Dark Incubation Research Articles

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.

Biodegradation of Terrigenous Organic Matter in a Stratified Large-Volume Water Column: Implications of the Removal of Terrigenous Organic Matter in the Coastal Ocean.

Large amounts of terrigenous organic matter (TOM) are delivered to the ocean every year. However, removal processes of TOM in the ocean are still poorly constrained. Here, we report results from a 339-day dark incubation experiment with a unique system holding a vertically stratified freshwater-seawater column. The quality and quantity of dissolved organic matter (DOM), RNA-based size-fraction microbial communities, and environmental factors were high-frequency-monitored. Microbial processes impacted TOM composition, including an increased DOM photobleaching rate with incubation time. The mixed layer had changed the bacterial community structure, diversity, and higher oxygen consumption rate. A two-end member modeling analysis suggested that estimated nutrient concentrations and prokaryotic abundance were lower, and total dissolved organic carbon was higher than that of the measured values. These results imply that DOM biodegradation was stimulated during freshwater-seawater mixing. In the bottom layer, fluorescent DOM components increased with the incubation time and were significantly positively related to highly unsaturated, oxygenated, and presumably aromatic compound molecular formulas. These results suggest that surfaced-derived TOM sinking leads to increased DOM transformation and likely results in carbon storage in the bottom water. Overall, these results suggest that microbial transforming TOM plays more important biogeochemical roles in estuaries and coastal oceans than what we know before.

Occurrence of Sclerotinia sclerotiorum Causing Sclerotinia Root Rot onAmerican Ginseng in Northeastern China.

American ginseng (Panax quinquefolium) is a valuable medicinal plant that is commercially cultivated in China. In May 2020, Sclerotinia root rot of American ginseng was observed on 4-year-old plants in Fusong County in northeastern China, which is the most important part of the country for American ginseng cultivation. The pathogen only infected the tuberous ginseng roots, with sclerotia tightly attached to the root surface. Infected roots, which were brownish and had a watery soft rotted appearance (Fig. 1), eventually became hollow and filled with sclerotia. There were no significant changes to the aboveground plant parts during the initial infection stage, but as the disease progressed, the foliage became discolored and wilted because of the damaged roots. More than 31% of the plants in a 30-ha field were infected. Symptomatic roots were collected and sclerotia were removed from the diseased tissue, immersed in 1% NaClO for 1 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After an incubation in darkness at 20 °C for 2-3 days, 21 suspected Sclerotinia isolates were obtained. Isolates JH1 and JH2 were randomly selected for identification. On PDA, colonies produced sparse, white, and cottony aerial mycelia (i.e., wool-like appearance), with septate, branched, and hyaline hyphae. Within 4 days of incubation, the PDA surface was covered with white hyphae. Small and white sclerotial primordia formed 3 days later and were irregularly distributed in the middle and along the edge of the Petri dish. After maturing, the hardened and black sclerotia had an irregular shape and size, ranging from 1.4 × 1.5 to 4.1 × 7.5 mm (n = 50). Most of the sclerotia developed separately, with approximately 15-25 per plate (Fig. 2). On the basis of their morphology, the isolates were initially identified as Sclerotinia sp. (Mordue and Holliday 1976; Kohn 1979). Using the JH1 and JH2 rDNA internal transcribed spacer (ITS) region (GenBank accession no. MZ031405 and MZ031406) and the aspartyl protease gene specific to S. sclerotiorum (MZ292709 and MZ292710) in GenBank as queries, BLAST searches revealed that the sequences were respectively 99%-100% similar to S. sclerotiorum sequences KF859933 and AF271387. The primer pairs for amplifying the ITS region and the aspartyl protease gene were respectively ITS4/ITS5 (White et al. 1990) and SSaspr F/SSaspr R (Abd-Elmagid et al. 2013). The pathogenicity of JH1 and JH2 was evaluated using healthy plants. The roots of 4-year-old ginseng plants were washed, wiped with 75% alcohol, and transferred to flower pots containing sterile sand and sorghum grain (10:1 v/v) infested with 10-day-old isolates. For both isolates, 12 plants were inoculated, with four plants per pot. Control plants were transferred to flower pots containing sorghum grain lacking fungus. The inoculated samples were incubated in a greenhouse (12 h photoperiod and 25 °C) for 25 days before they were examined. The test was repeated twice. The inoculated roots exhibited the same symptoms as those observed in the field, whereas the controls remained symptomless. The same fungus was reisolated from all infected roots and resequencing results confirmed its identity. To the best of our knowledge, this is the first report of S. sclerotiorum causing Sclerotinia root rot on American ginseng in China. Because this disease is detrimental to the production of American ginseng, effective management strategies will need to be developed.

