Beatrix Alsanius

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.

Cell-wall degrading enzymes in slow ®lters of closed hydroponic systems

Summary Slow ®ltration is an effective, reliable and economically reasonable technique for disinfestation of nutrient solution in closed irrigation systems. However, the problematic control of its ef®cacy is a major shortcoming of this technique. The ef®cacy of slow ®lters against pathogenic microorganisms is caused by a complex combination of mechanical, physical-chemical and biological factors. Due to the importance of biological factors, parameters describing biological processes are possible factors for predicting the ef®cacy of slow ®lters. The natural occurrence, induction and signi®cance of cell wall-degrading enzymes (CWDE, such as protease, chitinase, cellulase and b-1,3-glucanase) in slow ®lters were investigated. On non-treated slow ®lters of rockwool, proteolytic and chitinolytic activity was enhanced mainly in the top ®lter layer compared with the nutrient solution. For cellulase and b-1,3-glucanase activity, no accumulation could be found. Induction of all CWDE was possible in situ with a preparation of Fusarium oxysporum f. sp. cyclaminis. Statistical analysis revealed that enzyme activity on rockwool was correlated with the ef®cacy of slow ®lters (r2.=.0.495). Chitinase had the strongest in¯uence, followed by protease and cellulase, whereas b-1,3-glucanase had no signi®cance. Hence, enzyme activity represents one parameter for predicting the prevailing ef®cacy of slow ®lters.

Leaf Microbiota of Strawberries as Affected by Biological Control Agents

The increasing use of biological control agents (BCAs) against Botrytis cinerea in strawberry raises the question of whether there are any undesirable impacts of foliar applications of BCAs on nontarget microorganisms in the phyllosphere. Therefore, our objective was to investigate this issue within a field study. Strawberry plants were repeatedly sprayed with three BCAs-namely, RhizoVital 42 fl. (Bacillus amyloliquefaciens FZB42), Trianum-P (Trichoderma harzianum T22), and Naturalis (Beauveria bassiana ATCC 74040)-to suppress Botrytis cinerea infections. Microbial communities of differentially treated leaves were analyzed using plate counts and pyrosequencing and compared with the microbial community of nontreated leaves. Plate count results indicate that the applied Bacillus and Trichoderma spp. survived in the strawberry phyllosphere throughout the strawberry season. However, no significant impacts on the leaf microbiota could be detected by this culture-dependent technique. Pyrosequencing of internal transcribed spacer ribosomal RNA and 16S RNA sequences revealed a change in fungal composition and diversity at class level after the introduction of T. harzianum T22 to the phyllosphere, whereas the bacterial composition and diversity was not affected by either this Trichoderma preparation or the other two BCAs. Our results suggest that pyrosequencing represents a useful method for studying microbial interactions in the phyllosphere.

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.

Bacterial amelioration of ferulic acid toxicity to hydroponically grown lettuce (Lactuca sativa L.)

Ferulic acid (FA) is released from plant roots and by decomposition of plant residues and may be involved in allelopathic interactions. We isolated bacteria from the recirculating nutrient solution of a closed, hydroponic lettuce culture using nutrient media supplemented with 1.0 mM FA. The isolates were tested for their capacity to degrade FA in concentrations up to 200 μM. Isolates p208, p210 and p307 showed the highest degradation rates and were therefore used for single- and multiple-strain inoculation in two factorial experiments where lettuce (Lactuca sativa L. cv. Grand Rapids) plants were grown gnotobiotically for 2 weeks in nutrient solution with or without 200 μM FA. When isolate p208 or multiple strains were added, no FA was detectable at the end of the experiments. In the absence of FA, no significant effects of the bacterial treatments could be found with respect to plant dry weight. However, in the presence of FA, isolate p210 increased shoot dry weight and the multiple-strain treatment increased root and shoot dry weights in the first experiment. In the second experiment, isolate p210 neither affected the concentration of FA nor plant dry weights. Isolate p208 and the multiple-strain treatment reduced the negative effect of FA on lateral root lengths and root hair formation in both experiments. Finally, we conclude that bacteria with the capacity to degrade FA and to ameliorate phytotoxic effects of FA were present in the nutrient solution of a commercial hydroponic lettuce culture.

