Abstract: Twenty endophytic bacteria were isolated from surface-sterilized stems and roots of
cucumber plants. After removal of potential siblings and human pathogens, the
remaining seven strains were identified based on their 16S rDNA as Pseudomonas
fluorescens (2 strains) and P. putida (5 strains). Three strains, namely P. fluorescens
CS1, P. fluorescens CR2 and P. putida CR3, were able to suppress tomato foot and root
rot (TFRR). Special attention was paid to the characterization of the BIOLOG carbon
oxidation profiles of the isolated pseudomonads in order to identify nutrients which
might be important for their endophytic lifestyle. Comparative analysis of the profiles
of these seven strains with those of seven rhizospheric Pseudomonas spp. revealed
that endophytes were able to oxidize L-arabinose and 2,3-butanediol significantly
more often than the rhizospheric group. An independent growth experiment
performed in tubes using L-arabinose and 2,3-butanediol as sole carbon sources
showed the same results as seen using BIOLOG for L-arabinose, but not for 2,3-
butanediol. Since L-arabinose is one of the most abundant sugars in xylem of
cucumber plants and was not detected in their rhizosphere, our data suggest that
utilization of L-arabinose might be a trait contributing to the endophytic lifestyle of the
isolated Pseudomonas endophytes.
Abstract: Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on agricultural and ornamental plants, is important for predicting disease outbreaks. Since both pathogenic and non-pathogenic strains of Fox are ubiquitous and are able to colonize plant roots, detection of Fox DNA in plant material is not the ultimate proof of an ongoing infection which would cause damage to the plant. We followed the colonization of tomato plants by strains Fox f. sp. radicis-lycopersici ZUM2407 (a tomato foot and root rot pathogen), Fox f. sp. radicis-cucumerinum V03-2g (a cucumber root rot pathogen) and Fox Fo47 (a well-known non-pathogenic biocontrol strain). We determined fungal DNA concentrations in tomato plantlets by quantitative PCR (qPCR) with primers complementary to the intergenic spacer region (IGS) of these three Fox strains. Two weeks after inoculation of tomato seedlings with these Fox strains, the DNA concentration of Forl ZUM2407 was five times higher than that of the non-compatible pathogen Forc V03-2g and 10 times higher than that of Fo47. In 3-week-old plantlets the concentration of Forl ZUM2407 DNA was at least 10 times higher than those of the other strains. The fungal DNA concentration, as determined by qPCR, appeared to be in good agreement with data of the score of visible symptoms of tomato foot and root rot obtained 3 weeks after inoculation of tomato with Forl ZUM2407. Our results show that targeting of the multicopy ribosomal operon results in a highly sensitive qPCR reaction for the detection of Fox DNA. Since formae speciales of Fox cannot be distinguished by comparison of ribosomal operons, detection of Fox DNA is not evidence of plant infection by a compatible pathogen. Nevertheless, the observed difference in levels of plant colonization between pathogenic and non-pathogenic strains strongly suggests that a concentration of Fox DNA in plant material above the threshold level of 0.005% is due to proliferation of pathogenic Fox.
Abstract: Thirty endophytic bacteria were isolated from various plant species growing near Saint-Petersburg, Russia. Based on a screening for various traits, including plant-beneficial properties and DNA fragment patterns, potential siblings were removed. The remaining isolates were taxonomically identified using 16S rDNA sequences and potential human and plant pathogens were removed. The remaining strains were tested for their ability to promote radish root growth and to protect tomato plants against tomato foot and root rot. One strain, Bacillus subtilis HC8, isolated from the giant hogweed Heracleum sosnowskyi Manden, significantly promoted plant growth and protected tomato against tomato foot and root rot. Metabolites possibly responsible for these plant-beneficial properties were identified as the hormone gibberellin and (lipo)peptide antibiotics respectively. The antibiotic properties of strain HC8 are similar to those of the commercially available plant-beneficial strain Bacillus amyloliquefaciens FZB42. However, thin layer chromatography profiles of the two strains differ. It is speculated that endophytes such as B. subtilis HC8 contribute to the fast growth of giant hogweed.
