Identification of a Chitooligosaccharide Mechanism against Bacterial Leaf Blight on Rice by In Vitro and In Silico Studies.

This study focuses on a commercial plant elicitor based on chitooligosaccharides (BIG®), which aids in rice plant growth and disease resistance to bacterial leaf blight (BLB). When the pathogen (Xoo) vigorously attacks rice that has suffered yield losses, it can cause damage in up to 20% of the plant. Furthermore, Xoo is a seed-borne pathogen that can survive in rice seeds for an extended period. In this study, when rice seeds were soaked and sprayed with BIG®, there was a significant increase in shoot and root length, as well as plant biomass. Furthermore, BIG®-treated rice plants showed a significant reduction in BLB severity of more than 33%. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) analysis was used to characterize BIG®'s mechanism in the chemical structure of rice leaves. The SR-FTIR results at 1650, 1735, and 1114 cm-1 indicated changes in biochemical components such as pectins, lignins, proteins, and celluloses. These findings demonstrated that commercial BIG® not only increased rice growth but also induced resistance to BLB. The drug's target enzyme, Xoo 1075 from Xanthomonas oryzae (PDB ID: 5CY8), was analyzed for its interactions with polymer ingredients, specifically chitooligosaccharides, to gain molecular insights down to the atomic level. The results are intriguing, with a strong binding of the chitooligosaccharide polymer with the drug target, revealing 10 hydrogen bonds between the protein and polymer. Overall, the computational analysis supported the experimentally demonstrated strong binding of chitooligosaccharides to the drug target.

Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize.

Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma-plant interactions resulting in induction of defence responses.

Efficacy of Chitosan Nanoparticle Loaded-Salicylic Acid and -Silver on Management of Cassava Leaf Spot Disease.

Leaf spot is one of the most important cassava diseases. Nanotechnology can be applied to control diseases and improve plant growth. This study was performed to prepare chitosan (CS) nanoparticle (NP)-loaded salicylic acid (SA) or silver (Ag) by the ionic gelation method, and to evaluate their effectiveness on reducing leaf spot disease and enhancing the growth of cassava plants. The CS (0.4 or 0.5%) and Pentasodium triphosphate (0.2 or 0.5%) were mixed with SA varying at 0.05, 0.1, or 0.2% or silver nitrate varying at 1, 2, or 3 mM to prepare three formulations of CS-NP-loaded SA named N1, N2, and N3 or CS-NP-loaded Ag named N4, N5, and N6. The results showed that the six formulations were not toxic to cassava leaves up to 800 ppm. The CS-NP-loaded SA (N3) and CS-NP-loaded Ag (N6) were more effective than the remaining formulations in reducing the disease severity and the disease index of leaf spot. Furthermore, N3 at 400 ppm and N6 at 200, 400, and 800 ppm could reduce disease severity (68.9-73.6% or 37.0-37.7%, depending on the time of treatment and the pathogen density) and enhance plant growth more than or equal to commercial fungicide or nano-fungicide products under net-house conditions. The study indicates the potential to use CS-NP-loaded SA or Ag as elicitors to manage cassava leaf spot disease.

Chitosan Nanoparticles-Based Ionic Gelation Method: A Promising Candidate for Plant Disease Management.

By 2050, population growth and climate change will lead to increased demand for food and water. Nanoparticles (NPs), an advanced technology, can be applied to many areas of agriculture, including crop protection and growth enhancement, to build sustainable agricultural production. Ionic gelation method is a synthesis of microparticles or NPs, based on an electrostatic interaction between opposite charge types that contains at least one polymer under mechanical stirring conditions. NPs, which are commonly based on chitosan (CS), have been applied to many agricultural fields, including nanopesticides, nanofertilizers, and nanoherbicides. The CS-NP or CS-NPs-loaded active ingredients (Cu, saponin, harpin, Zn, hexaconazole, salicylic acid (SA), NPK, thiamine, silicon, and silver (Ag)) are effective in controlling plant diseases and enhancing plant growth, depending on the concentration and application method by direct and indirect mechanisms, and have attracted much attention in the last five years. Many crops have been evaluated in in vivo or in greenhouse conditions but only maize (CS-NP-loaded Cu, Zn, SA, and silicon) and soybean (CS-NP-loaded Cu) were tested for manage post flowering stalk rot, Curvularia leaf spot, and bacterial pustule disease in field condition. Since 2019, five of eight studies have been performed in field conditions that have shown interest in CS-NPs synthesized by the ionic gelation method. In this review, we summarized the current state of research and provided a forward-looking view of the use of CS-NPs in plant disease management.

Resistance Induction by Salicylic Acid Formulation in Cassava Plant against Fusarium solani.

