Host-Driven Selection, Revealed by Comparative Analysis of Xanthomonas Type III Secretion Effectoromes, Unveils Novel Recognized Effectors.

Xanthomonas species are specialized plant pathogens, often exhibiting a narrow host range. They rely on the translocation of effector proteins through the type III secretion system to colonize their respective hosts. The effector arsenal varies among Xanthomonas spp., typically displaying species-specific compositions. This species-specific effector composition, collectively termed the effectorome, is thought to influence host specialization. We determined the plant host-derived effectoromes of more than 300 deposited genomes of Xanthomonas species associated with either Solanaceae or Brassicaceae hosts. Comparative analyses revealed clear species-specific effectorome signatures. However, Solanaceae or Brassicaceae host-associated effectorome signatures were not detected. Nevertheless, host biases in the presence or absence of specific effector classes were observed. To assess whether host-associated effector absence results from selective pressures, we introduced effectors unique to Solanaceae pathogens to Xanthomonas campestris pv. campestris (Xcc), and effectors unique to Brassicaceae pathogens to Xanthomonas euvesicatoria pv. euvesicatoria (Xeue), and evaluated if these introductions hindered virulence on their respective hosts. Introducing the effector XopI into Xcc reduced virulence on white cabbage leaves without affecting localized or systemic colonization. Introducing the XopAC or XopJ5 effectors into Xeue reduced virulence and colonization on tomato but not on pepper. Additionally, XopAC and XopJ5 induced a hypersensitive response on tomato leaves when delivered by Xeue or through Agrobacterium-mediated transient expression, confirming recognition in tomato. This study demonstrates the role of host-derived selection in establishing species-specific effectoromes, identifying XopAC and XopJ5 as recognized effectors in tomato.

Belowground fungal volatiles perception in okra (Abelmoschus esculentus) facilitates plant growth under biotic stress.

Microbial volatile organic compounds (mVOCs) have great potential in plant ecophysiology, yet the role of belowground VOCs in plant stress management remains largely obscure. Analysis of biocontrol producing VOCs into the soil allow detailed insight into their interaction with soil borne pathogens for plant disease management. A root interaction trial was set up to evaluate the effects of VOCs released from Trichoderma viride BHU-V2 on soil-inhabiting fungal pathogen and okra plant growth. VOCs released into soil by T. viride BHU-V2 inhibited the growth of collar rot pathogen, Sclerotium rolfsii. Okra plants responded to VOCs by increasing the root growth (lateral roots) and total biomass content. VOCs exposure increased defense mechanism in okra plants by inducing different enzyme activities i.e. chitinase (0.89 fold), ß-1,3-glucanase (0.42 fold), peroxidase (0.29 fold), polyphenol oxidase (0.33 fold) and phenylalanine lyase (0.7 fold) when inoculated with S. rolfsii. In addition, T. viride BHU-V2 secreted VOCs reduced lipid peroxidation and cell death in okra plants under pathogen inoculated condition. GC/MS analysis of VOCs blend revealed that T. viride BHU-V2 produced more number of antifungal compounds in soil medium as compared to standard medium. Based on the above observations it is concluded that okra plant roots perceive VOCs secreted by T. viride BHU-V2 into soil that involved in induction of plant defense system against S. rolfsii. In an ecological context, the findings reveal that belowground microbial VOCs may play an important role in stress signaling mechanism to interact with plants.

Seed biopriming with antagonistic microbes and ascorbic acid induce resistance in tomato against Fusarium wilt.

Seed biopriming is an emerging technique to enhance seed germination under stress conditions. An integrated approach of tomato seed biopriming with ascorbic acid, Trichoderma asperellum BHU P-1 and Ochrobactrum sp. BHU PB-1 was applied to observe the response against wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici (FOL). Tomato seeds bioprimed with the aforementioned application expressed augmented seed germination and activated of defense response. Seed germination was recorded higher (80 %) at low concentration (1 pM) of ascorbic acid as compared to high concentration of 1 mM (41 %). Combination of both ascorbic acid and antagonistic microbe treatments (T5 & T6) significantly reduced disease incidence (up to 28 %) in tomato plants at 10 days. T5 and T6 treated plants exhibited higher accumulation of total phenol content and increased activity of Phenylammonia lyase (PAL), Peroxidase (PO), Chitinase (Chi) and Polyphenol oxidase (PPO) as compared to control (T1) plants. ROS formation in the form of H2O2 was also found to be reduced in combined treatment. Histochemical analysis revealed that phenylpropanoid pathway (lignin deposition) was more activated in combined priming treatment plants as compared to individual treatment upon challenge inoculation with FOL. Transcript expression analysis of defense genes confirmed the up-regulation of PAL (2.1 fold), Chi (0.92 fold), Pathogenesis related proteins (PR) (1.58 fold) and Lipoxygenase (Lox) (0.72 fold) in T6 treatment as compared to T1 treatment plants at 96 h. This study reveals that ascorbic acid treatment with antagonistic microbes through seed priming effectively induced seed germination and elicited defense mechanism to control wilt disease in tomato plants.

Antibacterial and biofilm inhibition activity of biofabricated silver nanoparticles against Xanthomonas oryzae pv. oryzae causing blight disease of rice instigates disease suppression.

