Physiological and molecular responses of a resistant and susceptible wheat cultivar to the fungal wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most economically destructive wheat foliar diseases. In this study, we explore the physiological and molecular changes elicited in two wheat cultivars with divergent responses (Taichung 29 = susceptible, and Shafir = resistant) upon infection by Z. tritici. Our aim is to uncover novel insights into the intricate mechanisms that govern wheat defense against Z. tritici infection. Our quantitative histopathological study showed that H2O2 accumulated in the resistant cultivar to a higher degree compared to the susceptible cultivar at the biotrophic and switching phase. Additionally, we combined qPCR with a targeted quantitative HPLC technique to evaluate the expression profiles of 13 defense-related genes and profile the polyphenolic compounds induced differentially in the STB susceptible and resistant cultivar. Our finding indicated that five out of 13 genes were strongly up-regulated in the resistant cultivar compared with that of the susceptible one at eight days post-inoculation (dpi), corresponding to the transition phase present in the infection process of Z. tritici. Finally, our targeted HPLC analysis demonstrated that the traced phenolic compounds were highly elevated in the susceptible cultivar infected by Z. tritici compared with that of the resistant cultivar. In conclusion, our comprehensive analysis unveils a robust defense response in the resistant wheat cultivar Shafir, characterized by heightened H2O2 accumulation, significant up-regulation of key defense-related genes during the transition phase, and a distinct profile of polyphenolic compounds, shedding light on the intricate mechanisms contributing to its resistance against Z. tritici, thereby providing valuable insights for the development of more resilient wheat varieties.

Fungal endophytes Fusarium solani SGGF14 and Alternaria tenuissima SGGF21 enhance the glycyrrhizin production by modulating its key biosynthetic genes in licorice (Glycyrrhiza glabra L.).

AIMS: To identify promising fungal endophytes that are able to produce glycyrrhizin and enhance it in licorice and the mechanisms involved. METHODS AND RESULTS: Fifteen fungal endophytes were isolated from Glycyrrhiza glabra L. rhizomes among which SGGF14 and SGGF21 isolates were found to produce glycyrrhizin by 4.29 and 2.58 µg g-1 dry weight in the first generation of their culture. These isolates were identified as Fusarium solani and Alternaria tenuissima, respectively, based on morphological characteristics and sequence analysis of internal transcribed spacer, TEF1, ATPase, and CAL regions. Subsequently, G. glabra plants were inoculated with these fungal isolates to examine their effect on glycyrrhizin production, plant growth parameters and the expression of key genes involved in glycyrrhizin pathway: SQS1, SQS2, bAS, CAS, LUS, CYP88D6, and CYP72A154. Endophytes were able to enhance glycyrrhizin content by 133%-171% in the plants. Natural control (NC) plants, harboring all natural endophytes, had better growth compared to SGGF14- and SGGF21-inoculated and endophyte-free (EF) plants. Expression of SQS1, SQS2, CYP88D6, and CYP72A154 was upregulated by inoculation with endophytes. LUS and CAS were downregulated after endophyte inoculation. Expression of bAS was higher in SGGF21-inoculated plants when compared with NC, EF, and SGGF14-inoculated plants. CONCLUSIONS: Two selected fungal endophytes of G. glabra can produce glycyrrhizin and enhance glycyrrhizin content in planta by modulating the expression of key genes in glycyrrhizin biosynthetic pathway.

Effect of common horsetail extract on growth characteristics, essential oil yield and chemical compositions of basil (Ocimum basilicum L.).