Factors Controlling the Spatial Distribution of Dissolved Organic Matter With Changes in the C/N Ratio From the Upper to Lower Reaches of the Ishikari River, Japan

Dissolved organic matter (DOM), particularly dissolved organic nitrogen (DON), is an important source of energy and/or organic nutrients for heterotrophic microorganisms in rivers. Although various factors controlling the quantity and quality of stream and riverine DOM have been extensively studied, DON has been under-researched compared to dissolved organic carbon (DOC). The spatial distribution of DOC and DON concentrations with respect to the C/N ratio and DOM optical properties was investigated in the Ishikari River and its tributaries in Hokkaido, northern Japan. Here, the upper reaches are forested and the middle and lower reaches are encompassed by agricultural land, in particular paddy fields. Furthermore, dark incubation experiments were conducted using filtered riverine water (<0.7 µm) to determine the bioavailability of DOC and DON (particularly due to small microorganisms) considered as a factor controlling the spatial distribution. In the mainstream, DOC and DON concentrations increased with river flow in the upper and middle reaches and remained unchanged in the lower reaches. The C/N ratio of bulk DOM decreased continuously from the upper reaches to lower reaches. The optical properties exhibited changes in the DOM characteristics in terms of higher molecular weight and higher aromaticity from the upper to middle reaches, suggesting that flooded paddy fields largely altered the riverine DOM concentration and composition. In the lower reaches, the C/N ratio of the bulk DOM decreased with the river flow. However, according to principal component analysis, no changes were observed in the optical properties with river flow, suggesting that the C/N ratio of bulk DOM changed owing to in situ biological activity in the river. DOC biodegradation was observed at four sites in the upper and middle reaches but not at the two sites in the lower reaches. However, the DON concentration during the dark incubation experiments at all sites did not differ significantly, which implies that microbial degradation, particularly by small microorganisms, is a factor that decreased the C/N ratio of bulk DOM in the upper and middle reaches. In contrast, large microorganisms possibly degraded C-rich DOM to decrease the C/N ratio of bulk DOM in the lower reaches of the Ishikari River.

Tandem mass tag-based proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under ultraviolet-B and dark stress.

Mulberry leaves have been used in traditional Chinese medicine due to their antioxidant, antidiabetic, and antihyperlipidemic properties. A previous study showed that ultraviolet-B radiation followed by dark incubation could improve the contents of active ingredients in mulberry leaves, such as moracin N and chalcomoracin. The endoplasmic reticulum (ER) serves as a protein quality control center and the location for protein synthesis, which is involved in the response to the environmental stress in plants. To investigate the mechanisms in response to ultraviolet-B radiation followed by dark incubation (UV + D), ER proteomics was performed on mulberry leaves. The ER protein markers, glucose-regulated protein (GRP78), and calnexin (CNX), were significantly higher in the ER fraction than in the total protein fraction, indicating that the ER was purified. Compared to the control, the abundance of protein disulfide isomerase, UDP-glucose glycoprotein glucosyltransferase, CNX, and calreticulin proteins decreased, while of the abundance of heat shock-related proteins increased under stress. P450 enzyme system-related proteins and ribosomal proteins showed significant increases. These results suggest that under UV + D stress, mulberry leaves activated the cell redox and ER quality control systems, enhancing protein synthesis and weakening N-glycan biosynthesis in the ER to resist the damage.

Survival and recovery of planktonic organisms in prolonged darkness and their implications on ballast water management

Understanding the population dynamics of transported organisms in the ship's ballast tanks during shipping operations and their ability to survive and colonize in the receiving environment (destination) can aid in developing effective ballast water treatment and management strategies. In the present study a natural plankton pool harvested from a tropical coast was used to study their survival in prolonged darkness and their regrowth potential upon re-illumination in laboratory conditions. In darkness, phytoplankton density and chlorophyll-a biomass decreased with increase in nutrient levels attributing to cell lysis. However, when subjected to vital staining using SYTOX® green, about 60% of phytoplankton survived in the dark tanks. Upon re-illumination after 36 days of incubation in darkness, >80% of recovered phytoplankton were pennate diatoms. On the other hand, zooplankton failed to survive/recover during the experiments when incubated in the dark. Less than 30% of the live zooplankton stained using Neutral Red in the dark tanks failed to recover during experiments. The data obtained clearly shows that prolonged dark incubation may promote the growth of opportunistic organisms and may alter their invasive potential through time and cause serious negative impacts to the receiving environment when transported by ballast water.

