Biological control of bacterial spot disease and plant growth-promoting effects of lactic acid bacteria on pepper

In our previous studies, we observed the biological control effect of lactic acid bacteria strains (LABs) KLF01, KLC02 and KPD03 against different plant pathogenic bacteria in vitro against Ralstonia solanacearum, and strains KLF01 and KLC02 against Pectobacterium carotovorum under greenhouse and field experiments, respectively. In this study, we observed the efficacy of these bacteria against bacterial spot pathogen (Xanthomonas campestris pv. vesicatoria) and their plant growth-promoting activities in pepper (Capsicum annuum L. var. annuum), under greenhouse and field conditions. LABs significantly (P < 0.05) reduced bacterial spot on pepper plants in comparison to untreated plants in both the greenhouse and the field experiments. The plant growth-promoting effect of LABs on pepper varied; some strains had a significant effect on growth promotion (P < 0.05) compared with untreated plants, while some showed no significant effect in the greenhouse and field experiments. Additionally, LABs were able to colonise roots, produce indole-3-acetic acid (IAA), siderophores and solubilise phosphate. These findings indicate that application of LABs could provide a promising alternative for the management of bacterial spot disease in pepper plants and could therefore be used as a healthy plant growth-promoting agent.     

Studies on a Quantitative Analysis of Oryzalides and Oryzalic Acids in Rice Plants by GC-SIM

A procedure for the quantitative analysis of oryzalides and oryzalic acids in leaves of rice cultivars was established by using GC-MS-selective ion monitoring (GC-SIM), 17-deuterated oryzalide B being used as an internal reference. The total contents of four compounds of oryzalides and oryzalic acids in the leaves at three different growth stages of Norin-27, i.e., seedling, tillering, and ripening, showed values of 5.3, 17.9, and 37.9 μg/g, respectively. The contents of oryzalides and oryzalic acids in each organ, i.e., leaf, stem, ear, and root, showed values of 35.0 and 1.7 μg/g for the former two, respectively, and were not detectable for the latter two. A preliminary analysis of Norin-27 and Rantai emas leaves inoculated with an incompatible and compatible strain of Xanthomonas campestris pv. oryzae showed a greater accumulation of oryzalides and oryzalic acids, especially in the lesion area, than the figures for healthy leaves. Mechanical injury by a needle-bundle also increased the total amounts.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Age as a factor in induction of systemic acquired resistance in tomato against bacterial speck by aqueous fruit extracts of Azadirachta indica

In the present study, the effect of aqueous fruit extracts of Azadirachta indica on activity of Peroxidase (POX) at different ages of Tomato (Lycopersicon esculentum) leading to induction of systemic resistance against Pseudomonas syringae pv. tomato was evaluated. For this evaluation, four ages, that is, 6, 8, 10 and 12?weeks of plants were selected. A single leaf at the third node from base of each plant was treated either singly or with different combinations of Neem extract and pathogen. Samples were collected at an interval of 24?h for up to five days and after two weeks of the treatment from both treated and untreated nodes. The change in the activity of defence enzyme POX and expression of its isoforms was studied. The results demonstrate that systemic acquired resistance induced by the Neem fruit extract increases as the plant matures but it is not only the limiting factor.     

Stage-specific induction of systemic acquired resistance by fruit extracts of Azadirachta indica

In the present study, the efficacy of aqueous fruit extract of Azadirachta indica A. Juss. (Neem) on induction of systemic acquired resistance (SAR) in tomato against Pseudomonas syringae pv. tomato through enhancement in the activity of polyphenol oxidase (PPO) at different ages was studied. Plants at 6, 8, 10 and 12?weeks of age were selected. A single leaf at the third node from the base of each plant was treated either singly or with different combinations of the pathogen and Neem fruit extract. Samples were collected from the non-treated leaves at an interval of 24?h up to five?days for enzyme assay and after two?weeks for disease development from both treated and untreated plants. The results demonstrate that the neem fruit extract could induce additional PPO isoforms both locally as well as systemically. The PPO activity was observed to be elevated in both the treated and non-treated leaves leading to induction of SAR. The induction of SAR enhances with the increase in the age of the plant.     

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.     

Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.