Petiole Tissues Research Articles

Study on obtaining of hairy root of Panax vietnamensis by using rol gene transformation via Agrobacterium rhizogenes

Having the higher saponin content in comparison with other plants of the genus Panax, Panax vietnamensis has been considered as one of the most valuable medical plants in Vietnam. Using Agrobacterium rhizogenes to transfer rol gene to increase hairy root formation is a high potential method in producing secondary metabolites in this plant. The experiment was conducted on 2 different kinds of tissues of in vitro seedling including leaves and petioles. These tissues were used as source of infection. The purpose of this experiment is to induct hairy root by using Agrobacterium rhizogenes to transfer rol gene to these tissues. Leaves and petioles of in vitro plantlets were infected and incubated in 30 mins, then cultured for 72 hrs. The result showed that the hairy root induction rate and the number of roots were higher in petiole tissues compared with those in leaf tissues (14.8%, 2.8% and 3.3%, 1.6% respectively). Hairy root induction in petiole tissues was in 5 weeks and that in leaves tissues was in 6 weeks. The presence of rolC in produced hair roots was confirmed by PCR. Secondary metabolite analysis of transgenic plants also showed the presence of MR2, Rb1 and Rg1 which are typically active elements inPanax vietnamensis. Liquid culture with shaking is the most suitable condition for proper hairy root development. The success of promoting root formation in our study will be potential to produce saponin in industrial scale.

Pseudomonas Blight Caused by Pseudomonas syringae on Raspberry in California.

In February 2013 in coastal California (Santa Cruz County), plantings of red raspberry (Rubus idaeus var. strigosus) exhibited symptoms of a previously undocumented disease. Initial symptoms were small (less than 5 mm wide), angular, water-soaked lesions on leaf and petiole tissues of recently emerged foliage. Lesions were observable from both adaxial and abaxial leaf surfaces. As disease progressed, lesions enlarged and coalesced, resulting in significant dark brown to black blighting of the foliage. The foliage of severely affected plants was stunted and wilted. The disease affected 5 ha and incidence was approximately 30%. Cream-colored bacterial colonies were isolated from surface disinfested symptomatic tissue that was macerated and streaked onto King's medium B (KMB) and sucrose peptone agar (SPA). Fungi were not recovered from any tissue that was surface disinfested and placed into acidified potato dextrose agar. Four representative strains were fluorescent on KMB and gram-negative based on lysis by KOH. Strains were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices but induced a hypersensitive response in tobacco (Nicotiana tabacum L. cv. Samsun); strains thus belonged to Lelliot's LOPAT group 1, P. syringae (3). All four strains had identical DNA fragment-banding patterns generated by repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer (4). The pattern generated was different than all P. syringae pathovars in genomospecies 1 including P. syringae pv. syringae. According to multilocus sequence analysis conducted by previously described methods, the strains are most closely related to P. syringae pv. aceris and P. syringae pv. solidagae in genomospecies 1 (1). Potted raspberry plants were used to test four strains for pathogenicity. Inoculum was prepared by growing the bacteria on SPA for 48 h and suspending the bacteria in sterile distilled water (SDW) for a final concentration of approximately 107 CFU/ml. Suspensions were sprayed until runoff onto three replicate plants per strain. Control plants were sprayed with SDW until runoff. Plants were enclosed in plastic bags for 24 h and then maintained in a greenhouse (23 to 25°C). After 7 to 8 days, water soaked lesions developed on all inoculated plants; lesions later turned dark brown and appeared similar to symptoms observed in the field. Plants treated with water developed no symptoms. Bacteria re-isolated onto KMB from symptomatic tissues were fluorescent and appeared identical to the bacteria used to inoculate the plants; two selected re-isolated strains were identical to the original strains according to rep-PCR, fluorescence, and LOPAT reactions. The experiment was repeated and disease development and recovery of fluorescent strains on KMB was identical to the first experiment. To our knowledge, this is the first report of Pseudomonas blight of raspberry, caused by P. syringae, in California. Affected plants initially were stunted in growth but later in the summer exhibited no lasting effects from the disease. Pseudomonas blight has been reported in the Pacific Northwest region of the United States, the British Columbia region of Canada, and Serbia (2).

Lead heavy metal toxicity induced changes on growth and antioxidative enzymes level in water hyacinths [Eichhornia crassipes (Mart.)

