Novel Pyrazole-4-Carboxamide Derivatives Containing Oxime Ether Group as Potential SDHIs to Control Rhizoctonia solani.

In the search for novel succinate dehydrogenase inhibitor (SDHI) fungicides to control Rhizoctonia solani, thirty-five novel pyrazole-4-carboxamides bearing either an oxime ether or an oxime ester group were designed and prepared based on the strategy of molecular hybridization, and their antifungal activities against five plant pathogenic fungi were also investigated. The results indicated that the majority of the compounds containing oxime ether demonstrated outstanding in vitro antifungal activity against R. solani, and some compounds also displayed pronounced antifungal activities against Sclerotinia sclerotiorum and Botrytis cinerea. Particularly, compound 5e exhibited the most promising antifungal activity against R. solani with an EC50 value of 0.039 µg/mL, which was about 20-fold better than that of boscalid (EC50 = 0.799 µg/mL) and 4-fold more potent than fluxapyroxad (EC50 = 0.131 µg/mL). Moreover, the results of the detached leaf assay showed that compound 5e could suppress the growth of R. solani in rice leaves with significant protective efficacies (86.8%) at 100 µg/mL, superior to boscalid (68.1%) and fluxapyroxad (80.6%), indicating promising application prospects. In addition, the succinate dehydrogenase (SDH) enzymatic inhibition assay revealed that compound 5e generated remarkable SDH inhibition (IC50 = 2.04 µM), which was obviously more potent than those of boscalid (IC50 = 7.92 µM) and fluxapyroxad (IC50 = 6.15 µM). Furthermore, SEM analysis showed that compound 5e caused a remarkable disruption to the characteristic structure and morphology of R. solani hyphae, resulting in significant damage. The molecular docking analysis demonstrated that compound 5e could fit into the identical binding pocket of SDH through hydrogen bond interactions as well as fluxapyroxad, indicating that they had a similar antifungal mechanism. The density functional theory and electrostatic potential calculations provided useful information regarding electron distribution and electron transfer, which contributed to understanding the structural features and antifungal mechanism of the lead compound. These findings suggested that compound 5e could be a promising candidate for SDHI fungicides to control R. solani, warranting further investigation.

Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes.

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

Evaluation of Phoma sp. Biomass as an Endophytic Fungus for Synthesis of Extracellular Gold Nanoparticles with Antibacterial and Antifungal Properties.

The aim of our study was to examine the different concentrations of AuNPs as a new antimicrobial substance to control the pathogenic activity. The extracellular synthesis of AuNPs performed by using Phoma sp. as an endophytic fungus. Endophytic fungus was isolated from vascular tissue of peach trees (Prunus persica) from Baft, located in Kerman province, Iran. The UltraViolet-Visible Spectroscopy (UV-Vis spectroscopy) and Fourier transform infrared spectroscopy provided the absorbance peak at 526 nm, while the X-ray diffraction and transmission electron microscopy images released the formation of spherical AuNPs with sizes in the range of 10-100 nm. The findings of inhibition zone test of Au nanoparticles (AuNPs) showed a desirable antifungal and antibacterial activity against phytopathogens including Rhizoctonia solani AG1-IA (AG1-IA has been identified as the dominant anastomosis group) and Xanthomonas oryzae pv. oryzae. The highest inhibition level against sclerotia formation was 93% for AuNPs at a concentration of 80 µg/mL. Application of endophytic fungus biomass for synthesis of AuNPs is relatively inexpensive, single step and environmentally friendly. In vitro study of the antifungal activity of AuNPs at concentrations of 10, 20, 40 and 80 µg/mL was conducted against rice fungal pathogen R. solani to reduce sclerotia formation. The experimental data revealed that the Inhibition rate (RH) for sclerotia formation was (15, 33, 74 and 93%), respectively, for their corresponding AuNPs concentrations (10, 20, 40 and 80 µg/mL). Our findings obviously indicated that the RH strongly depend on AuNPs rates, and enhance upon an increase in AuNPs rates. The application of endophytic fungi biomass for green synthesis is our future goal.

