Effect of cliff connectivity on the genetic population structure of a rock-dwelling land snail species with frequent self-fertilization

To examine the effect of habitat connectivity on the genetic differentiation in the frequently selfing gastropod Chondrina avenacea we assessed RAPD profiles in 12 subpopulations from continuous cliffs and in 12 subpopulations from small, naturally fragmented cliffs in the Swiss Jura mountains. Subpopulations of C. avenacea on continuous cliffs showed a smaller number of polymorphic RAPD loci and a lower gene diversity (Nei's h = 0.049) than subpopulations on fragmented cliffs (h = 0.078). Genetic distance was not correlated with geographical distance between subpopulations. G_ST analysis indicated strong isolation effects. Neither the number of polymorphic loci nor Nei's h was correlated with local population density. Our analysis indicates that the local population structure of C. avenacea is a combined result of frequent self-fertilization, genetic drift and low dispersal. Similar findings have been reported in studies on spatial population structure of plants. We suggest that future management actions (selective tree cutting, and controlled access of sport climbers) should also consider small, isolated limestone cliffs to preserve the genetic diversity of C. avenacea.     

Specific PCR-based marker for detection of pathogenic groups of Fusarium oxysporum f. sp. cucumerinum in India

For the detection of Fusarium oxysporum f. sp. cucumerinum pathogenic groups, a specific PCR-based marker was developed. Specific random amplified polymorphic DNA (RAPD) markers which identified in four pathogenic groups I, II, III, and IV were cloned into P^Gem-Teasy vector. Cloned fragments were sequenced, and used for developing sequence characterized amplified regions (SCAR) primers for detection of pathogenic groups. F. oxysporum f. sp. cucumerinum isolates belonging to four pathogenic groups in India, cucumber nonpathogenic F. oxysporum, F. oxysporum f. sp. moniliforme and melonis, Fusarium udum, and isolate of Alternaria sp. were tested using developed specific primers. A single 1.320 kb, 770 bp, 1.119 kb, and 771 bp fragment were amplified from pathogenic group I, II, III, and IV isolates, respectively. Results showed the PCR based marker, which used in this research work, could detect up to 1 ng of fungal genomic DNA. The specific SCAR primers and PCR technique developed in this research easily detect and differentiate isolates of each F. oxysporum f. sp. cucumerinum pathogenic groups.     

Comparison of the chemistry and diversity of endophytes isolated from wild-harvested and greenhouse-cultivated yerba mansa (Anemopsis californica)

With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 μg/mL, and three inhibited growth of Pseudomonas aeruginosa by ≥20% at 200 μg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional 14 known compounds were identified using LC–MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi.     

Laboratory and semi-field evaluation of Beauveria bassiana (Ascomycota: Hypocreales) against the lettuce aphid,Nasonovia ribisnigri (Hemiptera: Aphididae)

The lettuce aphid, Nasonovia ribisnigri (Mosley), is an economically important pest of lettuce worldwide. The entomopathogenic fungus Beauveria bassiana strain GHA has recently been reported as a potential biocontrol candidate for use against the lettuce aphid. This study provides information on the mortality inflicted by B. bassiana when applied against different life stages of the lettuce aphid under laboratory conditions and how fungus infection affects the aphid fecundity. In addition, temporal changes in persistence of fungus inoculum applied to foliage of young lettuce plants under semi-field conditions was analysed. Immature life stages were generally the least susceptible to fungal infection and the susceptibility of all stages was dose-dependent, with the highest mortality occurring at the highest dose. B. bassiana significantly affected the rate of nymph production by the lettuce aphid, with the highest effect seen when the alatoid fourth instar of N. ribisnigri was inoculated with B. bassiana. The persistence of B. bassiana conidia on lettuce foliage was not influenced by leaf position. Within 5 days, the cumulative percentage decline in the conidial population was 38% which declined further to 92% and 99% on day 11 and 20 post-spraying, respectively. In accordance, the infectivity to second instar lettuce aphid nymphs of B. bassiana conidia deposited on leaves declined according to an exponential decay model predicting an intercept of 0.59 ± 0.03 (S.E), a reduction in aphid mortality at a rate of 11% with each increasing day after fungal application and a fungus half-life of 6.34 ± 0.69 days.     

