Anne D. van Diepeningen

Effects of cattle feeding regimen and soil management type on the fate of Escherichia coli O157:H7 and Salmonella enterica serovar typhimurium in manure, manure-amended soil, and lettuce

ABSTRACT Survival of the green fluorescent protein-transformed human pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was studied in a laboratory-simulated lettuce production chain. Dairy cows were fed three different roughage types: high-digestible grass silage plus maize silage (6:4), low-digestible grass silage, and straw. Each was adjusted with supplemental concentrates to high and low crude protein levels. The pathogens were added to manure, which was subsequently mixed (after 56 and 28 days for E. coli O157:H7 and Salmonella serovar Typhimurium, respectively) with two pairs of organically and conventionally managed loamy and sandy soil. After another 14 days, iceberg lettuce seedlings were planted and then checked for pathogens after 21 days of growth. Survival data were fitted to a logistic decline function (exponential for E. coli O157:H7 in soil). Roughage type significantly influenced the rate of decline of E. coli O157:H7 in manure, with the fastest decline in manure from the pure straw diet and the slowest in manure from the diet of grass silage plus maize silage. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although decline was significantly faster in the manure derived from straw than in the manure from the diet of grass silage plus maize silage. The pH and fiber content of the manure were significant explanatory factors and were positively correlated with the rate of decline. With E. coli O157:H7 there was a trend of faster decline in organic than in conventional soils. No pathogens were detected in the edible lettuce parts. The results indicate that cattle diet and soil management are important factors with respect to the survival of human pathogens in the environment.

Relation between soil health, wave-like fluctuations in microbial populations, and soil-borne plant disease management

A healthy soil is often defined as a stable soil system with high levels of biological diversity and activity, internal nutrient cycling, and resilience to disturbance. This implies that microbial fluctuations after a disturbance would dampen more quickly in a healthy than in a chronically damaged and biologically impoverished soil. Soil could be disturbed by various processes, for example addition of a nutrient source, tillage, or drying-rewetting. As a result of any disturbance, the numbers of heterotrophic bacteria and of individual species start to oscillate, both in time and space. The oscillations appear as moving waves along the path of a moving nutrient source such as a root tip. The phase and period for different trophic groups and species of bacteria may be shifted indicating that succession occurs. DGGE, Biolog and FAME analysis of subsequent populations in oscillation have confirmed that there is a cyclic succession in microbial communities. Microbial diversity oscillates in opposite direction from oscillations in microbial populations. In a healthy soil, the amplitudes of these oscillations will be small, but the background levels of microbial diversity and activity are high, so that soil-borne diseases will face more competitors and antagonists. However, soil-borne pathogens and antagonists alike will fluctuate in time and space as a result of growing plant roots and other disturbances, and the periods and phases of the oscillations may vary. As a consequence, biological control by members of a single trophic group or species may never be complete, as pathogens will encounter varying populations of the biocontrol agent on the root surface. A mixture of different trophic groups may provide more complete biological control because peaks of different trophic groups occur at subsequent locations along a root. Alternatively, regular addition of soil organic matter may increase background levels of microbial activity, increase nutrient cycling, lower the concentrations of easily available nutrient sources, increase microbial diversity, and enhance natural disease suppression.

Evaluation of molecular and genetic approaches to generate glucoamylase overproducing strains of Aspergillus niger.

To evaluate the possibility of improving glucoamylase (GLA) production in Aspergillus niger strains carrying multiple copies of the GLA encoding gene (glaA), additional glaA copies were introduced either by genetic recombination or retransformation. For strains to be used in such experiments a genetic analysis was first carried out. The results of this analysis clearly revealed that in each transformant integration had occurred at a chromosome corresponding to a single linkage group (LG). The GLA production per gene copy showed considerable variation in these strains, indicating a clear effect of the site of integration on gene expression. Introduction of additional gene copies by genetic recombination experiments was carried out for different combinations of strains, carrying glaA copies in different chromosomes. The introduction of additional glaA gene copies by genetic recombination did not result in a considerable increase in GLA production compared to the parental strains. In some strains recombination resulted in genetic instability, observed by the frequent loss of glaA copies. Also, retransformation of multi-copy glaA strains did not result in an increase in GLA production. In several strains even a decrease in GLA production was found after retransformation. Southern analysis of these transformants suggested that newly introduced gene copies were heavily rearranged, which partly explains why GLA production was not increased. Further analysis of one such transformant provided evidence that the overexpression of the glaA gene is limited by the amount of trans-acting regulatory protein(s) available.

