Dark Incubation Research Articles

Meta-Topolin mediated in vitro propagation in an ornamentally important crop Iris × hollandica Tub. cv. professor Blaauw and genetic fidelity studies using SCoT markers

Dutch iris is a commercially important bulbous ornamental crop. Its high demand in global floriculture market necessitates the production of its high-quality planting material. In the present investigation, an efficient in vitro propagation system has been developed for Iris × hollandica Tub. cv. Professor Blaauw (Dutch iris) using meta-Topolin (mT) for the first time. Effect of various concentrations of BAP, Kn, and mT (0, 0.5, 1.0, 1.5 and 2 mg L−1) along with varying photoperiods (16 h light and dark incubation for 1, 2, 3 and 4 weeks) on in vitro shoot induction from the twin scale explants was studied. Of the cytokinins tested, different doses of mT has resulted in better shoot induction response from twin scale explants than BAP and Kn. Photoperiod duration has also affected shoot induction response significantly. Along with dark incubation for 1 week, cytokinin mT at 1.0 mg L−1 in MS medium has resulted in maximum shoot induction response (91.63%) with increased emergence of micro shoots (4.83 shoots/explant with average shoot length of 5.02 cm). Efficacy of BAP and mT alone or in combination with auxins for in vitro shoot multiplication was also compared. The synergistic effect of cytokinin-auxin in multiplication medium comprising MS + 1.0 mg L−1 mT + 0.25 mg L−1 NAA resulted in considerably higher number of shoots (17.53) with mean shoot length (7.06 cm) and maximum number of bulblets (2.74). Positive effect of increased sucrose concentration (90 g L−1) alone or with paclobutrazol (5 mg L−1) on in vitro bulblet formation and bulblet size was observed respectively. The superiority of mT over BAP was also found during in vitro rhizogenesis. Shoots raised on the mT medium were healthy and long enough, thus showed better rooting response (63.83%) on ½ MS medium + 0.5 mg L−1 IAA after 4 weeks of incubation. About 89.16% survival rate was recorded for in vitro raised plantlets under ex vitro conditions. Analysis of clonal fidelity of thirteen in vitro regenerated plants was done using SCoT markers. Out of 36 primers, 13 primers showed clearly scorable monomorphic bands, thus displaying genetic uniformity among in vitro regenerated plantlets. This mT mediated protocol can be routinely used for the rapid large scale production of this valuable floriculture crop.

Detection of Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphoid B, and Shigella dysentery in live Bacillus licheniformis products using propidium monoazide-real-time-quantitative polymerase chain reaction.

To eliminate the influences of excipients and interference of dead bacterial DNA on the detection of Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphoid B, and Shigella dysentery in live Bacillus licheniformis capsules, a polymerase chain reaction (PCR) method with high sensitivity and specificity was established. By combining bromide with propidium monoazide (PMA) -real-time quantitative PCR (qPCR) with microporous membrane filtration, excipients were removed, the filtrate was collected, and the bacteria were enriched using the centrifugal method. The optimal PMA working concentration, dark incubation time, and exposure time were determined. Specific E. coli, P. aeruginosa, S. paratyphoid B, and S. dysentery primers were selected to design different probes and a multiplex qPCR reaction system was established. The PMA-qPCR method was verified using different concentrations of dead and live bacteria. This method is efficient and accurate and can be widely applied to the detection of aforementioned pathogenic bacterial strains in live Bacillus licheniformis products.

Can alpine plant species "bank" on conservation?: Using artificial aging to understand seed longevity.

PremiseTo conserve native plants, many institutions are turning toward ex‐situ conservation methods, such as storage in seed banks; however, not all seeds are able to survive in seed bank conditions, or may not in the long term. Experimental aging has shown that alpine species lose viability more quickly than low‐elevation species. Furthermore, the germination requirements for rare species are largely unknown, but are a necessary first step in understanding storage behavior and viability decline.MethodsFive alpine species were subjected to germination and accelerated aging experiments to understand their longevity in storage. For the accelerated aging experiment, the seeds were rehydrated in a dark incubator and subsequently placed in a drying oven. Following the aging process, the seeds were placed into previously determined germination conditions.ResultsAll species had p 50 values of <13.7 days, which is the threshold to consider a species short lived. These results suggest that we cannot haphazardly store seeds and assume that all species will survive for decades.DiscussionAccelerated aging experiments are not a perfect measure of seed longevity, and true longevity needs to be empirically determined. However, this experimental method allows us to predict which species may be short lived and whether alternative ex‐situ conservation methods might be needed beyond conventional seed banking.

