The aim of the present work was to test known bacterial plant growth-promoting strains for their ... more The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumberbeneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they
Pseudomonas fluorescens PCL1751 is a rod-shaped Gram-negative bacterium isolated from the rhizosp... more Pseudomonas fluorescens PCL1751 is a rod-shaped Gram-negative bacterium isolated from the rhizosphere of a greenhouse-grown tomato plant in Uzbekistan. It controls several plant root diseases caused by Fusarium fungi through the mechanism of competition for nutrients and niches (CNN). This mechanism does not rely on the production of antibiotics, so it avoids the concerns of resistance development and is environmentally safe. Additionally, this bacterium promotes plant growth by alleviating salt stress for its plant host. To investigate the genetic mechanisms that may explain these observations, we determined the complete genome sequence of this bacterium, examined its gene content, and performed comparative genomics analysis with other Pseudomonas strains. The genome of P. fluorescens PCL1751 consisted of one circular chromosome that is 6,143,950 base-pairs (bp) in size; no plasmid was found. The annotation included 19 rRNA, 70 tRNA, and 5,534 protein-coding genes. The gene content...
Seventeen percent of cucumber plants grown in a Uzbek greenhouse were diseased. The major cucumbe... more Seventeen percent of cucumber plants grown in a Uzbek greenhouse were diseased. The major cucumber and tomato pathogens of Uzbek agricultural soils were identified as strains of Fusarium solani. Fifty two beneficial bacteria from collections of our institutes were screened for their ability to promote growth and/or to control diseases caused by F. solani on cucumber and tomato plants. The five best strains were used in large scale greenhouse trials. Four out of five strains significantly controlled cucumber foot and root rot, reducing the percentage of diseased plants from 54% in the negative control to between 10 and 29% in bacterized plants. All five strains increased the dry weight, by 29 up to 62%. In two consecutive years all five strains significantly increased the plant height (by 4 to 15%) as well as the fruit yield (by 12 to 32%). Tests of plant-beneficial traits suggest that auxin production, antibiosis and competition for nutrients and niches are mechanisms involved in th...
The aim of the present work was to test known bacterial plant growth-promoting strains for their ... more The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumberbeneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they
ABSTRACT Commercialization of plant growth stimulating and biocontrol microbes plays a significan... more ABSTRACT Commercialization of plant growth stimulating and biocontrol microbes plays a significant role in providing environmentally friendly and efficient alternatives for chemicals used in agriculture and horticulture. This chapter describes principles of the manufacturing process, including mass production and formulation, of microbes and specifies existing inoculation techniques. Moreover, since evaluation of the efficacy of products is essential for estimation of their commercial potential and because it is also required for their registration, rules and approaches used for the official efficacy tests based mostly on guidelines of the European Plant Protection Organization are included. In order to be placed on the market, microbiological plant protection products and biostimulants/biofertilizers have to be registered. Therefore, also requirements for registration of microbial products in different countries are described and discussed.
Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on 43 agr... more Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on 43 agricultural and ornamental plants, is important for predicting disease outbreaks. Since both 44 pathogenic and non-pathogenic strains of Fox are ubiquitous and are able to colonize plants,
Thirty endophytic bacteria were isolated from various plant species growing near Saint-Petersburg... more 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 plantbeneficial 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.
Biocontrol of tomato foot and root rot by Pseudomonas bacteria in stonewool, Jun 1, 2005
Curriculum vitae Shamil Zavdatovich Validov was born on the 5th of March 1971 in Kazan, Republic ... more Curriculum vitae Shamil Zavdatovich Validov was born on the 5th of March 1971 in Kazan, Republic Tatarstan, Russian Federation. He attended high school No 111. In 1988 he entered the Kazan State University. In 1993 he received his Master degree in genetics. In the same year he joined the Laboratory of Plasmid Biology at the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciencies in Biology Centre of Excellence in Puschino near Moscow. In 2001, within the framework of a NWO-sponsored cooperation between The ...
The influence of stonewool substrate on the exudation of the major soluble carbon nutrients and o... more 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.
The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bac... more 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. radicis-lycopersici 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.
Aims: Tomato foot and root rot (TFRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (F... more 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 Grampositive 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.
Pseudomonas fluorescens strain WCS365. Incubation of Forl microconidia in tomato root exudate sti... more 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.
Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl ( Fusarium ... more 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 'competition for nutrients and niches', 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.
Although bacteria from the genus Collimonas have demonstrated in vitro antifungal activity agains... more 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.