Efficient degradation of tannic acid by black Aspergillus species

A set of aspergillus strains from culture collections and wild-type black aspergilli isolated on non-selective media were used to validate the use of media with 20% tannic acid for exclusive and complete selection of the black aspergilli. The 20% tannic acid medium proved useful for both quantitative and qualitative selection of all different black aspergilli, including all recognized species: A. carbonarius, A. japonicus, A. aculeatus, A foetidus, A. heteromorphus, A. niger, A. tubingensis and A. brasiliensis haplotypes. Even higher concentrations of tannic acid can be utilized by the black aspergilli suggesting a very efficient tannic acid-degrading system. Colour mutants show that the characteristic ability to grow on high tannic acid concentrations is not causally linked to the other typical feature of these aspergilli, i.e. the formation of brown-black pigments. Sequence analysis of the A. niger genome using the A. oryzae tannase gene yielded eleven tannase-like genes, far more than in related species. Therefore, a unique ecological niche in the degradation of tannic acid and connected nitrogen release seems to be reserved for these black-spored cosmopolitans.

Potato brown rot incidence and severity under different management and amendment regimes in different soil types

Ralstonia solanacearum race 3 biovar 2, the causative agent of potato brown rot (bacterial wilt), is an economically important disease in tropical, subtropical and temperate regions of the world. In view of previous reports on suppression of the disease by organic amendments, and the expansion of organic agriculture, it was timely to compare the effects of organic and conventional management and various amendments on brown rot development in different soils (type: sand or clay; origin: Egypt or the Netherlands). Brown rot infection was only slightly reduced in organically compared to conventionally managed sandy soils from Egypt, but organic management significantly increased disease incidence and pathogen survival in Dutch sandy and clay soils, which correlated with high DOC contents in the organic Dutch soils. There was no correlation between disease incidence or severity and bacterial diversity in the potato rhizosphere in differently managed soils (as determined by 16S DGGE). NPK fertilization reduced bacterial wilt in conventional Egyptian soils but not in Dutch soils. Cow manure amendment significantly reduced disease incidence in organic Dutch sandy soils, but did not affect the bacterial population. However, cow manure did reduce densities of R. solanacearum in Egyptian sandy soils, most probably by microbial competition as a clear shift in populations was detected with DGGE in these and Dutch sandy soils after manure amendment. Amendment with compost did not have a suppressive effect in any soil type. The absence of a disease suppressive effect of mineral and organic fertilization in Dutch clay soils may be related to the already high availability of inorganic and organic nutrients in these soils. This study shows that the mechanism of disease suppression of soil-borne plant pathogens may vary strongly according to the soil type, especially if quite different types of soil are used.

DGGE fragments oscillate with or counter to fluctuations in cultivable bacteria along wheat roots

Previously, we showed that bacterial populations oscillate in response to a moving substrate source such as a root tip, resulting in moving wavelike distributions along roots. For this article, we investigated if bacterial communities fluctuate as a whole or if there is a succession in bacterial composition from peak to peak or within peaks. Rhizosphere microbial communities along roots of wheat Triticum aestivum L. were studied in detail (20–25 rhizosphere and bulk soil samples along the total root length) in two related soils by colony enumeration and culture-independent DNA analysis. Similar to our previous findings, the numbers of copiotrophic and oligotrophic bacteria oscillated with significant harmonics along each root, independent of soil moisture or lateral roots. Shifts in amplified eubacterial 16S rDNA fragments from denaturing gradient gel electrophoresis (DGGE) analysis were detected along the roots. The most abundant and intensively amplified fragments fluctuated in phase with colony-forming unit (CFU) oscillations; fewer amplified fragments with less intensive bands fluctuated out of phase or were restricted to certain root zones. The bacterial species richness along the root was negatively correlated with the numbers of oligotrophic bacterial CFUs. Discriminant analyses on DGGE patterns distinguished between soil types, rhizosphere and bulk soil, and waxing and waning phases in the oscillations along roots. Bacterial compositions shifted within oscillations but were repeated from oscillation to oscillation, supporting the idea that the most abundant bacterial taxa were growing and dying over time and consequently in space, whereas other taxa counterfluctuated or hardly responded to the substrate supplied by the passing root tip.

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with terminal inverted repeats and encodes a DNA and a RNA polymerase. Here we test whether pAL2-1 homologs cause rapid aging independent of intrinsic and external conditions. We first analyzed a natural population of P. anserina and in 40% of the 112 isolates we detected pAL2-1 homologous plasmids. Though the lifespan varied considerably among the strains, plasmid-infected wild-type strains are on average shorter lived than plasmid-free strains and typically show a reduced lifespan extending effect of calorie restriction (CR). However, interesting exceptions were found, inviting further study. To further investigate the effect of pAL2-1 homologs under various conditions, we constructed and analyzed isogenic lines with and without the plasmid. We found that the presence of pAL2-1 homologs did not significantly affect growth rate as suggested by the population analysis, but reduced lifespan under all conditions. This effect was particularly clear for the lifespan extending conditions tested (CR, low temperature, antibiotics) supporting the idea that pAL2-1 homologs are additional senescence factors independent of the intrinsic senescence determinants.

Mitochondrial recombination increases with age in Podospora anserina

With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function.

Natural variation of heterokaryon incompatibility gene het-c in Podospora anserina reveals diversifying selection

In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition.

The het-c heterokaryon incompatibility gene in Aspergillus niger.

Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent.