Characterization of Pythium chondricola associated with red rot disease of Pyropia yezoensis (Ueda) (Bangiales,Rhodophyta) from Lianyungang,China

Pyropia yezoensis(formerly Porphyra yezoensis) is an economically important red alga that is cultured extensively in China. The red rot disease occurs commonly during Pyropia cultivation,causing serious economic losses. An incidence of red rot disease was found in a P. yezoensis farm from mid-November to mid-December 2015 at Lianyungang, Jiangsu Province, China. Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen, leading to red rot symptoms. The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1(cox1). In artificial infection experiments on the P. yezoensis blades, the P. chondricola isolate was able to cause the same characteristic histopathology seen in natural infections. P. chondricola grew well at a wide range of temperatures in the range 8-31℃, salinities at 0-45 and pH 5-9. In an orthogonal test used to determine the effects of environmental factors(temperature, salinity, and zoospore concentration)on infection, the data revealed that temperature was the most important factor to affect red rot disease development, with the optimal conditions for disease expansion being 20℃, 35 salinity, and a zoospore concentration of 10~6 zoospores/mL. The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia, which will provide support to maintain the healthy development of the Pyropia industry in China.     

Identification and characterization of a Dna J gene from red alga Pyropia yezoensis(Bangiales,Rhodophyta)

Members of the Dna J family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length Dna J c DNA sequence from expressed sequence tags of Pyropia yezoensis( Py Dna J) via rapid identification of c DNA ends. This c DNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified Dna J proteins, such as a heat shock protein 40/Dna J from Pyropia haitanensis. The relative m RNA expression level of Py Dna J was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative m RNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that Py Dna J is an authentic member of the Dna J family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

Effects of Methyl Jasmonate on the Composition of Volatile Compounds in Pyropia yezoensis

Volatile organic compounds in marine algae have been reported to comprise characteristic flavor of algae and play an important role in their growth, development and defensive response. Yet their biogeneration remain largely unknown. Here we studied the composition of volatile compouds in Pyropia yezoensis and their variations in response to methyl jasmonate(MeJA) and diethyldithiocarbamic acid(DIECA) treatment using gas chromatography-mass spectrometry(GC-MS). A total of 44 compounds belonging to the following chemical classes(n) were identified, including aldehydes(11), alcohols(8), acids and esters(6), alkanes(5), ketones(5), alkenes(3), and S-or N-containing miscellaneous compounds(6). External treatment with plant hormone MeJA increased the content of 1-dodecanol, 4-heptenal, and 2-propenoic acid-2-methyl dodecylester, but decreased the content of phytol, 3-heptadecene, 2-pentadecanone, and isophytol. When pretreated with DIECA, an inhibitor of the octadecanoid pathway leading to the biosynthesis of endogeneous jasmonates and some secondary metabolites, phytol and isophytol were increased, while 4-heptenal, 1-dodecanol, and 2-propenoic acid-2-methyl dodecylester were decreased, both of which were negatively correlated with their variations under MeJA treatment. Collectively, these results suggest that MeJA does affect the volatile composition of P. yezoensis, and the octadecanoid pathway together with endogenous jasmonate pathway may be involved in the biosynthesis of volatile compounds, thereby providing some preliminary envision on the composition and biogeneration of volatile compounds in P. yezoensis.     

Pyropia yezoensis can utilize CO2 in the air during moderate dehydration

Pyropia yezoensis,an intertidal seaweed,experiences regular dehydration and rehydration with the tides.In this study,the responses of P.yezoensis to dehydration and rehydration under high and low CO 2 concentrations((600–700)×10-6 and(40–80)×10-6,named Group I and Group II respectively)were investigated.The thalli of Group I had a signifi cantly higher effective photosystem II quantum yield than the thalli of Group II at 71% absolute water content(AWC).There was little difference between thalli morphology,total Rubisco activity and total protein content at 100% and 71% AWC,which might be the basis for the normal performance of photosynthesis during moderate dehydration.A higher effective photosystem I quantum yield was observed in the thalli subjected to a low CO 2 concentration during moderate dehydration,which might be caused by the enhancement of cyclic electron fl ow.These results suggested that P.yezoensis can directly utilize CO 2 in ambient air during moderate dehydration.     

Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD) in Pyropia yezoensis. To prevent GSD from development and spread, an ef fective method to detect the pathogen at early GSD infection stages need to be established. In this research, PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein) and the dnaN gene(encoding β sliding clamp of DNA polymerase Ⅲ protein) to detect P. marina with three primer pairs pws-dnaA 2(Forward, 5′-ACCGCATTAACGAACTACTCGTG-3′; Reverse, 5′-TGCCATTACCTACAGCATGG-3′), pcs-dnaN 2(Forward, 5′-CTTACAACGTTATCAGCGGC-3′; Reverse, 5′-GTTGAGTATTAAGTGATTGAGTAAGC-3′) or pws-dnaN 3(Forward, 5′-ACTTACAACGTTATCAGCGGC-3′; Reverse, 5′-ACTGCTGTTTGAGTCTGCTAAC-3′). Three PCR methods corresponding to the three primer pairs su ? ciently distinguished P. marina from 22 bacterial species, thus resulting in detection limits of 4 to 4×10 2 CFU cells or 2.37×10 1 to 2.37×10 3 fg of P. marina DNA per PCR reaction. In an arti?cial infection experiment of P. yezoensis infected with P. marina, all established PCRs successfully detected P. marina at early GSD infection stages. The results show that the established PCRs are speci?c and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.     

Construction of high-density genetic linkage map of Pyropia yezoensis(Bangiales,Rhodophyta) and identification of red color trait QTLs in the thalli

Pyropia yezoensis is an important macroalga because of its extensive global distribution and economic importance. Color is an important trait in the thalli of P. yezoensis, it is also an effective marker to identify the hybridization in genetic breeding. In this study, a high-density genetic linkage map was constructed based on high-throughput single nucleotide polymorphism(SNP) markers, and used for analyzing the quantitative trait loci(QTLs) of red color trait in the thalli of P. yezoensis. The conchospore undergoes meiosis to develop into an ordered tetrad, and each cell has a haploid phenotype and can grow into a single individual. Based on this theory, F1 haploid population was used as the mapping population. The map included 531 SNP markers, 394.57 cM long on average distance of 0.74 cM. Collinear analysis of the genetic linkage map and the physical map indicated that the coverage between the two maps was 79.42%. Furthermore, QTL mapping identified six QTLs for the chromosomal regions associated with the red color trait of the thalli. The value of phenotypic variance explained(PVE) by an individual QTL ranged from 4.71%–63.11%. And QTL qRed-1-1, with a PVE of 63.11%, was considered the major QTL. Thus, these data may provide a platform for gene and QTL fine mapping, and marker-assisted breeding in P. yezoensis in the future.     

Involvement of cyclic electron flow in irradiance stress responding and its potential regulation of the mechanisms in Pyropia yezoensis

Pyropia yezoensis, belongs to the genus of P orphyra before 2011, inhabit on intertidal zone rocks where irradiation changes dramatically, implying that the seaweed has gained certain mechanisms to survive a harsh environment. Based on the photosynthetic parameters with or without the inhibitors determined by a Dual-PAM-100 apparatus, we investigated the photosynthetic performance and the changes in electron fl ow that occurred during the algae were stressed with dif ferent light intensities previously. When the irradiation saturation was approaching, the CEF around PS I became crucial since the addition of inhibitors usually led to an increase in non-photochemical quenching. The inhibitor experiments showed that there were at least three dif ferent CEF pathways in Py. yezoensis and these pathways compensated each other. In addition to maintaining a proper ratio of ATP/NAD(P)H to support effi cient photosynthesis, the potential roles of CEF might also include the regulation of dif ferent photoprotective mechanisms in Py. yezoensis. Under the regulation of CEF, chlororespiration is thought to transport electrons from the reduced plastoquinone(PQ) pool to oxygen in order to mitigate the reduction in the electron transfer chain. When irradiation was up to the high-grade stress conditions, the relative value of CEF began to decrease, which implied that the NADP+ pool or PQ + pool was very small and that the electrons were transferred from reduced PS I to oxygen. The scavenging enzymes might be activated and the water-water cycle probably became an ef fective means of removing the active oxygen produced by the irradiation stressed Py. yezoensis. We believe that the dif ferent mechanisms could make up the photoprotective network to allow Py. yezoensis for survival in a highly variable light stress habitat, which may enlighten scientists in future studies on irradiance stress in other algae species.     

