Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Endophytic fungi from Vitis labrusca L. ('Niagara Rosada') and its potential for the biological control of Fusarium oxysporum

We investigated the diversity of endophytic fungi found on grape (Vitis labrusca cv. Niagara Rosada) leaves collected from Salesopolis, SP, Brazil. The fungi were isolated and characterized by amplified ribosomal DNA restriction analysis, followed by sequencing of the ITS1-5.8S-ITS2 rDNA. In addition, the ability of these endophytic fungi to inhibit the grapevine pathogen Fusarium oxysporum f. sp herbemontis was determined in vitro. We also observed that the climatic factors, such as temperature and rainfall, have no effect on the frequency of infection by endophytic fungi. The endophytic fungal community that was identified included Aporospora terricola, Aureobasidium pullulans, Bjerkandera adusta, Colletotrichum boninense, C. gloeosporioides, Diaporthe helianthi, D. phaseolorum, Epicoccum nigrum, Flavodon flavus, Fusarium subglutinans, F. sacchari, Guignardia mangiferae, Lenzites elegans, Paraphaeosphaeria pilleata, Phanerochaete sordida, Phyllosticta sp, Pleurotus nebrodensis, Preussia africana, Tinctoporellus epiniltinus, and Xylaria berteri. Among these isolates, two, C. gloeosporioides and F. flavus, showed potential antagonistic activity against F. oxysporum f. sp herbemontis. We suggest the involvement of the fungal endophyte community of V. labrusca in protecting the host plant against pathogenic Fusarium species. Possibly, some endophytic isolates could be selected for the development of biological control agents for grape fungal disease; alternatively, management strategies could be tailored to increase these beneficial fungi.

Gibberellic acid in vegetative and reproductive development of Phalaenopsis orchid hybrid genus

The flower industry represents about one billion dollars in Brazil and the development of techniques aimed at flowering control is required. This study evaluated the influence of gibberellic acid (GA3) on the vegetative and reproductive development of young plants of Phalaenopsis FSNT 'Dai-Itigo' hybrid pink color. The application of GA3 was made by foliar sprays at concentrations of 0, 125, 250, 500 and 1,000 mg L -1. The length of leaves increased significantly when using GA3 at low concentrations, but leaf width decreased. The application of GA3 at 125 mg L -1 showed the best results for the promotion of flowering and flower quality of this orchid hybrid. In this treatment, about 50% of plants treated with GA3 flowered about 6-12 months before the plants that were non-treated with this plant growth regulator. The quality of flowering and flowers was best with 125 mg L -1 GA3.

Germination, carbohydrate composition and vigor of cryopreserved Caesalpinia echinata seeds

The present study investigated the germination and vigor of Caesalpinia echinata (Brazilwood) seeds stored at negative temperatures. Recently harvested seeds were cryopreserved at -18º or -196ºC and periodically evaluated for germination, seed vigor and carbohydrate composition. The temperatures did not influence the germination percentages or vigor. The germination percentage decreased from 88% in recently harvested seeds to 60% after 730 days of storage. The different temperature and storage times tested did not affect the vigor seed germination as indicated by the measures of plant growth and survival. The different temperatures used did not cause changes in the carbohydrate composition. The tegument cell walls were rich in lignin, arabinose and xylose. The cytoplasm of the cotyledons and embryos had high levels of glucose, fructose, and sucrose. The cryopreservation technique here presented was effective in the conservation of Brazilwood seeds for the medium term.

Biorregulador na composição química e na produtividade de grãos de soja

O objetivo do presente trabalho foi avaliar a composição química e a produtividade de grãos de soja, em resposta à aplicação de biorregulador na cultura da soja. Para tanto, sementes de soja da cultivar BRS 246 RR foram semeadas no mês de outubro dos anos agrícolas de 2007/2008 e 2008/2009, no delineamento experimental em blocos completos com os tratamentos casualizados, com quatro repetições. Os tratamentos, arranjados em esquema fatorial, foram compostos pela combinação do tratamento de sementes com o biorregulador (sem e com 0,500 L 100 kg-1 de sementes) e cinco doses do produto (0; 0,125; 0,250; 0,375 e 0,500 L ha-1) aplicadas via foliar, em dois estádios de desenvolvimento da cultura (V5 ou R3). Utilizou-se um biorregulador líquido da Stoller do Brasil Ltda., denominado de Stimulate®, composto por três reguladores vegetais na seguinte concentração: 0,005% do ácido indolbutirico - IBA (análogo de auxina), 0,009% de cinetina (citocinina) e 0,005% de ácido giberélico - GA3 (giberelina). O uso do biorregulador influenciou no incremento da produtividade; os teores de óleo e proteínas foram alterados pela ação do biorregulador, com tendência de favorecimento do conteúdo proteico.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