To investigate the effect of horsetail extract containing high silicon on morphological traits, growth, content, and compositions of essential oil of sweet basil (Ocimum basilicum L.) an experiment turned into carried out in the shape of a randomized complete block design with three replications. Foliar treatment of horsetail extract with zero, 0.5, 1, and 2% concentrations was applied on 6-8 leaf plants. The assessed traits include plant height, number of leaves per plant, number of sub-branches, leaf area index, plant fresh weight, plant dry weight, total anthocyanin, the content of total phenol and total flavonoid, antioxidant activity, essential oil content, and compounds were measured. The findings demonstrated that the increase of silicon-containing horsetail extract enhanced the improved increase in growth and phytochemical trait values. The use of horsetail extract in the 2% treatment increased plant height, the number of leaves per plant, the number of sub-branches, leaf area index, fresh weight, and dry weight of the plant by 49.79, 45.61, 91.09, 99.78, 52.78 and 109.25%, respectively, compared to the control. The highest content of total phenol (2.12 mg GAE/g DW), total flavonoid (1.73 mg RE/g DW), total anthocyanin (0.83 mg C3G/g DW), and antioxidant activity (184.3 µg/ml) was observed in the 2% extract treatment. The content of essential oil increased with increasing the concentration of horsetail extract, so the highest amount of essential oil was obtained at the concentration of 2%, which increased by 134.78% compared to the control. By using GC-MS, the essential oil was analyzed. The main components of the essential oil include methyl eugenol (12.93-25.93%), eugenol (17.63-27.51%), 1,8-cineole (15.63-20.84%), linalool (8.31-19.63%) and (Z)-caryophyllene (6.02-14.93%). Increasing the concentration of horsetail extract increased the compounds of eugenol, 1,8-cineole, and linalool in essential oil compared to the control, but decreased the compounds of methyl eugenol and (Z)-caryophyllene. Foliar spraying of horsetail extract, which contains high amounts of silicon, as a stimulant and biological fertilizer, can be a beneficial ingredient in increasing the yield and production of medicinal plants, especially in organic essential oil production.

Development and Optimization of Culture Medium for the Production of Glabridin by Aspergillus eucalypticola: An Endophytic Fungus Isolated from Glycyrrhiza glabra L. (Fabaceae).

Glabridin is a well-known active isoflavone found in the root of licorice (Glycyrrhiza glabra L.) that possess a wide range of biological activity. Plant cells, hairy roots, and fungal endophytes cultures are the most important alternative methods for plant resources conservation and sustainable production of natural compounds, which has received much attention in recent decades. In the present study, an efficient culture condition was optimized for the biomass accumulation and glabridin production from fungal endophyte Aspergillus eucalypticola SBU-11AE isolated from licorice root. Type of culture medium, range of pH, and licorice root extract (as an elicitor) were tested. The results showed that the highest and lowest biomass production was observed on PCB medium (6.43 ± 0.32 g/l) and peptone malt (5.85 + 0.11 g/l), respectively. The medium culture PCB was produced the highest level of glabridin (7.26 ± 0.44 mg/l), while the lowest level (4.47 ± 0.02 mg/l) was obtained from the medium peptone malt. The highest biomass (8.51 ± 0.43 g/l) and glabridin (8.30 ± 0.51 mg/l) production were observed from the PCB medium adjusted with pH = 6, while the lowest value of both traits was obtained from the same medium with pH = 7. The highest production of total glabridin (10.85 ± 0.84 mg/l) was also obtained from the culture medium treated with 100 mg/l of the plant root extract. This information can be interestingly used for the commercialization of glabridin production for further industrial applications.

Stb6 mediates stomatal immunity, photosynthetic functionality, and the antioxidant system during the Zymoseptoria tritici-wheat interaction.

This study offers new perspectives on the biochemical and physiological changes that occur in wheat following a gene-for-gene interaction with the fungal pathogen Zymoseptoria tritici. The Z. tritici isolate IPO323, carries AvrStb6, while ΔAvrStb6#33, lacks AvrStb6. The wheat cultivar (cv.) Shafir, bears the corresponding resistance gene Stb6. Inoculation of cv. Shafir with these isolates results in two contrasted phenotypes, offering a unique opportunity to study the immune response caused by the recognition of AvrStb6 by Stb6. We employed a variety of methodologies to dissect the physiological and biochemical events altered in cv. Shafir, as a result of the AvrStb6-Stb6 interaction. Comparative analysis of stomatal conductance demonstrated that AvrStb6-Stb6 mediates transient stomatal closures to restrict the penetration of Zymoseptoria tritici. Tracking photosynthetic functionality through chlorophyll fluorescence imaging analysis demonstrated that AvrStb6-Stb6 retains the functionality of photosynthesis apparatus by promoting Non-Photochemical Quenching (NPQ). Furthermore, the PlantCV image analysis tool was used to compare the H2O2 accumulation and incidence of cell death (2, 4, 8, 12, 16, and 21 dpi), over Z. tritici infection. Finally, our research shows that the AvrStb6-Stb6 interaction coordinates the expression and activity of antioxidant enzymes, both enzymatic and non-enzymatic, to counteract oxidative stress. In conclusion, the Stb6-AvrStb6 interaction in the Z. tritici-wheat pathosystem triggers transient stomatal closure and maintains photosynthesis while regulating oxidative stress.

