Quality management for vital forest tree seedlings : Final report, Project no: P33854-1

The work concerns development of a prototype molecular tests to identify vitality status of conifer seedlings. The work is done by NSure, Holland, Dalarna University and SUAS. In case for spruce, a successful validation experiment has been performed to validate the identified frost tolerance and vitality genes. Multiple indicators were identified that can be used to either reinforce the existing ColdnSure test, but also for development of a vitality test. The identified frost tolerance and vitality genes for pine still need to be validated. NSure together with Dalarna University aim to perform a validation next season. Multiple LN indicators were identified in spruce that can be used to determine the effectiveness of a LN treatment, but they are not yet validated. In spruce and pine hardly any scientific research is performed to study the effect of a LN treatment, particularly not at molecular level. Therefore NSure together with Dalarna Research Station want to apply for a project. Within this project, we would be able to develop the tests further.

Protection of spruce seedlings against pine weevil attacks by treatment of seeds or seedlings with nicotinamide, nicotinic acid and jasmonic acid

Sustainable methods are required to protect newly planted tree seedlings from insect herbivore attack. To this end, here Norway spruce (Picea abies (L.) Karst.) seeds were treated with 2.5 mM nicotinamide (NIC), 2.5 mM nicotinic acid (NIA), 3 mM jasmonic acid (JA) or 0.2 mM 5-azacytidine (5-Aza), and 6-month-old seedlings grown from these seeds were planted at a reforestation area in central Sweden. Attack by pine weevils (Hylobius abietis) was reduced by 50 per cent by NIC treatment, 62.5 per cent by JA treatment and 25 per cent by 5-Aza treatment, when compared with seedlings grown from untreated seeds. Watering 18-month-old spruce seedlings with 2 mM NIC or 2 mM NIA did reduce attack during the first season in the field by 40 and 53 per cent, respectively, compared with untreated plants. Girdling was also reduced by the different treatments. Analysis of conifer seedlings treated with 5-Aza points at a possible involvement of epigenetic mechanisms in this defensive capacity. This is supported by a reduced level of DNA methylation in the needles of young spruce seedlings grown in a greenhouse from NIC-treated seeds. Seed treatment for seedling defense potentiation is simple, inexpensive and also a new approach for forestry with many potential applications.

A machine learning approach for recognising woody plants on railway trackbeds

The purpose of this work in progress study was to test the concept of recognising plants using images acquired by image sensors in a controlled noise-free environment. The presence of vegetation on railway trackbeds and embankments presents potential problems. Woody plants (e.g. Scots pine, Norway spruce and birch) often establish themselves on railway trackbeds. This may cause problems because legal herbicides are not effective in controlling them; this is particularly the case for conifers. Thus, if maintenance administrators knew the spatial position of plants along the railway system, it may be feasible to mechanically harvest them. Primary data were collected outdoors comprising around 700 leaves and conifer seedlings from 11 species. These were then photographed in a laboratory environment. In order to classify the species in the acquired image set, a machine learning approach known as Bag-of-Features (BoF) was chosen. Irrespective of the chosen type of feature extraction and classifier, the ability to classify a previously unseen plant correctly was greater than 85%. The maintenance planning of vegetation control could be improved if plants were recognised and localised. It may be feasible to mechanically harvest them (in particular, woody plants). In addition, listed endangered species growing on the trackbeds can be avoided. Both cases are likely to reduce the amount of herbicides, which often is in the interest of public opinion. Bearing in mind that natural objects like plants are often more heterogeneous within their own class rather than outside it, the results do indeed present a stable classification performance, which is a sound prerequisite in order to later take the next step to include a natural background. Where relevant, species can also be listed under the Endangered Species Act.

