Responses of ectomycorrhizal Populus tremuloides and Betula papyrifera seedlings to salinity

Roots of trembling aspen (Populus tremuloides Michx.) and paper birch (Betula papyrifera Marsh.) seedlings were inoculated with Hebeloma crustuliniforme or Laccaria bicolor and treated with 25 mM NaCl for 6 weeks. Both tree species appeared to be relatively tolerant of the applied NaCl treatment and did not develop visible leaf symptoms that are characteristic of salt injury. Salt treatment reduced total dry weights in aspen and birch, but did not significantly affect transpiration rates and root hydraulic conductance. Salt-treated ectomycorrhizal aspen maintained higher root hydraulic conductance compared with non-mycorrhizal plants. Na and Cl concentrations increased in shoots and roots of mycorrhizal and non-mycorrhizal aspen and birch in response to NaCl treatment. Roots of NaCl-treated aspen inoculated with H. crustuliniforme had over twofold higher concentrations of Na compared with non-mycorrhizal NaCl-treated plants. Similarly to aspen, Na and Cl concentrations increased in roots and shoots of NaCl-treated birch seedlings. However, in birch, there were no significant differences in Na and Cl concentrations between mycorrhizal and non-mycorrhizal plants. The results suggest that salt exclusion by the ectomycorrhizal associations is host-specific or/and that the processes leading to salt exclusion are activated in ectomycorrhizal plants by a threshold salt level which may vary between plant species.     

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

ABSTRACT: BACKGROUND: Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall resynthesis and cell division. RESULTS: This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 uM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (+/-3.27) in controls to 65.3% (+/-4.60). Protoplasts isolated from callus grown in 100 uM AIP developed cell walls by day 2, had a division rate of 28.5% (+/-3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. CONCLUSIONS: This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated propotoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.     

Isolation and characterization of microsatellite markers for red elm (Ulmus rubra Muhl.) and cross-species amplification with Siberian elm (Ulmus pumila L.)

Ulmus pumila is an elm species, non-native to the USA that hybridizes with Ulmus rubra. In order to study the genetic structure and hybridization patterns between these two elm species, we developed 15 primer pairs for microsatellite loci in U. rubra and tested their cross-amplification in U. pumila. All 15 primers amplified in both species, 11 of which possessed species-specific alleles. Eight loci were polymorphic in U. pumila and eight in U. rubra, each with two to eight alleles per locus. In addition, five primer pairs previously developed in U. laevis and U. carpinifolia (syn. U. minor) cross-amplified and showed polymorphic loci in U. pumila and/or U. rubra. These markers will facilitate the study of genetic structure and gene flow between U. rubra and exotic, invasive U. pumila.     

Susceptibility of 32 elm species and hybrids (Ulmus spp.) to the elm leaf beetle (Coleoptera: Chrysomelidae) under field conditions in Arizona

We evaluated elm leaf beetle, Pyrhalta luteola (Müller) (Coleoptera: Chrysomelidae), defoliation of 32 elm species or hybrids (taxa) established under field conditions in Holbrook, AZ. Percentage of defoliation, number of eggs, and number of larvae were estimated on randomly selected 15-cm shoot lengths annually in July, from 1996 to 2001. The following nine taxa consistently sustained 15-46% mean overall defoliation: 1) Siberian elm, U. pumila L.; 2) 'Dropmore' elm, U. pumila; 3) 'Camperdownii' elm, U. glabra Huds.; 4) 'Regal' elm, U. glabra x U. carpinifolia Gledisch x U. pumila); 5) 'Sapporo Autumn Gold' elm (U. pumila x U. japonica Sang.); 6) 'New Horizon' elm (U. pumila x U. japonica); 7) 'Charisma' elm [(U. japonica x U. wilsoniana Schneid.) x (U. japonica x U. pumila)]; 8) 'W2115-1' elm (U. parvifolia Jacq. x U. procera Salisb.); and 9) 'Homestead' elm [(U. hollandica Mill. x U. carpinifolia) x (U. pumila-racemosa Dieck x U. carpinifolia)]. Percentage of defoliation was significantly low on four Chinese elm (U. parvifolia) cultivars ('Allee', 'Athena', 'Glory'/lace bark, and 'Kings Choice'). Percentage of defoliation was also low on seven Asian elms (including U. chemnoui Cheng, U. bergmaniana Sneid., U. szechuanica Fang, and species of the U. davidiana Planch. complex [U. davidiana, U. japonica, U. wilsoniana, and U. propinqua Koidz.]) and the American elm (U. americana L.) 'Valley Forge'. Percentage of defoliation and the number of eggs or larvae per plant were highly correlated. The results of this study are generally consistent with results of past laboratory screening trials.     

