Jane Debode

Biosurfactants are involved in the biological control of Verticillium microsclerotia by Pseudomonas spp.

Aims:  To examine the effect of previously described bacterial antagonists on the viability of Verticillium microsclerotia in vitro and to elucidate the possible modes of action of bacterial strains in the suppression of Verticillium microsclerotia viability.

Detection of Multiple Verticillium Species in Soil Using Density Flotation and Real-Time Polymerase Chain Reaction

Wet sieving of soil samples, followed by plating on semi-selective medium and microscopic analysis, is the most commonly used technique to quantify microsclerotia-forming Verticillium species in soil. However, the method is restricted to small samples, does not allow easy differentiation between species, and takes several weeks to complete. This study describes an alternative method to test 100-g soil samples for three Verticillium species (V. tricorpus, V. dahliae, and V. longisporum) using density flotation-based extraction of microsclerotia followed by new real-time polymerase chain reaction (PCR) assays. Primers for these real-time PCR assays were designed to the ribosomal DNA internal transcribed spacer for V. tricorpus and the β-tubulin gene for V. dahliae + V. longisporum and V. longisporum. Tests with artificially and naturally infested soils showed that the new method is reproducible and sensitive (0.1 to 0.5 microsclerotia/g soil), allows differentiation among the three species, and can be completed in one day. The results of the new method and the wet-sieving method were highly correlated for V. tricorpus (R2 = 0.78), but not for V. dahliae/V. longisporum, probably due to the loss of germinability of V. dahliae/V. longisporum microsclerotia during prolonged dry storage of the soil.

Characterisation of fungal pathogens causing basal rot of lettuce in Belgian greenhouses

Basal rot is a common disease in lettuce greenhouses. A 3-year study on the diversity of pathogens associated with basal rot in Belgium was carried out. A total of 150 isolates were collected originating from 56 greenhouses. Four pathogens appeared to be involved. Rhizoctonia solani was found to be the causal agent at 23 locations, Sclerotinia spp. at 14, Botrytis cinerea at 17 and Pythium spp. at seven. The isolates of R. solani were further characterised to anastomosis groups and subgroups using morphological characteristics, pectic zymogram and PCR-RFLP. Five anastomosis groups could be distinguished: AG1-1B, AG4 HGI, AG10, AG2-1, AG2-1 Nt and AG3, with isolates of AG4 HGI and AG1-1B being the most prevalent and the most aggressive. Sclerotinia sclerotiorum was found at 13 locations, while S. minor was found at only one location. Based on ITS-sequencing Pythium isolates were assigned to three different species. At 20°C, isolates of all pathogens were able to cause lesions on detached lettuce leaves, except isolates of R. solani AG3 and AG2-1 Nt. A correlation could be found between the occurrence of the pathogens and the growing season. Botrytis cinerea was the most common pathogen in winter, whereas R. solani was most frequently isolated in summer. Sclerotinia spp. and Pythium spp. were isolated in spring, summer and autumn. The information obtained in this study will be most useful in the development of an alternative control strategy for causal agents of basal rot.

Chitin Mixed in Potting Soil Alters Lettuce Growth, the Survival of Zoonotic Bacteria on the Leaves and Associated Rhizosphere Microbiology

Chitin is a promising soil amendment for improving soil quality, plant growth, and plant resilience. The objectives of this study were twofold. First, to study the effect of chitin mixed in potting soil on lettuce growth and on the survival of two zoonotic bacterial pathogens, Escherichia coli O157:H7 and Salmonella enterica on the lettuce leaves. Second, to assess the related changes in the microbial lettuce rhizosphere, using phospholipid fatty acid (PLFA) analysis and amplicon sequencing of a bacterial 16S rRNA gene fragment and the fungal ITS2. As a result of chitin addition, lettuce fresh yield weight was significantly increased. S. enterica survival in the lettuce phyllosphere was significantly reduced. The E. coli O157:H7 survival was also lowered, but not significantly. Moreover, significant changes were observed in the bacterial and fungal community of the lettuce rhizosphere. PLFA analysis showed a significant increase in fungal and bacterial biomass. Amplicon sequencing showed no increase in fungal and bacterial biodiversity, but relative abundances of the bacterial phyla Acidobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, and Proteobacteria and the fungal phyla Ascomycota, Basidiomycota, and Zygomycota were significantly changed. More specifically, a more than 10-fold increase was observed for operational taxonomic units belonging to the bacterial genera Cellvibrio, Pedobacter, Dyadobacter, and Streptomyces and to the fungal genera Lecanicillium and Mortierella. These genera include several species previously reported to be involved in biocontrol, plant growth promotion, the nitrogen cycle and chitin degradation. These results enhance the understanding of the response of the rhizosphere microbiome to chitin amendment. Moreover, this is the first study to investigate the use of soil amendments to control the survival of S. enterica on plant leaves.

