The Adsorption of a Polygalacturonase Isoenzyme of Botryodiplodia theobromae on Plant Tissues and the Implication on the Pathogenicity of the Fungus

The polygalacturonase isoeazyme (PG 3) of Botryodiplodia theobromae extracted from rotted sweet potato was adsorbed by sweet potato, potato, carrot, bean stem and tomato fruit to various degrees. Adsorption was greater with sweet potato and tomato fruit tissues. Carrots, bean stem and potato absorbed the enzyme to more or less the same degree. The enzyme was not adsorbed on tomato stem. A spore/mycelial suspension of B. theobromae infected the test tissues to various degrees. The enzyme completely macerated sweet potato roots, potato tubers and tomato fruits within 5 h while the bean stem and onion tissues were little affected by the enzyme. The tomato stem was neither infected by the fungus nor macerated by the enzyme.     

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA,a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.     

Factors Affecting Survival of Clavibacter michiganensis subsp. sepedonicus in Water

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non‐sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non‐mucoid strain was also studied in sterile ditch water and simulated ‘drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non‐culturable state.     

Plant uptake of bicarbonate as measured with the11C isotope

Summary ¹¹C which is cyclotron produced by¹⁴N(P, )¹¹C(half-life 20.1M) was use as a tracer of bicarbonate to determine its movements from a nutrient solution through roots to stems and leaves of bush bean plants (Phaseolus vulgaris L. var. Improved Tendergreen). The short time involved and the high solution pH minimized the need for use of the Hederson Hasselbach equation for activity correction. Quantities of¹¹C did move into roots, stems and leaves with a sharp decreasing gradient (root/stem=14.5, stems/leaves=11.7) More¹¹C moved into plants with KHCO₃ than with NaHCO₃. The (NH₄)₂SO₄ enhanced¹¹C uptake and KNO₃ than with competition indicated possibility of some uptake of HCO ₃ ^– . In an experiment withGalenia pubescens (Eckl. and Zeyh.) Druce, the¹¹C was more readily moved to stems and leaves than in bush bean indicating substantial uptake of HCO ₃ ^– .     

Common Bean Reaction to Fusarium oxysporum f. sp. Phaseoli, the Cause of Severe Vascular Wilt in Central Africa

During the September‐December season of 1990, severe symptoms of Fusarium wilt were for the first time observed on a popular climbing bean (Phaseolus vulgaris L.) cultivar. G 2333. introduced within the previous 5 years. Seventy‐three bean genotypes were screened for resistance lo the disease, using artificial inoculation. The effect of inoculation density on the reaction of four selected genotypes was also investigated. Of the 29 climbing bean genotypes evaluated, 19 were resistant, including 11 of the 15 pre‐release or released cultivars. Of the 44 bush bean cultivars evaluated, 28 were resistant, five were intermediate and 11 were susceptible. All susceptible cultivars showed vascular discoloration. In both susceptible and resistant genotypes, the fungus spread almost equally from the entry points in inoculated roots to the base of the plants, but colonization and vertical spread within the vascular system were markedly less in resistant than in susceptible cultivars. At 20 and 30 cm above soil level, the fungus was only recovered from susceptible cultivars. Increasing inoculum density from 10² to 10⁷ conidia/ml did not affect the resistance of cultivars RWR 950 and G 685 but. in the susceptible cultivars G 2333 and MLB‐48‐89 A. it resulted in early appearance, high incidence and severity of the disease.     

Seasonal respiration of foliage, fine roots, and woody tissues in relation to growth, tissue N, and photosynthesis

