Bacterial Pustule Disease Of Soybean Research Articles

Cu-Chitosan Nanoparticles Stimulated Defence Responses against Bacterial Pustule Disease in Soybean Crop

Aim: Assessment of Cu-chitosan nanoparticles on controlling Bacterial disease by enhancing the activity of different antioxidant and defence enzymes in soybean.
 Study Design: Randomized block Method
 Place and Duration of Study: Department of Molecular biology and Biotechnology (MBBT), RCA, Maharana Pratap University of Agriculture and Technology, Udaipur Sub humid southern plain region of India between 2017-2019.
 Methodology: We synthesized, characterized and tested the efficacy of Copper-chitosan Nanoparticles against Bacterial Pustule disease in Soybean. Activity of defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)] were estimated after treating the plants with various concentrations of Cu-chitosan nanoparticles.
 Results: Three treatments of Cu-chitosan treatments (0.02, 0.06 and 0.10%) were found most efficient in disease control in both laboratory and net house condition) experiments. Cu-chitosan Nanoparticles application to soybean plants significantly enhanced the activity of defence enzymes (PAL and PPO) and antioxidant enzymes (SOD and POD) from 1.1 to 2.29 folds.
 Conclusion: Cu-chitosan NPs developed in this study were effective in controlling disease by enhancing the activity of different defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)]. This could be an important nonmaterial research in the field of agriculture due to better compatibility of biodegradable Cu-chitosan nanoparticles with biological control. In this study, the effect of Cu-chitosan nanoparticles to boost defence responses against Xanthomonas axonopodis pv. Glycine  were evaluated.

​Cu-Chitosan Nanoparticle Induced Plant Growth and Disease Resistance Efficiency of Soybean [Glycine max (L.)]

Background: Inefficient and excessive use of inorganic fertilizers and pesticides causes environmental risks. Concurrent with the recent increase in agricultural productivity, agricultural systems are now also recognized to be a significant source of environmental damage. Chitosan is a biocompatible, biodegradable and nontoxic polymer with various applications. Methods: In the present investigation, the efficacy of Cu-chitosan nanoparticles (NPs) to plant growth promoting activity and antibacterial activity against bacterial pustule disease of soybean were evaluated. NPs treatments exhibited growth promoting effect in terms of plant height, root length, root weight, nodule number, weight of nodule and number of pods per plant in pot experiments. In field experiment, plant height, root length, root weight, nodule number, weight of nodule and number of pods per plant weight were enhanced in NPs treatments. Result: In control plants (water treated+inoculation) showed average disease severity 66.0%. All plants treated with 0.02 to 0.12% Cu-chitosan NPs showed significant antibacterial activity, express lower disease severity 50.0% to 33.3% and 55.3% to 34.0% in pot and in field condition respectively.

Antibacterial mechanism of Biochanin A and its efficacy for the control of Xanthomonas axonopodis pv. glycines in soybean.

Xanthomonas axonopodis pv. glycines (Xag) is a hazardous pathogen able to cause bacterial pustule disease in soybean, reducing crop yield and quality. Although flavonoids rutin and genistein are known to play an important role in soybean defence, soybean is only able to produce Biochanin A in low concentration. In this work, Biochanin A was found to produce higher antibacterial activity against Xag in comparison with genistein (minimum inhibitory concentration < 100 μg/mL). Biochanin A was able to inhibit DNA synthesis and flagella formation in Xag, and altered the composition of the bacterial membrane. These effects reduced swimming motility, extracellular protease activity and biofilm formation. Further, Biochanin A was tested for the control of Xag in soybean leaves, showing similar, or even higher, inhibitory ability in comparison with some products commonly used for the control of this pathogen. The antibacterial properties of Biochanin A against Xag have been studied for the first time, revealing new insights on the potential applications of this isoflavonoid for the management of bacterial pustule disease. © 2020 Society of Chemical Industry.

A LysR-Type Transcriptional Regulator LcrX Is Involved in Virulence, Biofilm Formation, Swimming Motility, Siderophore Secretion, and Growth in Sugar Sources in Xanthomonas axonopodis Pv. glycines

Xanthomonas axonopodis pv. glycines (Xag) is a Gram-negative bacterium that causes bacterial pustule disease in soybean. To acclimate to new environments, the expression of genes in bacteria is controlled directly or indirectly by diverse transcriptional factors. Among them, LysR type transcriptional regulators are well-characterized and abundant in bacteria. In a previous study, comparative proteomic analysis revealed that LysR type carbohydrate-related transcriptional regulator in Xag (LcrX) was more abundant in XVM2, which is a minimal medium, compared with a rich medium. However, the functions of LcrX in Xag have not been characterized. In this study, we generated an LcrX-overexpressing strain, Xag(LcrX), and the knockout mutant strain, XagΔlcrX(EV), to elucidate the functions of LcrX. Bacterial multiplication of Xag(LcrX) in soybean was significantly impaired, indicating that LcrX is related to virulence. Comparative proteomic analysis revealed that LcrX is mainly involved in carbohydrate metabolism/transport and inorganic ion transport/metabolism. Based on the results of proteomics analysis, diverse phenotypic assays were carried out. A gel electrophoresis mobility shift assay demonstrated that LcrX specifically bound to the putative promoter regions of genes encoding putative fructose 1,6-bisphosphatase and protease. Through a 96-well plate assay under various conditions, we confirmed that the growth of Xag(LcrX) was dramatically affected in the presence of various carbon sources, while the growth of XagΔlcrX(EV) was only slightly changed. Biofilm formation activity was reduced in Xag(LcrX) but enhanced in XagΔlcrX(EV). The production of siderophores was also decreased in Xag(LcrX) but not altered in XagΔlcrX(EV). In contrast, LcrX was not associated with exopolysaccharide production, protease activity, or bacterial motility. These findings provide new insights into the functions of a carbohydrate-related transcriptional regulator in Xag.

Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Draft genome sequence of Xanthomonas axonopodis pv. glycines 8ra possessing transcription activator-like effectors used for genetic engineering

Xanthomonas axonopodis pv. glycines 8ra is a causal agent of bacterial pustule disease in soybean. This bacterium possesses transcription activator-like (TAL) effectors which are useful for genetic/protein engineering applications in higher organisms including plants and humans. Here, we report that the draft genome sequence consists of 5,337,885-bp double-stranded DNA encoding 4674 open reading frames (ORFs) in 13 different contigs. This genome sequence would be useful in applications of TAL effectors in genetic engineering and in elucidating virulence factors against plants.

Metabolic stress-induced programmed cell death in Xanthomonas

Xanthomonas campestris pv. glycines (Xcg), an etiological agent of the bacterial pustule disease of soybean, displayed nutritionally regulated caspase-dependent programmed cell death (PCD). Experiments showed that Xcg was under metabolic stress during PCD, as evident from the intracellular accumulation of NADH and ATP. Further, the accumulation of reactive oxygen species (ROS), as confirmed by 2',7'-dichlorofluorescein diacetate labeling, electron spin resonance spectroscopy, and scopoletin assay, was also observed along with the activation of caspase-3. ROS scavengers such as dimethylsulfoxide, glutathione, n-propyl gallate, and catalase significantly inhibited caspase biosynthesis as well as its activity, eventually leading to the inhibition of PCD. The presence of a sublethal concentration of an electron transport chain uncoupler, 2,4-dinitrophenol, was found to reduce the ROS generation and the increase in the cell survival. These results indicated that Xcg cells grown in a protein-rich medium experienced metabolic stress due to electron leakage from the electron transport chain, leading to the generation of ROS and the expression as well as the activation of caspase-3, and resulting in PCD. A bacterial DNA gyrase inhibitor, nalidixic acid, was also found to inhibit PCD. Gyrase, which regulates DNA superhelicity, and consequently DNA replication and cell multiplication, appears to be involved in the process.

Involvement of caspase-3-like protein in rapid cell death of Xanthomonas.

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the aetiological agent of bacterial pustule disease of soybean, as well as some other strains of Xanthomonas including X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961, exhibited post-exponential rapid cell death (RCD) in Luria-Bertani (LB) medium. RCD was not displayed by Xanthomonas strains while growing in starch medium. Addition of starch to LB culture of XcgAM2 at any point of incubation during the exponential growth was found to arrest the onset of RCD. RCD in this organism was found to be associated with the synthesis of an endogenous enzyme similar to human caspase-3, a known marker of apoptosis in eukaryotes. On sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) the XcgAM2 caspase appeared to run along a 55 kDa protein molecular weight marker. The caspase-3-like protein was detected in all Xanthomonas strains tested. RCD was not detected in Escherichia coli cultures in LB medium. The caspase-3-like enzyme activity or pro-tein was also found to be absent in this bacterium. Caspase-3-like protein or Xanthomonas caspase was detected only in the cells of XcgAM2 growing in LB medium and not in those growing in starch medium. The Xanthomonas caspase protein appeared in cells at around 4 h of incubation, and peaked at around 24 h, before finally disappearing at around 54 h of incubation. However, caspase enzyme activity was detected only 12-13 h after incubation and peaked around 18-20 h. Addition of starch at the beginning or during the period of exponential growth in LB cultures of XcgAM2 terminated the synthesis of this protein. It is presumed that starch acted as the repressor of biosynthesis of the Xanthomonas caspase, thereby preventing the organism from undergoing RCD. The cells undergoing RCD also displayed the other markers of eukaryotic apoptosis. These included binding of annexin V to plasma membrane of cells undergoing RCD and the presence of nicked DNA in culture supernatant as evidenced by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay. Caspase-negative mutants of XcgAM2 did not display post-exponential RCD. The importance of RCD in Xanthomonas life cycle is not yet clear, however the phenomenon appears to have similarities with eukaryotic apoptosis.

Rapid cell death in Xanthomonas campestris pv. glycines.

