Long-term heathland restoration on former grassland: The results of a 17-year experiment

European lowland heaths have declined by up to 80% due to land use change and lack of management. There has been considerable research into the restoration of this threatened habitat. However, long-term outcomes of restoration are poorly understood, especially in situations where past agricultural land use imposes severe constraints on community re-assembly. In 1989 a large-scale experiment was established to examine the effectiveness of five treatments to restore heathland on formerly productive grassland: (i) natural regeneration; (ii) herbicide application to facilitate regeneration; (iii) cultivation and application of seed-rich heathland vegetation; (iv) soil removal and incorporation of heathland topsoil; and (v) heathland translocation. After 17 years the pH of the unamended agricultural soil remained significantly higher than that of the adjacent heathland. All treatments showed different trajectories of vegetation change in the long-term. Natural colonisation by heathland species was slow due to seed limitation, resulting in formation of an acid grassland community. Heathland community assembly was not facilitated by destruction of the initial grassland with herbicide. Incorporation of topsoil had an intermediate effect on pH reduction. This may explain the subsequent failure of the plant community to assemble in the anticipated proportions, and the dominance of leguminous scrub species (Ulex spp.). Turf translocation was effective in reducing pH to the required range and restoring the heathland community in the long-term. However, this technique should only be considered as a means of ‘rescue’ when habitat destruction is otherwise unavoidable. The only practical and sustainable means of increasing heathland extent on former farmland is the application of seed-bearing vegetation cut as part of routine management. However, this technique needs refining in order to establish the full range of characteristic heathland species.     

Use of aliette,a basipetally translocated antimicrobial compound,to enhance rhizophere colonization and nodulation by root-nodule bacteria

Summary A method was developed to improve the colonizing ability of inoculated strains of root-nodule bacteria using aliette (aluminum tris-O-ethyl phosphonate), a basipetally translocated fungicide. Aliette applied to seeds of alfalfa inoculated with an aliette-resistant strain of Rhizobium meliloti increased the numbers of R. meliloti in the rhizosphere after 3 but not 37 days, increased the number of nodules, and with some seed treatments, increased the growth of alfalfa. The enhanced colonization by R. meliloti as a result of seed treatment with aliette lasted for at least 31 days for alfalfa, although plant weights did not increase, Colonization by R. meliloti was further enhanced if seeds and foliage were treated with the fungicide. Coating seeds or sparaying the foliage with aliette also increased the number and weight of nodules and nitrogenase activity in soybeans inoculated with an aliette-resistant strain of Bradyrhizobium japonicum. The stimulation of B. japonicum in the rhizosphere and of nodulation was evident with successive plantings of soybeans if the seeds for each planting were treated with the chemical, but aliette did not increase the yield of inoculated soybeans in the subsequent plantings. With only the seeds of the first planting of inoculated soybeans treated with aliette, the numbers of B. japonicum in the rhizosphere of subsequent plantings were only occasionally greater and the numbers of nodules on the later plantings were not increased. We suggest that root colonization, nodulation, and N₂ fixation by Rhizobium and Bradyrhizobium may be enhanced by the use of basipetally translocated antimicrobial compounds together with root-nodule bacteria that are resistant to those compounds.     

Electrophoretic variation as a measure of speciesdifferentiation between four species of the genus Lolium

Isozyme electrophoresis was carried out on 423 accessionsbelonging to four species of the genus Lolium toassess the genetic variation within and between the species. Fourenzyme systems (acid phosphatase, glutamate oxaloacetatetransaminase, phospho-gluco isomerase and superoxidedismutase) were used to array allelic diversity at fivepolymorphic loci. Nei's genetic distance analysis andmultivariate analyses were computed and the taxonomic position ofeach species is discussed as a result of these computations.Electrophoresis is shown to clearly differentiate the inbreedingspecies, L. temulentum fromthe outcrossing species, L.perenne, L.multiflorum and L.rigidum, and to separate the species within theout-breeding group. The taxonomic results are discussed inrelation to an earlier paper on morphological variation, concludingthat electrophoresis is a valid taxonomic tool showing distinctseparations between the species, but that current plant breeding andagricultural practises may be increasing the amount of hybridisationbetween the species than occurred in the past.     

