Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

Relations of the semi-persistent viruses, parsnip yellow fleck and anthriscus yellows, with their vector, Cav arietta aegopodii

Studies were made of the relations of parsnip yellow fleck virus (PYFV) and its helper virus, anthriscus yellows (AYV), with their aphid vector, Cavariella aegopodii. Apterous insects were more efficient vectors than alates; apterous nymphs were as efficient as apterous adults. C. aegopodii never transmitted PYFV in the absence of AYV, but aphids carrying both viruses infected some test plants with one or other virus alone. C. aegopodii that fed first on a source of AYV and then on a source of PYFV transmitted both viruses to test plants, but aphids that fed on the sources in the reverse order transmitted only AYV. Test plants receiving some aphids from a source of AYV, and others from a source of PYFV, became infected only with AYV. C. aegopodii acquired AYV or the AYV/PYFV complex from plants in a minimum acquisition access time (AAT) of 10–15 mm and inoculated the viruses to test plants in a minimum inoculation access time (IAT) of 2 min. Increasing either AAT or IAT, or both, to 1 h or longer increased the frequency of transmission of each virus. Starving the insects before the acquisition feed on AYV or AYV/PFYV sources did not affect transmission. Aphids already carrying AYV acquired PYFV from plants in a minimum AAT of only 2 min; they acquired and inoculated PYFV in a minimum total time of 12 min. The data suggest that AYV is confined to deeply lying tissues whereas PYFV is distributed throughout the leaf. C. aegopodii transmitted both PYFV and AYV in a semi-persistent manner: the aphids retained both viruses for up to 4 days but lost them on moulting. Neither virus was passed to progeny of viruliferous adults. Earlier results suggesting that AYV is a persistent virus may have been caused by contamination of the AYV culture with carrot red leaf virus.     

The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii

Transmission of parsnip yellow fleck virus (PYFV) by the aphid Cavariella aegopodii occurs only when the aphids are also carrying the helper virus, anthriscus yellows (AYV). None of five other viruses tested was able to act as helper. In experiments in which aphids were allowed to feed through membranes on crude or treated extracts from infected plants, aphids already carrying AYV acquired PYFV, but virus-free aphids failed to acquire either AYV or PYFV. PYFV was not transmitted by insects injected with haemolymph from aphids carrying both viruses, or with purified preparations of PYFV. PYFV was transmitted when AYV-carrying aphids, except those whose stylets had been removed, were contaminated externally with PYFV preparations. Ultraviolet irradiation of infected leaves did not prevent aphids from acquiring AYV, presumably because it is confined to deeply-lying tissues. AYV-carrying aphids could acquire PYFV from u.v.-irradiated leaves after acquisition access times of 2 h but not after feeds of only 2 or 15 min (which are adequate on unirradiated leaves), suggesting that PYFV is present in all parts of the leaf. No ‘helper agent’ distinct from AYV itself was detected in these experiments or in experiments on minimum acquisition feeding time or maximum period of persistence in the aphid. U.v.-inactivated PYFV competed with infective PYFV for retention sites in AYV-carrying aphids, whereas AYV apparently did not. It is suggested that there is no helper agent for PYFV, other than AYV particles. The possibility that there is one for AYV is not excluded.     

Purification, properties and transmission of parsnip yellow fleck, a semi-persistent, aphid-borne virus

Parsnip yellow fleck virus (PYFV) is the commonest cause of virus-like symptoms in parsnip plants in Britain: it is sap-transmissible but systemically infects few species outside the Umbelliferae. It has isometric particles 29–31 mμ in diameter, a sedimentation coefficient of 167s, and loses infectivity in sap after dilution to 10^-3-10^-4, heating for 10 min at 57·5–65°C, or storage at room temperature for 4–7 days. Two isolates, from parsnip and Anthriscus sylvestris respectively, are only distantly serologically related. The aphid Cavariella aegopodii transmits PYFV in a semi-persistent manner from A. sylvestris but not from parsnip. Transmission by aphids apparently depends on the presence in A. sylvestris or other source plants of a second virus, anthriscus yellows (AYV), which is persistent in the vector and not manually transmissible. PYFV was therefore not transmitted by aphids from manually inoculated plants or from parsnip or other plants immune to AYV. In controlled experiments, C. aegopodii transmitted PYFV (both A. sylvestris and parsnip isolates) from chervil plants inoculated separately with PYFV and AYV, but not from plants inoculated only with PYFV.     

Evaluation of leaf analysis as a guide to nitrogen and phosphorus fertilization of potato (Solanum tuberosum L.)

Summary Recently matured leaf samples were collected, at 45, 60 and 75 days after planting, from potato (Solanum tuberosum L.) plants of cultivar Kufri sindhuri grown with varying levels of nitrogen (0, 60, 120, 180 and 240 kg N/ha) and phosphorus (0, 60, 120 and 180 kg P₂O₅/ha) on loam soil at Pantnagar. They were separated into petiole, midrib and leaf-lets and analysed for NO₃-N content. Petiole samples were also analysed for PO₄-P content. Nitrogen application increased the NO₃-N content of all the leaf components. P application increased the PO₄-P content in petiole. NO₃-N content and PO₄-P content in leaf tissues were positively correlated with final tuber yield. The association of NO₃-N content of petiole with the final tuber yield was very consistant. Hence this proved to be the best indicator tissue for reflecting the nitrogen status of the plant, particularly at 45 days after planting. NO₃-N content of midrib, at this stage, was also a good indication of nitrogen nutrition status of the plant. PO₄-P content of petiole at 45 days after planting was a good indication of nutritional status of plant with respect to phosphorus. The critical concentration of NO₃-N in petioles of 45 days old plants was in the range of 9100–9600 ppm. The corresponding range for midrib was 3000 to 3900 ppm. The critical concentration of PO₄-P for petioles of 45 days old plants was 2250 ppm.Publication No 796 under journal series of the G.B. Pant University of Agriculture and Technology, Experiment Station, Pantnagar.Junior Agronomist, Indian Institute of Horticultural Research, Bangalore-6, India.Junior Agronomist, Indian Institute of Horticultural Research, Bangalore-6, India.     

Effects, distribution and uptake of silicon in banana (Musa spp.) under controlled conditions

Three contrasted genotypes of Musa spp. (M. acuminata cv Grande Naine, M. acuminata spp. Banksii and M. balbisiana spp. Tani) were grown for 6 weeks under optimal conditions in hydroponics and were submitted to a wide range of Si supply (0–1.66 mM Si) to quantify the Si uptake and distribution in banana, as well as the effect of Si on banana growth. The level of Si supply did not affect plant growth, nor the rate of water and nutrient uptake. The rate of Si uptake and the Si concentration in plant tissues increased markedly with the Si supply. At the highest Si concentrations (1.66 mM), silicon absorption was essentially driven by mass flow of water (passive transport). However, at lower Si concentrations (0.02–0.83 mM), it was higher than its uptake by mass flow and caused the depletion of silicon in the nutrient solution, suggesting the existence of active processes in silicon transport. The distribution of silicon among shoot organs (pseudostem < petiole and midrib < young lamina < old leaf) confirmed the major role of transpiration in silicon accumulation and was not dependent on silicon supply. However, other mechanisms of transport might be operating in the roots and in the petiole and midrib of young leaves, whose silicon concentration was unexpectedly high at low Si supply (0.02 mM) compared to higher levels of Si. The three genotypes did not exhibit consistent differences in their responses to silicon supply.     

