Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

Assessment of Plant Growth Promotion Potential of Endophytic Bacterium B. subtilis KU21 Isolated from Rosmarinus officinalis.

The current study aimed to evaluate the plant growth-promoting (PGP) potential of endophytic strain Bacillus subtilis KU21 isolated from the roots of Rosmarinus officinalis. The strain exhibited multiple traits of plant growth promotion viz., phosphate (P) solubilization, nitrogen fixation, indole-3-acetic acid (IAA), siderophore, hydrogen cyanide (HCN), lytic enzymes production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. The isolate also exhibited antagonistic activity against phytopathogenic fungi, i.e., Fusarium oxysporum, Fusarium graminiarum, and Rhizoctonia solani. The P-solubilization activity of B. subtilis KU21 was further elucidated via detection of glucose dehydrogenase (gdh) gene involved in the production of gluconic acid which is responsible for P-solubilization. Further, B. subtilis KU21 was evaluated for in vivo growth promotion studies of tomato (test crop) under net house conditions. A remarkable increase in seed germination, plant growth parameters, nutrient acquisition, and soil quality parameters (NPK) was observed in B. subtilis KU21-treated plants over untreated control. Hence, the proposed module could be recommended for sustainable tomato production in the Northwest Himalayan region without compromising soil health and fertility.

Revealing the diversity of Jojoba-associated fungi using amplicon metagenome approach and assessing the in vitro biocontrol activity of its cultivable community.

Jojoba shrubs are wild plants cultivated in arid and semiarid lands and characterized by tolerance to drought, salinity, and high temperatures. Fungi associated with such plants may be attributed to the tolerance of host plants against biotic stress in addition to the promotion of plant growth. Previous studies showed the importance of jojoba as jojoba oil in the agricultural field; however, no prior study discussed the role of jojoba-associated fungi (JAF) in reflecting plant health and the possibility of using JAF in biocontrol. Here, the culture-independent and culture-dependent approaches were performed to study the diversity of the jojoba-associated fungi. Then, the cultivable fungi were evaluated for in-vitro antagonistic activity and in vitro plant growth promotion assays. The metagenome analysis revealed the existence of four fungal phyla: Ascomycota, Aphelidiomycota, Basidiomycota, and Mortierellomycota. The phylum Ascomycota was the most common and had the highest relative abundance in soil, root, branch, and fruit samples (59.7%, 50.7%, 49.8%, and 52.4%, respectively). Alternaria was the most abundant genus in aboveground tissues: branch (43.7%) and fruit (32.1%), while the genus Discosia had the highest abundance in the underground samples: soil (24%) and root (30.7%). For the culture-dependent method, a total of 14 fungi were isolated, identified, and screened for their chitinolytic and antagonist activity against three phytopathogenic fungi (Fusarium oxysporum, Alternaria alternata and Rhizoctonia solani) as well as their in vitro plant growth promotion (PGP) activity. Based on ITS sequence analysis, the selected potent isolates were identified as Aspergillus stellatusEJ-JFF3, Aspergillus flavus EJ-JFF4, Stilbocrea sp. EJ-JLF1, Fusarium solani EJ-JRF3, and Amesia atrobrunneaEJ-JSF4. The endophyte strain A. flavus EJ-JFF4 exhibited the highest chitinolytic activity (9 Enzyme Index) and antagonistic potential against Fusarium oxysporum, Alternaria alternata, and Rhizoctonia solani phytopathogens with inhibitory percentages of 72, 70, and 80 respectively. Also, A. flavus EJ-JFF4 had significant multiple PGP properties, including siderophore production (69.3%), phosphate solubilization (95.4 µg ml-1). The greatest production of Indol-3-Acetic Acid was belonged to A. atrobrunnea EJ-JSF4 (114.5 µg ml-1). The analysis of FUNGuild revealed the abundance of symbiotrophs over other trophic modes, and the guild of endophytes was commonly assigned in all samples. For the first time, this study uncovered fungal diversity associated with jojoba plants using a culture-independent approach and in-vitro assessed the roles of cultivable fungal strains in promoting plant growth and biocontrol. The present study indicated the significance of jojoba shrubs as a potential source of diverse fungi with high biocontrol and PGP activities.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Biocontrol potential of Bacillus subtilis RH5 against sheath blight of rice caused by Rhizoctonia solani.

The sheath blight disease of rice caused by Rhizoctonia solani is widely prevalent and one of the most destructive diseases, affecting rice cultivation and loss worldwide. In the present study, a set of twenty Bacillus isolates from saline soil of Uttar Pradesh were tested for their biocontrol activity against R. solani with the aim to obtain a potential strain for the control of sheath blight disease toward ecofriendly and sustainable agriculture. The results of dual-culture assay and scanning electron microscopic studies showed that the strain RH5 exhibited significant antagonistic activity (84.41%) against the fungal pathogen R. solani. On the basis of 16S rDNA sequencing analysis, the potential biocontrol strain RH5 was identified as Bacillus subtilis. Furthermore, the strain RH5 was characterized by different plant growth-promoting (PGP) activities and induction of defense-related enzymes in rice plants against R. solani. The strain RH5 posses various PGP attributes (indole acetic acid, siderophore, hydrogen cyanide production and phosphate, Zn, K solubility), hydrolytic enzymatic (chitinase, protease, cellulase, xylanase) activity, and presence of antimicrobial peptide biosynthetic genes (bacylisin, surfactin, and fengycin), which support the strain for efficient colonization of hyphae and its inhibition. Finally, the results of the greenhouse study confirmed that strain RH5 significantly increased plant growth and triggered resistance in rice plants through the production of defense-related antioxidant enzymes.