Effects of silicon dioxide, zinc oxide and titanium dioxide nanoparticles on Meloidogyne incognita, Alternaria dauci and Rhizoctonia solani disease complex of carrot.

Foliar spray of silicon dioxide (SiO2 NPs), zinc oxide (ZnO NPs) and titanium dioxide (TiO2 NPs) nanoparticles were used for the management of Meloidogyne incognita, Alternaria dauci and Rhizoctonia solani disease complex of carrot. Foliar spray of SiO2 NPs/ZnO NPs or TiO2 NPs increased plant growth attributes, chlorophyll and carotenoid of carrot. Foliar spray of 0.10 mg ml-1 SiO2 NPs caused the highest increase in plant growth, chlorophyll and carotenoid content of leaves followed by spray of 0.10 mg ml-1 ZnO NPs, 0.05 mg ml-1 SiO2 NPs, 0.05 mg ml-1 ZnO NPs, 0.10 mg ml-1 TiO2 NPs and 0.05 mg ml-1 TiO2 NPs. Use of SiO2 NPs caused a higher reduction in root galling, nematode multiplication and disease indices followed by ZnO NPs and TiO2 NPs. Two principal components analysis showed a total of 97.84% overall data variance in plants inoculated with single pathogen and 97.20% in plants inoculated with two or more pathogens. Therefore, foliar spray of SiO2 NPs appears interesting for the management of disease complex of carrot.

Antifungal Activity and Action Mechanism Study of Coumarins from Cnidium monnieri Fruit and Structurally Related Compounds.

The increasing resistance of plant diseases caused by phytopathogenic fungi highlights the need for highly effective and environmentally benign agents. The antifungal activities of Cnidium monnieri fruit extracts and five isolated compounds as well as structurally related coumarins against five plant pathogenic fungi were evaluated. The acetone extract, which contained the highest amount of five coumarins, showed strongest antifungal activity. Among the coumarin compounds, we found that 4-methoxycoumarin exhibited stronger and broader antifungal activity against five phytopathogenic fungi, and was more potent than osthol. Especially, it could significantly inhibit the growth of Rhizoctonia solani mycelium with an EC50 value of 21 µg mL-1 . Further studies showed that 4-methoxycoumarin affected the structure and function of peroxisomes, inhibited the ß-oxidation of fatty acids, decreased the production of ATP and acetyl coenzyme A, and then accumulated ROS by damaging MMP and the mitochondrial function to cause the cell death of R. solani mycelia. 4-Methoxycoumarin presented antifungal efficacy in a concentration- dependent manner in vivo and could be used to prevent the potato black scurf. This study laid the foundation for the future development of 4-methoxycournamin as an alternative and friendly biofungicide.

Enhanced Fungicidal Efficacy by Co-Delivery of Azoxystrobin and Diniconazole with Cauliflower-Like Metal-Organic Frameworks NH2-Al-MIL-101.

Long-term use of a single fungicide increases the resistance risk and causes adverse effects on natural ecosystems. Controlled release formulations of dual fungicides with different modes of action can afford a new dimension for addressing the current issues. Based on adjustable aperture and superhigh surface area, metal-organic frameworks (MOFs) are ideal candidates as pesticide release carriers. This study used Al3+ as the metal node and 2-aminoterephthalic acid as the organic chain to prepare aluminum-based metal-organic framework material (NH2-Al-MIL-101) with "cauliflower-like" structure and high surface area of 2359.0 m2/g. Fungicides of azoxystrobin (AZOX) and diniconazole (Dini) were simultaneously encapsulated into NH2-Al-MIL-101 with the loading content of 6.71% and 29.72%, respectively. Dual fungicide delivery system of AZOX@Dini@NH2-Al-MIL-101 demonstrated sustained and pH responsive release profiles. When the maximum cumulative release rate of AZOX and Dini both reached about 90%, the release time was 46 and 136 h, respectively. Furthermore, EC50 values as well as the percentage of inhibition revealed that AZOX@Dini@NH2-Al-MIL-101 had enhanced germicidal efficacy against rice sheath blight (Rhizoctonia solani), evidenced by the synergistic ratio of 1.83. The present study demonstrates a potential application prospect in sustainable plant protection through co-delivery fungicides with MOFs as a platform.

