Search

Phytophthora parasitica is a devastating plant pathogen that has a wide host range. As a new approach, silver nanoparticles (AgNPs) were assessed to control it. Previously AgNPs were shown to inhibit mycelial growth, zoospore production and germination, and germ tube elongation. However, the mechanism(s) of bioactivity of AgNPs on changes in metabolic patterns in P. parasitica has not been completely resolved. P. parasitica was exposed to AgNPs at several time points, RNA-Sequences were extracted, and then selected key genes in several of the pathways were verified for the level of expression using qRT-PCR. For RNA-Seq, the reads were mapped to the P. parasitica genome INRA 310.3. Principle component analysis illustrated low variation among biological replicates and emphasized the reproducibility of results. In this paper, seven candidate genes identified by RNA-Seq were evaluated for their expression using qRT-PCR. Interestingly, qRT-PCR confirmed that the genes that were significantly altered were found to be involved in major cellular pathways such as glutathione metabolism and ribosome biogenesis. qRT-PCR also revealed heat shock protein, ABC transporter, and Glutathione-S-Transferase (GST) genes were significantly affected. These genes are related to oxidative stress. Expression of GST and heat-shock protein significantly increased at 1 hr post-exposure to AgNPs, indicating a rapid response. The analysis based on qRT-PCR makes it evident that key genes involved in many major pathways were significantly altered on exposure to AgNPs.

Protection of crop plants from phytopathogens through endophytic bacteria is a newly emerged area of biocontrol. In this study, endophytic bacteria were isolated from the rhizosphere of Cannabis sativa. Based on initial antimicrobial screening, three (03) bacteria Serratia marcescens MOSEL-w2, Enterobacter cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 were selected. Antimicrobial assays of these selected bacteria against Phytophthora parasitica revealed that E. cloacae MOSEL-w7 and Paenibacillus sp. MOSEL-w13 possessed strong activity against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4–400 µg mL−1). P. parasitica hyphae treated with ethyl acetate extract of E. cloacae MOSEL-w7 resulted in severe growth abnormalities compared to control. The extracts were further evaluated for in vivo detached-leaf assay against P. parasitica on the wild type tobacco. Application of 1% ethyl acetate bacterial extract of S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus sp. MOSEL-w13 reduced P. parasitica induced lesion sizes and lesion frequencies by 60–80%. HPLC based fractions of each extract also showed bioactivity against P. parasitica. A total of 24 compounds were found in the S. marcescens MOSEL-w2, 15 compounds in E. cloacae MOSEL-w7 and 20 compounds found in Paenibacillus sp. MOSEL-w13. LC–MS/MS analyses showed different bioactive compounds in the bacterial extracts such as Cotinine (alkylpyrrolidine), l-tryptophan, l-lysine, l-Dopa, and l-ornithine. These results suggest that S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 are a source of bioactive metabolites and could be used in combination with other biocontrol agents, with other modes of action for controlling diseases caused by Phytophthora in crops. They could be a clue for the broad-spectrum biopesticides for agriculturally significant crops.

In a preliminary plant-based microbiome study, diverse bacterial taxa were identified from different medicinal plants using 16S rRNA gene sequencing. Based on initial antimicrobial screening, eight (8) bacterial endophytes in six (6) different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Arthrobacter, and Delftia, from four important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata, and Calendula arvensis were selected for further analyses. Antimicrobial assays revealed that Pseudomonas taiwanensis MOSEL-RD23 has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis MOSEL-RD23and Bacillus flexus MOSEL-MIC5 inhibited the growth of Phytophthora parasitica by more than 80%. Ethyl acetate extracts of Streptomyces alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, Enterobacter hormaechei MOSEL-FLS1, and Bacillus tequilensis MOSEL-FLS3, and Delftia lacustris MB322 displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition of more than 80% inhibition in vitro against P. parasitica at different concentrations (4–400 µg/mL). Bacterial extracts showing strong antimicrobial activity were selected for bioactivity-driven fractionation and showed anti-Phytophthoral activity in multiple fractions and different peaks observed in UV–Vis spectroscopy. In the detached-leaf assay against P. parasitica on tobacco, 1% ethyl acetate bacterial extract of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1, B. tequilensis MOSEL-FLS3, and D. lacustris MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Overall, P. taiwanensis MOSEL-RD23 showed positive activities for all the assays. Analyzing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for the sustainable production of crops.