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.

Chemical Composition, Algicidal, Antimicrobial, and Antioxidant Activities of the Essential Oils of Taiwania flousiana Gaussen.

Taiwania flousiana (T. flousiana) Gaussen is a precious wood in the family Taxodiaceae. This study investigated the chemical components of the essential oil from the stem bark of T. flousiana and its algicidal, antifungal, and antioxidant properties. Sixty-nine compounds representing 89.70% of the stem bark essential oil were identified by GC-MS. The essential oil showed strong anti-algae, anti-bacteria, and anti-fungus activities against the tested species, and antioxidant activities. The IC50 values of the essential oil against chlorophyll a, chlorophyll b, and the total chlorophyll of Spirogyra communis (a species of algae), 24-96 h after the treatment, ranged from 31.77 to 84.92 µg/mL, while the IC50 values of butachlor ranged from 40.24 to 58.09 µg/mL. Ultrastructure changes revealed by the transmission electron microscopy indicated that the main algicidal action sites were the chloroplast and cell wall. The essential oil showed antifungal activities on Rhizoctonia solani (EC50 = 287.94 µg/mL) and Colletotrichum gloeosporioiles (EC50 = 378.90 µg/mL). It also showed bactericidal activities on Ralstonia solanacearum and Staphylococcus aureus, with zones of inhibition (ZOIs) being 18.66 and 16.75 mm, respectively at 40 µg/disk. Additionally, the essential oil possessed antioxidant activity estimated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (IC50 = 33.51 µg/mL; IC50 value of the positive control ascorbic acid was 7.98 µg/mL). Thus, the essential oil of this plant might be used as a possible source of natural bioactive molecules in agrochemical industry as well as in food and cosmetic industries.

Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen Rhizoctonia solani AG1-IA.

Rhizoctonia solani AG1-IA is a soil-borne necrotrophic pathogen that causes devastating rice sheath blight disease in rice-growing regions worldwide. Sclerotia play an important role in the life cycle of R. solani AG1-IA. In this study, RNA sequencing was used to investigate the transcriptomic dynamics of sclerotial development (SD) of R. solani AG1-IA. Gene ontology and pathway enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the functions and pathways of differentially expressed genes (DEGs). Six cDNA libraries were generated, and more than 300 million clean reads were obtained and assembled into 15,100 unigenes. In total, 12,575 differentially expressed genes were identified and 34.62% (4353) were significantly differentially expressed with a FDR ≤ 0.01 and |log2Ratio| ≥ 1, which were enriched into eight profiles using Short Time-series Expression Miner. Furthermore, KEGG and gene ontology analyses suggest the DEGs were significantly enriched in several biological processes and pathways, including binding and catalytic functions, biosynthesis of ribosomes, and other biological functions. Further annotation of the DEGs using the Clusters of Orthologous Groups (COG) database found most DEGs were involved in amino acid transport and metabolism, as well as energy production and conversion. Furthermore, DEGs relevant to SD of R. solani AG1-IA were involved in secondary metabolite biosynthesis, melanin biosynthesis, ubiquitin processes, autophagy, and reactive oxygen species metabolism. The gene expression profiles of 10 randomly selected DEGs were validated by quantitative real-time reverse transcription PCR and were consistent with the dynamics in transcript abundance identified by RNA sequencing. The data provide a high-resolution map of gene expression during SD, a key process contributing to the pathogenicity of this devastating pathogen. In addition, this study provides a useful resource for further studies on the genomics of R. solani AG1-IA and other Rhizoctonia species.

Complete genome sequence of Burkholderia sp. JP2-270, a rhizosphere isolate of rice with antifungal activity against Rhizoctonia solani.

Burkholderia sp. JP2-270, a bacterium with a strong ability to inhibit the growth of Rhizoctonia solani, was isolated from the rhizosphere of rice. The phylogenetic analysis based on 16S rRNA gene revealed that JP2-270 belonged to Burkholderia cepacia complex. Here, we present the complete genome sequence of Burkholderia sp. JP2-270, which consists of three circular chromosomes (Chr1 3,723,585 bp, Chr2 3,274,969 bp, Chr3 1,483,367 bp) and two plasmids (Plas1 15,126 bp, Plas2 428,263 bp). A total of 8193 protein coding genes were predicted in the genome, including 67 tRNA genes, 18 rRNA genes and 4 ncRNA genes. In addition, mutation analysis of Burkholderia sp. JP2-270 revealed that the gene bysR (DM992_17470), encoding a lysR-type transcriptional regulator, was essential for the antagonistic activity of Burkholderia sp. JP2-270 against R. solani GD118 in vitro and in vivo. Identification of regulatory gene associated with antagonistic activity will contribute to understand the antagonistic mechanism of Burkholderia sp. JP2-270.