Christopher M. M. Franco

Isolation and Identification of Actinobacteria from Surface-Sterilized Wheat Roots

ABSTRACT This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.

Hydrophobic properties and chemical characterisation of natural water repellent materials in Australian sands

Water-repellency in non-wetting sands is due to hydrophobic waxes present on the surface of sand grains and contained in particulate organic matter present in these sands. This study investigates the physico-chemical characteristics of these natural waxes and compares them to waxes extracted from potential original source materials. Non-polar and polar hydrophobic wax extracts were obtained from whole non-wetting sand, and its individual constituents, and associated organic matter. These included the sand fraction, the intrinsic particulate organic matter, tree litter, eucalyptus leaves, bark, lucerne and lupin plants, and fungi and actinomycetes isolated from these sands. Waxes were characterised for their hydrophobic properties and composition of their chemical constituents. The hydrophobicities of the waxes were assessed by measuring the water-repellency induced after treating acid washed sand with wax extracts. Non-polar and polar wax extracts of the tree litter displayed hydrophobic properties that were similar to the corresponding waxes isolated from non-wetting sand and intrinsic particulate organic matter. Unlike these plant-derived waxes, the microbial wax extracts possessed different hydrophobic properties. Characterisation of the components of the extracted waxes by gas chromatography-mass spectroscopy (GC-MS) analysis revealed a strong similarity in the composition of waxes isolated from non-wetting sand, tree litter and other plant material. The major components found were unbranched and branched C16 to C36 fatty acids and their esters, alkanes, phytanols, phytanes, and sterols. Some of these components were not detected in the microbial waxes. Unextracted samples, as well as wax extracts of non-wetting sand, intrinsic particulate organic matter, tree litter and fresh plant material were further analysed by solution and solid state Nuclear Magnetic Resonance spectroscopy which revealed the relative content of the different chemical species present.

Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins

The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells.

Marine Sponge Derived Natural Products between 2001 and 2010: Trends and Opportunities for Discovery of Bioactives

Marine sponges belonging to the phylum Porifera (Metazoa), evolutionarily the oldest animals are the single best source of marine natural products. The present review presents a comprehensive overview of the source, taxonomy, country of origin or geographical position, chemical class, and biological activity of sponge-derived new natural products discovered between 2001 and 2010. The data has been analyzed with a view to gaining an outlook on the future trends and opportunities in the search for new compounds and their sources from marine sponges.

Studies on non-wetting sands. I: The role of intrinsic particulate organic matter in the development of water-repellency in non-wetting sands

The bulk of the organic matter in sands from the south-east of South Australia is present as discrete particles which constitute 4-6% of the mass of the sand. Hydrophobic particulate organic matter was separated from non-wetting sand obtained from two sites in the south-east of South Australia and characterized with respect to size and ability to induce water-repellency. When heated with both a wettable acid washed sand and a natural sand (non-wetting sand washed free of particulate organic matter), the intrinsic particulate organic matter induced strong water-repellence. The degree of hydrophobicity created was higher with the natural-washed sand than the acid-washed sand which indicated that a precoated surface, even one with a low initial hydrophobicity, has a strong enhancing effect. Particulate organic matter, especially the larger size fractions, acted as a reservoir of waxes or hydrophobic materials which diffused onto the surfaces of sand grains during heating, and particularly during wetting/heating/drying cycles. Thus, intrinsic particulate organic matter plays a substantial role in the development of water-repellency in sandy soils. In addition to hydrophobic waxes which diffuse out under environmental conditions prevalent in the field, water-repellency increases significantly when these hydrophobic particles interact with the natural precoated hydrophobic surface of the sand.

Visualization of an Endophytic Streptomyces Species in Wheat Seed

ABSTRACT Endophytic filamentous actinobacteria were isolated from surface-sterilized roots of wheat plants. Endophytic colonization of germinating wheat seed was examined using one of these endophytes, Streptomyces sp. strain EN27, tagged with the egfp gene. Endophytic colonization was observed from a very early stage of plant development with colonization of the embryo, endosperm, and emerging radicle.

Integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera suspension cultures

Abstract In a suspension culture of Vitis vinifera cells, anthocyanin biosynthesis was enhanced, whereas the cell growth was inhibited, following either the addition of jasmonic acid or light irradiation. The maximum anthocyanin accumulation of 13.8 CV (color value)/g-FCW (fresh cell weight) occurred on day 7 when jasmonic acid was added to the cultures at a final concentration of 20 μM on day 0. This represented an 8.5-fold increase compared with the control culture in the dark. Following the continuous light irradiation of 8000–8300 lux, the maximum anthocyanin accumulation reached was 6.8 CV/g-FCW on day 10, which was 4.8-fold that of the control. A process that combined jasmonic acid treatment and light irradiation resulted in a significant synergistic enhancement of anthocyanin accumulation up to 22.62 CV/g-FCW on day 7. This value was 13.9-fold that of the control. As a result, the maximum anthocyanin production of 2200 CV/l was achieved on day 10, representing a 5.8-fold increase compared with the control.

Effect of microbial inoculants on the indigenous actinobacterial endophyte population in the roots of wheat as determined by terminal restriction fragment length polymorphism.

ABSTRACT The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.

Actinobacterial endophytes for improved crop performance

Increasing numbers of endophytic bacteria are being isolated and identified revealing a rich vein of microbial interaction within a variety of crop plants. In addition, cultivation-independent studies have exposed a broader diversity, with many of the species belonging to culturable genera. Microscopic evidence of endophytic colonisation has been shown in some cases, proving ‘true’ endophytic status and providing an understanding of how these microorganisms gain entry and deploy through their host.