Accumulation of mansonones E and F in seedlings of Ulmus americana in response to inoculation with Ophiostoma ulmi

Summary The accumulation of mansonones E and F was investigated in Ulmus americana L. seedlings 5 weeks after inoculation with three aggressive and three non-aggressive isolates of Ophiostoma ulmi (Buism.) Nannf. The three non-aggressive isolates stimulated significantly more mansonone E and F accumulation than the three aggressive isolates of O. ulmi. Mansonone induction also varied within both the aggressive and the non-aggressive groups. Aggressive and non-aggressive isolates were recovered in equal frequencies from the inoculation wounds, whereas the aggressive isolates were recovered more frequently than the non-aggressive isolates 15 cm and 25 cm up the seedlings' stem. Vascular browning in the outer xylem of the seedlings correlated with mansonone E and F accumulation. Mansonone accumulation in U. americana seedlings is therefore associated with vascular browning and a reduction in fungal spread.     

Increased photosynthetic activities and thermostability of photosystem II with leaf development of elm seedlings (Ulmus pumila) probed by the fast fluorescence rise OJIP

Experiments were conducted to investigate the photosynthetic activity and thermostability of photosystem II (PSII) in elm seedling (Ulmus pumila) leaves from initiation to full expansion. During leaf development, photosynthesis, measured as CO₂ fixation, increased gradually and reached a maximum value when leaves were fully developed. In parallel with the increase of carbon assimilation, chlorophyll content increased. The chlorophyll a fluorescence measurements showed that the maximum quantum yield of PSII primary photochemistry (φ_po), the efficiency with which the energy of trapped excitons is converted into the electron transport beyond Q_A (Ψ_o) and the quantum yield of electron transport beyond Q_A (φ_Eo) increased gradually. The low light experiments confirmed these results independently. When subjected to heat stress, young leaves exhibited progressively lower φ_po and maximal fluorescence (F_m) values with considerably higher minimal fluorescence (F_o) than mature leaves, demonstrating that PSII in newly initiating leaves is more sensitive to heat stress. Further analysis revealed that PSII structure in newly initiating leaves showed a robust alteration under heat stress, which was reflected by the clear K phase in the OJIP curves. Therefore, we suggest that the enhanced thermostability of PSII in the case of leaf growth might be associated with an improvement of the stability of the oxygen-evolving complex (OEC) to heat stress during leaf development.     

Effects of induced water stress on leaf trichome density and foliar nutrients of three elm (Ulmus) species: implications for resistance to the elm leaf beetle

Seedlings of three elm species with variable susceptibility to the elm leaf beetle (Pyrrhalta luteola Müller) (Coleoptera: Chrysomelidae) were subjected to three water stress treatments (no stress, low stress, and high stress) in a greenhouse experiment. The species tested were Ulmus pumila L. (Siberian elm = highly susceptible), U. parvifolia Jacq. (Chinese elm = resistant), and U. americana L. (American elm = intermediate). The seedlings were analyzed for changes in the levels of selected host traits (trichome density, foliar concentration of nitrogen [N], phosphorus [P], potassium [K], calcium [Ca], magnesium [Mg], iron [Fe], and manganese [Mn]), some of which had previously been implicated in resistance to the elm leaf beetle. Density of leaf abaxial surface trichomes (simple, bulbous, and total trichomes) and foliar Fe and Mg concentrations increased significantly in the highly susceptible Siberian elms under water stress. In contrast, stress reduced trichome density in the moderately susceptible American elms, but it had no effect on levels of foliar mineral nutrients. The stress treatments had no influence on host traits in the resistant Chinese elms. The results suggest that environmental stress can alter plant traits that are likely involved in determining resistance of elms to the elm leaf beetle.     