The endophyte Verticillium Vt305 protects cauliflower against Verticillium wilt.

To investigate the interaction between cauliflower and the isolate VerticilliumVt305, obtained from a field suppressive to Verticillium wilt of cauliflower, and to evaluate the ability of VerticilliumVt305 to control Verticillium wilt of cauliflower caused by V. longisporum.

Effect of fungicides on epiphytic yeasts associated with strawberry

We studied the effect of two commonly used fungicides on the epiphytic yeast community of strawberry. Greenhouse and field experiments were conducted applying Switch (cyprodinil plus fludioxonil) or Signum (boscalid plus pyraclostrobin) to strawberry plants. Yeasts on leaves and fruits were assessed on treated and untreated plants at several time points via plating and denaturing gradient gel electrophoresis (DGGE) analysis. The yeast counts on plates of the treated plants were similar to the control plants. Unripe fruits had 10 times larger yeast concentrations than ripe fruits or leaves. Some dominant yeast types were isolated and in vitro tests showed that they were at least 10 times less sensitive to Switch and Signum as compared with two important fungal strawberry pathogens Botrytis cinerea and Colletotrichum acutatum, which are the targets for the fungicide control. DGGE analysis showed that the applied fungicides had no effect on the composition of the yeast communities, while the growing system, strawberry tissue, and sampling time did affect the yeast communities. The yeast species most commonly identified were Cryptococcus, Rhodotorula, and Sporobolomyces. These results point toward the potential applicability of natural occurring yeast antagonists into an integrated disease control strategy for strawberry diseases.

Real-time PCR mediated monitoring of Fusarium foetens in symptomatic and non-symptomatic hosts

Fusarium foetens is a recently described aggressive vascular pathogen of Begonia x hiemalis. Since 2004, it has caused severe losses for Begonia growers in Northern Europe and North America. F. foetens is likely to be of exotic origin. Little is known about the accumulation of the fungus in Begonia plants before and during symptom expression and about its host range. We have optimised a molecular detection method for F. foetens by only using the plant part containing the largest amount of the pathogen and by optimising the tissue maceration and DNA extraction techniques. This allowed a reliable detection limit of 2310 spore equivalents per plant and a theoretical detection limit of as low as 84 to 167 spore equivalents per plant. Using this method, we demonstrated exponential accumulation of F. foetens DNA in Begonia roots, resulting in symptoms at a threshold of approximately 107 spore equivalents and levelling off at 109 spore equivalents per plant. The observed rate of accumulation and the amount of pathogen DNA in non-symptomatic plants can be combined to determine whether the cuttings were infected after delivery at the Begonia nursery and to calculate the estimated timing of symptom development. To test the host range, we applied the optimised molecular detection technique. During these tests, only Begonia x hiemalis plants became symptomatic, but many other plant species supported growth of the pathogen. This information can be used to aid pathogen control and has implications for pest risk assessment.

First report of Pilidium concavum causing tan-brown rot on strawberry fruit in Belgium.

In April 2010, pink-orange spore masses that later turned brown were observed on 7 to 50% of the transplant lots during a routine screening of Belgian strawberry (Fragaria × ananassa, cv. Elsanta) for the latent presence of Colletotrichum acutatum using the petiole freeze method (4). These spore masses contained hyaline, canoe-shaped to allantoid conidia (mean size 7.5 × 1.8 μm), which is not consistent with C. acutatum spore morphology. Subsequently, a spore mass was transferred onto potato dextrose agar (PDA) and a gray-to-brown colony with whitish, aerial mycelium was produced, which is also not consistent with C. acutatum isolates. To identify the fungus, the ITS1-5.8S-ITS2 rDNA region was amplified by PCR and sequenced. The 485-bp region was 100% identical to that of Pilidium concavum specimen voucher BPI 1107275 (GenBank Accession No. AY487094). P. concavum (Desm.) Höhn. (synanamorph Hainesia lythri; teleomorph Discohainesia oenotherae) is a pathogen of strawberry causing tan-brown rot of fruit and is a common secondary invader of roots and dead strawberry plant parts (3). A recent strain of P. concavum from strawberry, isolate UPL 50, obtained from Brazil (L. Zambolim, Univ. Fed. de Viçosa, personal communication) showed similar colony, microscopic (mean spore size of 6.8 × 1.8 μm), and molecular (ITS sequence 98% identical to that of P. concavum specimen voucher BPI 1107275) features as the Belgian isolate. Pathogenicity tests were conducted on mature strawberry fruits by submerging 15 fruits per isolate for 3 min in a conidial suspension (2 × 106 conidia ml-1 of water) obtained from a 2-week-old colony on PDA. Controls were submerged in sterile distilled water. The inoculated fruits were incubated in a moist chamber at 25°C. Sunken, yellowish brown lesions with pink and later orange-brown spore masses were observed starting 3 days after inoculation on 88 and 94% of the fruit for the Brazilian and Belgian isolate, respectively. The control fruits remained healthy. The fungal isolates were reisolated from symptomatic fruits and their identity was confirmed based on morphological features. During a strawberry field survey in July 2010 in Sint-Truiden (Belgium), lesions typical of those described above were observed on eight strawberry fruits (cv. Elsanta). The fungus was isolated from the symptomatic tissue of two fruits and characterized as described above. Since P. concavum was latently present on strawberry transplants and caused disease on the fruits in the field, we conclude that P. concavum is a potential threat for Belgian strawberry production. Moreover, no strawberry cultivars with resistance to the pathogen have been reported. The disease has previously been reported on strawberry in South America and Poland (1,2), but to our knowledge, this is the first report of P. concavum on strawberry in Belgium. Although the spore and colony morphology of P. concavum is different from C. acutatum, the spore masses of P. concavum can easily be confused with the spore masses of C. acutatum when using the freeze method. This suggests the need for microscopic analysis of these spore masses during routine analyses. References: (1) L. Cedeno et al. Interciencia 26:113, 2001. (2) U. P. Lopes et al. New Dis. Rep. 21:7, 2010. (3) J. L. Maas. Compendium of Strawberry Diseases. The American Phytopathological Society St. Paul, MN, 1998. (4) J. C. Mertely and D. E. Legard. Plant Dis. 88:407, 2004.