Autotrophic respiration may regulate how ecosystem productivity responds to changes in temperature, atmospheric [CO₂] and N deposition. Estimates of autotrophic respiration are difficult for forest ecosystems, because of the large amount of biomass, different metabolic rates among tissues, and seasonal variation in respiration rates. We examined spatial and seasonal patterns in autotrophic respiration in a Pinus strobus ecosystem, and hypothesized that seasonal patterns in respiration rates at a common temperature would vary with [N] for fully expanded foliage and fine roots, with photosynthesis for foliage, and with growth for woody tissues (stems, branches, and coarse roots). We also hypothesized that differences in [N] would largely explain differences in maintenance or dormant‐season respiration among tissues. For April–November, mean respiration at 15 °C varied from 1.5 to 2.8 μmol kg^?1 s^?1 for fully expanded foliage, 1.7–3.0 for growing foliage, 0.8–1.6 for fine roots, 0.6–1.1 (sapwood) for stems, 0.5–1.8 (sapwood) for branches, and 0.2–1.5 (sapwood) for coarse roots. Growing season variation in respiration for foliage produced the prior year was strongly related to [N] (r² = 0.94), but fine root respiration was not related to [N]. For current‐year needles, respiration did not covary with [N]. Night‐time foliar respiration did not vary in concert with previous‐day photosynthesis for either growing or fully expanded needles. Stem growth explained about one‐third of the seasonal variation in stem respiration (r² = 0.38), and also variation among trees (r² = 0.43). We did not determine the cause of seasonal variation in branch and coarse root respiration, but it is unlikely to be directly related to growth, as the pattern of respiration in coarse roots and branches was not synchronized with stem growth. Seasonal variations in temperature‐corrected respiration rates were not synchronized among tissues, except foliage and branches. Spatial variability in dormant‐season respiration rates was significantly related to tissue N content in foliage (r² = 0.67), stems (r² = 0.45), coarse roots (r² = 0.36), and all tissues combined (r² = 0.83), but not for fine roots and branches. Per unit N, rates for P. strobus varied from 0.22 to 3.4 μmol molN^?1 s^?1 at 15 °C, comparable to those found for other conifers. Accurate estimates of annual autotrophic respiration should reflect seasonal and spatial variation in respiration rates of individual tissues.     

Cadmium loading into potato tubers: the roles of the periderm, xylem and phloem

The mobility of Cd in potato plants (Solanum tuberosum) was examined using both short‐term radioisotopic labelling with ¹⁰⁹Cd and long‐term growth experiments in soil supplemented with Cd, with an emphasis on the pathways through which Cd is taken up by tubers. Split‐pot experiments showed that tubers and their associated stolons and stolon roots contribute only a minor fraction to the overall Cd absorption by the plant. Most of the Cd was absorbed by the basal roots. ¹⁰⁹Cd absorbed from the soil was rapidly exported to other parts of the plant, especially the stem, with significant amounts appearing in the tubers within 30 h. Application of ¹⁰⁹Cd to leaves showed that Cd can be rapidly distributed via the phloem to all tissues. The results suggest that unlike Ca, Cd has high mobility in plants in both xylem and phloem, and that stems may have an important role in transfer between these two pathways.     

Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA Based PCR Fragments

The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desirée) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 10³ to 10⁵ CFU g⁻¹ of fresh plant tissue. Dilution plating revealed that a range of bacterial types dominated these populations. Dominant isolates fell into the α and γ subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group. Different representatives of the Firmicutes were also found. The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp. (including P. aureofaciens, P. corrugata, and P. putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes. Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue. The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil. Following stem injection, the S. maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE. However, only S. maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots. S. paucimobilis was never recovered from the plant irrespective of the mode of introduction. The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE. The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots. Evidence was obtained for the endophytic occurrence of a range of organisms falling into the α, β, and γ subgroups of the Proteobacteria as well as in the Firmicutes. Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms. However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.     

Variation in the development of stem nematodes, Ditylenchus dipsaci, in susceptible and resistant crop plants

Dicotyledonous plants were reliably inoculated with stem nematodes by placing drops of 1.3% carboxymethylcellulose containing the surface sterilised worms between the cotyledons or in the leaf axils of recently emerged seedlings raised in pots of cool, moist soil. Inoculating onion leaves, bean stems and potato tubers or potato leaves gave variable results and inoculating onion, tulip and narcissus bulbs and lucerne and red clover stems was usually unsuccessful. An attempt to characterise 67 stem nematode populations by the reactions of a number of different plants failed for lack of useful differential hosts. Lucerne was, however, resistant to all but lucerne populations. Multiplication of stem nematode populations varied greatly between cultivars of lucerne or red clover. Some cultivars were resistant to some populations of lucerne or red clover stem nematodes and susceptible to others. These differences could not be ascribed to differences in viability of the nematodes or to differences in success of the inoculations. They indicate the presence of different pathotypes or biotypes in different populations of a so-called ‘host race’ and indicate the need for new resistant cultivars to be tested against a range of populations before they are released for general use. Amongst lucerne cultivars tested, Vertus was resistant to some lucerne stem nematode populations and susceptible to others. The supposedly resistant lucerne cultivars Euver and Lifeuil were as susceptible as was Europe. Amongst red clover cultivars tested, Redhead, Kühn, Changins and Mt Calme were susceptible, Britta, Lucrum and Temara were the least resistant, Renova, Rittinova and Quin were intermediate and susceptible to one or more of the populations tested but Norseman and especially Sabtoron were very resistant.     