Stimulatory and inhibitory effects of light on Cereus repandus (Cactaceae) seed germination are strongly dependent on spectral quality

AbstractIn small seeds, light often promotes germination and longer-term exposure to darkness reduces light sensitivity. In cacti inhabiting harsh environments, a rapid response to light exposure is potentially advantageous for seedling establishment. We exposed dark-imbibed seeds of the cactus Cereus repandus to doses of red (RED) light and far-red (FR) light. The seeds exhibited positive photoblastism to RED light. Although the initial levels of germination varied between seed lots, the sensitivity to increasing the RED dose did not. As little as 5 min per day for 4 d was sufficient to saturate the light requirement for germination. The effects of RED light were reversed by FR exposure as long as the interval between RED and FR did not extend to 2 d, by which time the seeds had ‘committed’ to germinate. Dark incubation for 1–2 weeks prior to RED exposure reduced light sensitivity in two seed lots, such that RED only promoted around 20% germination. Phytochrome is assumed to mediate the reversibility of the RED:FR response. High sensitivity to light spectral quality suggests that seeds of C. repandus are able to germinate quickly in high-quality microsites, but seed burial or shading may commit the seeds to form a soil seed bank. The light characteristics of the germination trait in this species are typical of many small seeded species of the drylands.

The dominant role of sunlight in degrading winter dissolved organic matter from a thermokarst lake in a subarctic peatland

Abstract. Dissolved organic matter (DOM) leaching from thawing permafrost may promote a positive feedback on the climate if it is efficiently mineralized into greenhouse gases. However, many uncertainties remain on the extent of this mineralization, which depends on DOM lability that is seemingly quite variable across landscapes. Thermokarst peatlands are organic-rich systems where some of the largest greenhouse gas (GHG) emission rates have been measured. At spring turnover, anoxic waters release the GHG accumulated in winter, and the DOM pool is exposed to sunlight. Here, we present an experiment where DOM photoreactivity and bioreactivity were investigated in water collected from a thermokarst lake in a subarctic peatland during late winter (after 6 months of darkness). We applied treatment with or without light exposure, and manipulated the bacterial abundance with the aim to quantify the unique and combined effects of light and bacteria on DOM reactivity at ice-off in spring. We demonstrate that sunlight was clearly driving the transformation of the DOM pool, part of which went through a complete mineralization into CO2. Up to 18 % of the initial dissolved organic carbon (DOC, a loss of 3.9 mgC L−1) was lost over 18 d of sunlight exposure in a treatment where bacterial abundance was initially reduced by 95 %. However, sunlight considerably stimulated bacterial growth when grazers were eliminated, leading to the recovery of the original bacterial abundance in about 8 d, which may have contributed to the DOC loss. Indeed, the highest DOC loss was observed for the treatment with the full bacterial community exposed to sunlight (5.0 mgC L−1), indicating an indirect effect of light through the bacterial consumption of photoproducts. Dark incubations led to very limited changes in DOC, regardless of the bacterial abundance and activity. The results also show that only half of the light-associated DOC losses were converted into CO2, and we suggest that the rest potentially turned into particles through photoflocculation. Sunlight should therefore play a major role in DOM processing, CO2 production and carbon burial in peatland lakes during spring, likely lasting for the rest of the open season in mixing surface layers.

