraging and transport of resources. These networks have been analyzed recently using graph theory, enabling the development of models that seek to predict functional traits. However, attention has focused mainly on mature colonies. Here, we report the extraction and analysis of the graph corresponding to Trichoderma atroviride mycelia only a few hours after conidia germination. To extract the graph for a given mycelium, a mosaic conformed of multiple bright-field, optical microscopy images is digitally processed using freely available software. The resulting graphs are characterized in terms of number of nodes and edges, average edge length, total mycelium length, hyphal growth unit, maximum edge length and mycelium diameter, for colonies between 8 h and 14 h after conidium germination. Our results show that the emerging hyphal network grows first by hyphal elongation and branching, and then it transitions to a stage where hyphal-hyphal interactions become significant. As a tangled hyphal network develops with decreasing hyphal mean length, the mycelium maintains long (∼2 mm) hyphae—a behavior that suggests a combination of aggregated and dispersed architectures to support foraging. Lastly, analysis of early network development in Podospora anserina reveals striking similarity with T. atroviride, suggesting common mechanisms during initial colony formation in filamentous fungi.

Coming of age: Annual onset of coral reproduction is determined by age rather than size

Constraints on organisms possessing a unitary body plan appear almost absent from colonial organisms. Like unitary organisms, however, coral colonies seemingly delay reproduction until reaching a critical size. Elucidating ontogenetic processes, such as puberty and aging are complicated by corals' modular design, where partial mortality and fragmentation lead to distortions in colony size-age relationships. We explored these enigmatic relations and their influence on reproduction by fragmenting sexually mature colonies of five coral species into sizes below the known size at first reproduction, nurturing them for prolonged periods, and examining their reproductive capacity and trade-offs between growth rates and reproductive investment. Most fragments were reproductive regardless of their size, and growth rates hardly affected reproduction. Our findings suggest that once the ontogenetic milestone of puberty is reached, corals retain reproductive capacity irrespective of colony size, highlighting the key role that aging may have in colonial animals, which are commonly considered non-aging.

Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling

BackgroundIn the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the “amoeboid angiogenesis” in the absence of proteases activity.MethodsWe first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies.ResultsOur data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn’t boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF.ConclusionsWe underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

EDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin-antitoxin system.

In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin-antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment.

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene

BackgroundAstrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections.ResultsTo establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36–33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest “MPN survival” [5.5 ± 5.9% (0.12–12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES.ConclusionsThe optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Post-embryonic development of the female reproductive system of the Neotropical termite Silvestritermes euamignathus (Isoptera: Termitidae: Syntermitinae)

In termites, the maturation of the female reproductive system is progressive following post-embryonic development. In Silvestritermes euamignathus, the imaginal line is characterized by five nymphal instars that develop into the imago, but it can deviate in some instars to neotenic reproductives. In order to understand the rate of development of the female reproductive system throughout post-embryonic development, we analyzed the morphology of the reproductive system of nymphs, characterizing the stage of development and comparing it with neotenics and primary queens. We also followed embryonic development and compared eggs from neotenics and primary queens from incipient and mature colonies. Our results showed that gonadal development follows the post-embryonic instars, and previtellogenic oocytes are present in third-instar nymphs and are retained for the next two successive nymphal instars. The full maturation of the ovaries with vitellogenic oocytes requires molting to either imagos or neotenics. Eggs from neotenics follow the same embryonic development and each stage presents similar volume when compared to those of mature primary queens. Eggs of primary queens from incipient colonies are greater in volume than those of mature primary queens and of neotenics, suggesting an investment in egg quality rather than quantity during colony foundation.

