Antimicrobial Activity of Agastache Honey and Characterization of Its Bioactive Compounds in Comparison With Important Commercial Honeys.

There is an urgent need for new effective antimicrobial agents since acquired resistance of bacteria to currently available agents is increasing. The antimicrobial activity of Mono-floral Agastache honey produced from Australian grown Agastache rugosa was compared with the activity of commercially available honeys derived from Leptospermum species and with Jarrah honey for activity against clinical and non-clinical strains of Staphylococcus aureus (methicillin-susceptible and methicillin-resistant strains), Pseudomonas aeruginosa, and Escherichia coli. The minimum inhibitory concentration (MIC) for Agastache honey was in the range of 6-25% (w/v) for all species examined. The MICs for Leptospermum honeys were generally similar to those of Agastache honey, but MICs were higher for Super manuka and Jarrah honeys and lower for Tea tree honey. Staphylococci were more susceptible to all honeys than Pseudomonas aeruginosa and Escherichia coli. Pretreatment of honey with catalase increased the bacterial growth at MIC of Tea tree honey (35%), Super Manuka (15%), Jarrah honeys (12%), and Agastache honey (10%), indicating variable contributions of hydrogen peroxide to antimicrobial activity. Manuka and Jelly bush honeys retained their antimicrobial activity in the presence of catalase, indicating the presence of other antimicrobial compounds in the honey. An LC-MS/MS method was developed and used to identify possible antimicrobial phenolic compounds in Agastache honey and flowers, and five commercial honeys. The chemical markers characteristic of Agastache honey and honeys of Leptospermum origin were phenyllactic acid and methyl syringate. Overall, the bioactive compounds with antimicrobial and antioxidant activity in Agastache honey suggested a possible use for topical application and in wound care.

Antimicrobial Activity of Tulsi (Ocimum tenuiflorum) Essential Oil and Their Major Constituents against Three Species of Bacteria.

In recent years scientists worldwide have realized that the effective life span of any antimicrobial agent is limited, due to increasing development of resistance by microorganisms. Consequently, numerous studies have been conducted to find new alternative sources of antimicrobial agents, especially from plants. The aims of this project were to examine the antimicrobial properties of essential oils distilled from Australian-grown Ocimum tenuiflorum (Tulsi), to quantify the volatile components present in flower spikes, leaves and the essential oil, and to investigate the compounds responsible for any activity. Broth micro-dilution was used to determine the minimum inhibitory concentration (MIC) of Tulsi essential oil against selected microbial pathogens. The oils, at concentrations of 4.5 and 2.25% completely inhibited the growth of Staphylococcus aureus (including MRSA) and Escherichia coli, while the same concentrations only partly inhibited the growth of Pseudomonas aeruginosa. Of 54 compounds identified in Tulsi leaves, flower spikes, or essential oil, three are proposed to be responsible for this activity; camphor, eucalyptol and eugenol. Since S. aureus (including MRSA), P. aeruginosa and E. coli are major pathogens causing skin and soft tissue infections, Tulsi essential oil could be a valuable topical antimicrobial agent for management of skin infections caused by these organisms.

Subtracted diversity array identifies novel molecular markers including retrotransposons for fingerprinting Echinacea species.

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.

Fingerprinting the Asterid species using subtracted diversity array reveals novel species-specific sequences.

BACKGROUND: Asterids is one of the major plant clades comprising of many commercially important medicinal species. One of the major concerns in medicinal plant industry is adulteration/contamination resulting from misidentification of herbal plants. This study reports the construction and validation of a microarray capable of fingerprinting medicinally important species from the Asterids clade. METHODOLOGY/PRINCIPAL FINDINGS: Pooled genomic DNA of 104 non-asterid angiosperm and non-angiosperm species was subtracted from pooled genomic DNA of 67 asterid species. Subsequently, 283 subtracted DNA fragments were used to construct an Asterid-specific array. The validation of Asterid-specific array revealed a high (99.5%) subtraction efficiency. Twenty-five Asterid species (mostly medicinal) representing 20 families and 9 orders within the clade were hybridized onto the array to reveal its level of species discrimination. All these species could be successfully differentiated using their hybridization patterns. A number of species-specific probes were identified for commercially important species like tea, coffee, dandelion, yarrow, motherwort, Japanese honeysuckle, valerian, wild celery, and yerba mate. Thirty-seven polymorphic probes were characterized by sequencing. A large number of probes were novel species-specific probes whilst some of them were from chloroplast region including genes like atpB, rpoB, and ndh that have extensively been used for fingerprinting and phylogenetic analysis of plants. CONCLUSIONS/SIGNIFICANCE: Subtracted Diversity Array technique is highly efficient in fingerprinting species with little or no genomic information. The Asterid-specific array could fingerprint all 25 species assessed including three species that were not used in constructing the array. This study validates the use of chloroplast genes for bar-coding (fingerprinting) plant species. In addition, this method allowed detection of several new loci that can be explored to solve existing discrepancies in phylogenetics and fingerprinting of plants.