We compared surface properties, metabolic capping and antibacterial activity of silver nanoparticles, synthesized through extracts of cell cultures of Fagonia indica and its naturally grown form. Extracts from cell cultures (produced with thidiazuron (TDZ) or melatonin (MLN)) were compared to the naturally grown whole plant extracts (WPEs) for their reducing potential, and their effects on physical and biochemical properties of the biosynthesized silver nanoparticles. UV-Vis spectroscopy revealed that the surface plasmon resonance peaked at λ = 415 nm for MLN-AgNPs, λ = 430 nm for TDZ-AgNPs and λ = 460–465 nm for WPE-AgNPs. Transmission electron microscopy and energy dispersive X-rays of AgNPs showed that compared to WPE-AgNPs (mean diameter = 22 nm), extracts from MLN- and TDZ-induced cell cultures produced particles with spherical shapes and smaller diameters (i.e. mean diameter = 15 nm and 19 nm, respectively). Size distribution analysis also showed that TDZ-AgNPs were nearer to a symmetric distribution in terms of diameter (skewness = 0.80) as compared to WPE-AgNPs (skewness = 0.9) and MLN-AgNPs (skewness = 1.4). Furthermore, MLN-induced cell culture extracts produced AgNPs in higher concentration (210 μg mL−1) compared to AgNPs from TDZ-induced cell culture extracts (160 μg mL−1) and WPE (138 μg mL−1). Two-way comparisons of LC-MS/MS profiles of TDZ-AgNPs, MLN-AgNPs, and WPE-AgNPs revealed differences in their secondary metabolite profiles, which might account for differences in their differential response in bio-fabrication, and size distribution. Activity against different pathogenic bacterial strains, Escherichia coli, Bacillus cereus, Xanthomonas citri, Agrobacterium tumefaciens, Streptomyces griseus, and Erwinia carotovora suggested that MLN-AgNPs were more effective compared to TDZ- and WPE-AgNPs. These results indicated that phytohormones induced cell cultures can enhance the production, physical and biochemical properties of AgNPs.

Attenuating the Taxol yield of Aspergillus terreus with the subculturing and storage were the technical challenges that prevent this fungus to be a novel platform for industrial Taxol production. Thus, the objective of this study was to unravel the metabolic machineries of A. terreus associated with attenuation of Taxol productivity, and their restoring potency upon cocultivation with the Podocarpus gracilior microbiome. The Taxol yield of A. terreus was drastically reduced with the fungal subculturing. At the 10th subculture, the yield of Taxol was reduced by four folds (78.2 µg/l) comparing to the original culture (268 µg/l), as authenticated from silencing of molecular expression of the Taxol-rate limiting enzymes (GGPPS, TDS, DBAT and BAPT) by qPCR analyses. The visual fading of A. terreus conidial pigmentation with the subculturing, revealing the biosynthetic correlation of melanin and Taxol. The level of intracellular acetyl-CoA influx was reduced sequentially with the fungal subculturing, rationalizing the decreasing on Taxol and melanin yields. Fascinatingly, the Taxol biosynthetic machinery and cellular acetyl-CoA of A. terreus have been completely restored upon addition of 3% surface sterilized leaves of P. gracilior, suggesting the implantation of plant microbiome on re-triggering the molecular machinery of Taxol biosynthesis, their transcriptional factors, and/or increasing the influx of Acetyl-CoA. The expression of the proteins of 74.4, 68.2, 37.1 kDa were exponentially suppressed with A. terreus subculturing, and strongly restored upon addition of P. gracilior leaves, ensuring their profoundly correlation with the molecular expression of Taxol biosynthetic genes. From the proteomic analysis, the restored proteins 74.4 kDa of A. terreus upon addition of P. gracilior leaves were annotated as ribosome biogenesis proteins YTM and microtubule-assembly proteins that belong to WD40 superfamily. Thus, further ongoing studies for molecular cloning and expression of these genes with strong promotors in A. terreus, have been initiated, to construct a novel platform of metabolically stable A. terreus for sustainable Taxol production. Attenuating the Taxol yield of A. terreus with the multiple-culturing and storage might be due to the reduction on main influx of acetyl-CoA, or downregulation of ribosome biogenesis proteins that belong to WD40 protein superfamily.

Taxol is a blockbuster anticancer drug that commercially synthesized from 10-decaetylbaccatin III intermediate of Taxus baccata, however, this technology has been challenged by reproducibility and association of unwanted compounds. Recently, endophytic fungi with fast growing and feasibility of molecular engineering raise the hope for sustainable production of Taxol, unfortunately the loss of their productivity with multiple subculturing is the main challenge to authenticate this approach industrially. Thus, searching for novel fungal isolates with molecular and metabolic functional stability for Taxol production was the objective of this work. Among the experimented fungal isolates, Aspergillus flavipes, a soil-dwelling saprophyte, gave the highest Taxol yield (185 μg/l). Metabolically, A. flavipes had a strong stability for Taxol production till the 10th subcultures (185-160 μg/l) on the same cultural conditions. The yield of Taxol by A. flavipes was increased by 5 folds (850 μg/ L) with fluconazole (1.0 μg/ml) addition to 5 days old culture. The expression of Taxol biosynthetic genes ts, dbat, bapt and transcriptional factor pbcR was significantly increased by 8–10 folds upon fluconazole addition that correlates with Taxol yield. HSP70 was induced by 13 folds with addition of fluconazole to cultures of A. flavipes. Thus, Taxol biosynthesis could be transcriptionally regulated by pbcR and post-translationally modified by HSP70 chaperones. To the best of knowledge, this is the first report exploring the potency of saprophytic A. flavipes for Taxol production with metabolic stability with multiple subculturing.