Responses of grass pea seedlings to salinity stress in in vitro culture conditions

Physiological and molecular mechanisms of adaptation to abiotic stresses of grass pea (Lathyrus sativus L.) are still poorly understood. Responses of four genotypes of grass pea to salinity stress in tissue culture conditions were investigated at early seedling growth stages. Salinity stress was induced in the agar media by adding 0, 50, 100 and 200 mM of NaCl. Germination and seedling emergence percentage was not significantly affected by 50 and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered level of these parameters. Generally, exposure to NaCl stress significantly reduced length of grass pea seedling organs (root and shoot) but did not influence the content of dry weight in shoots and increased it in the roots in two cases. Increasing salt concentration decreased integrity of cellular membranes both in root and shoot tissues. Higher accumulation of phenolic compounds and significant changes in activity of antioxidant enzymes (peroxidase and catalase) were observed in the roots but not in the shoots. Similarly, the content of proline increased mostly in the roots from moderate (100 mM) salinity conditions. Adverse conditions did not resulted in alterations in photosynthetic pigments content of any tested genotypes. The better performance of shoots than roots may result from in vitro conditions in which experiments were conducted.

     

Influence of a Ceratocystis ulmi Toxin on Water Relations of Elm (Ulmus americana)

Water-soluble glycopeptides isolated from cultures of Ceratocystis ulmi have been reported to be toxins involved in Dutch elm disease. The influence of the glycopeptides on the water relations of Ulmus americana seedlings was tested by placing cut stems in glycopeptide preparations. After 4 hours in 200 micrograms per milliliter toxin the stem conductance of the seedlings was reduced by 79% and the leaf water potential was reduced by 3 bars to that at which the seedlings wilted, the stomata closed, and transpiration decreased. Decrease in stem conductance as the mode of action of the toxin was further confirmed by forcing toxin through the stem and petiole of elm and measuring the effects on stem conductance. High molecular weight dextrans were found to mimic the action of toxin on stem and petiole conductance, and their ability to do so was found to be correlated with their molecular weight. As low as 4 micrograms of toxin or dextrans were found to measurably decrease the stem and petiole conductance of elms. Disruption of the water-conducting system of elms and other plants by small quantities of high molecular weight compounds may be a factor in diseases with wilting symptoms.     

Anatomical and nutritional factors associated with susceptibility of elms (Ulmus spp.) to the elm leaf beetle (Coleoptera: Chrysomelidae)

A wide range of susceptibility exists across elm (Ulmus) species and hybrids to the elm leaf beetle, Pyrrhalta luteola (Müller) (Coleoptera: Chrysomelidae). We evaluated various elm species, hybrids, or cultivars (taxa) growing in an experimental plantation in the city of Holbrook, AZ, for leaf anatomical (toughness and trichome density) and nutritional (minerals and sugars) traits that may be associated with host resistance. Leaf toughness and percentage of defoliation (susceptibility) were not correlated. However, we found weak negative correlations between percentage of defoliation and density of trichomes on the leaf abaxial surface. Of the 11 leaf nutrients examined, concentrations of iron and phosphorus correlated inversely with percentage of defoliation. The remaining nine traits did not show any correlation with percentage of defoliation. We concluded that individual anatomical and nutritional traits of elm species/hybrids do not seem to create a strong barrier to elm leaf beetle defoliation. However, the results from a stepwise multiple regression analysis indicated that collectively, these traits may play an important role in determining susceptibility.     

