First Report of Leaf and Stem Downy Blight of Longan Seedlings Caused by Peronophythora litchii in Taiwan.

Fruit downy blight caused by Peronophythora litchii Chen ex Ko et al. is an important disease of litchi (Litchi chinensis Sonn.) in Taiwan, especially in rainy seasons. Previous records indicate litchi as the only natural host of P. litchii, but this pathogen was found on seedlings of longan (Euphoria longana Lamarck) in 2000. Young seedlings of longan that had emerged in a litchi orchard near Caotun, Nantou County, showed symptoms of droopy leaves and leaf blight. Water-soaked lesions appeared on young leaves, which turned into brown, round or irregular lesions (about 3 to 5 cm long). Diseased leaves withered and collapsed eventually. Dark brown lesions were found on stems of some infected seedlings but none of the infected seedlings were killed. Also, no symptoms were found on mature leaves. The pathogen produced numerous sporangia on sporangiophores on diseased leaves under humid conditions. The disease on young seedlings was observed again in another litchi orchard at Caotun, Nantou County, in 2003. P. litchii was consistently isolated from diseased leaves. Two isolates from colonized longan seedlings, Tari 20250 collected in 2000 and Tari 23301 collected in 2003, were used for further studies. Both isolates produced large numbers of sporangia on long sporangiophores when cultured on 5% V8 agar (5% V8 juice, 0.02% CaCO3, and 1.5% agar). Sporangia produced on the same sporangiophores matured almost simultaneously. Sporangiophores 240 to 1,600 µm (mean 623 µm) branched dichotomously two to eight times. Sporangia were oval or lemon-shaped with semispherical papilla and deciduous with very short pedicels (2 to 5 µm). The dimension was 25 to 55 (35.25) × 15 to 27.5 (21.2) µm for sporangia and 0.5 to 1 (0.55) µm for pedicels. The length/breadth (L/B) ratio of sporangia was 1.3 to 2.14 (1.67). Both isolates produced numerous oospores on 5% V8 agar cultures in darkness. Artificial inoculation tests were done by spraying 5 mL of sporangial suspension (1,000 sporangia/mL) on each longan seedling without wounding. Results showed that both longan isolates of P. litchii were pathogenic on young longan seedlings, causing symptoms similar to those observed on leaves and stems of naturally infected longan seedlings in litchi orchards. Also, both longan isolates of P. litchii caused downy blight on fruits of litchi (L. chinensis var black leaf) by artificial inoculation tests. Moreover, a P. litchi isolate from litchi caused symptoms of leaf blight on young longan seedlings. P. litchii was reisolated from the infected longan tissues. The ribosomal internal transcribed spacer (ITS) sequence confirmed that the longan isolate Tari 20250 (GenBank Accession No. JQ814693) was 100% identical to other P. litchii isolates (GenBank Accession Nos. Gu111613 to Gu111615). To our knowledge, this is the first report of longan as a natural host of P. litchii. The study also suggests that P. litchii on volunteer longan seedlings in litchi orchards may be a potential source of inoculum for fruit downy blight of litchi. References: (1) C. C. Chen. Special Publ. Coll. Agric., Natl. Taiwan Univ. 10:1, 1961. (2) W. H. Ko et al. Mycologia 70:380, 1978.

First Report of Phytophthora cambivora Causing Leaf and Stem Blight and Root Rot on Taiwan Cherry (Prunus campanulata) in Taiwan.

