First Report on the Association of a 16SrII-A Phytoplasma with Sesame (Sesamum indicum) Exhibiting Abnormal Stem Curling and Phyllody in Taiwan.

Sesame (Sesamum indicum L.), an annual plant, is grown as an oilseed crop and the seeds are used in bakery products in Taiwan. In June 2013, plants exhibiting symptoms including phyllody and abnormal stem curling were observed in sesame fields in Pitou Township, Changhua County, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas associated with sesame exhibiting phyllody, witches'-broom, or virescence have been classified as strains of 16SrI-B in Myanmar (GenBank Accession No. AB558132), 16SrII-A in Thailand (JN006075), 16SrII-D in Oman (EU072505) and India (KF429486), 16SrIV-C in Iran (JF508515), and 16SrVI-A (KF156894) and 16SrIX (KC139791) in Turkey (1). Three symptomatic and four asymptomatic plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy (TEM) revealed clusters of phytoplasma cells ranging from 300 to 800 nm in diameter only in phloem sieve elements of stems of three symptomatic and two asymptomatic plants. Comparable tissues from two other symptomless plants were devoid of phytoplasma cells. Total DNA was extracted with a modified CTAB method (2) from plant tissues (100 mg each) including stem, leaf, petiole, and root from the same plants used for TEM work. Analyses by a nested PCR using universal primer pairs P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT/5'-CGTCCTTCATCGGCTCTT) followed by R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG/5'-TGACGGGCGGTGTGTACAAACCCCG) were performed to detect putative phytoplasma DNA (3). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, only from the three symptomatic and two asymptomatic plant tissues that had phytoplasma cells in their sieve elements. It is likely that these two asymptomatic plants were in the early stage of infection before symptoms became noticeable. The nested PCR products (1.2 kb) amplified from the symptomatic plants were cloned separately and sequenced (GenBank Accession Nos. KF923391, KF923392, and KF923393). BLAST analysis of the sequences revealed that they shared 99.2% sequence identity with strains reported from India and Thailand (KF429486 and JN006075), which were classified to the 16SrII-D and 16SrII-A subgroups, respectively. Moreover, iPhyClassifier software (4) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences shared 99.4% identity with that of the 'Candidatus Phytoplasma australasiae' (Y10097) and the RFLP patterns were identical to that of the 16SrII-A subgroup, indicating the Taiwanese strain is a 'Ca. P. australasiae'-related strain. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing phyllody and abnormal stem curling on sesame in Taiwan. The occurrence of phytoplasma on sesame could have direct implications for the cultivation of this economically important oilseed plant and the bakery industry in Taiwan. References: (1) M. Catal et al. Plant Dis. 97:835, 2013. (2) T. M. Fulton et al. Plant Mol. Biol. Rep. 13:207, 1995. (3) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of a 16SrI Group Phytoplasma Associated with Roselle (Hibiscus sabdariffa) Wrinkled Leaves and Phyllody Disorder in Taiwan.

Roselle (Hibiscus sabdariffa L.), an annual plant with acidic taste, has been used for making juice, jelly, and other baking additives in Taiwan. In September 2013, symptoms including phyllody and wrinkled leaves were observed on roselle plants in a field in Tantsu Township, Taichung County, Taiwan. Incidence of the infected plants was estimated to be greater than 80% within a single field. A phytoplasma was recently reported as the causal agent of roselle phyllody and reddening of leaves in India and classified as a group 16SrV-D strain (1). Samples including stems, flowers, and leaves were collected from four symptomatic and one asymptomatic roselle plants from the field. Transmission electron microscopy revealed clusters of phytoplasma cells ranging from 400 to 750 nm in diameter only in phloem sieve elements of petioles and stems of symptomatic plants. These cells were not observed in asymptomatic plants. Total DNA was extracted from plant tissues (100 mg each) including stems, petioles, and mid veins of leaves by a modified CTAB method (2). Analyses by a nested PCR assay using universal primer pairs P1/P7 followed by R16F2n/R16R2 were performed to detect putative phytoplasma (1). Each primer pair amplified a single PCR product 1.8 kb and 1.2 kb long, respectively, only from tissues of the four symptomatic plants. The nested PCR products (1.2 kb) amplified from three independent symptomatic plants were cloned separately and sequenced by automatic DNA sequencing method with ABI3730 DNA Analyzer (Applied Biosystems) at the Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (GenBank Accession Nos. KF923397, KF923398, and KF923399). BLAST analysis of the sequences revealed that they shared 99.8% sequence identity with those of 16SrI group phytoplasma strains, e.g., garlic yellows phytoplasma, torenia yellows phytoplasma, and periwinkle leaf yellowing phytoplasma (AB750363, FJ437568, and GU361754). Moreover, i PhyClassifier software (3) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequences derived from the symptomatic roselle samples. The 16S rDNA sequences shared 99.6% identity with those of the 'Candidatus Phytoplasma asteris' reference strain (M30790) and the RFLP patterns were identical to that of the 16SrI group. However, this strain may represent a new subgroup because the shared similarity coefficient was only 0.94, which is within the values set for a new subgroup (3). Taken together, these results indicate the phytoplasma infecting roselle in Taiwan is a 'Ca. P. asteris'-related strain belonging to the 16SrI group. To our knowledge, this is the first report of a 16SrI group phytoplasma causing wrinkled leaves and phyllody on roselle in Taiwan. The occurrence of phytoplasma on roselle could have direct implication for the bakery and juice industries in Taiwan. References: (1) C. Biswas et al. Phytoparasitica 41:539, 2013. (2) I. Echevarría-Machado et al. Mol. Biotechnol. 31:129, 2005. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

First Report of a 16SrII-A Subgroup Phytoplasma Associated with Purple Coneflower (Echinacea purpurea) Witches'-Broom Disease in Taiwan.