BackgroundLead (Pb) heavy metal pollution in water bodies is one of the serious problems across the world. This study was designed to find out the effect of Pb toxicity on physiological and biochemical changes in Eichhornia crassipes (water hyacinth) seedlings.ResultsThe plant growth was significantly inhibited (50%) at 1000 mg/L Pb concentration. Accumulation of Pb was higher in root than in shoot tissues. The maximum level of Pb accumulation was noticed in roots (5.45%) followed by petiole (2.72%) and leaf tissues (0.66%). Increasing the Pb concentration gradually decreased the chlorophyll content. Intracellular distribution of Pb was also studied using SEM-EDX, where the Pb deposition was observed in both root and leaf tissues. MDA content increased in both the leaf and root tissues up to the 400 mg/L Pb treatment and slightly decreased at higher concentrations. The activity of antioxidative enzymes, such as APX and POX, positively correlated with Pb treatment, while in the case of SOD and CAT enzymes increased up to 800 mg/L treatment and then slightly decreased at higher concentration in both leaf and root tissues.ConclusionsThese results suggest that water hyacinth plants have efficient mechanism to tolerate Pb toxicity, as evidenced by an increased level of antioxidative enzymes. Results clearly indicate that water hyacinth is a feasible plant for hyperaccumulation of heavy metals from polluted wetlands.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0054-6) contains supplementary material, which is available to authorized users.

First Report of ‘Candidatus Liberibacter solanacearum’ in Carrot in France

In summer 2012, carrot (Daucus carota L.) plants displaying symptoms of leaf yellowing, stunting and proliferation of dwarfed shoots with bushy tops, and a dense hairy growth of secondary roots were observed. Symptomatic carrots were collected from three fields used for seed production and located in Region Centre of France near Orléans. The presence of psyllids (Psyllidae) in one of the fields was reported but they were not clearly identified. Fifty percent of the field was infected. Due to a large amount of plant debris, the harvested seeds were difficult to separate and the germination rate was low (from 10 to 77%), rendering them unmarketable. The symptoms observed were similar to those described for carrots infected by Aster yellows phytoplasma and 'Candidatus Liberibacter solanacearum' in Europe (3). Total DNA was extracted from petiole and root tissue of 16 symptomatic and 6 asymptomatic carrots (cv. Amsterdam, CAC3075), 2 samples of black nightshade leaves (Solanum nigrum) collected from the same fields, and 2 samples of carrot plants (cv Berlicum) grown in a high containment greenhouse, using a cetyl trimethyl ammonium bromide (CTAB) buffer extraction method. All DNA extracts were tested for phytoplasmas (1) and for 'Ca. L. solanacearum' by real-time PCR (2). DNA extracts were also tested for 'Ca. L. solanacearum' by PCR using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rpIJ/rpIL ribosomal protein genes, respectively (4). DNA from greenhouse carrot plants yielded no amplicon with all PCR. Phytoplasma was not detected in any of the tested samples. However, amplification was observed with the real-time PCR assay for 'Ca. L. solanacearum' (2) for all DNA samples extracted from symptomatic and asymptomatic field carrots (cycle threshold [ct] values between 16.75 and 30.59), and from S. nigrum (ct between 31.62 and 33.25). For field carrot DNA, a 1,168-bp 16S rDNA fragment and a 669-bp rpIJ/rpIL fragment were amplified whereas DNA from S. nigrum yielded no amplicon. Four amplicons obtained from these PCR assays with both primer pairs from symptomatic carrot samples were sequenced directly (Beckmann Coulter Genomics, Grenoble, France). BLAST analysis of the 16S rDNA sequences (KF357911) showed 99% nucleotide identity to those of 'Ca. L. solanacearum' amplified from carrot in Finland (GU373049). The rpIJ/rpIL nucleotide sequences (KF357912) were 99% identical to sequences of the analogous rpIJ/rpIL 'Ca. L. solanacearum' ribosomal protein gene from carrot in Spain (JX308305). These results confirmed the presence of 'Ca. L. solanacearum' in all symptomatic and asymptomatic carrot sampled in Region Centre, France. To our knowledge, this is the first report of this pathogen in carrot in France. These results, in addition to those previously obtained (4), suggest a wider distribution of 'Ca. L. solanacearum' than previously reported in Europe and should lead plant health managers to consider this pathogen as an emerging threat.

