A.M. Semenov

In search of biological indicators for soil health and disease suppression.

Abstract While soil quality encompasses physical and chemical besides biological characteristics, soil health is primarily an ecological characteristic. Ecosystem health has been defined in terms of ecosystem stability and resilience in response to a disturbance or stress. We therefore, suggest that indicators for soil health could be found by monitoring responses of the soil microbial community to the application of different stress factors at various intensities. The amplitude of a response and time to return to the current state before application of stress could serve as measures of soil health. Root pathogens are an integral part of soil microbial communities, and the occurrence of epiphytotics forms an indication of an ecosystem in distress. Disease suppression can be viewed as a manifestation of ecosystem stability and health. Thus, indicators for soil health could possibly also function as indicators for disease suppressiveness. Previously suggested indicators for soil health and disease suppression have mainly been lists of variables that were correlated to more or less disturbed soils (ranging from conventional to organic agricultural soils, grassland and forest soils) or to conduciveness to disease. We suggest a systematic ecological approach to the search for indicators for soil health and disease suppression, namely, measuring biological responses to various stress factors and the time needed to return to the current state.

Short-Term Wavelike Dynamics of Bacterial Populations in Response to Nutrient Input from Fresh Plant Residues

The objectives of the research were to investigate short-term dynamics of bacterial populations in soil after a disturbance in the form of fresh organic matter incorporation and to investigate how these dynamics are linked to those of some environmental parameters. To reach these objectives, soil bacterial populations, mineral nitrogen, pH, and redox potential (ROP) were monitored daily for 1 month after incorporation of clover-grass (CG) plant material in microcosm experiments. Colony-forming units (CFUs) and direct microscopic counts of FDA-stained and FTTC-stained bacteria increased immediately after incorporation of the plant material, dropped within 2 days, and fluctuated thereafter. Harmonics analysis demonstrated that there were significant wavelike fluctuations with three or four significant peaks within 1 month after incorporation of clover-grass material. Peaks in CFUs were 1–2 days ahead of those in direct counts. Ammonium (NH4) concentrations increased from the start of the experiments until nitrification commenced. Nitrate (NO3) concentrations dropped immediately after plant incorporation, and then rose monotonically until the end of the experiments. There were no wavelike fluctuations in NH4 and NO3 concentrations, so that bacterial fluctuations could not be attributed to alternating mineral N shortages and sufficiencies. pH levels rose and declined with NH4 levels. ROP dropped shortly before NH4 concentrations rose, and increased before NH4 concentrations decreased; there were no regular fluctuations in ROP, so that temporary oxygen shortages may not have been responsible for the observed fluctuations in bacterial populations. Thus, for the first time, regular wavelike dynamics were demonstrated for bacterial populations after perturbation by addition of fresh organic matter to soil, and several potential reasons for the death phase of the fluctuations could be excluded from further consideration.

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.

Wavelike distributions of infections by an introduced and naturally occurring root pathogen along wheat roots.

Previously, we showed that copiotrophic and oligotrophic bacteria fluctuate as moving waves along roots. These waves probably originate as a result of growth and death cycles at any location where a moving nutrient source passed. In this study, we placed sclerotia of Rhizoctonia solani AG8 along growing roots of wheat and showed that the proportions of root sections from which R. solani was isolated fluctuated with distance from the root tip. Similarly, proportions of root sections from which naturally occurring Pythium spp. were isolated fluctuated with distance from the root tip. Fourier analysis showed that these fluctuations constituted significant waves. Cross-correlation analyses demonstrated that there were negative correlations between R. solani infections and colony forming units of copiotrophic bacteria at the time of inoculation at the same locations on the root (lag = 0 cm), indicating that infection by R. solani could have been inhibited by these bacteria. There was a positive correlation between Pythium infections and copiotrophic bacteria at a lag of 6 cm along the roots. It therefore appears that Pythium infection took place shortly after the initial peak in copiotrophic bacteria following the passage of the root tip.

Short-Term Fluctuations of Sugar Beet Damping-Off by Pythium ultimum in Relation to Changes in Bacterial Communities After Organic Amendments to Two Soils

Previously, oscillations in beet seedling damping-off by Pythium ultimum, measured as area under the disease progress curve (AUDPC), were demonstrated after incorporation of organic materials into organic and conventional soils. These periodic fluctuations of P. ultimum infections were cross-correlated with oscillations of copiotrophic CFU at lags of 2 to 4 days. For this article, we investigated whether bacterial communities and microbial activities fluctuated after a disturbance from incorporation of organic materials, and whether these fluctuations were linked to the short-term oscillations in AUDPC of beet seedling damping-off and bacterial populations (CFU) in soil. Soil microbial communities studied by polymerase chain reaction-DGGE analysis of 16S DNA after isolation of total DNA from soil and microbial activities measured as CO(2) emission rates were monitored daily for 14 days after addition of grass-clover (GC) or composted manure (CM) into organic versus conventional soils. Similar to our previous findings, AUDPC and density of copiotrophic bacteria oscillated with time. Fluctuations in species richness (S), Shannon diversity index (H), and individual amplicons on DGGE gels were also detected. Oscillations in AUDPC were positively cross-correlated with copiotrophic CFU in all soils. Oscillations in AUDPC were also positively cross-correlated with 19 to 35% of the high-intensity DNA fragments in soils amended with GC but only 2 to 3% of these fragments in CM-amended soils. AUDPC values were negatively cross-correlated with 13 to 17% of the amplicons with low average intensities in CM-amended soils, which were not correlated with densities of copiotrophic CFU. CO(2) emission rates had remarkable variations in the initial 7 days after either of the soil amendments but were not associated with daily changes in AUDPC. The results suggest that infection by P. ultimum is hampered by competition from culturable copiotrophic bacteria and some high-intensity DGGE amplicons, because AUDPC is cross-correlated with these variables at lags of 1 to 4 days. However, negative cross-correlations with low-intensity DNA fragments indicate that P. ultimum infection could also be suppressed by antagonistic bacteria with low densities that may be nonculturable species, especially in CM amended soil. The organic soil generally had lower AUDPC values, higher bacterial diversity, and negative cross-correlations between AUDPC and low-intensity DNA fragments (after CM amendment), indicating that specific bacteria that do not attain high densities may contribute to P. ultimum suppression in organic soils.

