Study of thiabendazole resistance and volatile organic compounds production of Penicillium expansum strains

The years of excessive use of thiabendazole to control Penicillium expansum has induced the development of resistance. Sensitivity of fourty eight strains collected from orchards and packinghouses in Emilia Romagna to pure and commercial TBZ was determined in vitro on TBZ amended medium (400μg/mL). Out of 48 strains, 35 were thiabendazole-sensitive (S) and 13 were thiabendazole-resistant (R). Microtiter assay adapted to P. expansum, showed EC50 values ranging from 54 to 320 μg/mL for ten TBZ-resistant strains. At the highest dose (50 μg/mL), resistant strains growth was not inhibited and the reported MICs value were >1000 μg/mL. Therefore, preliminary screening combined with microtiter assay, can be a good strategy to test susceptibility to TBZ. Mutations in the β-tubulin gene were studied on amino acid sequences from residue 167 to residue 357 of 10 P. expansum strains. Mutation at codon 198 was associated with TBZ-resistance. However, its absence in 3 resistant strains can be explained by the involvement of other mechanisms. Moreover, a P. expansum strain LB8/99 showed good antifungal effect against some fungal pathogens through double petri dish assay. It inhibited both mycelium growth and conidia germination of B. cinerea, C. acutatum, and M. laxa, and reduced significantly by 53% and 18% respectively P. expansum. Three major VOCS: geosmin, phenethyl alcolhol (PEA) and an unknown substance were identified by GC-MS analysis. Consistent fumigation of fungal pathogens with PEA (1230 mg/mL), inhibited both conidia germination and mycelium growth of all pathogens, except conidia germination of P. expansum that was reduced by 90% with respect to control. While, the concentration of PEA produced naturally by LB8/99 was ineffective in controlling the pathogens and seemed to have a synergic or additive effect with the other VOCS. Investigations to study the biofumigant effect of LB8/99 on other commodities like seeds and seedlings are in progress.

Recruitment limitation constrains local species richness and productivity in dry grassland

Species coexistence and local-scale species richness are limited by the availability of seeds and microsites for germination and establishment. We conducted a seed addition experiment in seminatural grassland at three sites in southern Switzerland and repeated the experiment in two successive years to evaluate various circumstances under which seed limitation and establishment success affect community functioning. A collection of 144000 seeds of 22 meadow species including grasses and forbs of local provenance was gathered, and seeds were individually sown in a density that resembled natural seed rain. The three communities were seed limited. Three years after sowing, single species varied in emergence (0–50%), survival (0–69%), and establishment rates (0–27%). One annual and 13 perennial species reached reproductive stage. Low establishment at one site and reduced growth at another site indicated stronger microsite limitation compared to the third site. Recruitment was influenced by differences in abiotic environmental conditions between sites (water availability, soil minerals) and by within-site differences in biotic interaction (competition). At the least water-limited site, sowing resulted in an increase in phytomass due to establishment of short-lived perennials in the second and third years after sowing. This increase persisted over the following two years due to establishment of longer-lived perennials. After sowing in a wetter year with higher phytomass, however, productivity did not increase, because higher intensity of competition in an early phase of establishment resulted in less vigorous plants later on. Due to the generally favorable weather conditions during this study, sowing year had a small effect on numbers of established individuals over all species. Recruitment limitation can thus constrain local-scale species richness and productivity, either by a lack of seeds or by reduced seedling growth, likely due to competition from the established vegetation.

Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

Niche differentiation between diploid and hexaploid Aster amellus

The maintenance of separated diploid and polyploid populations within a contact zone is possible due to both prezygotic and postzygotic isolation mechanisms. Niche differentiation between two cytotypes may be an important prezygotic isolating mechanism and can be studied using reciprocal transplant experiments. We investigated niche differentiation between diploid and hexaploid Aster amellus in their contact zone in the Czech Republic. Diploid populations are confined to habitats with low productivity, whereas hexaploid populations occur in habitats with both low and high productivity. Thus, we chose three diploid populations and six hexaploid populations, three in each of the two different habitat types. We analyzed habitat characteristics and carried out reciprocal transplant experiments in the field using both seeds and adult plants. Sites of diploid and hexaploid populations differed significantly in vegetation and soil properties. The mean number of juveniles was higher at sites of home ploidy level than at sites of foreign ploidy level, suggesting niche differentiation between the two cytotypes. On the other hand, transplanted adult plants survived at all sites and juvenile plants were able to establish at some sites of the foreign cytotype. Furthermore, the mean number of juveniles, survival, and flowering percentages were higher at home sites than at foreign sites, indicating local adaptation. We conclude that niche differentiation between the two cytotypes and local adaptation within each cytotype may contribute to the maintenance of diploid and hexaploid populations of A. amellus in their contact zone. Moreover, further factors, such as differences in flowering phenology and exclusion of minority cytotypes, should also be considered.

