Investigation of the Biocontrol Potential of Two Ash Endophytes against Hymenoscyphus fraxineus Using In Vitro Plant–Fungus Dual Cultures

Development of effective biocontrol procedures using ash endophytes to combat an ash pathogen Hymenoscyphus fraxineus would be an appropriate contribution to the ongoing effort to protect European ash stands against ash decline. In this study we investigated the biocontrol potential of two ash endophytes, Thielavia basicola and Minimidochium sp., against H. fraxineus using in vitro plant-fungus and fungus-fungus dual cultures approach in three biocontrol models. The tests aimed to determine whether the endophytes show antagonism toward Fraxinus excelsior and F. pennsylvanica, to assess the level of antagonism of the endophytes toward H. fraxineus and to identify potential secondary metabolites induced by the presence of H. fraxineus. The results that dual culture experiments modeled according to our design may be a very useful tool to precisely study biocontrol potential of fungi, i.e., without the impact of environmental factors. Such experiments also enable the selection of most resistant ash genotypes and rapid propagation, producing large numbers of pathogen-free seedlings. It should be noted, however, that both of the endophytes tested in the dual cultures strongly inhibited the growth of H. fraxineus. Their growth under the influence of callus/seedlings was also inhibited. Comparison of HPLC profiles showed that the presence of H. fraxineus in the post-culture medium induced the production of an unknown secondary metabolite in this species. Such results suggest that some of the plant–fungus combinations examined in this study may have potential to be developed as biocontrol methods, thus increasing the survivability of ash stands under natural conditions.

Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

Inhibition of the fungus Colletotrichum lindemuthianum in the presence of different potassium sorbate concentrations

One of the major problems of in vitro plant cultivation is caused by contaminations such asfungal, which represent great losses for plant cultivation laboratories. In view of the problem about contamination and gaps in efficient disinfection protocols, the present study aimed to evaluate the inhibition of the fungus Colletotrichum. lindemuthianum in the presence of the potassium sorbate food preservative at different concentrations (0 g L-1; 0.076 g. L-1; 0.15 g. L-1; 0.30 g. L-1; 0.45 g. L-1; 0.60 g. L-1 and 0.75 g. L-1). The analyses of the Percentage of Micelal Growth Inhibition (ICP) and the growth at 24 and 72 hours at different concentrations were quantified. It was observed that Potassium Sorbate was effective in the analysis of ICP at different concentrations in relation to control, with greater effectiveness for T4 to T7. The highest percentages of inhibition (T5 to T7) presenting an inhibition rate of mean mycelial growth of 17.5 % in relation to the control for the 72h period, indicating, therefore, that with further studies this preservative can be used to combat fungal microorganisms in vitro culture.