On reproduction in red algae: futher research needed at the molecular level

[EN]Multicellular red algae (Rhodophyta) have some of the most complex life cycles known in living organisms. Economically valuable seaweeds, such as phycocolloid producers, have a triphasic (gametophyte, carposporophyte, and tetrasporophyte) life cycle, not to mention the intricate alternation of generations in the edible “sushi-alga” nori. It is a well-known fact that reproductive processes are controlled by one or more abiotic factor(s), including day length, light quality, temperature, and nutrients. Likewise, endogenous chemical factors such as plant growth regulators have been reported to affect reproductive events in some red seaweeds. Still, in the genomic era and given the high throughput techniques at our disposal, our knowledge about the endogenous molecular machinery lags far behind that of higher plants.

Salinity Effect on Horticultural Crops: Morphological, Physiological, and Biomolecular Elements of Salinity Stress Response

Among abiotic stresses, high salinity stress is the most severe environmental stress. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. Salt tolerance in plants is a multifarious phenomenon involving a variety of changes at molecular, organelle, cellular, tissue as well as whole plant level. In addition, salt tolerant plants show a range of adaptations not only in morphological or structural features but also in metabolic and physiological processes that enable them to survive under extreme saline environments. The main objectives of my dissertation were understanding the main physiological and biomolecular features of plant responses to salinity in different genotypes of horticultural crops that are belonging to different families Solanaceae (tomato) and Cucurbitaceae (melon) and Brassicaceae (cabbage and radish). Several aspects of crop responses to salinity have been addressed with the final aim of combining elements of functional stress response in plants by using several ways for the assessment of plant stress perception that ranging from destructive measurements (eg. leaf area, relative growth rate, leaf area index, and total plant fresh and dry weight), to physiological determinations (eg. stomatal conductance, leaf gas exchanges, water use efficiency, and leaf water relation), to the determination of metabolite accumulation in plant tissue (eg. Proline and protein) as well as evaluation the role of enzymatic antioxidant capacity assay in scavenging reactive oxygen species that have been generated under salinized condition, and finally assessing the gene induction and up-down regulation upon salinization (eg. SOS pathway).

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Biochar in perennial crops: nutritional, agronomical and environmental implications

Biochar is the solid C-rich matrix obtained by pyrolysis of biomasses, currently promoted as a soil amendment with the aim to offset anthropogenic C emissions, while ameliorating soil properties and growth conditions. Benefits from biochar seem promising, although scientific understandings are beginning to be explored. In this project, I performed a suite of experiments in controlled and in field conditions with the aims to investigate the effect of biochar on: a) the interaction with minerals; b) Fe nutrition in kiwifruit; c) soil leaching, soil fertility, soil CO2 emissions partitioning, soil bacterial profile and key gene expression of soil nitrification-involved bacteria; d) plant growth, nutritional status, yield, fruit quality and e) its physical-chemical changes as affected by long-term environmental exposure. Biochar released K, P and Mg but retained Fe, Mn, Cu and Zn on its surface which in turn hindered Fe nutrition of kiwifruit trees. A redox reaction on the biochar surface exposed to a Fe source was elucidated. Biochar reduced the amount of leached NH4+-N but increased that of Hg, K, P, Mo, Se and Sn. Furthermore, biochar synergistically interacted with compost increasing soil field capacity, fertility, leaching of DOC, TDN and RSOC, suggesting a priming effect. However, in field conditions, biochar did not affect yield, nutritional status and fruit quality. Actinomadura flavalba, Saccharomonospora viridis, Thermosporomyces composti and Enterobacter spp. were peculiar of the soil amended with biochar plus compost which exhibited the highest band richness and promoted gene expression levels of Nitrosomonas spp., Nitrobacter spp. and enzymatic-related activity. Environmental exposure reduced C, K, pH and water infiltration of biochar which instead resulted in a higher O, Si, N, Na, Al, Ca, Mn and Fe at%. Oxidation occurred on the aged biochar surface, it decreased progressively with depth and induced the development of O-containing functional groups, up to 75nm depth.