Development of organelle single nucleotide polymorphism(SNP) markers and their application for the identification of cytoplasmic inheritance patterns in Pyropia yezoensis(Bangiales,Rhodophyta)

The genus Pyropia contains several important cultivated species.Genetic research in nori species has mainly focused on the cell nucleus,with few studies on organelles(chloroplast and mitocho ndria).Due to the high copy numbers of organelles in cells,which influence the development and traits of algae,it is necessary to study their genetic mechanism.In this study,the marine red alga yezoensis,an important economic macroalga,was selected as the study object.To investigate organelle(chloroplast and mitochondria) inheritance in P yezoensis,the wild type RZ(maternal strain) was cro ssed with the red mutant HT(paternal strain) and 30 color-sectors from 11 F1 gametophytic blades were examined.The complete chloroplast and mitochondrial genomes of the red mutant(HT) were assembled for the first time.One reliable and stable single nucleotide polymorphism(SNP) loci filtrated by bioinformatics analysis was used as a molecular marker for chloroplast and mitochondrial DNA,respectively,in subsequent experiments.PCR amplification and sequence analysis showed that the haplotypes of color-sectors detected were consistent with those of the maternal parent,confirming that both chloroplast and mitochondrial genomes were inherited maternally in P.yezoensis.The inheritance pattern of organelles in P.yezoensis can be used to guide the hybridization and breeding of nori.Additionally,the organelle SNP markers developed in this study can be applied in subsequent genetic research.     

The first complete organellar genomes of an Antarctic red alga,Pyropia endiviifolia:insights into its genome architecture and phylogenetic position within genus Pyropia(Bangiales,Rhodophyta)

Pyropia species grow in the intertidal zone and are cold-water adapted. To date, most of the information about the whole plastid and mitochondrial genomes(ptDNA and mtDNA) of this genus is limited to Northern Hemisphere species. Here, we report the sequencing of the ptDNA and mtDNA of the Antarctic red alga Pyropia endiviifolia using the Illumina platform. The plastid genome(195 784 bp, 33.28% GC content) contains 210 protein-coding genes, 37 tRNA genes and 6 rRNA genes. The mitochondrial genome(34 603 bp, 30.5% GC content) contains 26 protein-coding genes, 25 tRNA genes and 2 rRNA genes. Our results suggest that the organellar genomes of Py. endiviifolia have a compact organization. Although the collinearity of these genomes is conserved compared with other Pyropia species, the genome sizes show significant differences, mainly because of the different copy numbers of rDNA operons in the pt DNA and group II introns in the mtDNA. The other Pyropia species have 2–3 distinct intronic ORFs in their cox 1 genes, but Py. endiviifolia has no introns in its cox 1 gene. This has led to a smaller mtDNA than in other Pyropia species. The phylogenetic relationships within Pyropia were examined using concatenated gene sets from most of the available organellar genomes with both the maximum likelihood and Bayesian methods. The analysis revealed a sister taxa affiliation between the Antarctic species Py. endiviifolia and the North American species Py. kanakaensis.     

Effect of cytokinins and auxins on the growth of free-living conchocelis ofPorphyra yezoensis

IAA 3-Indolylacetic acid, NAA a-Naphthylacetic acid and cytokinins in PESI culture medium were used in a study on the effects of plant hormones on the growth of free-living conchocelis ofPorphyra yezoensis which showed that its growth in medium with cytokinins, IAA and NAA was more rapid than that in medium with non—phytohormones; that the optimal concentrations for promoting growth were 10 μg/L for IAA and ZA (Zeatin), and 0.1 μg/L for BA 6-Benzyl amino purine and KIN 6-Furfurylamino- purine. Mix use of NAA, IAA and cytokinins, NAA/ZA 1–1000/1 μg/L, NAA/BA 10/1-1000 μg/L, NAA/KIN 1/1-1000 μg/L promoted growth. IAA/ZA 0.1-1/0.1-1 μg/L; IAA/BA 0.1-1/0.1-10 μg/L IAA/KIN 1/0.1-1000 μg/L also promoted growth. Contribution No. 2335 from the Institute of Oceanology, Chinese Academy of Sciences. Contribution No. 94 from the Experimental Marine Biology Lab Qingdao, China. The research was supported by a grant from the Chinese Academy of Sciences.     