Aerenchyma develops by cell lysis in roots AND CELL SEPARATION IN LEAF PETIOLES IN Sagittaria lancifolia (Alismataceae)

Aerenchyma gas spaces are important for plants that survive flooding because these spaces provide an internal pathway for oxygen transport to the root zone. The objective of this study was to characterize the development of aerenchyma gas spaces in Sagittaria lancifolia L., a dominant species in freshwater wetlands adjacent to the Gulf of Mexico. Tissue at different developmental stages was collected from hydroponically grown plants, embedded in plastic, and sections were observed with a light microscope. In S. lancifolia roots, lysigeny (cell lysis) produced gas spaces that increased in volume from the root meristem to the most mature root tissue. Shoot aerenchyma occurred in the large petioles of S. lancifolia and through the blade midrib, but not in the laminar portion of the blade. In contrast to the roots, gas spaces in the petiole were formed by schizogeny (cell separation during development). Shoot initials produced cells that formed interlocking cylinders in the cortex and diaphragm cells that bridged the central portion of the cylinders. Division and expansion of both these cell types increased the diameter of the cylinders and created schizogenous gaps between diaphragm layers that produced large gas spaces in mature tissue. Therefore, aerenchyma development occurs by two different processes in S. lancifolia.     

PETIOLE VASCULARIZATION OF LUXEMBURGIEAE (OCHNACEAE)

Sections through the petiole and blade midrib and sometimes the node of 20 genera of Luxemburgieae have revealed variations in vascularization which appear significant in determining the phylogeny of the tribe. The most primitive group are the pentacarpellate genera; they are characterized by multilacunar nodes and numerous leaf traces which mostly remain free to the leaf apex; some of these become medullary bundles in the petiole. The more advanced bicarpellate genera have multilacunar or trilacunar nodes, still with many leaf traces in the case of Wallacea. These traces fuse in the blade, resulting in a siphonostele. The most advanced genera are tricarpellate and have trilacunar nodes with only three traces; these traces fuse, forming a siphonostele. There are no medullary bundles present in this last group of genera.     

COMPARATIVE STUDIES OF THE NODAL AND VASCULAR ANATOMY IN THE NEOTROPICAL CYATHEACEAE. III. NODAL AND PETIOLE PATTERNS; SUMMARY AND CONCLUSIONS

Comparative studies of the nodal and vascular anatomy in the Cyatheaceae are discussed as they relate to the taxonomy and phylogeny of the family. There is in the Cyatheaceae (excluding Metaxya and Lophosoria) a basic nodal pattern consisting of four major phases of leaf trace separations. Abaxial traces arise from the leaf gap margins, and the last abaxial traces from each side of the gap are larger and undergo numerous divisions. Distally adaxial traces separate from the gap margins, and the last adaxial traces are usually larger and undergo multiple divisions. In addition, medullary bundles frequently become petiole strands of the adaxial arc in the petiole. Rarely, cortical bundles form petiole strands in the abaxial arc in the petiole. Leaf gaps of the squamate genera of the Cyatheaceae are fusiform and possess prominent lateral constrictions which result from medullary bundle fusions and the separation of leaf traces. A characteristic petiole pattern is found in all members of the Cyatheaceae. There is an increase in the complexity of the petiole vascular tissue which results in a gradation from the undivided strand in Metaxya, to the three-parted petiole pattern in Lophosoria, and finally to the much-dissected petiole vascular tissue in the advanced genera. Nodal and vascular anatomy data basically support Tryon's phyletic scheme for the family. The Sphaeropteris-Alsophila-Nephelea line shows certain tendencies toward increased complexity of nodal and vascular anatomy, whereas the Trichipteris-Cyathea-Cnemidaria line shows the same anatomical and morphological characters in a direction of increased simplification or reduction.     

Effect of Excision on Leaf Water Potential

Improvements of thermocouple hygrometric techniques for in situleaf water potential measurement in the field now allows forcontinuous monitoring of water potential in response to an externalperturbation, such as leaf excision. Using Citrus jambhiri plants,measured leaf water potentials of completely excised leaf portionsimmediately increased when the petiole was excised or incisionswere made either transverse or parallel to the midrib. Incisionsparallel to the midrib were on the side nearest the hygrometeror opposite it if preceded by a petiole excision. Midrib incisionswere 100–150 mm long with the nearest cut edge being 20–50mm from the hygrometer cavity. All excisions were such thatleaf tissue was removed from the leaf with water potential onone of the leaf portions being measured continuously prior toand after excising. The peak increase in measured water potentialof the excised leaf portions ranged between 20 kPa and 80 kPabut averaged 50 kPa. In uncovered leaves, particularly underfield conditions with the associated high evaporative demand,measured leaf water potential declined rapidly after the initialincrease. The increase in measured water potential immediatelyfollowing various types of excision was confirmed for dark andlight conditions (laboratory and field respectively) using bothpsychrometric and dewpoint modes and occurred for secondaryexcisions, but to a lesser extent. Discovery of this phenomenonimplies that water potential measured on detached leaves maynot always represent accurately in situ leaf water potential. Key words: Leaf water potential, Thermocouple hygrometers, Leaf excision effects     

AN ANALYSIS OF LEAF ELONGATION IN XANTHIUM PENSYLVANICUM PRESENTED IN RELATIVE ELEMENTAL RATES

Maksymowych , Roman . (Villanova U., Villanova, Pa.) An analysis of leaf elongation in Xanthium pensylvanicum presented in relative elemental rates . Amer. Jour. Bot. 49(1): 7–13. Illus. 1962.—Xanthium plants were grown vegetatively, and leaves, whose developmental stages were specified by a previously described leaf plastochron index (L.P.I.), were marked with India ink along the midrib and photographed during 3 successive days. The relative elemental rates of elongation, d(dX/dpl)/dX were estimated during the whole course of development. The pattern of elongation was not constant but was changing with increasing plastochron age of the leaf. The elements of a young leaf of L.P.I. 0.75 elongated with a constant relative rate. In older leaves, the d(dX/dpl)/dX values were progressively declining toward the tip of the lamina. After L.P.I. 6.3 the only increment in length was due to the elongation of the elements of the petiole. The pattern of growth distribution is discussed in terms of relative elemental rates with respect to cell division and cell elongation in various portions of the lamina and is correlated with the basipetal trend of tissue differentiation in the developing Xanthium leaf.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): MORPHOLOGY AND ANATOMY

Mature field- and growth-chamber-grown leaves of Populus deltoides Bartr. ex Marsh. were examined with light and scanning electron microscopes to determine their vasculature and the spatial relationships of the various orders of vascular bundles to the mesophyll. Three leaf traces, one median and two lateral, enter the petiole at the node. Progressing acropetally in the petiole these bundles are rearranged and gradually form as many as 13 tiers of vascular tissue in the petiole at the base of the lamina. (Most leaves contained seven vertically stacked tiers.) During their course through the midrib the tiers “unstack” and portions diverge outward and continue as secondary veins toward the margin on either side of the lamina. As the midvein approaches the leaf tip it is represented by a single vascular bundle which is a continuation of the original median bundle. Tertiary veins arise from the secondary veins or the midvein, and minor veins commonly arise from all orders of veins. All major veins–primaries, secondaries, intersecondaries, and tertiaries–are associated with rib tissue, while minor veins are completely surrounded by a parenchymatous bundle sheath. The bundle sheaths of tertiary, quaternary, and portions of quinternary veins are associated with bundle-sheath extensions. Minor veins are closely associated spatially with both ad- and abaxial palisade parenchyma of the isolateral leaf and also with one or two layers of paraveinal mesophyll that extend horizontally between the veins. The leaves of growth-chamber-grown plants had thinner blades, a higher proportion of air space, and greater interveinal distances than those of field-grown plants.     