Evaluation of <Emphasis Type="Italic">Phaseolus acutifolius</Emphasis> A. Gray plant introductions under high temperatures in a controlled environment

Heat susceptibility is a cause of yield loss in common bean (Phaseolus vulgaris L.) in temperate and tropical lowland growing regions. Interspecific hybridization with related species that include tepary bean (Phaseolus acutifolius A. Gray) can potentially be used to transfer heat tolerance traits to common bean. This study evaluated 25 tepary bean plant introductions (PIs) following exposure to high-temperature (35 °C day/32 °C night) and control temperature (27 °C day/24 °C night) during reproductive development. These 25 PIs were selected for high yield potential in a 14 h photoperiod greenhouse environment. High temperature when compared to non-stress treatment resulted in a mean reduction in seed yield of 88.8% among tepary beans and caused 100% yield loss in common beans. Twelve P. acutifolius PIs exhibited negligible yield under heat-stress when compared to control treatment. Although yields of these accessions were low under very high-temperature conditions, PI 200902, PI 312637, PI 440785, PI 440788, and PI 440789 exhibited the highest yield component stability under the high temperature treatment.     

Analysis of the contribution of Mesoamerican and Andean gene pools to European common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) germplasm and strategies to establish a core collection

Common bean (Phaseolus vulgaris L.) was introduced in Europe from both Mesoamerican and Andean centres of origin. In this study, a collection including 544 accessions from all European regions showed that the Andean phaseolin types ‘T’ (45.6%) and ‘C’ (30.7%) prevailed over the Mesoamerican ones ‘S’ (23.7%), and accessions with cuboid seed shape (34.9%), maroon coat darker colour seed (44.3%), uniform seed colour (69.6%) were the most frequent. European accessions with phaseolin ‘S’ showed a significantly larger average seed size compared to those from America in the same phaseolin class while those presenting ‘T’ and ‘C’ phaseolin did not. This suggests that, during crop expansion in Europe, sampling or selection favoured the large-seeded races within the Mesoamerican ‘S’ gene pool or, possibly, introgression from Andean germplasm did occur. A core collection was developed using sampling approaches based on the information available in the genebank databases and on phaseolin patterns. Four sampling strategies were used: simple random sampling, and three random-stratified samplings, by logarithm of frequency of accessions by country, by European region, and by phaseolin pattern, respectively. Two sampling strategies resulted in core collections significantly different for phaseolin electrophoretic patterns from the whole collection. Stratification by phaseolin patterns increased the frequency of ‘S’ types (‘C’ type = 33%, ‘T’ type = 5.7% and ‘S’ type = 31.3%). The core collections were validated using seven seed characters, and no significant difference was observed in all strategies. This first developed European bean core collection will help to assess the contribution of the two American gene pools to the European germplasm and their relative importance for breeding purposes.     

Use of GIS for Optimizing a Collecting Mission for a Rare Wild Pepper (<Emphasis Type="Italic">Capsicum flexuosum</Emphasis> Sendtn.) in Paraguay

This paper presents an effective method for prioritizing areas within a country for acquisition of germplasm of a crop gene pool for ex situ conservation. The method was applied to the rare wild pepper species, Capsicum flexuosum Sendtn., in south-east Paraguay. A model to prioritize areas for collecting germplasm was constructed by combining (1) a prediction of the species' geographic distribution based on the climate at previous collection points, (2) the distribution of forest margins (the species' natural habitat) and (3) areas accessible by road. The model was then tested in the field by visiting 20 sites having both high and low predicted probability of occurrence of C. flexuosum. Six new populations were found, representing a significant improvement over two previous collecting missions for the species in the same region, undertaken without the use of GIS targeting. Using the most optimistic analysis of model performance, C. flexuosum was found at five out of seven points predicted to harbour the species and not found at four of five points predicted not to harbour the species. The model was then improved by the use of higher resolution climate surfaces. It is recommended that future explorers use more recent and higher resolution satellite images to locate suitable habitats. The method is replicable for different species in different geographic regions and is offered as a means of optimizing efficiency in financially constrained, national plant genetic resources programs.     