Novel Pyridyl-Oxazole Carboxamides: Toxicity Assay Determination in Fungi and Zebrafish Embryos.

Eight novel pyridyl-oxazole carboxamides were evaluated against fungi and displayed good fungicidal activities against Botrytis cinereal and Rhizoctonia solani. Preliminary bioassay results indicated that at 100 mg/L, compounds 6a-6e, 6g and 6h exhibited 100% fungicidal activities against Botrytis cinerea, and the compound 6b to Rhizoctonia solani at 100%. Then, the zebrafish embryo acute toxicity test was performed to assess the toxicity of 6b and 6c. A series of malformations appeared, when the zebrafish embryos were exposed to 6b and 6c, such as delayed yolk sac resorption, significant shortening of body length, pericardial edema, bending spine, lack of melanin, heart hemorrhage, head hemorrhage, delayed swim sac development, yolk malformation and head malformation. In addition, the acute toxicity of 6b to zebrafish embryo is 4.878 mg/L, and 6c is 6.257 mg/L.

Comparative Genome Analysis Reveals Differences in Biocontrol Efficacy According to Each Individual Isolate Belonging to Rhizospheric Fluorescent Pseudomonads.

Biocontrol fluorescent pseudomonads produce a number of antibiotic organic compounds, including 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, and phenazine. We previously classified rhizospheric fluorescent pseudomonads harboring antibiotic biosynthetic gene clusters into 10 operational taxonomic units (OTUs). In the present study, we report the complete genome sequences of selected strains from these OTUs. The genetic diversity of antibiotic biosynthetic gene clusters and their surrounding sequences correlated with the OTU classification. In comparisons of the biocontrol activity and distribution of antibiotic biosynthetic gene clusters, we found that the pyrrolnitrin biosynthetic gene cluster more effectively controlled the growth of Rhizoctonia solani.

The Wall-Associated Receptor-Like Kinase TaWAK7D Is Required for Defense Responses to Rhizoctonia cerealis in Wheat.

Sharp eyespot, caused by necrotrophic fungus Rhizoctonia cerealis, is a serious fungal disease in wheat (Triticum aestivum). Certain wall-associated receptor kinases (WAK) mediate resistance to diseases caused by biotrophic/hemibiotrophic pathogens in several plant species. Yet, none of wheat WAK genes with positive effect on the innate immune responses to R. cerealis has been reported. In this study, we identified a WAK gene TaWAK7D, located on chromosome 7D, and showed its positive regulatory role in the defense response to R. cerealis infection in wheat. RNA-seq and qRT-PCR analyses showed that TaWAK7D transcript abundance was elevated in wheat after R. cerealis inoculation and the induction in the stem was the highest among the tested organs. Additionally, TaWAK7D transcript levels were significantly elevated by pectin and chitin treatments. The knock-down of TaWAK7D transcript impaired resistance to R. cerealis and repressed the expression of five pathogenesis-related genes in wheat. The green fluorescent protein signal distribution assays indicated that TaWAK7D localized on the plasma membrane in wheat protoplasts. Thus, TaWAK7D, which is induced by R. cerealis, pectin and chitin stimuli, positively participates in defense responses to R. cerealis through modulating the expression of several pathogenesis-related genes in wheat.

Survivability and controlled release of alginate-microencapsulated Pseudomonas fluorescens VUPF506 and their effects on biocontrol of Rhizoctonia solani on potato.