Unexpected Dissociation of Photoresponsive UV-ON DNA Carrying p-tert-Butyl Azobenzene under UV Light Irradiation

Abstract A hybridization behavior of Vis-OFF/UV-ON type photoresponsive DNA tethering p-tert-butyl azobenzene (pDNA) was investigated using fluorescence and absorbance measurement. We found that the partial dissociation of pDNA duplex into single strands occurred during UV irradiation, whereas incubation in dark after UV irradiation induced duplex formation. This feature is commonly considered to be important for an experimental design of photo-controllable system utilizing azobenzene.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Orychophragmus violaceus in China.

Orychophragmus violaceus, belonging to the Brassicaceae family, is widely grown in many provinces of China as an ornamental plant and also as a green manure crop. In December 2019, field investigations showed that a leaf spot disease occurred on O.violaceus with 50% to 80% incidence in Huize City, Yunnan Province of China. Infected leaves showed symptoms of small black point spots in the early stage of onset. The lesions are distributed throughout the leaves and finaly expand to 10-15 mm in diameter after 10-15 days of onset. At this time, the lesions are gray to black, and some have round patterns, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. The leaves with typical lesion symptoms were sampled and photographed, and then subjected to isolate and characterize the pathogen. Six pure cultures (HEYA2; HEYA4; HEYC6; HEYD7; HEYD8; HEYD10) were obtained by single-hyphae isolation. On PCA medium, colony can reach 27 mm after 7d, at 25°C in darkness. Aerial hypha is cottony with white to pale gray color, while the colony reverse is fawn to dark. on V8 medium, conidiophore solitary or clustered, erect or knee-curved, occasionally branched, pare brown, separated, 82-130 × 5-9 µm. Conidia are solitary, straight or slightly curved, inverted rod-shaped, pare brown to brown, with 6-10 transverse septa, 0-5 oblique and longitudinal septa, columnar beak, conidial bodies (47.7-)69.3-103.8(-119.6)(11.2-)16.6-23.6(-27.8) µm. Beak septum, pare brown, (29.2-)34.4-72.4(-101.3)(4.2-)6.6-9.5(-11.3) µm. Morphologically these isolates resembled species belonging to genus Alternaria (Simmons, 2007). Genomic DNA of each culture was quickly extracted from mycelia using QS method (Chi et al. 2009). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and translation elongation factor -1α (TEF -1α) genes were amplified according described procedures (White et al. 1990; Berbee et al. 1999; Liu et al. 1999；Sung et al. 2007; Carbone & Kohn 1999). The sequences obtained in this study were deposited in GenBank with accession numbers: MW867245, MW867246, MW867247, MW867248, MW867249, MW867250, MW882913, MW882914, MW882915, MW882916, MW882917, MW882918, MW882919, MW882920, MW882921, MW882922, MW882923, MW882924, MW882925, MW882926, MW882927, MW882928, MW882929, MW882930. Phylogenetic analysis was conducted with combined sequences of the four loci, using the maximum likelihood method and the maximum parsimony method. In the phylogenetic tree, the six isolates and Alternaria brassicae (CBS 116528) clustered together with high bootstrap support values (MLBS=100; MPBS= 100). Based on both morphological characters and phylogenetic results, the isolates were identified as Alternaria brassicae. Pathogenicity test of isolate HEYA2 was carried out on the detached leaves in a dark thermostat incubator at 25°C. Five pots per leaf were inoculated with mycelia plugs (5 mm in diameter), another five pots were inoculated with pure agar plugs and used as the negative control. In addition, conidia suspension (105 conidia/ml) of isolate HEYD8 were sprayed on 3-month-old healthy plants grown in a greenhouse at 22 °C-28 °C. The plants sprayed with sterilized water were used as negative controls. The test was conducted three times. After 5-7 days, the leaves inoculated with other the conidia suspension or the mycelium plugs showed brown necrotic lesions that are similar to the symptoms observed in the field, but the controls remained healthy. The pathogen was reisolated and confirmed to be A. brassicae, completing Koch's postulates. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on Orychophragmus violaceus in China.