Noninvasive genetic assessment of brown bear population structure in Bulgarian mountain regions

The Balkans are one of the last large refugia for brown bear (Ursus arctos) populations in Europe, and Bulgaria, in particular, contains relatively large areas of suitable brown bear habitat and a potential population of more than 600 individuals. Despite this, the majority of brown bear research remains focused on bear populations in Central and Western Europe. We provide the first assessment of genetic population structure of brown bears in Bulgaria by analysing tissue samples (n = 16) as well as samples collected with noninvasive genetic methods, including hair and faecal samples (n = 189 and n = 163, respectively). Sequence analysis of a 248 base pair fragment of the mitochondrial control region showed that two highly divergent mitochondrial European brown bear lineages form a contact zone in central Bulgaria. Furthermore, the analysis of 13 polymorphic microsatellite markers identified 136 individuals and found substantial genetic variability (H_e = 0.74; N_A = 8.9). The combination of both genetic markers revealed the presence of weak genetic substructure in the study area with considerable degrees of genetic admixture and the likely presence of migration corridors between the two subpopulation in the Rhodope Mountains and Stara Planina as evidenced from the genetic detection of two male long-distance dispersers. A detailed assessment from densely collected samples in the Rhodope Mountains resulted in a population size estimate of 315 (95% CI = 206–334) individuals, indicating that not all available habitat is presently occupied by bears in this region. Efficient management plans should focus on preserving connectivity of suitable habitats in order to maintain gene flow between the two Bulgarian brown bear subpopulations.     

Restocking a keystone species in a biodiversity hotspot: Recovering the European rabbit on a landscape scale

Rabbit populations in Iberia remain at low densities in several areas in which their endangered predators still coexist, and the recovery of these populations is therefore urgent if the integrity of Iberian Mediterranean ecosystems is to be maintained. The enhancement of wild rabbit populations has been attempted through the use of in situ extensive rabbit captive breeding enclosures (restocking plots), which reduce mortality caused by terrestrial predators and dispersal movements and permit the breeding of young individuals which can then naturally disperse to settle in the surrounding areas. However, their effectiveness, the role of its size, the optimal habitat management that should be promoted around them and the habitat features remains uncertain. Here, we show results from a four year study of an ambitious rabbit restocking plan on a landscape scale. We measured rabbit abundance in a vast area in which thirty-two restocking plots were built to create an initial rabbit population for further dispersion, in addition to an intensive habitat management program. We also compared rabbit abundance between managed and unmanaged UTM cells of 2.5 km × 2.5 km. Our results showed that rabbit abundance was three times higher in managed cells, but four years after restocking, rabbit abundances had only reached the threshold needed to support stable Iberian lynx presence (at least 10 latrines per km^?1) in 9 of the 23 managed cells. Rabbit abundance was strongly affected by habitat treatment and scrub coverage. The increase of shelter was useful in low cover areas but ineligible in places with high scrub cover, where the increase of refuge plus scrub clearing to create pastures improve rabbit abundance more effectively. In the light of our results, restocking plots should be built only in places with suitable habitat, whereas pastures should be created in dense scrublands and refuge in low cover areas.     

Re-establishment of Calluna vulgaris (L.) Hull in an eight-year grazing experiment on upland acid grassland

Upland heathland is an internationally important habitat but a large area in the UK has been degraded to acid grassland by intensive livestock grazing. Re-establishment of dwarf shrubs, particularly Calluna vulgaris (L.) Hull, is a key objective for restoring heathland on these sites. A replicated plot-scale experiment was set up to examine effects of disturbance and seed addition on C. vulgaris establishment in a Nardus stricta L. grassland under three grazing regimes: sheep only (1.5 ewes ha^?1 for 10 months per year); cattle only (0.5 heifers ha^?1 in summer only); and, the cattle regime combined with sheep (1.0 ewes ha^?1 for 10 months per year). Early results of the experiment have been reported previously but it was not known if these results were an indication of the longer-term restoration success. Here we evaluate the success of the restoration methods (disturbance, seeding treatments and grazing regime) eight years after the treatments began. In seeded plots, young C. vulgaris plants had greatest above-ground height, dry weight and shoot length if grazing was excluded or the cattle-only regime was applied. C. vulgaris cover was greatest, and increased most, in plots that had been disturbed, seeded and ungrazed or subjected to the cattle-only regime. The vegetation in these plots also became more similar to reference sites with 50% or more cover of C. vulgaris. The invasive Juncus effusus L. was more frequent in disturbed and grazed plots but less frequent in plots with C. vulgaris established from added seed. Previous results that showed the benefits of disturbance and seeding treatments were still valid but changes in the vegetation composition were still occurring and longer-term studies will be needed to determine when grazing regimes including sheep might be reintroduced.     

