Plant neighbours can make or break the disease transmission chain of a fungal root pathogen.

Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission. Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development. Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host-neighbour interactions changed which mechanisms steered disease transmission rate. Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.

Lost in diversity: the interactions between soil-borne fungi, biodiversity and plant productivity.

There is consensus that plant species richness enhances plant productivity within natural grasslands, but the underlying drivers remain debated. Recently, differential accumulation of soil-borne fungal pathogens across the plant diversity gradient has been proposed as a cause of this pattern. However, the below-ground environment has generally been treated as a 'black box' in biodiversity experiments, leaving these fungi unidentified. Using next generation sequencing and pathogenicity assays, we analysed the community composition of root-associated fungi from a biodiversity experiment to examine if evidence exists for host specificity and negative density dependence in the interplay between soil-borne fungi, plant diversity and productivity. Plant species were colonised by distinct (pathogenic) fungal communities and isolated fungal species showed negative, species-specific effects on plant growth. Moreover, 57% of the pathogenic fungal operational taxonomic units (OTUs) recorded in plant monocultures were not detected in eight plant species plots, suggesting a loss of pathogenic OTUs with plant diversity. Our work provides strong evidence for host specificity and negative density-dependent effects of root-associated fungi on plant species in grasslands. Our work substantiates the hypothesis that fungal root pathogens are an important driver of biodiversity-ecosystem functioning relationships.

Feeding preference as a main determinant of microscale patchiness among terrestrial nematodes.

Soil biota are responsible for essential ecosystem services such as carbon storage, nutrient cycling and water retention. However, assessment of the condition of soil biota is hampered by an overwhelming level of diversity. With representatives in all trophic levels of the food web, nematode communities can be used as bioindicators. Accurate assessment of nematode assemblages requires insight into the distribution of specimens with distinct food preferences. With the availability of taxon-specific quantitative PCR assays, distribution patterns of multiple nematode groups can be investigated simultaneously. Here, microscale patchiness of 45 nematode taxa was studied on 12 sampling sites (each with four adjacent microplots) located on arable fields or semi-natural grasslands ('system'), and on marine, river clay or sandy soils ('soil type'). From each microplot, five composite samples were collected. Contrary to our expectations, an increase in the number of cores per composite sample did not result in more accurate measurements, and apparently the levels of microscale patchiness of the taxa are low compared to what has been reported for oligophagous plant-parasites. System and soil type did not affect microscale distribution. To investigate the level of patchiness in more detail, detection probability (DP) and variability of abundances were calculated. Common and widespread bacterivorous and fungivorous taxa had DP ≥ 90%, confirming low level of microscale patchiness. With DPs of 40%-70%, predators and most omnivores showed degrees of local clustering. An overview of mean variabilities of abundances is presented that offers insight into how feeding preferences impact the microscale distribution both between and within trophic groups.

Ecology of Fungal Plant Pathogens.

Fungal plant pathogens are ubiquitous and highly diverse. Key to their success is high host density, which notably is the case in agroecosystems. Several hypotheses related to the effects of plant pathogens on plant diversity (the Janzen-Connell hypothesis, the dilution effect hypothesis) and the phenomenon of higher biomass in plant mixtures (i.e., overyielding) can all be explained by the quantitative interplay between host and pathogen density. In many agroecosystems, fungal plant pathogens cause great losses, since in monocultures diseased plants cannot be replaced by healthy plants. On the other hand, in natural ecosystems fungal plant pathogens shape the succession of vegetation and enhance the biodiversity of forests and grasslands. When pathogens are introduced into areas outside their natural range, they may behave differently, causing severe damage. Once introduced, changes may occur such as hybridization with other closely related pathogens or host shifts, host jumps, or horizontal gene transfer. Such changes can be hazardous for both agricultural and natural ecosystems.

Heterodera schachtii nematodes interfere with aphid-plant relations on Brassica oleracea.