Gastropod Seed Dispersal: An Invasive Slug Destroys Far More Seeds in Its Gut than Native Gastropods

Seed dispersal is one of the most important mechanisms shaping biodiversity, and animals are one of the key dispersal vectors. Animal seed dispersal can directly or indirectly be altered by invasive organisms through the establishment of new or the disruption of existing seed dispersal interactions. So far it is known for a few gastropod species that they ingest and defecate viable plant seeds and consequently act as seed dispersers, referred to as gastropodochory. In a multi-species experiment, consisting of five different plant species and four different gastropod species, we tested with a fully crossed design whether gastropodochory is a general mechanism across native gastropod species, and whether it is altered by the invasive alien slug species Arion lusitanicus. Specifically, we hypothesized that a) native gastropod species consume the seeds from all tested plant species in equal numbers (have no preference), b) the voracious invasive alien slug A. lusitanicus – similarly to its herbivore behaviour – consumes a higher amount of seeds than native gastropods, and that c) seed viability is equal among different gastropod species after gut passage. As expected all tested gastropod species consumed all tested plant species. Against our expectation there was a difference in the amount of consumed seeds, with the largest and native mollusk Helix pomatia consuming most seeds, followed by the invasive slug and the other gastropods. Seed damage and germination rates did not differ after gut passage through different native species, but seed damage was significantly higher after gut passage through the invasive slug A. lusitanicus, and their germination rates were significantly reduced.

Tree size and fecundity influence ballistic seed dispersal of two dominant mast-fruiting species in a tropical rain forest

Seed production, seed dispersal, and seedling recruitment are integral to forest dynamics, especially in masting species. Often these are studied separately, yet scarcely ever for species with ballistic dispersal even though this mode of dispersal is common in legume trees of tropical African rain forests. Here, we studied two dominant main-canopy tree species, Microberlinia bisulcata and Tetraberlinia bifoliolata (Caesalpinioideae), in 25 ha of primary rain forest at Korup, Cameroon, during two successive masting events (2007/2010). In the vicinity of c. 100 and 130 trees of each species, 476/580 traps caught dispersed seeds and beneath their crowns c. 57,000 pod valves per species were inspected to estimate tree-level fecundity. Seed production of trees increased non-linearly and asymptotically with increasing stem diameters. It was unequal within the two species’ populations, and differed strongly between years to foster both spatial and temporal patchiness in seed rain. The M. bisulcata trees could begin seeding at 42–44 cm diameter: at a much larger size than could T. bifoliolata (25 cm). Nevertheless, per capita life-time reproductive capacity was c. five times greater in M. bisulcata than T. bifoliolata owing to former’s larger adult stature, lower mortality rate (despite a shorter life-time) and smaller seed mass. The two species displayed strong differences in their dispersal capabilities. Inverse modelling (IM) revealed that dispersal of M. bisulcata was best described by a lognormal kernel. Most seeds landed at 10–15 m from stems, with 1% of them going beyond 80 m (<100 m). The direct estimates of fecundity significantly improved the models fitted. The lognormal also described well the seedling recruitment distribution of this species in 121 ground plots. By contrast, the lower intensity of masting and more limited dispersal of the heavier-seeded T. bifoliolata prevented reliable IM. For this species, seed density as function of distance to traps suggested a maximum dispersal distance of 40–50 m, and a correspondingly more aggregated seedling recruitment pattern ensued than for M. bisulcata. From this integrated field study, we conclude that the reproductive traits of M. bisulcata give it a considerable advantage over T. bifoliolata by better dispersing more seeds per capita to reach more suitable establishment sites, and combined with other key traits they explain its local dominance in the forest. Understanding the linkages between size at onset of maturity, individual fecundity, and dispersal capability can better inform the life-history strategies, and hence management, of co-occurring tree species in tropical forests.