Precocious in vitro flowering in threatened ornamental orchid, Dendrobium ovatum – Decoding the causal factors

Dendrobium has been the focus of floral industries for decades. Wild Dendrobiums serve as germplasm for creating hybrids in the cut flower industry and are priced high. But, fewer reports exist on the wild tropical species of Dendrobium. The present study reports in vitro flowering in Dendrobium ovatum - an epiphytic orchid species endemic to the oriental tropics. The seedlings are raised from the protocorms and protocorm-like bodies. This research also highlights the traits displayed by the in vitro flowers of D. ovatum. An optimized modified MS medium was developed to enhance the flowering in the orchid species. The pre-treatment methods, notably dark incubation for 12 h and prechilling at 15 °C for 2 h, demonstrated the synthesis of pharmacologically relevant bioactives viz., ‘Moscatilin’ and ‘Resveratrol’. The tissue culture system devised can decode the enigmatic reproductive biology and its underlying molecular regulation of D. ovatum. This strategy could be emulated in other horticultural, medicinal and food crops for producing industrially relevant phytochemicals.

Phytoplankton Responses to Bacterially Regenerated Iron in a Southern Ocean Eddy.

In the Subantarctic sector of the Southern Ocean, vertical entrainment of iron (Fe) triggers the seasonal productivity cycle but diminishing physical supply during the spring to summer transition forces microbial assemblages to rapidly acclimate. Here, we tested how phytoplankton and bacteria within an isolated eddy respond to different dissolved Fe (DFe)/ligand inputs. We used three treatments: one that mimicked the entrainment of new DFe (Fe-NEW), another in which DFe was supplied from bacterial regeneration of particles (Fe-REG), and a control with no addition of DFe (Fe-NO). After 6 days, 3.5 (Fe-NO, Fe-NEW) to 5-fold (Fe-REG) increases in Chlorophyll a were observed. These responses of the phytoplankton community were best explained by the differences between the treatments in the amount of DFe recycled during the incubation (Fe-REG, 15% recycled c.f. 40% Fe-NEW, 60% Fe-NO). This additional recycling was more likely mediated by bacteria. By day 6, bacterial production was comparable between Fe-NO and Fe-NEW but was approximately two-fold higher in Fe-REG. A preferential response of phytoplankton (haptophyte-dominated) relative to high nucleic acid (HNA) bacteria was also found in the Fe-REG treatment while the relative proportion of diatoms increased faster in the Fe-NEW and Fe-NO treatments. Comparisons between light and dark incubations further confirmed the competition between picophytoplankton and HNA for DFe. Overall, our results demonstrate great versatility by microorganisms to use different Fe sources that results in highly efficient Fe recycling within surface waters. This study also encourages future research to further investigate the interactions between functional groups of microbes (e.g. HNA and cyanobacteria) to better constraint modeling in Fe and carbon biogeochemical cycles.

Marine sediment as a likely source of methyl and ethyl iodides in subpolar and polar seas

The surface ocean is an important source of longer-lived volatile organic iodine compounds, such as methyl and ethyl iodide. However, their production in deep waters is poorly constrained. Here, we combine shipborne seawater and sediment core analyses to investigate the temporal and vertical distribution of methyl and ethyl iodide concentrations in the Bering and Chukchi Seas in July 2017 and 2018, and Funka Bay, Japan, between March 2018 and December 2019. We find the highest methyl and ethyl iodide concentrations in sediment porewaters and at the sediment surface. In Funka Bay, concentrations in the sediment and bottom waters strongly increased following the spring bloom and subsequent settling of phytodetritus to the seafloor. Dark incubation experiments suggest that diatom aggregates are a source of methyl and ethyl iodide to the ambient water. Our findings suggest that phytodetritus at the seafloor is a potential source of volatile organic iodine compounds to the water column in polar and subpolar coastal oceans.

Effects of incubation lighting with green or white light on brown layers: hatching performance, feather pecking and hypothalamic expressions of genes related with photoreception, serotonin, and stress systems