Preserving the efficacy of plant probiotic bacteria in soil is a major challenge to the biological control of plant diseases. The microencapsulation technique is an important step in preserving the viability and activity of probiotics in adverse environmental conditions. The main objective of this study was to choose an appropriate coating for probiotic encapsulation. For this purpose, the survivability and controlled release of Pseudomonas fluorescens VUPF506 encapsulated with alginate (Alg) combined with whey protein concentrate (WPC), carboxymethyl cellulose (CMC), and peanut butter (PB) were evaluated. Moreover, the encapsulated cells were evaluated to control for Rhizoctonia solani in potato plants under in vivo conditions. The results showed that all tested wall material maintained more than 80% of the bacterial cells. The Alg-WPC microcapsules provided a better controlled release over two months. Interestingly, the greenhouse experiment also revealed that the treatment of potato plants with Alg-WPC microcapsules was the most effective treatment, suppressing 90% of the pathogen. The results showed that Alg-WPC is the most promising combination to improve the survivability of P. fluorescens VUPF506. Moreover, it can be used as a fertilizer due to its content of valuable amino acids.

Lipopeptide production by Bacillus atrophaeus strain B44 and its biocontrol efficacy against cotton rhizoctoniosis.

OBJECTIVES: An assay was conducted to show the comparisons the effects of nine metal ions on antagonistic metabolites (lipopeptides, siderophores and gibberellins) by Bacillus atrophaeus strain B44 using well-diffusion assays, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, chrome azurol S plus mannitol salt agar (CAS-MSA) tests, and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. This assay is also designed to demonstrate the biocontrol efficacy of B44 against cotton rhizoctoniosis using pot culture tests. RESULTS: Both the lipopeptide yield and the antimicrobial activity of B44 increase with the MnSO4, MgSO4, CaCO3, and CuSO4 treatments and either have no effect or decreased lipopeptide yield and antimicrobial activity with the FeSO4, K2HPO4, KCl, KH2PO4 and ZnSO4 treatments. The medium containing MgSO4 has no significant effect on either the lipopeptide yield or antimicrobial activity. MALDI-TOF-MS analysis shows a broad range of m/z peaks, indicating that strain B44 produces a complex mixture of iturin, surfactin, and fengycin lipopeptides. Gibberellin production by strain B44 varies greatly depending on the culture medium, and the siderophore production is not significantly affected by the culture medium. Pot tests show that lipopeptide production affects the disease control efficacy of strain B44. CONCLUSION: The biocontrol efficacy of B. atrophaeus strain B44 is related to the lipopeptide yield. Moreover, B. atrophaeus strain B44 significantly increases the size of cotton seedlings, which is related to the GA3 concentration.

Characterization of the major antifungal extrolite from rice endophyte Lysinibacillus sphaericus against Rhizoctonia solani.

Sheath blight of rice caused by Rhizoctonia solani is regarded as one of the most widely distributed diseases of rice, and is one of the major production constraints for rice in India and most rice-growing countries of Asia. Biological control of plant diseases using antagonistic bacteria is now considered as a promising alternative to the use of hazardous chemical fungicides or bactericides. Several bacterial endophytes have been reported to support growth and improve the health of the plants and therefore, may be important as biocontrol agents. In the present study, putative antifungal metabolites were extracted from rice foliage endophyte Lysinibacillus sphaericus KJ872548 by solvent extraction methods and purified using HPTLC techniques. Separated bands were subjected to assess the in vitro antagonistic activity toward rice sheath blight pathogen Rhizoctonia solani using a dual culture method. Partially purified active fraction B2 obtained from HPTLC analysis showed the highest percentage of inhibition (76.9%). GC MS and FTIR analyses of B2 revealed the compound as1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester, a strong antifungal volatile organic compound. Light microscopic analysis of the fungal mycelium from the dual culture plate of both culture filtrate and 1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester disclosed strong mycolytic activity as evident by mycelial distractions and shrinkage. This is the first report on antifungal production by endophytic Lysinibacillus sphaericus against R. solani, the rice sheath blight pathogen. The findings of this study biologically prospect the endophyte L. sphaericus as an inexpensive broad spectrum bioagent for eco-friendly, economic and sustainable agriculture.