Plant density-dependent variations in bioactive markers and root yield in Australian-grown Salvia miltiorrhiza Bunge.

The plant density-dependent variations in the root yield and content, and the yield of biomarkers in Australian grown Salvia miltiorrhiza Bunge, a commonly used Chinese medicinal herb for the treatment of cardiovascular diseases, were investigated in a field trial involving six different plant densities. The key biomarker compounds cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantified by a validated RP-HPLC method, and the root yields were determined per plant pair or unit area. There were significant variations (p<0.05) in the root yields and contents and the yields of the biomarkers between the different plant densities. Positive linear correlations were observed between the contents of the three tanshinones, whereas negative linear correlations were revealed between the contents of the tanshinones and salvianolic acid B. The highest root yield per plant pair was achieved when the plants were grown at 45×30 cm or 45×40 cm, whereas the highest root production par unit area was obtained for a plant density of 30×30 cm. The highest contents of the three tanshinones and the most abundant production of these tanshinones per unit area were achieved when the plants were grown at 30×30 cm. However, the highest content of salvianolic acid B was found for a density of 45×40 cm, while its highest yield per unit area was obtained for densities of 30×40 cm or 45×30 cm. The findings suggest that the plant density distinctly affects the root yield and content and the yield of tanshinones and salvianolic acid B in Australian grown S. miltiorrhiza, which may be used as a guide for developing optimal agricultural procedures for cultivating this herb.

Detection of Panax quinquefolius in Panax ginseng using 'subtracted diversity array'.

BACKGROUND: Food adulteration remains a major global concern. DNA fingerprinting has several advantages over chemical and morphological identification techniques. DNA microarray-based fingerprinting techniques have not been used previously to detect adulteration involving dried commercial samples of closely related species. Here we report amplification of low-level DNA obtained from dried commercial ginseng samples using the Qiagen REPLI-g Kit. Further, we used a subtracted diversity array (SDA) to fingerprint the two ginseng species, Panax ginseng and Panax quinquefolius, that are frequently mixed for adulteration. RESULTS: The two ginseng species were successfully discriminated using SDA. Further, SDA was sensitive enough to detect a deliberate adulteration of 10% P. quinquefolius in P. ginseng. Thirty-nine species-specific features including 30 P. ginseng-specific and nine P. quinquefolius-specific were obtained. This resulted in a feature polymorphism rate of 10.5% from the 376 features used for fingerprinting the two ginseng species. The functional characterization of 14 Panax species-specific features by sequencing revealed one putative ATP synthase, six putative uncharacterized proteins, and two retroelements to be different in these two species. CONCLUSION: SDA can be employed to detect adulterations in a broad range of plant samples.

HPLC profiles and biomarker contents of Australian-grown Salvia miltiorrhiza f. alba roots.

Salvia miltiorrhiza f. alba (Baihua Danshen) is a Chinese medicinal herb commonly used for treating cardiovascular disease. It has been grown in Australia, although the quality of its main medicinal part (dried root) has not been assessed. In this study, we investigated HPLC profiles and biomarker contents of Australian-grown S. miltiorrhiza f. alba roots. Patterns of HPLC profiles were established in MeOH, and aqueous extracts in terms of number of common characteristic peaks and their relative retention times. The contents of three tanshinone biomarkers (cryptotanshinone (3), tanshinone I (1), and tanshinone IIA (2)) were significantly higher (p<0.05) in the roots of one-year-old plants than those of two-year-old plants. In contrast, salvianolic acid B (4) content was significantly higher in the roots of two-year-old plants than in those of one-year-old plants. The findings suggest that the biomarker contents in Australian-grown S. miltiorrhiza f. alba roots vary with the growth periods of the plants, which may be important in determining the optimal harvest time for the plant roots with targeted levels of tanshinones and salvianolic acid B (4).

Seasonal variations in bioactive marker contents in Australian-grown Salvia miltiorrhiza roots.