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response to P. parasitica infection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) among P. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 h post inoculation and 5521 genes were differentially expressed at 48 h post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. Many R genes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response to P. parasitica infection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks to P. parasitica.

Protection of crop plants from phytopathogens through endophytic bacteria is a newly emerged area of biocontrol. In this study, endophytic bacteria were isolated from the rhizosphere of Cannabis sativa. Based on initial antimicrobial screening, three (03) bacteria Serratia marcescens MOSEL-w2, Enterobacter cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 were selected. Antimicrobial assays of these selected bacteria against Phytophthora parasitica revealed that E. cloacae MOSEL-w7 and Paenibacillus sp. MOSEL-w13 possessed strong activity against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4-400 µg mL

In a preliminary plant-based microbiome study, diverse bacterial taxa were identified from different medicinal plants using 16S rRNA gene sequencing. Based on initial antimicrobial screening, eight (8) bacterial endophytes in six (6) different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Arthrobacter, and Delftia, from four important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata, and Calendula arvensis were selected for further analyses. Antimicrobial assays revealed that Pseudomonas taiwanensis MOSEL-RD23 has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis MOSEL-RD23and Bacillus flexus MOSEL-MIC5 inhibited the growth of Phytophthora parasitica by more than 80%. Ethyl acetate extracts of Streptomyces alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, Enterobacter hormaechei MOSEL-FLS1, and Bacillus tequilensis MOSEL-FLS3, and Delftia lacustris MB322 displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition of more than 80% inhibition in vitro against P. parasitica at different concentrations (4-400 µg/mL). Bacterial extracts showing strong antimicrobial activity were selected for bioactivity-driven fractionation and showed anti-Phytophthoral activity in multiple fractions and different peaks observed in UV-Vis spectroscopy. In the detached-leaf assay against P. parasitica on tobacco, 1% ethyl acetate bacterial extract of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1, B. tequilensis MOSEL-FLS3, and D. lacustris MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Overall, P. taiwanensis MOSEL-RD23 showed positive activities for all the assays. Analyzing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for the sustainable production of crops.

This study describes ZnO NPs biosynthesis using leaf extracts of Verbena officinalis and Verbena tenuisecta. The extracts serve as natural reducing, capping and stabilization facilitators. Plant extracts phytochemical analysis, revealed that V. officinalis showed higher total phenolic and flavonoid content (22.12 and 6.38 mg g (−1) DW) as compared to V. tennuisecta (12.18 and 2.7 mg g (−1) DW). ZnO NPs were characterised by ultraviolet–visible spectroscopy, Fourier transform infrared, X‐ray diffraction, scanning electron microscope, transmission electron microscopy (TEM) and energy dispersive X‐ray. TEM analysis of ZnO NPs reveals rod and flower shapes and were in the range of 65–75 and 14–31 nm, for V. tenuisecta and V. officinalis, respectively. Bio‐potential of ZnO NPs was examined through their leishmanicidal potential against Leishmania tropica. ZnO NPs showed potent leishmanicidal activity with 250 µg ml(−1) being the most potent concentration. V. officinalis mediated ZnO NPs showed more potent leishmanicidal activity compared to V. tenuisecta mediated ZnO NPs due to their smaller size and increased phenolics doped onto its surface. These results can be a step forward towards the development of novel compounds that can efficiently replace the current medication schemes for leishmaniasis treatment.

Twenty-four endophytic fungal isolates recovered from Podocarpus gracilior were screened for Taxol biosynthetic potency. Among these endophytes, three isolates of Aspergillus terreus EFB108, EFB59, EFB14 recovered from P. gracilior cork, leaves and twigs, respectively, were reported as potent Taxol producers. The chemical identity of A. terreus extracted Taxol was verified from the HPLC, NMR and FTIR analyses, with strong cytotoxic activity for HEPG2 and MCF7 cell lines (IC50 4.5–6.5 nM). Upon nutritional of A. terreus EFB108 (MF377552), the maximum yield of Taxol (265 μg/L) was obtained by growing the fungus on MID medium of initial pH 8.0, with 0.6% asparagine and 2.4% xylose, incubated for 21 days. Taxol yield of A. terreus was increased by ∼2.4 folds (432 μg/L) upon addition of surface sterilized P. gracilior leaves. The yield of Taxol by A. terreus was not affected by the dichloromethane plant extract negating the presence of direct signals inducing Taxol biosynthesis. So, the intimate physical interaction of endogenous endophytes of P. gracilior with the fungus, could be essential for triggering the biosynthesis of Taxol by A. terreus. This is the first report describing the feasibility of A. terreus, endophytes of P. gracilior, for Taxol production that could be a good industrial platform.