Caesalpinia spinosa (Caesalpiniaceae) leaves: anatomy, histochemistry, and secondary metabolites

Caesalpiniaspinosa (Molina) Kuntze is an extensively used plant in the industry and folk medicine, and it is characterized by containing high quantities of tannins in its pods. Ultrastructure and chemical aspects of C.spinosa leaves were analyzed to identify their properties and the function of their specialized cells. Leaflets are amphistomatic with an internal bifacial structure. Subspherical idioblasts occur on the subepidermal area of leaflets and are immersed in the parenchymatous tissue of rachis and petiole. Histochemical analyses showed that tannins are widespread on the parenchymatous tissue of leaflets, rachis, and petioles, and subspherical idioblasts present a lipophilic nature containing essential oils and lipids. Glandular trichomes occurring on the rachis and petiole bear phenolic compounds. GC–MS analysis reveals the presence predominantly of monoterpenes in the leaf essential oil. The detected compounds may be related to the antimicrobial- and antioxidant activity of C. spinosa extracts.

Transposon based activation tagging in diploid strawberry and monoploid derivatives of potato.

Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds. As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5% in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85% reliable in strawberry and potato, respectively), although the effective concentration differed (0.5% for strawberry and 0.03% for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.

Measurement of the concentration of nutrients in grapevine petioles by attenuated total reflectance Fourier transform infrared spectroscopy and chemometrics

Background and Aims We determined the effectiveness of attenuated total reflectance Fourier transform infrared spectroscopy as a rapid and low-cost method for measuring the concentration of nutrients in grapevine petioles. Methods and Results Attenuated total reflectance Fourier transform infrared spectra were recorded for 392 dried and ground petiole samples collected from Chardonnay, Semillon and Shiraz vineyards in the Riverina between 2003 and 2010. Predictive models of nutrient concentration obtained with standard reference methods were developed using partial least squares regression. Good predictive models were produced for all macronutrients, with R2 values for nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca) and sulfur (S) of 0.945, 0.915, 0.951, 0.961, 0.940 and 0.849, respectively. For sodium (Na) and the micronutrients iron (Fe), manganese (Mn), boron (B), copper (Cu) and zinc (Zn), R2 values were 0.773, 0.750, 0.743, 0.630, 0.612 and 0.835, respectively. When ranked according to the residual predictive deviation, expressed as the ratio of the standard error of performance to the calibration set standard deviation, values of 5.4, 3.8, 3.1, 3.8 and 3.5 were obtained for N, P, K, Mg and Ca, respectively; intermediate values of 2.4, 2.0 and 1.8 were obtained for S, Zn and Na, while Fe, Mn, B, Cu had a value of 1.3 or below. Conclusion All macronutrients in grapevine petiole tissue can be determined by attenuated total reflectance Fourier transform infrared spectroscopy with sufficient accuracy to assess grapevine nutritional status against standard interpretive ranges. Certain micronutrients can also be determined, although the predictive models for these elements were not as robust as models for the macronutrients. Significance of Study The study presents a rapid, low-cost and easy-to-implement technique suited to the widespread adoption and routine analysis of plant tissue for the management of grapevine nutrition in the Australian winegrape vineyards.

Modification of serological techniques and their evaluation for detection of potato viruses in seed certification related activities

Development of alternative serological techniques to ELISA for detection of potato viruses offers advantages for monitoring virus incidence and for seed potato certification systems. Several trials showed that multiplex tissue print immunoassay (TPIA) and dot blot immunoassay (DBIA) might represent fast, practical, and sensitive alternatives for the detection of: Potato leaf roll virus (PLRV), Potato virus S (PVS), Potato virus X (PVX) and Potato virus Y (PVY), from green and/or tuber tissues. In TPIA, the specific precipitation patterns in infected tissues of leaf petioles or stem cross sections, observed with each virus, allowed identification of the specific virus or mixed infections in a single multiplex assay. For detection of PVY in green tissues, DBIA was shown to be over 50 times more sensitive than ELISA. TPIA and ELISA from the tuber stem end or from eyes might be used for rapid detection of PVY and PVS in seed potato tubers without prior germination. PVS was evenly distributed in potato tuber tissue, while PVY was localized in the vascular tissue beneath the epidermis, with irregular distribution along the periphery of the potato tuber. For laboratories in developing countries lacking time and facilities for tests based on tuber germination, monitoring for PVS and PVY using TPIA in tuber tissue may be a suitable alternative to ELISA.

