Individual Spores Research Articles

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Sphaerocarpos ibericus sp. nov. (Sphaerocarpaceae), a new European liverwort species that includes S. stipitatus auct. eur. non Lindenb.

ABSTRACT Introduction Specimens of Sphaerocarpos collected during 2022 from the central Iberian Peninsula did not match any previously known species of this genus. Examination of other herbarium material revealed three identical specimens from three different localities in Spain. These were also morphologically identical to Portuguese specimens previously identified as S. stipitatus, a southern hemisphere and Himalayan species that in fact is different from these Portuguese plants. Methods The morphology of the newly collected specimens was compared with that of the known European species plus descriptions of South African plants of Sphaerocarpos stipitatus. A phylogenetic analysis based on the nuclear gene 26S and plastid psbA and rbcL regions was performed to investigate its relationships in the genus. Key results and conclusions Based on morphological and molecular data, we describe the plant as a new species of bryophyte, Sphaerocarpos ibericus M.Infante, Luceño, Quirós-de-la-Peña, Míguez, J.Muñoz & Heras, hitherto an Iberian endemic. It is diagnosed by having cylindrical female involucres with a partially bistratose wall whose inner cells are finger-like and arranged in more-or-less definite ribs in the medial part of the involucre, and the distal part of its spores covered with complete areolae larger at the centre of the spores than on their sides, gradually becoming incomplete and with the remaining lamellae transforming into ridges towards the line where the individual spores of the tetrad are joined. According to published illustrations, Portuguese specimens identified as S. stipitatus pertain indeed to the new species, and therefore S. stipitatus auct. eur. non Lindenb. should be removed from the European liverwort flora.

Elaborate Report of Allium sativum Clove Brown Rot Caused by Fusarium solani in China.

In September 2023, brown rot disease was observed on cloves of garlic (Allium sativum) variety "Zipi-1" (purple skin) collected from Jinxiang County, China during scientific research at the Beijing Academy of Agricultural and Forestry Sciences after being planted in nutrient soil for approximately 2 weeks in a growth chamber maintaining 22℃, 60% relative humidity, and 16 hours of light. Out of the 90 garlic cloves investigated, 18 showed signs of decay, characterized by a brown color and rot, with the surface covered in blue and white mold layers. Six symptomatic cloves were collected for isolating the pathogen using the method described in a previous study (Wu et al. 2021). After 2 d of incubation, individual spores were harvested from the fungal colonies and recultured. Single-spore cultures growing on PDA medium appeared white and flocculent when viewed from the top, and yellowish-brown when viewed from the bottom. After 5 d of cultivation, the colonies had a diameter of approximately 5.8 cm and microscopic examination revealed that the mycelium had a diameter of about 9-13 µm (Fig. S1a, b and c). Isolate As1 produced three types of spores: oval-shaped chlamydospores with a diameter of approximately 6 µm, while spindle-shaped microconidia and sickle-shaped macroconidia measuring approximately 6-7 × 20-30 µm and 8 × 50 µm, respectively (Fig. S1d and e). The mycelial characteristics and reproductive structures of the isolates fit the morphological description of Fusarium solani (Xie et al. 2022). To confirm the identification, TEF1, RPB1 and RPB2 regions of the genome were amplified from three separate isolates (As1, As2, and As3) using EF1/EF2, RPB1-Fa/G2R, RPB2-5F2/7cR, and RPB2-7cF/11aR primer pairs (O'Donnell et al. 2022). The results indicated that the sequences of the three isolates were completely identical. Furthermore, the BLASTn comparison results of the TEF1 (OR916018, 710bp, 100%), RPB1 (OR916019, 1797bp, 99.8%), and RPB2 (OR916020, 1874bp, 100%) sequences in the FUSARIUM-ID v.3.0 database revealed that As1 was identified as F. solani species complex 5 (O'Donnell et al. 2022). To assess the pathogenicity of As1, the surface of healthy garlic cloves (n = 30) was spread with 106 microconidia/mL As1 suspension, while a control group (n = 30) was inoculated with sterile water. All inoculated cloves were placed in an artificial climate chamber under same conditions described above. After 10 d, all inoculated cloves exhibited rot symptoms consistent with those of the initially infected cloves identified in September 2023, while the control plant cloves remained asymptomatic (Fig. S2). Based on morphological and molecular characters (TEF1, RPB1, and RPB2), the reisolated pathogen from diseased plants was identical to the As1 isolate used for inoculation, and the disease assays were repeated twice. Fusarium spp. has been reported as the causal agent of garlic rot disease in several countries such as Mexico, America, and Russia (Gálvez and Palmero 2022). Tai (1979) previously published a report on the presence of F. solani in garlic; however, the content in the book is rather basic, lacking detailed information on the isolation, identification, and the potential for causing garlic diseases, whether postharvest or during growth. Our research can be considered a supplement and improvement to the study by Tai (1979) and lays the groundwork for future studies on management strategies to combat plant diseases caused by F. solani.

Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Influence of Temperature, Light, and H2O2 Concentration on Microbial Spore Inactivation: In‐Situ Raman Spectroscopy Combined with Optical Trapping

To gain insight on chemical sterilization processes, the influence of temperature (up to 70 °C), intense green light, and hydrogen peroxide (H2O2) concentration (up to 30% in aqueous solution) on microbial spore inactivation is evaluated by in‐situ Raman spectroscopy with an optical trap. Bacillus atrophaeus is utilized as a model organism. Individual spores are isolated and their chemical makeup is monitored under dynamically changing conditions (temperature, light, and H2O2 concentration) to mimic industrially relevant process parameters for sterilization in the field of aseptic food processing. While isolated spores in water are highly stable, even at elevated temperatures of 70 °C, exposure to H2O2 leads to a loss of spore integrity characterized by the release of the key spore biomarker dipicolinic acid (DPA) in a concentration‐dependent manner, which indicates damage to the inner membrane of the spore. Intensive light or heat, both of which accelerate the decomposition of H2O2 into reactive oxygen species (ROS), drastically shorten the spore lifetime, suggesting the formation of ROS as a rate‐limiting step during sterilization. It is concluded that Raman spectroscopy can deliver mechanistic insight into the mode of action of H2O2‐based sterilization and reveal the individual contributions of different sterilization methods acting in tandem.

Arbuscular Mycorrhizal Fungi (AMF) diversity and population in UPV Nature Trail, Miagao, Iloilo, Philippines

Abstract. De Asis EPT, Demetillo FMM, Paguntalan DP. 2023. Arbuscular Mycorrhizal Fungi (AMF) diversity and population in UPV Nature Trail, Miagao, Iloilo, Philippines. Biodiversitas 24: 6692-6703. Arbuscular Mycorrhizal Fungi (AMF) are soil microorganisms that form the most prevalent mutualistic associations with plant species. Studies revealed its global distribution in various terrestrial habitats, but available local literature remains few. This study investigated AMF diversity in the University of the Philippines Visayas (UPV) Nature Trail, Miagao, Iloilo, Philippines. AMF spores were isolated using wet sieving, decanting, and centrifugation. A total of 2815 individual spores were documented, with 114 distinct morphospecies representing 16 genera, nine families, and four orders classified. Glomus clarum, Acaulospora sp.7, and Scutellospora pellucida tallied the highest abundance across all points. Scutellospora pellucida (FOC=83.3%) recorded the highest species richness, followed by Ambispora sp. (FOC=77.78%), then Acaulospora mellea and Glomus australe (FOC=72.22%). Overall, Shannon-Weiner diversity index (H=3.6019) and Simpson diversity index (D=0.0420) revealed a very high AMF diversity in the trail, while the Pielou evenness index (J=0.7605) suggested a semi-balanced number of individuals among each morphospecies. A more abundant, even, and diverse, albeit not statistically significant, AMF community was recorded in forested areas compared to non-forested areas of the trail. Results generally suggest that the AMF community is diverse and abundant regardless of the location, ecosystem type, and faunal association.