Scutellaria or skullcap is a genus containing approximately 350 species of flowering plants, many of which are sold and marketed for their medicinal value. Flavonoids found in Scutellaria spp. have demonstrated anti-inflammatory, antiviral, sedative, antithrombotic and antioxidant affects. Baicalin, a flavonoid produced by Scutellaria spp., is an important compound used to treat anxiety. Scutellaria spp. have potential as commercially valuable specialty crops based on their visual and medicinal properties; however, a lack of commercial production techniques for successful cultivation of this genus precludes adoption by most growers. The influence of plant production techniques on flowering and baicalin synthesis is undocumented; thus, empirical research is needed for development of commercial production protocol. Objectives of this research were to investigate the effect of nutrient application rate and plant available water on growth and baicalin synthesis in Scutellaria arenicola and Scutellaria integrifolia, two common species of Scutellaria found in eastern United States. To accomplish these objectives, S. arenicola and S. integrifolia were cultivated in a greenhouse and subjected to one out of four nutrient application rates and one out of two volumetric water content rates. Results demonstrated that synthesis of baicalin, the main flavonoid of the Scutellaria genus that contributes to its reported medicinal benefits, occurred in both species. Fertilization rate and volumetric water content were found to influence both plant growth and baicalin concentration in S. integrifolia. In contrast to results observed for S. integrifolia, volumetric soil water and nutrient application rate did not influence plant growth in S. arenicola. Scutellaria spp. cultivated in the greenhouse had similar concentrations of baicalin to those harvested from the wild, undisturbed natural habitats. Results from this investigation will assist in development of commercial production protocol for these species and provides the first foundational research that has reported the presence of baicalin, a high value medicinal compound in S. arenicola. Key words: Skullcap, medicinal, flavonoid, cultivation, production.

Biological control is an eco-friendly strategy for mitigating and controlling plant diseases with negligible effects on human health and environment. Biocontrol agents are mostly isolated from field crops, and microbiomes associated with wild native plants is underexplored. The main objective of this study was to characterize the bacterial isolates associated with Smilax bona-nox L, a successful wild plant with invasive growth habits. Forty morphologically distinct bacterial isolates were recovered from S. bona-nox. Based on 16S rRNA gene sequencing, these isolates belonged to 12 different genera namely Burkholderia, Pseudomonas, Xenophilus, Stenotrophomonas, Pantoea, Enterobactriaceae, Kosakonia, Microbacterium, Curtobacterium, Caulobacter, Lysinibacillus and Bacillus. Among them, Pseudomonas sp. EA6 and Pseudomonas sp. EA14 displayed the highest potential for inhibition of Phytophthora. Based on sequence analysis of rpoD gene, these isolates revealed a 97% identity with a Pseudomonas fluorescence strain. Bioactivity-driven assays for finding bioactive compounds revealed that crude proteins of Pseudomonas sp. EA6 inhibited mycelial growth of P. parasitica, whereas crude proteins of Pseudomonas sp. EA14 displayed negligible activity. Fractionation and enzymatic analyses revealed that the bioactivity of Pseudomonas sp. EA6 was mostly due to glucanolytic enzymes. Comparison of chromatographic profile and bioactivity assays indicated that the secreted glucanolytic enzymes consisted of β-1,3 and β-1,4 glucanases, which acted together in hydrolyzing Phytophthora cell walls. Since the biological activity of the crude glucanolytic extract was >60-fold higher than the purified β-1,3 glucanase, the glucanolytic enzyme system of Pseudomonas sp. EA6 likely acts synergistically in cell wall hydrolysis of P. parasitica.

The genus Artemisia has been utilized worldwide due to its immense potential for protection against various diseases, especially malaria. Artemisia absinthium, previously renowned for its utilization in the popular beverage absinthe, is gaining resurgence due to its extensive pharmacological activities. Like A. annua, this species exhibits strong biological activities like antimalarial, anticancer and antioxidant. Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures. This review elaborates on the traditional medicinal uses of Artemisia species and explains current trends to establish cell cultures of A. annua and A. absinthium for enhanced production of medicinally important secondary metabolites.

Impatiens downy mildew is a highly destructive disease of Impatiens walleriana, and economically important bedding ornamental crop. This disease is caused by a recently emerged pathogen Plasmopara obducens. Since both the host and pathogen are relatively less studied, there are only a few genomic resources available for both I. walleriana and P. obducens. In this study, we have analyzed transcriptional changes in I. walleriana in response to P. obducens infection during different stages of disease development. Our main goal was to identify candidate genes that may be involved in I. walleriana susceptibility to P. obducens. Since the genome of I. walleriana is not available publicly, we constructed and optimized a de novo transcriptome assembly consisting of 73,022 transcripts. Differential expression analysis based on this optimized de novo transcriptome assembly revealed 3,000 to 4,500 differentially expressed transcripts (DETs) at 0 hr, 12 hr, 48 hr, 120 hr, and 240 hr time points after infection. Functional annotation of these DETs revealed that numerous plant stress responsive genes are activated and deactivated throughout the infection cycle. Genes in the calcium signaling pathways, receptor-like kinases (RLKs) including 10 disease resistance associated RLK transcripts, powdery mildew resistance genes (MLO), and many other plant stress related genes were predominantly differentially expressed in I. walleriana in response to P. obducens. Analyses reported here provides molecular insights into the disease susceptibility mechanism of the Impatiens downy mildew, and lays out a strong foundation for future studies aimed at improving downy mildew resistance in I. walleriana.

The Arabidopsis thaumatin-like protein 1 (ATLP1), which belongs to the PR5 family of pathogenesis-related proteins, is induced by pathogen attacks and by systemic acquired resistance (SAR)-inducing compounds. To test whether the overexpression of ATLP1 will enhance fungal resistance in transgenic plants, the cDNA of this gene under the control of a constitutive promoter was transferred into Solanum tuberosum Cv. Desiree. The expression of the introduced gene was confirmed by Northern and Western blot analyses. Western blot analysis performed with intercellular fluid (ICF) showed that ATLP1 is secreted to apoplast. Several independent transgenic lines with high-level expression of the ATLP1 were assayed for resistance against early blight (Alternaria solani) and late blight (Phytophthora infestans). The rate of Alternaria lesions development was significantly reduced in the ATLP1 transgenic lines as compared to a control line. Percent reductions in area under the disease progress curve (AUDPC) values for the ATLP1 transgenic lines as compared to control line ranged between 52 and 82%. In response to P. infestans, infection efficiency (IE), lesion size (LS) and sporulation capacity (SC) were significantly reduced in ATLP1 transgenic lines as compared to a control line. On the average, IE, LS and SC were reduced by 18, 22 and 20%, respectively, for all transgenic lines with a maximum reduction of 25% in IE, 25% in LS and 32% in SC. Resistance assays against P. infestans using whole plants showed 40-70% reduction in symptoms as compared to control. These results suggest that constitutive expression of a heterologous ATLP in potato confers enhanced resistance against early and late blights.

In a preliminary DNA-based microbiome studies, diverse culturable and unculturable bacterial taxa were identified in the roots and rhizospheres of different medicinal plants. In this report, culturable endophytic bacteria were isolated from four economically important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata and Calendula arvensis. On the basis of initial antimicrobial screening, nine bacterial species in seven different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Pantoea, Pseudarthrobacter and Delftia, were selected for further analyses. These bacteria were identified using 16S rRNA gene sequencing. Antimicrobial assays of these selected bacteria revealed that Pseudomonas taiwanensis has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis inhibited growth of P. parasitica more than 80%. Ethyl acetate extracts of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1 and B. tequilensis MOSEL-FLS3 and D. lacustris MB322 also displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4 µg/mL – 400 µg/mL). Bacterial extracts showing higher bioactivity (>80% inhibition in vitro) were selected for detached-leaf assay against P. parasitica on tobacco. In detached-leaf assay, application of 1% ethyl acetate bacterial extract of MOSE L-RD3, MOSEL-RD23, MOSEL-FLS1, MOSEL-FLS3 and MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Over all P. taiwenensis MOSEL-RD23 showed positive activities for all the assays. Analysing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for sustainable production of crops.

Thermal stress induces a wide array of morphological and physiological changes in potato affecting its development and economic yield. Response to thermal stress in plants is mostly regulated by heat shock factors (hsfs). The current study aimed at improving heat tolerance by transforming potato plant with heat shock factor, HsfA1d, using Agrobacterium. Gateway cloning strategy was adopted for isolation of HsfA1d from Arabidopsis thaliana and cloning into plant expression vector. The target gene was introduced into potato by infecting internodal explants with Agrobacterium strain GV3101 carrying pGWB402Ω-HsfA1d construct. Upon exposure to heat stress, the wild-type plants turned yellowish, whereas no phenotypic effect on transgenic plants was observed. Expression of HsfA1d in transgenic plants was increased by 5.8-fold under thermal stress compared to room temperature. Transgenic plants exhibited 6-fold increase in the expression of downstream HSP70 under thermal stress compared to wild-type plants. Both chlorophyll a and b were significantly decreased in wild-type plants while no such decrease was recorded in transgenic plants under thermal stress. Heat stress was found to have no significant effect on carotenoid pigments of both wild-type and transgenic plants. Significantly lower electrolyte leakage from transgenic plants was witnessed compared to wild type upon exposure to thermal stress. Transgenic plants accumulated significantly higher proline content compared to wild-type plants under heat stress. It is concluded that HsfA1d plays a vital role in plant thermotolerance and hence can be effectively used to enhance the resistance of crop plants against heat stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-01043-6) contains supplementary material, which is available to authorized users.

Phytophthora parasiticais one of the most widespreadPhytophthoraspecies, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response toP. parasiticainfection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) amongP. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 hours post inoculation and 5521 genes were differentially expressed at 48 hours post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. ManyRgenes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response toP. parasiticainfection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks toP. parasitica.