Taiwan cherry or Formosan cherry (Prunus campanulata Maxim.) is a beautiful ornamental tree that is native to Taiwan. In spring 2005, a severe disease was observed on 1- to 3-year-old seedlings of Taiwan cherry in a garden in Tungshih, Taichung, Taiwan. Infected plants showed symptoms of greenish water-soaked spots on leaves that became dark brown, 2 to 3 cm in diameter. Infected leaves withered and fell to the ground in 3 to 5 days and young shoots showed symptoms of withering and drooping. Infected roots showed symptoms of necrosis. Severely infected plants eventually died. A Phytophthora sp. was isolated consistently from diseased samples of Taiwan cherry and associated soil. Six isolates of Phytophthora, of the A1 mating type (1), were isolated from single zoospores. Two of these isolates, Tari 25141 (deposited as BCRC34932 in Bioresource Collection and Research Center, Shinchu, Taiwan) and Tari 25144 (BCRC34933), were used for pathogenicity tests on 1-year-old seedlings of Taiwan cherry to fulfill Koch's postulates. Inoculation was done by placing a cotton swab containing zoospore suspension on leaves or stem, or by soaking seedlings in the zoospore suspension. Inoculated seedlings were kept in a greenhouse at 20 to 25°C for 30 days and examined for appearance of symptoms. Results showed that both isolates were pathogenic on seedlings of Taiwan cherry, causing symptoms similar to those observed on naturally infected seedlings. The temperature range for growth of the six isolates of Phytophthora was 8 to 32°C with optimum temperature at 24°C. The linear growth rate was 72 mm per day on V8A culture (5% V8 vegetable juice, 0.02% CaCO3, and 2% Bacto agar) at 24°C. The colonies on potato dextrose agar produced sparse aerial mycelia with conspicuous radiate patterns. Sporangia were sparse on V8A agar blocks, but abundant when the agar blocks were placed in water under continuous white fluorescent light (average 2,000 lux) for 2 days. Sporangiophores branched sympodially. Sporangia were pear shaped, nonpapillate and nondeciduous, 50 to 75 (62) × 30 to 48 (40) µm, with a length/width ratio of 1.2 to 2.2 (1.6). New internal nested proliferate sporangia were formed inside the empty sac of old matured sporangia after releasing zoospores. No chlamydospores were formed on V8A. Hyphal swellings with distinctive irregular catenulation were produced on V8A and in water. The pathogen was stimulated to form its own oospores by the A2 tester using the method described by Ko (1). Oogonia were 28 to 50 (40) µm in diameter with smooth or irregularly protuberant walls. Oospores were mostly aplerotic and 18 to 42 (31) µm in diameter. Antheridia were amphigynous, mostly two-celled, and 10 to 42 (29) × 12 to 24 (19) µm. The sequence of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of isolate Tari 25141 (GenBank Accession No. GU111589) was 831 bp and had 99% sequence identity with a number of Phytophthora cambivora isolates such as GenBank Accession Nos. HM004220 (2), AY787030, and EF486692. Based on the morphological characteristics of sporangia and sexual structures and the molecular analysis of ITS sequences, the pathogen from Taiwan cherry was identified as P. cambivora (Petri) Buis. To our knowledge, this is the first report of P. cambivora on native Taiwan cherry in Taiwan and, so far, no other natural hosts have been reported. References: (1) W. H. Ko. J. Gen. Microbiol. 116:459, 1980. (2) P. W. Reeser et al. Mycologia 103:225, 2011.

Relationship between bone turnover and density with teriparatide, denosumab or both in women in the DATA study.

BACKGROUND: While changes in biochemical markers of bone turnover (BTM) have been reported to predict changes in bone mineral density (BMD), the relationship between changes in BMD and BTMs with combined antiresorptive/anabolic therapy is unknown. METHODS: In the DATA study, 94 postmenopausal osteoporotic women (ages 51-91) received either teriparatide 20-mcg SC daily, denosumab 60-mg SC every 6months, or both for 2years. Pearson's correlation coefficients (R) were calculated to determine the relationship between baseline and early changes in BTMs (as well as serum sclerostin) and 2-year changes in BMD. RESULTS: In women receiving teriparatide, baseline BTMs did not correlate with 2-year BMD changes though 12-month increases in osteocalcin and P1NP were associated with 2-year increases in spine BMD. In women receiving denosumab, spine and hip BMD gains correlated with both baseline and changes in P1NP and C-telopeptide. In women receiving combined teriparatide/denosumab, while both baseline and decreases in P1NP were associated with spine BMD gains, distal radius increases were associated with less CTX suppression. Neither baseline nor changes in serum sclerostin correlated with BMD in any treatment group. SUMMARY AND CONCLUSIONS: In women treated with teriparatide or denosumab, early BTM changes (increases and decreases, respectively) predict 2-year BMD gains, especially at the spine. In women treated with combined teriparatide/denosumab therapy, BMD increases at the distal radius were associated with less suppression of bone turnover. These results suggest that efficacy of combination therapy at cortical sites such as the radius may depend on residual bone remodeling despite RANKL inhibition.