Identification of a Novel Bioactive Phenazine Derivative and Regulation of phoP on Its Production in Streptomyces lomondensis S015.

Natural phenazines are a class of multifunctional secondary metabolites of bacteria that play an important role in the biocontrol of plant pathogens. In this paper, a novel bioactive phenazine derivative was isolated from Streptomyces lomondensis S015 through silica gel chromatography and preparative high-performance liquid chromatography (HPLC). The structure was identified as 1-carboxyl-6-formyl-4,7,9-trihydroxy-phenazine (CFTHP) by NMR spectroscopy in combination with ultraperformance liquid chromatography & mass spectrometry (UPLC-MS). CFTHP could inhibit Pythium ultimum, Rhizoctonia solani, Septoria steviae, and Fusarium oxysporum f. sp. niveum with minimal inhibitory concentration (MIC) values of 16, 32, 16, and 16 µg/mL, respectively. A global regulatory gene phoP could positively regulate CFTHP biosynthesis since its production was 3.0-fold enhanced by phoP overexpression and inhibited by phoP deletion in Streptomyces lomondensis S015. These studies illustrated the potential of CFTHP as a promising biopesticide and provided a reference for phenazine production improvement.

Design, Synthesis, and Antifungal Evaluation of Cryptolepine Derivatives against Phytopathogenic Fungi.

Inspired by the widely antiphytopathogenic application of diversified derivatives from natural sources, cryptolepine and its derivatives were subsequently designed, synthesized, and evaluated for their antifungal activities against four agriculturally important fungi Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, and Sclerotinia sclerotiorum. The results obtained from in vitro assay indicated that compounds a1-a24 showed great fungicidal property against B. cinerea (EC50 < 4 µg/mL); especially, a3 presented significantly prominent inhibitory activity with an EC50 of 0.027 µg/mL. In the pursuit of further expanding the antifungal spectrum of cryptolepine, ring-opened compound f1 produced better activity with an EC50 of 3.632 µg/mL against R. solani and an EC50 of 5.599 µg/mL against F. graminearum. Furthermore, a3 was selected to be a candidate to investigate its preliminary antifungal mechanism to B. cinerea, revealing that not only spore germination was effectively inhibited and the normal physiological structure of mycelium was severely undermined but also detrimental reactive oxygen was obviously accumulated and the normal function of the nucleus was fairly disordered. Besides, in vivo curative experiment against B. cinerea found that the therapeutic action of a3 was comparable to that of the positive control azoxystrobin. These results suggested that compound a3 could be regarded as a novel and promising agent against B. cinerea for its valuable potency.

Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungus Trichoderma atroviride reveals a role in conidiation and antagonism.

Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T. atroviride interactions with the prey fungi Botrytis cinerea or R. solani. Phenotypic analysis of agl1 deletion strains (Δagl1) showed reduced conidiation compared to T. atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δagl1 strains display reduced antagonism towards B. cinerea and R. solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T. atroviride.

Major latex protein-like encoding genes contribute to Rhizoctonia solani defense responses in sugar beet.