Xanthomonas campestris pv. glycines strain AM2 (XcgAM2), the etiological agent of bacterial pustule disease of soybean, exhibited post-exponential rapid cell death (RCD) in LB medium. X. campestris pv. malvacearum NCIM 2310 and X. campestris NCIM 2961 also displayed RCD, though less pronouncedly than XcgAM2. RCD was not observed in Pseudomonas syringae pv. glycines, or Escherichia coli DH5alpha. Incubation of the post-exponential LB-grown XcgAM2 cultures at 4 degrees C arrested the RCD. RCD was also inhibited by the addition of starch during the exponential phase of LB-growing XcgAM2. Protease negative mutants of XcgAM2 were found to be devoid of RCD behavior observed in the wild type XcgAM2. While undergoing RCD, the organism was found to transform to spherical membrane bodies. The presence of membrane bodies was confirmed by using a membrane specific fluorescent label, 1,6-diphenyl 1,3,5-hexatriene (DPH), and also by visualizing these structures under microscope. The membrane bodies of XcgAM2 were found to contain DNA, which was devoid of the indigenous plasmids of the organism. The membrane bodies were found to bind annexin V indicative of the externalization of membrane phosphatidyl serine. Nicking of DNA in XcgAM2 cultures undergoing RCD in LB medium was also detected using a TUNEL assay. The RCD in XcgAM2 appeared to have features similar to the programmed cell death in eukaryotes.

An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.

Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean.

Characterization and Plasmid Profile of Xanthomonas campestris pv. Glycines from Maharashtra, India

Abstract Xanthomonas campestris pv. glycines, (Xcg), the causative agent of the bacterial pustule disease of soybean was isolated and characterized. On susceptible soybean the pathogenic isolates displayed characteristic chlorotic lesions around the site of infection within 48 h of inoculation. The pathogenic isolates were found to contain two cryptic plasmids. A smaller plasmid of 1.5 kb and a larger one of size about 25 kb. SDS‐PAGE profile of the soluble proteins of the pathogenic isolatess, howed a different pattern compared to that of the non‐pathogenic isolates.

Bacterial Pustule Disease of Soybean: Microscopy of Pustule Development in a Susceptible Cultivar

Bacterial Pustule Disease of Soybean: Microscopy of Pustule Development in a Susceptible Cultivar

Purification and characterization of Xanthomonas campestris pv. glycines exopolysaccharide

The exopolysaccharides (EPS) of virulent and avirulent strains of Xanthomonas campestris pv. glycines, causal agent of bacterial pustule disease of soybean, and one strain of the soybean non-pathogen X. c. pv. campestris were isolated, purified, and their compositions compared. EPS produced by X. c. pv. glycines in a completely defined medium appears to be identical to the well-characterized EPS produced by X. c. pv. campestris (commonly referred to as xanthan gum). The EPS of all strains was composed of the carbohydrates glucose, mannose and glucuronic acid with acetyl and pyruvyl substituents present. Permethylation analyses indicated EPS preparations had identical hexose substitution patterns. Avirulent strains of X. c. pv. glycines produced as much or more acidic EPS as did virulent strains in vitro. None of the EPS preparations were active as elicitors of the soybean pterocarpanoid phytoalexin glyceollin as determined by a soybean cotyledon bioassay.

Accumulation of isoflavonoids and isoflavone glucosides after inoculation of soybean leaves with Xanthomonas campestris pv. glycines and pv. campestris and a study of their role in resistance

Pathogenic and nonpathogenic strains of Xanthomonas campestris pv. glycines (causal agent of bacterial pustule disease of soybean) or a strain of the soybean nonpathogen X. campestris pv. campestris were tested for their ability to grow and induce accumulation of isoflavonoids (glyceollin, coumestrol, daidzein, formononetin and genistein) and isoflavone glucosides (daidzin, genistin and ononin) in leaves of soybean cv. Clark (susceptible to bacterial pustule) and cv. Clark 63 (resistant to bacterial pustule due to the presence of the recessive rpx gene pair). Growth in vivo of nonpathogenic strains of pv. glycines and the strain of pv. campestris was highly restricted in cv. Clark while bacterial growth kinetics of a pathogenic strain of pv. glycines in the susceptible cv. Clark and in the resistant cv. Clark 63 were similar. No or very low levels (<50 μg g −1 fresh weight) of isoflavonoids including glyceollin accumulated by 4 days after inoculation of leaves of cvs Clark or Clark 63 with bacterial strains which showed restricted growth in vivo. In general, higher levels of isoflavone glucosides accumulated, but similar levels of isoflavone glucosides were found after inoculation with bacterial strains which were restricted or not restricted in their growth in vivo. In addition, the isoflavone glucosides were not inhibitory towards the xanthomonads in in vitro bioassays. Additional compounds of unknown identity with antibacterial activity also accumulated in certain interactions, but these compounds did not appear to be involved in resistance either. Resistance to pathogenic strains of pv. glycines conditioned by the rpx gene pair in cv. Clark 63 appears unusual in that bacterial growth is not restricted, but rather symptomology is reduced with a lowering of the number of pustular lesions accompanied by aborted growth and early necrosis of the pustules

A New Bacterial Agglutinin from Soybean

A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca(2+), Mn(2+) and Mg(2+) did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants.