Melatonin alleviates drought impact on growth and essential oil yield of lemon verbena by enhancing antioxidant responses, mineral balance, and abscisic acid content.

Melatonin has recently emerged as a multifunctional biomolecule with promising aspects in plant stress tolerance. The present study examined the effects of foliar-sprayed melatonin (0, 100, and 200 µM) on growth and essential oil yield attributes of lemon verbena (Lippia citriodora) under water-shortage (mild, moderate and severe). Results revealed that melatonin minimized drought effects on lemon verbena, resulting in improved growth and essential oils yield. Drought impositions gradually and significantly reduced several growth parameters, such as plant height and biomass, whereas melatonin application revived the growth performance of lemon verbena. Melatonin protected the photosynthetic pigments and helped maintain the mineral balance at all levels of drought. Melatonin stimulated the accumulation of proline, soluble sugars and abscisic acid, which were positively correlated with a better preservation of leaf water status in drought-stressed plants. Melatonin also prevented oxidative damages by enhancing the superoxide dismutase, ascorbate peroxidase and catalase activities. Furthermore, increased levels of total phenolic compounds, chicoric acid, caffeic acid and chlorogenic acid, as well as ascorbate and total antioxidant capacity in melatonin-sprayed drought-stressed plants indicated that melatonin helped verbena plants to sustain antioxidant and medicinal properties during drought. Finally, melatonin treatments upheld the concentrations and yield of essential oils in the leaves of lemon verbena regardless of drought severities. These results provided new insights into melatonin-mediated drought tolerance in lemon verbena, and this strategy could be implemented for the successful cultivation of lemon verbena, and perhaps other medicinal plants, in drought-prone areas worldwide.

Diversity and Spatiotemporal Distribution of Fungal Endophytes Associated with Citrus reticulata cv. Siyahoo.

Endophytic fungi are characterized as microorganisms found within internal tissues of living plants without any immediate, overtly negative effects. The present study was carried out to isolate, taxonomically characterize and determine the spatiotemporal distribution of endophytic fungi associated with leaf, stem, trunk, and root of mandarin (Citrus reticulata cv. Siyahoo). To do so, the sampling program was done seasonally in four geographically isolated mandarin growing areas of Hormozgan province of Iran, including Siyahoo, Ahmadi, Sikhoran, and Roudan. In total, 702 fungal isolates were obtained from leaf, stem, trunk, and root of healthy mandarin trees divided into 26 distinct morphotypes based on morphological characteristics. The morphotypes were taxonomically characterized through phylogenetic analysis of the ITS1-5.8S-ITS4 rDNA region sequences. Accordingly, 10 different fungal orders from 5 fungal classes were identified, i.e., Saccharomycetes (Saccharomycetales), Eurotiomycetes (Eurotiales), Dothideomycetes (Capnodiales, Pleosporales, Dothideales), and Sordariomycetes (Diaporthales, Hypocreales, Microascales, Togniniales), all from Ascomycota, which represented 97.2% and Ustilaginomycetes (Ustilaginales) from Basidiomycota which represented 2.8% of the isolates. The Aureobasidium pullulans, Penicillium citrinum, and Dothideomycetes sp. were the most frequent isolates. The trunk and leaf showed the highest and lowest total colonization frequency and species richness of endophytic fungi, respectively, in all sampling periods. The results showed that the colonization frequency of endophytes in Hormozgan province was higher in autumn than that in spring, winter, and summer. The trunk showed the maximum diversity of endophytes over all seasons. The Shannon-Wiener (H') and Simpson indices had significant correlation with sampling cites and tissue type and the maximum value of Shannon and Simpson indices (H' = 3.05 and 1 - D = 0.94) was found in the specimens collected from Siyahoo. In conclusion, the three factors (season, location, and tissue type) all in together could determine fungal endophyte composition of C. reticulata.

Molecular Identification of Fungal Endophytes of Medicinal Plant Citrullus colocynthis (L.) Schrad as a Medicinal Plant: Role of Tissue Type and Sampling Location on the Diversity.

Endophytic fungi are an important group of organisms in association with plants which are able to colonize all plant internal tissues and improve their fitness. The present research aims to isolate and identify endophytic fungi of Citrullus colocynthis plant and then investigate the effects of sampling location and tissue type on the fungal endophyte diversity of this plant. To do so, a sampling program was done in 11 geographically isolated C. colocynthis growing areas of Hormozgan province, Iran. For molecular identification of endophytic fungi of C. colocynthis, the internal transcribed spacer region (ITS1-5.8S-ITS4), as a universal DNA barcode marker for fungi, was amplified using primer sets. Totally, 12 taxa (Alternaria solani, Cladosporium halotolerans, Setosphaeria rostrata, Aspergillus niger, A. allahabadii, A. terreus, A. occultus, A. cristatus, Penicillium chrysogenum, Talaromyces purpureogenus, Fusarium sp., and Pseudozyma flocculosa) were isolated. Our findings also showed that the diversity of fungal endophytes isolated from C. colocynthis was affected by the tissue type and sampling site. Accordingly, the leaves and seeds were found to have the highest and lowest rates of endophyte colonization and richness in all sampling seasons, respectively. Simpson's diversity index of 0.8165 in root tissue indicated the high diversity of endophytes in this organ. In addition, Shannon's diversity index in the root (1.846) was higher than that in the other organs. The highest Shannon's and Simpson's indices were observed in Khoon Sorkh and Minab regions. Generally, at least two factors (region and type of tissue) played the most important roles in determining the composition of fungal endophytes in C. colocynthis.