Lysine 2-hydroxyisobutyrylation orchestrates cell development and aflatoxin biosynthesis in Aspergillus flavus.

Lysine 2-hydroxyisobutyrylation (Khib ) is a recently identified post-translational modification (PTM) that regulates numerous cellular metabolic processes. In pathogenic microorganism, although glycolysis and fungal virulence are regulated by Khib , its potential roles in fungi remain to be elusive. Our preliminary results showed that levels of Khib fluctuate over time in Aspergillus flavus, which frequently contaminates crops and produces carcinogenic aflatoxins. However, the perception of Khib function in A. flavus is limited, especially in mycotoxin-producing strains. Here, we performed a comprehensive analysis of Khib in A. flavus, and 7156 Khib sites were identified in 1473 proteins. Notably, we demonstrated that Khib of AflM, a key enzyme in aflatoxin biosynthesis, affected conidia production and sclerotia formation. Furthermore, aflM deletion impaired aflatoxin biosynthesis, and more importantly, strains in which Khib was mimicked by K to T mutation at K49, K179 and K180 sites showed reduced aflatoxin production compared with wild type and ΔaflM complementation strains. These results indicate that Khib at these sites of AflM negatively regulates aflatoxin biosynthesis in A. flavus. In summary, our study revealed the potential roles of Khib in A. flavus, and particularly shed light on a new way to regulate aflatoxin production via Khib .

Inhibition of the Aspergillus flavus Growth and Aflatoxin B1 Contamination on Pistachio Nut by Fengycin and Surfactin-Producing Bacillus subtilis UTBSP1.

In this study, the treatment of pistachio nuts by Bacillus subtilis UTBSP1, a promising isolate to degrade aflatoxin B1 (AFB1), caused to reduce the growth of Aspergillus flavus R5 and AFB1 content on pistachio nuts. Fluorescence probes revealed that the cell free supernatant fluid from UTBSP1 affects spore viability considerably. Using high-performance liquid chromatographic (HPLC) method, 10 fractions were separated and collected from methanol extract of cell free supernatant fluid. Two fractions showed inhibition zones against A. flavus. Mass spectrometric analysis of the both antifungal fractions revealed a high similarity between these anti-A. flavus compounds and cyclic-lipopeptides of surfactin, and fengycin families. Coproduction of surfactin and fengycin acted in a synergistic manner and consequently caused a strong antifungal activity against A. flavus R5. There was a positive significant correlation between the reduction of A. flavus growth and the reduction of AFB1 contamination on pistachio nut by UTBSP1. The results indicated that fengycin and surfactin-producing B. subtilis UTBSP1 can potentially reduce A. flavus growth and AFB1 content in pistachio nut.

Evaluation of genetic variability, rust resistance and marker-detection in cultivated Artemisia dracunculus from Iran.

