Effects of defoliation caused by the leaf miner<Emphasis Type="Italic"> Cameraria ohridella</Emphasis> on wood production and efficiency in<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis> growing in north-eastern Italy

The leaf miner Cameraria ohridella causes premature defoliation of Aesculus hippocastanum trees. Repeated defoliation has been reported to cause decrease in radial growth of trees and a progressive decline due to reduced production and allocation of photosynthates. Our study represents an attempt to estimate the impact of C. ohridella on annual wood increments and the hydraulic properties of the wood as well as on the dry mass of seeds. Twenty-two adult horse chestnut trees were selected, four of which had been chemically treated to prevent attack (controls). All other trees were heavily infested. The ground cover (GC) of each tree, measured from monthly hemispherical photographs, revealed that infested trees were completely defoliated in September and the slope of the GC-to-measurement dates relationship (named GC decrease index) was positively related to the number of mines per leaf. Anatomical observations showed that infested trees produced more wood per year than controls through more false rings with wider xylem conduits and, hence, with higher conductive area and theoretical flow. In fact, the theoretical flow was positively related to the defoliation intensity. In contrast, the allocation of photosynthates to seeds was strongly reduced in infested trees with respect to controls (up to 50% less). The hypothesis was advanced that horse chestnut trees reacted to C. ohridella attacks by increasing the hydraulic efficiency of the wood, thus ameliorating the water and nutrient supply to leaves between the spring and mid-summer and, therefore, compensating, at least partly, the reduced leaf lifespan.     

Impact of the leaf miner <Emphasis Type="Italic">Cameraria ohridella</Emphasis> on whole-plant photosynthetic productivity of <Emphasis Type="Italic">Aesculus hippocastanum</Emphasis>: insights from a model

The leaf miner Cameraria ohridella causes premature defoliation of Aesculus hippocastanum trees. In order to assess the whole-plant loss of productivity caused by the parasite, we monitored seasonal changes of leaf gas exchange and leaf area losses in horse chestnut trees freely infested or chemically treated to prevent moth infestation (controls). Data were integrated in a model and the annual loss of net primary productivity (NPP) was calculated for infested trees with respect to controls. Measurements showed marked vertical stratification of C. ohridella attacks, with lower crown strata being more infested than higher ones. Leaf gas exchange was maximum between May and early June, but it strongly decreased starting from mid-June even in controls. Model calculations showed that NPP loss of infested trees was about 30% on an annual basis (when the first moth attack is recorded at the end of April). Model simulations showed that postponing the start day of attack would have important positive effects on plants NPP. For example, if the start day of attack was postponed to 20 May, the annual loss of NPP would be about 15%. Our study suggests that A. hippocastanum trees attacked by C. ohridella are not facing serious risks of decline, especially if methods are adopted to postpone the start day of attack (e.g. removal of fallen leaves in autumn). Our data do not support the view that plants need to be totally protected from the parasite by application of insecticides.     

Effects of defoliation by horse chestnut leafminer (<Emphasis Type="Italic">Cameraria ohridella</Emphasis>) on reproduction in<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis>

In large parts of Europe horse chestnut trees ( Aesculus hippocastanum) suffer from severe defoliation by an alien invasive species, the specialist leaf mining moth Cameraria ohridella (Lepidoptera; Gracillariidae). Heavily infested trees have a drastically shortened period for photosynthesis. We quantified the effect of moth infestation on reproduction of horse chestnut trees in two different cities in central Europe, Bern and Munich. C. ohridella negatively affected seed and fruit weight of A. hippocastanum at both locations. In Munich, seed weight of heavily damaged trees was reduced by almost half. However, the number of seeds per fruit, the number of fruits per inflorescence, and the number of inflorescences per tree did not change due to herbivory. We conclude that C. ohridella mining affects seed quality but not seed quantity. The reduced seed weight may severely impair growth and survival of horse chestnut seedlings and thus may endanger the long-term persistence of A. hippocastanum in its endemic forests in south-east Europe.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Biocontrol of <Emphasis Type="Italic">Cameraria ohridella</Emphasis> by insectivorous birds in different landscape contexts

The horse chestnut leaf miner (HCLM) Cameraria ohridella Deschka and Dimic (Lepidoptera, Gracillariidae) is a novel but significant pest in Europe. Current control measures are either inefficient or environmentally harmful. Tits (Parus spp.) open the mines and prey on HCLM, but the biocontrol efficiency of this behaviour has not yet been quantified. We installed bird nesting-boxes in a biennial field study on four sites close to Brunswick (Germany). On the same sites, we counted HCLM pupae, larvae, opened and closed mines, and parasitised larvae and pupae in leaves collected from horse chestnut (Aesculus hippocastanum L., Hippocastanaceae) trees with and without bird exclusion. In both years, the HCLM number and the proportion of closed mines were higher in bird exclusion trees, particularly on sites with high abundance of tits. Hence, we suggest including the facilitation of birds, particularly tits, in future HCLM biocontrol strategies.     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

Secondary embryogenesis in androgenic embryo cultures of <Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.