Reaction of Certain Solanaceous and Asteraceous Plant Species to Inoculation with Phytophthora infestans in Cameroon

Experiments were conducted to detect potential hosts of Phytophthora infestans, causal agent of potato late blight among weeds occurring in Cameroon. Isolates of P. infestans isolated from garden huckleberry (Solanum scabrum), potato (S. tuberosum) and tomato (S. lycopersicon) were inoculated on detached leaves of 12 solanaceous and 14 asteraceous species collected from the potato agroecosystem in the western highlands of Cameroon. Isolates of P. infestans from huckleberry and potato infected the same host plants as well as gboma eggplant (S. macrocarpon) and two asteraceous weeds; Billy goatweed (Ageratum conyzoides) and Dichrocephala (Dichrocephala integrifolia). Inoculum from potato caused late blight symptoms on haemorrhage plant (Aspilia africana); while inoculum from tomato resulted in late blight on worowo (Solanecio biafrae). This is the first report of late blight infection on S. macrocarpon, A. conyzoides, Sol. biafrae and Asp. africana in Cameroon. The research results indicate that some asteraceous and solanaceous weeds may be alternative hosts of P. infestans in the potato agroecosystem.     

The suitability of some natural and artificial substrates as oviposition sites for the insidious flower bug, Orius insidiosus

A variety of natural and artificial substrates were tested for their suitability as oviposition sites for the predatory anthocorid bug Orius insidiosus. The bugs oviposited into models of beans constructed from Parafilm, but none of the eggs laid into these models hatched. In choice tests, beans stems were preferred to both bean pods and potato sprouts. Hatching rates at 100% r.h. were highest (59%) for bean pods and significantly lower for both potato sprouts (39%) and bean stems (29%). At 60% r.h., hatching rates for bean pods (39%), bean stems (34%) and potato sprouts (33%) were similar. At 30% r.h. hatching rates for all three substrates were less than 15%. The results suggest that bean stems can be used successfully as an alternative to bean pods for the mass rearing of these biological control agents.     

Detection of Ralstonia solanacearum in Soil and Weeds from Commercial Tomato Fields Using Immunocapture and the Polymerase Chain Reaction

A detection assay for Ralstonia solanacearum in soil and weeds was developed by combining immunocapture and the polymerase chain reaction (IC‐PCR). Anti‐R. solanacearum polyclonal antibodies were produced in a white female rabbit and Dynal^® super‐paramagnetic beads were coated with purified immunoglobulinG (IgG). Using IC‐PCR, the 718 bp target DNA was amplified at a detection threshold of approximately 10⁴ colony‐forming units (CFU) bacteria per millilitre of suspension. DNA was not amplified in soil suspensions derived from autoclaved and non‐autoclaved soils, which contained R. solanacearum at 1–10⁵ CFU/g soil. However, a positive PCR result was obtained when bacteria in the soil suspensions were first enriched in nutrient broth. IC‐PCR detected R. solanacearum in tomato stems 24 h after inoculation by stem puncture with a suspension containing approximately 10⁵ CFU/ml. IC‐PCR detected the bacterium in 28 of 55 (51%) weeds and 10 of 32 (31%) soil samples. Of the weeds, Physalis minima, Amaranthus spinosus and Euphorbia hirta had the highest incidence of infection. R. solanacearum was not detected in soil taken from fallow fields, but it was discovered in some weed species. Symptomless tomato and pepper plants collected from the fields in which tomato bacterial wilt had previously occurred were found to contain R. solanacearum. These discoveries suggest that weeds and latent hosts may play a role in the survival of R. solanacearum between cropping cycles.     