Simultaneous Removal of Nitrate and Sulfate from Greenhouse Wastewater by Constructed Wetlands

This study evaluated the effectiveness of C-enriched subsurface-flow constructed wetlands in reducing high concentrations of nitrate (NO) and sulfate (SO) in greenhouse wastewaters. Constructed wetlands were filled with pozzolana, planted with common cattail (), and supplemented as follows: (i) constructed wetland with sucrose (CW+S), wetland units with 2 g L of sucrose solution from week 1 to 28; (ii) constructed wetland with compost (CW+C), wetland units supplemented with a reactive mixture of compost and sawdust; (iii) constructed wetland with compost and no sucrose (CW+CNS) from week 1 to 18, and constructed wetland with compost and sucrose (CW+CS) at 2 g L from week 19 to 28; and (iv) constructed wetland (CW). During 28 wk, the wetlands received a typical reconstituted greenhouse wastewater containing 500 mg L SO and 300 mg L NO. In CW+S, CW+C, and CW+CS, appropriate C:N ratio (7:3.4) and redox potential (-53 to 39 mV) for denitrification resulted in 95 to 99% NO removal. Carbon source was not a limiting factor for denitrification in C-enriched constructed wetlands. In CW+S and CW+CS, the dissolved organic carbon (DOC)/SO ratios of 0.36 and 0.28 resulted in high sulfate-reducing bacteria (SRB) counts and high SO removal (98%), whereas low activities were observed at DOC/SO ratios of 0.02 (CW) to 0.11 (CW+C, CW+CNS). On week 19, when organic C content was increased by sucrose addition in CW+CS, SRB counts increased from 2.80 to 5.11 log[CFU+1] mL, resulting in a level similar to the one measured in CW+S (4.69 log[CFU+1] mL). Consequently, high sulfate reduction occurred after denitrification, suggesting that low DOC (38-54 mg L) was the limiting factor. In CW, DOC concentration (9-10 mg L) was too low to sustain efficient denitrification and, therefore, sulfate reduction. Furthermore, the high concentration of dissolved sulfides observed in CW+S and CW+CS treated waters were eliminated by adding FeCl.

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.

Control of Botrytis cinerea in strawberries by biological control agents applied as single or combined treatments

There is little knowledge about the effects of simultaneously applied biological control agents (BCAs) on the incidence of grey mould (Botrytis cinerea) in strawberries under field conditions. Our objective was, therefore, to investigate the use of three BCAs (Bacillus amyloliquefaciens FZB42, Aureobasidium pullulans DSM 14940 and DSM 14941 and Beauveria bassiana ATCC 74040) with respect to their efficacy against B. cinerea in field grown strawberries following single compared to combined application. In two subsequent years (2011 and 2012), improved control of B. cinerea resulted from combined BCA treatments, whereas the single BCAs were not efficient. Efficient combinations of BCA treatments were not the same in 2011 and 2012, however. The development of B. cinerea was reduced by single treatments of B. amyloliquefaciens and A. pullulans as well as by most combined BCA treatments during storage for one of two fruit samples in 2011. In 2012, however, no effects on B. cinerea during storage resulted from BCA treatments. The results of the present study suggest that the use of combined BCA treatments only cannot overcome inconsistent efficacies of BCAs against strawberry grey mould under field conditions. Further investigations on the interactions between the BCAs in combined BCA treatments as well as between the BCAs and the pathogen in dependence on multiple factors prevailing in the phyllosphere, however, need to be included to tailor combined BCA treatments with constant efficacies in different years.

Effect of polysaccharides in slow filters integrated into closed hydroponic greenhouse systems

Summary Slow filtration is a widespread and environmentally sound method for the disinfestation of nutrient solutions in closed hydroponic systems. The efficacy of slow filters can be explained in part by straining and adsorption of pathogens onto filter material surfaces. Both mechanisms are influenced by choice of filter material, flow rate of the nutrient solution, biomass and extent of extracellular polymers on the filter skin. In a 2-year study, the total polysaccharide content and enzyme activity of the filter skin, and filter efficacy were monitored. Closed hydroponic systems using the nutrient film technique connected to slow filters were run with or without weekly inoculation with a fungal cell wall preparation (10.5 g m–2 of filter surface). This organic matter amendment increased the amount of total polysaccharide and enzyme activity within the filter skin. The fungal cell wall preparation did not accumulate, but was degraded rapidly. The total polysaccharide content (tPS; in mg g–1) of the filter skin increased linearly with time at 34.50 mg g–1 + 0.26 mg g–1 d–1 × days (d) (R2 = 0.556; P = 0.02) for untreated filters, and at 43.40 mg g–1 + 0.70 mg g–1 d–1 × days (d) (R2 = 0.826; P = 0.000) for treated filters. The extracellular polysaccharide content in the filter skin, determined after 16 and 20 weeks of slow filter operating time, ranged from 87.9 – 109.7 mg g–1 mineral wool (rockwool) sampled. The amount of extracellular polysaccharide was not influenced by the total polysaccharide content of the filter skin, by enzyme activity, or by addition of the fungal cell wall preparation. There was a trend towards higher filter efficacies when fungal cell wall preparations were added to the filter skin (treated: 98.3% ± 0.96; untreated: 97.9% ± 1.8); however, on the basis of the present data, no correlation between the assessed parameters and filter efficacies were observed.