Eusociality and the transition from biparental to alloparental care in termites

AbstractIn eusocial organisms, cooperative brood care within a colony represents a situation where the ancestral parental care duties have shifted away from the reproductive parent(s) towards their offspring. The shift to alloparental care was often instrumental in the initial emergence of eusociality, as it ultimately contributed to the establishment of the reproductive division of labour.Remarkably, eusocial taxa such as ants and termites, which still display an ancestral independent colony foundation phase, must go through an obligatory parental care period, as a temporary subsocial family unit. In termites specifically, an incipient colony inherently remains a woodroach family unit until alloparental care is established. Colony foundation success can then be limited by a series of factors that may include environmental, behavioural, symbiotic and physiological constraints.In this study, 450 incipient termite colonies (Coptotermes gestroi) were established to investigate the timing of physiological changes in founders during the transition from biparental to alloparental care. Results showed that the finite initial internal nutritional resources that alates carry during the dispersal flight are a primary limiting factor for successful colony establishment. TheCoptotermesqueen and king must rapidly establish (<150 days) their first cohort of offspring to reach alloparental care or simply run out of resources and die. Alates, therefore, carry just enough internal resources to produce the first few alloparents (< 15 workers) to prime the system towards colony ergonomic growth, with a definitive shift to solely reproductive functions.Eusocial insect primary reproductive traits were optimized for three successive functions within the life cycle of a colony: alate dispersal (sexual reproduction), colony foundation (parental care) and colony growth (increased egg production towards colony maturity). However, results suggest that trade‐offs involving these functions appear to primarily favour dispersal ones (quantity vs. quality of alates), as founder(s) carry minimal resources and have no room for parental care inefficiency and as they then fully rely on their alloparents for further reproductive output.The transition towards alloparental care during colony foundation of eusocial insects may, therefore, reflect on the initial evolutionary transition from ancestral subsociality to eusociality.Read the freePlain Language Summaryfor this article on the Journal blog.

P506 First case of cutaneous mycosis caused by Nannizziopsis sp. in Chelonia

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe present report characterized a case of severe superficial and deep mycoses in a yellow-bellied slider (Trachemys scripta scripta) caused by Nannizziopsis sp. detected by histopathology and fungal culture. A female, 4-year-old, yellow-bellied slider (Trachemys scripta scripta) was presented for ulcerative and proliferative, chronic, skin lesions. White to pink, slightly round, with fuzzy borders, 0.5-2.5 cm in diameter, multifocal, proliferative, and ulcerative lesions were evident mainly on the left forelimb, right forelimb, pericloacal, chin, and oral cavity. The left forelimb was severely affected by a nodular subcutaneous mass.Several courses of disinfectant therapy, antibiotic, and antifungal treatments were given with no clinical improvements.Cytological smears of the ulcerative skin lesion revealed macrophages, heterophils, and fragments of septate hyphae. To investigate the cause of the lesions, the left forelimb was excised, fixed in 10% neutral buffered formalin, and submitted for histopathological examination. Biopsies and swabs from left forelimb, right forelimb, chin, and oral lesions were submitted for microbiological analysis.Samples were cultured on Sabouraud Dextrose Agar (SDA) and incubated at 30°C for 10 days. Histopathological examination showed severe pyogranulomatous dermatitis and myositis. Numerous septate, irregularly branched hyphae were evident within the pyogranulomas. Grocott methenamine silver stain (GMS) and Periodic acid-Schiff (PAS) confirmed the presence of filamentous fungi in the examined sections.Pure cultures of the same fungal strain were obtained from all the samples. Mature colonies on SDA showed a powdery aspect and a white felted surface. Microscopically, the hyphae were straight and branched, and conidia were clavate, sessile, or on short stalks.Based on both macroscopic and microscopic features the fungal strain was identified as Nannizziopsis sp.Susceptibility profile of the fungal isolate was tested for griseofulvin, miconazole, fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole. Amplification and sequencing of ITS, 18S, and SSU gene regions have been performed to identify the fungal isolate. The sequences were aligned with the most similar sequences of established species deposited in GenBank in order to define levels of relatedness. Sequences showed that the isolate clustered with Nannizziopsiaceae. The sequence similarities of the isolate were 99% to N. arthrosporioides in ITS (535/543 bps), 99% to N. vriesii in 18S, and SSU (1648/1649 and 970/973 bps, respectively).Based on the histopathological, microbiological, and molecular analysis, Nannizziopsis sp. was the primary cause of severe deep mycoses in the present turtle. Mycoses caused by Chrysosporium-related fungi are an emerging infectious disease affecting reptiles. The fungi of this group were collectively identified as Chrysosporium anamorph of Nannizziopsis vriesii (CAVN) complex by their morphological characteristics. Recent molecular taxonomy studies have now re-classified Chrysosporium-related fungi into three genera: Nannizziopsis, Paranannizziopsis, and Ophidiomyces.Until now, nine species have been isolated and identified from reptiles. Dermatomycosis associated with Nannizziopsis spp. is frequently reported in lizards, terrestrial, and aquatic snakes, and crocodiles, with classical cutaneous ulcerative lesions. Cutaneous mycosis is common in turtles, however, to our knowledge this is the first case of Nannizziopsis cutaneous mycosis in Chelonia.

