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.

Interaction of Salmonella with E. coli and Proteus spp. in Biofilm Formation

Aims: Investigate the interaction of Salmonella spp. with E. coli and Proteus spp. in biofilm formation as mono and dual-species at different time durations Experimental Design: Salmonella, Proteus, and E. coli were isolated from Broiler chicken meat, and the biofilm-forming ability of these organisms were studied. Place and Duration of Study: The study was conducted at the Laboratory of Livestock Production, Faculty of Agricultural Sciences, Sabaragamuwa University of Sri Lanka, from 2019 December to 2020 May. Methodology: This study investigated the biofilm-forming ability of Salmonella as a mono species and its interaction with E. coli and Proteus in the process of biofilm formation. Microorganisms used for this study were isolated from broiler chicken meat. Biofilm was quantified using a microtitre plate assay. The interaction effects were tested at the temperature of 280C in different time durations (up to 120 hours). Results: Salmonella 1 and Proteus monocultures showed significantly higher biofilm-forming ability than Salmonella 3 isolate at all tested time points. At 120 hr, additionally to the salmonella 1 and Proteus isolates E. coli also formed significantly higher biofilms than Salmonella 3. However, Salmonella 3 was the lowest biofilm former as mono biofilm at all tested time durations. Salmonella 1 interaction with Salmonella 3 isolates formed less biofilms than Salmonella 1 mono biofilm at 48hr and 72hr correspondingly. Salmonella 1 and its interactions with Salmonella 3, Proteus, E. coli showed similar biofilm-forming abilities without significant differences at all other tested time points. Specifically, Salmonella 3 interaction with Salmonella 1 as dual biofilm showed higher biofilm-forming ability than Salmonella 3 mono biofilm at all tested time points. Tested isolates and their interaction achieved the highest biofilm formation at numerous time points. In fact, at 48hr, Salmonella 3 isolates and its interaction of Proteus, E. coli, and Salmonella 1 interaction with Proteus attained their highest biofilm formation abilities. The highest biofilm formation was achieved by Salmonella 1 isolate as mono biofilm and Salmonella 1 interaction with E. coli as dual biofilm at 72hr. Biofilm-forming trend of respective isolates and interactions showed numerous patterns at tested time durations. Specifically, E. coli rapidly enhanced its biofilm-forming ability as monoculture from 24 hr to 120 hr. Proteus, Salmonella 3 as monocultures, Salmonella 3 interaction with Proteus and E. coli as dual cultures showed progressive biofilm development from 24 hr to 48 hr. Salmonella 1 monoculture and its interaction with Salmonella 3, E. coli as dual biofilm improved their biofilm-forming ability from 24 hr to 72 hr. Similar to Salmonella 3 interaction with Proteus, Salmonella 1 interaction with Proteus also increased its biofilm-forming ability from 24 hr to 48 hr. Conclusions: This study concluded that there is a variation among isolates and their combinations in forming the biofilms, where there is an enhancement of biofilm in dual-species over the mono-species in some interaction, and there is a reduction in biofilm formation by dual-species with some combinations. Further, this concluded that Salmonella is interacting with other commonly found bacteria such as Proteus and E. coli in biofilm formation.

Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the importance of the QS regulation in the bacteria-yeast interactions.

Phytophthora heterospora sp. nov., a New Pseudoconidia-Producing Sister Species of P. palmivora

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica. Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora, indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

Diffusible Compounds Produced by Hanseniaspora osmophila and Gluconobacter cerinus Help to Control the Causal Agents of Gray Rot and Summer Bunch Rot of Table Grapes