Differences of Three Ampeloviruses' Multiplication in Plant May Explain Their Incidences in Vineyards.

Grapevine leafroll ampeloviruses have been recently grouped into two major clades, one for Grapevine leafroll associated virus (GLRaV) 1 and 3 and another one grouping GLRaV-4 and its variants. In order to understand biological factors mediating differential ampelovirus incidences in vineyards, quantitative real-time polymerase chain reactions were performed to assess virus populations in three grapevine varieties in which different infection status were detected: GLRaV-3 + GLRaV-4, GLRaV-3 + GLRaV-4 strain 5, and GLRaV-4 alone. Specific primers based on the RNA-dependent RNA polymerase (RdRp) domains of GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 were used. Absolute and relative quantitations of the three viruses were achieved by normalization of data to the concentration of the endogenous gene actin. In spring, the populations of GLRaV-4 and GLRaV-4 strain 5 were 1.7 × 104 to 5.0 × 105 genomic RNA copies/mg of petiole tissue whereas, for GLRaV-3, values were significantly higher, ranging from 5.6 × 105 and 1.0 × 107 copies mg-1. In autumn, GLRaV-4 and GLRaV-4 strain 5 populations increased significantly, displaying values for genome copies between 4.1 × 105 and 6.3 × 106 copies mg-1, whereas GLRaV-3 populations displayed a less pronounced boost but were still significantly higher, ranging from 4.1 × 106 to 1.6 × 107 copies mg-1. To investigate whether additional viruses may interfere in the quantifications the small RNA populations, vines were analyzed by Ion Torrent high-throughput sequencing. It allowed the identification of additional viruses and viroids, including Grapevine virus A, Hop stunt viroid, Grapevine yellow speckle viroid 1, and Australian grapevine viroid. The significance of these findings is discussed.

Pathogen and Circadian Controlled 1 (PCC1) Protein Is Anchored to the Plasma Membrane and Interacts with Subunit 5 of COP9 Signalosome in Arabidopsis

The Pathogen and Circadian Controlled 1 (PCC1) gene, previously identified and further characterized as involved in defense to pathogens and stress-induced flowering, codes for an 81-amino acid protein with a cysteine-rich C-terminal domain. This domain is essential for homodimerization and anchoring to the plasma membrane. Transgenic plants with the ß-glucuronidase (GUS) reporter gene under the control of 1.1 kb promoter sequence of PCC1 gene display a dual pattern of expression. At early post-germination, PCC1 is expressed only in the root vasculature and in the stomata guard cells of cotyledons. During the transition from vegetative to reproductive development, PCC1 is strongly expressed in the vascular tissue of petioles and basal part of the leaf, and it further spreads to the whole limb in fully expanded leaves. This developmental pattern of expression together with the late flowering phenotype of long-day grown RNA interference (iPCC1) plants with reduced PCC1 expression pointed to a regulatory role of PCC1 in the photoperiod-dependent flowering pathway. iPCC1 plants are defective in light perception and signaling but are not impaired in the function of the core CO-FT module of the photoperiod-dependent pathway. The regulatory effect exerted by PCC1 on the transition to flowering as well as on other reported phenotypes might be explained by a mechanism involving the interaction with the subunit 5 of the COP9 signalosome (CSN).

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures.

BackgroundPlant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.ResultsPhenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.ConclusionsAs a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.

Culturable bacterial endophytes isolated from Mangrove tree (Rhizophora apiculata Blume) enhance seedling growth in Rice.