Effect of infection by Metarhizium anisopliae (Hypocreales: Clavicipitaceae) on the feeding and oviposition of the pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) on different host plants

The effect of fungal infection by Metarhizium anisopliae on feeding and oviposition of adult Liriomyza huidobrensis was examined on three host plants, faba bean (Vicia faba), French bean (Phaseolus vuklgaris) and snow pea (Pisum sativum) in the laboratory. Flies were contaminated with dry conidia and allowed to feed and oviposit on the different host plants. Mortality in L. huidobrensis varied between 14% and 20% in the controls and between 77% and 100% in fungal treatments 120 h post-infection for the three host plants. L. huidobrensis made more punctures (47.3–52.6 cm^?2) in the control than in the fungal treatments (23.1–26.9 cm^?2) for the three host plants. The cumulative average number of punctures cm^?2/female by L. huidobrensis was higher in the controls than in fungal treatments from 72 h post-treatment in faba bean (12.2 vs. 8.2) and French bean (14.8 vs. 8.9), and from 48 h post-inoculation in snow pea (8.5 vs. 5.7). Female L. huidobrensis laid more eggs in the control (0.6–6.1) than in fungal treatments (0.2–1.5) across the host plants tested. The cumulative mean number of eggs cm^?2/female was significantly higher in the controls than in fungal treatments from 48 h post-treatment in faba bean (0.4 vs. 0.2) and French bean (0.1 vs. 0), and 96 h post-inoculation in snow pea (0.2 vs. 0.1). The host plant did not affect the average total number of punctures but had a significant effect on egg laying, with faba bean harboring greater number of eggs in both control and fungal treatments. A proper timeline application of the fungus before onset of feeding and oviposition peaks will be crucial in field suppression of the pest using M. anisopliae. In addition, a great consideration must be given to the target host plants prior to application of the fungus.     

Crossing the border? Structure of the red deer (Cervus elaphus) population from the Bavarian–Bohemian forest ecosystem

Anthropogenic impact such as overhunting and habitat fragmentation has reduced the total red deer population (Cervus elaphus) across Europe. In Germany remaining subpopulations are even confined to designated areas with limited or no gene flow among them. Red deer populations inhabiting the Bavarian–Bohemian forest ecosystem had been divided by a fortified State border between Germany and former Czechoslovakia. To assess red deer genetic diversity more than two decades after the removal of the fortifications, we analysed a population from the Bavarian Forest National Park, Germany, and one from the National Park ?umava, Czech Republic, using 11 microsatellite loci and a 910 bp long section of the mitochondrial control region (mtDNA). Bayesian analyses of microsatellite allele frequencies favoured the presence of a single population in the Bavarian-Bohemian forest ecosystem over other population genetic structures. This admixture was supported by a lack of population pairwise differentiation between German and Czech red deer microsatellite genotypes in the analysis of molecular variance (AMOVA, F_ST = 0.009, p = 0.383). Contrastingly, AMOVA revealed a highly significant matrilinear differentiation of mtDNA between the two samples (Φ_ST = 0.285, p = 0.002), whereby German red deer belonged predominantly to haplogroup A (western Europe) and Czech red deer predominantly to haplogroup C (eastern Europe). In combination, these findings indicated a high degree of philopatry by does and extensive gene flow across the former border mediated by stags. They also identified the Bavarian–Bohemian forest ecosystem as part of a suture zone between western and eastern European red deer matrilines.     

