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Loline alkaloids produced by Epichloee spp. are known to deter feeding by insect herbivores while also serving as a significant carbon source for certain epiphytic bacteria on tall fescue leaves. In this study we examined the role of loline alkaloids in attracting certain bacteria to the rhizosphere of tall fescue plants that harbor loline producing fungal endophytes. Population studies were used to compare the fitness of known loline catabolizing strains to other rhizosphere bacteria. Pyrosequencing of 16S rRNA fragments compared the composition of bacterial communities inhabiting the endophyte infected tall fescue (Festuca arundinacea) rhizosphere to those of endophyte free fescue plants. Rhizosphere population studies demonstrated that loline catabolizing strains Burkholderia ambifaria 7R and Pseudomonas aureofaciens outcompete and suppress the growth of non-loline catabolizing strains. Pyrosequencing of 16S rRNA fragments showed greater percentages of certain plant growth promoting bacteria in rhizosperes seeded with B. ambifaria 7R than non-inoculated soils. Rhizospheres of endophyte infected plants showed higher species richness (Shannon diversity index=4.03) over endophyte free rhizospheres (Shannon diversity index=3.08) and a greater percentage of Firmicutes. The differences in microbial community composition between endophyte-infected and endophyte-free rhizospheres suggest that the presence of fungal endophytes influences microbial community structure. Loline alkaloid production may be one proxy by which the fungal endophyte shapes microbial communities, as evidenced by increased fitness of loline catabolizing bacteria in the tall fescue rhizosphere.

This work was aimed to study the opportunity of transgenic oilseed rape (Brassica napus L.), expressing the gene of antimicrobial peptide cecropin P1 (cecP1) to be inhabited with associative microorganisms Methylobacterium mesophilicum and Pseudomonas aureofaciens. Previously, resistance was demonstrated to the microbial pathogens:Erwinia carotovora B15, Pseudomonas syringae, Fusarium oxysporum, and Botrytis cinerea. Analysis of the fatty acid composition of transgenic plant seeds showed an increase in the proportion of unsaturated fatty acids. Plants studied showed increased photosynthetic activity in oxidative stress induced by paraquat instead of in oxidative stress and UV radiation compared to control ones. A study of the interaction of transgenic plants with the associative microorganisms showed that bacteria were located in all initially colonized plants, as well as in plants obtained after several passages of microproliferation. Accordingly, transgenic plants, containing the cecP1 gene were actively colonized by associative bacteria, indicating their safety for associative bacteria, indicating their safety for beneficial microorganisms.

Pseudomonas aureofaciens strain 30-84 is a biocontrol agent against take all disease of wheat (Gaumannomyces graminis var. tritici). The PhzR/PhzI quorum sensing (QS) system regulates phenazine biosynthesis while the CsaR/CsaI QS system regulates aspects of the cell surface. We initiated studies on the effects of mutations in these two QS systems on cell adhesion/ biofilm formation by strain 30-84. A static adhesion plate assay was used to quantify bacterial adhesion to polystyrene surfaces. Surprisingly, these experiments showed that phzR and phzI mutants and phenazine structural mutants phzB adhered less well than the wild type or csaR and csaI mutants. Addition of purified N-acyl homoserine lactones restored adhesion/biofilm formation to phzI, csaI and phzI/csaI double mutants but not to phzB mutants, supporting the role of QS in adhesion/biofilm formation in strain 30-84. Further, the addition of TLC-purified phenazines (2-OH-PCA, PCA) restored adhesion/biofilm formation to all phenazine mutants, including phzB. Addition of phenazines to the phzR mutant did not restore wild type adhesion levels, indicating this QS system controls other traits involved in adhesion. These results support a new hypothesis that phenazines play additional roles for the producing bacterium beyond antibiosis and competition.