Spore variability in Hepaticae: a case study on four short-lived Riccia L. species

ABSTRACT Introduction In the present study, we tested the variability of spore morphology in freshly collected plants of Riccia bifurca Hoffm., R. glauca L., R. sorocarpa Bisch. and R. warnstorfii Limpr. ex Warnst. at the individual and population scale, and examined the taxonomical power of distinct characters that are used in the literature for species delimitation in the genus Riccia. Methods Spore size, number of areolae across the distal spore side, and wing width were determined from permanent preparations of spores, using three individuals of each of the four tested species. Statistical analyses, including power analyses, were carried out, in addition to evaluation using light microscopy images. Key results Our results suggest that individual spore characteristics are subject to high variability at the individual and population level. Owing to the high dispersion of the collected data, the boundaries for these characteristics are obscured across different species. Conclusions Generally, although the specific morphology of the spores does differ across Riccia species, several characters are too strongly specified in the literature and should be used only in a statistical context and in combination with additional, often qualitative, characters.

First Report of Fusarium tricinctum Causing Root Rot on Chinese dwarf cherry (Cerasus humilis) in China.

Chinese dwarf cherry (Cerasus humilis) is a perennial small shrub indigenous to northern China, highly regarded for its calcium-rich fruit known as 'calcium fruit'. These fruits have a remarkable capacity to aid in human calcium absorption. Additionally, they contain beneficial flavonoids that hold promise for applications in the healthcare industry (Wang et al., 2018). In July 2020, a concerning development occurred on the farms in Jingtai County (37.48o N, 103.82o E), Baiyin City, Gansu Province in China. Approximately 20 to 30% of C. humilis at the full culture stage exhibited symptoms of root rot, including brownish leaves, rotten roots, and plant mortality, leading to a decrease of over 30% in calcium fruit yield. To identify the pathogen, the surface of symptomatic roots was sterilized with 3% NaOCl for 2 minutes, followed by 70% ethanol for 30 seconds, and rinsed three times with sterile water. Tissue sections (5×5mm) from the margin of the necrotic lesion were cut and cultured on potato dextrose agar (PDA) medium, and incubated for 7 days at 25℃. Five pure culture isolates were obtained from individual spores. Initially, the isolates exhibited abundant white aerial mycelia that turned light pink on the third day. Macroconidia were falciform, two to five septate, straight or slightly curved, and measuring 20.1 to 32.5×2.2 to 3.8 μm (n=50). Napiform microconidia were oval-ellipsoid, non-septate, and measuring 6.2 to 9.3×4.2 to 5.8 μm (n=50). Based on these morphological characteristics, the fungus was tentatively identified as Fusarium species (Leslie and Summerell, 2006). To confirm the identification, the internal transcribed spacer region (ITS) and the translation elongation factor (EF1α) of the isolate CH-2 were partially amplified and sequenced using the primers ITS1/ITS4 and EF2T/EF3 (Li et al., 2013, Yang et al., 2022). Upon comparison with the sequences in GenBank, 857 bp ITS sequence showed 100% homology to Fusarium tricinctum isolate QY3-1 (GenBank accessions no. MZ572963.1), and 665 bp EF1α sequence showed 99.7% homology to F. tricinctum strain TQC-C2 (GenBank accessions no. KF939493.1). The resulting sequences were deposited into GenBank with accession nos. OQ581576 and OQ848462 respectively. A maximum likelihood (ML) phylogenetic analysis based on combined partial ITS and EF1α data set was conducted via ML bootstrapping using MEGA 11. According to morphology and phylogenetic analysis, the isolate was identified as F. tricinctum (Wang et al., 2022). For a pathogenicity test, a pot experiment was conducted in a greenhouse with a temperature range of 20-27℃ and 60% relative humidity. Roots of C. humilis were immersed in a spore suspension (1×107 conidia/ml) of isolate F. tricinctum CH-2 for approximately 5 minutes. Subsequently the treated roots were planted in pots filled with sterilized field soil, while roots dipped in sterilized water were used as the control. The experiment considered of ten pots for the inoculation treatment and six pots for the control treatment. All pots were maintained in the greenhouse. After 15 days, it was observed that 80% of the inoculated plants displayed symptoms consistent with the field observations, indicating successful infection. In contrast, plants in the control treatment did not exhibit any symptoms. The same fungal pathogen as F. tricinctum CH-2 was reisolated from the diseased root tissue and confirmed through morphological and molecular assays, thereby satisfying Koch's postulates. This is the first documented report of F. tricinctum causing root rot in C. humilis in China. This disease has the potential to become one of the most significant diseases affecting C. humilis in China.