Typha × glauca dominance and extended hydroperiod constrain restoration of wetland diversity

Urban wetlands typically have few plant species. In wetlands designed to improve water quality, nutrient-rich water and highly variable water levels often favor aggressive, flood-tolerant plants, such as Typha × glauca (hybrid cattail). At Des Plaines River Wetlands Demonstration Site (Lake Co., IL), we assessed T. × glauca dominance and plant community composition under varying hydroperiods in a complex of eight constructed wetlands. Plots flooded for more than 5 weeks during the growing season tended to be dominated by T. × glauca, while plots flooded fewer days did not. Plots with high cover of T. × glauca had low species richness (negative correlation, R² = 0.72, p < 0.001). However, overall species richness of the wetland complex was high (94 species), indicating that wetlands in urbanizing landscapes can support many plant species where T. × glauca is not dominant. T. × glauca-dominated areas resisted the establishment of a native plant community. Removing T. × glauca and introducing native species increased diversity initially, but did not prevent re-invasion. Although 12 of the 24 species we seeded became established in our cleared plots, T. × glauca rapidly re-invaded. In year 1, T. × glauca regained an average of 11 ramets m⁻², and its density doubled in year 2. The likelihood of planted species surviving decreased as duration of inundation increased, and in both seeded and planted plots, graminoids had greater survivorship through year 2 than forbs across a range of water levels. Within 4 years, however, T. × glauca was the most common plant, present in 92% of the cleared plots. Simply removing T. × glauca and adding propagules to an urban wetland is not sufficient to increase diversity.     

Modifying orchard planting sites conserves entomopathogenic nematodes,reduces weevil herbivory and increases citrus tree growth,survival and fruit yield

In Florida, a root weevil pest of citrus, Diaprepes abbreviatus, is more damaging and attains higher population density in some orchards on fine textured, poorly drained “flatwoods” soils than in those on the deep, coarse sandy soils of the central ridge. Previous research revealed that sentinel weevil larvae were killed by indigenous entomopathogenic nematodes (EPNs) at significantly higher rates in an orchard on the central ridge, compared to one in the flatwoods. We hypothesized that filling tree planting holes in a flatwoods orchard with sandy soil from the central ridge would provide a more suitable habitat for EPNs, thereby reducing weevil numbers and root herbivory. Fifty trees were planted in oversized planting holes filled with coarse sand and 50 trees were planted in native soil in a split plot design where whole plots were species of introduced EPNs and split plots were soil type. Each of Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, or no EPNs were introduced into the rhizospheres in 10 plots of each soil type. During four years, EPN numbers in soil samples and the relative abundance of seven species of nematophagous fungi associated with nematodes were measured three times using real-time PCR. The efficacy of EPNs against sentinel weevil larvae was also measured three times by burying caged weevils in situ. EPN species richness (P = 0.001) and diversity (P = 0.01) were always higher in sand than native soil. Soil type had no effect on numbers of EPNs in samples, but EPNs were detected more frequently (P = 0.01) in plots of sandy soil than native soil in 2011. Two nematophagous fungi species, Paecilomyces lilacinus and Catenaria sp. were significantly more abundant in nematode samples from sandy soil on all three sampling dates. Efficacy of EPNs against weevil larvae was greater in sandy soil inoculated with S. diaprepesi (P = 0.03) in June 2010 and in all treatments in sandy soil in May 2011 (P = 0.03). Sixty-eight percent more adult weevils (P = 0.01) were trapped emerging from native soil during two years than from sandy soil. By May 2011, the cumulative number of weevils emerging from each plot was inversely related (P = 0.01) to the numbers of EPNs detected in plots and to EPN efficacy against sentinels. Three trees in sandy soil died as a result of root herbivory compared to 21 trees in native soil. Surviving trees in sandy soil had trunk diameters that were 60% larger (P = 0.001) and produced 85% more fruit (P = 0.001) than those in native soil. Although it is not possible to characterize all of the mechanisms by which the two soil treatments affected weevils and trees, substitution of sand for native soil was an effective means of conserving EPNs and shows promise as a cultural practice to manage D. abbreviatus in flatwoods citrus orchards with a history of weevil damage to trees.     