Endozoochory by slugs can increase bryophyte establishment and species richness

Herbivory can affect plant community composition and diversity by removing biomass and reducing light competition. Herbivory may particularly benefit low growing species such as bryophytes, which are frequently limited by light competition. Gastropods are important herbivores of seed plants and cryptogams, furthermore, they can disperse propagules such as seeds and spores via endozoochory. However, whether gastropod herbivory can reduce the dominance of vascular plants and thereby promote the germination and establishment of endozoochorously dispersed bryophyte spores has never been tested experimentally. Moreover, it is unclear whether these possible interacting effects can influence bryophyte species richness. Here, we tested for endozoochorous spore dispersal by slugs, in combination with sowing of vascular plants, in a fully factorial common garden experiment. Enclosures contained either slugs previously fed with bryophyte sporophytes, control slugs, or no slugs. After 21 days the bryophyte cover was on average 2.8 times higher (3.9 versus 1.4) and after eight months the bryophyte species richness 2.6 times higher (5.8 versus 2.2) in enclosures containing slugs previously fed with bryophyte sporophytes than in the other treatments. Furthermore, after eight months high vascular plant cover reduced bryophyte diversity. On average enclosures without seed sowing harboured 1.6 times more bryophyte species than the ones with seed sowing (4.2 versus 2.6), indicating competitive effects of vascular plants on bryophytes. Our findings suggest that slugs are important dispersal vectors for bryophytes and that they can increase bryophyte populations and maintain bryophyte diversity by reducing the dominance of vascular plants.

Enhancing Food Sovereignty and Food Security through the Revitalization of Indigenous Knowledge: Experiences from the Bolivian Andes

Endogenous development is defined as development that values primarily locally available resources and the way people organized themselves for that purpose. It is a dynamic and evolving concept that also embraces innovations and complementation from other than endogenous sources of knowledge; however, only as far as they are based on mutual respect and the recognition of cultural and socioeconomic self-determination of each of the parties involved. Experiences that have been systematized in the context of the BioAndes Program are demonstrating that enhancing food security and food sovereignty on the basis of endogenous development can be best achieved by applying a ‘biocultural’ perspective: This means to promote and support actions that are simultaneously valuing biological (fauna, flora, soils, or agrobiodiversity) and sociocultural resources (forms of social organization, local knowledge and skills, norms, and the related worldviews). In Bolivia, that is one of the Latin-American countries with the highest levels of poverty (79% of the rural population) and undernourishment (22% of the total population), the Program BioAndes promotes food sovereignty and food security by revitalizing the knowledge of Andean indigenous people and strengthening their livelihood strategies. This starts by recognizing that Andean people have developed complex strategies to constantly adapt to highly diverse and changing socioenvironmental conditions. These strategies are characterized by organizing the communities, land use and livelihoods along a vertical gradient of the available eco-climatic zones; the resulting agricultural systems are evolving around the own sociocultural values of reciprocity and mutual cooperation, giving thus access to an extensive variety of food, fiber and energy sources. As the influences of markets, competition or individualization are increasingly affecting the life in the communities, people became aware of the need to find a new balance between endogenous and exogenous forms of knowledge. In this context, BioAndes starts by recognizing the wealth and potentials of local practices and aims to integrate its actions into the ongoing endogenous processes of innovation and adaptation. In order to avoid external impositions and biases, the program intervenes on the basis of a dialogue between exogenous, mainly scientific, and indigenous forms of knowledge. The paper presents an analysis of the strengths and weaknesses of enhancing endogenous development through a dialogue between scientific and indigenous knowledge by specifically focusing on its effects on food sovereignty and food security in three ‘biocultural’ rural areas of the Bolivian highlands. The paper shows how the dialogue between different forms of knowledge evolved alongside the following project activities: 1) recuperation and renovation of local seeds and crop varieties (potato – Solanum spp., quinoa – Chenopodium quinoa, cañahua – Chenopodium pallidicaule); 2) support for the elaboration of community-based norms and regulations for governing access and distribution of non-timber forest products, such as medicinal, fodder, and construction plants; 3) revitalization of ethnoveterinary knowledge for sheep and llama breeding; 4) improvement of local knowledge about the transformation of food products (sheep-cheese, lacayote – Cucurbita sp. - jam, dried llama meat, fours of cañahua and other Andean crops). The implementation of these activities fostered the community-based livelihoods of indigenous people by complementing them with carefully and jointly designed innovations based on internal and external sources of knowledge and resources. Through this process, the epistemological and ontological basis that underlies local practices was made visible. On this basis, local and external actors started to jointly define a renewed concept of food security and food sovereignty that, while oriented in the notions of well being according to a collectively re-crafted world view, was incorporating external contributions as well. Enabling and hindering factors, actors and conditions of these processes are discussed in the paper.

