Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Control of the Verticillium Wilt on Tomato Plants by Means of Olive Leaf Extracts Loaded on Chitosan Nanoparticles

In this research, a new ecofriendly and sustainable fungicide agent, with the ability to control Verticillium wilt, was developed. To this purpose, a green extract of olive leaf (OLE) was prepared by ultrasound-assisted extraction (UAE) and characterized in terms of polyphenol content and antioxidant activity. Then, OLE was loaded in chitosan nanoparticles (CTNPs) to combine the antifungal activity of CTNPs and phenolic compounds to obtain an important synergic effect. Nanoparticles were synthetized using the ionic gelation technique and characterized in terms of sizes, polydispersity index, Z-potential, encapsulation efficiency, and release profile. Qualitative and quantitative analyses of OLE were performed by the HPLC method. OLE-loaded CTNPs exhibited good physicochemical properties, such as a small size and positive surface charge that significantly contributed to a high antifungal efficacy against Verticillum dahliae. Therefore, their antifungal activity was evaluated in vitro, using the minimal inhibition concentration (MIC) assay in a concentration range between 0.071 and 1.41 mg/mL. Free OLE, blank CTNPs, and OLE-loaded CTNPs possessed MIC values of 0.35, 0.71, and 0.14 mg/mL, respectively. These results suggest an important synergic effect when OLE was loaded in CTNPs. Thereafter, we tested the two higher concentrations on tomato plants inoculated with V. dahliae, where no fungal growth was observed in the in vitro experiment, 0.71 and 1.41 mg/mL. Interestingly, OLE-loaded CTNPs at the higher concentration used, diminished the symptoms of Verticillium wilt in tomato plants inoculated with V. dahliae and significantly enhanced plant growth. This research offers promising results and opens the possibility to use OLE-loaded CTNPs as safe fungicides in the control strategies of Verticillium wilt at open field.

Dynamic characteristics and Functional Analysis Provide new insights into the role of GauERF105 in resistance against Verticillium wilt in Cotton

Verticillium wilt is the most devastating disease of cotton and it results in huge yield losses every year in the fields. The underlying mechanisms of VW in cotton are not well explored yet. In the current approach we used the transcriptome data from G. australe in response to Verticillium wilt attack to mine the ERF TFs and prove their potential role in resistance against VW attack in cotton. We identified 23 ERFs in total, and on the basis of expression at different time points i.e., 24h, 48h and 72h post inoculation and selected GauERF105 for further validation. We performed VIGS in cotton and over expression in Arabidopsis respectively. Moreover, DAB and trypan staining also suggests that the impact of disease was more in the wildtype as compared to transgene lines. On the basis of our results, we confirmed that GauERF105 is the key candidate and playing a key role for defending cotton against VW attack. Current finding might be helpful for generating resistance germplasm in cotton and it will be beneficial to recover the yield losses in field.

Calcium-Dependent Protein Kinase GhCDPK28 Was Dentified and Involved in Verticillium Wilt Resistance in Cotton

Verticillium dahliae is a soil-borne fungus that causes vascular wilt through the roots of plants. Verticillium wilt caused by V. dahliae is one of the main diseases in cotton producing areas of the world, resulting in huge economic losses. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. Calcium-dependent protein kinases (CDPKs) play a pivotal role in plant innate immunity, including regulation of oxidative burst, gene expression as well as hormone signal transduction. However, the function of cotton CDPKs in response to V. dahliae stress remains unexplored. In this study, 96, 44 and 57 CDPKs were identified from Gossypium hirsutum, Gossypium raimondii and Gossypium arboretum, respectively. Phylogenetic analysis showed that these CDPKs could be divided into four branches. All GhCDPKs of the same clade are generally similar in gene structure and conserved domain arrangement. Cis-acting elements related to hormones, stress response, cell cycle and development were predicted in the promoter region. The expression of GhCDPKs could be regulated by various stresses. Gh_D11G188500.1 and Gh_A11G186100.1 was up-regulated under Vd0738 and Vd991 stress. Further phosphoproteomics analysis showed that Gh_A11G186100.1 (named as GhCDPK28-6) was phosphorylated under the stress of V. dahliae. Knockdown of GhCDPK28-6 expression, the content of reactive oxygen species was increased, a series of defense responses were enhanced, and the sensitivity of cotton to V. dahliae was reduced. Moreover, overexpression of GhCDPK28-6 in Arabidopsis thaliana weakened the resistance of plants to this pathogen. Subcellular localization revealed that GhCDPK28-6 was localized in the cell membrane. We also found that GhPBL9 and GhRPL12C may interact with GhCDPK28-6. These results indicate that GhCDPK28-6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