Measurement of nuclear DNA content of the genus <Emphasis Type="Italic">Trifolium</Emphasis> L. as a measure of genebank accession identity

The collection, identification and maintenance of genebank accessions of the genus Trifolium is a major task because of the large number of genera and their occasional morphological similarity. We investigated whether the measurement of nuclear DNA content can serve as an additional criterion for identification of mislabeled accessions. Relative nuclear DNA content was determined by flow cytometry measurements for a total of 151 genebank accessions of 23 Trifolium species with notable agronomical value. Among 23 species analyzed, 15 were found to possess a uniform relative nuclear DNA content, with intraspecific variability of the majority of analyzed species lower than 5%. Within six Trifolium species, 1–2 atypical accessions with outstanding differences in relative DNA content, chromosome number and morphological features were found. In T. hybridum and partially in T. ambiguum these outstanding differences could be ascribed to variations in accession ploidy level. For the remaining atypical accessions, the determined nuclear DNA content, chromosome number and morphological features were not related to those characteristic of the species. Additionally taxonomic identity of atypical accessions was determined using ITS region sequencing and morphological observations. We propose flow cytometric measurements of nuclear DNA content as simple and reliable technique which can be used at seedling stage to verify identity and genetic stability of Trifolium genebank accessions.     

Use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil

A novel greenhouse based soil tilting table apparatus was used to investigate the potential for movement of the protozoan pathogen Cryptosporidium parvum both through and across a low permeability soil following the application of contaminated livestock waste to land. Soil blocks supported at an angle of 7.5% by the soil table were inoculated at one end with oocyst seeded slurry and subsequently irrigated at regular intervals over a 70-day period. Movement of the pathogen in runoff was demonstrated for at least 21 days and in one case in excess of 70 days from the time of inoculation. Water was also lost following percolation down through the soil profile and significant numbers of oocysts were also lost via this route, average numbers leached decreasing from 8.36±0.56×10⁶ at day 1 to 2.27±0.73×10⁴ at day 70. At the end of the study cores were removed from the soil blocks to determine the location of oocysts remaining within the soil. Numbers decreased down through the soil profile and as the distance from the point of inoculation increased so that 70 cm from the point of inoculation no oocysts could be detected in the soil at any depth. This implies that oocysts contained in runoff stay in the aqueous phase and do not precipitate out onto the soil surface, suggesting that even if the distances travelled are increased there may still be a significant pollution threat.     

Aspergillus flavus transformation of glucosinolates to nitriles by an arylsulfatase and a β-thio-glucosidase

Biofumigation is a biocontrol method that uses volatile compounds to combat soil pathogens. We investigated a biofumigation process based on the green manure of Brassicaceae. These plants contain the glucosinolate-myrosinase system which releases inhibitory compounds such as isothiocyanate into the soil. However, the biocontrol effectiveness is often lower than expected, possibly due to microbial transformation of the isothiocyanates. In order to identify the possible function of microorganisms, their interaction with glucosinolates and glucosinolate-derived products was investigated. We report the ability of a soil isolate of Aspergillus flavus to grow in liquid culture and convert 2-propenyl and 2-phenylethyl glucosinolate and their desulfo-derivatives, to nitriles. This finding would suggest the existence of an arylsulfatase and a β-thio-glucosidase, different from myrosinase, which could direct glucosinolate conversion in soil towards nitriles rather than isothiocyanates. If confirmed in soil, our observations could at least partially explain the low concentrations of isothiocyanates after biofumigation.     