Fusarium root rot caused by the soil-borne fungus Fusarium solani is one of the most important fungal diseases of cassava in Thailand, resulting in high yield losses of more than 80%. This study aimed to investigate if the exogenous application of salicylic acid formulations (Zacha) can induce resistance in cassava against Fusarium root rot and observe the biochemical changes in induced cassava leaf tissues through synchrotron radiation based on Fourier-transform infrared (SR-FTIR) microspectroscopy. We demonstrated that the application of Zacha11 prototype formulations could induce resistance against Fusarium root rot in cassava. The in vitro experimental results showed that Zacha11 prototype formulations inhibited the growth of F. solani at approximately 34.83%. Furthermore, a significant reduction in the disease severity of Fusarium root rot disease at 60 days after challenge inoculation was observed in cassava plants treated with Zacha11 at a concentration of 500 ppm (9.0%). Population densities of F. solani were determined at 7 days after inoculation. Treatment of the Zacha11 at a concentration of 500 ppm resulted in reduced populations compared with the distilled water control and differences among treatment means at each assay date. Moreover, the SR-FTIR spectral changes of Zacha11-treated epidermal tissues of leaves had higher integral areas of lipids, lignins, and pectins (1,770-1,700/cm), amide I (1,700-1,600/cm), amide II (1,600-1,500/cm), hemicellulose, lignin (1,300-1,200/cm), and cellulose (1,155/cm). Therefore, alteration in defensive carbohydrates, lipids, and proteins contributed to generate barriers against Fusarium invasion in cassava roots, leading to lower the root rot disease severity.

Plant systemic acquired resistance compound salicylic acid as a potent inhibitor against SCF (SKP1-CUL1-F-box protein) mediated complex in Fusarium oxysporum by homology modeling and molecular dynamics simulations.

Fusarium oxysporum causes significant economic losses in many crop plants by causing root rot, necrosis, and wilting symptoms. Homology and molecular dynamics studies are promising tools for the detection in F. oxysporum of the systemic resistance compound, salicylic acid, for control of the SKP1-CUL1-F-box protein complex. The structure of SKP1-CUL1-F-box subunit Skp1 from F. oxysporum is produced by Modeler 9v7 for the conduct of docking studies. The Skp1 structure is based on the yeast Cdc4/Skp1 (PDB ID: 3MKS A) crystal structure collected by the Protein data bank. Applying molecular dynamic model simulation methods to the final predicted structure and further evaluated by 3D and PROCHECK test programmers, the final model is verified to be accurate. Applying GOLD 3.0.1, SCF Complex Skp1 is used to prevent stress-tolerant operation. The SKP1-CUL1-F-box model is predicted to be stabilized and tested as a stable docking structure. The predicted model of the SCF structure has been stabilized and confirmed to be a reliable structure for docking studies. The results indicated that GLN8, LYS9, VAL10, TRP11, GLU48, ASN49 in SCF complex are important determinant residues in binding as they have strong hydrogen bonding with salicylic acid, which showed best docking results with SKP1-CUL1-F-box complex subunit Skp1 with docking score 25.25KJ/mol. Insilco studies have been used to determine the mode of action of salicylic acid for Fusarium control. Salicylic acid hinders the SKP1-CUL1-F-box complex, which is important in protein-like interactions through hydrogen bodings. Results from docking studies have shown that the best energy for SKP1-CUL1-F-box was salicylic acid.Communicated by Ramaswamy H. Sarma.

Investigation of bioactive compounds from Bacillus sp. against protein homologs CDC42 of Colletotrichum gloeosporioides causing anthracnose disease in cassava by using molecular docking and dynamics studies.

Manihot esculenta, commonly called cassava, is an economically valuable crop and important staple food, grown in tropical and subtropical regions of the world. Demand for cassava in the food and fuel industry is growing worldwide. However, anthracnose disease caused by Colletotrichum gloeosporioides severely affects cassava yield and production. The bioactive molecules from Bacillus are widely used to control fungal diseases in several plants. Therefore, in this study, bioactive compounds (erucamide, behenic acid, palmitic acid, phenylacetic acid, and ß-sitosterol) from Bacillus megaterium were assessed against CDC42, a key protein for virulence, from C. gloeosporioides. Structure of the CDC42 protein was generated through the comparative homology modeling method. The binding site of the ligands and the stability of the complex were analyzed through docking and molecular dynamics simulation studies, respectively. Furthermore, a protein interaction network was envisaged through the STRING database, followed by enrichment analysis in the WebGestalt tool. From the enrichment analysis, it is apparent that bioactive from B. megaterium chiefly targets the MAP kinase pathway that is essential for filamentous growth and virulence. Further exploration through experimental studies could be advantageous for cassava improvement as well as to combat against C. gloeosporioides pathogen.

Reproduction number and sensitivity analysis of cassava mosaic disease spread for policy design.

We develop a mathematical model for the dynamics of Cassava Mosaic Disease (CMD), which is driven by both planting of infected cuttings and whitefly transmission. We use the model to analyze the dynamics of a CMD outbreak and to identify the most cost-effective policy for controlling it. The model uses the reproduction number $ \mathscr{R}_0 $ as a threshold, calculated using the Next-Generation Method. A locally-asymptotically-stable disease-free equilibrium is established when $ \mathscr{R}_0 < 1 $, proved by the Routh-Hurwitz criterion. The globally-asymptotically-stable disease-free and endemic-equilibrium points are obtained using Lyapunov's method and LaSalle's invariance principle. Our results indicate that the disease-free equilibrium point is globally-asymptotically-stable when $ \mathscr{R}_0 \leq 1 $, while the endemic-equilibrium point is globally-asymptotically-stable when $ \mathscr{R}_0 > 1 $. Our sensitivity analysis shows that $ \mathscr{R}_0 $ is most sensitive to the density of whitefly. Numerical simulations confirmed the effectiveness of whitefly control for limiting an outbreak while minimizing costs.