Single cell ICP-MS to evaluate the interaction behaviour for Cd, Ce and U with Streptomyces coelicolor spores

Streptomyces are important soil bacteria used for bioremediation of metal-contaminated soils, however, it is still unknown how metal-selective Streptomyces are and which mechanisms are involved during their capture. In this work, we exposed S. coelicolor spores to environmentally relevant concentrations (0.1, 1, 10, 100 μM) of Ce, U and Cd in solid medium for one week to investigate the uptake behaviour of hyphae in the newly formed spores. Additionally, metal adsorption onto the spores was explored by incubating inactive, ungerminated spores for one day in aqueous metal solution. The spore-washing treatment was key to distinguishing between strongly spore-associated (e.g. incorporation; Tris-EDTA buffer) and weakly spore-associated metals (Tris buffer alone minus Tris-EDTA). Single cell (sc) ICP-MS was used to quantify metal-associated content in individual spores. Our results revealed element-specific adsorption onto inactive spores showing that out of the total metal exposure, both strongly (Ce: 58%; U: 54%; Cd: 28%) and weakly (Ce: 12%; U: 1%; Cd: 18%) adsorbed metals occur. However, scICP-MS showed that from metal-amended solid medium, only Ce and U were strongly spore-associated (averages 0.040 and 0.062 fg spore−1 for 10 μM exposures, respectively) while Cd was below the limit of detection (< 0.006 fg spore−1). We propose that hyphae only metabolically interact with Ce in a controlled manner but uncontrolled with U, as 66–73% Ce and only 2–4% U were inherited from adsorbed content. We conclude that Streptomyces spore-metal interaction starts with a relevant adsorption step of Ce, U and Cd as presented for aqueous conditions. If spores start to germinate, hyphae are capable of effectively encapsulating Ce and U, but not Cd. This study brings light into the still unknown field of metal interactions with Streptomyces and applied understanding for more efficient and metal-specific use of Streptomyces in bioremediation of metal-polluted soils.

New insights into the effects of UV light on individual Nosema bombycis spores, as determined using single-cell optical approaches.

Nosema bombycis (Nb) is a pathogen causing pebrine in sericulture. Ultraviolet (UV) light exposure is a common physical disinfection method, but the mechanisms underlying UV-based disinfection have only been studied at the population level. In this study, changes in and germination of UV-irradiated spores were observed using Raman tweezers and phase-contrast imaging to evaluate the effects of UV radiation on Nb spores at the single-cell level. We found that irradiation caused the complete leakage of trehalose from individual spores. We also found that more spores leaked as the UV dose increased. There was no significant loss of intracellular biomacromolecules and no marked changes in the peaks associated with protein secondary structures. Low-dose radiation promoted spore germination and high-dose radiation decreased the germination rate, while the germination time did not undergo significant alterations. These results suggest that UV radiation disrupts the permeability of the inner membrane and alters the spore wall, thereby affecting the ability of the spore to sense and respond to extracellular stimuli, which further triggers germination and reduces or stops spore germination. This study provides new insights into the molecular mechanisms underlying conventional disinfection measures on microsporidian spores.

Real-Time Monitoring of H2O2 Sterilization on Individual Bacillus atrophaeus Spores by Optical Sensing with Trapping Raman Spectroscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H2O2, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H2O2. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H2O2 shows that the formation of reactive oxygen species from H2O2 is the rate-limiting factor of oxidative spore death.

A lab-on-a-chip utilizing microwaves for bacterial spore disruption and detection

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolinic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