Effects of BAP and Lighting Duration on Banana (Musa paradisiaca cv. Raja Bulu) Micropropagation

This study aims to determine the best concentration of 6-Benzilaminopurine (BAP) and lighting duration for Raja Bulu banana cultivar micropropagation. The explant was taken from Pagubugan Village, Cilacap Regency, Central Java. The experiment was arranged as a Randomized Completely Block Design (RCBD) with two factors. The first factor was BAP, with concentration used were 0; 2.5; 5; 7.5 mg/L and the addition of Indole-3-acetic acid (IAA) 0.5 mg/L for all treatment. The second factor was lighting treatment. All explants were put in dark incubation for two weeks followed by light duration treatment: 12 hours of light/day and 16 hours of light/day for the next two weeks. The addition of BAP increased the number of shoots, but did not affect time for shoot initiation, shoot height, number of leaves and number of roots. The best BAP concentration was 2.5 mg/L with an average number of 3.2 shoots/explant. Scalps were formed in the treatment of BAP 5 mg/L and 7 mg/L after four weeks of incubation and has the potential to become new shoots. Nevertheless, the lighting duration did not affect all observed variables.

Assessing the Contribution of Seasonality, Tides, and Microbial Processing to Dissolved Organic Matter Composition Variability in a Southeastern U.S. Estuary

Uncovering which biogeochemical processes have a critical role controlling dissolved organic matter (DOM) compositional changes in complex estuarine environments remains a challenge. In this context, the aim of this study is to characterize the dominant patterns of variability modifying the DOM composition in an estuary off the Southeastern U.S. We collected water samples during three seasons (July and October 2014 and April 2015) at both high and low tides and conducted short- (1 day) and long-term (60 days) dark incubations. Samples were analyzed for bulk DOC concentration, and optical (CDOM) and molecular (FT-ICR MS) compositions and bacterial cells were collected for metatranscriptomics. Results show that the dominant pattern of variability in DOM composition occurs at seasonal scales, likely associated with the seasonality of river discharge. After seasonal variations, long-term biodegradation was found to be comparatively more important in the fall, while tidal variability was the second most important factor correlated to DOM composition in spring, when the freshwater content in the estuary was high. Over shorter time scales, however, the influence of microbial processing was small. Microbial data revealed a similar pattern, with variability in gene expression occurring primarily at the seasonal scale and tidal influence being of secondary importance. Our analyses suggest that future changes in the seasonal delivery of freshwater to this system have the potential to significantly impact DOM composition. Changes in residence time may also be important, helping control the relative contribution of tides and long-term biodegradation to DOM compositional changes in the estuary.

Cold Stress-induced Seed Germination and Biosynthesis of Polyphenolics Content in Medicinally Important Brassica rapa

Background:Brassica rapa is an annual to biennial herb, cultivated worldwide, and mostly adapted to temperate climates. It is commonly cultivated in many countries as edible vegetable and for the production of vegetable oil for growing population. However, the fluctuation in temperature greatly affect the plant growth and development and the production of bioactive compounds. Thus the current study aim to investigate the effect of cold stress on seed germination, biomass gain, and biosynthesis of polyphenolics content in medicinally important Brassica rapa. Methods: In order to investigate cold stress on seed germination, biomass gain, and biosynthesis of polyphenolics content, the authenticated seeds of Brassica rapa were collected from PCSIR Labs Complex, Peshawar-Pakistan. The surface sterilized seeds were keep under control temperature to germinate and then the geminated seeds were treated with cold stress of 72 hours (3 days), 144 hours (6 days), 216 hours (9 days) and 288 hours (12 days). Results: In this study, 72 hours (T1=3 Days) of cold stress was found to be the best treatment for radicle development (152 mm) as compared to control (95 mm). However, 144 hours of cold stress (T2=6 Days) suppressed radicle development (69 mm) compared to the control. The cold treatments (T3=216 and T4=288 hours) also reduced radicle development. The radicle length in the dark was very elongated (105 mm) compared to control. Furthermore, the minimum cold stress of 72 hours promoted plumule development (55 mm) compared to the control (42 mm). However, other cold treatments reduced the growth of plumule. The seeds placed in dark incubation produced maximum plumule length (115 mm) compared to control and other treatments. The increasing doses of cold stress gradually reduced biomass accumulation. Currently, 72 hours and dark incubation improved biomass accumulation. The cold treatment of 72 hours induced maximum production of phenolics content (2.61 mg/g-DW). However, cold stress of 216 hours produced 0.892 mg/g-DW flavonoids content. Conclusion: Our study found that duration of cold stress is inversely proportional to the radicle and the plumule length of the plantlets. Also we found that cold stress of 72 hours (3 days) promoted seed germination while 288 hours of cold stress reduced germination in Brassica rapa. Finally our study suggests further improvement of Brassica species and its use as medicinal herb and need to understand the molecular mechanism involve during cold stress to protect plants from such abiotic stresses.

First Report of Anthracnose of American Ginseng caused by Colletotrichum sojae in Northeast China.