Thidiazuron promotes adventitious shoot regeneration from pothos (Epipremnum aureum) leaf and petiole explants

Summary Regeneration of adventitious shoots of pothos (Epipremnum aureum Linden and Andre) ‘Jade’ was obtained using leaf and petiole explants preprated from shoot tips of 3-yr-old greenhouse-grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), 6-(4-hydroxy-3-methy-trans-2-butenyl-amino)purine (zeatin) or N-isopentenylaminopurine (2iP) individually with α-naphthaleneacetic acid (NAA) in 18 combinations. Callus was initiated from cut surface and along the midrib or major vein of leaf sections. Shoot regeneration from leaf and petoole explants occurred in 30d on medium containing 1, 5 or 10μM TDZ with 0.5 or 1.0μM NAA except petioles on medium with 10 μM TDZ and 1.0 μM NAA where regeneration failed. More time (50d) was needed for shoot regeneration when explants were cultured on medium containing either 2iP or zeatin with NAA. Regeneration frequencies were up to 20% and 50% for leaf and petiole explants, respectively. Shoot numbers per responding explant attained 30 for leaf and petiole explants on medium containing TDZ but only one to four on medium containing either 2iP or zeatin. These results indicate that TDZ is a more effective cytokinin for in vitro regeneration of pothos than either zeatin or 2iP.. Shoots elongated readily and rooted well on MS basal medium, without plant growth regulators. Plantlets acclimatized rapidly and grew vigorously in the greenhouse after transfer to pots containing a commerecial potting medium.     

Adventitious shoot regeneration fromFagus sylvatica leaf explantsin vitro

Summary Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.     

Distribution and structural aspects of extrafloral nectaries in Cedrela fissilis (Meliaceae)

The occurrence of extrafloral nectaries (EFNs) in Meliaceae has been reported for some genera, but little anatomical data are available. Therefore, to determine the distribution and structural aspects of EFNs, Cedrela fissilis Vell. leaves in different stages of development were collected, fixed, and processed for light and scanning electron microscopy. On the petiole, rachis and petiolule, EFNs were found to be arranged predominantly towards the abaxial surface, while their occurrence in leaflet blades was restricted to the abaxial surface of the major veins, noticeably on the midrib. Basal leaflets displayed few EFNs; however, we observed an increase towards the leaf's apex. The leaf can contain more than 300 inconspicuous EFNs, which show secretory activity throughout the leaf's life. Two EFN morphotypes were visible: flattened or elevated, both circular or slightly elliptical and similar in origin and tissue composition. The secretory tissue is embedded in the rachis cortex or in the major veins of the leaf blade and EFNs are not vascularized. The EFN secretory pole shows a uniseriate epidermis with compactly arranged cells and a thin cuticle; stomata and trichomes are absent. The observation of ant visits at these structures reinforces the assumption that EFNs mediate ant–plant interactions and play a protective role against herbivores throughout the life of a leaf.     

The Spatial Distribution of Alkaloids in Psychotria prunifolia (Kunth) Steyerm and Palicourea coriacea (Cham.) K. Schum Leaves Analysed by Desorption Electrospray Ionisation Mass Spectrometry Imaging

Introduction

Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI‐MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques.

Objectives

To apply DESI‐MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC–MS and histochemical analyses.

Methodology

Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI‐MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC–MS/MS analysis performed using multiple‐reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer.

Results

Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10‐hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10‐hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface.

Conclusion

Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC–MS analysis and histochemical results. The DESI‐MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd.     

Comparisons of Acids in Smoke of Lamina and Midrib of Flue-cured Tobacco Leaves

Acids of lamina and midrib cigarette smoke were converted into trimethylsilyl derivatives and they were analyzed with glass capillary column gas chromatography. Then compositional differences of acids between lamina and midrib cigarette smoke were discussed. The concentrations of organic acids were higher for lamina cigarette smoke than for midrib cigarette smoke. Large concentration difference were found in formic, acetic, propionic, lactic, glycolic, furoic, benzoic, phenylacetic, fumalic and m-hydroxybenzoic acid. Succinic and methylsuccinic acid were similar in lamina smoke and in midrib smoke.

A large amount of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one(amino-carbonyl reaction product) was identified for the first time in lamina smoke.