Seasonal variations in contents of bioactive markers in Australian-grown Salvia miltiorrhiza roots were investigated in a two-year field trial. Cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantitatively determined by reversed-phase (RP) HPLC. Similar accumulation patterns were observed for the three tanshinones throughout the trial period, although roots harvested in the first year was found to contain relatively higher contents of these compounds. In contrast, the content of salvianolic acid B was peaked at 250 days after planting in the first year, and subsequently maintained at a plateau level in the second-year period. Linear correlations between the contents of individual tanshinones were observed, but not between those of tanshinones and salvianolic acid B. The findings suggest that tanshinones and salvianolic acid B have different accumulation patterns in Australian-grown Salvia miltiorrhiza roots, which should be critically considered for optimum harvesting of the roots for pharmaceutical applications.

Effectiveness of an innovative prototype subtracted diversity array (SDA) for fingerprinting plant species of medicinal importance.

The accurate identification of medicinal plants is becoming increasingly important due to reported concerns about purity, quality and safety. The previously developed prototype subtracted diversity array (SDA) had been validated for the ability to distinguish clade-level targets in a phylogenetically accurate manner. This study represents the rigorous investigation of the SDA for genotyping capabilities, including the genotyping of plant species not included during the construction of the SDA, as well as to lower classification levels including family and species. The results show that the SDA, in its current form, has the ability to accurately genotype species not included during SDA development to clade level. Additionally, for those species that were included during SDA development, genotyping is successful to the family level, and to the species level with minor exceptions. Twenty polymorphic SDA features were sequenced in a first attempt to characterize the polymorphic DNA between species, which showed that transposon-like sequences may be valuable as polymorphic features to differentiate angiosperm families and species. Future refinements of the SDA to allow more sensitive genotyping are discussed with the overall goal of accurate medicinal plant identification in mind.

Cultivar variations of Australian-grown Danshen (Salvia miltiorrhiza): bioactive markers and root yields.

Danshen (Salvia miltiorrhiza Bunge, Lamiaceae) is a commonly used and highly valued Chinese medicinal herb grown widely in China. In the present work, we studied cultivar variations of Australian-grown Danshen in order to select optimal cultivars for local herbal production. Root yields of seven cultivars, V1-V7, were monitored in a one-year field trial, and bioactive markers, including cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B, were quantitatively determined using a validated RP-HPLC method. Significant variations were found in root yields, root production efficiencies, and contents of the bioactive marker compounds. Linear correlations were observed among the contents of three tanshinones but not among those of tanshinones and salvianolic acid B. Among the cultivars, V6 was the best cultivar for production of tanshinones, and V4 and V5 were best for production of salvianolic acid B. The findings indicate that it is possible to achieve optimal root yields, and high contents of tanshinones and salvianolic acid B by selecting specific Danshen cultivars.

Transcriptional profiling of chickpea genes differentially regulated in response to high-salinity, cold and drought.

BACKGROUND: Cultivated chickpea (Cicer arietinum) has a narrow genetic base making it difficult for breeders to produce new elite cultivars with durable resistance to major biotic and abiotic stresses. As an alternative to genome mapping, microarrays have recently been applied in crop species to identify and assess the function of putative genes thought to be involved in plant abiotic stress and defence responses. In the present study, a cDNA microarray approach was taken in order to determine if the transcription of genes, from a set of previously identified putative stress-responsive genes from chickpea and its close relative Lathyrus sativus, were altered in chickpea by the three abiotic stresses; drought, cold and high-salinity. For this, chickpea genotypes known to be tolerant and susceptible to each abiotic stress were challenged and gene expression in the leaf, root and/or flower tissues was studied. The transcripts that were differentially expressed among stressed and unstressed plants in response to the particular stress were analysed in the context of tolerant/susceptible genotypes. RESULTS: The transcriptional change of more than two fold was observed for 109, 210 and 386 genes after drought, cold and high-salinity treatments, respectively. Among these, two, 15 and 30 genes were consensually differentially expressed (DE) between tolerant and susceptible genotypes studied for drought, cold and high-salinity, respectively. The genes that were DE in tolerant and susceptible genotypes under abiotic stresses code for various functional and regulatory proteins. Significant differences in stress responses were observed within and between tolerant and susceptible genotypes highlighting the multiple gene control and complexity of abiotic stress response mechanism in chickpea. CONCLUSION: The annotation of these genes suggests that they may have a role in abiotic stress response and are potential candidates for tolerance/susceptibility.

Construction and validation of a prototype microarray for efficient and high-throughput genotyping of angiosperms.