Amino Acid Export in Developing Arabidopsis Seeds Depends on UmamiT Facilitators

Essential amino acids cannot be synthesized by humans and animals. They often are limiting in plant-derived foods and determine the nutritional value of a given diet [1]. Seeds and fruits often represent the harvestable portion of plants. In order to improve the amino acid composition of these tissues, it is indispensable to understand how these substrates are transported within the plant. Amino acids result from nitrogen assimilation, which often occurs in leaves, the source tissue. They are transported via the vasculature, the xylem, and the phloem into the seeds, the so-called sink tissue, where they are stored or consumed. In seeds, several tissues are symplasmically isolated [2, 3], i.e., not connected by plasmodesmata, channels in the cell walls that enable a cytoplasmic continuum in plants [4]. Consequently, amino acids must be exported from cells into the apoplast and re-imported many times to support seed development. Several amino acid importers are known, but exporters remained elusive [5, 6]. Here, we characterize four members of the plant-specific UmamiT transporter family from Arabidopsis, related to the amino acid facilitator SIAR1 and the vacuolar auxin transporter WAT1 [7, 8]. We show that the proteins transport amino acids along their (electro)chemical potential across the plasma membrane. In seeds, they are found in tissues from which amino acids are exported. Loss-of-function mutants accumulate high levels of free amino acids in fruits and produce smaller seeds. Our results strongly suggest a crucial role for the UmamiTs in amino acid export and possibly a means to improve yield quality.

Pollen and macrofossils profiles and age determination from three cores in Northeastern Kansas, U.S.A.

Two marshes near Muscotah and Arrington, Atchison County, northeastern Kansas, yielded a pollen sequence covering the last 25,000 yrs of vegetation development. The earliest pollen spectra are comparable with surface pollen spectra from southern Saskatchewan and southeastern Manitoba and might indicate a rather open vegetation but with some pine, spruce, and birch as the most important tree species, with local stands of alder and willow. This type of vegetation changed about 23,000 yrs ago to a spruce forest, which prevailed in the region until at least 15,000 yrs ago. Because of a hiatus, the vegetation changes resulting in the spread of a mixed deciduous forest and prairie, which was present in the region from 11,000 to 9,000 yrs ago, remain unknown. Prairie vegetation, with perhaps a few trees along the valleys, covered the region until about 5,000 yrs ago, when a re-expansion of deciduous trees began in the lowlands.

Rock magnetic properties and radiocarbon dates of samples obtained on Ile de Possession, South Indian Ocean

A 6200 year old peat sequence, cored in a volcanic crater on the sub-Antarctic Ile de la Possession (Iles Crozet), has been investigated, based on a multi-proxy approach. The methods applied are macrobotanical (mosses, seeds and fruits) and diatom analyses, complemented by geochemical (Rock-Eval6) and rock magnetic measurements. The chronology of the core is based on 5 radiocarbon dates. When combining all the proxy data the following changes could be inferred. From the onset of the peat formation (6200 cal yr BP) until ca. 5550 cal yr BP, biological production was high and climatic conditions must have been relatively warm. At ca. 5550 cal yr BP a shift to low biological production occurred, lasting until ca. 4600 cal yr BP. During this period the organic matter is well preserved, pointing to a cold and/or wet environment. At ca. 4600 cal yr BP, biological production increased again. From ca. 4600 cal yr BP until ca. 4100 cal yr BP a 'hollow and hummock' micro topography developed at the peat surface, resulting in the presence of a mixture of wetter and drier species in the macrobotanical record. After ca. 4100 cal yr BP, the wet species disappear and a generally drier, acidic bog came into existence. A major shift in all the proxy data is observed at ca. 2800 cal yr BP, pointing to wetter and especially windier climatic conditions on the island probably caused by an intensification and/or latitudinal shift of the southern westerly belt. Caused by a stronger wind regime, erosion of the peat surface occurred at that time and a lake was formed in the peat deposits of the crater, which is still present today.