Biocontrol involves harnessing disease-suppressive microorganisms to improve plant health. Disease suppression by biocontrol agents is the sustained manifestation of interactions among the plant, the pathogen, the biocontrol agent, the microbial community on and around the plant, and the physical environment. Even in model laboratory systems, the study of biocontrol involves interactions among a minimum of three organisms. Therefore, despite its potential in agricultural applications, biocontrol is one of the most poorly understood areas of plant-microbe interactions. The complexity of these systems has influenced the acceptance of biocontrol as a means of controlling plant diseases in two ways. First, practical results with biocontrol have been variable. Thus, despite some stunning successes with biocontrol agents in agriculture, there remains a general skepticism born of past failures (Cook and Baker, 1983; Weller, 1988). Second, progress in understanding an entire system has been slow. Recently, however, substantial progress has been made in a number of biocontrol systems through the application of genetic and mathematical approaches that accommodate the complexity. Biocontrol of soilborne diseases is particularly complex because these diseases occur in the dynamic environment at the interface of root and soil known as the rhizosphere, which is defined as the region surrounding a root that is affected by it. The rhizosphere is typified by rapid change, intense microbial activity, and high populations of bacteria compared with non-rhizosphere soil. Plants release metabolically active cells from their roots and deposit as much as 20% of the carbon allocated to roots in the rhizosphere, suggesting a highly evolved relationship between the plant and rhizosphere microorganisms. The rhizosphere is subject to dramatic changes on a short temporal scale-rain events and daytime drought can result in fluctuations in salt concentration, pH, osmotic potential, water potential, and soil particle structure. Over longer temporal scales, the rhizosphere can change due to root growth, interactions with other soil biota, and weathering processes. It is the dynamic nature of the rhizosphere that makes it an interesting setting for the interactions that lead to disease and biocontrol of disease (Rovira, 1965, 1969, 1991; Hawes, 1991; Waisel et al., 1991). The complexity of the root-soil interface must be accommodated in the study of biocontrol, which must involve whole organisms and ultimately entire communities, if we are to understand the essential interactions in soil in the field. The challenge in elucidating mechanisms of biocontrol is in reducing the complexity to address tractable scientific questions. One of the most effective approaches toward the identification of critical variables in a complex system has been genetics. The study of mutants can be conducted in simplified laboratory systems or in the field, thus making accessible the examination of particular genetic changes and the associated biochemical characteristics in the real world. This review presents recent advances in our understanding of the biocontrol of root diseases. We emphasize research aimed at enhancing our understanding of the biology of the interactions that result in disease suppression. It is this understanding that will make possible the practical use of microorganisms in the management of plant disease in agroecosystems. Numerous recent reviews present comprehensively the variety of microbial biocontrol agents (Chet, 1987; Weller, 1988; Whipps and Lumsden, 1991; O'Sullivan and O'Gara, 1992; Cook, 1993; Goldman et al., 1994; Cook et al., 1995; Lumsden et al., 1995). In this discussion of current and future directions in biocontrol, our goal is to present key themes in the discipline, drawing on the bacteria Pseudomonas and Bacillus and the fungi Trichoderma and Gliocladium as examples representing a range of life strategies and mechanisms of disease suppression. We address the principles of interactions of the biocontrol agent with the pathogen, the host plant, and the microbial community, illustrating each principle with some well-studied examples of successful biocontrol agents (DBO).

This study included eight bacterial isolates originating from the apple phyllosphere or soil environment that were previously selected using the pear fruitlet test (Mikiciński 2017). Identification of these isolates based on phenotypic assays and DNA analysis showed that five of them belonged to species for which an antagonistic activity against Erwinia amylovora and the protective capacity of apple and pear against fire blight were not previously demonstrated. These were L16 identified as Pseudomonas vancouverensis, 3 M as Pseudomonas chlororaphis subsp. aureofaciens, 35 M – Pseudomonas congelans, 43 M – Enterobacter ludwigii, and 59 M – Pseudomonas protegens. Investigation of the biotic relationships between the tested strains and E. amylovora showed that 3 M, 35 M and 59 M inhibited the growth of the pathogen on five out of six media used (NAS, KB, LB, R2A, NAG), but 43 M did not do so on any of these media. Strain L16 did not inhibit the growth of the pathogen on LB or R2A medium. In contrast, all strains grown on medium 925 stimulated the growth of the pathogen, which showed no growth without co-cultivation with these strains. The experiments on apple trees and detached apple branches showed the ability of the tested bacteria to protect flowers at medium to high levels, depending on the experiment (55–93%). In some cases, this protection was even higher than that of the copper product used for comparison. In studies assessing the bacterial ability to protect shoots of M.26, the highest efficacy was observed for strains 35 M (96%) and 43 M (93%) but on ‘Gala Must’ all tested strains showed 100% of efficacy.

In agroecosystems, most isotopic investigations of NO3- involve the use of tracers that are artificially enriched in 15N. Although the dual isotope composition of NO3---delta (15)N and delta (18)O is especially beneficial for understanding the origin and fate of NO3-, its use for KCl-extractable soil NO3- has been hampered by the lack of a suitable analytical technique. Our objective was to test whether the denitrifier method, whereby NO3- is reduced to N2O before mass spectrometric analysis, can be used to determine the N and O isotopic composition of NO3- from 2 M KCl soil extracts. Several internationally accepted NO3-standards were dissolved in 2 M KCl, the conventional extractant for soil inorganic N, and inoculated with the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985). The standard deviation of the NO3- standards analyzed did not exceed 0.2 ppt for delta (15)N and 0.3 ppt for delta (18)O values. After appropriate corrections, differences between our measured and consensus delta (15)N and delta (18)O values of standard NO3- generally were within the standard deviations given for the consensus values. Both delta (15)N and delta (18)O values were reproducible among separate analytical runs. The method was also tested on genuine 2 M KCl extracts from unfertilized and fertilized soils. Depending on N fertilization, the soils had distinct delta (15)N and delta (18)O values, which were attributed to amendment with NH4NO3 fertilizer. Hence, our data indicate that the denitrifier method provides a fast, reliable, precise, and accurate way of simultaneously analyzing the natural abundances of (15)N and (18)O in KCl-extractable soil NO3-.