The aim of this study was to evaluate the effect of 16L:8D photoperiod with green (GREEN) or white (WHITE) lights during incubation on hatching performance, blood melatonin, corticosterone, and serotonin levels, hypothalamic expressions of genes related to photoreception, serotonin, and stress systems in layers in relation with feather pecking behavior. Dark incubation (DARK) was the control. Eggs (n = 1,176) from Brown Nick breeders in 2 batches (n = 588/batch) were incubated in the experiment. A total of 396 female chicks and 261 hens were used at rearing and laying periods until 40 wk. Incubation lighting did not affect hatchability, day-old chick weight, and length, but resulted in a more synchronized hatch as compared with the DARK. The effect of incubation lighting on blood hormones was not significant except for reduced serotonin in the GREEN group at the end of the experiment. There was no effect of incubation lighting on gentle, severe, and aggressive pecking of birds during the early rearing period. From 16 wk, GREEN hens showed increased gentle pecking with increasing age. WHITE hens had the highest gentle pecking frequency at 16 wk while they performed less gentle but higher severe and aggressive pecks at 24 and 32 wk. At hatching, the hypothalamic expression of CRH, 5-HTR1A, and 5-HTR1B was higher for the WHITE group compared with both GREEN and DARK, however, 5-HTT expression was higher in GREEN than WHITE which was similar to DARK. Except for the highest VA opsin expression obtained for WHITE hens at 40 wk of age, there was no change in hypothalamic expression levels of rhodopsin, VA opsin, red, and green opsins at any age. Although blood hormone levels were not consistent, results provide preliminary evidence that incubation lighting modulates the pecking tendencies of laying hens, probably through the observed changes in hypothalamic expression of genes related to the serotonin system and stress. Significant correlations among the hypothalamic gene expression levels supplied further evidence for the associations among photoreception, serotonin, and stress systems.

Photodynamic inactivation of pathogenic Gram-negative and Gram-positive bacteria mediated by Si(IV) phthalocyanines bearing axial ammonium units.

The photodynamic inactivation (PDI) of microorganisms has gained interest as an efficient option for conventional antibiotic treatments. Recently, Si(IV) phthalocyanines (SiPcs) have been highlighted as promising photosensitizers (PSs) to the PDI of microorganisms due to their remarkable absorption and emission features. To increase the potential of cationic SiPcs as PS drugs, one novel (1a) and two previously described (2a and 3a) axially substituted PSs with di-, tetra-, and hexa-ammonium units, respectively, were synthesized and characterized. Their PDI effect was evaluated for the first time against Escherichia coli and Staphylococcus aureus, a Gram-negative and a Gram-positive bacterium, respectively. The photodynamic treatments were conducted with PS concentrations of 3.0 and 6.0μM under 60min of white light irradiation (150mW.cm-2). The biological results show high photodynamic efficiency for di- and tetra-cationic PSs 1a and 2a (6.0μM), reducing the E. coli viability in 5.2 and 3.9 log, respectively (after 15min of dark incubation before irradiation). For PS 3a, a similar bacterial reduction (3.6 log) was achieved but only with an extended dark incubation period (30min). Under the same experimental conditions, the photodynamic effect of cationic PSs 1a-3a on S. aureus was even more promising, with abundance reductions of ca. 8.0 log after 45-60min of PDI treatment. These results reveal the high PDI efficiency of PSs bearing ammonium groups and suggest their promising application as a broad-spectrum antimicrobial to control infections caused by Gram-negative and Gram-positive bacteria.

The relationship between photosystem II regulation and light-dependent hydrogen production by microalgae.

Certain microalgae species are capable of light-dependent hydrogen production under conditions of dark anaerobic incubation or nutrient deprivation. From the biotechnological point of view, this phenomenon is a process of synthesizing the energy carrier H2 while consuming light energy. Here, we overview the functional connection between the photosynthetic machinery and light-dependent hydrogen production and assess the physiological significance of this process. We characterize events involved in PSII downregulation, as well as the relationship between PSII regulation mechanisms and hydrogen generation. We suggest that the light-dependent hydrogen production forms part and parcel of the sophisticated regulatory network ensuring adaptation of microalgae to such environmental stresses as anaerobiosis or nutrient deprivation.

Massive loss and microbial decomposition in reproductive biomass of Zosteramarina