Genome-Wide Identification and Characterization of GhCOMT Gene Family during Fiber Development and Verticillium Wilt Resistance in Cotton

Caffeic acid O-methyltransferases (COMTs) play an essential role in lignin synthesis procession, especially in the plant’s phenylalanine metabolic pathway. The content of COMT genes in cotton and the relationship between their expression patterns have not been studied clearly in cotton. In this study, we have identified 190 COMT genes in cotton, which were classified into three groups (I, II and III), and mapped on the cotton chromosomes. In addition, we found that 135 of the 190 COMT genes result from dispersed duplication (DSD) and whole-genome duplication (WGD), indicating that DSD and WGD were the main forces driving COMT gene expansion. The Ka/Ks analysis showed that GhCOMT43 and GhCOMT41 evolved from GaCOMT27 and GrCOMT14 through positive selection. The results of qRT-PCR showed that GhCOMT13, GhCOMT28, GhCOMT39 and GhCOMT55 were related to lignin content during the cotton fiber development. GhCOMT28, GhCOMT39, GhCOMT55, GhCOMT56 and GhCOMT57 responded to Verticillium Wilt (VW) and maybe related to VW resistance through lignin synthesis. Conclusively, this study found that GhCOMTs were highly expressed in the secondary wall thickening stage and VW. These results provide a clue for studying the functions of GhCOMTs in the development of cotton fiber and VW resistance and could lay a foundation for breeding cotton cultivates with higher quantity and high resistance to VW.

The Respiratory Burst Oxidase Homolog Protein D (GhRbohD) Positively Regulates the Cotton Resistance to Verticillium dahliae

Verticillium wilt, mainly caused by a soil-inhabiting fungus Verticillium dahliae, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to V. dahliae, and found early differentially expressed protein burst oxidase homolog protein D (GhRbohD). However, the role of GhRbohD-mediated ROS in cotton defense against V. dahliae needs further investigation. In this study, we analyzed the function of GhRbohD-mediated resistance of cotton against V. dahliae in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of GhRbohD was significantly upregulated after V. dahliae inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H2O2 and Ca2+ contents of GhRbohD-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of GhRbohD-silenced cotton’s defense against V. dahliae. The expression levels of resistance-related genes were downregulated in GhRbohD-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of GhRbohD enhanced resistance of transgenic Arabidopsis to V. dahliae challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that GhRbohD activates ROS production to positively regulate the resistance of plants against V. dahliae.

Effect of Olive Mill Wastewater of in the Control of Olive Verticillium Wilt Caused by Verticillium dahlia Kleb

Muhrez, K.A., M.Z. Tawil and B. Barhoum. 2021. Effect of Olive Mill Wastewater of in the Control of Olive Verticillium Wilt Caused by Verticillium dahlia Kleb. Arab Journal of Plant Protection, 39(4): 273-280. https://doi.org/10.22268/AJPP-39.4.273280 This research aimed to evaluate the application of olive mill wastewater in controlling Verticillium dahliae the causal agent of olive wilt disease on olive trees. This study was carried out on 2 years old olive local variety (Khderi) plants planted in pots after it has been artificial inoculated with V. dahliae, and severity was recorded within 90 days. Olive mill wastewater (omww) efficacy was estimated at the end of the experiment. Severity was increased from 8.89% before treatment to 28.89% 60 days after treatment, and decreased to 13.33% 90 days after treatment with omww 450 ml/l. Disease severity increased from 11.11% before treatment to 37.78% 60 days after treatment and decreased to 35.56% 90 days after treatment with omww 300 ml/l. Whereas, disease severity increased from 17.78% before treatment to 40% 90 days after treatment with azoxystrobin compared to the inoculated control where disease severity increased from 13.33% before treatment to 100% 90 days after treatment. Omww 450 ml/l had the highest efficacy of 80% followed by azoxystrobin 70%, and omww 300 ml/l 57.33%. It can be concluded that olive mill wastewater has a potential application as a bio-fungicide for the control of olive verticillium wilt disease. Keywords: Olive mill waste waters, Verticillium dahlia, olive, Fungicide.