In plants, subcellular fluctuations in Ca2+ ion concentration are among the earliest responses to biotic and abiotic stresses. Calmodulin, which is a ubiquitous Ca2+ ion sensor in eukaryotes, plays a major role in translating these Ca2+ signatures to cellular responses by interacting with numerous proteins located in plasma membranes, cytoplasm, organelles and nuclei. In this report, we show that one of the Phytophthora RXLR effector, Avrblb2, interacts with calmodulin at the plasma membrane of the plant cells. Using deletion and single amino acid mutagenesis, we found that calmodulin binds to the effector domain of Avrblb2. In addition, we show that most known homologs of Avrblb2 in three different Phytophthora species interact with different isoforms of calmodulin. Type of amino acids at position 69 in Avrblb2, which determines Rbi-blb2 resistance protein-mediated defense responses, is not involved in the Avrblb2-calmodulin interaction. Using in planta functional analyses, we show that calmodulin binding to Avrblb2 is required for its recognition by Rpi-blb2 to incite hypersensitive response. These findings suggest that Avrblb2 by interacting with calmodulin interfere with plant defense associated Ca2+ signaling in plants.

Chinese elm (Ulmus parvifolia Jacq.) is native to China, Korea, and Japan and was introduced to the U.S. to replace the American elm, which is highly susceptible to Dutch elm disease. Cultivar Emmer II trademark Allee elm (or Allee elm) is preferred by nurseries for its rich green foliage and beauty of bark. In the summer of 2011, a new disease was observed on Allee elm at a tree farm in FL. Approximately 1% of elms in the same farm and in residential areas in central Florida had similar canker-like symptoms consisting of tan to orange patches of decomposed and loose bark. These symptoms were observed on the main trunks often extending into branches of affected trees. Cankered sections of the trunk were often several feet in length and penetrated the wood to a depth just under the bark into the phloem. To isolate the causal organism, cankers were gathered from 7 trees and tissues from the margin of 1 to 3 cankers per tree were surface sterilized in 1% sodium hypochlorite, plated on PDA, and incubated at 22°C under a 16-h/8-h light/dark cycle for 7 days. Colonies displayed white, fluffy mycelium with sporadic black acervuli containing aggregated conidia. Conidia were 5-celled with two or more apical appendages or hairs; the three central cells were dark brown and the two outer cells were hyaline (1). Based on conidial morphology, these isolates were putatively identified as Pestalotiopsis spp. To identify the species, the rDNA internal transcribed spacer (ITS) region was sequenced for two field isolates (GenBank Accessions JX999998 and JX999999). A BLASTn search in GenBank revealed 100% identity to Pestalotiopsis mangiferae ITS (JX305704.1). To test Koch's postulates, experiments were performed in the field and greenhouse. A mycelial plug of isolate 11-40 was grown on PDA, inoculated on wounded trunk of healthy 18-month-old Allee elms (n = 48) in the field and 7-month-old Allee elms (n = 12) in the greenhouse. Both experiments were set up as a randomized complete block design. The trunk of each tree was wounded with a scalpel to a depth of 5 mm, the wound was inoculated with a 5-mm2 agar plug from a 7-day PDA culture, and the inoculated wound was wrapped with grafting tape. Plants that served as negative controls [n = 20 (field experiment) and n = 12 (greenhouse)] were mock-inoculated with a sterile PDA plug without mycelial growth. After 4 to 6 months, symptoms consisting of loose or fallen off bark developed on all pathogen-inoculated trees but not on control trees. Control wounds healed with no expansion beyond the original 5 mm inoculation zone, whereas Pestalotiopsis-inoculated cankers expanded to 3 to 8 cm in each direction in 6 months. Pestalotiopsis (confirmed by conidial morphology and ITS sequencing) was reisolated from pathogen-inoculated trees but not from control trees. These experiments were repeated with similar results at least three times, each consisting of 15 replications (greenhouse) and 12 replications (field) with additional Pestalotiopsis isolates. Many other Pestaliopsis spp. have been reported on other shade trees through the U.S. and the world. To our knowledge, this is the first report of P. mangiferae on an Ulmus species in the U.S. and the world. Since elm canker mainly affects the trunk, a featured characteristic of the Allee elm, it can potentially result in economic loss to the ornamental industry. References: (1) Y. Ko et al. Plant Dis. 91:1684, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Epothilones are secondary metabolites produced by Sorangium cellulosum with powerful antiproliferative activity against tumor cells by stabilizing their microtubule arrays, arresting their cellular division at G2-M phase. Unfortunately, the lower yield of epothilone is the challenge for its higher accessibility, thus, searching for alternative sources with promising epothilone producing potency is the prospective. Endophytic fungi are the potential repertoire for bioactive metabolites, thus exploring the epothilone producing potency of endophytic fungi of medicinal plants was objective. Thirty-two fungal isolates were recovered from the tested medicinal plants and their potency to produced epothilone have been assessed using the TLC, HPLC and molecular markers epoA, epoC and epoK. Aspergillus fumigatus EFBL, an endophyte of Catharanthus roseus, was the potent epothilone producer (21.5 μg/g biomass) as revealed from the chromatographic analyses and PCR of molecular markers. The chemical identity of extracted epothilone was verified from the HPLC, NMR, FTIR and LC-MS analyses as epothilone B analogue. The putative epoA gene from A. fumigatus was amplified using RT-PCR with the conservative corresponding primers to the active-sites of S. cellulosum. The amplicons of epoA was 517 bp displayed 98 % similarity with A. fumigatus PKS-NRPS domains, and 40 % similarity with epoA of S. cellulosum. From the in silico analyses, Val506, Ala605 and Ser630 are the conservative amino acids of epoA protein of A. fumigatus and S. cellulosum. Epothilone B from A. fumigatus displayed a strong antiproliferative activity against HepG-2, MCF-7 and LS174 T as revealed from the IC50 values 6.4, 8.7 and 10.21 μM, respectively. The productivity of epothilone B from A. fumigatus was optimized by surface response methodology with Plackett-Burman and Faced Centered Central Composite. With the Plackett-Burman design, the yield of epothilone (54.4-60.1 μg/g biomass) by A. fumigatus was increased by about 2.8-3.0 folds comparing to non-optimized cultures (21.5 μg/ g biomass). From the FCCD design, sucrose, tryptone and incubation time being the highest significant variables medium components affecting the epothilone yield of A. fumigatus. This is the first report exploring the feasibility of endophytic fungi for epothilone producing potency, that could be a novel platform for industrial production of epothilone.