The biomass of Zostera marina in the temperate coastal zone has seasonal variations in their dry weight, with a peak during the spring season when a large section of the shoots develops reproductive organs. However, the period of high biomass is only short-term, after which large numbers of reproductive shoots drastically disappeared. Dislodgement is an important fate of the reproductive biomass. The fraction of biomass that reaches the deep sea contributes to carbon sequestration, however the processes of dislodgement and transportation of reproductive shoots of Z. marina has been rarely studied. Here, based on a monitoring survey of two Z. marina meadows, we estimated the net loss of biomass, including their reproductive one. In addition, microbial decomposition was assessed by dark incubation to evaluate the fate of the dislodged biomass. The apparent loss of biomass from the Z. marina meadows in the “Declining Period”, defined with the most rapid decrease in biomass, accounted for 82–100% of the biomass at the initial time-point of this period. By subtracting the biomass production from the apparent loss, we calculated the net loss of biomass in the Declining Period, accounting for 14–81% of the annual production. Approximately half of the biomass loss in the Declining Period was attributed to reproductive biomass, suggesting that dislodgement of reproductive shoots is an important fate of photosynthetic products of Z. marina. The carbon to nitrogen (CN) ratio of the reproductive shoots was found to be significantly higher than that of vegetative shoots. Since nitrogenous materials are relatively labile, we expected a lower bioavailability of reproductive shoots. However, microbial decomposition did not reflect the CN ratio, probably due to the heterogeneity of nitrogenous materials and/or due to the contribution of carbohydrates consisting mostly of non-nitrogenous compounds.

Metabolic Profiling of Averrhoa carambola Fruit Extract using UHPLC-ESI-QTOF-MS and Determination of the Concentration of Essential Elements using MP-AES

A rapid and sensitive ultra-high-performance liquid chromatography-electrospray ionization quadrupole time of flight mass spectrometry (UHPLC-ESI-QTOF-MS) based method was developed to separate and identify 44 phenolics and non-phenolics present in Averrhoa carambola fruit in three different stages of its growth. 5 anthocyanins, 9 phenolic acids, 3 flavones, 5 flavanols, 3 flavanones, amino acids, vitamins and other phenolic and non-phenolic compounds were identified. The identification and quantification of 27 bioactive compounds from methanolic star fruit extract are being reported for the first time. All the metabolites were quantified by normalizing the area under the peaks with that of the internal standard hydrocortisone. The extraction of metabolites by using a homogeniser (bead beater) was found to be the best method for the extraction of metabolites. Four essential microelements (Zn, Fe, Mn, Cr) and three macroelements (K, Mg, Na) were quantified from star fruit using MP-AES. The zinc concentration (6.988 mg/100 g) in star fruit was found to be higher than in many cereals, vegetables and fruits. The methanolic star fruit extract showed excellent DPPH scavenging activity of 93% after 30 min of incubation in dark for the dry weight sample concentration of 50 mg/ml. In conclusion, the UHPLC-ESI-QTOF-MS based method developed and MP-AES results presented here can be used in the quality control of food supplements and medicines.

Ecological Impacts of Aged Freshwater Biofilms on Estuarine Microbial Communities Elucidated Through Microcosm Experiments: A Microbial Invasion Perspective.

Inadvertent introductions of alien species via biofilms as a vector released through ballast water are of environmental importance, yet their consequences are not much known. In the present study, biofilm communities developed in an inland freshwater port under in situ and dark conditions were subjected to long-term dark incubations. Subsequently, the impact of these aged biofilms as vectors on estuarine water column communities were evaluated using microcosm experiments in the laboratory. Variations in biofilm and planktonic microbial communities were quantified using quantitative PCR.Upon prolonged dark incubation, a shift in bacterial diversity with an increase in tolerant bacterial communities better adapted to stress was observed. Actinobacteria were the dominant taxa in both aged biofilms upon dark incubations. The laboratory studies indicated that on exposure of these biofilms to estuarine water, resuscitation of Vibrio alginolyticus, V. parahaemolyticus, and V. cholerae from a dormant state existing in these biofilms to culturable form was observed. Moreover, the results revealed that both the biofilm types can pose a threat to the environment, but the degree of risk can be attributed to the imbalance caused by significant changes in the surrounding estuarine microbial communities. Consequently, this may result in either proliferation or decline of some genera with different metabolic potential and resuscitation of pathogenic forms not present earlier, thereby influencing the ecology of the environment. Quantifying these effects in the field using biofilm metagenomes with an emphasis on virulent species and understanding traits that enable them to adapt to changing environments is a way forward.