The population density and structure of complexes of soil microscopic fungi in the rhizosphere and rhizoplane of spring wheat ( Triticum aestivum L.), plant damage by root rot and leaf diseases, and crop yield were determined in a stationary field experiment on a silty loamy soddy-podzolic soil (Albic Retisol (Loamic, Aric)) in dependence on the soil tillage technique: (a) moldboard plowing to 20–22 cm and (b) non-inversive tillage to 14–16 cm. The results were treated with the two-way ANOVA method. It was shown that the number of fungal propagules in the rhizosphere and rhizoplane of plants in the variant with non-inversive tillage was significantly smaller than that in the variant with plowing. Minimization of the impact on the soil during five years led to insignificant changes in the structure of micromycete complexes in the rhizosphere of wheat. The damage of the plants with root rot and leaf diseases upon non-inversive tillage did not increase in comparison with that upon plowing. Wheat yield in the variant with non-inversive tillage was insignificantly lower than that in the variant with moldboard plowing. The application of biopreparations based on the Streptomyces hygroscopicus А4 and Pseudomonas aureofaciens BS 1393 resulted in a significant decrease of plant damage with leaf rust.

Pseudomonas aureofaciens (30-84) is a phenazine producing bacterium and reported as asuccessful biocontrol agent of some plant fungal pathogens. In the present study, the possibility of biological control of cotton damping-off caused by Rhizoctonia solani (AG-4) through phenazine production by the 30-84 strain, was investigated. In the search for the development of bioformulations of Pa (m) (PhzR–) and Pa (w) (PhzR+) strains of 30-84, four new carriers including soybean meal (SM), cottonseed meal (CM), rice bran (RB), and talc powder (TAL) were selected. The efficacy of bacterial formulations in reducing disease incidence was evaluated in four intervals (15, 30, 45, and 60 days after sowing), and compared with each bacterial suspension efficacy under green-house conditions. The results revealed that organic carriers were more effective than talc powder. It was also found that all the bioformulations were more efficient than each bacterial suspension. The most effective in reducing disease incidence was Pa (w) + RB. In contrast, Pa (m), Pa (m) + TAL, and Pa (m) + RB did not significantly suppress the disease in comparison with the infested control. Thus, phenazine production as a main biocontrol mechanism of P. aureofaciens (30-84) may be affected by the kind of carriers used for the bioformulation development.

Due to the importance of the biological control of plant diseases, testing and introducing new biocontrol-active microorganisms is a major concern among plant pathologists. The causal agent of cotton seedling damping-off disease is . In this regard, we tried to investigate the antagonistic activities of ( ) 30–84 (phenazine producing wild type and non-phenazine producing mutant) strains on , in comparison with some isolates of under both (laboratory) and (greenhouse) conditions. In the laboratory experiment, the inhibitory effects of all the bacteria, on the growth of , were evaluated using the dual culture procedure. Results showed that five isolates of along with both strains of significantly inhibited the growth of . Effective bacterial antagonists were then evaluated in a greenhouse experiment where cotton seeds were coated with their suspensions and were sown in pasteurised field-soil. The soil had been pre-inoculated with a virulent isolate of . The efficacy of the bacterial antagonists was evaluated by counting the number of surviving seedlings in different treatments, at 15 and 60 days after sowing, for determining pre- and post-emergence damping-off incidence. According to the results of the greenhouse experiment, at both intervals, two isolates of along with both strains of caused significant increases in the number of healthy seedlings, in comparison with the untreated control, and a commonly used fungicide (carboxin-thiram). The efficacy of phenazine producing a wild type strain of was higher than its non-phenazine producing mutant, indicating that phenazine plays an important role in the antagonistic activity of . Effective bacterial antagonists were then studied for their antagonistic mechanisms. The results showed that all four bacteria employed different mechanisms. The bacteria produced siderophore, and volatile metabolites and non-volatile metabolites, in their antagonistic activities. The results of this study suggest that may be a new biocontrol agent for controlling cotton seedling mortality disease.

Pseudomonas aureofaciens PA147-2 shows antifungal activity toward a variety of plant pathogenic fungi. We have been investigating the molecular mechanisms underlying the fungal inhibition, and during these studies it was observed that the use of pLAFR3-based cosmids for in trans complementation of mutants lacking antifungal activity is hindered by cosmid instability. It was hypothesised that the cosmid stability could be improved by inactivation of recA. The recA gene of PA147-2 was cloned and shown to complement recA mutants of E. coli, restoring RecA-dependent functions when expressed in trans. Two recA mutants of PA147-2 were constructed. Both of these mutants show sensitivity to DNA damage. Cosmid pPS2122 restores antifungal activity to a mutant by allele exchange, but is unstable in trans. The stability of pPS2122 is shown to be improved in a recA mutant of PA147-2 with respect to the wild type.