Disease resistance induced by nonantagonistic endophytic Streptomyces spp. on tissue-cultured seedlings of rhododendron

Of 82 strains of endophytic actinomycetes isolated from rhododendron plants, 12 were not antagonistic against Pestalotiopsis sydowiana, which is the causal agent of Pestalotia disease. Of these 12, MBR-37 and MBR-38 (identified as Streptomyces spp.) grew on IMA-2 medium. Tissue-cultured seedlings of rhododendron treated with these nonantagonistic strains showed less wilting and/or smaller lesions to P. sydowiana, although the degree of resistance was a little lower than that conferred by antagonistic Streptomyces galbus strain R-5. These seedlings accumulated the anthocyanin(s), suggesting that resistance induced by these strains could depend on activated defense responses associated with the phenylpropanoid pathway rather than with antibiosis.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

A single gene transfer of gibberellin biosynthesis gene cluster increases gibberellin production in a Fusarium fujikuroi strain with gibberellin low producibility

Fusarium fujikuroi, the causative agent of bakanae disease in rice, produces many kinds of secondary metabolites. Recently, two phylogenetic subgroups (F and G groups) of Japanese F. fujikuroi have been identified and found to have differences in their gibberellin (GA) and fumonisin production. G-group F. fujikuroi produces large amounts of GA, but is a fumonisin nonproducer. F-group produces large amounts of fumonisin, but is a GA low or nonproducer. We investigated the cause of low GA production in the F-group. Genetic mapping suggests that low GA production in the F-group strain Gfc0825009 is due to a GA gene cluster for GA biosynthesis. Analysis of the nucleotide and amino acid sequences of the genes in the GA gene cluster showed >98.4% homology between the F-group strain Gfc0825009 and the G-group strain Gfc0801001. Following a 7-day culture under low nitrogen conditions, we found that expression of P450-1, P450-4, and P450-2 in the cluster increased in the G-group strain and not in the F-group strain. We hypothesized that complementation by GA genes in the G-group strain would be required to increase GA production in the F-group strain. However, we found that this occurred with a single gene complementation of DES, P450-1, P450-4, or P450-2. Simultaneous increase in the expression of P450-1, P450-4, and P450-2 were detected in the complementary transformants. Moreover, the same phenomenon was observed by reintegration of its own P450-1. Our results suggest the presence of unknown regulatory mechanisms of the GA gene cluster in F. fujikuroi.     

A promising strain of endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. for biological control of cucumber anthracnose

In a survey for effective biocontrol agents of Colletotrichum orbiculare, causal agent of cucumber anthracnose, 43 (MBCu series) and 135 (MBPu series) endophytic actinomycete strains were recovered from surface-sterilized organs of cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) plants, respectively. The strains were cultured with C. orbiculare on IMA-2 agar medium to determine their in vitro antagonistic ability. Eleven strains that strongly inhibited hyphal growth of the pathogen were selected as potent antagonists. Detached cotyledons of cucumber were soaked in a spore suspension of an antagonistic strain 1 day before challenge-inoculation with the pathogen, and six of these antagonists (MBCu-32, 36, 42, 45, and 56, and MBPu-75) significantly reduced the number and size of the lesions on the cotyledons compared to the untreated control. In the same way, these six strains inhibited lesion development on attached leaves of 3-week-old cucumber seedlings. Strain MBCu-56, the most suppressive of the strains, was selected for further tests. Its suppressiveness increased as concentrations increased; pretreatment of leaves with the strain at 10⁷, 10⁸ and 10⁹ cfu/ml suppressed disease by 72, 79 and 93%, respectively. These results strongly indicated that MBCu-56 has strong potential for controlling cucumber anthracnose. Based on the taxonomic characteristics and 16S rDNA sequence, MBCu-56 was identified as Streptomyces sp. With scanning electron microscopy, the substrate mycelia of the strain were seen to colonize the surface of leaves above the cuticle. Some hyphae also penetrated and grew underneath the cuticle.     

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.     