Phosphatic metabolism in dark- and light-adapted rat retinas

This study defines retinal phosphatic metabolites and their adjustment to illumination in rat retinas under conditions that preserve retinal function. Metabolic data are measured using high-performance liquid chromatography (HPLC) and 31P nuclear magnetic resonance (31P NMR) spectroscopy after 10 min of light exposure in vivo compared with retinas from dark-adapted rats. Multiple high-energy and low-energy phosphatic metabolites of intermediary metabolism were quantified. The concentration of the high-energy phosphate adenosine triphosphate (ATP) remained unchanged from dark- to light-adaptation. Under the same conditions the concentrations of the high-energy phosphates guanosine triphosphate (GTP) and creatine phosphate increased, whereas the inorganic phosphate decreased. Comparing dark-adapted controls with retinas light-adapted either in vitro or in vivo, the evidence is consistent with a light-dependent increase in GTP and a decrease in cyclic guanosine monophosphate. Although cyclic adenosine monophosphate (cAMP) levels were lower in retinas light-adapted in vivo than in the dark-adapted controls, this did not seem to be an effect of light, as cAMP levels decreased similarly after 10 min incubation in dark or light in parallel with recovery of ATP/adenosine diphosphate ratios. This study: (1) reports on retinal metabolic changes with adjustment in illumination, (2) provides baseline measurements of retinal phosphatic metabolites in whole retinas, and (3) reports on the validity of chromatographic and spectroscopic methods used for studying retinal metabolism establishing a high correlation among measurements made using HPLC and 31P NMR.

Occurrence of Neopestalotiopsis clavispora Causing Leaf Spot on Dendrobium officinale in China.

Dendrobium officinale Kimura L., an endangered orchid plant, is a rare and precious Chinese herb and widely used to prepare Chinese traditional medicine (Zheng et al. 2005). In August 2021, significant indications of an unknown leaf spot disease were observed on greenhouse-grown D. officinale in Yueqing of Wenzhou (28.39°N, 121.04°E), Zhejiang Province, China, the main producing location of this orchid plant. Approximately twenty percent of plants surveyed showed typical infection symptoms. Initially, the symptoms appeared as small, circular black spots. As the disease developed, the center of the lesions was sunken with a black border. To determine the causal agent, 10 symptomatic plant samples were collected and all pieces from symptomatic plant leaves were used for isolating pathogen. Tissues between healthy and necrotic area were cut into pieces (5 × 5 mm, n=10), disinfected with 10% sodium hypochlorite for 1 minute, rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar medium (PDA) for 1 piece per plate, and incubated at 25℃ in a dark biochemical incubator. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. Twelve same fungal isolates were obtained from all symptomatic leave fragments, then DDO11 was chosen as a representative isolate for further study. The colonies showed white aerial mycelium after 5 days culture at 25°C on PDA. Black viscous acervuli appeared and scattered on the surface of the colony after 8-12 days culture. Conidia were spindle shape, five cells, four septa, average 29.3 × 8.5 μm (n = 30; length × width). The apical and basal cells were lighter in color, and most of them were hyaline. The middle three cells were darker in color, and mostly brown. There are 2 to 4 colorless and transparent unbranched accessory filaments at the top, 32.5 µm in average length, and the basal cell has a small appendage, 9.2 µm in average length, n=30. For fungal identification to species level, Internal transcribed spacer (ITS) region, β-tubulin gene (TUB2) and translation elongation factor-1α (TEF-1α) were amplified (Qiu et al. 2020), respectively. The ITS, TUB2 and TEF-1α gene sequences of the representative isolate DDO11 were deposited in NCBI GenBank nucleotide database with accession numbers OK631881, OK655895 and OK655896, respectively. BLASTn analysis respectively showed 100%, 100% and 99.6% nucleotide sequence identity with Neopestalotiopsis clavispora strain accessions MG729690, MG740736 and MH423940, which indicated that the pathogen belonged N. clavispora. A maximum-likelihood phylogenetic analysis based on multi-locus sequence (ITS, TUB2, and TEF-1α) using MEGA X showed the similar result (Kumar et al. 2018). To verify pathogenicity, thirty 1-year-old healthy D. officinale plants of cultivar Yandang1 were used for inoculation tests. Spores of N. clavispora DDO11 were produced on PDA for 7 days at 28°C and washed with sterile distilled water, and the concentrations were adjusted to 1 × 106 spores/ml using a hemocytometer. Fifteen surface disinfected healthy plants were inoculated by spraying the suspension (2 ml, 1 × 106 spores/ml) and covered with plastic bags for 24 h, and another 15 plants treated with sterile distilled water were used as control. The plants were placed in a humidified chamber (>95% relative humidity) at 25°C for 48 h after inoculation and kept in a growth chamber (Kiangnan, China) at 25°C with 12-h day/night cycle for 8 days (Cao et al. 2019). All inoculated leaves showed symptoms identical to those observed in the field. No disease occurred on the controls. The Neopestalotiopsis isolate was reisolated from the symptomatic leaves, and species identification was confirmed by the morphological and molecular method described above. N. clavispora has been reported to cause diseases on a variety of plants all over the world, such as strawberry (Gilardi et al. 2019), blue berry (Shi et al. 2021), Syzygium cumini (Banerjee et al. 2020), Macadamia (Qiu et al. 2020), and so on. To the best of our knowledge, this is the first report of N. clavispora causing leaf spot on D. officinale in China. This report will help us to recognize the leaf spot disease of D. officinale and establish a foundation for future studies on N. clavispora to address effective management strategies.