American ginseng (Panax quinquefolium) is a medicinal plant that is commercially cultivated in China. Anthracnose is a devastating disease of American ginseng, with annual production losses exceeding 20%. In July 2019, anthracnose of American ginseng was observed on 3-year-old plants in Fusong County, Jilin Province, China, the most important region of American ginseng. Round or irregular-shaped, brown, sunken and necrotic lesions (5 to 11 mm in diameter), occasionally with a concentric ring or surrounded by brown halos, were detected on leaves (Fig. 1). Multiple lesions gradually coalesced, eventually causing yellowing and wilting. More than 36% of plants in a 30-ha field were infected. Symptomatic leaves (n=16) were collected and the diseased tissue was cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After incubation in darkness at 25°C for 4 days, 15 suspected Colletotrichum single-spore isolates purified in water agar were obtained. The isolate XTJ2 was randomly selected for identification. On PDA, colonies were white to gray, occasionally mixed with gray-black strips, and the reverse was similar to the surface. Colonies on nutrient-poor agar (SNA) were flat, thin, floccose, with an entire margin, whitish to pale gray with the same colors on the reverse. The conidia were hyaline, smooth-walled, straight with a rounded base and apex, ranging from 11.1 to 21.2 × 4.0 to 5.5 μm (n=100), with length/width =3.5. Conidia were initially aseptate, but became septate with age. Setae were dark brown with a slightly acute tip, 2 to 3-septa, and 31.5 to 81.6 μm long. Appressoria were rarely observed, brown, smooth-walled, oval, bullet-shaped or irregular. Chlamydospores were not observed. The isolate was initially identified as Colletotrichum sp. (Damm et al. 2019). Initial BLAST searches of XTJ2 sequences of the rDNA internal transcribed spacer region (GenBank accession no. MW048745), a partial glyceraldehyde-3-phosphate dehydrogenase (MW053381), chitin synthase 1 (MW053382), histone H3 (MW053383), actin (MW053384) and beta-tubulin (MW053385) in GenBank showed that the sequences were respectively 100% similar to Colletotrichum sojae sequences: NR_158358, MG600810, MG600860, MG600899, MG600954 and MG601016 (Carbone and Kohn 1999; Crous et al. 2004;Guerber et al. 2003). The identity of XTJ2 was confirmed by constructing a phylogenetic tree combining all loci, which grouped the isolate and the type strain of C. sojae into one clade (Fig. 2). The sequences of all isolates were genetically identical to the XTJ2 sequences. For pathogenicity tests, 15 healthy 3-year-old plants grown in five pots were spray-inoculated with the XTJ2 conidial suspension (1×105 spores/mL), and the same number of plants were sprayed with water as the control. This experiment was repeated twice. Plants were kept in a greenhouse (28°C, natural light, and 85% relative humidity) under clear plastic bags. After 10 days, inoculated leaves exhibited symptoms that were similar to those observed in the field, whereas the controls were symptomless. The same fungus was recovered and sequenced, and its identity was confirmed by a phylogenetic analysis. This is the first report of C. sojae causing anthracnose of American ginseng in China, being a potential threat to the production of this culture. More studies on the epidemiology of this disease are needed to improve disease management.

Leaf Spot Disease Caused by Pestalotiopsis kenyana on Zanthoxylum schinifolium in Sichuan Province, China.