Aboveground and belowground herbivore species modify plant defense responses differently. Simultaneous attack can lead to non-additive effects on primary and secondary metabolite composition in roots and shoots. We previously found that aphid (Brevicoryne brassicae) population growth on Brassica oleracea was reduced on plants that were infested with nematodes (Heterodera schachtii) prior (4 weeks) to aphid infestation. Here, we examined how infection with root-feeding nematodes affected primary and secondary metabolites in the host plant and whether this could explain the increase in aphid doubling time from 3.8 to 6.7 days. We hypothesized that the effects of herbivores on plant metabolites would depend on the presence of the other herbivore and that nematode-induced changes in primary metabolites would correlate with reduced aphid performance. Total glucosinolate concentration in the leaves was not affected by nematode presence, but the composition of glucosinolates shifted, as gluconapin concentrations were reduced, while gluconapoleiferin concentrations increased in plants exposed to nematodes. Aphid presence increased 4-methoxyglucobrassicin concentrations in leaves, which correlated positively with the number of aphids per plant. Nematodes decreased amino acid and sugar concentrations in the phloem. Aphid population doubling time correlated negatively with amino acids and glucosinolate levels in leaves, whereas these correlations were non-significant when nematodes were present. In conclusion, the effects of an herbivore on plant metabolites were independent of the presence of another herbivore. Nematode presence reduced aphid population growth and disturbed feeding relations between plants and aphids.

Testing the paradox of enrichment along a land use gradient in a multitrophic aboveground and belowground community.

In the light of ongoing land use changes, it is important to understand how multitrophic communities perform at different land use intensities. The paradox of enrichment predicts that fertilization leads to destabilization and extinction of predator-prey systems. We tested this prediction for a land use intensity gradient from natural to highly fertilized agricultural ecosystems. We included multiple aboveground and belowground trophic levels and land use-dependent searching efficiencies of insects. To overcome logistic constraints of field experiments, we used a successfully validated simulation model to investigate plant responses to removal of herbivores and their enemies. Consistent with our predictions, instability measured by herbivore-induced plant mortality increased with increasing land use intensity. Simultaneously, the balance between herbivores and natural enemies turned increasingly towards herbivore dominance and natural enemy failure. Under natural conditions, there were more frequently significant effects of belowground herbivores and their natural enemies on plant performance, whereas there were more aboveground effects in agroecosystems. This result was partly due to the "boom-bust" behavior of the shoot herbivore population. Plant responses to herbivore or natural enemy removal were much more abrupt than the imposed smooth land use intensity gradient. This may be due to the presence of multiple trophic levels aboveground and belowground. Our model suggests that destabilization and extinction are more likely to occur in agroecosystems than in natural communities, but the shape of the relationship is nonlinear under the influence of multiple trophic interactions.

Influence of aerobic and anaerobic conditions on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in Luria-Bertani broth, farm-yard manure and slurry.

The influence of aerobic and anaerobic conditions on the survival of the enteropathogens Escherichia coli O157:H7 and Salmonella serovar Typhimurium was investigated in microcosms with broth, cattle manure or slurry. These substrates were inoculated with a green fluorescent protein transformed strain of the enteropathogens at 10(7) cells g(-1) dry weight. Survival data was fitted to the Weibull model. The survival curves in aerobic conditions generally showed a concave curvature, while the curvature was convex in anaerobic conditions. The estimated survival times showed that E. coli O157:H7 survived significantly longer under anaerobic than under aerobic conditions. Survival ranged from approximately. 2 weeks for aerobic manure and slurry to more than six months for anaerobic manure at 16 °C. On average, in 56.3% of the samplings, the number of recovered E. coli O157:H7 cells by anaerobic incubation of Petri plates was significantly (p < 0.05) higher in comparison with aerobic incubation. Survival of Salmonella serovar Typhimurium was not different between aerobic and anaerobic storage of LB broth or manure as well as between aerobic and anaerobic incubation of Petri dishes. The importance of changes in microbial community and chemical composition of manure and slurry was distinguished for the survival of E. coli O157:H7 in different oxygen conditions.

Reduction of rare soil microbes modifies plant-herbivore interactions.

Rare species are assumed to have little impact on community interactions and ecosystem processes. However, very few studies have actually attempted to quantify the role of rare species in ecosystems. Here we compare effects of soil community assemblages on plant-herbivore interactions and show that reduction of rare soil microbes increases both plant biomass and plant nutritional quality. Two crop plant species growing in soil where rare microbes were reduced, had tissues of higher nutritional quality, which theoretically makes them more susceptible to pest organisms such as shoot-feeding aphids and root-feeding nematodes. Reduction of rare microbes increased aphid body size in the absence of nematodes; nematodes always reduced aphid body size independent of the soil microbial community. This study is the first to show that rare soil microbes are not redundant but may play a role in crop protection by enhancing aboveground and belowground plant defence. It remains to be tested whether these are direct effects of rare soil microbes on plants and herbivores, or indirect effects via shifts in the microbial soil community assemblages.