Effect of Salicylic AcidFormulations on Induced Plant Defense against Cassava Anthracnose Disease.

This study was to investigate defense mechanisms on cassava induced by salicylic acid formulation (SA) against anthracnose disease. Our results indicated that the SA could reduce anthracnose severity in cassava plants up to 33.3% under the greenhouse condition. The ß-1,3-glucanase and chitinase enzyme activities were significantly increased at 24 hours after inoculation (HAI) and decrease at 48 HAI after Colletotrichum gloeosporioides challenge inoculation, respectively, for cassava treated with SA formulation. Synchrotron radiation-based Fourier-transform infrared microspectroscopy spectra revealed changes of the C=H stretching vibration (3,000-2,800 cm-1), pectin (1,740-1,700 cm-1), amide I protein (1,700-1,600 cm-1), amide II protein (1,600-1,500 cm-1), lignin (1,515 cm-1) as well as mainly C-O-C of polysaccharides (1,300-1,100 cm-1) in the leaf epidermal and mesophyll tissues treated with SA formulations, compared to those treated with fungicide carbendazim and distilled water after the challenged inoculation with C. gloeosporioides. The results indicate that biochemical changes in cassava leaf treated with SA played an important role in the enhancement of structural and chemical defense mechanisms leading to reduced anthracnose severity.

Identification of Salicylic Acid Mechanism against Leaf Blight Disease in Oryza sativa by SR-FTIR Microspectroscopic and Docking Studies.

The present study was to investigate the application and mechanism of salicylic acid (SA) as SA-Ricemate for the control of leaf blight disease using a Synchrotron Radiation-based Fourier-Transform Infra-Red (SR-FTIR) microspectroscopy and docking studies. After treating rice plants cv. KDML 105 with SA-Ricemate, the leaves were inoculated with Xanthomonas oryzae pv. oryzae, the causal agent of leaf blight, and disease severity were assessed. The leaves were also used to detect changes in endogenous SA content. The results indicated that SA-Ricemate, as an activated compound, reduced disease severity by 60% at three weeks post-inoculation and increased endogenous content by 50%. The SR-FTIR analysis of changes in the mesophyll of leaves (treated and untreated) showed that the groups of lipids, pectins, and proteins amide I and amide II occurred at higher values, and polysaccharides were shown at lower values in treated compared to untreated. Besides, docking studies were used to model a three-dimensional structure for Pathogenesis-related (PR1b) protein and further identify its interaction with SA. The results showed that ASP28, ARG31, LEU32, GLN97, and ALA93 are important residues that have strong hydrogen bonds with SA. The docking results showed that SA has a good interaction, confirming its role in expression.

Expression and purification of biologically active Trichoderma virens proteinaceous elicitor Sm1 in Pichia pastoris.

The beneficial fungus Trichoderma virens secretes a small cysteine-rich protein (Sm1) that induces defense responses in dicot and monocot plants and is a member of the cerato-platanin family. Purification of Sm1 from T. virens results in low protein yield limiting the application of this protein for crop disease protection to small-scale assays. To increase the yield of Sm1, we cloned the sm1 gene in the pPIC9K vector for transformation into the AOX1 locus of Pichia pastoris strain GS115. Transformants of P. pastoris were selected based on the presence of the vector insert as indicated by PCR analysis and the ability to secrete high levels of the rSm1 protein. The optimal incubation period and methanol concentrations for induction were determined for production of rSm1 in shake flasks. One Pichia transformant was estimated to express approximately 55 mg/l of rSm1 after 4 days culture in a 1% final concentration of methanol. The secreted rSm1 was purified by ammonium sulfate precipitation, ion exchange chromatography and gel column chromatography. SDS-PAGE and Western blot analysis revealed that the purified rSm1 expressed in Pichia was recognized by anti-Sm1 polyclonal antibody. The protein sequence was verified by ESI/MS/MS analysis of a tryptic digest of the rSm1. Greater than 90% peptide coverage was obtained and determined to be identical to the predicted sequence. The MALDI/TOF/MS analysis revealed the molecular mass of rSm1 to be 13.1 kDa, which is higher than native Sm1 (12.6 kDa). Edman sequencing of the purified protein revealed an N-terminal extension of six amino acids (EAEAYV). The extension is the result of insufficient activity of the Ste13 protease preventing efficient cleavage of the spacer (EAEA) downstream of the Kex2 cleavage site. Maize (cv. Silver Queen) treated with rSm1 or native Sm1 demonstrated the induction of two defense genes. Enhanced production of this elicitor has implications for the treatment of specialty crops to promote disease resistance.