Tiletamine-zolazepam-xylazine immobilization of American marten (Martes americana)

The effectiveness of tiletamine-zolazepam (Telazol) and xylazine as an immobilizing combination for American martens (Martes americana) was evaluated. Fifteen martens were intramuscularly injected on 19 occasions using a 3:2 mixture of tiletamine-zolazepam (3.2+/-0.6 mg/kg [mean +/- SD]) and xylazine (2.1+/-0.4 mg/kg) at Pictured Rocks National Lakeshore, Michigan (USA) during May to October 2002-2003. Mean induction time was 2.5+/-1.8 min; mean recovery time was 70.8+/-31.9 min. There was no relation between the amount (mg/kg) of tiletamine-zolazepam-xylazine injected and induction (r(2)=0.08, P=0.26). However, there was an inverse relation (r(2)=0.28, P<0.01) between dosage and time to first effect of immobilants. Time to recovery increased (r(2)=0.21, P=0.05) with increased dosage. Mean heart rate, respiratory rate, and body temperature declined through 10 min postinduction (P<0.05). No mortality occurred and no short-term adverse effects were observed in recaptured individuals. In conclusion, a 3:2 mixture of tiletamine-zolazepam/xylazine is a safe and effective immobilizing agent for martens when conducting non-surgical field procedures. Immobilizing martens with 4.2 mg/kg tiletamine-zolazepam and 2.8 mg/kg xylazine should provide < or =30 min of handling time and allow full recovery in about 70 min.     

Resistance of temperate Chinese elms (Ulmus spp.) to feeding by the adult elm leaf beetle (Coleoptera: Chrysomelidae)

Newly acquired Ulmus species from the temperate regions of China growing at The Morton Arboretum, Lisle, IL, were evaluated in laboratory bioassays for feeding preference and suitability for larvae and adults of the elm leaf beetle, Pyrrhalta luteola (Müller). Larval and adult no-choice and adult multiple-choice feeding studies revealed that U changii, U. lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis were the least suitable for feeding and reproduction by the elm leaf beetle. Ulmus wallichiana and the highly preferred U pumila were more suitable for feeding and reproduction. Suitability had no significant effect on male and female longevity. The least suitable species of U. changii, U. lanceaefolia, U. prunifolia, U. pseudopropinqua, and U. taihangshanensis show promise for future elm breeding programs.     

松嫩草原榆树疏林对不同干扰的响应

榆树疏林是中国东北松嫩草原沙地植被演替的顶极群落,其对维持物种多样性,保持水土,涵养水源等都发挥着重要的功能。过去20a间,榆树疏林在人为干扰(农耕和放牧)下,其群落结构和物种组成发生了巨大变化。在1983年和2004年,对松嫩草原不同人为干扰下榆树疏林群落结构和物种组成等进行调查,结果显示:农耕和放牧一方面使松嫩草原榆树疏林群落多物种消失、生物量和多样性显著下降、植被结构简单化;另一方面,也是导致建群种——家榆低矮化,灌木化的主要因素。     

Growth response of Afzelia africana Sm. seedlings to ectomycorrhizal inoculation in a nutrient-deficient soil

 The growth and mineral nutrition responses of seedlings of two provenances of Afzelia africana Sm. from Senegal and Burkina Faso, inoculated with four ectomycorrhizal (ECM) fungi (Scleroderma spp. and an unidentified isolate) from the same regions were assessed in a pot experiment in a savanna ECM-propagule-free soil deficient in NPK. There was little variation in the ability of the different fungal species to colonize roots of either provenance of A. africana or to produce external hyphal in soil. Root colonization by ECM fungi and their hyphal development were not related to mineral nutrition or ECM dependency. Differences in P, N, Mg and Ca concentrations in the leaves of inoculated and non-inoculated Afzelia seedlings were not always associated with production of biomass. Only leaf K concentration increased in both provenances after ECM inoculation. However, the Burkina Faso provenance responded better to inoculation with the two fungal isolates than the Senegal provenance in terms of biomass production. This was due to stimulation of root dry weight of the Burkina Faso provenance. Therefore, the hypothesis arises that non-nutritional rather than nutritional effects explain the contribution of ECM inoculation to the growth of A. africana seedlings. Accepted: 27 April 1999