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing.

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3-/4- on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Association analysis and molecular tagging of phytochemicals in the endangered medicinal plant licorice (Glycyrrhiza glabra L.).

Licorice (Glycyrrhiza glabra L.) is a medicinal plant species valued in many countries in Asia and Europe for its phytochemical characteristics. Licorice biodiversity is becoming threatened nowadays in Iran due to increasing demand and a drastic decline of its natural habitats. Therefore, licorice domestication would be necessary in the near future, and molecular breeding would help to introduce genotypes suitable for cultivation. The present study was carried out with 170 individual licorice plants sampled in the wild in 59 localizations in 21 provinces of Iran. The association of 436 polymorphic AFLP markers, produced by 15 primer combinations (EcoRI/MseI), with six phenotypic phytochemical traits was studied. The AMOVA analysis show gene diversity among and within localizations. The population structure analysis identified two main sub-populations with significant genetic variation. Significant associations were identified between three markers (E3/M40-4, E34/M4-12 and E12/M31-15) and glycyrrhizin concentration, and between four markers (E11/M34-12, E11/M34-15, E9/M7-29, and E9/M7-30) and phenolic compounds contents. Markers detected can be useful in the domestication of licorice as well as in breeding programs. Licorice sampled in four localizations (KBA1, KBA2, SKh2 and Fa1) were found to be superior in terms of glycyrrhizin and antioxidants content, and therefore they can be considered as elite genotypes which could be included in the domestication process.

Assessment of genetic diversity in Salvadora persica L. based on inter simple sequence repeat (ISSR) genetic marker.

Studies on the genetic variation in marginal populations and differentiation between them are essential for assessment of best gene conservation strategies and sampling schemes. In this study, ISSR markers were used to establish the level of genetic relationships and polymorphism 50 genotypes of Salvadora persica collected from 6 different regions of Hormozgan province. The ISSR analysis with 9 anchored primers also generated 105 scorable loci, of which 85 were polymorphic (80.95%). Parameters of genetic diversity and its partitioning were calculated. The genetic analysis demonstrated that S. persica maintain relatively high genetic diversity (PIC was 0.63, Na was 1.27 and Ho and He were 0.15 and 0.17 respectively). The coefficient of genetic differentiation among populations based on FST equaled 0.20. Genetic identities between population's pairs were high (mean I = 0.88). These values are high as compared with other widespread congener species. Cluster analysis based on the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) revealed 3 main clusters for the ISSR data. The levels of genetic diversity maintained within populations of S. persica indicate that an appropriate sampling design for ex situ safeguarding should capture the majority of genetic diversity found within these taxa to help ensure the long term viability of this species. Furthermore, it could be inferred that ISSR markers are suitable tools for the evaluation of genetic diversity and relationships within the Salvadora persica.

Effect of drought stress on growth parameters, osmolyte contents, antioxidant enzymes and glycyrrhizin synthesis in licorice (Glycyrrhiza glabra L.) grown in the field.

Glycyrrhiza glabra L. (licorice) is a medicinal species rich in the specialised plant metabolite glycyrrhizin. It has been previously proposed that drought, which is increasing in importance due to the climatic change and scarcity of water resources, can promote the synthesis of glycyrrhizin. The effects of slight, moderate and intense drought (70, 35 and 23% of the regular irrigation, respectively) on growth parameters, osmolyte content, oxidative stress markers, antioxidant enzymes, glycyrrhizin biosynthesis genes and root glycyrrhizin concentration and contents, have been assessed in five Iranian licorice genotypes grown in the field. Drought decreased progressively biomass and leaf relative water contents, and increased progressively osmolyte (proline, glycine-betaine and soluble sugars) concentrations in leaves and roots. Drought caused oxidative stress in leaves, as indicated by lipid peroxidation and hydrogen peroxide concentrations, and increased the activities of antioxidant enzymes in leaf extracts (catalase, peroxidase, superoxide dismutase and pholyphenoloxidase). Drought promoted the synthesis of glycyrrhizin, as indicated by the increases in the expression of the glycyrrhizin biosynthesis pathway genes SQS1, SQS2, bAS, CYP88D6, CYP72A154 and UGT73, and increased the root concentrations of glycyrrhizin with drought in some genotypes. However, the large decreases in root biomass caused by drought led to general decreases in the amount of glycyrrhizin per plant with moderate and intense drought, whereas the slight drought treatment led to significant decreases in glycyrrhizin content in only one genotype. Under intense drought two of the genotypes were still capable to maintain half of the control glycyrrhizin yield, whereas in the other three genotypes glycyrrhizin yield was 22-33% of the control values. Results indicate that under intense drought, with only 23% of the normal water dose being applied, an appropriate choice of genotype can still lead to acceptable glycyrrhizin yields.