Background:Endophytic bacteria do have several potential applications in medicine and in other various sectors of biotechnology including agriculture. Bacterial endophytes need to be explored for their potential applications in agricultural biotechnology. One of the potential applications of bacterial endophytes in agricultural is to enhance the growth of the agricultural crops. Hence, this study was undertaken to explore the plant growth promoting potential application of bacterial endophytes.Objective:The objective of this study was to examine the effect of endophytic bacteria from mangrove tree (Rhizophora apiculata Blume) for their efficacy in promoting seedling growth in rice.Materials and Methods:Eight endophytic bacterial isolates (EBIs) isolated from twig and petiole tissues of the mangrove were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequence homology. Separately, surface sterilized paddy seeds were treated with cell-free broth and cell suspension of the EBIs. Rice seedlings were analyzed by various bioassays and data was recorded.Results:The gene sequences of the isolates were closely related to two genera namely, Bacillus and Pantoea. Inoculation of EBIs from R. apiculata with rice seeds resulted in accelerated root and shoot growth with significant increase in chlorophyll content. Among the isolates, Pantoea ananatis (1MSE1) and Bacillus amyloliquefaciens (3MPE1) had shown predominance of activity. Endophytic invasion was recognized by the non-host by rapid accumulation of reactive oxygen species (ROS) and was counteracted by the production of hydrogen peroxide (H2O2) and lipid peroxide. The results demonstrated that EBIs from mangrove tree can increase the fitness of the rice seedlings under controlled conditions.Conclusion:These research findings could be useful to enhance the seedling growth and could serve as foundation in further research on enhancing the growth of the rice crop using endophytic bacteria.

Comparative analysis of the biodiversity of fungal endophytes in insect-induced galls and surrounding foliar tissue

Insect-induced galls are abnormal plant growths that can provide food and shelter to their inhabitants, resulting in stressed plant tissue that may alter the conditions for the colonization or proliferation of endophytic fungi. We investigated the effect gall formation has on fungal endophyte communities and diversity. Using three closely-related gall-forming aphid species that specialize on poplars, we characterized fungal endophyte diversity in galls and surrounding petiole and leaf lamina tissue. A total of 516 fungal endophyte samples were isolated from 272 tissue samples (32 leaves, 31 petioles, and 209 galls), resulting in 23 distinct morphotypes. Despite sharing a common host plant and often forming spatially contiguous galls, the endophyte profiles within the galls of each aphid species were distinct, not only from the galls of the other species, but also from surrounding plant tissue. These results suggest that insect galls can affect the composition of fungal endophyte species in plant tissues, by altering either the colonization or proliferation of their endophytic mycobiota. Likewise, fungal endophytes may be important in the ecology and evolution of insect galls.

Verticillium Wilt of Redbud in China Caused by Verticillium dahliae.

Chinese redbud (Cercis chinensis Bunge), a member of the Fabaceae, is an important ornamental plant native to China with reported desirable medicinal effects, including stimulating blood circulation, detumescence, and detoxification (1). In October 2011, wilt symptoms of gradual leaf yellowing, wilting, scorching (marginal browning), and twig dieback were observed on plants in Yangling, Shaanxi, China. The incidence of diseased plants was about 20% in two main areas (about 20 ha in Zijingshan Park in Zhengzhou, Henan Province; and about 3,000 ha in Taiping National Forest Park in Xi'an, Shaanxi Province). Wilted leaves of diseased plant senesced and died, but defoliation was not observed. Brown discoloration was observed in vascular tissues of petioles, twigs, and stems of diseased plants, sometimes in a ring pattern. The symptoms were often restricted to the lower part of the tree or a few branches. To identify the causal agent, six twigs (each approximately 50 mm in diameter and 10 cm long) sampled from an infected tree in Yangling were rinsed in running water, surface-sterilized with 75% ethanol for 2 min, rinsed in sterilized water three times, dried, cut into 1 cm long segments, and the segments put onto potato dextrose agar (PDA) medium. A fungal isolate was recovered from diseased vascular tissues of each sample when cultured on PDA in the dark at 25°C. After 5 days, colonies changed from white to black as a result of production of microsclerotia. Microscopic observation revealed that conidiophores were hyaline and verticillate, with three to four phialides at each node. Conidia were ellipsoidal, hyaline, single-celled, and 2.5 to 7.5 × 1.25 to 4.5 μm. On the basis of these morphological characteristics, the fungus was identified as Verticillium dahliae (3). To prove Koch's postulates, the roots of 10 healthy, 1-year-old C. chinensis plants were each irrigated in a greenhouse with 50 ml of a conidial suspension (1.0 × 107 spores/ml) of an isolate recovered from an infected plant (2); five control plants were inoculated similarly with sterilized water. Fifteen days after inoculation, the same wilt symptoms observed on the original plants had developed on 9 of the 10 inoculated plants, whereas the control plants remained healthy. The pathogen was recovered 15 days after inoculation by isolating from petiole and stem tissues of symptomatic plants, but was not isolated from the control plants. The internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA was PCR-amplified with primers ITS1 and ITS4 (4), and sequenced. BLAST analysis of the ITS sequence (GenBank Accession No. AB735536) showed 100% homology with that of an isolate of V. dahliae (FJ572050). To our knowledge, this is the first report of Verticillium wilt on C. chinensis in China.

