The Ethylene Biosynthesis Genes ACS2 and ACS6 Modulate Disease Severity of Verticillium dahliae.

Verticillium dahliae is one of the most destructive soilborne plant pathogens since it has a broad host range and there is no chemical disease management. Therefore, there is a need to unravel the molecular interaction between the pathogen and the host plant. For this purpose, we examined the role of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs) of Arabidopsis thaliana upon V. dahliae infection. We observed that the acs2, acs6, and acs2/6 plants are partially resistant to V. dahliae, since the disease severity of the acs mutants was lower than the wild type (wt) Col-0 plants. Quantitative polymerase chain reaction analysis revealed that acs2, acs6, and acs2/6 plants had lower endophytic levels of V. dahliae than the wt. Therefore, the observed reduction of the disease severity in the acs mutants is rather associated with resistance than tolerance. It was also shown that ACS2 and ACS6 were upregulated upon V. dahliae infection in the root and the above ground tissues of the wt plants. Furthermore, the addition of 1-aminocyclopropane-1-carboxylic acid (ACC) and aminooxyacetic acid (AOA), the competitive inhibitor of ACS, in wt A. thaliana, before or after V. dahliae inoculation, revealed that both substances decreased Verticillium wilt symptoms compared to controls irrespectively of the application time. Therefore, our results suggest that the mechanism underpinning the partial resistance of acs2 and acs6 seem to be ethylene depended rather than ACC related, since the application of ACC in the wt led to decreased disease severity compared to control.

Rhizosphere-enriched microbes as a pool to design synthetic communities for reproducible beneficial outputs.

Composts represent a sustainable way to suppress diseases and improve plant growth. Identification of compost-derived microbial communities enriched in the rhizosphere of plants and characterization of their traits, could facilitate the design of microbial synthetic communities (SynComs) that upon soil inoculation could yield consistent beneficial effects towards plants. Here, we characterized a collection of compost-derived bacteria, previously isolated from tomato rhizosphere, for in vitro antifungal activity against soil-borne fungal pathogens and for their potential to change growth parameters in Arabidopsis. We further assessed root-competitive traits in the dominant rhizospheric genus Bacillus. Certain isolated rhizobacteria displayed antifungal activity against the tested pathogens and affected the growth of Arabidopsis, and the Bacilli members possessed several enzymatic activities. Subsequently, we designed two SynComs with different composition and tested their effect on Arabidopsis and tomato growth and health. SynCom1, consisting of different bacterial genera, displayed negative effect on Arabidopsis in vitro, but promoted tomato growth in pots. SynCom2, consisting of Bacilli, didn't affect Arabidopsis growth, enhanced tomato growth and suppressed Fusarium wilt symptoms. Overall, we found selection of compost-derived microbes with beneficial properties in the rhizosphere of tomato plants, and observed that application of SynComs on poor substrates can yield reproducible plant phenotypes.

Disruption and Overexpression of the Gene Encoding ACC (1-Aminocyclopropane-1-Carboxylic Acid) Deaminase in Soil-Borne Fungal Pathogen Verticillium dahliae Revealed the Role of ACC as a Potential Regulator of Virulence and Plant Defense.

It has been suggested that some microorganisms, including plant growth-promoting rhizobacteria, manipulate the level of ethylene in plants by degrading 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, into α-ketobutyrate and ammonia, using ACC deaminase (ACCd). Here, we investigated whether ACCd of Verticillium dahliae, a soil-borne fungal pathogen of many important crops, is involved in causing vascular wilt disease. Overexpression of the V. dahliae gene encoding this enzyme, labeled as ACCd, significantly increased virulence in both tomato and eggplant, while disruption of ACCd reduced virulence. Both types of mutant produced more ethylene than a wild-type (70V-WT) strain, although they significantly differed in ACC content. Overexpression strains lowered ACC levels in the roots of infected plants, while the amount of ACC in the roots of plants infected with deletion mutants increased. To test the hypothesis that ACC acts as a signal for controlling defense, roots of WT and Never-ripe (Nr) tomato plants were treated with ACC before V. dahliae inoculation. Plants pretreated with ACC displayed less severe symptoms than untreated controls. Collectively, our results suggest a novel role of ACC as a regulator of both plant defense and pathogen virulence.