High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang,an endangered species of China

Genetic diversity and genetic differentiation of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang, an endangered species of China, were analyzed using random amplified polymorphic DNA (RAPD) markers. Totally, 165 stable and clearly scored RAPD bands were achieved from 12 primers. The genetic diversity of S. chinensis was high (P% = 68.84; h = 0.2421 ± 0.1978; I = 0.3615 ± 0.2789), whereas that at the population level was relatively low (P% = 14.49; h = 0.0578 ± 0.0167; I = 0.0843 ± 0.0236). High genetic differentiation among populations was detected based on Shannon's information index (0.7668), Nei's gene diversity (0.7613) and AMOVA (0.8183). This might be explained by its survival in refugia during the last glaciation in southeastern China with origin from a widespread continental progenitor. The 10 populations from different geographical sites could be clustered into two groups. Low gene flow due to mixed mating system and anthropogenic activities likely played important roles in shaping the population genetic structure of the species.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Comparative research of chemical constituents,antifungal and antitumor properties of ether extracts of Panax ginseng and its endophytic fungus

The chemical compositions and bioactivities of ether extracts of an endophytic fungus Paecilomyces sp. isolated from Panax ginseng were reported, and the comparative analysis of the constituents, antifungal and antitumor properties of the ether extracts from this fungus and its host ginseng were also conducted. By means of GC/MS technique, 51 compounds of Panax ginseng and 38 compounds of Paecilomyce sp. were determined. It is attractive that the extracts derived from Paecilomyce sp. and ginseng samples contained the same compound falcarinol, a natural pesticide and anti-cancer agent. The ether extracts of Paecilomyce sp., tested at 7.8 μg/ml, completely inhibited the visible growth of Pyricularia oryzae. Furthermore, both extracts were tested against four human pathogenic fungi and showed the IC₈₀ of Paecilomyce sp. was 4 μg/ml against Trichophyton rubrum, equally to the control. Finally, the in vitro antitumor experience showed that the most of the IC₅₀ values were all being below 20 μg/ml.     

Molecular detection of Coccidioides spp. from environmental samples in Baja California: linking Valley Fever to soil and climate conditions

Coccidioidomycosis is an important human fungal infection of American deserts and nearby semi-arid regions with highly endemic areas distributed along the United States-Mexico border. Despite the increasing incidence in the last 20 yr, reports of positive isolations of the causal agent, Coccidioides spp. from environmental samples have been scarce. To resolve this paradox, it is extremely important to first identify the fundamental ecological niche of this fungus. Soil samples (n = 90) including those from heteromyids’ active burrows, latrines and other mammals’ dens were collected using an oriented sampling method from areas of Baja California, Mexico previously predicted as putative endemic “hotspots”. The total genomic DNA obtained from the collected samples was subjected to a nested PCR followed by a diagnostic PCR designed to amplify the internal transcribed spacer (ITS) 2 region of Coccidioides spp. From the 42 amplicons obtained and sequenced (37 from Valle de las Palmas (VDP) and five from San Jose de la Zorra (SJZ)), 32 were confirmed to belong to Coccidioides spp. No Coccidioides spp. were found in soils collected in Ensenada.VDP and SJZ have different soil characteristics but share a Mediterranean climate having less than 250 mm of precipitation per year, as well as a dry period of at least 6 months. The development of Coccidioides spp. is probably related to the structure of the microbial population adapted to these conditions in the semi-arid-mediterranean ecotone.     

Faunal utilization of constructed intertidal oyster (Crassostrea rivularis) reef in the Yangtze River estuary,China