Abstract: Several microbes promote plant growth, and many microbial products that stimulate plant growth have been marketed. In this review we restrict ourselves to bacteria that are derived from and exert this effect on the root. Such bacteria are generally designated as PGPR (plant growth- promoting rhizobacteria). The beneficial effects of these rhizobacteria
on plant growth can be direct or indirect. This review begins with describing the conditions under which bacteria live in the rhizosphere. To exert their beneficial effects, bacteria usually must colonize the root surface efficiently. Therefore, bacterial traits required for root
colonization are subsequently described. Finally, several mechanisms by which microbes can act beneficially on plant growth are described. Examples of direct plant growth promotion that are discussed include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation, and (d ) plant stress control. Mechanisms of biological control by which
rhizobacteria can promote plant growth indirectly, i.e., by reducing the level of disease, include antibiosis, induction of systemic resistance, and competition for nutrients and niches.
Abstract: Pseudomonas putida strain PCL1760 was isolated previously from the avocado rhizosphere, using an enrichment method for competitive tomato root tip colonizers that selects for biological control strains which act through the biological control mechanism ââcompetition for nutrients and nichesâ (CNN). Here we demonstrate that strain PCL1760 showed significant biological control of tomato foot and root rot (TFRR), a disease caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), in eight independent laboratory experiments in stonewool substrate. Furthermore, its activity in stonewool was also tested in an industrial setup. The presence of Forl appeared to decrease seed germination. The additional presence of the biological control strain partly restored the germination level. Introduction of P. putida PCL1760 resulted in significant biological control of TFRR. PCR quantification revealed that the biological control
strain decreased the amount of Forl DNA in tomato plant tissue significantly. We conclude that the results of this trial show that P. putida strain PCL1760, which acts through the new mechanism CNN, controls TFRR also under industrial stonewool conditions.
Abstract: Soil salinization is increasing steadily in many parts of the world and causes major problems for plant productivity. Under these stress conditions, root associated
beneficial bacteria can help improve plant growth and nutrition. In this study, salt-tolerant bacteria from the rhizosphere of Uzbek wheat with potentially beneficial traits were isolated and characterized.
Eight strains which initially positively affect the growth of wheat plants in vitro were investigated in detail. All eight strains are salt tolerant and have some of the following plant growth-beneficial properties: production of auxin, HCN, lipase or protease and
wheat growth promotion. Using sequencing of part of the 16S rDNA, the eight new isolates were identified as Acinetobacter (two strains), Pseudomonas
aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterobacter hormaechei, Pantoae agglomerans and Alcaligenes faecalis. All these strains are potential human pathogens. Possible reasons for why these bacteria present in the rhizosphere and establish there are discussed.
Abstract: Fusarium oxysporum f.sp.radicis-licopersici (Forl) is a soilborne pathogenic fungus which can cause tomato foot and root rot (TFRR). Tomato root exudate is a good source of nutrients for both Forl and the TFRR-suppressing biocontrol bacterium Pseudomonas fluorescens strain WCS365. Incubation of Forl microconidia in tomato root exudate
stimulates their germination. This phenomenon is observed, to a lesser extent, upon incubation in plant nutrient solution supplemented with citrate or glucose, the major organic acid and sugar components, respectively, of tomato root exudate. Here we
show that induction of germination of microconidia is significantly reduced in the presence of P. fluorescens WCS365 in all tested media. Scanning electron microscopy revealed that P. fluorescens WCS365 colonizes developing hyphae. Efficient colonization correlates with low nutrient availability. Eventually, new microconidia are formed. The presence
of P. fluorescens WCS365 reduces the number of newly formed microconidia. This reduction does not depend on physical contact between bacteria and hyphae. We discuss that the ability of P. fluorescens WCS365 to slow down the processes of
microconidia germination and development of new microconidia of the phytopathogen, and therefore the ability to reduce fungal dissemination, is likely to contribute to the biocontrol efficacy of this strain.