Herbicidal effects under field conditions of Ailanthus altissima bark extract,which contains ailanthone

Extracts of Ailanthus altissima stem bark were evaluated for herbicidal effects under field conditions in two outdoor trials. Previous investigations had shown A. altissima bark, extracted with methanol, yielded a strongly phytotoxic extract that contained ailanthone as one of the major herbicidal compounds. The first field trial investigated the level of activity and selectivity of the extract. A. altissima bark extract was sprayed post-emergence onto 17 species of weeds and crops at rates of 366, 177, 93, 47, 23, and 0 kg ha^–1. These application rates provided herbicidal activity equivalent to 4.5, 2.2, 1.1, 0.6, 0.3, and 0.0 kg of pure ailanthone per hectare, based on the results of a laboratory bioassay of extract and pure ailanthone. Strong herbicidal effects were observed within several days. Even the lowest rate caused mortality and injury in excess of 50% for nine of the 17 species, and a significant reduction in shoot biomass for 13 species. The second field trial tested the ability of bark extract to control weeds under field conditions with horticultural crops (bush bean, cauliflower, sweet corn, tomato). A. altissima bark extract was sprayed post-emergence at rates of 99, 50, 26, 13, and 0 kg ha^–1, providing herbicidal activity equivalent to 1.1, 0.6, 0.3, 0.14, and 0.0 kg of pure ailanthone per hectare. Extract treatment provided partial weed control (greatest reduction in weed biomass was 40%), but also caused serious crop injury. Bush bean was the only crop that showed a significant increase in shoot biomass and fruit yield, compared to the non-weeded control. None of the crops, regardless of application rate, showed a level of shoot biomass or fruit yield comparable to the hand-weeded control. The herbicidal effects of A. altissima bark extract declined within the first few weeks after application, supporting previous evidence that ailanthone is rapidly degraded under field conditions.     

Studies on the Diagnosis of Bacterial Ring Rot of Potatoes

The eggplant test, described by Olsson (1976) and Lelliott and Sellar (1976) as a means of detecting Corynebacterium sepedonicum, the causal agent of bacterial ring rot, has been tested for its reliability. Eggplants inoculated with a pure suspension of bacterial cells developed typical chlorotic symptoms at a minimum concentration of 2 × 10³ cells/ml inoculum. Whereas with an inoculum which consisted of potato tissue equally mixed with the bacterial suspension, the first symptoms developed at a concentration of 10⁴ cells/ml. Reisolation of the pathogen by the IFAS-test or on YDC-medium was possible following innculation at a concentration of 2 × 10³ cells/ml. It was mostly possible to isolate the bacteria in pure culture. Further solanaceous host species tested in comparison with the eggplant did not develop typical symptoms following inoculation with C. sepedonicum. On this account, the eggplant test should currently be considered as the most suitable biological assay to detect C.sepedonicum.     

Characterization of blackleg and soft rot from potato in northern Thailand

Blackleg and soft rot of potato cause economic loss through reduced yield and quality. The causal agents of bacterial blackleg and soft rot of potato were identified based on biological data and sequence analyses of the 16S rDNA gene. Between 2016 and 2018, diseased potato stems and tubers were collected in Chai Prakan District, Chiang Mai Province, and Chiang Khum District, Pa Yao Province. The symptoms included black stem lesions, soft rot on tubers, wilting, break down of the stem vascular ring and foliar yellowing. Of 13 bacterial isolates, five were identified as Pectobacterium carotovorum subsp. brasiliense, four‐Dickeya dadantii, two‐Pseudomonas putida and two‐Bacillus altitudinis. Pathogenicity tests of P. carotovorum subsp. brasiliense and D. dadantii resulted in lower leaves turning yellow and wilting followed by blackleg symptoms on lower stems and maceration of tuber tissue. Symptoms caused by P. putida were yellowing and wilting of leaves. B. altitudinis caused yellowing of the lower leaves and wilting followed by drying of leaf tissue. This is a first report of these bacterial pathogens causing blackleg and soft rot of potato in Thailand.     