Gray and summer bunch rot are important diseases of table grapes due to the high economic and environmental cost of their control with synthetic fungicides. The ability to produce antifungal compounds against the causal agents Botrytis, Aspergillus, Penicillium, and Rhizopus of two microorganisms isolated from table grapes and identified as Hanseniaspora osmophila and Gluconobacter cerinus was evaluated. In dual cultures, both biocontrol agents (together and separately) inhibited in vitro mycelial growth of these pathogens. To identify the compounds responsible for the inhibitory effect, extractions were carried out with organic solvents from biocontrol agents separately. Through dual cultures with pathogens and pure extracts, only the hexane extract from H. osmophila showed an inhibitory effect against Botrytis cinerea. To further identify these compounds, the direct bioautography technique was used. This technique made it possible to determine the band displaying antifungal activity at Rf = 0.05–0.2. The compounds present in this band were identified by GC-MS and compared to the NIST library. The most abundant compounds, not previously reported, corresponded to alkanes, ketones, alcohols, and terpenoids. H. osmophila and G. cerinus have the potential to control the causal agents of gray and summer bunch rot of table grapes.

MICROEVOLUTION OF DESULFOVIBRIO VULGARIS CO-CULTURED WITH METHANOSARCINA BARKERI REVEALED BY GENOME RE-SEQUENCING AND SINGLE-CELL RT-QPCR ANALYSIS

An integrative approach of adaptive laboratory evolution, whole-genome sequencing and single-cell analysis was used to explore mechanisms related to establishment and maintenance of syntrophic interaction between sulfate-reducing Desulfovibrio vulgaris and methanogen Methanosarcina barkeri. Adaptive laboratory evolution of the D. vulgaris and M. barkeri dual-cultures under two different concentrations of electron donor lactate (38 mM and 50 mM) was conducted by propagating continuously for 50 transfers (~200 generations). Physiological analysis showed that, compared with the initial dual-cultures, the adapted dual-cultures (E38 and E50) have increased growth rates (1.1-fold and 1.2 -fold) and higher biomass yields (3.0-fold and 3.8-fold) on 38 mM and 50 mM lactate, respectively. Whole-genome re-sequencing of D. vulgaris in the adapted dual-cultures revealed 11 and 12 mutations in the D. vulgaris genomes of E38 and E50 dual-cultures, respectively, among which 4 mutations were found in both adapted dual-cultures. RT-qPCR analysis showed that the expression levels of 8 mutated genes were gradually up-regulated in D. vulgaris along with the evolution process. In addition, their heterogeneity was found decreased along with the evolution, as revealed by single-cell RT-qPCR analysis, reflecting adjustments of both gene expression and gene heterogeneity to the gradually established syntrophic relationship.

Interpretation of the Heroine’s Emotional Reconstruction in The Ghost Train

Paul Yee’s imaginary myth The Ghost Train mainly relates the experience of the heroin Choon-yi who is born with the drawing talent that helps those deceased Chinese labors return to China with the guidance of her father in her dream. On the road, she has confronted with many Chinese-Canadian cultural conflicts. Thus, the article will analyze the process of her emotional reconstruction from the aspects of her inner rebellion against the Canadian hegmonism, adaption and integration of the dual cultures, revealing how she accomplished a journey of salvation for nationalities and a pilgrim of seeking herself-growth.

Biological Control of Pythium aphanidermatum, the Causal Agent of Tomato Root Rot by Two Streptomyces Root Symbionts