Sugar beets are attacked by several pathogens that cause root damages. Rhizoctonia (Greek for "root killer") is one of them. Rhizoctonia root rot has become an increasing problem for sugar beet production and to decrease yield losses agronomical measures are adopted. Here, two partially resistant and two susceptible sugar beet genotypes were used for transcriptome analysis to discover new defense genes to this fungal disease, information to be implemented in molecular resistance breeding. Among 217 transcripts with increased expression at 2 days post-infection (dpi), three resistance-like genes were found. These genes were not significantly elevated at 5 dpi, a time point when increased expression of three Bet v I/Major latex protein (MLP) homologous genes BvMLP1, BvMLP2 and BvML3 was observed in the partially resistant genotypes. Quantitative RT-PCR analysis on diseased sugar beet seedlings validated the activity of BvMLP1 and BvMLP3 observed in the transcriptome during challenge by R. solani. The three BvMLP genes were cloned and overexpressed in Arabidopsis thaliana to further dissect their individual contribution. Transgenic plants were also compared to T-DNA mutants of orthologous MLP genes. Plants overexpressing BvMLP1 and BvMLP3 showed significantly less infection whereas additive effects were seen on Atmlp1/Atmlp3 double mutants. The data suggest that BvMLP1 and BvMLP3 may contribute to the reduction of the Rhizoctonia root rot disease in sugar beet. Impact on the defense reaction from other differential expressed genes observed in the study is discussed.

Bioengineered chitosan-magnesium nanocomposite: A novel agricultural antimicrobial agent against Acidovorax oryzae and Rhizoctonia solani for sustainable rice production.

Chitosan is a potent biopolymer having promising antimicrobial properties against phytopathogens. Recently, engineered nanomaterials (ENMs) have gained much attention due to their potential application in the plant disease management. In this study, we reported the green synthesis of chitosan-magnesium (CS-Mg) nanocomposite and its antimicrobial activity against two rice pathogens namely Acidovorax oryzae and Rhizoctonia solani for the first time. The green MgO nanoparticles synthesized by using a native Bacillus sp. strain RNT3, were used to fabricate CS-Mg nanocomposite utilizing one-pot synthesis method. The synthesis of CS-Mg nanocomposite was further confirmed by using UV-vis spectroscopy, whereas, FTIR and XRD analysis showed the capping of CS-Mg nanocomposites by different functional groups together with their crystalline structure, respectively. Besides, SEM and TEM images revealed the spherical shape along with the particles size ranging from 29 to 60 nm. Moreover, EDS analysis confirmed the elemental purity of nanocomposite. The CS-Mg nanocomposite showed remarkable antimicrobial activity against A. oryzae and R. solani and significantly inhibited the growth as compared to non-treated control. The ultrastructure studies showed damaged structure of cell wall and internal cellular organelles after treatment with 100 µg mL-1 CS-Mg nanocomposite. The results of this study indicated that CS-Mg nanocomposite-based antimicrobial agents could be considered as promising nanopesticides against phytopathogens in plant disease management.

Molecular level changes during suppression of Rhizoctonia solani growth by humic substances and relationships with chemical structure.

Elucidation of the inhibitory effects of humic substances (HSs) on phytopathogenic fungi and the underlying molecular mechanisms are highly important for improved biocontrol. In this study, we investigated the growth suppression, morphological characteristics, transcriptomic sequence, and radical signals of Rhizoctonia solani following HS addition (50 mg/L). Through mycelial cultured experiment, mycelia growth of R. solani had been suppressed with HS addition, and the inhibition rate was 24.88 ± 0.11% compared to the control. Field emission-scanning electron microscopy showed increased and superimposed branching mycelial growth, with a shriveled appearance. RNA samples of R. solani cultured with or without HSs were both extracted to examine the sequence on molecular level by Illumina HiSeq sequencing platform. RNA sequencing analysis revealed 175 differentially expressed genes (DEGs; 111 upregulated and 64 downregulated) between the HSs treatment and control. The upregulated unigenes were annotated and significantly enriched to three molecular processes: vitamin B6 metabolism, ABC transporters, and glutathione metabolism, while the downregulated unigenes were annotated to carbohydrate metabolism, but not significantly enriched. Real time-quantitative polymerase chain reaction analysis showed that the unigenes related to hexokinase, glucose-6-phosphate isomerase, glutathione synthase, and glutathione reductase were significantly decreased (by 60.03%, 70.70%, 60.33%, and 57.59%, respectively), while those related to glutathione S-transferase were significantly increased (2.66-fold). The electron paramagnetic resonance spectra showed that HSs induced increased the intensity of radical signals of R. solani in a cultured system increased by 59.56% compared to CK (without HSs addition). Network analysis based on DEGs expression and the chemical structure of HSs revealed that the carbonyl moiety in HSs formed the most links with nodes of the DEGs (sum of the links of positive and negative effects = 70), implicating this structure as the active fraction responsible for the inhibitory effect. This study provides molecular and chemical evidence of the biofungicidal activity of HSs with the potential for practical application.