This report investigates population structure and genetic variability of Phytophthora spp. isolated from botanically diverse plants in Florida. Internal transcribed spacer-based molecular phylogenetic analyses indicate that Phytophthora isolates recovered from ornamental plants in Florida represent a genetically diverse population and that a majority of the isolates belong to Phytophthora nicotianae (73.2%), P. palmivora (18.7%), P. tropicalis (4.9%), P. katsurae (2.4%), and P. cinnamomi (0.8%). Mating type analyses revealed that most isolates were heterothallic, consisting of both mating type A1 (25.2%) and mating type A2 (39.0%), and suggesting that they could outcross. Fungicide sensitivity assays determined that several isolates were moderate to completely insensitive to mefenoxam. In addition, several isolates were also moderately insensitive to additional fungicides with different modes of action. However, correlation analyses did not reveal occurrence of fungicide cross-resistance. These studies suggest that a genetically diverse Phytophthora population infects ornamental crops and the occurrence of mefenoxam-insensitive Phytophthora populations raises concerns about disease management in ornamentals. Mitigating fungicide resistance will require prudent management strategies, including tank mixes and rotation of chemicals with different modes of actions.

Unlike chemical synthesis, biological synthesis of nanoparticles is gaining tremendous interest, and plant extracts are preferred over other biological sources due to their ample availability and wide array of reducing metabolites. In this project, we investigated the reducing potential of aqueous extract of Artemisia absinthium L. for synthesizing silver nanoparticles (AgNPs). Optimal synthesis of AgNPs with desirable physical and biological properties was investigated using ultra violet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDX). To determine their appropriate concentrations for AgNP synthesis, two-fold dilutions of silver nitrate (20 to 0.62 mM) and aqueous plant extract (100 to 0.79 mg ml(-1)) were reacted. The results showed that silver nitrate (2mM) and plant extract (10 mg ml(-1)) mixed in different ratios significantly affected size, stability and yield of AgNPs. Extract to AgNO3 ratio of 6:4v/v resulted in the highest conversion efficiency of AgNO3 to AgNPs, with the particles in average size range of less than 100 nm. Furthermore, the direct imaging of synthesized AgNPs by TEM revealed polydispersed particles in the size range of 5 to 20 nm. Similarly, nanoparticles with the characteristic peak of silver were observed with EDX. This study presents a comprehensive investigation of the differential behavior of plant extract and AgNO3 to synthesize biologically stable AgNPs.

Correction for ‘Variation in surface properties, metabolic capping, and antibacterial activity of biosynthesized silver nanoparticles: comparison of bio-fabrication potential in phytohormone-regulated cell cultures and naturally grown plants’ by Tariq Khan et al., RSC Adv., 2020, 10, 38831–38840, DOI: 10.1039/D0RA08419K.