Innate and acquired components of oligophagy in the herbivorous lepidopteran,Manduca sexta

Innate and acquired aspects of oligophagy were investigated in the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), a species normally restricted to members of the family Solanaceae. Larvae were reared in the laboratory on solanaceous species tobacco (Nicotiana tabacum L.), strawberry groundcherry (Physalis pruinosa L.), and potato (Solanum tuberosum L.), as well as the non‐solanaceous foods cowpea [Vigna sinensis (L.) (Fabaceae)], rapeseed [Brassica napus L. (Brassicaceae)], and artificial diet. Feeding assays were conducted using the above plants plus corkscrew vine [Vigna caracalla (L.) Verdc. (Fabaceae)], lettuce [Lactuca sativa L. (Asteraceae)], and moist glass‐fiber filter paper. Larval feeding was characterized using two camera‐monitored assays: (1) a quantitative no‐choice disc test that determines consumption over time, delays in initiation of feeding, and % of larvae rejecting the food, and (2) a novel no‐choice emigration test that measures how soon a larva abandons a whole plant. Experimental results from both assays revealed that larvae displayed high levels of acceptance of Solanaceae regardless of whether they had been reared on solanaceous or non‐solanaceous food. We conclude that solanaceous oligophagy in M. sexta is primarily innate and does not require (but may be strengthened by) previous feeding experience on Solanaceae. In contrast, larvae tested on non‐solanaceous plants or moist filter paper showed large variation in both acceptance of foods and emigration times that were strongly dependent on the food on which they had been reared (analogous to the ‘induction of preference’ of earlier literature). Two types of induction were identified: an increase in acceptability of a plant of (1) the same species as the one on which the larva was reared, or (2) a related species. These discoveries both challenge and expand our current understanding of oligophagy in the tobacco hornworm.     

EFFECT OF PICLORAM ON TRANSPORT IN YOUNG BEAN,MESQUITE, AND HUISACHE PLANTS

The effect of 4-amino,3,5,6-trichloropicolinic acid (picloram) on transport from leaves to the roots was studied using young bean (Phaseolus vulgaris L.), mesquite (Prosopis juliflora var. velutina (Woot.) Sarg.), and huisache (Acacia farnesiana (L) Willd.) plants. The only picloram treatments which were effective in enhancing transport of ¹⁴C-assimilate or ¹⁴C-picloram to the roots were those made to the shoots or roots one day or more before application of the label to the shoots. The enhancement of transport was not evident when un-labeled picloram or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or both, were applied at the same time as the ¹⁴C-label. Enhancement of transport was to the more mature stem or root tissues. Inorganic nitrogen applied to nitrogen-deficient bean plants also increased transport of ¹⁴C-assimilate to the roots, especially the rate.     

Factors affecting the distribution of cadmium,copper and lead and their effect upon yield and zinc content in bush beans (Phaseolus vulgaris L.)

Summary Concentrations of Cd, Pb and Cu in the roots, stems and leaves of bulgarian bush beans (Phaseolus vulgaris L.) were determined for plants grown in various soils of increasing levels of contamination of these metals. Most of each heavy metal absorbed by plants was retained in roots. Concentrations of Cd, Pb and Cu in roots increased in response to soil concentrations, whereas, in stems, only Cd and Pb concentrations increased and Cu concentration was relatively constant. It is thought that Cu transport to the stele was metabolically controlled, whereas Cd and Pb reached the stem by leakage across non suberised areas of the endodermis. Uptake of heavy metals was associated with a decrease in zinc content in plants and a decrease in yield. By regression analysis decrease in both zinc content and plant yield could be best related to Cd content in stems. Possible reasons for these effects are discussed.     

Carbon isotope composition of current-year shoots from Fagus sylvatica in relation to growth, respiration and use of reserves

Temporal variations in the stable carbon isotope composition (δ¹³C) of leaves and current‐year stems were examined in beech trees over one year. The δ¹³C of both tissues were equal in the bud stage and started to diverge during growth, with values decreasing by 2·5 and 4·5‰ for stems and leaves, respectively. The dynamics of the δ¹³C and content of non‐structural sugars were also assessed. The beginning of the growth period was characterized by a decrease in starch content and high starch δ¹³C values. Later in the season, the δ¹³C of leaf soluble sugars progressively decreased from the end of May and the δ¹³C of stem sucrose was at least 1·5‰ higher than that of leaves. The δ¹³C of CO₂ respired by stem tissue increased during stem growth and exhibited large seasonal variations ( from ?22·1 to ?26·3‰). These values generally fell between those of starch and total organic matter. The results of the study showed that the δ¹³C of stems is altered by two apparent fractionation steps: one during sugar transfer from leaves to stems and one during stem respiration. These results may have implications for analysis of isotopic signals in tree rings and forest ecosystems.