First Report of Petiole (Rachis) Blight of Washingtonia filifera Caused by Phoma glomerata in Greece.

In July 2007, a severe petiole (rachis) blight disease was observed on several California fan palms (Washingtonia filifera) in the vicinity of Heraklion (Crete), Greece. Typical symptoms included discolored (brown to reddish-brown), reversed V-shaped lesions on the petiole bases of the oldest (lowest) leaves, and elongated yellow to dark-brown stripes along the petiole. The lesions progressively expanded and penetrated the petioles, resulting in gradual discoloration (from tan to brown-black) of the internal petiole tissues, including the vascular tissue. The bases of infected petioles occasionally became fragile and burst open, while the corresponding leaf blades were characterized initially by yellowing and one-sided or uneven wilt and, later, desiccation and death with the entire leaves curving downwards. The disease gradually moved upward to younger leaves, severely debilitating but rarely killing the infected trees. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) plates from sections of diseased petioles, forming dense, dark green colonies with abundant light to dark brown, subglobose pycnidia (diameter ranging between 36.4 to 177.4 μm, and averaging 99.4 μm, n = 50) on the agar surface or immersed in the medium. Chlamydospores and numerous dictyochlamydospores were also observed, with the latter being initially light to dark brown and later becoming black. The numerous conidia were hyaline, ovoid to ellipsoid, and single-celled. Their dimensions were 5.3 to 7.3 × 2.4 to 4.9 μm, averaging 6.5 × 3.2 μm (n = 100). The ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (3), were amplified with PCR from total DNA extracted from two representative isolates, and sequenced (GenBank Accession Nos. KC802086 to KC802087). Using BLASTn, both sequences were 100% identical to Phoma glomerata ITS sequences (FJ427018, FJ427011, AF126816). Based on morphological and molecular analyses, the pathogen was identified as Phoma glomerata (Corda) Wollenw. & Hochapfel, also known as Peyronellaea glomerata (Corda) Goid. ex Togliani or Coniothyrium glomeratum Corda (1,2). To prove pathogenicity and fulfill Koch's postulates, petioles of the older leaves of eight W. filifera 2-year-old seedlings were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface), inoculated with agar discs from a 2-week-old PDA culture of the fungus, and sealed with Parafilm. For controls, sterile PDA plugs were placed on the artificial wounds of five more seedlings. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Petiole blight and leaf necrosis symptoms-identical to those observed in the infected plants-were evident 5 weeks post-inoculation, and P. glomerata was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. This is the first report of petiole blight of a palm species caused by P. glomerata in Greece. Due to the extensive use of palms as ornamentals in Greece, the occurrence of P. glomerata can potentially cause economic loss to the local ornamental industry.

Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)

Digitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.

Next-Generation Sequencing-Based Transcriptional Profiling of Sacred Lotus “China Antique”

Rhizomes are underground stems that serve various purposes including vegetative propagation, invasion of new territory, and bioactive compound synthesis and storage. An important rhizomatous plant is sacred lotus (Nelumbo nucifera), which is prized in Asia as a medicine and a food. RNA-seq and total transcriptome analysis of rhizomes and other lotus tissues was applied to identify genes involved in rhizome growth, development and metabolism. Root, petiole, rhizome internode, and leaf tissues were used for single-read RNA-seq analysis. Two whole transcriptome paired-end read libraries from rhizome apical tip and elongation zone tissues were also generated in order to survey gene expression profiles. In this analysis, 22,803 genes were expressed: 20,476 in rhizome apical meristem and elongation zone, 17,171 in rhizome internode, 16,656 in leaf, 19,457 in root, and 16,845 in petiole. Gene ontology (GO) analysis indicated that “other membrane”, “nucleotide binding”, and “other cellular processes” were highly represented in the expressed genes. A total of 231 genes displayed rhizome-specific expression including several transcription factors, protein kinases, cytochromes P450 and a sulfate transporter. GOseq analysis showed that genes in the “molecular function” GO category and several genes related to cell proliferation based on KEGG IDs were preferentially up-regulated in rhizome tissue. In addition, 1,251 possible exon-skipping events were observed in 1,149 gene models. These results provide valuable insight into gene expression profiles and regulation in sacred lotus, and the identified rhizome-specific genes provide insight into important processes involved in the biology and development of sacred lotus rhizomes.

