Evaluation of the biocontrol activity of Frateuria defendens-derived metabolites against mollicutes.

Frateuria defendens is a candidate biocontrol agent that has been shown to reduce phytoplasma-related disease symptoms in grapevines and periwinkle plants. While a crude filtrate prepared from F. defendens can inhibit mollicute growth, the specific growth parameters for this bacterium, necessary to enhance this protective inhibitory response, remain unknown. Moreover, the separation of filtrate preparations from bacterial cells via centrifugation and filtration is laborious and time-consuming. As such, the present study was conducted to define the optimal growth conditions associated with maximal inhibitory activity of F. defendens and to establish a better approach to separating these bacterial cells from their secreted metabolites. To conduct these analyses, F. defendens was cultured in a range of media types, while associated inhibitory effects were tested in vitro using Spiroplasma melliferum as a model mollicute bacterium, and in planta using phytoplasma-infected periwinkle plantlets. These analyses revealed F. defendens growth patterns change based upon media composition, with filtrates prepared from a specific rich medium (S-medium) exhibiting beneficial activities, including the inhibition of S. melliferum and enhanced plant growth. When F. defendens cells were grown within semi-permeable, membrane-coated Small Bioreactor Platform (SBP) capsules, they could be more readily separated from the secreted metabolite fraction, obviating the need for filtration and/or centrifugation. This study is the first to have reported the use of SBP capsules to separate bacterial cells from their secreted metabolites under sterile conditions while retaining the ability of these metabolites to inhibit S. melliferum growth and to benefit the host plant. The results highlight promising new approaches to the effective biocontrol of phytoplasma-driven diseases in grapevines and other economically important plant species.

Antimicrobial Activity of Metabolites Secreted by the Endophytic Bacterium Frateuria defendens.

Candidatus Phytoplasma, the causative agent of yellows disease, inflicts substantial damage on several hundred plant species including perennials and annual plants. The endophytic bacterium Frateuria defendens reduces the symptoms of yellows disease in a number of agricultural crops. One possible mode of action is that the bacterium secretes antimicrobial metabolites. To test this hypothesis, the substances secreted by the endophyte during 10 days of growth in an artificial medium were identified by GC-MS (gas chromatography-mass spectrometry). Synthetic analogues to these substances were then used on periwinkle, a nurse culture plant infected by phytoplasma. Phytoplasma quantities were evaluated by quantitative PCR, and disease symptoms were monitored and recorded. It was found that specific compounds identified by the biochemical analysis caused a significant reduction in both the titer of phytoplasma and the disease symptoms in periwinkle when compared to untreated infected plants. Further research is required to examine the potential of these compounds as an effective treatment against yellows disease.

Frateuria defendens sp. nov., bacterium isolated from the yellows grapevine's disease vector Hyalesthes obsoletus.

A Dyella-like bacterium was previously isolated from the planthopper Hyalesthes obsoletus (Hemiptera). Based on its 16S rRNA gene sequence, strain DHoT was assigned to the family Rhodanobacteraceae with Dyella and Frateuria as its closest relatives. The closest 16S rRNA gene sequences were Frateuria aurantia DSM 6220T (98.2 %), Dyella thiooxydans ATSB10T (98 %), Dyella terrae JS14-6T (97.8 %) and Dyella marensis CS5-B2T (97.8 %). Strain DHoT is a Gram-negative, aerobic, motile, yellow-pigmented, rod-shaped bacterium. Strain DHoT cells grew well at 28-30 °C and at pH 6.5-7.5 on a nutrient agar plate. DNA-DNA hybridization showed that the relatedness between strain DHoT and D. jiangningensis strain SBZ3-12T, and F. aurantia DSM 6220T was 42.7 and 42.6 %, respectively. Ubiquinone Q-8 was the predominant respiratory quinone, and the major fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. In silico analysis based on phylogenetics and sequence identity at the nucleotide and protein levels suggests that Frateuria is the closest known relative of strain DHoT. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain DHoT was designated as a novel species of the genus Frateuria, for which the name Frateuria defendens sp. nov. is proposed. The type strain is DHoT (=NCCB 100648T; =DLBT=DSM 106169T).