Antimicrobial activity of silver nanoparticles (AgNPs) has been well documented in earlier studies. As their efficient role in combating phytopathogens has begun recently, there is a huge scope to explore their effectiveness in agriculture. Considering the strong antifungal activity of biosynthesized AgNPs (as reported in our previous study), our main aim is to elucidate their antibacterial activity against bacterial plant pathogens to authenticate their wide range of agricultural applications. The present manuscript highlights the potential role of biosynthesized AgNPs against Xanthomonas oryzae pv. oryzae (Xoo) causing disastrous sheath blight disease of rice worldwide. We observed strong antibacterial activity of biosynthesized AgNPs (size ~ 12 nm) against Xoo at 20, 30 and 50 µg/mL concentrations. The significant inhibitory impact of AgNPs on biofilm formation by Xoo was noted even at the lower dose of 5 µg/mL (p = 0.001). Maximum biofilm inhibition (p = 0.000) was caused at 50 µg/mL concentration of AgNPs in comparison to control. Furthermore, disease suppression by biosynthesized AgNPs was authenticated under greenhouse conditions. Foliar spray of AgNPs significantly reduced the blight symptoms in rice sheaths as shown by 9.25% DLA (% Diseased leaf area) as compared to 33.91% DLA in Xoo inoculated rice plants. Altogether, our data suggest that biosynthesized AgNPs based nanoformulation can be applied for successful management of blight disease of rice. In addition, the antibiofilm strategies instigated by AgNPs can be exploited against a wide range of bacterial phytopathogens. In light of rapidly emerging antibiotic-resistant microbial strains, the current work provides an alternate effective platform for the application of nanoformulation for augmenting sustainability in the agriculture.

Modulation in phenolic root exudate profile of Abelmoschus esculentus expressing activation of defense pathway.

Phenolics play a key role in communication between plants and microbes in the rhizosphere. In this study, shikimic, gallic, fumaric, ferulic, vanillic acid and quercetin in root exudates of Abelmoschus esculentus act as chemoattractants of endophytic Alcaligenes faecalis strains, BHU 12, BHU 16 and BHU M7. In vitro chemotaxis assay showed that BHU 12 expressed highest chemotactic movement (CFU ∼50×1012) towards A.esculentus root exudates followed by BHU 16 and BHU M7 (CFU∼ 9×1012), thereby confirming their ability to colonize the host rhizoplane region. However, BHU 16 expressed highest biofilm formation ability followed by BHU 12 and BHU M7. Assessment of chemotactic and biofilm formation potential towards individual phenolic acids revealed BHU 12 to be maximally attracted towards 1µM shikimic acid (2×1015) while BHU 16 towards 1mM vanillic acid (6.5×1012) and BHU M7 towards 1mM ferulic acid (3.5×1012), thereby confirming the phenolic acid components responsible for particularly attracting the endophytic isolates. Upon colonization, the endophytic isolates modified the phenolic profiles of root exudates in planta in a manner so as to plausibly attract more of the beneficial rhizospheric microbiota as well as self-fortification against pathogenic microbes. This hypothesis was verified by monitoring the changes in phenolic components of A. esculentus root exudate owing to S. rolfsii infection, a disastrous soil-borne pathogen. Thus, on the whole, the work provides intricate details of plant-endophyte interactions for biotic stress management through careful manipulation of root exudates, thereby aiding in sustainable agriculture.

Biochemical and histochemical analyses revealing endophytic Alcaligenes faecalis mediated suppression of oxidative stress in Abelmoschus esculentus challenged with Sclerotium rolfsii.

Sclerotium rolfsii is a highly aggressive pathogen that causes huge economic losses, especially in temperate climates. Alcaligenes faecalis, particularly in endophytic form, has rarely been used to control this fungus. In this study, endophytic Alcaligenes sp. strain BHU 12, BHU 16 (isolated from Abelmoschus esculentus leaf) and BHU M7 (isolated from Andrographis paniculata leaf) were reported to trigger a wide range of host defenses in Okra plant against the collar-rot pathogen S. rolfsii. Endophytic colonization of the strains in ten days old plants was assessed through re-isolation of the rif-tagged strains on rifampicin augmented nutrient agar media. The ability of the endophytic strains to induce systemic defense responses in above-ground organs was assessed by collecting leaf tissues of the Okra plants grown under non-gnotobiotic conditions at different time intervals post seedling bacterization with the endophytic biocontrol agents. The pathogen challenged unprimed plants exhibited flaccidity of the stem and leaves at 48 h post infection (hpi) in contrast to the bioprimed and challenged plants. Biochemical and histochemical analyses explained the above phenomenon as activation of phyto-peroxidases leading to an increased metabolism of the reactive oxygen species (ROS), accompanied by activation of the phenylpropanoid network and a subsequent enhancement in plant phenolics. Interestingly, though the maximum increase in the defense pathways was observed in treatments with native endophytes of Okra plant, yet the enhancement in antioxidant pathway due to A. paniculata borne endophytes was also quite significant. Thus, this work clearly demonstrates how Okra plants respond to the "non-hostile" colonization of bacterial endophytes and how induced defense response can contribute to the biocontrol activity of the endophytic strains.