Characterization of the stem anatomy of the Eocene fern Dennstaedtiopsis aerenchymata (Dennstaedtiaceae) by use of confocal laser scanning microscopy

Permineralization provides the most faithful known mode of three-dimensional preservation of the morphology and cellular anatomy of fossil plants. Standard optical microscopic documentation of such structures can provide only an approximation of their true three-dimensional form and is incapable of revealing fine-structural (<300 nm) details, deficiencies that can be addressed by the use of confocal laser scanning microscopy (CLSM). To demonstrate the usefulness of CLSM in such studies, we compare confocal laser scanning micrographs and optical photomicrographs of the permineralized tissues of rhizomes and petioles of the Eocene fern Dennstaedtiopsis aerenchymata preserved in cherts of the Clarno Formation of Oregon, USA, and the Allenby Formation (Princeton chert) of British Columbia, Canada. The laser-induced fluorescence detected by CLSM produces crisp high-resolution images of the three-dimensionally permineralized tissues of Dennstaedtiopsis aerenchymata. Tissues analyzed include the epidermis and epidermal hairs, cortex, aerenchyma, endodermis, vascular tissue, and pith-for each of which, CLSM yields results superior to those of standard optical microscopy. CLSM and previous Raman spectroscopic analyses of the same specimens provide evidence consistent with original biochemistry. Use of CLSM to characterize the morphology and cellular anatomy of permineralized fossil plants can provide accurate data in two and three dimensions at high spatial resolution, information that can be critically important to taxonomic, taphonomic, and developmental interpretations. Results presented here from this first detailed CLSM-based study of permineralized plant axes indicate that this nonintrusive, nondestructive technique should be widely applicable in paleobotany.

칼라의 무름병 저항성 품종 선발을 위한 검정 방법 개발

This experiment was carried out to establish the screening methods for the selection of Zantedeschia aethiopica cultivars resistant to calla lily soft rot caused by Pectobacterium carotovorum subsp. carotovorum. A center of leaf and petiole tissues of four Z. aethiopica cultivars were wounded and inoculated with three different inoculum concentrations, , , and , of EccNHRI-21 isolate. And they were evaluated at 4, 18, and 26 hours after inoculation. The lesion of maceration was developed well in the leaf tissues inoculated with concentration. And evaluation of resistance in 18 hours after inoculation was correlated with field resistance positively. Using this method, ten Z. aethiopica commercial cultivars and four wild types were screened. 'Crowbrough' and 'White Cutie' were selected as highly resistant genotypes and 'Mont Blanc' and 'Silky White' as resistant genotypes. 'Wedding March' and 'Kiwi Blush' were evaluated as moderate resistant and the others including 'Childsiana' were susceptible. And all of four wild types were evaluated as more than moderate resistant. In spite of control through cultural, biological and chemical methods, the use of resistant cultivars is most efficient to overcome calla lily soft rot. This newly developed screening method was helpful for breeding new varieties resistant to calla lily soft rot.

Molecular detection and identification of phytoplasma associated with pepper witches’ broom in China

Pepper witches’ broom (PWB) disease was observed in a field in Yangling, Shaanxi Province, China. The result of mechanical inoculation test for this disease was negative. Phytoplasma-like bodies were observed in ultrathin sections of petiole tissues of symptomatic samples. 16S rRNA gene and tuf gene of phytoplasma were amplified from the total DNA of symptomatic samples. Phylogeny analysis of the 16S rRNA gene and tuf gene suggested that the pepper witches’ broom associated phytoplasma belongs to the subgroup 16SrI-B, which was confirmed by the RFLP analysis of the 16S rRNA gene. The phytoplasma subgroup 16SrI-B was also detected in the vector Cicadella viridis trapped from the infected field. To our knowledge, this is the first report of 16SrI-B phytoplasma causing pepper witches’ broom in China.