Sammar Khalil

Suppression of disease in tomato infected by Pythium ultimum with a biosurfactant produced by Pseudomonas koreensis

The use of biosurfactants is a promising alternative in biological control of zoospore-producing oomycetes, which are a major plant pathogen world-wide in a wide variety of crops. Oomycetes are of particular concern in closed hydroponic cultivation systems. The present study investigated the efficacy of a biosurfactant produced by Pseudomonas koreensis and added as a crude extract against the oomycete Pythium ultimum in hydroponic tomato cultivation. A significant reduction in disease was observed. Biosurfactant addition did not affect the indigenous root microflora when evaluated as sole carbon source utilisation. Chemical analysis, using electrospray hybrid mass spectrometry (ESI-MSMS), of the biosurfactant indicated it to be lokisin, a cyclic lipopeptide. These results confirm that biosurfactants are important in developing sustainable biological control strategies for oomycetes.

Characterization of biosurfactant-producing strains of fluorescent pseudomonads in a soilless cultivation system.

The use of biosurfactants is a promising alternative in biological control of zoospore-producing plant pathogens. In the present study, biosurfactant production by the indigenous population of fluorescent pseudomonads in a soilless plant cultivation system was studied during the growing season. A total of 600 strains was screened and of these 18.5% were observed to produce biosurfactants. Production of both antibiotics and biosurfactant was uncommon among the isolated strains. A selective effect of the cultivation system filter was observed on the biosurfactant-producing strains and these strains were only occasionally observed after the filter, despite having a significantly higher motility than the nonbiosurfactant-producing strains. The majority of biosurfactant-producing strains were isolated from the filter skin, which suggests that this is a suitable surface for inoculation with biocontrol strains.

Utilisation of Carbon Sources by Pythium, Phytophthora and Fusarium Species as Determined by Biolog® Microplate Assay

This study examined the metabolic activity of pure cultures of five root pathogens commonly found in closed hydroponic cultivation systems (Phytophthora cryptogea (PC), Phytophthora capsici (PCP), Pythium aphanidermatum (PA), Fusarium oxysporum f.sp. radicis-lycopersici (FORL) and Fusarium solani (FS)) using sole carbon source utilisation in order to develop effective biocontrol strategies against these pathogens. Aliquots of 150 µL of the mycelial suspension were inoculated in each well of GN2 microtitre plates. On the basis of average well colour development and number of positive wells, the pathogens were divided into two groups, (i) PA and FORL and (ii) PC, PCP and FS. Group (i) was characterised by a short lag-phase, a rapid exponential phase involving almost all carbon sources offered and a long stationary phase, while group (ii) had a more extended lag-phase and a slower utilisation rate of the carbon sources offered. The three isolates in group (ii) differed significantly during their exponential phase. The lowest utilisation rate of carbon sources and number of sources utilised was found for PCP. Of the major group of carbon sources, six carbohydrates, three carboxylic acids and four amino acids were rapidly used by all isolates tested at an early stage. The carbon sources gentibiose, α-D-glucose, maltose, sucrose, D-trehalose, L-aspartic acid, L-glutamic acid, L-proline persisted to the end of the exponential phase.Moreover, similarities between the metabolic profiles of the tested pathogen and the those of the resident microflora could also be found. These findings are of great importance as regards the role of the resident microflora in the biocontrol.

Production of biosurfactants and antibiotics by fluorescent pseudomonads isolated from a closed hydroponic system equipped with a slow filter

The presence of antibiotic- and biosurfactant-producing strains of fluorescent pseudomonads in a closed hydroponic system equipped with a slow filter was investigated. A total of 271 strains of pseudomonads were isolated before the filter, from the filter skin and from the effluent. Production of biosurfactants was determined using the drop-collapse method. The ability of the strains to inhibit the growth of the plant pathogens Pythium ultimum, Phytophthora cryptogea and Fusarium oxysporum was determined using dual culture plating. The influence of carbon sources on production was determined for selected strains, which also were identified to species level. Production of antibiotics or biosurfactants was observed to be a common trait among the fluorescent pseudomonads within the closed hydroponic system and it was affected by the filter. Pythium ultimum was the pathogen that was most sensitive to antibiotics produced by the fluorescent pseudomonads. The results indicated a strong influence of nutritional resources on antibiotic and biosurfactant production.

Effects of growing medium on the interactions between biocontrol agents and tomato root pathogens in a closed hydroponic system.

Summary Three commercial biocontrol agents [Trichoderma polysporum plus T. harzianum (Binab T), Gliocladium cantenulatum (Gliomix), and Streptomyces griseoviridis (Mycostop)] were evaluated for their ability to reduce root diseases caused by Pythium aphanidermatum, Phytophthora cryptogea, or Fusarium oxysporum f. sp. radicis-lycopersici in hydroponically-grown tomatoes. Studies were performed using two types of growing medium, peat or pumice, under controlled conditions in a closed irrigation system. The level of disease control varied depending on the biocontrol agent, growing medium, and pathogen. In pumice, amendment with Binab T, Gliomix, or Mycostop reduced the levels and disease incidence of all three pathogens. However, Mycostop had no significant effect on the level of any of the three pathogens in peat, although Binab T and Gliomix both achieved successful biocontrol. In both growth media, biocontrol of F. oxysporum f. sp. radicis-lycopersici was poor compared with that of P. aphanidermatum or P. cryptogea. Overall, tomato plant growth increased after the introduction of any of the biocontrol agents, in the presence of any of the three pathogens, compared with the untreated controls.