Abstract: Aims: Tomato foot and root rot (TFRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive rootcolonizing bacteria from total rhizosphere soil samples.
Methods and Results: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains.
Conclusions: The new biocontrol strains, six Gram-negative and one Gram-positive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus.
Significance and Impact of the Study: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.
Abstract: Although bacteria from the genus Collimonas have demonstrated in vitro antifungal activity against many different fungi, they appeared inactive against the plant-pathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersici (Forl), the causal agent of tomato
foot and root rot (TFRR). Visualization studies using fluorescently labelled organisms showed that bacterial cells attached extensively to the fungal hyphae under nutrient-poor conditions but not in glucose rich Armstrong medium. Collimonas fungivorans was
shown to be as efficient in colonizing tomato root tips as the excellent colonizer Pseudomonas fluorescens strain WCS365. Furthermore, it appeared to colonize the same sites on the root as did the phytopathogenic fungus. Under greenhouse conditions in potting soil, C. fungivorans performed as well in biocontrol of TFRR as the well-established biocontrol strains P. fluorescens WCS365 and Pseudomonas chlororaphis
PCL1391. Moreover, under biocontrol conditions, C. fungivorans did not attach to Forl hyphae colonizing plant roots. Based on these observations, we hypothesize that C. fungivorans mainly controls TFRR through a mechanism of competition for nutrients
and niches rather than through its reported mycophagous properties, for which attachment of the bacteria to the fungal hyphae is assumed to be important
Abstract: The influence of stonewool substrate on the exudation of the major soluble carbon nutrients and of the auxin precursor tryptophane for Pseudomonas biocontrol agents was studied. To this end, the composition of the organic acids and sugars,
as well that of tryptophane, of axenically collected exudates of seed, seedlings, and roots of tomato, cucumber, and sweet pepper was determined. The major results were as follows. i) The total amount of organic acid is much higher than that of
total sugar. ii) Exudation of both organic acids and sugars increases during plant growth. iii) Citric, succinic, and malic acids represent the major organic acids, whereas fructose
and glucose are the major sugars. iv) Compared with glass beads as a neutral substrate, stonewool substantially stimulates exudation of organic acids and sugars. v) It appeared that enhanced root-tip-colonizing bacteria isolated previously from the rhizosphere of tomato and cucumber grow much better in minimal medium with citrate as the sole carbon source than other, randomly selected rhizobacteria do. This indicates that the procedure which selects for excellent root-tip colonizers enriches for strains which utilize the major exudate carbon source citrate. vi) The content of L-tryptophane,
the direct precursor of auxin, is approximately 60- fold higher in seedling exudates of tomato and sweet pepper than in cucumber seedling exudates, indicating a higher possibility of plant growth stimulation after inoculation with auxin-producing rhizobacteria for tomato and sweet pepper crops than for cucumber. However, the biocontrol strain
Pseudomonas fluorescens WCS365, which is able to convert tryptophane into auxin, did not stimulate growth of these three crops. In contrast, this strain did stimulate growth of
roots of radish, a plant which exudes nine times more tryptophane than tomato does.
Abstract: The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused
severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate
revealed that the presence of F. oxysporum f. sp. radicislycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic
acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased,
mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.
Abstract: Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl (
Fusarium oxysporum f.sp. radicis-lycopersici). Several bacteria have been described to be able to control the disease,using different mechanisms. Here we describe a
method that enables us to select, after application of a crude rhizobacterial mixture on a sterile seedling, those strains that reach the root tip faster than our best tomato root colonizer tested so far, the Pseudomonas fluorescens biocontrol strain WCS365.
Of the five tested new isolates, four appeared to be able to reduce the number of diseased plants. Analysis of one of these strains, P. fluorescens PCL1751, suggests that it controls the disease through the mechanism âcompetition for nutrients and nichesâ, a mechanism novel for biocontrol bacteria. Moreover, this is the first report describing a method to enrich for biocontrol strains from a crude mixture of rhizobacteria.