Purple coneflower (Echinacea purpurea), widely grown as an ornamental and medicinal plant, is a perennial flowering plant that is native to eastern North America. In July 2011, symptoms indicative of phytoplasma disease, including floral virescence, phyllody, and witches'-broom (WB), were observed to be affecting plants in coneflower fields in Wufeng, Taichung City, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas previously associated with purple coneflower WB disease have all been classified as aster yellows group (16SrI) strains (GenBank Accession Nos. EU333395, AY394856, EU416172, and EF546778) except for pale purple coneflower (Echinacea pallida) WB in Australia, which was identified as a subgroup 16SrII-D member (2). Three diseased plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy revealed clusters of phytoplasma cells ranging from 170 to 490 nm in diameter in phloem sieve elements of virescent and phylloid flowers and stems from diseased plants. Comparable tissues from symptomless plants were devoid of phytoplasma. Total DNA was extracted from plant tissue samples (50 to 100 mg each) including stems, leaves, and flowers by a modified CTAB method (1) from three symptomatic plants as well as from three asymptomatic coneflower plants seedlings. Analyses by a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 were performed to detect putative phytoplasma (2). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, from diseased plant tissues only. The nested PCR products (1.2 kb) amplified from phylloid flowers of the three diseased plants were cloned separately and sequenced (GenBank Accession Nos. JN885460, JN885461, and JN885462). Blast analysis of the sequences revealed a 99.7 to 99.8% sequence identity with those of Echinacea WB phytoplasma strain EWB5 and EWB6 (GenBank Accession Nos. JF340076 and JF340080), which reportedly belonged to the 16SrII-D subgroup (2). Moreover, iPhyClassifier software (3) was used to perform sequence comparison and generate the virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences share a 99.4 to 99.5% similarity with that of the 'Candidatus Phytoplasma australasiae' reference strain (Y10097) and the RFLP patterns are identical to that of the 16SrII-A subgroup. Taken together, these results indicated that the phytoplasma infecting purple coneflower in Taiwan is a 'Ca. Phytoplasma australasiae'-related strain and belongs to the 16SrII-A subgroup. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing WB disease on purple coneflower in Taiwan. The occurrence of phytoplasma on purple coneflower could have direct implication for the economically important ornamental, medicinal plant, and floral industry in Taiwan, especially to the growers and breeders that eagerly promote the purple coneflower industry. References: (1) T. M. Fulton et al. Plant Mol. Biol. Rep. 13:207, 1995. (2) T. L. Pearce et al. Plant Dis. 95:773, 2011. (3) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Motor equivalence and self-motion induced by different movement speeds.

This study investigated pointing movements in 3D asking two questions: (1) Is goal-directed reaching accompanied by self-motion, a component of the joint velocity vector that leaves the hand's movement unaffected? (2) Are differences in the terminal joint configurations among different speeds of reaching motor equivalent (i.e., terminal joint configurations differ more in directions of joint space that do not produce different pointer-tip positions than in directions that do) or non-motor equivalent (i.e., terminal joint configurations differ equally or more in directions of joint space that lead to different pointer-tip positions than in directions that do not affect the pointer-tip position). Subjects reached from an identical starting joint configuration and pointer-tip location to targets at slow, moderate, and fast speeds. Ten degrees of freedom of joint motion of the arm were recorded. The relationship between changes in the joint configuration and the three-dimensional pointer-tip position was expressed by a standard kinematic model, and the range- and null subspaces were computed from the associated Jacobian matrix. (1) The joint velocity vector and (2) the difference vector between terminal joint configurations from pairs of speed conditions were projected into the two subspaces. The relative length of the two components was used to quantify the amount of self-motion and the presence of motor equivalence, respectively. Results revealed that reaches were accompanied by a significant amount of self-motion at all reaching speeds. Self-motion scaled with movement speed. In addition, the difference in the terminal joint configuration between pairs of different reaching speeds revealed motor equivalence. The results are consistent with a control system that takes advantage of motor redundancy, allowing for flexibility in the face of perturbations, here induced by different movement speeds.

Regulation of the expression of angiotensin-converting enzyme 2 by polyunsaturated fatty acids in porcine adipocytes.

Using suppression subtractive hybridization technique, we found that 2 novel genes (AEUG1 and AEUG3) were highly expressed in the adipocytes compared with preadipocytes. We then identified that these 2 genes were both angiotensin-converting enzyme 2 (ACE2). We applied 3'RACE (rapid amplification of cDNA end), 5'RACE, and PCR to obtain the full-length porcine ACE2 cDNA sequence. Because eicosanoids derived from PUFA are involved in regulating blood pressure, we hypothesized that PUFA can regulate the expression of the vasodilator ACE2. Preadipocytes from Landrace pigs were induced to differentiate for 4 d, then treated with 50 µM of different PUFA, CLA, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or stearic acid (18:0). Addition of EPA or 18:0 for 48 h did not change the ACE2 mRNA abundance, whereas the treatments of arachidonic acid, CLA, and DHA significantly decreased ACE2 mRNA abundance after 48 h (P ≤ 0.05). Treatment with PUFA did not change (P > 0.05) type I and type II angiotensin receptor mRNA abundance. To further understand how PUFA metabolites affect ACE2 mRNA expression, we inhibited individual enzymes that are involved in eicosanoid production. We found that 3 individual eicosanoid pathway enzyme inhibitors recovered the PUFA effect on the expression of ACE2, indicating these pathways are involved in mediating the PUFA function. In conclusion, we obtained the full-length porcine ACE2 cDNA sequence and found PUFA could downregulate the expression of ACE2 through its metabolites without changing the expression of their receptor in porcine adipocytes.

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.