Effect of some mutagenic compounds on lipase production by fluconazole-treated Candida catenulata

Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes widely applied in industry and medicine. Our previous study showed that Candida catenulata produced much higher lipase activity in presence of 150 mg fluconazole than that in untreated control. Consequently in this study, optimization of fluconazole-treated C. catenulata for maximum lipase production was carried out. Fluconazole-treated C. catenulate cultured on medium containing 2.5% Tween 80 as carbon source, 0.7% yeast extract as nitrogen source, and 10 mM CuSO4.5H2O. pH 5.5, dark incubation at 37 °C under dark agitation for 3 days expressed the optimum physiological variables for maximum lipase production. Transmission electron microscopy (TEM) of the native cell (control) of C. catenulata declared a typical morphology of Candida cell with a uniform central density, typical structured nucleus, and a cytoplasm with several elements of endomembrane system and enveloped by a regular intact cell wall. The TEM photomicrograph of 150 mg fluconazole-treated cells of C. catenulata showed that the ultrastructures of treated C. catenulata displayed many noticeable morphological changes. The effect of mutagens [sodium azide (SA), ethidium bromide (EtBr), ethanol (EtOH)] was assayed on the lipase activity from C. catenulata. All mutagenic compounds with different concentrations decreased the lipase activity. Yeast specific activity, protein concentrations, and dry biomass gradually reached their highest decline at mutagen-elevated concentrations. Combination of lower concentrations of mutagen (0.1 µg/ml EtBr + 0.5 mM SA + 10% EtOH, Comb-Fl) exerted synergistic inhibition action and reduced C. catenulata virulent factor (lipase) over their single treatment. When 150 mg fluconazole was added to the mutagen’s combination mixture (Comb + Fl), an increase in the production of the virulence factor lipase by C. catenulata was detected. This action indicated the antagonistic effect of fluconazole over the mutagen combination mixture in increasing the fungal pathogenicity.

SWATH-based quantitative proteomic analysis of Morus alba L. leaves after exposure to ultraviolet-B radiation and incubation in the dark