Effects of conifer sawdust, hardwood sawdust, and peat on soil properties and a barefoot conifer seedling development

Organic amendments are commonly used to improve tree nursery soil conditions for increased seedling growth. However, few studies compare organic amendments effects on soil conditions, and fewer compare subsequent effects on seedling growth. The effects of three organic amendments on soil properties and seedling growth were investigated at the USDA Forest Service J.W. Toumey Nursery in Watersmeet, MI. Pine sawdust (red pine, Pinus resinosa), hardwood sawdust (maple, Acer spp. and aspen, Populus spp.), and peat were individually incorporated into a loamy sand nursery soil in August, 2006, and soil properties were sampled periodically for the next 14 months. Jack (Pinus banksiana), red, and white pine (Pinus strobus) were sown into test plots in June, 2007 and sampled for growth responses at the end of the growing season. It is hypothesized; pine sawdust and peat can be used as a satisfactory soil amendment to improve soil conditions and produce high quality seedlings, when compared to hardwood sawdust in bareroot nursery soils. This study has the potential to reduce nursery costs while broadening soil amendment options. The addition of peat and pine sawdust increased soil organic matter above control soil conditions after 14 months. However, hardwood sawdust-amended soils did not differ from control soils after same time period. High N concentrations in peat increased total soil N over the other treatments. Similarly, the addition of peat increased soil matric potential and available water over all other treatments. Seedlings grew tallest with the largest stem diameter, and had the largest biomass in both control soil and soil amended with peat, compared to either sawdust treatment. Seedlings grown in peat-amended soils had higher N concentrations than those grown in soils treated with pine sawdust, though neither was different from seedlings grown in control or hardwood sawdust-amended soils. Overall, peat is a well suited organic soil amendment for the enhancement of soil properties, but no amendments were able to increase one-year seedling growth over control soils.

(Table 2) Survivorship of transplanted seedlings of Picea glauca inside and outside three tree islands in 1994, 1995, 2009 and after 15 years

The northern boundary of boreal forest and the ranges of tree species are expected to shift northward in response to climate warming, which will result in a decrease in the albedo of areas currently covered by tundra vegetation, an increase in terrestrial carbon sequestration, and an alteration of biodiversity in the current Low Arctic. Central to the prediction of forest expansion is an increase in the reproductive capacity and establishment of individual trees. We assessed cone production, seed viability, and transplanted seedling success of Picea glauca (Moench.) Voss. (white spruce) in the early 1990s and again in the late 2000s at four forest stand sites and eight tree island sites (clonal populations beyond present treeline) in the Mackenzie Delta region of the Northwest Territories, Canada. Over the past 20 years, average temperatures in this region have increased by 0.9 °C. This area has the northernmost forest-tundra ecotone in North America and is one of the few circumpolar regions where the northern limit of conifer trees reaches the Arctic Ocean. We found that cone production and seed viability did not change between the two periods of examination and that both variables decreased northward across the forest-tundra ecotone. Nevertheless, white spruce individuals at the northern limit of the forest-tundra ecotone produced viable seeds. Furthermore, transplanted seedlings were able to survive in the northernmost sites for 15 years, but there were no signs of natural regeneration. These results indicate that if climatic conditions continue to ameliorate, reproductive output will likely increase, but seedling establishment and forest expansion within the forest-tundra of this region is unlikely to occur without the availability of suitable recruitment sites. Processes that affect the availability of recruitment sites are likely to be important elsewhere in the circumpolar ecotone, and should be incorporated into models and predictions of climate change and its effects on the northern forest-tundra ecotone.

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem-limited bacteria (Candidatus Liberibacter spp.) associated with citrus `huanglongbing` by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pera (Rutaceae) by using the electrical penetration graph (EPG-DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem-limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G-like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8-h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non-probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.