Zanthoxylum schinifolium Sieb. et Zucc, a species of prickly ash, is one of the main economic plants in China and mainly grown in Southwest China. The planting area of Z. schinifolium accounts for more than 70% of the total area of prickly ash, and one of the largest plantings of Z. schinifolium is located in Jianyang City (Sichuan) with the area of 6.67 km2. Since 2018, Z. schinifolium, located in Jianyang City, have developed leaf spot disease, with approximately 50% showing disease symptoms. At the beginning of the occurrence, yellow-brown lesions formed on the leaves; in the later stages, the area of the lesions expanded. At the severe stage, multiple lesions merged into one large, dead spot, and the plants failed to blossom and bear fruit. The samples were collected from typical symptoms of Z. schinifolium leaves in Jianyang City. A total of 20 leaf samples were collected from 5 Z. schinifolium plants (4 leaves per plant), and were cut into small pieces of 2 × 2mm at the junction of infected and healthy tissues. These tissues were surface-disinfested for 30 s in 3% sodium hypochlorite and the for 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in a dark incubator at 25°C. Morphological observation was performed on 18 recovered isolates, 15 of which were described as Pestalotiopsis sp. The colonies were incubated on PDA at 25°C for 7 days and reached a diameter of 80-90 mm. The colonies were white with undulating edges and were similar in colors on the reverse side. After colony culture at 25°C for 10 days, gregarious black conidiomata were scattered on the mycelial mats. The conidia and appendages of the samples were measured by Leica Application Suite X 3.4.1.17822 (20 conidia per isolate), and the sizes of which were consistent with the description from Maharachchikumbura et al. Based on morphological observations, the isolates were identified as Pestalotiopsis kenyana Maharachch., K.D. Hyde & Crous. PCR was performed with primers ITS1/ITS4 for the ITS region, primers D1/D2 for the large subunit ribosomal RNA gene (LSU), primers 5f2/7cr for the RNA polymerase II second largest subunit (RPB2), primers Bt2a/Bt2b for the β-tubulin gene (TUB), and primers EF1-526F/EF2-567R for the translation elongation factor 1-alpha gene (TEF). The Sanger-sequenced PCR products were sequenced and blasted in GenBank, and the sequences showed that ITS: 99.17% (594 out of 599 bp), LSU: 100% (909 out of 909 bp), RPB2: 99.17% (832 out of 832 bp), TUB: 100% (774 out of 774 bp), TEF: 100% (485 out of 485 bp) with the type specimen of P. kenyana CBS 442.67 (ITS: GenBank accession NR147549.1, LSU: MH870724.1, PRB2: MH554958.1, TUB: KM199395.1, TEF: KM199502.1). Representative sequences were deposited in GenBank (ITS: MT509798; LSU: MT509800; RPB2: MT522448; TUB: MT522450; TEF: MT522449). To fulfill Koch's postulates, leaves on fifteen one-year-old healthy potted Z. schinifolium plants were sterilized by 75% ethanol cotton balls, and were rinsed by sterile water for three times. Then each leaf was punctured with sterile needles for two wounds (five leaves per plant). The wounds were inoculated by placing 8 mm mycelial plugs obtained from the periphery of 7-day-old single-spore cultures. An equal number of plants were wounded with the same method, and were respectively inoculated with sterile water and PDA plugs without mycelium as controls. All plants were placed in a growth chamber at 25°C under 90% relative humidity. After 7 days, all mycelial-inoculated leaves of the plants showed symptoms identical to those described above, whereas the control plants remained symptom free. P. kenyana was re-isolated from the infected leaves and confirmed to be the same as the inoculated pathogen through analyses of morphological characteristics and molecular techniques. The pathogenicity test was repeated three times with similar results. To our knowledge, this is the first report of P. kenyana as a causal agent of leaf spot disease on Z. schinifolium in China. These findings will aid the development of better preventive measures in accordance with the emergence of this new pathogen.

Episodic Nutrient Addition Affects Water Column Nutrient Processing Rates in River‐to‐Lake Transitional Zones

AbstractStorm‐driven nutrient loading from tributaries can fuel eutrophication in nearshore and open water areas of lentic ecosystems. However, nutrient processing in river‐to‐lake transitional zones can substantially alter the amount and composition of nutrients transported to lakes from upstream surface waters. We measured the removal of nutrients and dissolved organic carbon (DOC) from the water column in the Fox rivermouth (Green Bay, Lake Michigan) to evaluate the response of rivermouth plankton to episodic nutrient enrichment. Light and dark water column incubations (8–12 hr) were conducted on four occasions from April through September to measure changes in dissolved nitrogen (N), phosphorus (P), and DOC concentrations in three locations along the Fox rivermouth. Two incubation experiments were conducted on consecutive days, (a) under ambient nutrient concentrations, and (b) under experimentally enriched N and P concentrations. Spatial and temporal variation was observed in nutrient uptake rates, but light incubations consistently had higher nutrient uptake rates than dark incubations. Nutrient enrichment increased total dissolved P and total dissolved N uptake and DOC release in light incubations, but only increased total dissolved P uptake in dark incubations. Moreover, nutrient uptake ratios (N:P) decreased from ambient to nutrient enriched conditions and indicated preferential P uptake by phytoplankton communities in light conditions. Our study substantiates that rivermouths can process nutrients bound for downstream ecosystems and demonstrates the potential of plankton communities to dynamically increase net uptake rates in response to episodic nutrient enrichment.

Biodegradation and Photodegradation of Vegetation-Derived Dissolved Organic Matter in Tidal Marsh Ecosystems

Tidal wetlands are a significant source of dissolved organic matter (DOM) to coastal ecosystems, which impacts nutrient cycling, light exposure, carbon dynamics, phytoplankton activity, microbial growth, and ecosystem productivity. There is a wide variety of research on the properties and sources of DOM; however, little is known about the characteristics and degradation of DOM specifically sourced from tidal wetland plants. By conducting microbial and combined UV exposure and microbial incubation experiments of leachates from fresh and senescent plants in Chesapeake Bay wetlands, it was demonstrated that senescent material leached more dissolved organic carbon (DOC) than fresh material (77.9 ± 54.3 vs 21.6 ± 11.8 mg DOC L−1, respectively). Degradation followed an exponential decay pattern, and the senescent material averaged 50.5 ± 9.45% biodegradable DOC (%BDOC), or the loss of DOC due to microbial degradation. In comparison, the fresh material averaged a greater %BDOC (72.6 ± 19.2%). Percent remaining of absorbance (83.3 ± 26.7% for fresh, 90.1 ± 10.8% for senescent) was greater than percent remaining DOC, indicating that colored DOM is less bioavailable than non-colored material. Concentrations of DOC leached, %BDOC, and SUVA280 varied between species, indicating that the species composition of the marsh likely impacts the quantity and quality of exported DOC. Comparing the UV + microbial to the microbial only incubations did not reveal any clear effects on %BDOC but UV exposure enhanced loss of absorbance during subsequent dark incubation. These results demonstrate the impacts of senescence on the quality and concentration of DOM leached from tidal wetland plants, and that microbes combined with UV impact the degradation of this DOM differently from microbes alone.

Effect of curcumin-encapsulated Pluronic® F-127 over duo-species biofilm of Streptococcus mutans and Candida albicans.

To assess the effect of curcumin-encapsulated Pluronic® F-127 (Cur-Plu) during antimicrobial photodynamic therapy (aPDT) over duo-species biofilm of Streptococcus mutans and Candida albicans. Thermal analysis, optical absorption, and fluorescence spectroscopy were evaluated. Minimum inhibitory concentration (MIC) and minimum bactericidal/fungal concentration were obtained. The biofilms were cultured for 48h at 37°C and treated according to the groups: P + M + L + (photosensitizer encapsulated with Pluronic® F-127 + light); P + D + L + (photosensitizer incorporated in 1% DMSO + light); P - M + L + (no Pluronic® F-127 + light); P - D + L + (1% DMSO + light); P - L + (Milli-Q water + light); P + M + L - (photosensitizer encapsulated with Pluronic® F-127 no light); P + D + L - (photosensitizer in 1% DMSO, no light); P - M + L - (Pluronic® F-127 no light); P - D + L - (1% DMSO, no light); P - L - (Milli-Q water, no light; negative control group); CHX (0.2% chlorhexidine, positive control group); and NYS (Nystatin). Dark incubation of 5min was used. The groups that received aPDT were irradiated by blue LED (460nm, 15J/cm2). Cell viability of the biofilms was performed by colony-forming units (CFU/mL) and confocal microscopy. Two-way ANOVA followed by Tukey's post hoc test was used at a significance level of 5%. P + D + L + and P + M + L + groups exhibited better log-reduction for both Candida albicans and Streptococcus mutans biofilms than P - M + L + , P - L + , and P - D + L + experimental groups. Furthermore, P + M + L + and P + D + L + showed greater reduction for Candida albicans than for Streptococcus mutans. aPDT mediated by Cur-Plu can be a potential strategy for biofilm control against duo-species biofilm of Streptococcus mutans and Candida albicans.

Short-wavelength blue light contributes to the pyroptosis of human lens epithelial cells (hLECs)

PurposeThe purpose of this study is to examine the effect of short-wavelength blue light (SWBL) on cultured human lens epithelial cells (hLECs). The pathogenesis of cataracts after SWBL exposure is discussed. MethodsHLE-B3 hLECs were randomly divided into 3 groups: the NC group, which was grown in a dark incubator; the acetyl (Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) treatment group; and the SWBL exposure group. After SWBL (2500 lux) irradiation (for 8, 16, 24, and 32 h), caspase-1 and gasdermin D (GSDMD) expression levels in HLE-B3 hLECs were examined using ELISA, immunofluorescence staining, and Western blotting analyses. Double-positive staining of hLECs for activated and inhibited caspase-1 was used to determine pyroptosis in HLE-B3 hLECs. ResultsSWBL led to hLEC death, but a caspase-1 inhibitor suppressed cell death. The flow cytometry results also confirmed the dose-dependent effect of SWBL irradiation on the pyroptotic death of hLECs. Caspase-1 and GSDMD expression levels in all hLEC groups changed with blue light exposure times (8, 16, 24, and 32 h) and were higher in the AC-YVAD-CMK and SWBL exposure groups than in the NC group. The immunofluorescence results revealed higher GSDMD-N expression in the cell membrane of both the AC-YVAD-CMK and SWBL exposure groups than in the NC group. ConclusionsBased on the data, SWBL induces pyroptotic programmed cell death by activating the GSDMD signalling axis in HLE-B3 hLECs. These results provide new insights into the exploitation of new candidates for the prevention of cataracts.