Damping-off caused by Pythium aphanidermatum, as one of the most infamous plant diseases, is considered as a significant disease that causes severe damages in greenhouse and field crops in a vast range of hosts especially vegetables. The application of chemical fungicides as a common method to control this disease poses negative side effects on humans and the environmental components. Actinobacteria, especially members of the genus Streptomyces, are fascinating biocontrol agents and plant growth-promoting rhizobacteria, which exhibit safer alternative managements to control this disease. The present study aims to explore for bioactives soil Streptomyces isolates that are able to control P. aphanidermatum, which is the causal agent of damping-off in tomato. Out of a total of 116 actinobacteria isolates collected from the soil, 53 have showed an antagonistic activity against P. aphanidermatum, as deduced through in vitro dual cultures. Based on in vitro Petri plate seedling-bioassays (IPSB), from the 53 tested isolates in dual cultures, two isolates coded as H2 and H3, considered as the most bioactive agents, were selected to assess their biocontrol performances against P. aphanidermatum in the Sandwich bed-mix technique in greenhouse experiments. In vivo greenhouse statistical studies were performed to compare seven treatments using completely randomized design experiments. Metalaxyl fungicide was applied as the chemical-control treatment. To evaluate biocontrol efficiencies of the two Streptomyces symbionts, disease incidence recorded throughout the course of experiment and criteria of number of leaves, length of the plants, plant fresh and dry weights, were measured at the end of experiment and analyzed statistically. The resulted disease incidences for all treatments indicated that the two Streptomyces strains had PGPR activity, and they were effective in decreasing disease incidence and improving plant performances regarding number of leaves, height, and plant fresh and dry weights. Based on the phylogenetic analysis of the partial sequences of the 16S rRNA gene, the strain H2 revealed a close relation to six Streptomyces species, namely S. badius, S. caviscabies, S. globisporus, S. parvus, S. sindenensis, and S. griseoplanus, with 99.9% similarity. The strain H3 also indicated a close relation of the same similarity to two species, namely S. flavogriseus and S. pratensis. In overall, collected data dictated that the two bioactive root symbiont Streptomyces strains effectively controlled the damping-off disease caused by P. aphanidermatum.

Micromycetes antagonistic potential in vitro against Alternaria sp., a pathogenic strain associated with grapes

Fungi from the genus Alternaria Nees are new harmful grapes mycopathogens; they are a part of the necrotic leaf spot pathocomplex. The disease intensive development leads to more than 50% necrotisation of the leaf surface, leaving no leaf on the plant by the end of the growing season. An alternative to chemical protection against the disease is the use of biocontrol agents. For this purpose, the screening of fungal antagonist strains from the collection of the laboratory of the biological method of the Federal scientific center «V.S. Pustovoit All-Russian Research Institute of Oil crops» (FSBSI «FSC «ARRIOC») was carried out in relation to the causative agent of necrotic leaf spot of grapes Alternaria sp. by the dual cultures method on two nutrient media – potato-sucrose agar and Rudakov’s agar. Of the 27 fungal strains that were screened for the antagonistic activity to 425-3 Alternaria sp. 17 strains showed the activity. Of these, 12 strains competed for feeding area and exhibited hyperparasitism. Strains A-1 and I-3 Basidiomycota, Tr-1 Trichothecium sp. and all strains of Trichoderma sp. showed the greatest antagonistic activity on both media.

Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum

Wheat scab caused by F. graminearum is a highly destructive disease that leads to yield reduction and mycotoxin contamination of grains. In this study, an endophytic bacterium of strain YB-130 was isolated from surface sterilized wheat spikes with scab symptoms and identified as Bacillus velezensis by whole genome annotation, 16S rRNA gene and average nucleotide identities analysis. The whole-genome sequence of strain YB-130 was obtained by PacBio sequencing. 88 putative Carbohydrate-Active Enzymes and 12 gene clusters encoding for secondary metabolites were identified in the YB-130 genome, including one gene cluster for the synthesis of lanthipeptide only found in strain YB-130 genome. In dual cultures, strain YB-130 significantly inhibited the growth of F. graminearum PH-1 and other eight fungal plant pathogens, indicating a broad antifungal activity. Furthermore, strain YB-130 was able to significantly inhibit spore morphology and hyphal development of F. graminearum PH-1. Strain YB-130 also reduced deoxynivalenol production by F. graminearum PH-1 in dual cultures, possibly due to its ability to suppress the expression of tri5, tri3, and tri8 that are required for deoxynivalenol production in F. graminearum. Overall, B. velezensis YB-130 is a promising biological control agent of both F. graminearum infection and mycotoxin production.