Biocontrol of Rhizoctonia solani (Kühn) and Fusarium solani (Marti) causing damping-off disease in tomato with Azotobacter chroococcum and Pseudomonas fluorescens.

BACKGROUND AND OBJECTIVE: The Damping-off disease is one of the most reasons for low productively of tomato in the world, especially in Iraq. In the current study, two types of bacteria (Azotobacter chroococcum and Pseudomonas fluorescens) were used to evaluate their efficacy in inhibiting the growth of pathogenic fungi Rhizoctonia solani and Fusarium solani and protecting the seeds of tomato and increasing their germination percentage. MATERIALS AND METHODS: Dual culture technique and Food poisoning technique were used to study the effect of bacteria on the growth of fungi understudy, and study the effect of bacterial filtrates on germination of tomato seeds. RESULTS: A. chroococcum showed the strongest antagonistic activity followed by P. fluorescens with the percentage of inhibition ranging between 72.9-77.1 and 69.5-70.3% for R. solani and F. solani respectively after 7 days of incubation. The effect of A. chroococcum and P. fluorescens filtrates were increased and also increased the inhibition of growth of fungi understudy, A. chroococcum filtrate also showed the strongest inhibitory effect followed by P. fluorescens with the percentage of inhibition ranging between 86.0-87.0 and 83.0-83.5% for R. solani and F. solani respectively at 20% concentration of filtrate. The percentage of seeds germination reached 90% in the treatment of A. chroococcum filtrate and 80% in the treatment of P. fluorescens filtrate. CONCLUSION: It can be concluded that the filtrates of A. chroococcum and P. fluorescens have antifungal properties against R. solani and F. solani and provided a high protection and increasing tomato seeds germination percentage.

Genome Mining and Evaluation of the Biocontrol Potential of Pseudomonas fluorescens BRZ63, a New Endophyte of Oilseed Rape (Brassica napus L.) against Fungal Pathogens.

Endophytic bacteria hold tremendous potential for use as biocontrol agents. Our study aimed to investigate the biocontrol activity of Pseudomonas fluorescens BRZ63, a new endophyte of oilseed rape (Brassica napus L.) against Rhizoctonia solani W70, Colletotrichum dematium K, Sclerotinia sclerotiorum K2291, and Fusarium avenaceum. In addition, features crucial for biocontrol, plant growth promotion, and colonization were assessed and linked with the genome sequences. The in vitro tests showed that BRZ63 significantly inhibited the mycelium growth of all tested pathogens and stimulated germination and growth of oilseed rape seedlings treated with fungal pathogens. The BRZ63 strain can benefit plants by producing biosurfactants, siderophores, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and ammonia as well as phosphate solubilization. The abilities of exopolysaccharide production, autoaggregation, and biofilm formation additionally underline its potential to plant colonization and hence biocontrol. The effective colonization properties of the BRZ63 strain were confirmed by microscopy observations of EGFP-expressing cells colonizing the root surface and epidermal cells of Arabidopsis thaliana Col-0. Genome mining identified many genes related to the biocontrol process, such as transporters, siderophores, and other secondary metabolites. All analyses revealed that the BRZ63 strain is an excellent endophytic candidate for biocontrol of various plant pathogens and plant growth promotion.