Modeling the Effect of Temperature on Ginger and Turmeric Rhizome Sprouting

Ginger and turmeric are tropical plant species with medicinal, beverage, and edible uses. Both species are typically propagated using seed rhizomes that often lack uniformity when sprouting, ultimately affecting the transplant growth and quality. Our objectives were to (1) develop a model to predict the effect of temperature on rhizome sprouting and transplant growth and (2) characterize the morphological factors affecting the sprouting of ginger and turmeric rhizomes. Two experiments were conducted where the rhizomes were placed in plastic bags with a moist substrate inside dark incubator chambers. Five temperature treatments (21, 25, 27, 30, and 32 °C) were used for calibrating the model, and six temperature treatments (14, 20, 25, 30, 35, and 40 °C) were used in the validation phase. The number of days for rhizomes to develop 1- and 5-cm sprouts were counted; after which, the total number of sprouts, total leaf length, and root quality were measured. A nonlinear regression analysis was used to develop temperature–response curves. Ginger and turmeric had optimal sprouting temperatures of 27.5 and 30.1 °C, respectively. Temperatures close to the optimal reduced the time to sprout and to subsequently reach the transplant stage. No sprouting was observed at 14 °C, and the minimum temperature to develop 5-cm sprouts was estimated at slightly above 17 °C in both species. Temperatures above 32 °C resulted in tissue damage and rhizome loss. The results from this study show the potential to produce uniform ginger and turmeric transplants using temperature treatments that accelerate sprouting.

Benthic nutrients and oxygen fluxes at the water sediment interface in a pearl farming atoll (Ahe, Tuamotu, French Polynesia)

Benthic exchanges of oxygen and nutrient at the sediment-water interface were investigated under light and dark conditions at 5 selected sites in a sub-tropical atoll. Mean oxygen fluxes were - 1316.5 ± 242.0 μmol m−2 h−1 and mean effluxes of oxygen under light conditions were 2231.7 ± 626.4 μmol m−2 h−1, presumably due to microphytobenthos present at the sediment-water interface. The consequences of this high related productivity was a systematic consumption of nutrients (DIN, PO4 and Si(OH)4) during almost all light incubations, contrasting with the effluxes of nutrients during dark incubations. Our results suggest that the sediments were net autotrophic and the oxygen balance in favor of microbenthic production when compared to community demand. Diurnal rates of gross benthic primary productivity were high (3423 ± 1192 μmol m−2 h−1) which emphasize the role of microphytobenthos in maintaining the oxygen reservoir in tropical lagoons.

Megafire affects stream sediment flux and dissolved organic matter reactivity, but land use dominates nutrient dynamics in semiarid watersheds.

Climate change is causing larger wildfires and more extreme precipitation events in many regions. As these ecological disturbances increasingly coincide, they alter lateral fluxes of sediment, organic matter, and nutrients. Here, we report the stream chemistry response of watersheds in a semiarid region of Utah (USA) that were affected by a megafire followed by an extreme precipitation event in October 2018. We analyzed daily to hourly water samples at 10 stream locations from before the storm event until three weeks after its conclusion for suspended sediment, solute and nutrient concentrations, water isotopes, and dissolved organic matter concentration, optical properties, and reactivity. The megafire caused a ~2,000-fold increase in sediment flux and a ~6,000-fold increase in particulate carbon and nitrogen flux over the course of the storm. Unexpectedly, dissolved organic carbon (DOC) concentration was 2.1-fold higher in burned watersheds, despite the decreased organic matter from the fire. DOC from burned watersheds was 1.3-fold more biodegradable and 2.0-fold more photodegradable than in unburned watersheds based on 28-day dark and light incubations. Regardless of burn status, nutrient concentrations were higher in watersheds with greater urban and agricultural land use. Likewise, human land use had a greater effect than megafire on apparent hydrological residence time, with rapid stormwater signals in urban and agricultural areas but a gradual stormwater pulse in areas without direct human influence. These findings highlight how megafires and intense rainfall increase short-term particulate flux and alter organic matter concentration and characteristics. However, in contrast with previous research, which has largely focused on burned-unburned comparisons in pristine watersheds, we found that direct human influence exerted a primary control on nutrient status. Reducing anthropogenic nutrient sources could therefore increase socioecological resilience of surface water networks to changing wildfire regimes.