Transition of entheropathogenic and saprotrophic bacteria in the niche cycle: Animals-excrement-soil-plants-animals

The possibility of transition of saprotrophic and enteropathohenic bacterial populations following the chain of naturally related habitats—fodder-animal gastrointestinal tract (GIT)-animals excrement-soil-plants and again animals with a cyclic formation—has been investigated quantitatively. All bacteria used in the experiments have been shown to successfully overcome all the mechanical, physical-chemical, and biological barriers in the food chain and to come out into the environment with a quite high number. It has been demonstrated that the same bacterial population can pass the whole cycle without additional introduction of similar populations from the outside.

Daily dynamics of bacterial numbers, CO2 emissions from soil and relationships between their wavelike fluctuations and succession of the microbial community

The daily dynamics of the number of copiotrophic and oligotrophic bacteria (in colony-forming units) and CO2 emissions from cultivated soils after short- and long-term disturbances were studied for 25–27 days in a microfield experiment. The relationship of the wavelike fluctuations of the bacterial number and CO2 emission with the succession of the soil microbial community was determined by the polymerase chain reaction method—denaturing gradient gel electrophoresis (PCR-DGGE). Short-term disturbances involved the application of organic or mineral fertilizers, pesticides, and plant residues to the soils of different plots. The long-term effect was a result of using biological and intensive farming systems for three years. The short-term disturbances resulted in increased peaks of the bacterial number, the significance of which was confirmed by harmonics analysis. The daily dynamics of the structure of the soil microbial community, which was studied for 27 days by the DGGE method, also had an oscillatory pattern. Statistical processing of the data (principal components analysis, harmonics and cross-correlation analyses) has revealed significant fluctuations in the structure of microbial communities coinciding with those of the bacterial populations. The structure of the microbial community changed within each peak of the dynamics of the bacterial number (but not from peak to peak), pointing to the cyclical character of the short-term succession. The long-term effects resulted in a less intense response of the microbiota—a lower rate of CO2 emission from the soil cultivated according to the organic farming system.

Translocation of bacteria from animal excrements to soil and associated habitats

The population dynamics of Salmonella enterica var. Typhimurium MAE 110 gfp, Escherichia coli O157:H7 gfp, and Pseudomonas fluorescens 32 gfp were investigated in their introduction to cattle excrements and subsequent entering the soil, plants of cress (Lepidium sativum L.), and migration through the gastroenteric tract of French snails (Helix pomatia L.). The survival of these bacteria in the excrements and soil was investigated at cyclically changing (day-night, 25–15 °C) and constant (18 °C) temperatures. The cyclically changing temperature adversely affected the survival of E. coli O157:H7 gfp, and P. fluorescens but did not influence S. enterica var. Typhimurium. All the bacteria and, especially, the analogues of enteropathogens showed high survival in the cattle and snail excrements, soil, and on the plants under the gradual decrease in their population. On the cress plants grown in a mixture of cattle excrements and soil, an increase in the number of the introduced bacteria was observed.

Daily dynamics of cellulase activity in arable soils depending on management practices

The daily dynamics of cellulase activity was studied during 27 days by the cellophane membrane method on soils managed using the conventional high-input farming system (application of mineral fertilizers and pesticides) and the biological conservation farming system (application of organic fertilizers alone) in a microfield experiment. The regular oscillatory dynamics of the cellulase activity were revealed and confirmed by the harmonic (Fourier) analysis. The oscillatory dynamics of the cellulase activity had a self-oscillatory nature and was not directly caused by the disturbing impacts of both the uncontrolled (natural) changes in the temperature and moisture (rainfall) and the controlled ones (the application of different fertilizers). The disturbing impacts affected the oscillation amplitude of the cellulase activity but not the frequency (periods) of the oscillations. The periodic oscillations of the cellulase activity were more significant in the soil under the high-input management compared to the soil under the biological farming system.

Estimation of the Effects of Earthworms and Initial Substrates on the Bacterial Community in Vermicomposts

The effects of earthworms and initial substrates on the structural and functional compositions of the bacterial community in vermicomposts have been estimated by the method of denaturing gradient gel electrophoresis (DGGE) of amplified bacterial 16S rRNA gene fragments and by the kinetic method for assessing the physiological state of bacterial communities. ANOVA of the data on structural characteristics of the community (DGGE data) has demonstrated that the nature of the composted substrate has the major effect on the structure of the bacterial community. The associated variance for this factor accounts for 53% and exceeds the effect of earthworms (29%). A functional characterization of the bacterial community (data on the physiological state of bacteria as judged from their growth on different organic compounds) suggests that the presence of earthworms activates the bacterial community by increasing the variable characterizing the physiological state of bacteria (r0). As has been shown, it is impossible to predict the effect of earthworms on the particular bacterial taxa, because the effects of similar signs (increase, decrease, or the absence of changes in all the variants) just for the three analyzed types of substrates have only been observed for 15% of the operational taxonomic units.