Biofilm formation and flocculation potential analysis of halotolerant Bacillus tequilensis and its inoculation in soil to mitigate salinity stress of chickpea.

Application of beneficial microbes in soil is an important avenue to control plant stresses. In this study, the salinity tolerance of halotolerant bacteria (Bacillus tequilensis) was investigated and the bacterium was inoculated in the soil to mitigate salinity stress. The results revealed the highest floc yield and biofilm formation ability of B. tequilensis at 100 mM NaCl concentration. Fourier transformed infrared spectroscopy depicted the presence of carbohydrates and proteins which binds with sodium ions (Na+) and provide tolerance against salinity. Using PCR, plant growth-promoting bacterial genes viz., 1-aminocyclopropane-1-carboxylate deaminase and pyrroloquinoline quinone were successfully amplified from the genome of B. tequilensis. In the saline soil, B. tequilensis was inoculated and chickpea plants were grown. The bacterial strain improved the physiology, biochemistry, and antioxidant enzyme activities of the chickpea plant under salt stress. Plants inoculated with B. tequilensis exhibited higher relative water content, higher photosynthetic pigments, lower levels of hydrogen peroxide (H2O2) and malondialdehyde, and improved enzymatic activity for the scavenging of reactive oxygen species. The findings of this study suggest the sustainable use of B. tequilensis to mitigate the salinity stress of chickpea and other crops. This bacterium not only helps in the alleviation of the toxic effects of salt but also increases plant growth along with a reduction in crop losses due to salinity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01280-1.

Exogenous inoculation of endophytic bacterium Bacillus cereus suppresses clubroot (Plasmodiophora brassicae) occurrence in pak choi (Brassica campestris sp. chinensis L.).

MAIN CONCLUSION: The presence of Bacillus cereus plays a key role in clubroot suppression and improves plant biomass in pak choi. B. cereus is reported for the first time as a novel biocontrol agent against clubroot. Plasmodiophora brassicae Woronin causes a devastating infectious disease known as clubroot that is damaging to cruciferous vegetables. This study aimed to isolate beneficial bacteria from the rhizosphere soil of pak choi (Brassica campestris sp. chinensis) and to evaluate the ability of the isolate to reduce the severity of clubroot. Strains obtained from the rhizosphere of symptomless pak choi were first selected on the basis of their germination inhibition rate and effects on the viability of P. brassicae resting spores. Eight bacterial isolates had inhibitory effects against the resting spores of clubroot causing pathogen. However, MZ-12 showed the highest inhibitory effect at 73.4%. Inoculation with MZ-12 enhanced the plant biomass relative to plants grown without MZ-12 as well as P. brassicae infected plants. Furthermore, enhanced antioxidant enzymatic activities were observed in clubroot-infected plants during bacterial association. Co-inoculation of the plant with both P. brassicae and MZ-12 resulted in a 64% reduction of gall formation in comparison to plants inoculated with P. brassicae only. Three applications of MZ-12 to plants infected with P. brassicae at 7, 14 and 21 days after seeding (DAS) were more effective than one application and repressed root hair infection. According to 16S rDNA sequence analysis, strain MZ-12 was identified as had a 100% sequence similarity with type strain Bacillus cereus. The findings of the present study will facilitate further investigation into biological mechanisms of cruciferous clubroot control.

HSP70-16 and VDAC3 jointly inhibit seed germination under cold stress in Arabidopsis.