Secondary somatic embryos appeared on the cotyledons and radiculi of embryos derived from suspension and anther cultures of Aesculus hippocastanum L. The highest number of secondary somatic embryos formed on a hormone-free medium.This work was supported by the Ministry of Science and Environment Protection of Serbia, grant N₀. 1573.     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Spatial leaf distribution and self-thinning exponent of<Emphasis Type="Italic"> Pinus banksiana</Emphasis> and<Emphasis Type="Italic"> Populus tremuloides</Emphasis>

A hypothesis that the pattern of spatial leaf distribution in forest canopies is numerically related to the exponent of the self-thinning relationship in even-aged monocultures was tested by evaluating the crown fractal dimension of Pinus banksiana (jack pine) and Populus tremuloides (quaking aspen) in Wood Buffalo National Park, Canada. Pure species stands that were considered the most dense for a given mean tree size were measured to establish the empirical self-thinning relationships. The value of the self-thinning exponent was estimated as –1.42 with 95% Confidence Interval (CI) (–1.47, –1.36) for Pinus banksiana, and –1.29 with 95% CI (–1.45, –1.14) for Populus tremuloides. For each species the box dimension of spatial leaf distribution was estimated from unit cylinders described by sequentially lowering in forest canopies, horizontal flaps of one of various diameters attached to the top of a height-measuring pole. The box dimension appeared as 1.95 (1.84, 2.06) for Pinus banksiana, and 2.24 (2.05, 2.43) for Populus tremuloides. By assuming that the box dimension is equivalent to the fractal dimension at the inter-population level, the self-thinning exponent was predicted to be –1.53 (–1.62, –1.45) for Pinus banksiana, and –1.33 (–1.45, –1.23) for Populus tremuloides. The empirical exponent was equivalent to that predicted from the box fractal dimension, as judged by the 95% CI of the dimensions. We conclude that spatial patterns of leaf distribution in forest canopies, as being characterized by the box fractal dimension, are closely related to the value of the self-thinning exponent in the dense monocultures of the species we examined.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Biomass production,photosynthesis, and leaf water relations of <Emphasis Type="Italic">Spartina alterniflora</Emphasis> under moderate water stress

The perennial smooth cordgrass, Spartina alterniflora, has been successfully introduced in salty ecosystems for revegetation or agricultural use. However, it remains unclear whether it can be introduced in arid ecosystems. The aim of this study was to investigate the physiological response of this species to water deficiency in a climate-controlled greenhouse. The experiment consisted of two levels of irrigation modes, 100 and 50% field capacities (FC). Although growth, photosynthesis, and stomatal conductance of plants with 50% FC were reduced at 90 days from the start of the experiment, all of the plants survived. The water-stressed plants exhibited osmotic adjustment and an increase in the maximum elastic modulus that is assumed to be effective to enhance the driving force for water extraction from the soil with small leaf water loss. An increase in the water use efficiency was also found in the water-stressed plants, which could contribute to the maintenance of leaf water status under drought conditions. It can be concluded that S. alterniflora has the capacity to maintain leaf water status and thus survive in arid environment.     

Complementary expression of<Emphasis Type="Italic"> AP-2</Emphasis> and<Emphasis Type="Italic"> AP-2rep</Emphasis> in ectodermal derivatives of<Emphasis Type="Italic"> Xenopus</Emphasis> embryos

In an attempt to define the pattern of developmental expression of AP-2rep and AP-2 in Xenopus embryos, we cloned a Xenopus AP-2rep cDNA. The AP-2rep message was localized in the organizer region at the gastrula stage whereas AP-2 was expressed ventro-laterally in the animal hemisphere. Later, AP-2rep was expressed in the entire neural tissue at the neurula stage while AP-2 was predominantly expressed in the cranial neural crest areas. The endogenous expression of AP-2 in the neural crest area was diminished by ectopic injection of AP-2rep RNA, suggesting a role for AP-2rep in the differentiation of neural tissues by restricting the expression of AP-2 in the Xenopus embryo.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