Morus alba is a woody shrub of the family Moraceae and used as traditional Chinese medicine for a long history. Ultraviolet-B (UV-B) radiation, as a kind of abiotic stress factor, affected the growth and secondary metabolism in M. alba. Previous studies indicated that the contents of several secondary metabolites such as moracin N, chalcomaricin were significantly increased under high level UV-B radiation and dark incubation in M. alba leaves. To reveal the response mechanism under UV-B radiation and dark incubation in M. alba leaves, SWATH-based quantitative proteomic analysis was performed. Totally, 716 proteins were identified and quantified in the control, UVB, and UVD groups. Among them, 123 proteins and 96 proteins were identified as differentially abundant proteins in UVB group and UVD groups, respectively. Proteins related to photosynthesis, amino acid biosynthesis, and tocopherol biosynthesis were significantly altered in UVB group, while proteins related to the biosynthesis of phenolic compounds were significantly altered in UVD group. In addition, the abundances of proteins involved in the ubiquitin-proteasome system (UPS) were significantly increased in both UVB and UVD groups, indicating that UPS combined with secondary mechanism participated in the resistance to UV-B radiation and dark incubation. The obtained results provide novel insight into the effects of high level UV-B radiation on M. alba leaves and on the strategies used for maximizing the chemical constituents and the medicinal value of the M. alba leaves.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D underlies lesion-mimic phenotype in dark-treated Arabidopsis pheide a oxygenase mutant leaves.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) encoding a plasma membrane NADPH oxidase is responsible for the lesion-mimic phenotype in detached Arabidopsis leaves with mutation of PHEIDE a OXYGENASE during extended darkness. Chlorophyll degradation is an indispensable process in leaf senescence, either age-dependent or dark-induced. Besides higher chlorophyll retention, a lesion-mimic phenotype (abbreviated as LMP afterwards) was exhibited in Arabidopsis leaves with mutation of PHEIDE a OXYGENASE (PaO) involved in chlorophyll degradation during dark incubation, but the associated mechanism remains elusive. We found that dark-treated pao leaves showed higher membrane damage and H2O2 accumulation, while scavenging H2O2 by its chemical scavenger diminished LMP. RBOHD which encodes NADPH oxidase was strikingly up-regulated in pao leaves during dark treatment. Chemical inhibition of NADPH oxidase or mutation of RBOHD in pao leaves suppressed LMP. Thus, our study suggests that up-regulated RBOHD transcription is responsible for the formation of LMP in dark-treated pao leaves and there may be a retrograde signaling pathway mediating upregulation of RBOHD which remains to be elucidated.

Linking Microbial Population Succession and DOM Molecular Changes in Synechococcus-Derived Organic Matter Addition Incubation.

ABSTRACTThe molecular-level interactions between phytoplankton-derived dissolved organic matter (DOM) and heterotrophic prokaryotes represent a fundamental and yet poorly understood component of the marine elemental cycle. Here, we investigated the degradation of Synechococcus-derived organic matter (SynOM) by coastal microorganisms using spectroscopic and ultrahigh-resolution mass spectrometry analyses coupled with high-throughput sequencing. The added SynOM showed a spectrum of reactivity during a 180-day dark incubation experiment. Along with the decrease in DOM bioavailability, the chemical properties of DOM molecules overall showed increases in oxidation state and aromaticity. Both the microbial community and DOM molecular compositions became more homogeneous toward the end of the incubation. The experiment was partitioned into three phases (I, II, and III) based on the total organic carbon consumption rates from 7.0 ± 1.0 to 1.0 ± 0.1 and to 0.1 ± 0.0 μmol C L−1 day−1, respectively. Diverse generalists with low abundance were present in all three phases of the experiment, while a few abundant specialists dominated specific phases, suggesting their diverse roles in the transformation of DOM molecules from labile and semilabile to recalcitrant. The changes of organic molecules belonging to CHO, CHNO, and CHOS containing formulas were closely associated with specific microbial populations, suggesting close interactions between the different bacterial metabolic potential for substrates and DOM molecular compositional characteristics. This study sheds light on the interactions between microbial population succession and DOM molecular changes processes and collectively advances our understanding of microbial processing of the marine elemental cycle.IMPORTANCE Phytoplankton are a major contributor of labile dissolved organic matter (DOM) in the upper ocean, fueling tremendous marine prokaryotic activity. Interactions between microorganisms and algae-derived DOM regulate biogeochemical cycles in the ocean, but key aspects of their interactions remain poorly understood. Under global warming and eutrophication scenarios, Synechococcus blooms are commonly observed in coastal seawaters, and they significantly influence the elemental biogeochemistry cycling in eutrophic ecosystems. To understand the interactions between Synechococcus-derived DOM and heterotrophic prokaryotes as well as their influence on the coastal environment, we investigated the degradation of DOM by coastal microbes during a 180-day dark incubation. We showed substantial DOM compositional changes that were closely linked to the developments of microbial specialists and generalists. Our study provides information on the interactions between microbial population succession and DOM molecular changes, thereby advancing our understanding of microbial processing of the marine DOM pool under the influence of climate change.