Uncertainty in coprophilous fungal spore concentration estimates

The abundance of coprophilous (dung-inhabiting) fungal spores (CFS) in sedimentary records is an increasingly popular proxy for past megaherbivore abundance that is used to study megaherbivore-vegetation interactions, timing of megaherbivore population declines and extinctions, and the introduction of domesticated herbivores. This method often relies on counting CFS alongside pollen and tracers of known concentration such as exotic pollen or synthetic microspherules. Prior work has encouraged reporting CFS abundances as accumulation rates (spores/unit2/year) or concentration (spores/unit3) instead of percentages relative to the total pollen abundance, because CFS percentages can be sensitive to fluctuations in pollen influx. In this work, we quantify the uncertainty associated with estimating concentration values at different total counts and find that high uncertainty is associated with concentration estimates using low to moderate total counts (n = 20 to 200) of individual fungal spore types and tracers. We also demonstrate the effect of varying tracer proportions, and find that larger tracer proportions result in narrower confidence intervals. Finally, the probability of encountering a CFS spore from a specific taxon occurring in moderate concentrations (1,000 spores/unit2) dramatically decreases after a low tracer count (∼50). The uncertainties in concentration estimates caused by calculating tracer proportion are a likely cause of the high observed variance in many CFS time series, especially when CFS or tracer concentrations are low. Thus, we recommend future CFS studies increase counts and report the uncertainty surrounding concentration values. For some records, reporting spore data as presence/absence rather than concentrations or counts is preferable, such as when performing high counts is not feasible.

Direct Observation of the Sporicidal Action of Hydrogen Peroxide on Bacillus atrophaeus Spores By Optical Trapping Raman Microscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is frequently used for chemical sterilization in aseptic food processing. Due to their inherent robustness, bacterial spores, i.e. spores of Bacillus atrophaeus, are commonly employed to evaluate the efficacy of the sterilization procedure [1]. Despite its widespread use, however, the details of the sporicidal action of H2O2 are not yet fully understood, with various mechanisms being discussed in the literature [2,3]. Here, we employ optical trapping Raman microscopy to elucidate the sporicidal mechanism of H2O2. Optical trapping Raman microscopy allows us to isolate, investigate, and manipulate individual spores from a sample (Fig. 1A). Combining optical microscopy with the real-time, label-free chemical information obtained from Raman spectroscopy directly reveals the sequence of steps that lead to oxidative spore death after H2O2 exposure (Fig. 1B).We find that spore degradation begins with the fast, cooperative release of dipicolinic acid (DPA), a key spore biomarker and a major component of spore’s core. This indicates a breach of the inner membrane surrounding the core. The onset of DPA release depends on the concentration of H2O2. DPA outflux is accompanied by the formation of oxidation products (mostly carbonyl compounds) and oxidation continues after exchange of DPA with the surrounding medium is complete. The remaining spore shell then slowly disintegrates and continues to shrink on a timescale of hundreds of seconds. Simultaneous observation of spore morphology and size by optical microscopy and particle tracking corroborates this three-step mechanism.The presented assay allows on-line monitoring of spore composition and morphology, which directly reveals the mechanism underlying the sporicidal action of H2O2. In addition, trapped spores can be exposed to different chemical or physical stimuli (temperature, light, chemical composition of the surrounding medium, etc.) to investigate a variety of industrially relevant sterilization conditions. Jildeh, Z. B., Wagner, P. H. & Schöning, M. J. Sterilization of Objects, Products, and Packaging Surfaces and Their Characterization in Different Fields of Industry: The Status in 2020. physica status solidi (a) 218, 2000732 (2021). Setlow, P. & Christie, G. What’s new and notable in bacterial spore killing. World J Microbiol Biotechnol 37, 144 (2021). Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514-525 (2006). Figure 1

Evaluation of copper uptake in individual spores of Streptomyces coelicolor and endogenic nanoparticles formation to modulate the secondary metabolism.

Copper modulates secondary metabolism in Streptomyces. Although the cytosolic copper concentration is controlled by several chaperones and transporters, the formation of copper nanoparticles (NPs) and its relation to the antibiotic production has never been established in the model Streptomyces coelicolor. In this work, state-of-the-art analytical tools are used to evaluate the incorporation of copper in individual spores of S. coelicolor at different exposure concentrations (40, 80, and 160 μM Cu). Among them, the use of single cell-inductively coupled plasma-mass spectrometry revealed incorporation levels in the range of 2 to 2.5 fg/spore (median) increasing up to 4.75 fg/spore at the upper exposure concentrations. The copper storage within the spores in the form of NPs was evaluated using a combination of single particle-inductively coupled plasma-mass spectrometry and transmission electron microscopy. The obtained data confirmed the presence of NPs in the range of 8 to 40 (mean size 21 nm) inside S. coelicolor spores. The presence of the NPs was correlated with the actinorhodin production in liquid non-sporulating cultures amended with up to 80 μM Cu. However, further increase to 160 μM Cu, yielded to a significant decrease in antibiotic production. Secondary metabolism is activated under stressful conditions and cytosolic copper seems to be one of the signals triggering antibiotic production. Particularly, NP formation might contribute to modulate the secondary metabolism and prevent for copper toxicity. This work describes, for first time, the formation of endogenous copper NPs in S. coelicolor and reveals their correlation with the secondary metabolism.