Evaluation of biocontrol agents for managing root diseases on hydroponically grown tomato

The effect of the bacterial strain Pseudomonas fluorescens 5.014 together with three commercial biocontrol products (Binab T, Gliomix and Mycostop) against root diseases caused by Pythium ultimum, P. aphanidermatum, Phytophthora cryptogea and Fusarium oxysporum f. sp. radicis-lycopersici was evaluated in the climate chamber for 4 weeks. The results indicated efficiency of all the biocontrol strains against P. ultimum and P. aphanidermatum. Effectiveness against F. oxysporum f. sp. radicis-lycopersici was only shown by the commercial products. BinabT and Mycostop were also effective against P. cryptogea. A growth promoting effect was indicated by the biocontrol strains after their application to the system.ZusammenfassungDie Wirkung des Bakterienstamms Pseudomonas fluorescens 5.014 und der drei kommerziell produzierten Antagonisten Binab T, Gliomix und Mycostop gegeüber den Wurzelerregern Pythium ultimum, P. aphanidermatum, Phytophthora cryptogea und Fusarium oxysporum f. sp. radicis-lycopersici wurde über 4 Wochen in Klimakammern untersucht. Alle Antagonisten wirkten gegenüber P. ultimum and P. aphanidermatum, während nur die drei kommerziellen Produkte F. oxysporum f. sp. radicis-lycopersici kontrollierten. BinabT and Mycostop wirkten auch gegenüber P. cryptogea. Eine wachstumsfördernde Wirkung der Antagonisten wurde ebenfalls beobachtet.

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350–990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5–09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5–09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity.

Influence of electrical conductivity on biological activity of Pythium ultimum and Binab T in a closed soilless system

The effects of electrical conductivity (EC) of the nutrient solution on the performance of the commercial biocontrol product Binab T against the root pathogen Pythium ultimum were studied in order to identify supporting factors to increase the efficiency and decrease the inconsistency of biocontrol agents in closed cultivation systems. Using a substrate-based growing system, the commercial product Binab T with the active strains Trichoderma polysporum plus T. harzianum was evaluated at four different EC levels (1.5, 2.5, 3.5 and 5.0 mS cm−1) over seven weeks in a controlled climate chamber environment, using tomato as the model plant. Addition of the biocontrol agent to the cultivation system caused a reduction in the level and incidence of pathogen attack that improved with increasing EC level from 2.5 mS cm−1 onward. Production of the cell wall-degrading enzymes cellulase and glucanase by Binab T increased with increasing EC level, independent of pathogen presence. In a control treatment with no artificial inoculation, enzyme activity decreased with increasing EC level. Increasing EC levels in the presence of Binab T also had a positive effect on plant biomass. The highest antagonistic effect of Binab T against Pythium ultimum, as indicated by enzyme activity, amount of pathogen in the system and disease incidence, occurred at 3.5 mS cm−1. The plant growth-promoting effect of Binab T also peaked at 3.5 mS cm−1.

Cross Response of Slow Filters to Dual Pathogen Inoculation in ClosedHydroponic Growing Systems

The efficacy of slow filters for horticultural purposes is suggested to be the result of multiple factors. Physical and biological properties of the filter column and the nutrient solution are likely to influence on filter performance. It has previously been shown that enzyme activity on the filter skin is a main parameter which will explain roughly 50% of filter efficacy. Since enzyme activity is enhanced by the addition of fungal cell walls to the filter skin, presence of a fungal root pathogen might affect enzyme activity and filter efficacy. In this study, slow filters integrated into closed NFT systems with a tomato crop were inoculated with mycelia from Pythium aphanidermatum. Enzyme activity, microbial colonisation, polysaccharide content and DNA content of the filter skin as well as microbial flora and total organic carbon in the nutrient solution were monitored. Tests of filter efficacy against Fusarium oxysporum f.sp. cyclaminis showed no significant influence on filter efficacy by the addition of P. aphanidermatum. Enzyme activities in the filter skin were not affected by the addition of P. aphanidermatum. A negative correlation was found between biofilm polysaccharide content and activity of xylanase activity on the filter skin. Filter efficacy was high (>99.9%) throughout the study. No damage to the crop as effect of pathogenic fungi was seen. We concluded that abundance of an oomycete pathogen in a closed hydroponic system does not affect filter efficacy, and that the filter skin microflora function, but not composition, is affected by this pathogen.

Effect of growing medium water content on the biological control of root pathogens in a closed soilless system

Summary The water content of the growing medium and its interaction with a biocontrol agent was studied in a closed soilless culture system in order to identify factors that could increase the efficiency and decrease the variability of biocontrol agents. Pumice was chosen as the growing medium because of its high water-holding capacity and in view of the high water content required for the growth of the root pathogen. The commercial biocontrol agent Binab T (active strains Trichoderma polysporum and T. harzianum) was evaluated at 50% and 70% water content. Studies were carried out in a controlled environment, in a climate chamber, using tomato as the model plant and Pythium ultimum as the model pathogen. Pathogen levels were affected by the water content of the medium and the biocontrol treatment, while the amount of biocontrol agent was not affected by the water content of the growing medium. The Trichoderma strains in Binab T were able to reduce the amount of pathogen and the incidence of disease at both water contents tested. The biocontrol activity of Binab T against P. ultimum, as indicated by the level of glucanase production, was affected by variations in the water content of the growing medium. The pathogen, the biocontrol agent, and the water content of the growing medium, all had an impact on the microbial communities resident in the system.