Dynamics in the Strawberry Rhizosphere Microbiome in Response to Biochar and Botrytis cinerea Leaf Infection

Adding biochar, the solid coproduct of biofuel production, to peat can enhance strawberry growth, and disease resistance against the airborne fungal pathogen Botrytis cinerea. Additionally, biochar can induce shifts in the strawberry rhizosphere microbiome. However, the moment that this biochar-mediated shift occurs in the rhizosphere is not known. Further, the effect of an above-ground infection on the strawberry rhizosphere microbiome is unknown. In the present study we established two experiments in which strawberry transplants (cv. Elsanta) were planted either in peat or in peat amended with 3% biochar. First, we established a time course experiment to measure the effect of biochar on the rhizosphere bacterial and fungal communities over time. In a second experiment, we inoculated the strawberry leaves with B. cinerea, and studied the impact of the infection on the rhizosphere bacterial community. The fungal rhizosphere community was stabilized after 1 week, except for the upcoming Auriculariales, whereas the bacterial community shifted till 6 weeks. An effect of the addition of biochar to the peat on the rhizosphere microbiome was solely measured for the bacterial community from week 6 of plant growth onwards. When scoring the plant development, biochar addition was associated with enhanced root formation, fruit production, and postharvest resistance of the fruits against B. cinerea. We hypothesize that the bacterial rhizosphere microbiome, but also biochar-mediated changes in chemical substrate composition could be involved in these events. Infection of the strawberry leaves with B. cinerea induced shifts in the bacterial rhizosphere community, with an increased bacterial richness. This disease-induced effect was not observed in the rhizospheres of the B. cinerea-infected plants grown in the biochar-amended peat. The results show that an above-ground infection has its effect on the strawberry rhizosphere microbiome, changing the bacterial interactions in the root-substrate interface. This infection effect on the bacterial rhizosphere microbiome seems to be comparable to, but less pronounced than the effect of biochar-addition to the peat. The biological meaning of these observations needs further research, but this study indicates that biochar and an above-ground pathogen attack help the plant to recruit rhizosphere microbes that may aid them in their plant growth and health.

Trichoderma-Inoculated Miscanthus Straw Can Replace Peat in Strawberry Cultivation, with Beneficial Effects on Disease Control

Peat based growing media are not ecologically sustainable and often fail to support biological control. Miscanthus straw was (1) tested to partially replace peat; and (2) pre-colonized with a Trichoderma strain to increase the biological control capacity of the growing media. In two strawberry pot trials (denoted as experiment I & II), extruded and non-extruded miscanthus straw, with or without pre-colonization with T. harzianum T22, was used to partially (20% v/v) replace peat. We tested the performance of each mixture by monitoring strawberry plant development, nutrient content in the leaves and growing media, sensitivity of the fruit to the fungal pathogen Botrytis cinerea, rhizosphere community and strawberry defense responses. N immobilization by miscanthus straw reduced strawberry growth and yield in experiment II but not in I. The pre-colonization of the straw with Trichoderma increased the post-harvest disease suppressiveness against B. cinerea and changed the rhizosphere fungal microbiome in both experiments. In addition, defense-related genes were induced in experiment II. The use of miscanthus straw in growing media will reduce the demand for peat and close resource loops. Successful pre-colonization of this straw with biological control fungi will optimize crop cultivation, requiring fewer pesticide applications, which will benefit the environment and human health.