Decomposition of willow-leaf litter in a short-rotation forest in relation to fungal colonization and palatability for earthworms

Summary The influence of leaf litter from three Salix spp. on fungal growth and microbial decomposition was studied using 1-mm-mesh litter-bags, and the effect on additional soil macrofaunal activity was studied by measuring litter disappearance from 4-mm-mesh bags and under 4-mm-mesh nets. Mineral macro-elements, water-and ethanol-extractable substances, lignin, and protein-precipitating substances (astringency) in the litter were determined, taking contaminating of the litter with soil particles into account. As expected, the litter disappeared more quickly from the large-mesh bags than from the small-mesh bags, which was attributed to earthworm activity. During the 1st year, the rate of leaf disappearance from both types of bags and under the nets was much higher for S. daphnoides than for S. viminalis and S. fragilis. The lower initial astringency, related to the tannin content, of the S. daphnoides litter might account for this difference. Tannin metabolites probably hampered both microbial decomposition and earthworm acceptability for some time also after the astringency was lost. Neither the content of macronutrients nor that of the other organic fractions studied can be assumed to have had any effect on weight losses due to microbial decomposition. Although, the S. daphnoides leaves initially contained the least amount of fungal mycelium (m g^-1 dry weight), the increase after contact with soil was most pronounced in this litter. The species composition of Fungi Imperfecti in the leaves of S. viminalis and S. daphnoides differed only for fresh litter, whereas the number of isolates was somewhat higher for S. daphnoides throughout the study. Similar seasonal variations in fungal composition occurred in both the S. viminalis and the S. daphnoides litter.     

Evaluation of a core collection of Brassica oleracea accessions for resistance to white rust of crucifers (Albugo candida) at the cotyledon stage

The identification of seedling resistance to white rust of crucifers was performed in a screening of a B. oleracea core collection with 400 accessions representing the genetic and geographic diversity of the species. Fifty seedlings per accession were tested against the Portuguese isolate Ac502 using the methodology and evaluation procedures developed by and . The percentage of resistant seedlings (%R) and the conventional rating criteria of the mean Disease Index (DI) based on the two different evaluation procedures of disease expression used, were compared and adopted as the criteria to rank the accessions for their interest as sources of resistance. A great variability of reactions was found between and within accessions of the core collection, ranging from complete resistance to full susceptibility. Sources of resistance were found namely among the cauliflowers, broccoli and tronchuda cabbages gene pools. Forty-seven accessions presented at least 20% of resistant seedlings. Nine accessions (the kales INRA18 and INRA62, the cauliflowers HRI4856, HRI4866 and HRI5424, the loose-head cabbage HRI11555, the savoy cabbage BRA848, the black broccoli HRI6318 and the Portuguese tronchuda cabbage ISA207) presented 50–78% of resistant seedlings and so they should be considered as potential and useful sources for direct use in breeding programs for white rust resistance. Fourteen inbred lines, representing the full range of disease expression, derived from resistant accessions of the core collection were also tested for resistance to other two Portuguese isolates (Ac503 and Ac504) and to a UK isolate. The results provided no evidence of differential reaction to the A. candida isolates tested.     

Use of NaCN to increase the growth of N2-fixing bacteria in a model spermosphere mimicked by sucrose and amino acids leached by seeds

In order to study the establishment of a spermosphere, the C₂H₂ reduction activity of N₂-fixing bacteria isolated from river sand was examined in a simulated spermosphere in the river sand which contained sucrose, an amino acid mixture, and CN^- released from plant seeds. The sand incubated with 10^-10 to 10^-9 mol CN^- 30 g sand^-1 exhibited higher C₂H₂ reduction activity than that without CN^-. The change in the most probable number of N₂ fixers with increasing quantities of CN^- roughly corresponded to that in C₂H₂ reduction activity. However, the most probable number of non-N₂-fixing bacteria decreased except for CN^--tolerant ones. Both C₂H₂ reduction activity and proliferation of the N₂ fixers isolated on a modified Burk's medium were almost similar to those in the bacteria in the sand. In contrast, the proliferation of some nonfixers decreased with an increasing CN^- concentration. C₂H₂ reduction activity of N₂ fixers cultured in combination with non-fixers exhibited a clear peak at 10^-7 M CN^- as for C₂H₂ reduction activity in the sand. We therefore speculate that cyanide evolved from seeds during a pregermination period may suppress the growth of general bacteria, but may promote the proliferation of N₂ fixers, thus contributing to the establishment of a spermosphere.