An intertidal oyster reef (～260 ha) was created by planting hatchery-reared seed oysters (Crassostrea rivularis) on an artificial concrete modular reef in the Deepwater Navigation Channel Regulation Project of the Yangtze River estuary. We examined the development of reef communities (oyster, barnacle and motile epibenthic macrofauna), characterized nekton use and assessed the habitat value of the constructed reef. The C. rivularis oyster population showed a rapid exponential increase with time and reached maximum density (3410 ± 241 ind./m²) and biomass (3175 ± 532 g/m²) after one year of restoration. The barnacle Balanus albicostatus was the most abundant sessile macrofauna and had a significantly greater density in the high intertidal zone than in the low intertidal zone (P < 0.05). The reef also supported diverse motile epibenthic macrofauna (11 mollusks, 11 crustaceans, 4 annelids and 2 fishes), and the reef-associated communities were numerically dominated by Neanthes japonica, Perinereis aibuhitensis, Nerita yoldi and Littorinopsis intermedia. A total of 50 nekton species (31 fishes, 9 shrimps and 10 crabs) utilized the constructed intertidal oyster reef, and grass shrimp Palaemon spp. dominated the nekton communities in term of abundance. Since the constructed intertidal oyster reef supports a variety of reef communities and abundant nektons, it should be recognized as an important and protective fish habitat in the Yangtze River estuary.     

Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.     

Modifying soil to enhance biological control of belowground dwelling insects in citrus groves under organic agriculture in Florida

An emerging organic citrus industry in Florida could benefit greatly from effective, non-conventional methods to mitigate losses from pests and diseases. We studied part of a soil food web in an organic orchard to learn ways to conserve and enhance biological control of insect pests by native entomopathogenic nematodes (EPNs). We evaluated two OMRI (Organic Materials Review Institute) approved cultural practices: (i) a mulch of commercially pelleted chicken manure, (ii) a commercial formulation of Purpureocillium lilacinus, and (iii) an un-amended control. Several soil nutrients (i.e. nitrogen, phosphate, and potassium) were affected by the amendments, but initial equilibrium values (T0) were restored by the last sampling time (T12). The plant parasitic nematode Tylenchulus semipenetrans increased in both treatments compared to the untreated control at T3 (P < 0.05). The oomycete Phytophthora nicotianae increased in the P. lilacinus plots at T1, marginally at T12, but decreased at T6 and T9. Steinernema diaprepesi, Heterorhabditis indica and Heterorhabditis zealandica were the only EPNs regularly detected in the orchard. Mulch increased numbers of H. zealandica at T6 and T9 (P < 0.05) and free living nematodes at T12 (P < 0.01). The nematophagous fungus (NF) P. lilacinus persisted in plots where it was augmented (P < 0.05), reaching a maximum level at T3 that was 17.5-fold greater than that in controls. Numbers of Paenibacillus sp. were directly related to both those of S. diaprepesi and Acrobeloides-group nematodes (P < 0.01), but inversely to the FLN counts (P < 0.05). The application of these two amendments did not produce strong changes in the EPN community but decreased the emergence from soil of adult Diaprepes abbreviatus, a root weevil pest. Thus, both amendments might contribute to citrus pest management under organic production.     

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55 mg/mL) and savory (0.32 mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40 mg/mL for thyme oil and 0.21 mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).     

Synthetic sheep urine alters fungal community structure in an upland grassland soil

Agricultural improvement (fertilisation, liming, intensification of grazing) of acidic upland pastures results in loss of indigenous flora and notable changes in microbial community structure. Such practices have recently raised concerns regarding the possible impacts on natural ecosystem biodiversity and functioning. The effects of synthetic sheep urine (SSU) and plant species on fungal community structure in upland grassland microcosms were investigated. Plant species typical of agriculturally unimproved (Agrostis capillaris) and improved (Lolium perenne) pastures were treated with low, medium or high concentrations of SSU, with harvests carried out 10 d and 50 d after SSU application. Root biomass was negatively affected by SSU addition whereas shoot biomass did not display any significant change. Fungal richness (number of operational taxonomic units) was negatively correlated with SSU concentration (p < 0.001), and also with time (p < 0.001).Multi-dimensional scaling plots revealed significant changes in fungal community composition, depending on concentration of SSU and plant species type, while canonical correspondence analysis also emphasised the importance of interacting environmental variables. In addition, SIMPER analyses supported the finding that shifts in fungal community composition under different SSU and plant treatments had occurred. Overall, while SSU appeared to be influential in determining fungal community structure, community changes were largely driven by interacting environmental factors. This study contributes to our understanding of the potential implications of intensified farming, in particular increased pressure from grazing animals, on fungal community structure in semi-natural grassland systems.