Manure-amended soil characteristics affecting the survival of E. coli O157:H7 in 36 Dutch soils.

The recent increase in foodborne disease associated with the consumption of fresh vegetables stresses the importance of the development of intervention strategies that minimize the risk of preharvest contamination. To identify risk factors for Escherichia coli O157:H7 persistence in soil, we studied the survival of a Shiga-toxin-deficient mutant in a set of 36 Dutch arable manure-amended soils (organic/conventional, sand/loam) and measured an array of biotic and abiotic manure-amended soil characteristics. The Weibull model, which is the cumulative form of the underlying distribution of individual inactivation kinetics, proved to be a suitable model for describing the decline of E. coli O157:H7. The survival curves generally showed a concave curvature, indicating changes in biological stress over time. The calculated time to reach the detection limit ttd ranged from 54 to 105 days, and the variability followed a logistic distribution. Due to large variation among soils of each management type, no differences were observed between organic and conventional soils. Although the initial decline was faster in sandy soils, no significant differences were observed in ttd between both sandy and loamy soils. With sandy, loamy and conventional soils, the variation in ttd was best explained by the level of dissolved organic carbon per unit biomass carbon DOC/biomC, with prolonged survival at increasing DOC/biomC. With organic soils, the variation in ttd was best explained by the level of dissolved organic nitrogen (positive relation) and the microbial species diversity as determined by denaturing gradient gel electrophoresis (negative relation). Survival increased with a field history of low-quality manure (artificial fertilizer and slurry) compared with high-quality manure application (farmyard manure and compost). We conclude that E. coli O157:H7 populations decline faster under more oligotrophic soil conditions, which can be achieved by the use of organic fertilizer with a relatively high C/N ratio and consequently a relatively low rate of nutrient release.

Estimating the stability of Escherichia coli O157:H7 survival in manure-amended soils with different management histories.

The objective of this study is to describe survival of Escherichia coli O157:H7 populations in manure-amended soils in terms of population stability, i.e. the temporal variation around the decline curve, in relation to soil characteristics indicative of soil health. Cow manure inoculated with E. coli O157:H7 was mixed with 18 pairs of organically and conventionally managed soils (10% of manure, kg kg(-1)). For four of the soil pairs, also three different manure densities (5%, 10% and 20%) were compared. All soil-manure mixtures were incubated for 2 months, and population densities of E. coli O157:H7 were quantified weekly. De-trending of survival data was done by modified logistic regression. The residual values were used to assess variation in the changes of E. coli O157:H7 populations by performing the approximate entropy (ApEn) procedure. The term irregularity is used to describe this variation in ApEn literature. On average, the decline of E. coli O157:H7 was more irregular in conventional and loamy soils than in organic and sandy soils (P < 0.05). Multiple regression analysis of irregularity of E. coli O157:H7 survival on 13 soil characteristics showed a positive relation with the ratio of copiotrophic/oligotrophic bacteria, suggesting greater instability at higher available substrate concentrations. Incremental rates of manure application significantly changed the irregularity for conventional soils only. Estimation of temporal variation of enteropathogen populations by the ApEn procedure can increase the accuracy of predicted survival time and may form an important indication for soil health.

Network formation by rhizomorphs of Armillaria lutea in natural soil: their description and ecological significance.

Armillaria lutea rhizomorphs in soil were mapped over areas of 25 m2 at a Pinus nigra (site I) and a Picea abies (site II) plantation. Rhizomorph density was 4.3 and 6.1 m m(-2) soil surface with 84% and 48% of the total rhizomorph length in the mapped area interconnected in a network at site I and site II, respectively. At site I there were only two network attachments to Pinus stumps, but at site II many more to Picea roots and stumps. Anastomoses of rhizomorphs resulted in cyclic paths, parts of the network that start and end at the same point. Connections between different rhizomorph segments were shown to allow gaseous exchange. The network at site I consisted of 169 rhizomorphs ('edges'), and 107 rhizomorph nodes ('vertices'). Disruption of two critical edges ('bridges') would lead to large parts (13% and 11%) being disconnected from the remainder of the mapped network. There was a low probability that amputation of a randomly chosen edge would separate the network into two disconnected components. The high level of connectedness may enhance redistribution of nutrients and provide a robust rhizomorph structure, allowing Armillaria to respond opportunistically to spatially and temporally changing environments.

Influence of temperature fluctuations on Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cow manure.