Relation between incidence of Fusarium graminearum in seeds, emergence and occurrence of giberela in wheat seedlings

In order to verify the behavior of 30 genotypes of wheat in relation to the emergence and incidence of giberela in wheat seedlings from seeds contaminated with F graminearum, experiments were carried out under laboratory and greenhouse conditions. In the laboratory, seeds were analyzed for health using freezer blotter test. In the greenhouse, seeds were sowed in plastic boxes filled with sand treated with methyl bromide. Statistical design was randomized blocks with 30 treatments, four replications of 50 seeds (200 seeds/treatment). Emergence of seedlings and giberela incidence were evaluated at seven, 14 and 21 days after sowing. Symptomatic seedlings were removed and submitted to humid chambers for 24 hours under laboratory conditions. There was no significant difference in the incidence of the pathogen in the emergence of seedlings. There was no correlation between the incidence of F graminearum in the genotypes and incidence of giberela in seedlings, nor between the incidence of giberela in seedlings and the incidence of the pathogen in the seeds.

Histological, physiological and molecular investigations of Fagus sylvatica seedlings infected with Phytophthora citricola

P>The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola.

Low glyphosate rates do not affect Citrus limonia (L.) Osbeck seedlings

BACKGROUND: Glyphosate is used to control weeds in citrus orchards, and accidental spraying or wind drift onto the seedlings may cause growth arrest owing to metabolism disturbance. Two experiments were carried out to investigate the effect of non-lethal rates (0, 180, 360 and 720 g Al ha(-1)) of glyphosate on four-month-old `Cravo` lime, Citrus limonia (L.) Osbeck, seedlings. Photosynthesis and the concentrations of shikimic acid, total free amino acids and phenolic acids were evaluated. RESULTS: Only transitory effects were observed in the! contents of shikimate and total free amino acids. No visual effects were observed. CONCLUSION: The present study showed that glyphosate at non-lethal rates, which is very usual when accidental spraying or wind drift occurs in citrus orchard, did not cause severe metabolic damage in `Cravo` lime seedlings. (C) 2009 Society of Chemical Industry

Diversity of endophytic bacteria from Eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans

The diversity and beneficial characteristics of endophytic microorganisms have been studied in several host plants. However, information regal-ding naturally, occurring seed-associated endophytes and vertical transmission among different life-history stages of hosts is limited. Endophytic bacteria were isolated from seeds and seedlings of 10 Eucalyptus species and two hybrids. The results showed that endophytic bacteria, Such as Bacillus, Enterococcus, Paenibacillus and Methylobacterium, are vertically transferred from seeds to seedlings. In addition, the endophytic bacterium Pantoea agglomerans was tagged with the gfp gene, inoculated into seeds and further reisolated from seedlings. These results suggested it novel approach to change the profile of the plants, where the bacterium is a delivery vehicle for desired traits. This is the first report of an endophytic bacterial community residing in Eucalyptus seeds and the transmission of these bacteria from seeds to seedlings. The bacterial species reported ill this work have been described as providing benefits to host plants. Therefore, we Suggest that endophytic bacteria can be transmitted vertically from seeds to seedlings, assuring the support of the bacterial community in the host plant.

The oleoresin secretory system in seedlings and adult plants of copaiba (Copaifera langsdorffii Desf., Leguminosae-Caesalpinioideae)

The ecological and economic importance of oleoresin produced by Copaifera langsdorffii is well established. This study aims to investigate the ontogeny, anatomy and ultrastructure of the internal glands of C. langsdorffii during plant development. Samples were processed for light and electron microscopy and a specific technique was applied to impregnate endomembranes. Internal secretory glands were observed in the hypocotyl, epicotyl and eophylls of seedlings, and in the primary stem, pulvinus, petiole, rachis and leaf blade of adult plants. Canals and cavities show differential distribution. They arise from ground meristem cells, and the lumen is first formed by schizogenesis followed by later schizolysigenous development. The dense cytoplasm of epithelial cells shows mitochondria, plastids without thylakoids, polyribosomes and endoplasmic reticulum. A periplastidial reticulum was also observed. Secretion is released by eccrine, granulocrine and holocrine processes. Lipophilic and hydrophilic compounds were histochemically detected in both canals and cavities, whereas resin was detected only in canals. The presence of these substances has been associated with plants` defences against dehydration, as well as against attacks from herbivores and pathogens, from seedling stage onwards. (C) 2011 Elsevier GmbH. All rights reserved.