First Report of Leaf Spot Disease on Microstegium vimineum Caused by Bipolaris setariae in China.

First Report of Leaf Spot Disease on Microstegium vimineum Caused by Bipolaris setariae in China.

First report of Fusarium solani Species Complex Causing Root Rot of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica), a native fruit tree to China, is a popular edible fruit with medicinal properties (Badenes et al. 2013). A 2016-2019 field survey of ~13,000 loquat trees in two orchards in Chongqing and Fujian provinces showed about 5 to 10% root rot disease incidence. The disease symptoms included leaf yellowing, wilting, rotting of main root, and cracking of lateral roots, eventually leading to defoliation and death. To determine the causative agent, diseased roots from six trees were collected, washed in tap water, cut into 2-3 mm pieces, and disinfected for 3 min in 75% (v/v) EtOH. After rinsing in sterilized water, the root pieces were soaked in 10% NaClO (w/v) for 5-10 min, rinsed thrice in sterile water, and plated on potato dextrose agar (PDA). After 7 days of incubation at 25°C, individual spores were collected from the fungal colonies and replated. Single spore cultures growing on PDA gave rise to woolly-cottony, cream-white colored aerial mycelium and a yellowish pigmented mycelium. The average colony growth rate was 8.6 mm day-1 (n=3). Microscopic observation of the mycelium revealed septate and hyaline hyphae and long cylindrical monophialides. Macroconidia were moderately curved, stout, 3-4 septate, measuring 20.79-48.70 μm × 4.16-10.14 μm (n=50). Microconidia produced from long phialides were kidney-shaped, 0-2 septate, and 5.72-17.28 μm × 2.29-6.51 μm (n=50) in size. The mycelial characteristics and reproductive structures of the isolates fit the morphological description of Fusarium sp. (Summerell et al. 2003). To confirm this identification, translation elongation factor (EF-1α) and RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) regions of the genome were PCR amplified from 3 separate isolates (R2, R4 and R5) using EF1/ EF2, RPB1-Fa/G2R, RPB2-5f2/7cR & RPB2-7cF/11aR primer pairs (O'Donnell et al. 2010) and sequenced. BLASTn comparison of the EF-1α (MT976167), RPB1 (MT967271) and RPB2 (MW233052) regions from isolate R4 showed 99% identity with the EF-1α (GU170620, 675/676 bp), RPB1 (KC808270, 1543/1545 bp) and RPB2 (MK4419902, 1637/1638 bp) sequences of Fusarium solani species complex (FSSC) in GenBank database. The same species level identification was also found using FUSARIUM-ID and FUSARIUM-MLDT databases. Two-year-old seedlings (n=3) of two different cultivars, 'Hunanzaoshu' and 'Huabai No. 1', growing in pots indoors at 25-27 °C were inoculated by drenching the soil with a conidial suspension of isolate R4 (40 mL, 106 conidia mL-1 obtained from 6-10 day old cultures). Control plants (n=3) were inoculated with sterilized water. At 20 days after inoculation (DAI) the leaves of inoculated plants became chlorotic and wilted, defoliated over time, and by 53 DAI 91.67% of plants died. The taproot and lateral roots of inoculated plants appeared brown to black in color and most lateral roots died and decomposed at 53 DAI, whereas the control plant roots remained healthy. All control plants remained symptomless. Based on morphological and molecular characters (TEF-1, RPB1 and RPB2), the re-isolated pathogen from diseased plants was identical to the R4 isolate used for inoculation and the disease assays were repeated thrice. FSSC was recently reported to cause fruit rot disease on loquat in Pakistan (Abbas et al. 2017). Identifying Fusarium solani species complex as a disease agent in Chinese loquat will assist in future development of improved germplasm for this important worldwide tree crop.