Composition of<Emphasis Type="Italic">Casuarina</Emphasis> leaf litter and its influence on<Emphasis Type="Italic">Frankia-Casuarina</Emphasis> symbiosis in soil

Plant needles ofCasuarina equisetifolia were collected and analyzed in parallel with soil analysis. In three strains ofFrankia—symbionts ofCasuarina—their infectivity and plant performance was determinedin vitro after soil amendment with different leaf litter concentrations. Only one strain was able to nodulate the plant at all litter concentrations (0.5, 3 and 5%) although the nodules were very small. However, all treated plants grew poorly; their growth was reduced by approximately 90% (for 5% litter concentration) compared to plants grown on untreated soil, on the basis of total dry mass. Inhibition of nodulation can be attributed to high concentrations of some elements and compounds that were either found inC. equisetifolia litter or originally found in soil (i.e. chloride, cyanide, copper, manganese and phenols). In general, plant growth decreased as more litter was added. Plant total nitrogen content was also reduced after increasing the litter concentration. The inhibitory effect of high litter concentrations was mainly on plant growth and to a lesser extent on plant nodulation byFrankia strains.     

Optimization of horse chestnut (<Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.) somatic embryo conversion

Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol) in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets (43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of converted plantlets was assessed by flow cytometry and no major ploidy changes were found.     

Injured and uninjured leaf photosynthetic responses after mechanical injury on <Emphasis Type="Italic">Nerium oleander</Emphasis> leaves,and <Emphasis Type="Italic">Danaus plexippus</Emphasis> herbivory on <Emphasis Type="Italic">Asclepias curassavica</Emphasis> leaves

Insect herbivory has variable effects on plant physiology; so greater understanding is needed about how injury alters photosynthesis on individual injured and uninjured leaves. Gas exchange and light-adapted leaf chlorophyll fluorescence measurements were collected from uninjured and mechanical partial leaf defoliation in two experiments with Nerium oleander (Apocynaceae) leaves, and one experiment with Danaus plexippus herbivory on Asclepias curassavica (Asclepiadaceae) leaves. Gas exchange impairment (lower photosynthetic rate (P _n ), stomatal conductance (g _s)) indicates water stress in a leaf, suggests stomatal limitations causing injury P _n impairment. The same pattern of gas exchange impairment also occurred on uninjured leaves opposite from injured leaves in both N. oleander experiments. This is an interesting result because photosynthetic impairment is rarely reported on injured leaves near injured leaves. No photosynthetic changes occurred in uninjured A. curassavica leaves opposite from D. plexippus-fed leaves. Partially defoliated leaves that had P _n and g _s reductions lacked any significant changes in intercellular leaf [CO₂], C _i. These results neither support, nor are sufficient to reject, stomatal limitations to photosynthesis. Manually imposed midrib vein severance in N. oleander experiment #1 significantly increased leaf C _i, indicating mesophyll limitations to photosynthesis. Maximal light-adapted leaf photochemical efficiency () and also non-photochemical quenching (q _N) were reduced by mechanical or insect herbivory to both study species, suggesting leaf trouble handling excess light energy not used for photochemistry. Midrib injury on N. oleander leaves and D. plexippus herbivory on A. curassavica leaves also reduced effective quantum yield (ΦPSII) and photochemical quenching (q _P); so reduced plastoquinone pools could lead to additional PSII reaction center closure.     

Arbuscular mycorrhizal fungi colonize decomposing leaves of<Emphasis Type="Italic"> Myrica parvifolia</Emphasis>,<Emphasis Type="Italic"> M. pubescens</Emphasis> and<Emphasis Type="Italic"> Paepalanthus</Emphasis> sp.

Hyphae and vesicles of arbuscular mycorrhizal fungi (AMF) were found within the decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. at three montane sites in Colombia. Hyphae, vesicles, and arbuscule-like structures were also found within scale-like leaves of the rhizomes of Paepalanthus sp. The litter found in the vicinity of the roots was divided into three decomposition layers. The highest AMF colonization occurred in the most decomposed leaves, which were in close association with roots. In contrast, there were no differences in AMF colonization of roots present in the different decomposition layers. Colonization of decomposing leaves by AMF did not differ between the two closely related species M. parvifolia and M. pubescens, nor between two sites (Guatavita and Zipacón, Colombia) differing in soil fertility. Occurrence of vesicles in decomposing leaves was correlated with abundant AMF extraradical hyphae among the leaves. We propose that AMF enter decomposing leaves mechanically through vascular tissue. As a consequence, AMF are well positioned to obtain and efficiently recycle mineral nutrients released by decomposer microorganisms before their loss by leaching or immobilization in soil.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.