Counterbalancing effects of competition for resources and facilitation against grazing in alpine snowbed communities

Alpine snowbeds are habitats where the major limiting factors for plant growth are herbivory and a small time window for growth due to late snowmelt. Despite these limitations, snowbed vegetation usually forms a dense carpet of palatable plants due to favourable abiotic conditions for plant growth within the short growing season. These environmental characteristics make snowbeds particularly interesting to study the interplay of facilitation and competition. We hypothesised an interplay between resource competition and facilitation against herbivory. Further, we investigated whether these predicted neighbour effects were species-specific and/or dependent on ontogeny, and whether the balance of positive and negative plant–plant interactions shifted along a snowmelt gradient. We determined the neighbour effects by means of neighbour removal experiments along the snowmelt gradient, and linear mixed model analyses. The results showed that the effects of neighbour removal were weak but generally consistent among species and snowmelt dates, and depended on whether biomass production or survival was considered. Higher total biomass and increased fruiting in removal plots indicated that plants competed for nutrients, water, and light, thereby supporting the hypothesis of prevailing competition for resources in snowbeds. However, the presence of neighbours reduced herbivory and thereby also facilitated survival. For plant growth the facilitative effects against herbivores in snowbeds counterbalanced competition for resources, leading to a weak negative net effect. Overall the neighbour effects were not species-specific and did not change with snowmelt date. Our finding of counterbalancing effects of competition and facilitation within a plant community is of special theoretical value for species distribution models and can explain the success of models that give primary importance to abiotic factors and tend to overlook interrelations between biotic and abiotic effects on plants.

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.

A comparison of ground cover and frequency estimation methods for post-harvest soil monitoring

The amount and type of ground cover is an important characteristic to measure when collecting soil disturbance monitoring data after a timber harvest. Estimates of ground cover and bare soil can be used for tracking changes in invasive species, plant growth and regeneration, woody debris loadings, and the risk of surface water runoff and soil erosion. A new method of assessing ground cover and soil disturbance was recently published by the U.S. Forest Service, the Forest Soil Disturbance Monitoring Protocol (FSDMP). This protocol uses the frequency of cover types in small circular (15cm) plots to compare ground surface in pre- and post-harvest condition. While both frequency and percent cover are common methods of describing vegetation, frequency has rarely been used to measure ground surface cover. In this study, three methods for assessing ground cover percent (step-point, 15cm dia. circular and 1x5m visual plot estimates) were compared to the FSDMP frequency method. Results show that the FSDMP method provides significantly higher estimates of ground surface condition for most soil cover types, except coarse wood. The three cover methods had similar estimates for most cover values. The FSDMP method also produced the highest value when bare soil estimates were used to model erosion risk. In a person-hour analysis, estimating ground cover percent in 15cm dia. plots required the least sampling time, and provided standard errors similar to the other cover estimates even at low sampling intensities (n=18). If ground cover estimates are desired in soil monitoring, then a small plot size (15cm dia. circle), or a step-point method can provide a more accurate estimate in less time than the current FSDMP method.

Quantifying the ecological benefits of lakeshore restoration in northern Wisconsin

Housing development has increased dramatically in the Midwest with a high concentration around lakes. This development plays an important role in the economy of Northwoods communities. However, poorly planned development has the potential to alter a lake’s ecological processes and integrity. Studies have documented the impacts of housing developments and reported dramatic, negative changes to the flora and fauna in Vilas County, Wisconsin. One component of my research included examining the previously unstudied effects of residential development on the abundance and diversity of medium to large-bodied mammals using lakeshore ecosystems. The results suggest that a higher diversity of mammals were detected on low-development lakes. Coyotes were the most numerous species detected with the majority encountered on low-development lakes. White-tailed deer and red fox were more abundant on high-development lakes as compared to low-development lakes. I concluded that high-development lakes are having a negative affect on the mammal community in this area. Recently, lakeshore restoration has occurred on privately owned property in Vilas County and elsewhere in the Northwoods, but little is known about the benefit, if any, from these restoration efforts. A partnership between government agencies and academia has launched a long-term research project investigating the ecological benefits of lakeshore restoration. I investigated the impacts of using down woody material (DWM) to increase the success of restoration projects. Specifically, I tested the hypothesis that down woody material would reduce the variation in soil temperature, retain soil moisture, and improve plant survival and growth rates. I randomly assigned three DWM coverage treatments (0%, 25%, and 50%) on 3 m × 3 m experimental plots (n = 10 per treatment). The mean maximum soil temperature, temperature variation, and change in soil moisture content were significantly lower in the 25% and 50% DWM plots. I found no difference in survival, but snowberry (Symphoricarpos albus) and Barren strawberry (Waldstenia fragaroides) growth was significant greater in the 25% and 50% DWM plots. DWM addition can be considered a useful technique to physically manipulate soil properties and improve plant growth. Finally, I provided baseline data on vegetation structure, bird and small mammal community diversity and abundance for three lakes targeted for restoration efforts and their paired reference lakes. This study is one of the first of it kind in the area and continuing to document the degree of change in subsequent years will provide insight into the way the local ecosystem functions and how ecological communities are structured.