Artemisia dracunculus L. (tarragon), a small shrubby perennial herb, is cultivated for the use of its aromatic leaves in seasoning, salads, etc., and in the preparation of tarragon vinegar. In the present work, genetic analysis of 29 cultivated individuals of this species was carried out employing 12 ISSR and 11 SRAP markers. A total of 59 (71.64%) and 79 (83.14%) polymorphic bands were detected by 12 ISSR primers and 11 SRAP primer pairs, respectively. High similarity for patterns of genetic diversity and clustering of individuals was observed using two ISSR and SRAP marker systems and combined data. Range of genetic similarity by ISSR markers was 0.14 to 0.95, by SRAP markers was 0.14 to 0.90, while this range varied from 0.18 to 0.91 by ISSR+SRAP. In the UPGMA cluster analysis (ISSR, SRAP and ISSR+SRAP), we always found two clusters, the first cluster included 22 individuals and the second contained seven individuals. The results demonstrated that both ISSR and SRAP methods were suitable for discriminating among the studied individuals and the SRAP markers were more efficient and preferable. The results of multiple regression analysis revealed statistically significant association between rust resistance and some molecular markers that they can provide clues for identification of the individuals with higher rust resistance. The molecular marker-based study of genetic diversity suggests that the germplasm studied representing the kind of variability would be a valuable genetic resource for future breeding. In addition, in situ conservation measures are recommended to preserve the valuable A. dracunculus genetic resources as the most effective and economical approach.

Effect of silver nanoparticles on Oryza sativa L. and its rhizosphere bacteria.

Silver nanoparticles (AgNPs) are widely used as antibacterial and antifungal agents in agriculture. Nevertheless, these nanoparticles with newborn properties pose a potential risk to the environment, Due to contact with crops and bacteria that are beneficial to the soil. This study is based on the examination of the phytotoxic effects of AgNPs on Oryza sativa L. and some of its rhizosphere bacteria, by physiological and biochemical assays. In order to study the complex interaction of the AgNPs life expectancy that are mixed with culture medium, the incubation time for the fresh mixture, 7, 14 and 21 days old of AgNPs, on the seedlings growth was investigated. Results indicated that plant's response to the treatment with AgNPs affected on the cell wall, and that with an increase in its concentration (up to 60 µg/mL). The obtained results of transmission electron microscopy (TEM) exhibited that those particles not only penetrated the cell wall, but they could also damage the cell morphology and its structural features. AgNPs treatment up to 30 µg/mL accelerated root growth and at 60 µg/mL was able to restrict a root's ability to grow. The 30 µg/mL treatment had significant effect on root branching and dry weight. In contrast, shoot growth was more susceptible to the effects of AgNPs treatment. The root content for total soluble carbohydrates and starch demonstrated that despite stable starch content, total soluble carbohydrates showed the tendency to significantly decline in response to AgNPs. However, induction of root branching and photosynthetic pigments can attributed to AgNPs stress based on evidence from the production of the reactive oxygen species (ROS) and local root tissue death. Nine isolates of the genus Bacillus selected and identified according to morphological and chemotaxonomic methods. The AgNPs treatment revolutionized the populations of bacteria as Bacillus thuringiensis SBURR1 was totally eliminated, and Bacillus amyloliquefaciens SBURR5 became the most populated one. Images from an electron microscope and the leakage of reducing sugars and protein through the bacterial membrane, similarly confirmed the "pit" formation mechanism of the AgNPs. Moreover the hypothesis from the growth curve study demonstrated that AgNPs may damage bacterium cell wall and transform them to protoplasts.

Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic taxol-producing fungus from Taxus baccata grown in Iran.

In this study, a total of 25 endophytic fungi were successfully isolated from the inner bark of Taxus baccata grown in Iran by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (ts) gene, which encodes the enzyme catalyzing the first committed step of taxol biosynthesis. Four of 25 isolated endophytic fungi isolates showed PCR positive for the ts gene. Subsequently, taxol and 10-deacetylbaccatin III (10-DAB III) were extracted from culture filtrates and mycelia of the PCR positive isolates and analyzed by high-performance liquid chromatography and mass spectrometry. The analysis showed that one isolate (SBU-16) produced taxol (6.9 ± 0.2 µg L(-1) ) and its intermediate compound, 10-DAB III (2.2 ± 0.1 µg L(-1) ). The isolate SBU-16 was identified as Stemphylium sedicola SBU-16, according to its morphological characteristics as well as the internal transcribed spacer nuclear rDNA gene sequence analysis. Interestingly, this is the first report of the genus Stemphylium as a taxol-producing taxon.