Optical Trapping Raman Spectroscopy As a Sensor / Actuator / Treatment System

In recent years, Raman spectroscopy has evolved into a broadly used analytical method in chemistry, life sciences, and medicine. Here, we combine Raman spectroscopy with optical trapping and optical microscopy to isolate and investigate individual micron-size biological particles from a sample. In this setup, Raman spectroscopy provides a label-free chemical fingerprint while light microscopy reveals the sample morphology. In addition, elastic light scattering as well as observation of diffusive movement in the optical trap’s potential offer information on particle size and refractive index. Single particles can be captured and manipulated for tens of minutes. During this time, the stably trapped particles can be exposed to a variety of chemical and physical stimuli such as heat, light of different wavelengths, or changes in the chemical composition of the particle environment through microfluidics (Fig. 1A).In this work, we employ this optical trapping Raman spectroscopy sensor / actuator / treatment system to study the effect of hydrogen peroxide (H2O2) on individual spores of Bacillus atrophaeus, an important model system to assess the efficacy of industrial sterilization processes. Interestingly, we find B. atrophaeus spores to be stable for more than 30 minutes while optically trapped even at elevated temperatures (> 70 °C). Exposure to H2O2 results in spore degradation in a concentration-dependent manner within several minutes and is accelerated by high temperatures. Raman spectroscopy indicates that spore degradation is characterized by the rupture of the spore core resulting in the fast release of dipicolinic acid, a major spore biomarker that is considered key for spore resistance to environmental stress (Fig. 1B). Oxidative degradation of the spore shell starts before core rupture and continues after dipicolinic acid release. Simultaneous observation of spore morphology corroborates this two-phase model. Illumination with shorter (< 550 nm) wavelength light exposes the spores to oxidative stress via the generation of reactive oxygen species in the spore environment by light, thereby mimicking the H2O2 sterilization process. In the presence of small amounts (0.5%) H2O2, spore lifetime is reduced to several seconds.With the presented system, we address spore activity down to the single cellular level in a multimodal manner: trapping and manipulation of single spores over several tens of minutes (actuator functionality), stimulation of single spores by temperature, illumination, and H2O2 exposure (treatment functionality) as well as on-line monitoring of spore activity (sensor functionality). Figure 1

Bioluminescence dynamics in single germinating bacterial spores reveal metabolic heterogeneity.

Spore-forming bacteria modulate their metabolic rate by over five orders of magnitude as they transition between dormant spores and vegetative cells and thus represent an extreme case of phenotypic variation. During environmental changes in nutrient availability, clonal populations of spore-forming bacteria exhibit individual differences in cell fate, the timing of phenotypic transitions and gene expression. One potential source of this variability is metabolic heterogeneity, but this has not yet been measured, as existing single-cell methods are not easily applicable to spores due to their small size and strong autofluorescence. Here, we use the bacterial bioluminescence system and a highly sensitive microscope to measure metabolic dynamics in thousands of B. subtilis spores as they germinate. We observe and quantitate large variations in the bioluminescence dynamics across individual spores that can be decomposed into contributions from variability in germination timing, the amount of endogenously produced luminescence substrate and the intracellular reducing power. This work shows that quantitative measurement of spore metabolism is possible and thus it opens avenues for future study of the thermodynamic nature of dormant states.

Modeling the impact of water activity, pH, and calcium propionate on the germination of single spores of Penicillium paneum

This study aimed to assess the effect of water activity (aw) (0.93–0.95), pH (5.1–5.8), and concentration of calcium propionate (0.25 and 0.41%) on the germination time of individual spores of Penicillium paneum. The germination of single spores was examined by capturing images at every hour using the Axio Observer.Z1 inverted microscope ( × 100) connected to the camera at 25 °C for 96 h. The results indicated that P. paneum germination time was significantly affected by aw and pH (ρ < 0.05). As most of the growth curves exhibited asymmetric shapes, the asymmetric model was fitted to germination data and the maximum percentage of viable spores (Pmax; %), shape parameter (d), and germination time (τ; hour) were obtained. τ -values ranged from 26.5 to 40.7 h. After the Monte Carlo simulation, a probability distribution of time for 10% of the P. paneum population (t10%) to germinate was obtained. The mean value of t10% was 25 h, ranging from 15 to 47 h (95% confidence interval). The variability in germination and growth of P. paneum single spores highlights the challenge of predicting and preventing this critical phenomenon.