Introduction of a putative biocontrol agent into a range of phytoplasma- and liberibacter-susceptible crop plants.

BACKGROUND: Phytoplasma, the causative agent of Bois Noir disease of grapevines, are vectored by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). A Dyella-like bacterium (DLB) isolated from H. obsoletus inhibits the growth of Spiroplasma melliferum, a cultivable relative of phytoplasma. Additional evidence suggests that DLB can reduce the symptoms of yellows disease in grapevine plantlets. The aim of this study was to test whether DLB could colonize a range of phytoplasma- and liberibacter-sensitive crop plants, and thus assess its potential agricultural use. RESULTS: Vitex agnus-castus, the preferred host plant of H. obsoletus was found to be a natural host of DLB, which was successfully introduced into a range of crop plants belonging to seven families. The most effective DLB application method was foliar spraying. Microscopy observation revealed that DLB aggregated on the leaf surface and around the stomata, suggesting that this is its route of entry. DLB was also present in the vascular tissues of plants, indicating that it moved systemically through the plant. CONCLUSIONS: DLB is a potential biocontrol agent and its broad spectrum of host plants indicates the possibility of its future use against a range of diseases caused by phloem-limited bacteria. © 2017 Society of Chemical Industry.

Dyella-Like Bacterium Isolated from an Insect as a Potential Biocontrol Agent Against Grapevine Yellows.

Yellows diseases, caused by phytopathogenic bacteria of the genus Phytoplasma, are a major threat to grapevines worldwide. Because conventional applications against this pathogen are inefficient and disease management is highly challenging, the use of beneficial bacteria has been suggested as a biocontrol solution. A Dyella-like bacterium (DLB), isolated from the Israeli insect vector of grapevine yellows (Hyalesthes obsoletus), was suggested to be an endophyte. To test this hypothesis, the bacterium was introduced by spraying the plant leaves, and it had no apparent phytotoxicity to grapevine. Fluorescent in situ hybridization analysis showed that DLB is colonizing grapevine phloem. Because phytoplasmas inhabit the same niche, DLB interactions with this phytopathogen were examined. When the isolate was introduced to phytoplasma-infected Chardonnay plantlets, morphological disease symptoms were markedly reduced. The mode of DLB action was then tested using bioinformatics and system biology tools. DLB genome analysis suggested that the ability to reduce phytoplasma symptoms is related to inhibition of the pathogenic bacterium. These results provide the first step in examining the potential of DLB as a biological control agent against phytoplasmas in grapevine and, possibly, other agricultural crops.

Bacterial associates of Hyalesthes obsoletus (Hemiptera: Cixiidae), the insect vector of bois noir disease, with a focus on cultivable bacteria.

The planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae) is an important vector of phytoplasma diseases in grapevine. In the current study, the bacterial community compositions of symbionts of this insect were examined. Two dominant bacterial lineages were identified by mass sequencing: the obligate symbiont Candidatus Sulcia, and a facultative symbiont that is closely related to Pectobacterium sp. and to BEV, a cultivable symbiont of another phytoplasma vector, the leafhopper Euscelidius variegatus. In addition, one bacterium was successfully isolated in this study - a member of the family Xanthomonadaceae that is most closely related to the genus Dyella. This Dyella-like bacterium (DLB) was detected by FISH analysis in H. obsoletus guts but not ovaries, and its prevalence in H. obsoletus increased during the fall, suggesting that it was acquired by the host through feeding. We found that DLB inhibits Spiroplasma melliferum, a cultivable relative of phytoplasma, suggesting that it is a potential candidate for biological control against phytoplasma in grapevines.

Draft Genome Sequence of a Dyella-Like Bacterium from the Planthopper Hyalesthes obsoletus.

We report here the draft genome sequence of a Dyella-like bacterium (DLB) isolated from Hyalesthes obsoletus, the insect vector of the uncultivable mollicute bacterium "Candidatus Phytoplasma." This isolate inhibits Spiroplasma melliferum, a cultivable mollicute. The draft genome of DLB consists of 4,196,214 bp, with a 68.6% G+C content, and 3,757 genes were predicted.