The relevance of seed size in modulating leaf physiology and early plant performance in two tree species

he size of seeds and the microsite of seed dispersal may affect the early establishment of seedlings through different physiological processes. Here, we examined the effects of seed size and light availability on seedling growth and survival, and whether such effects were mediated by water use efficiency. Acorns of Quercus petraea and the more drought-tolerant Quercus pyrenaica were sowed within and around a tree canopy gap in a sub-Mediterranean forest stand. We monitored seedling emergence and measured predawn leaf water potential (Ψpd), leaf nitrogen per unit area (Na), leaf mass per area, leaf carbon isotope composition (δ13C) and plant growth at the end of the first summer. Survival was measured on the next year. Path analysis revealed a consistent pattern in both species of higher δ13C as Ψpd decreased and higher δ13C as seedlings emerged later in the season, indicating an increase in 13C as the growing season is shorter and drier. There was a direct positive effect of seed size on δ13C in Q. petraea that was absent in Q. pyrenaica. Leaf δ13C had no effect on growth but the probability of surviving until the second year was higher for those seedlings of Q. pyrenaica that had lower δ13C on the first year. In conclusion, leaf δ13C is affected by seed size, seedling emergence time and the availability of light and water, however, leaf δ13C is irrelevant for first year growth, which is directly dependent on the amount of seed reserves.

Disentangling factors controlling fruit and seed removal by rodents in temperate forests

Fleshy fruits fall on to the ground together with cleaned seeds previously ingested by primary dispersers, offering a wide range of fruits and seeds to the ground foragers. Although nutritional properties strongly differ between fruits and seeds, this different seed presentation (cleaned seeds versus seeds within the pulp) has not been addressed in seed removal studies. This study reports on the removal of fruits versus their seeds in five fleshy-fruited species in a temperate forest. We found that rodents removed most of the seeds and partially consumed most of the fruits, preferring seeds to fruits. Rodents bit the fruits to extract the seeds, leaving most of the pulp. We found a preference ranking for the seeds (Sorbus aucuparia>Ilex aquifolium>Sorbus aria>Rosa canina>Crataegus monogyna) but no preferences were found for the fruits, probably due to their similarities in pulp constituents. Seed and fruit choice were affected by chemical and physical properties and not by their size. The presence of alternative and preferred seeds (nuts) delayed the encounter of the fruits and seeds and diminished their removal rates. We found that higher rodent abundance is not necessarily associated with higher removal rates of fleshy fruits. Rodent abundance, fruit size and seed size are minor factors in the removal of fleshy fruits and their seeds. This study underlines that scatter-hoarding rodents are important removers of fleshy fruits and their seeds, producing a differential seed removal depending on the seed presentation (with or without pulp), the nutritional properties of the seeds (but not of the fruits) and the presence of alternative food

Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana

An HPLC/GC–MS/MS technique (high-pressure liquid chromatography in combination with gas chromatography–tandem mass spectrometry) has been worked out to analyze indole-3-acetamide (IAM) with very high sensitivity, using isotopically labelled IAM as an internal standard. Using this technique, the occurrence of IAM in sterile-grown Arabidopsis thaliana (L.) Heynh. was demonstrated unequivocally. In comparison, plants grown under non-sterile conditions in soil in a greenhouse showed approximately 50% higher average levels of IAM, but the differences were not statistically significant. Thus, microbial contributions to the IAM extracted from the tissue are likely to be minor. Levels of IAM in sterile-grown seedlings were highest in imbibed seeds and then sharply declined during the first 24 h of germination and further during early seedling development to remain below 20–30 pmol g–1 fresh weight throughout the rosette stage. The decline in indole-3-aetic acid (IAA) levels during germination was paralleled by a similar decline in IAM levels. Recombinant nitrilase isoforms 1, 2 and 3, known to synthesize IAA from indole-3-acetonitrile, were shown to produce significant amounts of IAM in vitro as a second end product of the reaction besides IAA. NIT2 was earlier shown to be highly expressed in developing and in mature A. thaliana embryos, and NIT3 is the dominantly active gene in the hypocotyl and the cotyledons of young, germinating seedlings. Collectively, these data suggest that the elevated levels of IAM in seeds and germinating seedlings result from nitrilase action on indole-3-acetonitrile, a metabolite produced in the plants presumably from glucobrassicin turnover.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae), a narrow and endangered endemic species of SW Spain

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vázquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known. This species was catalogued in the ?Endangered? category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out an exhaustive census of A. gines-lopezii , and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%?60%) was increased to 97%?99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.