Identification and bioinformatics analysis of micro RNAs from the sporophyte and gametophyte of Pyropia haitanensis

Pyropia haitanensis(T.J.Chang et B.F.Zheng) N.Kikuchi et M.Miyata( Porphyra haitanensis) is an economically important genus that is cultured widely in China.P.haitanensis is cultured on a larger scale than Pyropia yezoensis,making up an important part of the total production of cultivated Pyropia in China.However,the majority of molecular mechanisms underlying the physiological processes of P.haitanensis remain unknown.P.haitanensis could utilize inorganic carbon and the sporophytes of P.haitanensis might possess a PCK-type C 4-like carbon-fixation pathway.To identify micro RNAs and their probable roles in sporophyte and gametophyte development,we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P.haitanensis.Five micro RNAs were identified that shared no sequence homology with known micro RNAs.Our results indicated that P.haitanensis might posses a complex s RNA processing system in which the novel micro RNAs act as important regulators of the development of different generations of P.haitanensis.     

Identification and characterization of a DnaJ gene from red alga <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> (Bangiales,Rhodophyta)

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis (PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.     

Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated(-73 0 generations,-two days each)to adapt freshwater BGll medium.A number of freshwater medium adapted colonyderived strains were obtained.The strains were verified phylogenetically to be N.oceanica LAMB0001 based on the 18 S ribosomal RNA gene.Freshwater-medium adapted strain(FA1)grew faster in the BG11 medium prepared with freshwater than wild-type N.oceanica grew in f/2 medium prepared with seawater.We assumed that(1)the expression patterns of the genes that expressed differentially between FA1 and the wild-type N.oceanica exposing to the BG11 medium(WT-F)have been reprogrammed;(2)the physiological processes in which these genes involved have been modified;and(3)a Gene Ontology(GO)term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up-or down-regulated if it was enriched simultaneously by up-or down-regulated DEGs between FA1 and WT-F,respectively.Under these assumptions,we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion,membrane and membrane integrity,material transportation,cell movement,and cellular signaling network.These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium.It is feasible to domesticate marine microalgae to a freshwater habitat,which may aid to modify their cultivation performances.     

APPLICATION OF CLAYS TO REMOVAL OF RED TIDE ORGANISMS Ⅰ.COAGULATION OF RED TIDE ORGANISMS WITH CLAYS

Coagulation of red tide organism cells with clays is discussed in theory and the effects of some fac-tors on it are studied.A quantitative model is presented which describes how the coagulation varies withpH in solution.According to the model,within the pH-range between pH_(zpc_a)and pH_(zpc_b),V_R is negativeand the coagulation is strong and strongest at pH=(pH_(zpc_a)+pH_(zpc_b))/2.It is also demonstrated that whenthe diameter of clay particles is close to that of cells,the collision probability is low,resulting in weakercoagulation.The model results have been corroborated by experiments and so are scientific and theoreticalbases for application.     

Identification of the toxic compounds produced by Sargassum thunbergii to red tide microalgae

The inhibitory effects of methanol extracts from the tissues of three macroalgal species on the growths of three marine red tide microalgae were assessed under laboratory conditions.Extracts of Sargassum thunbergii(Mertens ex Roth) Kuntz tissue had stronger inhibitory effects than those of either Sargassum pallidum(Turner) C.Agardh or Sargassum kjellmanianum Yendo on the growths of Heterosigma akashiwo(Hada) Hada,Skeletonema costatum(Grev.) Grev,and Prorocentrum micans Ehrenberg.Methanol extracts of S.thunbergii were further divided into petroleum ether,ethyl acetate,butanol,and distilled water phases by liquid-liquid fractionation.The petroleum ether and ethyl acetate fractions had strong algicidal effects on the microalgae.Gas chromatography-mass spectrometry analyses of these two phases identified nine fatty acids,most of which were unsaturated fatty acids.In addition,pure compounds of four of the nine unsaturated fatty acids had effective concentrations below 5 mg/L.Therefore,unsaturated fatty acids are a component of the allelochemicals in S.thunbergii tissue.     

APPLICATION OF CLAYS TO REMOVAL OF RED TIDE ORGANISMS Ⅲ.THE COAGULATION OF KAOLIN ON RED TIDE ORGANISMS

This article reports studies on the coagulation of kaolin(from Wuxian,Jiangsu,China)on various red tide organisms,and the observation for the first time that the coagulation of kaolin is much greaterthan that of montmorillonite so that kaolin is a more effective clay for removing red tide organisms.The authors’theoretical explanation and analysis by a mathematical-physical model prove that comparedto montmorillonite,kaolin has greater attraction for organism cells and therefore greater coagulation capabil-ity.This project’s studies on the effects of pH and acid-treatment show that the acid-treatment does not have much influence on the kaolin system;whereas the effect of pH on the kaolin system is the same asthat on the montmonrillonite system.     