Phytophthora spp are one of the most devastating pathogens to a wide range of crop plants. Chemical fungicides are commonly used for controlling Phytophthora diseases, however, due to environmental concerns and development of fungicide resistant Phytophthora strains, alternative low-risk method such as biological controls are the prospective approaches. In an in vitro bioactivity-driven screen of rhizosphere-inhabiting fungi, Aspergillus flavipes was identified as a strong inhibitor for growth of various species of Phytophthora. As well as, the crude extracellular extract of broth cultures of A. flavipes displayed a significant growth inhibition of various Phytophthora spp, with EC50 values ranged from 0.018−0.02 units. Bioactivity-driven fractionation analyses revealed that the antiphytophthoral activity was mostly reported in the ethyl acetate fraction. In addition to mycelial growth inhibition, the ethyl acetate fraction inhibited the zoospore germination and sporangial production in P. parasitica. Microscopic analyses of P. parasitica hyphae treated with A. flavipes ethyl acetate extract, revealed severe growth abnormalities such as irregular condensed hyphal aggregations, shorter hypha and thicker cells, in addition to the strongly inhibition of P. parasitica infection and pathogenicity to Nicotiana benthamiana. From the direct co-culturing, P. parasitica and its metabolites had no effect on over elicitation of the active metabolites of A. flavipes. The putative antiphytophthoral compounds from A. flavipes were chemically verified as 3-hydroxy-2′,4,4′,6′-tetramethoxychalocone, 7,3,4,5′-tetramethoxyflavanone, isovitexin and amodiaquine at concentrations 35.8, 9.1, 26.4 and 11.4%, respectively. The cultures of A. flavipes and its active extracts did not display any phytotoxicity to N. benthamiana and S. lycopersicum suggesting their potentially for controlling the diseases of Phytophthora spp.

Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37–40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0–8.0, with putative pI 5.0–5.5, higher structural denaturation at pH 4.0–5.5 and 9.5–12 as revealed from absorbance at λ280nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower Kr value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen.

Pothos is a tropical ornamental vine that has been grown commercially in the US for almost 100 years and is used for a variety of aesthetic purposes. This 5-page publication addresses identification and treatment of common diseases encountered during greenhouse production of pothos. Written by David J. Norman and G. Shad Ali and published by the UF/IFAS Plant Pathology Department, July 2018. http://edis.ifas.ufl.edu/pp340

Rhizoctonia solani causes destructive diseases in many crops throughout the world, resulting in significant yield and quality losses. Early detection of R. solani would facilitate deployment of timely disease management strategies and prevention of spread of asymptomatic infected plant material. The aim of this study was to develop a simple, rapid and sensitive loop-mediated isothermal amplification (LAMP) method for the detection of R. solani in plants and soil samples. LAMP primers, based on the internal transcribed spacer DNA sequence, were designed for detecting most anastomosis groups of R. solani. Using these primers, a LAMP protocol was developed, which in sensitivity tests was shown to detect very low levels of DNA of R. solani and R. zeae, but not R. oryzae. This LAMP protocol successfully detected R. solani on inoculated plant tissues and soil samples. For onsite application in the field, the LAMP protocol was implemented in a generic anti-biotin and anti-fluorescein antibody-based LFD. ...

Cystathionine γ-lyase (CGL) was purified to its electrophoretic homogeneity from Aspergillus carneus by various chromatographic approaches. The purified enzyme has four identical subunits of 52 kDa based on SDS and native PAGE analyses. To improve its structural stability, purified CGL was modified by covalent binding to polyethylene glycol moieties. The specific activity of free-CGL and PEG-CGL was 59.71 and 48.71 U/mg, respectively, with a PEGylation yield of 81.5 and 70.7% modification of surface ε-amino groups. Free- and modified CGL have the same pattern of pH stability (8.0-9.0). At 50°C, the thermal stability [half-life time (T1/2)] of PEG-CGL was increased by 40% in comparison to free-CGL. The activity of CGL was completely inhibited by hydroxylamine and Hg+2, with no effect by EDTA. Free-CGL (0.04 mM-1s-1) and PEG-CGL (0.03 mM-1s-1) have a similar catalytic efficiency to L-cystathionine as a substrate. The inhibition constant values of propargylglycine were 0.31 and 0.52 µM for the free- and PEG-CGL, respectively. By in vitro proteolysis, PEG-CGL retains >50% of its initial activity compared to 1/2 was 19.1 and 28.9 h for the Holo free-CGL and PEG-CGL, respectively, ensuring the role of PEGylation on shielding the CGL surface from proteolytic attack, reducing its antigenicity, and stabilizing its internal Schiff base. By external infusion of pyridoxal 5′-phosphate (10 µM), the T1/2 of free- and PEG-CGL was prolonged to 24 and 33 h, respectively, so dissociation of pyridoxal 5′-phosphate was one of the main causes of loss of enzyme activity. The biochemical and hematological responses of rabbits to free- and PEG-CGL were assessed, with relative similarity to the negative control, confirming the nil toxicity of enzymes. The titer of IgG was duplicated in response to free- versus PEG-CGL after 45 days. To the best of our knowledge, this is the first report concerned with purification and PEGylation of CGL from fungi, with higher affinity for L-cystathionine. With further molecular studies, CGL will be a promising enzyme against various cardiovascular diseases and antioxidant deficiency, as well as for generation of a neurotransmitter (H2S).

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.