The effects of four average temperatures (7, 16, 23 and 33 degrees C) and daily oscillations with three amplitudes (0, +/-4, +/-7 degrees C) on the survival of the enteropathogens Escherichia coli O157:H7 and Salmonella serovar Typhimurium were investigated in small microcosms. Manure was inoculated with a green fluorescent protein transformed strain of either pathogen at 10(7) cells g(-1) dry weight. Samples were collected immediately after inoculation, and 1 and 2 weeks after inoculation for E. coli O157:H7, and immediately and after 2 and 3 weeks for Salmonella serovar Typhimurium. Population densities were determined by dilution plating and direct counting. In addition, total bacterial CFUs were determined. Growth and survival data were fitted to a modified logistic model. Analysis of the estimated parameter values showed that E. coli O157:H7 survived for shorter periods of time and was more sensitive to competition by the native microbial community than Salmonella serovar Typhimurium. Survival of both pathogens significantly declined with increasing mean temperatures and with increasing amplitude in daily temperature oscillations. The results indicated that responses of enteropathogens to fluctuating temperatures cannot be deduced from temperature relationships determined under constant temperatures.

Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms.

Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx(1), and stx(2), which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.

Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.

The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca sativa cv. Tamburo) grown in an inoculated hydroponic and soil system. The second aim was to quantify the level of contamination with the use of a proper surface sterilization method. Silver nitrate was superior in reducing the number of viable bacteria on leave surfaces compared to sodium hypochlorite and ethanol. With the hydroponic system internal colonization of lettuce only occurred at high densities with S. Typhimurium MAE 119. With the soil system E. coli O157:H7, S. Typhimurium 110 and S. Typhimurium 119 were found at considerable densities in sterilized leaf samples (respectively, 3.95, 2.57 and 2.37 log cfu/g on average) with prevalences of 0.29, 0.23 and 0.15, respectively. No statistical differences were observed between the Salmonella strains. A negative correlation was observed between shoot weight and leaf contamination. The observed presence of the pathogens in lettuce, after thorough surface sterilization, demonstrates the possible presence of human pathogens in locations were they are unlikely to be removed by the actions of consumer washing and therefore pose a serious threat when occurring in field situations.

Effect of mixed and single crops on disease suppressiveness of soils.

ABSTRACT The effect of mixed cropping on disease suppressiveness of soils was tested for two cropping systems, Brussels sprouts-barley and triticale-white clover. Disease suppressiveness of field soils was evaluated in bioassays for the soilborne pathogens Rhizoctonia solani, Fusarium oxysporum f. sp. lini, and Gaeumannomyces graminis var. tritici. For both cropping systems, mixed cropping did not enhance disease suppressiveness of the soils. In some cases, soil cropped to barley alone was significantly more suppressive to F. oxysporum f. sp. lini than soils cropped to Brussels sprouts or the mixture of Brussels sprouts and barley. Analyses of the diversity of the indigenous bacterial and fungal microflora by denaturing gradient gel electrophoresis of amplified 16S- and 18S-rDNA fragments, respectively, revealed, in most cases, no significant differences between mixed and mono-cropped soils. In conclusion, in this study, mixed cropping of soils with Brussels sprouts and barley or with triticale and white clover did not enhance microbial diversity or disease suppressiveness of soils to three different soilborne plant pathogens.

Long-Term Effect of Biological Soil Disinfestation on Verticillium Wilt.

Biological soil disinfestation (BSD), involving incorporation of grass combined with plastic mulching, eliminates many soilborne pests and diseases through the creation of anaerobic conditions. BSD was compared at two locations with a nontreated control, Italian ryegrass amendment alone, and plastic mulch alone. After the soil treatments, plots were cropped with Acer platanoides and Catalpa bignonioides and grown for 4 years. Relative to the control, soil inoculum levels of Verticillium dahliae were reduced by 85% after BSD and did not increase for 4 years. Populations of Pratylenchus fallax, known for their interaction with V. dahliae, in the soil and in roots were reduced by 95 to 99%. The incidence of infection by V. dahliae was reduced by 80 to 90%. Verticillium wilt severity was significantly reduced in A. platanoides in all 4 years at one location and in the first 2 years at the other location, and significantly fewer plants died at one location. Shoot length and trunk width were larger after BSD compared with the control at one location. Market value of the crop in BSD plots was up to € 140,000 ha-1 higher for A. platanoides and up to € 190,000 ha-1 higher for C. bignonioides than in the untreated control. BSD is an effective, economically profitable, and environmentally friendly control method for tree nurseries.

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil.

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.