Morpho-anatomical studies of seeds and seedlings of wild indigo, ""anileira"", Indigofera-Leguminosae

(Morpho-anatomical studies of seeds and seedlings of wild indigo, ""anileira"", Indigofera-Leguminosae). The common name ""wild indigo"" specifies Indigofera will L., I. suffruticosa Mill. (legitimate name) and I. truxillensis Kunth (legitimate name) that are very similar due to their external morphology. This work analyzed diagnostic characteristics of seeds and seedlings of these species since such features are widely used in taxonomic approaches within Leguminosae. We studied surface features and morpho-anatomy of seeds and cotyledons with scanning electronic microscopy and light microscopy, and described seedling phases. Although seedlings are similar, seed characteristics (size, shape, surface ornamentation, shape of the hilum and embryo size) and cotyledon characteristics (shape, trichome ornamentation, organization of spongy parenchyma along central veins and metabolite distribution in the tissues) have diagnostic features for the species. I. anil differs from I. suffruticosa in its larger seeds and acuminate-margined cotyledons. I. truxillensis is recognized by its cylindrical seeds and kidney-shaped cotyledons with large oil drops. We assume that the characteristics examined, plus fruit morphology and foliar anatomy suggest that I. anil, I. suffruticosa and I. truxillensis should not be synonymized.

Dry matter production and allocation in Eucalyptus cloeziana and Eucalyptus argophloia seedlings in response to soil water deficits

Effects of soil water availability on seedling growth, dry matter production and allocation were determined for Gympie ( humid coastal) and Hungry Hills ( dry inland) provenances of Eucalyptus cloeziana F. Muell. and for E. argophloia Blakely ( dry inland) species. Seven-month-old seedlings were subjected to well-watered (100% field capacity, FC), moderate (70% FC) and severe (50% FC) soil water regimes in a glasshouse environment for 14 wk. There were significant differences in seedling growth, biomass production and allocation patterns between species. E. argophloia produced twice as much biomass at 100% FC, and more than three times as much at 70% and 50% FC than did either E. cloeziana provenance. Although the humid provenance of E. cloeziana had a greater leaf area at 100% FC conditions than did the dry provenance, total biomass production did not differ significantly. Both E. cloeziana provenances were highly sensitive to water deficits. E. argophloia allocated 10% more biomass to roots than did E. cloeziana. Allometric analyses indicated that relative biomass allocation patterns were significantly affected by genotype but not by soil water availability. These results have implications for taxon selection for cultivation in humid and subhumid regions.

Spatial and temporal changes in multiple hormone groups during lateral bud release shortly following apex decapitation of chickpea (Cicer arietinum) seedlings

Although the co-ordination of promotive root-sourced cytokinin (CK) and inhibitory shoot apex-sourced auxin (IAA) is central to all current models on lateral bud dormancy release, control by those hormones alone has appeared inadequate in many studies. Thus it was hypothesized that the IAA : CK model is the central control but that it must be considered within the relevant timeframe leading to lateral bud release and against a backdrop of interactions with other hormone groups. Therefore, IAA and a wide survey of cytokinins (CKs), were examined along with abscisic acid (ABA) and polyamines (PAs) in released buds, tissue surrounding buds and xylem sap at 1 and 4 h after apex removal, when lateral buds of chickpea are known to break dormancy. Three potential lateral bud growth inhibitors, IAA, ABA and cis-zeatin 9-riboside (ZR), declined sharply in the released buds and xylem following decapitation. This is in contrast to potential dormancy breaking CKs like trans-ZR and trans-zeantin 9-riboside 5'phosphate (ZRMP), which represented the strongest correlative changes by increasing 3.5-fold in xylem sap and 22-fold in buds. PAs had not changed significantly in buds or other tissues after 4 h, so they were not directly involved in the breaking of bud dormancy. Results from the xylem and surrounding tissues indicated that bud CK increases resulted from a combination synthesis in the bud and selective loading of CK nucleotides into the xylem from the root.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.