Until recently, the identification of plants relied on conventional techniques, such as morphological, anatomical and chemical profiling, that are often inefficient or unfeasible in certain situations. Extensive literature exists describing the use of polymerase chain reaction (PCR) DNA-based identification techniques, which offer a reliable platform, but their broad application is often limited by a low throughput. However, hybridization-based microarray technology represents a rapid and high-throughput tool for genotype identification at a molecular level. Using an innovative technique, a 'Subtracted Diversity Array' (SDA) of 376 features was constructed from a pooled genomic DNA library of 49 angiosperm species, from which pooled non-angiosperm genomic DNA was subtracted. Although not the first use of a subtraction technique for genotyping, the SDA method was superior in accuracy, sensitivity and efficiency, and showed high-throughput capacity and broad application. The SDA technique was validated for potential genotyping use, and the results indicated a successful subtraction of non-angiosperm DNA. Statistical analysis of the polymorphic features from the pilot study enabled the establishment of accurate phylogenetic relationships, confirming the potential use of the SDA technique for genotyping. Further, the technique substantially enriched the presence of polymorphic sequences; 68% were polymorphic when using the array to differentiate six angiosperm clades (Asterids, Rosids, Caryophyllids, Ranunculids, Monocots and Eumagnoliids). The 'proof of concept' experiments demonstrate the potential of establishing a highly informative, reliable and high-throughput microarray-based technique for novel application to sequence independent genotyping of major angiosperm clades.

Transcriptional profiling of chickpea genes differentially regulated by salicylic acid, methyl jasmonate and aminocyclopropane carboxylic acid to reveal pathways of defence-related gene regulation.

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes and grasspea (Lathyrus sativus L.) expressed sequence tags, chickpea responses to treatments with the defence signalling compounds salicylic acid (SA), methyl jasmonate (MeJA) and aminocyclopropane carboxylic acid (ACC) were studied in three chickpea genotypes with ranging levels of resistance to ascochyta blight [Ascochyta rabiei (Pass.) L.]. The experimental system minimised environmental effects and was conducted in reference design, where samples from untreated controls acted as references against post-treatment samples. Microarray observations were also validated by quantitative reverse transcription-polymerase chain reaction. The time-course expression patterns of 715 experimental microarray features resulted in differential expression of 425 transcripts. The A. rabiei resistant chickpea genotypes showed a more substantial range of defence-related gene induction by all treatments, indicating that they may possess stronger abilities to resist pathogens. Further, the involvement of SA, MeJA and ACC signalling was identified for the regulation of some important A. rabiei responsive transcripts, as well as cross-talk between these pathways. In the current study we also found evidence to suggest the involvement of A. rabiei-specific signalling mechanisms for the induction of several transcripts that were previously implicated in A. rabiei resistance. This study characterised the regulatory mechanisms of many chickpea transcripts that may be important in defence against various pathogens, as well as other cellular functions. These results provide novel insights to the molecular control of chickpea cellular processes, which may assist the understanding of chickpea defence mechanisms and allow enhanced development of disease resistant cultivars.

Functional genomics in chickpea: an emerging frontier for molecular-assisted breeding.

Chickpea is a valuable and important agricultural crop, but yield potential is limited by a series of biotic and abiotic stresses, including Ascochyta blight, Fusarium wilt, drought, cold and salinity. To accelerate molecular breeding efforts for the discovery and introgression of stress tolerance genes into cultivated chickpea, functional genomics approaches are rapidly growing. Recently a series of genetic tools for chickpea have become available that have allowed high-powered functional genomics studies to proceed, including a dense genetic map, large insert genome libraries, expressed sequence tag libraries, microarrays, serial analysis of gene expression, transgenics and reverse genetics. This review summarises the development of these genomic tools and the achievements made in initial and emerging functional genomics studies. Much of the initial research focused on Ascochyta blight resistance, and a resistance model has been synthesised based on the results of various studies. Use of the rich comparative genomics resources from the model legumes Medicago truncatula and Lotus japonicus is also discussed. Finally, perspectives on the future directions for chickpea functional genomics, with the goal of developing elite chickpea cultivars, are discussed.

Expression profiling of chickpea genes differentially regulated during a resistance response to Ascochyta rabiei.

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) expressed sequence tags (ESTs) and lentil (Lens culinaris Med.) resistance gene analogues, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, in which samples from mock-inoculated controls acted as reference against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye swap), the inclusion of negative controls and strict selection criteria for differentially expressed genes, including a fold change cut-off determined by self-self hybridizations, Student's t-test and multiple testing correction (P < 0.05). Microarray observations were also validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The time course expression patterns of 756 microarray features resulted in the differential expression of 97 genes in at least one genotype at one time point. k-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and A. rabiei-susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, Ca-binding protein and several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on the functional validation of the genes of interest.