Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Rapid detection of Pythium porphyrae in commercial samples of dried Porphyra yezoensis sheets by polymerase chain reaction

The fungal parasite Pythium porphyrae is the causative organism of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried Porphyra yezoensis sheets was achieved using the species-specific primers PP-1 (5′-TGTGTTCTGTGCT-CCTCTCG-3′) and PP-2 (5′-CCCAAATTGGTGTTGCCTCC-3′) with the polymerase chain reaction (PCR). The DNA sequence (707 bp) of PCR product was found to be identical to that amplified from ITS rDNA extracted from a type species of P. porphyrae (IFO 30800, The Institute of Fermentation, Osaka, Japan). Quantities of the product amplified varied with the time when samples were harvested after the occurrence of red rot disease in Porphyra farms. This simple, rapid, and inexpensive method should have great applications in furthering quality control and determination of quality ranking in the Porphyra processing industry.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Carbohydrate regulation of attachment, encystment, and appressorium formation by Pythium porphyrae (Oomycota) zoospores on Porphyra yezoensis (Rhodophyta)

Pythium porphyrae Takahashi et Sasaki, a facultative parasite of Porphyra spp., is the common microbial agent responsible for red rot disease of this red alga in Japan. Host infection by this species and other plant parasitic members of the Pythiaceae is initiated by motile biflagellate zoospores. Factors regulating host specificity and the initial steps involved in the infection process, consisting of attachment, encystment and appressorium formation, are not known. Zoospore encystment and appressorium formation of P. porphyrae were monitored by staining of the fungal cell walls using calcofluor. The zoospores infected only Porphyra spp. and Bangia atropurpurea (Roth) C. Agardh thalli, although they attached to, and encysted on, many other members of the Rhodophyceae (Stylonema alsidii[Zanardini] Drew, Gelidium elegans Kützing, Pterocladiella capillacea[Gmelin] Santelices et Hommersand, Carpopeltis affinis[Harvey] Okamura, Gloiosiphonia capillaris[Hudson] Carmichael in Berkeley, Grateloupia turuturu Yamada, Callophyllis adhaerens Yamada, Gracilaria spp., Lomentaria hakodatensis Yendo, Rhodymenia intricata[Okamura] Okamura, Griffithsia subcylindrica Okamura, Wrangelia tanegana Harvey, and Polysiphonia morrowii Harvey). No attachment or encystment was observed on the red alga Kappaphycus striatum (Schmitz) Doty ex Silva in Silva et al., the brown algae Undaria pinnatifida (Harvey) Suringar, Scytosiphon sp., and Sargassum thunbergii (Mertens ex Roth) Kuntze as well as members of the Ulvaceae (green algae). Sequential extraction of carbohydrates from Porphyra yezoensis Ueda thalli and the addition of diverse monosaccharides, polysaccharides, and amino acids to zoospore suspensions indicated that encystment and appressorium formation were induced only by sulfated galactans (porphyran, commercial agar, agarose, and carrageenans). Zoospore attachment and encystment on thalli of P. yezoensis was abolished by periodate oxidation of the thallus surface and was reduced by 80–90% after enzymatic removal of sulfated galactan (porphyran). It appears that the interaction of zoospore surface receptors with sulfated galactan (porphyran) determinants on the thallus surface induced specific attachment and encystment on Porphyra spp. thalli. Zoospores encysted, germinated, and formed appressoria on sulfated galactan films and in suspensions of this carbohydrate. Attachment and encystment were induced on commercial agar and agarose films, but appressoria were not induced on agarose films. Supplementation of agarose media with both cold and hot water fractions and with porphyran from P. yezoensis–induced appressoria implicated sulfated galactans (porphyran) in appressorium formation.     

Simultaneous infection by red rot and chytrid diseases in <Emphasis Type="Italic">Porphyra yezoensis</Emphasis>Ueda

This paper presents the results of a study on the diseases of Porphyra yezoensisUeda along the north coast of China, where red rot (Pythium porphyrae) and the chytrid Olpidiopsis sp. diseases were both found to be present. Infection by the mycelia of Pythium porphyraeand the thallus of Olpidiopsis sp. was studied in detail. At the early stage of infecton the mycelia of Pythium porphyraeand the fungus of chytrid can be found in host cells at the same time. In the middle and late stages of the complication, it mainly appears as red rot disease, toward the end appearing almost completely as red rot disease. The complication even can be found on the cells of fronds from the freeze-storage nori nets. However, the freeze-storage nets can help prevent spread of the infection and improve nori quality.     

Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release,development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta)

Growth at the restrictive temperature (42 degrees C) of Aspergillus nidulans B120, carrying the conditional-lethal mutation sod(VI)C1, was partially improved by the addition of 1.0 M sorbitol to the medium. The mutant grown at 42 degrees C, with osmotic stabilizer, showed abnormal hyphal morphology, a decrease in beta-1,3-glucan synthase activity as well as cell wall sugar content, but an increase in chitin synthase activity and N-acetyl-glucosamine content. The mutation also affected the secretion of extracellular protease. The temperature-dependent osmo-sensitive phenotype of a Saccharomyces cerevisiae alpha-COP mutation can be rescued by the A. nidulans sod(VI)C(+) gene. These results indicate that the sod(VI)C1 mutation affects proper processing of secretory proteins destined for the surface of cells or beyond.     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

Effect of DMSO on PCR of Porphyra yezoensis (Rhodophyta) gene

In order to improve the predictability ofresults of PCR with Porphyra yezoensisUeda genes, a study was made of possiblemodifications to the basic PCR protocol. DMSO used as an adjuvant considerablyincreased amplification efficiency andspecificity of PCR, the optimalconcentration being 5%. This protocolallowed for DNA templates with a high GCcontent to be amplified by PCR withoutproblem.     

Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda conchospore germlings (1–4-cell stages) were treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) for inducing mutations. Three kinds of color-mutated gametophytic blades, which were composed of the mutated cells wholly, sectorially or spottedly, were obtained; and most of them were sectorially variegated blades. The highest frequency of these mutated blades was 1.3%. Four different pigmentation mutant strains were obtained by regenerating single cells and protoplasts that were enzymatically isolated from the mutated sectors of the sectorially variegated blades. The mutants were relatively stable in color in both gametophytic blade and conchocelis phases. In the two phases, each mutant strain showed characteristic differences in the in vivo absorption spectra, and had different pigment contents of major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) as compared with the wild-type and with each other. The gametophytic blades from the four mutant lines showed significant differences in growth and photosynthetic rates, when they were cultured in the same conditions. By crossing the mutant with the wild-type, it was found that the color phenotypes of two mutants reported above, were resulted from two mutations in different genes, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of a cDNA encoding a homologue of actin from Porphyra yezoensis (Rhodophyta)

We report the nucleotide sequence of a cDNA encoding an actin from amarine red alga, Porphyra yezoensis Ueda. A cDNA clone wasisolated from a leafy gametophyte cDNA library and analyzed for the sequence.The clone contained an open reading frame for a protein of 373 amino acidswhichexhibits sequence similarity to known actins. The GC content of the thirdposition (83.9%) was much higher than that at the first (56.3%) and second(42.4%) positions. The actin forms a gene family in the P.yezoensis genome. Comparison of the deduced amino acid sequenceshowed higher similarity to the Florideophycidae Chondruscrispus (85%) than to the ProtoflorideophycidaeCyanidioschyzon merolae (70%). The mRNA was detected inboth the leafy gametophytes and filamentous sporophytes. The nucleotidesequence data reported in this paper will appear in theDDBJ/EMBL/GenBank databases under accession number AB039831.     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues

Forces exerted by hyphae of the phytopathogen Pythium graminicola and mammalian pathogen Pythium insidiosum were compared with the mechanical resistance of their hosts' tissues. Hyphal apices of both species exerted a mean force of 2 microN, corresponding to mean pressures of 0.19 microN microm(-2) (or MPa) for P. graminicola, and 0.14 microN microm(-2) for P. insidiosum. Experiments with glass microprobes showed that the epidermis of grass roots resisted penetration until the pressure applied at the probe tip reached 1-12 microN microm(-2). Previously published data show that mammalian skin offers even greater resistance (10-47 microN microm(-2)). Clearly, tissue strength exceeds the pressures exerted by hyphae of these pathogens, verifying that secreted enzymes must play a critical role in reducing the resistance of plant and animal tissues. It is presumed that hyphae are sufficiently powerful to bore through any obstacles remaining after enzyme action.     