Stochastic diffusion characterises early colony formation in Mediterranean coral Corallium rubrum

The colony formation in Mediterranean coral Corallium rubrum is initiated by a larva that metamorphoses into the first polyp of the emerging colony approximately two weeks after settlement. The primary polyp then sets up a slow process that eventually, at least after a few years, gives rise to a tree-like rigid colony structure on which other polyps flourish. For a mature colony, this axial skeleton provides support for new polyps. However, the first emergence of the characteristic axial skeleton can take two years or more from the larva stage. The early colony morphology, instead, is shaped exclusively by the polyps’ abundant deposition of sclerites, a magnesian calcite biomineral that has a different granularity from the distinctive red-coloured skeleton. With the appearance of the first polyp, a growing sclerite heap in a mesoglea layer provides a base for the emerging colony. In this paper, to elucidate the mechanical processes of early skeleton development in C. rubrum colonies, we present a computational model whereby the mesoglea layer provides a diffusion medium for the sclerites that the polyps deposit. We show that our stochastic model with three parameters captures the dynamic variability observed in measurements on living colonies. Our simulation results provide evidence for a diffusion process whereby the interplay between polyp budding and sclerite deposition are the main determinants of structure in early colony formation. Our model demonstrates that the frequency of budding events in an early colony can be described as a function of the available mesoglea surface whereas the number of polyps on the colony plays a secondary role in determining this frequency. We show that these model predictions are confirmed by direct observations on the colonies in our sample. Moreover, our results indicate that diffusion is a prevalent mechanism of colony development also at later stages of a colony’s life span.

First Report of Brown Leaf Spot of Rice Caused by Curvularia hawaiiensis in the United States.

Multiple diseases, including brown spot (Cochliobolus miyabeanus), leaf spot (Epicoccum sorghimum), and blast (Magnaporthe oryzae), can cause spot-like symptoms on the leaves of rice. In July 2021, a disease showing symptoms like brown spot was observed in an 8-hectare field of rice, with disease incidence of >30%, in Beaumont, Texas. Lesions started as small pinhead-size blackish spots on leaf tips or from the edges of leaf blades. The spots enlarged to become irregular (most) or oval brown spots with a slight chlorotic halo. Diseased leaves were collected, washed in running tap water and cut into small pieces. Pieces of the tissue were surface sterilized with 1%NaOCl for 2 min followed by 75% ethanol for 30 s and then washed in sterile distilled water three times with each time lasting for 1 min. The disinfected tissue pieces were air dried, placed on potato dextrose agar (PDA) medium and incubated at 25℃. Initially fungal colonies were hairy in texture with light dark brown center and whitish edge and dark brown pigmentation at the reverse side. Mature colonies turned to black in the center and dark brown toward the edge, with black at the reverse side after 2 or more weeks of incubation. Conidia were oval to narrowly oblong, rounded at the ends, with 2 to 6 distoseptate, and 15 to 35 × 6 to 10 µm in size. These morphological characteristics were similar to those described for Curvularia hawaiiensis (Aslam et al. 2019; Ellis 1971; Kusai et al. 2015). For molecular identification, DNA was extracted and the two different rRNA regions internal transcribed spacer (ITS) and large subunit (LSU), and the two genes RNA Polymerase II (RPB1) and translation elongation factor 1 alpha (EF1) of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 200), and RPB1 and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OK397200) was 98.27% identical to C. hawaiiensis (KP131943); the EF1 sequence (OK492159) was 99.78% identical to C. hawaiiensis (KC503942); the LSU sequence (OK397295) was 98.96% identical to multiple C. hawaiiensis (MN055715, MH160813, MH875853, etc.); the RPB1 sequence (OK492160) was 97.41% identical to C. hawaiiensis (JN992363). To evaluate pathogenicity, three rice plants (cv. Presidio) at the 3-leaf stage were spray inoculated with a conidial suspension of 1 x 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 25 to 30℃. After 2 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants while no symptoms developed on the control plants. The same fungus was consistently re-isolated from the diseased plants. The pathogenicity test was conducted three times with similar results. To our knowledge, this is the first report of brown leaf spot caused by C. hawaiiensis in rice in the United States. Curvularia species are frequently associated with rice grain and cause blackish discoloration symptoms on grain kernels. Recently, however, C. hawaiiensis has also been reported to cause brown leaf spot in Malaysia (Kusai et al. 2015) and Pakistan (Aslam et al. 2019). This research will help identify this disease from other leaf spot-like diseases and develop effective management strategies.