The aim of the present work was to test known bacterial plant growth-promoting strains for their ... more The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumberbeneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they
Pseudomonas fluorescens PCL1751 is a rod-shaped Gram-negative bacterium isolated from the rhizosp... more Pseudomonas fluorescens PCL1751 is a rod-shaped Gram-negative bacterium isolated from the rhizosphere of a greenhouse-grown tomato plant in Uzbekistan. It controls several plant root diseases caused by Fusarium fungi through the mechanism of competition for nutrients and niches (CNN). This mechanism does not rely on the production of antibiotics, so it avoids the concerns of resistance development and is environmentally safe. Additionally, this bacterium promotes plant growth by alleviating salt stress for its plant host. To investigate the genetic mechanisms that may explain these observations, we determined the complete genome sequence of this bacterium, examined its gene content, and performed comparative genomics analysis with other Pseudomonas strains. The genome of P. fluorescens PCL1751 consisted of one circular chromosome that is 6,143,950 base-pairs (bp) in size; no plasmid was found. The annotation included 19 rRNA, 70 tRNA, and 5,534 protein-coding genes. The gene content...
Seventeen percent of cucumber plants grown in a Uzbek greenhouse were diseased. The major cucumbe... more Seventeen percent of cucumber plants grown in a Uzbek greenhouse were diseased. The major cucumber and tomato pathogens of Uzbek agricultural soils were identified as strains of Fusarium solani. Fifty two beneficial bacteria from collections of our institutes were screened for their ability to promote growth and/or to control diseases caused by F. solani on cucumber and tomato plants. The five best strains were used in large scale greenhouse trials. Four out of five strains significantly controlled cucumber foot and root rot, reducing the percentage of diseased plants from 54% in the negative control to between 10 and 29% in bacterized plants. All five strains increased the dry weight, by 29 up to 62%. In two consecutive years all five strains significantly increased the plant height (by 4 to 15%) as well as the fruit yield (by 12 to 32%). Tests of plant-beneficial traits suggest that auxin production, antibiosis and competition for nutrients and niches are mechanisms involved in th...
The aim of the present work was to test known bacterial plant growth-promoting strains for their ... more The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumberbeneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they
ABSTRACT Commercialization of plant growth stimulating and biocontrol microbes plays a significan... more ABSTRACT Commercialization of plant growth stimulating and biocontrol microbes plays a significant role in providing environmentally friendly and efficient alternatives for chemicals used in agriculture and horticulture. This chapter describes principles of the manufacturing process, including mass production and formulation, of microbes and specifies existing inoculation techniques. Moreover, since evaluation of the efficacy of products is essential for estimation of their commercial potential and because it is also required for their registration, rules and approaches used for the official efficacy tests based mostly on guidelines of the European Plant Protection Organization are included. In order to be placed on the market, microbiological plant protection products and biostimulants/biofertilizers have to be registered. Therefore, also requirements for registration of microbial products in different countries are described and discussed.
Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on 43 agr... more Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on 43 agricultural and ornamental plants, is important for predicting disease outbreaks. Since both 44 pathogenic and non-pathogenic strains of Fox are ubiquitous and are able to colonize plants,
Thirty endophytic bacteria were isolated from various plant species growing near Saint-Petersburg... more 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 plantbeneficial 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.
Biocontrol of tomato foot and root rot by Pseudomonas bacteria in stonewool, Jun 1, 2005
Curriculum vitae Shamil Zavdatovich Validov was born on the 5th of March 1971 in Kazan, Republic ... more Curriculum vitae Shamil Zavdatovich Validov was born on the 5th of March 1971 in Kazan, Republic Tatarstan, Russian Federation. He attended high school No 111. In 1988 he entered the Kazan State University. In 1993 he received his Master degree in genetics. In the same year he joined the Laboratory of Plasmid Biology at the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciencies in Biology Centre of Excellence in Puschino near Moscow. In 2001, within the framework of a NWO-sponsored cooperation between The ...
The influence of stonewool substrate on the exudation of the major soluble carbon nutrients and o... more 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.
The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bac... more 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. radicis-lycopersici 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.
Aims: Tomato foot and root rot (TFRR), caused by Fusarium oxysporum f. sp. radicis-lycopersici (F... more 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 Grampositive 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.
Pseudomonas fluorescens strain WCS365. Incubation of Forl microconidia in tomato root exudate sti... more 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.
Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl ( Fusarium ... more 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 'competition for nutrients and niches', 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.
Although bacteria from the genus Collimonas have demonstrated in vitro antifungal activity agains... more 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.
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