Estimation of the degree of self-fertilization in Porphyra yezoensis (Bangiales,Rhodophyta)

Crosses between genotypically distinct thalli of the monoecious species Porphyra yezoensis were carried out using immature thallus fragments from green- and red-type color mutants and also wild-type thalli. As the genes governing the mutants are monogenic, recessive to the wild-type, and belong to the same linkage group, the degree of self-fertilization could be estimated based on the pigmentation of the resultant diploid conchocelis. The degree of self-fertilization in the cross between the green-type and the wild-type was 48.5–55.0%, and in the cross between the red-type and the wild-type was 45.1–56.5%. In the cross between the green- and red-type mutants, the degree of self-fertilization was 46.0–54.5% when the green-type was the female parent, and was 44.8–55.6% when the red-type was the female parent.     

紫菜腐霉激发子基因家族特征及其在感染过程中的作用

[背景]激发子(elicitin)是卵菌(Oomycetes)疫霉和腐霉分泌的可诱发宿主产生免疫反应的小分子化合物.[目的]鉴定紫菜腐霉激发子基因家族,分析其结构特征和在感染宿主过程中可能的作用机制.[方法]运用同源比对法筛查紫菜腐霉NBRC33253基因组中激发子基因家族成员,利用生物信息学工具分析激发子家族的理化性...     

Rapid DNA extraction from conchocelis and ITS-1 rDNA sequences of seven strains of cultivated Porphyra yezoensis (Bangiales, Rhodophyta)

In order to extract DNA rapidly from cultivated Porphyra, we extracted total DNA from conchocelis using the ISOPLANT II kit (Nippon Gene) without liquid nitrogen treatment or CsCl-gradient ultracentrifugation. By confirming the reproducibility of RAPD patterns, it is concluded that the quality of the extracted DNA is sufficient to use as a template for molecular investigation. Using this rapid method, the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions was amplified from seven strains of cultivated Porphyra, which had been maintained as free-living conchocelis by subculturing in the laboratory. From the amplified DNAs, the ITS-1 sequences were determined in order to identify the species and genetic relationship of the strains. The sequences were identical in the seven strains, and all the strains were identified as P. yezoensis. Furthermore, the gametophytic blades of these strains showed long linear or oblanceolate shapes in the laboratory culture. It was concluded that these strains are P. yezoensis form. narawaensis. This rapid DNA extraction method from conchocelis will be a powerful tool for phylogenetic analysis and for genetic improvement of cultivated Porphyra.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.     

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota)

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac 1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Acl, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Acl, 3, 4. Correspondence to: D. Bhattacharya     

Free amino acid contents of the gametophytic blades from the green mutant conchocelis and the heterozygous conchocelis in Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

Free amino acid contents in green mutant(G-1) blades and sectored F₁gametophytic blades with green andwild-type portions, which were developedfrom heterozygous conchocelis obtained by across between the wild type (0110) and thegreen mutant (G-1) of Porphyrayezoensis, were compared with those of thewild-type blades in laboratory culture. The contents of the major four free aminoacids (aspartic acid, glutamic acid,alanine and taurine) as well as those ofthe total free amino acids were highest inthe green mutant blades, intermediate inthe F₁ gametophytic blades, and lowestin the wild-type blades. A similar trendwas obtained in the blades developed frommonospores of the F₁ gametophyticblades. In addition, the green-typesectors also had a higher content of thefour major free amino acids and total freeamino acids compared with the wild-typesectors in the F₁ blades cultivated ata nori farm. The green mutant ischaracterized by higher contents of thefour major free amino acids compared withthe wild type, which has a higher growthrate. Hence, it is considered that thesectored F₁ gametophytic bladesproduced from the heterozygous conchocelishave both parental advantages (high freeamino acid contents and high growth rate)and compensate for both parentaldisadvantages. This seems to be one of thepossible ways of genetic improvement inregards to the taste of nori and stableproduction in Porphyra cultivation.