Rhizosphere Microbiome Recruited from a Suppressive Compost Improves Plant Fitness and Increases Protection against Vascular Wilt Pathogens of Tomato.

Suppressive composts represent a sustainable approach to combat soilborne plant pathogens and an alternative to the ineffective chemical fungicides used against those. Nevertheless, suppressiveness to plant pathogens and reliability of composts are often inconsistent with unpredictable effects. While suppressiveness is usually attributed to the compost's microorganisms, the mechanisms governing microbial recruitment by the roots and the composition of selected microbial communities are not fully elucidated. Herein, the purpose of the study was to evaluate the impact of a compost on tomato plant growth and its suppressiveness against Fusarium oxysporum f. sp. lycopersici (Foxl) and Verticillium dahliae (Vd). First, growth parameters of tomato plants grown in sterile peat-based substrates including 20 and 30% sterile compost (80P/20C-ST and 70P/30C-ST) or non-sterile compost (80P/20C and 70P/30C) were evaluated in a growth room experiment. Plant height, total leaf surface, and fresh and dry weight of plants grown in the non-sterile compost mixes were increased compared to the plants grown in the sterile compost substrates, indicating the plant growth promoting activity of the compost's microorganisms. Subsequently, compost's suppressiveness against Foxl and Vd was evaluated with pathogenicity experiments on tomato plants grown in 70P/30C-ST and 70P/30C substrates. Disease intensity was significantly less in plants grown in the non-sterile compost than in those grown in the sterile compost substrate; AUDPC was 2.3- and 1.4-fold less for Foxl and Vd, respectively. Moreover, fungal quantification in planta demonstrated reduced colonization in plants grown in the non-sterile mixture. To further investigate these findings, we characterized the culturable microbiome attracted by the roots compared to the unplanted compost. Bacteria and fungi isolated from unplanted compost and the rhizosphere of plants were sequence-identified. Community-level analysis revealed differential microbial communities between the compost and the rhizosphere, suggesting a clear effect of the plant in the microbiome assembly. Proteobacteria and Actinobacteria were highly enriched in the rhizosphere whereas Firmicutes were strongly represented in both compartments with Bacillus being the most abundant species. Our results shed light on the composition of a microbial consortium that could protect plants against the wilt pathogens of tomato and improve plant overall health.

Biodiversity and ochratoxin A profile of Aspergillus section Nigri populations isolated from wine grapes in Cyprus vineyards.

The objective of this study was to evaluate the biodiversity of Aspergillus section Nigri populations from Cyprus vineyards by morphological, toxigenic and phylogenetic analysis. Aspergillus section Nigri populations were isolated from grapes of the varieties 'Maratheftiko' and 'Cabernet Sauvignon' originating from six growing regions of Cyprus during 2010 and 2011 years. The isolation frequency of Aspergillus section Nigri from grape samples was 43.3% and a total of 284 isolates were selected for further analyses based on the macroscopic characteristics of black aspergilli. The isolates were characterized by sequencing analysis of the calmodulin gene in order to identify species responsible for ochratoxin A (OTA) production. The phylogenetic analysis showed that the isolates were grouped in three major clusters. The A. tubingensis cluster included 262 isolates (92.25%), the A. niger cluster included 15 isolates identified as A. niger (5.3%) and 6 isolates identified as A. welwitschiae (2.1%). One isolate was classified as A. carbonarius (0.35%) and was grouped in a cluster together with the reference isolates of A. carbonarius, A. sclerotioniger, A. sclerotiocarbonarius and A. ibericus. All the isolates were evaluated for their ochratoxigenic ability by HPLC coupled with a fluorescence detector (HPLC-FLD) and the positive isolates were re-examined using ultra high-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The Aspergillus carbonarius isolate produced an average quantity of 1436.1 ng OTA/g Czapek Yeast Agar (CYA); From the A. niger strains three isolates (20%) produced OTA and only one isolate from A. welwitschiae (16.7%) was proved ochratoxigenic with toxin production average at 23.9 ng/g and 9.1 ng/g CYA respectively. Grape must samples derived from the collected berries were also analyzed for OTA and none of the samples were found contaminated with the mycotoxin. The results showed that the geographic area and the meteorological conditions had no significant effect on the incidence and the distribution of black aspergilli in this 2-year project. However, absence of rainfall and low humidity during the harvesting period were critical for the low incidence of the ochratoxigenic A. carbonarius on grapes.