Another advantage of the method is that four out of five strains do not produce antifungal metabolites, which is preferential for registration as a commercial product.
Abstract: Most plant diseases are caused by fungi and oomycetes. Presently, the major method for controlling plant diseases is by the use of agrochemicals. However, this practice raises health and environmental concerns among consumers and politicians. An alternative for chemicals is the application of products based on natural enemies of the pathogen. Several of such Biological Control Agents (BCAs) with bacteria or fungi as the active ingredient are already in the market. In this review, we describe the discovery of such microbes as well as methods for their isolation. Using microscopy, we visualize biocontrol at the cellular level. Furthermore, we describe the role of root colonization by the BCA in biocontrol. Finally, mechanisms of biocontrol at the molecular level are described and the risk of resistance toward BCAs is discussed.
Abstract: Crop cultivation in salinated soils is one of the major challenges in agriculture: salinated areas are increasing worldwide and plants growing under saline or water-imbalance stress are more vulnerable to diseases caused by soil-borne pathogens. Biocontrol using salt-tolerant, plant-growth-promoting rhizobacteria (PGPR) to protect plant roots against high salinity as well as pathogens offers sustainable solutions for plant protection. Screening strategies for specific PGPRs are presented and assessed. Stenotrophomonas rhizophila is a model for a rhizosphere- and phylloplane-competent, salt-tolerant PGPR. The most effective strain S. rhizophila DSM 14405T showed biocontrol activity on various crops under salinated conditions in greenhouse and field trials in Uzbekistan. Besides, the strain DSM 14405T does not only show rhizosphere competence and antagonistic activity but also produces high amounts of osmoprotective substances allowing it to survive under saline conditions. New insights into its mode of action are presented from genomic information.
Abstract: The development of microbial inoculants for specific crops is a multistage process with participation of scientific researchers and industry. The process starts with isolation/selection of potential strains with desired properties such as plant growth stimulation, enhancement of availability of vitally important nutrients and therefore improvement of plant nutrition, amelioration of biotic and abiotic stress, degradation of pollutants, and biological control of phytopathogenic microbes. Subsequent assessment of the potentially promising strains for safety, the development of industrial production protocols and suitable formulations, registration, and marketing are the most costly and time consuming steps in product development. Elucidation of dominant properties of the strain will help to choose the correct strategy for positioning of the product on the market of microbial inoculants. The evaluation of examples of selected products based on Trichoderma harzianum T22, Bacillus subtilis FZB24, Bacillus amyloliquefaciens FZB42, and Pseudomonas chlororaphis MA342 provides important insights into successful transfer of academically gained knowledge in commercially successful microbial products used worldwide. Major challenges in development, registration, and marketing of microbial inoculants are discussed.
Abstract: Many plant-beneficial rhizobacteria have been described in the literature. These have been isolated from the plant root, where they usually live under conditions of nutrient starvation and at a low pH. In order to be beneficial, they usually need to colonize the root efficiently. Moreover, they have to multiply fast in order to be competitive with other organisms. To this end, traits such as chemotaxis to, and fast utilization of, the nutrients secreted by the root are required. These nutrients mainly consist of organic acids and sugars. Some plant-beneficial bacteria promote plant growth directly, e.g., by making nutrients available to the plant or by stimulating the growth of plants by production of hormones. Other plant-beneficial bacteria stimulate plant growth indirectly, e.g., by degrading environmental pollutants which inhibit plant growth or by controlling the growth of pathogens.
Commercialization of microbes is a complex and long-lasting process. Firstly, industry must see opportunities for making a profit. Secondly, the bacterium as well as the final product must be efficient and safe with respect to humans, animals, as well as nontarget organisms. Fast up scaled production of the organism against a reasonable price is another important prerequisite. Finally, the microbe must be formulated and packed in a form that is stable for many months and which is consistent with agricultural practice.
