Serological detection of red rot disease initiation stages of microbial pathogen, Pythium porphyrae (Oomycota) on Porphyra yezoensis

Pythium porphyrae (Oomycota), a pathogen causing red rot diseasein Porphyra spp., can at present only be detected when colonizationof the host thallus has already occurred and so it is often too late to takeappropriate disease control measures. The paper presents an account of an effective methdology for early detection of the disease. Since Py.porphyrae zoospores are the primary means of pathogen dispersal,polyclonal antibodies (Pabs) were raised against the surface components ofzoospores and encysted zoospores. Using these Pabs the disease initiationstages of the Pythium porphyrae were detected on the surface of Porphyra thalli by immunofluorescence assay. The specificity of theseantibodies and the efficacy of immunofluorescence assay in the detectionof red rot disease are discussed.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

An antifungal protein from the marine bacterium streptomyces sp. Strain AP77 is specific for Pythium porphyrae,a causative agent of red rot disease in Porphyra spp

A novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium, Streptomyces sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses. By using gel filtration, the molecular mass of SAP was estimated to be 160 kDa. Structural analysis of SAP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that SAP is composed of three heterologous protein subunits of 41.7 kDa (SAP1), 21.7 kDa (SAP2), and 18.7 kDa (SAP3) at a molar ratio of 1:1:5 (or 1:1:6). N-terminal amino acid sequence analysis and a homology search revealed that SAP1, SAP2, and SAP3 exhibit 64.3, 68.4, and 86.7% similarity to three Streptomyces coelicolor polypeptides, puromycin resistance protein (Pur8), a conserved hypothetical protein, and bacterioferritin, respectively. The MIC of purified SAP against Pythium porphyrae was determined to be 1.6 microg/disk, whereas no inhibitory effect was observed at concentrations up to 100 microg/disk against most of the fungal and bacterial strains tested; the only exception was relatively strong antifungal activity against Pythium ultimum (MIC, 6.3 microg/disk). In vitro and in vivo toxicity tests demonstrated that SAP showed no toxicity against Porphyra yezoensis cells, human normal dermal fibroblasts, and mice at doses up to 700 microg/ml (for 24 h), 250 microg/ml (for 12 h), and 75 mg/kg (for 35 days), respectively. SAP was labile when it was subjected to a heated-air drying treatment, which is a great advantage in food production procedures. These results indicated that Streptomyces sp. strain AP77 might be useful as a gene source for safe transgenic Porphyra breeding for tolerance to Pythium infection.     

Rapid isolation and sequence analysis of the beta-tubulin gene from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp) from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced 458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank databases under the accession number AY221630.     

Sensory and fatty acid analyses of two Atlantic species of Porphyra (Rhodophyta)

Sensory analyses were conducted to determine levels of consumer acceptability of Porphyra yezoensis, P. umbilicalis, and P. amplissima to select appropriate species for aquaculture development in Maine (USA). The subjects included children (n = 67) and adults (n = 84); the children participated in study design by helping to select the 9 point hedonic scale used in the affective sensory tests. Two substrates were used; Porphyra was baked in crackers and also used as a coating for popcorn. No significant differences (p > 0.5) in acceptability of one species over another were observed in either trial, which suggests that native Atlantic species of Porphyra such as P. amplissima and P. umbilicalis have developmental potential in foods for North American consumers. Fatty acids were analyzed in the taste test material and in freshly collected P. umbilicalis; eicosapentaenoic acid [EPA; 20:5 (n-3)] and palmitic acid were the most common fatty acids. Quantitative analysis of EPA determined that freshly collected (January 2005) P. umbilicalis contained 3.2 mg EPA g dry wt⁻¹ (74 mg EPA 100 g fresh wt⁻¹). This concentration is not high enough to make P. umbilicalis a primary source of daily omega-3 fatty acids, but the favorable n-3/n-6 ratio (2-3:1) in these species contributes to their nutritional value.     

甘蔗赤腐病内生拮抗菌YC89的筛选及鉴定

【目的】筛选防治甘蔗赤腐病(sugarcane red rot)的生防菌株。【方法】实验以前期分离获得的甘蔗内生细菌为目标菌,以甘蔗赤腐病的病原真菌镰孢炭疽菌(Colletotrichum falcatum Went.)为指示菌,采用平板对峙法筛选对该病菌有较强抑制作用的菌株,然后通过琼脂扩散法测定菌株代谢产物对抑菌活性的影响,并对具有较好拮抗效果的高效菌株进行抑菌广谱性分析并对其进行鉴定。最后通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对高效菌株YC89进行鉴定。【结果】经初筛筛选到抑菌带均大于1.60 cm的5株拮抗细菌,其中X22、W2、YC89抑菌带均高达1.87 cm。对初筛得到的5株内生菌进行复筛,结果所示菌株YC89、H1、X22、W2、YT93对镰孢炭疽菌的抑菌率都在75%以上,其中菌株YC89对该病菌的抑菌效果最好,其抑菌率为78%。菌株YC89的发酵液、上清液、过滤液及粗蛋白提取液对镰孢炭疽菌的生长有较强的抑制作用,且菌株YC89对玉米大斑病、甘蔗梢腐病、草莓灰霉病等7种病原菌也有较好的抑制效果。通过菌株鉴定结果,初步将YC89菌株鉴定为贝莱斯芽孢杆...     

Increasing the transient expression of GUS gene in Porphyra yezoensis by 18S rDNA targeted homologous recombination

In order to test whether 18S rDNA can influence positively GUS gene transient expression in the red alga Porphyra yezoensis, a targeting vector pQD-GUS was constructed containing a portion of the 18S rDNA of P. yezoensis and transformed it into the same strain protoplasts. The results showed that GUS protein activity was increased markedly with pQD-GUS compared to the parent pBS-GUS. It is suggested that this system can be used to enhance the expression of exogenous genes in transgenic P. yezoensis.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

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.     

Pythium aphanidermatum root rot of pawpaw (Carica papaya L.) in Nigeria

Root rot of pawpaw (Carica papaya L.) reported in Nigeria is caused byPythium aphanidermatum which was consistently isolated from diseased plant parts and highly pothogenic. Out of 16 different media tested, it grew best on corn-meal-agar (CMA) and CMA supplemented with cellulose and sucrose. The highest number of oospores/ml was on CMA with average diameter of 19.9±0.1 µm. The symptom is characterized by dark brown rot of roots, absence of secondary roots and disintegration of internal tissue of the main root. These cause the progressive decline of the aerial parts of the tree untill it dies.