Effects of Two Years of Teriparatide, Denosumab, or Both on Bone Microarchitecture and Strength (DATA-HRpQCT study).

CONTEXT: In postmenopausal osteoporosis, combining denosumab and teriparatide increases hip and spine bone mineral density more than either monotherapy. OBJECTIVE: The objective of the study was to determine the effects of 2 years of combination therapy on bone microarchitecture and estimated strength. DESIGN: This was an open-label, randomized controlled trial. PARTICIPANTS AND METHODS: We performed high-resolution peripheral quantitative computed tomography at the distal tibia and radius in 94 postmenopausal osteoporotic women randomized to 2 years of teriparatide 20 µg sc daily, denosumab 60 mg sc every 6 months, or both. RESULTS: Total volumetric bone mineral density (vBMD) at the radius and tibia, trabecular vBMD at the radius, and cortical vBMD at the tibia all increased more in the combination group than both monotherapy groups (P < .002 for all comparisons with combination). Cortical thickness at the tibia also increased more in the combination group (8.1% ± 4.3%) than both other groups (P < .001). Cortical porosity at both the radius and tibia increased progressively over the 24-month treatment period in the teriparatide group but was stable in both other groups (P < .001 teriparatide vs both other groups). Trabecular vBMD at the tibia increased similarly in all groups, whereas radius trabecular vBMD increased more in the combination group than the other groups (P < .01 for both comparisons). Finite element analysis-estimated strength improved or was maintained by all treatments at both the radius and tibia. CONCLUSIONS: Two years of combined teriparatide and denosumab improves bone microarchitecture and estimated strength more than the individual treatments, particularly in cortical bone. These findings suggest that this regimen may be beneficial in postmenopausal osteoporosis.

Differential expression of glycogen synthase kinase 3 genes during zebrafish embryogenesis.

Glycogen synthase kinase 3 (GSK-3) belongs to a highly conserved family of protein serine/threonine kinase whose members in high eukaryotes are involved in hormonal regulation, nuclear signaling, and cell fate determination. We have identified two zebrafish homologues related to mammalian GSK-3, ZGSK-3alpha and ZGSK-3beta. ZGSK-3alpha was expressed uniformly from cleavage onward, and later was found in many but not all tissues, especially in the central nervous system, spinal cord, somites and pronephric ducts. ZGSK-3beta was also transcribed maternally but the transcripts were not uniformly distributed during early cleavage stage. Most signals were concentrated in the inner part of the blastomeres. From midblastula stage onward, the ZGSK-3beta transcripts remained confined to inner parts of the deep cell layer. During shield stage, both epiblast and hypoblast expressed the transcripts. After late gastrulation, the signals were detected ubiquitously. During segmentation, prominent ZGSK-3beta signal was detected in head portion of the neural system. In the trunk, the expression was maintained in the neural tube and paraxial mesoderm and then became prominent in adaxial cells, followed by expression at the posterior region of somites. In pharyngula period ZGSK-3beta transcripts were distributed in similar regions as those of ZGSK-3alpha, namely, neural tissues of the head portion, spinal cord and somites.

Two-step cell disruption for the extraction of membrane-associated recombinant protein from Saccharomyces cerevisiae.