Recent insights into physiological responses to nutrients by the cylindrospermopsin producing cyanobacterium,Cylindrospermopsis raciborskii

The harmful cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. Studies have demonstrated some key attributes of this species which may explain its global dominance. It has a high level of flexibility with respect to light and nutrients, being capable of growth under low and variable light conditions. However, it is the strategy with respect to nutrient utilization that has received more attention. Unlike many bloom forming species, the dominance of this species is not simply linked to higher nutrient loads. In fact it appears that it is more competitive when phosphorus and nitrogen availability is low and/or variable. An important component of this flexibility appears to be the result of within-population strain variability in responses to nutrients, as well as key physiological adaptations. Strain variability also appears to have an effect on the population-level cell quota of toxins, specifically cylindrospermopsins(CYNs). Field studies in Australia showed that populations had the highest proportion of toxic strains when dissolved inorganic phosphorus was added, resulting in stoichiometrically balanced nitrogen and phosphorus within the cells. These strategies are part of an arsenal of responses to environmental conditions, making it a challenging species to manage. However, our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology and ecology of this species.     

Effects of elevated CO2 on the photosynthesis and nitrate reductase activity of Pyropia haitanensis (Bangiales,Rhodophyta) grown at different nutrient levels

Pyropia haitanensis,a commercially important species,was cultured at two CO2 concentrations(390×10- 6 and 700×10-6(parts per million)) and at low and high nutrient levels,to explore the effect of elevated CO2 on the species under nutrient enrichment. Results show that in CO2-enriched thalli,relative growth rate(RGR) was enhanced under nutrient enrichment. Elevated CO2 decreased phycobiliprotein(PB) contents,but increased the contents of soluble carbohydrates. Nutrient enrichment increased the contents of chlorophyll a(Chl a) and PB,while soluble carbohydrate content decreased. CO2 enrichment enhanced the relative maximum electronic transport rate and light saturation point. In nutrient-enriched thalli the activity of nitrate reductase(NRA) increased under elevated CO2. An instantaneous pH change in seawater(from 8.1 to 9.6) resulted in reduction of NRA,and the thalli grown under both elevated CO2 and nutrient enrichment exhibited less pronounced reduction than in algae grown at the ambient CO2. The thermal optima of NRA under elevated CO2 and/or nutrient enrichment shifted to a lower temperature(10–15°C) compared to that in ambient conditions(20°C). We propose that accelerated photosynthesis could result in growth increment. Nassimilation remained high in acidified seawater and refl ected increased temperature sensitivity in response to elevated CO2 and eutrophication.     

Effects of Methyl Jasmonate on the Composition of Volatile Compounds in <Emphasis Type="Italic">Pyropia yezoensis</Emphasis>

Volatile organic compounds in marine algae have been reported to comprise characteristic flavor of algae and play an important role in their growth, development and defensive response. Yet their biogeneration remain largely unknown. Here we studied the composition of volatile compouds in Pyropia yezoensis and their variations in response to methyl jasmonate (MeJA) and diethyldithiocarbamic acid (DIECA) treatment using gas chromatography-mass spectrometry (GC-MS). A total of 44 compounds belonging to the following chemical classes (n) were identified, including aldehydes (11), alcohols (8), acids and esters (6), alkanes (5), ketones (5), alkenes (3), and S- or N-containing miscellaneous compounds (6). External treatment with plant hormone MeJA increased the content of 1-dodecanol, 4-heptenal, and 2-propenoic acid-2-methyl dodecylester, but decreased the content of phytol, 3-heptadecene, 2-pentadecanone, and isophytol. When pretreated with DIECA, an inhibitor of the octadecanoid pathway leading to the biosynthesis of endogeneous jasmonates and some secondary metabolites, phytol and isophytol were increased, while 4-heptenal, 1-dodecanol, and 2-propenoic acid-2-methyl dodecylester were decreased, both of which were negatively correlated with their variations under MeJA treatment. Collectively, these results suggest that MeJA does affect the volatile composition of P. yezoensis, and the octadecanoid pathway together with endogenous jasmonate pathway may be involved in the biosynthesis of volatile compounds, thereby providing some preliminary envision on the composition and biogeneration of volatile compounds in P. yezoensis.