Food-entrainable circadian oscillator in goldfish: multiple daily feeding times and food-anticipatory activity

The existence of a food-entrainable circadian oscillator (FEO) was examined in goldfish Carassius auratus. Single goldfish were exposed to light–dark (LD) 12:12 (lights on 06:00–18:00 h) and fed at 12:00 h (phase I). The photoperiod regime was shifted to constant light (LL) (phase II). The fish were then fed at 06:00 or at 18:00 h (phase III), and all fish were fasted (phase IV). In both groups, three out of eight fish exhibited food-anticipatory activity (FAA) in phases II and III, the free-running period (τ) of locomotor activity was approximately 24.0 h, and circadian rhythms of locomotor activity started to free-run from the previous FAA in phase IV, indicating the existence of an FEO. Next, single goldfish were exposed to LD 12:12 and were fed twice daily at 12:00 and 00:00 h (phase I). The photoperiod regime was shifted to LL (phase II), and the fish were fasted (phase III). In phase II, FAA was observed in three out of six fish for both feeding times. In phase III, a circadian rhythm of locomotor activity was observed in five fish, and circadian rhythms started to free-run from the previous feeding time (either 12:00 or 00:00 h) in three fish. These results suggest that individual goldfish can remember two daily feeding times.     

Phylogeography of the Japanese eight-barbel loach <Emphasis Type="Italic">Lefua echigonia</Emphasis> from the Yamagata area of the Tohoku district,Japan

The Japanese eight-barbel loach Lefua echigonia, which is a freshwater fish native to Japan, is distributed from the Tohoku to Kinki districts and is divided into six regional populations according to mtDNA analysis. In this study, we investigated L. echigonia collected from several locations in Yamagata Prefecture and neighboring prefectures using mtDNA control region sequences and confirmed the spatial distribution pattern among the new regional population (Yamagata population). The new population was limited to the Mogami river system in the inland area of Yamagata Prefecture and is distinguished from other regional populations by high sequence divergences.     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

Seasonal potential fishing ground prediction of neon flying squid (Ommastrephes bartramii) in the western and central North Pacific

We explored the seasonal potential fishing grounds of neon flying squid (Ommastrephes bartramii) in the western and central North Pacific using maximum entropy (MaxEnt) models fitted with squid fishery data as response and environmental factors from remotely sensed [sea surface temperature (SST), sea surface height (SSH), eddy kinetic energy (EKE), wind stress curl (WSC) and numerical model‐derived sea surface salinity (SSS)] covariates. The potential squid fishing grounds from January–February (winter) and June–July (summer) 2001–2004 were simulated separately and covered the near‐coast (winter) and offshore (summer) forage areas off the Kuroshio–Oyashio transition and subarctic frontal zones. The oceanographic conditions differed between regions and were regulated by the inherent seasonal variability and prevailing basin dynamics. The seasonal and spatial extents of potential squid fishing grounds were largely explained by SST (7–17°C in the winter and 11–18°C in the summer) and SSS (33.8–34.8 in the winter and 33.7–34.3 in the summer). These ocean properties are water mass tracers and define the boundaries of the North Pacific hydrographic provinces. Mesoscale variability in the upper ocean inferred from SSH and EKE were also influential to squid potential fishing grounds and are presumably linked to the augmented primary productivity from nutrient enhancement and entrainment of passive plankton. WSC, however, has the least model contribution to squid potential fishing habitat relative to the other environmental factors examined. Findings of this work underpin the importance of SST and SSS as robust predictors of the seasonal squid potential fishing grounds in the western and central North Pacific and highlight MaxEnt's potential for operational fishery application.     

Changes in brain and pituitary GnRH levels during ovarian maturation in wild female Japanese flounder

To elucidate the role of gonadotropin-releasing hormone (GnRH) in gonadal maturation in wild female Japanese flounder Paralichthys olivaceus, we monitored changes in the levels of seabream GnRH (sbGnRH) in the olfactory bulb, telencephalon, hypothalamus, and pituitary during ovarian development together with changes in plasma levels of testosterone (T), estradiol-17β (E₂), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). Fish were caught offshore of the northern mainland of Japan in the Pacific Ocean at 3- to 4-week intervals between April and September by gill net. The netted fish were categorized into six groups based on ovarian stages: previtellogenic (April–early May), early yolk (April–late May), late yolk (late May–June), early spawning (June–August), late spawning (September), and termination (September) stages. The gonadosomatic index significantly increased from the previtellogenic to early spawning stages and decreased thereafter. In the olfactory bulb, no significant differences were observed in sbGnRH levels among the developmental stages. In contrast, sbGnRH levels in the telencephalon and hypothalamus were very high in the previtellogenic stage, lower in the early spawning stage, and relatively high in latter stages. sbGnRH levels in the pituitary were high in the previtellogenic stage and low in the early spawning stage. In addition, the relatively high levels of pituitary sbGnRH were found together with high plasma T, E₂, and DHP levels in fish in the late yolk stage. These results indicate that sbGnRH in the telencephalon, hypothalamus, and pituitary is involved in ovarian maturation and that sbGnRH may play an important role in the initiation of ovarian recrudescence in wild Japanese flounder.