Abscisic acid (ABA) transport plays a crucial role in seed germination under unfavourable conditions such as cold stress. Both heat shock protein 70 (HSP70) and voltage-dependent anion channel (VDAC) protein are involved in cold stress responses in Arabidopsis. However, their roles in seed germination with regard to ABA signaling remain unknown. Here we demonstrated that Arabidopsis HSP70-16 and VDAC3 jointly suppress seed germination under cold stress conditions. At 4°C, both HSP70-16 and VDAC3 facilitated the efflux of ABA from the endosperm to the embryo and thus inhibited seed germination. HSP70-16 interacted with VDAC3 on the plasma membrane and in the nucleus, and the interplay between HSP70-16 and VDAC3 activated the opening of the VDAC3 ion channel. Our work established a novel function of HSP70-16 in seed germination under cold stress and a possible association of VDAC3 activity with ABA transportation from endosperm to embryo under cold stress conditions. This study reveals that HSP70-16 interacts with VDAC3 and facilitates the opening of the VDAC3 ion channel, which influences ABA efflux from endosperm to embryo, thus negatively regulates seed germination under cold stress.

Phytotoxic effects of chemically synthesized copper oxide nanoparticles induce physiological, biochemical, and ultrastructural changes in Cucumis melo.

Nanotechnology has achieved great attention due to its impressive performance especially engineered nanoparticles (ENPs). Copper-based nanoparticles offer favorable development in the fabrication of agrochemicals including fertilizers and pesticides in the field of agriculture. However, their toxic impact on melon plants (Cucumis melo) still needs to be investigated. Therefore, the aim of the current work was performed to focus on the toxic impact of Cu oxide nanoparticles (CuONPs) in hydroponically grown Cucumis melo. Our results demonstrated that CuONPs with 75, 150, and 225 mg/L significantly (P<0.005) suppressed the growth rate and badly affect physiological and biochemical activities in melon seedlings. Also, results revealed remarkable phenotypical changes besides significantly reduced fresh biomass and decreased levels of total chlorophyll contents in a dose-dependent manner. Atomic absorption spectroscopy (ASS) analysis exhibited that C. melo treated with CuONPs accumulates NPs in the shoot. Moreover, exposure to higher CuONPs (75-225mg/L) significantly increased the reactive oxygen species (ROS) accumulation, malondialdehyde (MDA), and hydrogen peroxide (H2O2) level in the shoot and induced toxicity in melon root with an increase in electrolyte leakage. Furthermore, antioxidant enzyme peroxidase (POD) and superoxide dismutase (SOD) activity in the shoot significantly increased under exposure to higher CuONPs. Exposure to higher concentrations of CuONPs (225 mg/L) significantly deformed the stomatal aperture. Furthermore, reducing the number and abnormal size of palisade mesophyll and spongy mesophyll cells were investigated especially at high doses of CuONPs. Overall, our current work demonstrates that CuONPs of 10-40 nm size provide direct evidence for a toxic effect in C. melo seedlings. Our findings were expected to inspire the safe production of NPs and agrifood security. Thus, CuONPs prepared from toxic route and its bioaccumulation into our food chain through crop plants possess a serious threat to the ecological system.

Melatonin-mediated resistance to copper oxide nanoparticles-induced toxicity by regulating the photosynthetic apparatus, cellular damages and antioxidant defense system in maize seedlings.

The pollution of nanoparticles (NPs) has linked with severe negative effects on crop productivity. Thus, effective strategies are needed to mitigate the phytotoxicity of NPs. The aim of present study was to evaluate the efficacy of exogenously applied melatonin (MT) in mitigating the toxic effects of copper oxide nanoparticles (CuO NPs) from maize seedlings. Therefore, we comprehensively investigated the inhibitory effects of MT against CuO NPs-induced toxicity on morpho-physiological, biochemical and ultrastructural levels in maize. Our results show that CuO NPs (300 mg L-1) exposure displayed significantly reduction in all plant growth traits and induced toxicity in maize. Furthermore, 50 µM MT provided maximum plant tolerance against CuO NPs-induced phytotoxicity. It was noticed that MT improved plant growth, biomass, photochemical efficiency (Fv/Fm), chlorophyll contents (Chl a and Chl b), SPAD values and gas exchange attributes (stomatal conductance, net photosynthetic rate, intercellular CO2 concentration and transpiration rate) under CuO NPs stress. In addition, MT enhanced the antioxidant defense system and conferred protection to ultrastructural (mainly chloroplast, thylakoids membrane and plastoglobuli) damages and stomatal closure in maize plants subjected to CuO NPs stress. Together, it can be stated that the exogenous supply of MT improves the resilience of maize plants against the CuO NPs-induced phytotoxicity. Our current findings can be useful for the enhancement of plant growth and yield attributes in CuO NPs-contaminated soils. The reported information can provide insight into the MT pathways that can be used to improve crop stress tolerance in a challenging environment.

ZnO Nanoparticle-Mediated Seed Priming Induces Biochemical and Antioxidant Changes in Chickpea to Alleviate Fusarium Wilt.

Chickpea (Cicer arietinum L.) is one of the main pulse crops of Pakistan. The yield of chickpea is affected by a variety of biotic and abiotic factors. Due to their environmentally friendly nature, different nanoparticles are being synthesized and applied to economically important crops. In the present study, Trichoderma harzianum has been used as a stabilizing and reducing agent for the mycosynthesis of zinc oxide nanoparticles (ZnO NPs). Before their application to control Fusarium wilt of chickpea, synthesized ZnO NPs were characterized. X-ray diffraction (XRD) analysis revealed the average size (13 nm) of ZnO NPs. Scanning electron microscopy (SEM) indicated their spherical structure, and energy dispersive X-ray analysis (EDX) confirmed the oxide formation of ZnO NPs. Transmission electron microscopy (TEM) described the size and shape of nanoparticles, and Fourier transform infrared (FTIR) spectroscopy displayed the presence of reducing and stabilizing chemical compounds (alcohol, carboxylic acid, amines, and alkyl halide). Successfully characterized ZnO NPs exhibited significant mycelial growth inhibition of Fusarium oxysporum, in vitro. In a greenhouse pot experiment, the priming of chickpea seeds with ZnO NPs significantly increased the antioxidant activity of germinated plants and they displayed 90% less disease incidence than the control. Seed priming with ZnO NPs helped plants to accumulate higher quantities of sugars, phenol, total proteins, and superoxide dismutase (SOD) to create resistance against wilt pathogen. These nanofungicides were produced in powder form and they can easily be transferred and used in the field to control Fusarium wilt of chickpea.

Reprisal of Schima superba to Mn stress and exploration of its defense mechanism through transcriptomic analysis.

One of the most diverse protein families, ATP-binding cassette (ABC) transporters, play a role in disease resistance, heavy metal tolerance, and food absorption.Differentially expressed genes contribute in the investigation of plant defense mechanisms under varying stress conditions. To elucidate the molecular mechanisms involved in Mn metal stress, we performed a transcriptomic analysis to explore the differential gene expression in Schima superba with the comparison of control. A total of 79.84 G clean data was generated and 6558 DEGs were identified in response to Mn metal stress. Differentially expressed genes were found to be involved in defense, signaling pathways, oxidative burst, transcription factors and stress responses. Genes important in metal transport were more expressive in Mn stress than control plants. The investigation of cis-acting regions in the ABC family indicated that these genes might be targeted by a large variety of trans-acting elements to control a variety of stress circumstances. Moreover, genes involved in defense responses, the mitogen-activated protein kinase (MAPK) signaling and signal transduction in S. superba were highly induced in Mn stress. Twenty ABC transporters were variably expressed on 1st, 5th, and 10th day of Mn treatment, according to the qRT PCR data. Inclusively, our findings provide an indispensable foundation for an advanced understanding of the metal resistance mechanisms. Our study will enrich the sequence information of S. superba in a public database and would provide a new understanding of the molecular mechanisms of heavy metal tolerance and detoxification.

Functional Annotation of a Full-Length Transcriptome and Identification of Genes Associated with Flower Development in Rhododendronsimsii (Ericaceae).

Rhododendronsimsii is one of the top ten famous flowers in China. Due to its historical value and high aesthetic, it is widely popular among Chinese people. Various colors are important breeding objectives in Rhododendron L. The understanding of the molecular mechanism of flower color formation can provide a theoretical basis for the improvement of flower color in Rhododendron L. To generate the R.simsii transcriptome, PacBio sequencing technology has been used. A total of 833,137 full-length non-chimeric reads were obtained and 726,846 high-quality full-length transcripts were found. Moreover, 40,556 total open reading frames were obtained; of which 36,018 were complete. In gene annotation analyses, 39,411, 18,565, 16,102 and 17,450 transcriptions were allocated to GO, Nr, KEGG and COG databases, correspondingly. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with Protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT) and Coding Non Coding Index (CNCI) databases and observed 6170, 2265, 4084 and 1240 lncRNAs, respectively. Based on the results, most genes were enriched in the flavonoid biosynthetic pathway. The eight key genes on the anthocyanin biosynthetic pathway were further selected and analyzed by qRT-PCR. The F3'H and ANS showed an upward trend in the developmental stages of R. simsii. The highest expression of F3'5'H and FLS in the petal color formation of R. simsii was observed. This research provided a huge number of full-length transcripts, which will help to proceed genetic analyses of R.simsii. native, which is a semi-deciduous shrub.

PacBio Single-Molecule Long-Read Sequencing Reveals Genes Tolerating Manganese Stress in Schima superba Saplings.

Schima superba (Theaceae) is a subtropical evergreen tree and is used widely for forest firebreaks and gardening. It is a plant that tolerates salt and typically accumulates elevated amounts of manganese in the leaves. With large ecological amplitude, this tree species grows quickly. Due to its substantial biomass, it has a great potential for soil remediation. To evaluate the thorough framework of the mRNA, we employed PacBio sequencing technology for the first time to generate S. Superba transcriptome. In this analysis, overall, 511,759 full length non-chimeric reads were acquired, and 163,834 high-quality full-length reads were obtained. Overall, 93,362 open reading frames were obtained, of which 78,255 were complete. In gene annotation analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Non-Redundant (Nr) databases were allocated 91,082, 71,839, 38,914, and 38,376 transcripts, respectively. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT), and Coding Non-Coding Index (CNCI) databases and observed 8,551, 9,174, 20,720, and 18,669 lncRNAs, respectively. Moreover, nine genes were randomly selected for the expression analysis, which showed the highest expression of Gene 6 (Na_Ca_ex gene), and CAX (CAX-interacting protein 4) was higher in manganese (Mn)-treated group. This work provided significant number of full-length transcripts and refined the annotation of the reference genome, which will ease advanced genetic analyses of S. superba.

Cd-tolerant SY-2 strain of Stenotrophomonas maltophilia: a potential PGPR, isolated from the Nanjing mining area in China.

Microbial and plant assisted bioremediation is an emerging way for the remediation of soils polluted with heavy metals. To screen the cadmium tolerant bacteria, soil samples were collected from Nanjing mining area, China. The average cadmium content of the mine soil reached 45.71 mg/kg, which was indicating serious pollution and potential ecological risk. From the mine soil, six cadmium tolerant plant growth-promoting rhizobacteria (PGPR) were isolated. The isolated bacterial strain "SY-2" showed maximum cadmium tolerance and it was selected for further experimentation. This strain was identified as Stenotrophomonas maltophilia by 16S rRNA gene sequencing (GenBank accession number MG597057). SY-2 was found to tolerate maximum cadmium at 1.0 mM concentration. This strain also exhibited good adsorption capacity (up to 35.7%) of heavy metal at 0.5 mM concentration. The results of this study exhibited organic phosphorus solubilization (37.08 mg/L) and IAA biosynthesis (15.11 mg/L) ability of isolated S. maltophilia. Scanning electron microscopy (SEM) revealed cell shrinkage and the cell wall of S. maltophilia was very rough. Moreover, the energy dispersive X-ray (EDX) analysis endorsed the adsorption of Cd ions on the surface of biomass. FT-IR study described the presence of functional groups and the nature of chemical bonds, before and after cadmium stress. At 0.25 mM cadmium concentration, S. maltophilia treated seeds of Capsicum annuum L. developed 1.46 times longer roots than untreated seeds. The results of this study helped us to conclude that SY-2 strain of S. maltophilia possesses significant metal tolerance and bioremediation potential against cadmium. In the future, this strain can be used as a microbial remediation agent to detoxify heavy metals in contaminated soils. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02524-7s) contains supplementary material, which is available to authorized users.