The use of rDNA technology to express heterologous proteins has been very successful during the last several years. Choice of an expression host is very important in order to retain the biological activity of recombinant proteins. Baker's yeast, Saccharomyces cerevisiae, is a eucaryotic GRAS organism suitable for the expression of biologically active proteins. Specifically, hepatitis B surface antigen (HBsAg) is expressed in baker's yeast. Because the yeast cells need to be disrupted for the recovery of bioactive intracellular proteins and because the protein HBsAg is hydrophobic and has a tendency to become associated with cell membranes, the use of detergent increases the recovery yield. In order to remove most of the contaminants from yeast, a two-step disruption/extraction scheme has been developed that facilitates downstream processing. Furthermore, it also has the advantage of minimizing proteolytic actions on the recombinant protein by removing most of the contaminants and proteases into the supernatant during the first disruption step, while keeping the desired protein in the pellet fraction. Final recovery is then achieved by the extraction process. Parameters affecting the disruption/extraction processes have been discussed.

Induction and inhibition of cytochrome P-450-dependent monooxygenases in hamster tissues by ethanol.

The effects of ethanol on hamster hepatic and extrahepatic monooxygenases were determined in the present study. Chronic ethanol administration increased cytochrome P-450 (P-450) content and monooxygenase activities towards aniline, N-nitrosodimethylamine, and 7-ethoxyresorufin. In contrast, benzphetamine and benzo(a)pyrene oxidation rates were decreased 21-24% by ethanol. In kidney, ethanol pretreatment increased P-450 content, aniline and N-nitrosodimethylamine oxidation activities. In lung, ethanol ingestion selectively increased aniline hydroxylation without affecting other monooxygenase activities. Intestinal monooxygenase activity was refractory to ethanol induction. Immunoblotting of the microsomal proteins showed that ethanol induced a protein cross-reactive with rabbit antibody raised against human P-450 2E1 in hamster liver, kidney, and lung. Immunoblotting analysis using mouse monoclonal antibody 1-12-3 raised against scup P-450 1A1 revealed that ethanol induced an immunorelated protein in hamster liver, kidney, and lung. Induction of P-450 2E1 and 1A was not observed with intestinal protein blots. Immunoblotting analysis using mouse monoclonal antibody 2-66-3 against rat P-450 2B1 showed inhibition of an immunorelated protein in ethanol-treated hamster liver. The inhibitory effect on P-450 2B was not observed with extrahepatic tissues. These results suggest that ethanol has the ability to induce P-450s 2E1 and 1A and to inhibit P-450 2B in hamster tissues.

Tissue specificity of induction of hamster monooxygenase activity by ethanol.

The effects of ethanol on liver, kidney and intestine monooxygenases were studied using hamsters chronically fed with isocaloric control and ethanol-containing liquid diets. The inductive effects of ethanol on liver and kidney aniline hydroxylase activities began to approach plateau level after the animals were fed ethanol for two weeks. Intestinal aniline hydroxylation was refractory to ethanol induction. In control and ethanol-fed hamsters, CO-difference spectra of hepatic and extrahepatic microsomes differed in absorption maxima. Chronic alcohol consumption caused significant increases of cytochrome P-450 and cytochrome b5 contents of liver and kidney microsomes. The increases of the heme proteins were associated with the induction of aniline hydroxylase, N-nitrosodimethylamine demethylase and 7-ethoxycoumarin 0-deethylase activities. In contrast to the liver and kidney, intestinal microsomal cytochromes P-450 and b5 contents in ethanol-treated animals were lower than the controls. Ethanol pretreatment was without effect on intestinal monooxygenase activities toward the metabolism of aniline, N-nitrosodimethylamine, 7-ethoxycoumarin and benzo(a)pyrene. Gel electrophoresis of tissue microsomes from control and ethanol-treated hamsters revealed that ethanol treatment enhanced the intensity of the protein band(s) in the cytochrome P-450 molecular weight region in the liver and kidney, but not in the intestine. These results demonstrate that in hamsters the response of monooxygenase to ethanol may vary from tissue to tissue and it is difficult to make a generalization regarding the inducing property of ethanol. The differential effect on cytochrome P-450 may be an important factor in determining the interaction between ethanol and xenobiotic metabolism in animal tissues.

Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver, kidney, and lung.

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochrome b5 contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline. N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 and b5. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues.(ABSTRACT TRUNCATED AT 250 WORDS)