Evaluation of Metal Tolerance of Fungal Strains Isolated from Contaminated Mining Soil of Nanjing, China.

Rapidly increasing industry has resulted in greater discharge of hazardous chemicals in the soil. In the current study, soil samples were collected from Nanjing mine (32°09'19.29″ N 118°56'57.04″ E) and subjected to heavy metal analysis and microbe isolation. A total of 460 fungi were isolated, and five of these were yeast strains. Most of the strains exhibited tolerance to one metal. Five multimetal tolerant strains were selected and identified as Aspergillus sclerotiorum, Aspergillus aculeatus, Komagataella phaffii, Trichoderma harzianum, and Aspergillus niger. Isolated strains were grown in high concentrations of cadmium (Cd), chromium (Cr) and lead (Pb), for induced-tolerance training. The tolerance index (TI) revealed the highest Cd tolerance of novel K. phaffii strain at 5500 ppm (TI: 0.2). K. phaffii also displayed resistance at 4000 ppm against Cr (TI: 0.32) and Pb (TI: 0.32). In contrast, tolerance training for A. niger was not that successful. K. phaffii also displayed the highest bioaccumulation capacity for Cd (25.23 mg/g), Cu (21.63 mg/g), and Pb (20.63 mg/g) at 200 ppm. Scanning electron microscopy (SEM) explored the morphological changes in the mycelia of stressed fungi. Results of this study describe this delicate approach to be species and metal dependent and suggest a potential utilization of this fungal strain for the bioremediation of contaminated soils.

Resistance associated metabolite profiling of Aspergillus leaf spot in cotton through non-targeted metabolomics.

Aspergillus tubingensis is an important pathogen of economically important crops. Different biotic stresses strongly influence the balance of metabolites in plants. The aim of this study was to understand the function and response of resistance associated metabolites which, in turn are involved in many secondary metabolomics pathways to influence defense mechanism of cotton plant. Analysis of non-targeted metabolomics using ultra high performance liquid chromatography-mass spectrometry (UPLC-MS) revealed abundant accumulation of key metabolites including flavonoids, phenylpropanoids, terpenoids, fatty acids and carbohydrates, in response to leaf spot of cotton. The principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and partial least squares discriminant analysis (PLS-DA) score plots illustrated the evidences of variation between two varieties of cotton under mock and pathogen inoculated treatments. Primary metabolism was affected by the up regulation of pyruvate and malate and by the accumulation of carbohydrates like cellobiose and inulobiose. Among 241 resistance related (RR) metabolites, 18 were identified as resistance related constitutive (RRC) and 223 as resistance related induced (RRI) metabolites. Several RRI metabolites, identified in the present study were the precursors for many secondary metabolic pathways. These included phenylpropanoids (stilbenes and furanocoumarin), flavonoids (phlorizin and kaempferol), alkaloids (indolizine and acetylcorynoline) and terpenoids (azelaic acid and oleanolic acid). Our results demonstrated that secondary metabolism, primary metabolism and energy metabolism were more active in resistant cultivar, as compared to sensitive cultivar. Differential protein and fatty acid metabolism was also depicted in both cultivars. Accumulation of these defense related metabolites in resistant cotton cultivar and their suppression in susceptible cotton cultivar revealed the reason of their respective tolerance and susceptibility against A. tubingensis.